Historical And Global Treatment Of Trade Unionists History Essay

In the early 19th century the economic situation for the majority of the United Kingdom’s workers was dire. The influx of veterans from the Napoleonic wars seeking work and the loss of government contracts to supply the army had a major impact on the country’s economic woes. The position was particularly bad for the many thousands of agricultural workers living in abject poverty in the British countryside. Many farm labourers lived in tied houses on starvation level wages. For many the Enclosure Act had led to the loss of the common land which their families used to graze animals, the winter work of cereal threshing was under threat from the landowner’s purchase of threshing machines and the poor harvests of 1828 and 1829 resulted in rising food prices and falling wages.

The lives of the country’s industrial workers in the towns and cities were no better. Workers and their families lived in insanitary conditions and for the most part worked long hours in unsafe and unhealthy conditions.

The “Luddites” and “Machine Breakers”

The Luddite movement began when British textile artisans protested at the changes in production methods brought about during the industrial revolution. They saw their livelihoods being threatened by the introduction of large mills and their fellows being forced to work in degrading conditions.

In 1811 and 1812 mills and factory machinery were burned by handloom weavers and for a time action was so widespread that it led to clashes with the army and in 1812 a mass trial in York at which many were sentenced to death or transportation,

Although short lived the movement is indicative of the rising tide of English working class discontent in the early 19th century.

The “Swing Riots”

The Agricultural variant of the Luddite movement may be seen in the “Swing Riots”

In the autumn of 1830 unrest among farm workers erupted in a series of violent protests across southern and eastern England. Threshing machines were attacked and hayricks burnt. During the years 1830 and 1831 over 700 incidents were recorded. Greedy and unpopular employers were sent letters ordering them to provide better wages and conditions for their workers, the letters were signed by the fictitious “Captain Swing”. Some farmers were compelled to maintain a reasonable wage level due to these activities although they were certainly unpopular with their peers for doing so. It may perhaps be said that it is unfortunate that Swing did not exist, if he had the protests may have been better organised rather than the relatively random acts that took place and so may have been more effective in achieving their goals.

The response to the incidents was however very severe. The belief of the ruling classes was that good behaviour among the masses could only be ensured by the threat of horrendous punishments, 250 were sentenced to death, most had their sentence reduced to imprisonment or transportation, but 9 people were executed, this number included a 12 year old boy, and over 1000 were jailed or transported for their part in the riots. Even given the fear and distrust of the working classes by their supposed intellectual and moral superiors (which in many cases merely meant richer and better fed) the figures are staggering. The men and boys involved in the incidents were for the most part honest, hardworking citizens who were driven to desperate measures by the intransigence of those willing to watch their workers and their children slowly starve to death. In fact many of those convicted had done no more than attend a meeting of like minded individuals. The peasants’ revolt had been put down but the cause of the unrest had not been dealt with.

Friendly Societies

During the medieval period the Guilds provided organisation for tradesmen and artisans. Part of their function was to help provide mutual aid for the Guild members and their families in time of difficulty. They also sought to control wages and production levels and set the level of skills of their crafts.

When the unskilled masses sought similar protection they looked to the Friendly Societies for aid. Members would make small weekly or monthly contributions into a fund to help to provide financial support in time of sickness or to provide funeral benefits. These societies started in the late 17th century but saw a huge expansion in the 19th century when more and more unskilled workers gained employment in the mills and factories of post industrial revolution Britain.

The origin of the British trade union can therefore be seen in both of these organisations and it can be argued that they the result of an amalgamation of them both.

Tolpuddle

The area around Tolpuddle had been involved in the “Swing” incidents and this was to be a factor in the treatment of 6 tolpuddle men. At the time Tolpuddle was a small village in Dorset and no different from many others in the county, it was however, the home of George Loveless who history tells us was the leader of those 6 men who were to become known as the Tolpuddle Martyrs.

George Loveless was an intelligent man who had taught himself to read and write and had gained a sufficient standard of education and of theology to become a Methodist lay preacher. He was dismayed that no matter how hard he worked he was decreasingly able to support his family on his wages. Acting in a dishonest or illegal manner would not have been acceptable to him but he decided that this situation was unfair and unjust and that something had to be done about it. It is believed that George sought advice from one of the Friendly Societies and he and the other 5 men formed a union of their own in order to petition their employer for better pay and took an oath not to betray each other.

This was not the formation of a trade union in the modern sense but I believe the events that followed and the consequent public response led to the formation of the modern trade union movement.

The Tolpuddle men were eventually arrested and convicted of administering an unlawful oath. Being a member of a trade union was legal at this time so the magistrates (who had a vested interest in seeing an example being made of these men) convicted them using an archaic piece of naval law and they were sentenced to 7 years transportation to Australia. It is in the actions that took place in Britain to secure their release that we see the birth of effective trade union organisation.

The Action of The New Unions

The fledgling unions were quick to realise that the transportation of the “Tolpuddle Martyrs” was a direct attack on them and started to organise actions to bring about the men’s release.

Petitions were raised and meetings and demonstrations took place throughout the country. At the largest of these in 1834 up to 100,000 supporters marched through London to deliver a petition demanding the release of the 6 farm labourers from Dorset.

Eventually the government bowed to pressure and on 14th March 1836 it was agreed that the men would receive a full and free pardon.

It is within this process that we see the types of action still in use today by trade unions around the world. Mass demonstrations, petitions (although we are likely to use e-mail to gather signatures), political support being sought from M.P’s, etc. are all tools we would expect to see today. I can only look back in admiration at the skills of those early unionists who almost 200 years ago were able to raise such a high level of support and stir up such public outcry. It is indeed unfortunate that so little record remains of their speeches and letters.

Trade unions grew up in Europe and America from the mid 1800’s to the present day on broadly similar lines.

The Situation Today

Here in the United Kingdom today we enjoy legal protection from persecution on grounds of trade union membership and activity. Some of this protection dates back to the 1870’s and the influence of that early work done following the Tolpuddle incident cannot be ignored. Although every year cases arise of disregard of this protection which are then usually dealt with successfully by the unions and the courts, we are free to express ourselves and to take action without fear of reprisal or physical harm to ourselves and our families. Although here in Britain the catering company Gate Gourmet dismissed 670 trade unionists for taking industrial action. American giant WalMart have also offered financial inducements to UK employees to leave the union. Is this the case for our trade union brothers and sisters around the world? Globally it is true that most democratic countries offer protection of the rights of individuals to belong to a union, however, in all too many parts of the world there are horrendous human rights violations taking place every day against trade union activists.

According to data provided by the International Confederation of Free Trade Unions (ICFTU) in 2002,

10,000 unionists were sacked

4,000 unionists were arrested

1,000 unionists were injured

Over 200 unionists were killed.

These figures would seem to indicate that globally unions are under attack and membership is being discouraged in many parts of the world by unscrupulous employers often with the backing (albeit covertly in most cases) of their governments.

The European Situation

Across Central and Eastern Europe resistance to the formation of independent trade unions has been a common trend among both employers and the State. In Poland the ICTFU reported at least 7 cases of workers being intimidated until they left the union in 2006.

Coca Cola appears to have carried out some of more serious violations. Union members at the St. Petersburg branch faced unjustified reprimands, insults, threats of dismissal and obstruction of collective bargaining. At the Coca Cola plant in Volzhsky workers were subjected to anti-union harassment and told they would lose their jobs if they joined the union.

Turkey saw the violent repression of a demonstration of the teachers union with 10 arrests and 17 injured. The metal workers union saw 164 members dismissed and 275 forced to resign, while 50 oil workers union members were unfairly dismissed.

In the Russian Federation a trade unionist who had received death threats died when his house caught fire.

Attempts to move away from collective bargaining are becoming increasingly common throughout European employers.

Asia and the Pacific

In many countries in this region the process of taking strike action is so cumbersome that most strikes in the region are technically illegal.

Hundreds of striking workers were injured in Bangladesh when police attacked workers in 3 separate incidents. They were protesting at management ill treatment which included physical violence.

Police intervention in India led to 300 trade unionists being seriously injured.

Cambodia, South Korea and the Philippines saw workers, union leaders and activists injured with impunity in police actions.

Deaths of unionists occurred in Bangladesh, South Korea and the Philippines. In the Philippines Diosdado Fortuna, leader of the food and drug industry union was shot dead, Victoria Ramonte of the Andres Soriano College Employees union was stabbed to death,

Ricardo Ramos, President of the Sugar Workers Union was shot and killed and the local union president of a transport group also died at the hands of gunmen.

Women are frequently the target of anti-union activity in the region, this is due in main to the fact that many of the employees in the electronics, textiles, public sector and education are women.

Dozens of activists jailed in China in previous years are still incarcerated.

The government of Australia has shown itself to be one of the severely anti-union in the industrialised world. It has introduced legislation to restrict trade union organising and rights to collective bargaining. These laws gave employers powers to push workers into individual contracts and also removed their protection from unfair dismissal in many cases.

Africa

In several African countries no unions are allowed in the public and civil services, another problem is the difficulty of taking part in legal industrial action. The repression of the right to strike has led to fatalities on 3 occasions.

In South Africa strikes and protests have been broken up using stun guns, tear gas and rubber bullets. Unsurprisingly this has led to many injuries and deaths, ironically one of these occurred at a protest at the arrests and beatings that had taken place at earlier protests.

In the Nigerian oil industry 170 workers were dismissed for asking for better working conditions and the General Secretary of the Nigeria Labour Congress had his passport seized as he tried to board a plane to attend the International Labour Organisation (ILO) conference in Switzerland.

The leaders of the Zimbabwe Confederation of Trade Unions have received death threats, several faced physical attacks and many were arrested. A teacher’s union leader was tortured and other protesters badly beaten.

No independent trade unions are permitted in Sudan, Egypt or Libya.

The Middle East

This is an area of great unrest and turmoil, where the imprisonment and deaths of union activists has become an almost common occurrence to the horror of people around the world. In Iraq in 2006 there were several assassinations of union leaders along with some unsuccessful assassination attempts and at least 2 kidnappings. One union, the Union of Mechanics, Printing and Metalworkers, reported the deaths of at least 10 of its members.

Most recently in Iran the world became aware of the execution of Farzad Kamanger, a Kurdish teacher and trade unionist, imprisoned, beaten and convicted in a farcical trial where the courts “lost” his notes at the appeal hearing.

Despite global condemnation of the treatment he and fellow unionists received at the hands of the Iranian government Farzad was executed in May 2010.

On a brighter note, Qatar followed the example set by Bahrain and adopted a labour code which allows for the formation of independent trade unions but this law still falls below international labour standards. In Bahrain itself a law was passed to restrict legal strike action.

Due to the continued security issues in Israel and Palestine, it is very difficult for the Palestinian trade unions to carry out their normal duties.

There are no trade union rights in Saudi Arabia or Oman, the law there does not recognise the right to form trade unions.

The Americas

In the “land of the free” U.S. governments are very anti-union. Union busting is rife with concerted efforts being made at many employers to reduce and eliminate trade union membership.

In Canada the WalMart supermarket chain, smarting from the success of its workers in Jonquiere Quebec, in setting up the first WalMart employees union in North America, promptly closed the store. Elsewhere WalMart use intimidation, electronic spying and other measures to discourage the setting up of unions at its other branches.

In Colombia in 2006 at least 70 people were killed for their trade union activities and at least a further 260 received death threats. This was a significant decrease in deaths of activists but the alarming fact remains that trade unionists are being targeted by armed groups with impunity. Violence against women trade unionists steadily increased throughout 2006 and it is with good reason that people in the area say that to become a trade union activist is “to walk around with your tombstone on your back”. No words can adequately express the admiration trade unionists around the world should feel towards these brave men and women.

There have also been union members killed in Brazil and Honduras where Francisco Cruz Galeano of the National Trade Union Centre was shot 25 times.

Death threats remain a common occurrence in many South American countries.

In Ecuador members of the banana workers unions face many problems. At the San Jose plantation 44 workers were dismissed for forming a union and overall in 2006 a further 250 workers lost their jobs for trying to form a union and requesting collective bargaining.

In Peru a telecommunications company sacked 23 union members shortly after the union was formed and threatened others with dismissal and transferred others to lower paid jobs. Very similar tactics were used by 2 soft drinks multinational companies.

Some Mexican union members told the ICFTU that in the garments industry organising had to be carried out without the employer’s knowledge.

What Can Be Done?

I believe that if we could muster support of the levels seen in the 1830’s international pressure would lead to improvements of the rights and treatment of trade unionists worldwide. Many campaigns have been launched via the internet but perhaps because computers are impersonal they are not as effective as the massed marches of old.

Has our relatively comfortable existence made us apathetic to the suffering and problems of others or is it simply a case of our ignorance because these matters are not news worthy enough to receive prime time coverage? It could be that the legacy of the Thatcher years and the anti-union spin of the governments and media have left the average citizen cold to the suffering of workers in other parts of the globe. Very few people seem to be aware of the working conditions of their fellow human beings who provide us with the goods we crave in our consumer society. Some good work has been done by the makers of television documentaries highlighting the plight of sweat shop workers. It is unfortunate that these were not given more coverage in the press or shown on the main channels in place of the escapist soap operas.

By writing to our local politicians and raising their awareness of the strength of feeling about these issues we can push them to discuss the problems at government level. Ultimately public knowledge of the issues, brought to bear on local, national and global governments to change legislation and to influence employers will eventually change the situation.

The formation of global unions such as the partnership between the UK union Unite and the US Teamsters union may be able to help bring pressure to bear on the governments of the world to pass legislation to improve and to protect the rights of fellow trade unionists, but pressure must also be brought to bear by individuals who are prepared to speak out against injustice and to show at least some of the courage and determination shown by our forefathers and those still fighting to establish and defend their rights.

Books and other sources:

Fraser Hamish W. (1999) A History Of British Trade Unionism 1700-1998 Macmillan Press

International Confederation of Free Trade Unions 2006 Annual Survey of Violations of Trade Union Rights www.ifftu.org

BBC News www.bbc.co.uk

Tolpuddle Martyrs Museum www.tolpuddlemartyrs.org.uk

Logan John. U.S. Anti Union Consultants: A Threat to the Rights of British Workers T.U.C

This is Preview only. If you need the solution of this assignment, please send us email with the complete assignment title: ProfessorKamranA@gmail.com

Risks Of The Oil Spill In Floreffe Environmental Sciences Essay

 

In 1988 an Ashland Oil tank was being filled in Floreffe, Pennsylvania. During the fill, the tank collapsed, releasing three and a half million gallons of petroleum oil into water dikes. The resulting oil slick contaminated drinking water for over one million people. Contamination of the drinking water is not only a huge inconvenience, but also a health risk for all those who could consume the drinking water, in addition to a legal liability for Ashland Oil Inc.1

Environmental Risks: Oil does not blend with salt or fresh water. It then floats on the surface spreading out in a think layer. This is known as a slick. Most slicks are 0.01 mm or less thick. The slick can be spread further across the surface due to the weather, as well as water currents and waves. As times goes on the oil can breakdown and sink to the bottom of the ocean continuing to contaminate the body of water.2

The lengthy amount of time that oil can remain in the water source that it contaminated can then begin to damage the water’s wildlife. Ingestion of oil by fish will poison these animals if they survive the destruction of their habitat. Ingestion of either contaminated marine life or contaminated water could be very damaging to the health of any human relying on the water source.2

Measures to improve water safety: The Safe Drinking Water Act (SDWA) was established in 1974. Its purpose is “to protect public health by regulating the nation’s public drinking water supply.” This act applies to any public water system that is located within the Unites States. Over time and through many amendments the act now requires many different actions to protect the water source in addition to the actual drinking water. The Environmental Protection Agency (EPA) sets the standards used and regulated through the SDWA. The act includes a number of different possible contaminated that need to be watched for. They include chemicals, animal waste, pesticides, underground waste, human threats, and naturally substances. This includes oil contamination. The standards that are set by the EPA are enforceable by law. If water systems are not meeting the standards, action can and will be taken until the standards are met.3

In 1948 the Clean Water Act (CWA) was developed. It was amended to its current status in 1977. The CWA is used to regulate the amount of pollution that is disbursed into any U.S. water. The act also helps to regulate the standards for U.S. surface waters. The Clean Water Act allows the EPA to use pollution control programs. These programs include things such as setting standards for industry wastewater. The EPA was also able to make it against the law to disburse any form of pollution from a point source without a permit.4

Ashland Oil, Inc.: Ashland Oil was the largest oil refiner in the United States when this spill occurred. On January 2nd, 1988 one of Ashland’s oil tanks ruptured while it was being filled. The oil company itself constructed the tank that ruptured. It was constructed using steel that was 40 years old. Standards at the time allowed the use of old steel to build tanks as long as a permit was obtained, but like any other oil tank it had to meet the American Petroleum Industry standards set forth. These standards involve the use of a hydrostatic water method. This tank, however, was tested with alternative method that is only acceptable to use in areas where water is scarce. This meant that not only were the best methods available not used in testing the tank, but also a permit was never received in the first place to use the older material in the first place. This presented quite a few legal ramifications for the oil company.1 These ramifications included EPA standard violations, as well as SDWA and CWA violations.5

3. Key constituencies

Public—The general public expects to have clean drinking water and not be exposed to any contamination that may have occurred. Exposure to high levels of contamination, including oil contamination, can lead to severe public health risks and possible panic amongst the public if exposure is realized and there is not method of correction.

Oil tanking industry—This is the main focus because of the adverse impact the oil spill could have on other companies.

Governmental agencies in general—They are a central point in regulating public drinking water sources and making sure that the public’s safety is kept through these governmental regulations

Environmental Protection Agency—The EPA regulates the standards of public drinking water sources that are set by the Safe Water Drinking Act and the Clean Water Act.

Legal System—Violations of the Safe Water Drinking Act and Clean Water Act would be passed on to the appropriate governmental agency for any oil spill and resulting slick that resulted.

Core Public Health Disciplines Involved

This case is typical one that involved almost every public health discipline. These disciplines are:

Environmental Health: this is the main discipline in the case. Exposure to toxic and poisonous substances contaminated water, aquatic life, soil, and air.

Epidemiology: this discipline played an important role in establishing a source of exposure, characterizing the exposure and the disease that occurred, ascertaining trends of occurrence and risk of exposure. It also helps mapping the spill area and proposes appropriate community surveillance.

Health Administration/policy: this section helps in addressing legal issue relating to this spill and help formulating adequate response to the catastrophe. It definitely helps in formulating new laws to proactive react in any future spill that might occur or reinforce existing laws in order to provide clarity on the legal processes and actions.

Biostatistics: this section facilitates in detecting the causes, risks, and exposures of different factors. New investigation is needed detect additional parameters that can accelerate the contamination.

Health Education/Behavior: after the spill, education is important in providing the community or public proper information on the risks of the oil spill and the actions being taken to help preserve the quality of life of the public. Also, the discipline should prepare the educational materials for public.

Cross-discipline domains involved

Additional disciplines are involved in this case. These cross-discipline domains are:

Communication and Informatics: because the oil spill involved many regions and states in the U.S., coordination imposes to standardize and share information on oil spill as they happened elsewhere. Expertise at multiple states and regions levels should be oriented and targeted in order to provide corrective actions in a timely manner.

Diversity and Culture: public or community affected by the oil spill lived in three states and different regions within a state. Therefore, the methodology to address the problem should take into account such diversity.

Leadership: the surprise of the oil spill and the size of the spill created important pressure from affected community and the Ashland Oil staff. The person in charge should be able to supervise responsibilities and be ready to deal with various concerns involved.

Professionalism: Ashland oil staff was unprepared and unprofessional when it comes to deal with the oil spill. The staff showed an extremely poor leadership and professionalism such reacting late to the oil spill, providing false information about tank certification, and focusing on the company financial future instead of being concern about community and environmental health. In this case, public health professional should push for actions and solutions to defend public good.

Program Planning: View the damage of this oil spill in public and the environment; protocols should be set to minimize uncertainty and inconsistent of reaction. The impact of this type of catastrophe in community and wild life calls for effort. This effort should be devoted in getting some emergency planners that can time to time simulate an oil spill in other to test not only the employers’ readiness and community awareness but also the company’s equipment and automatism.

Public Health Biology: this section help in providing exposure risks and biological susceptibility.

C. External disciplines

1. Law:

In September 1988, Ashland Oil Company was indicted by a federal grand jury for negligently discharging oil into the Monongahela River in violation of section 311(b)(3) of the Clean Water Act. The Clean Water Act of 1972 (CWA) was designed to help enforce preservation of water quality. It is the principal federal statute protecting navigable waters and adjoining shorelines from pollution, and has formed the foundation for regulations detailing specific requirements for pollution prevention and response measures. Section 311 of the CWA addresses pollution from oil and hazardous substance releases, providing EPA and the U.S. Coast Guard with the authority to establish a program for preventing, preparing for, and responding to oil spills that occur in navigable waters of the United States.

In 1973, EPA issued the Oil Pollution Prevention regulation (codified at 40 CFR Part 112), to address the oil spill prevention provisions contained in the Clean Water Act of 1972. The regulation applies to non-transportation-related facilities that: (1) Have an aboveground storage capacity of more than 660 gallons in a single tank, an aggregate aboveground storage capacity of more than 1,320 gallons, or a total underground storage capacity of 42,000 gallons, and; (2) could reasonably be expected to discharge oil in harmful quantities into navigable waters of the United States.

This regulation requires each owner or operator of a regulated facility to prepare a spill prevention, control, and countermeasures (SPCC) Plan. It is required to address the facility's design, operation, and maintenance procedures established to prevent spills from occurring, as well as planned measures to control, contain, clean up and mitigate the effects of an oil spill that could affect navigable waters.

Following Ashland's massive oil spill, the EPA formed the SPCC Task Force to examine federal regulations governing oil spills from aboveground storage tanks. The Task Force recommended that certain provisions in the Oil Pollution Prevention regulation were clarified, as well as establishing additional technical requirements for regulated facilities, and to require the preparation of facility-specific response plans. In 1990, the Oil Pollution Act was enacted by Congress - one of the regulations requiring certain oil storage facilities to prepare facility response plans.

2. Political Science:

Is it legal to engage in the negligent conduct, which led to the oil discharge into the river? The main thrust of the implications of this case are that owner-operators of facilities with hazardous materials are legally bound to protect those resources from being dangerously mishandled. As a part of the oil industry, Ashland was had the responsibility to ensure that the tanker with oil was properly insured against problems. Three things that came under scrutiny in the preliminary investigation were: age of the oil tanker, testing that had been done before filling it, and whether a permit had been filed to use the tanker.

The tanker had been transported from Cleveland, and many were under the impression that it was a new tank. Upon more investigation, it was found that while the tank had been newly constructed, it was made out of 40-year-old steel. While this was a common industry practice, the application for a permit should have specifically stated that this was the case.

Adding further complications for Ashland was the fact that the permit had only been applied for, it had not been granted. Construction commenced with only a verbal commitment. Immediately after the oil spill, Ashland had provided documentation to show proof of permit - but it had actually been a statement from a different agency to acknowledge that construction was underway.

The final area of concern was the testing on the tank before filling. The American Petroleum Industry (API) has a standard 650 for proper testing of tanks. Ashland is required to use a hydrostatic method of testing - filling the tank with water to settle the foundation and to test the strength of the tank's welds. Instead of this mandated test, Ashland personnel filled the tank with only three feet of water to settle the foundation. They had sprayed oil on the welds inside the tank and then vacuum suctioned the outside to determine whether any oil could be pulled through possible leaks in the weld. This is the testing model presented by 650 for desolate locations with scarce water supplies.

Resource protection, under environmental law, helps to maintain the environmental balance of public resources. This includes public lands and rivers; mineral resources; solid waste management; historic preservation; and water pollution, among other areas. The government has taken responsibility for the enactment of laws, as well as the enforcement, for acts that threaten the environment. The National Environmental Policy Act (NEPA) is a major piece of federal legislation that oversees all federal actions that affect the quality of the human environment.

When the oil tanker collapsed, it caused a sequence of reactions that had a major impact outside the bounds of Ashland. It put the water supply of over a million people at risk. Because it was such a major environmental issue, putting the safety of citizens at risk, it became an issue of federal concern. The Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) created the Superfund for environmental cleanup. The EPA was dispatched immediately, and the federal government stepped in to coordinate the cleanup, using Ashland employees. The liability for cleanup was legally appointed to Ashland, since they were the owner-operators of the hazardous site.

D. Ethical Issues:

The actions taken by Ashland, including sidestepping the permit protocol and rushing through testing, have obvious legal ramifications. They were held accountable by law to do everything in their power to prevent such an accident from occurring.

Ashland Oil promoted a great deal of negligent in regards to the oil spill. Their negligence began with the construction of the tank and grew as the oil spill continued. The attempted to show no responsibility or blame for what had happened with the construction of the tank. Nor where they ready for an incident of this magnitude. The company showed malfeasance in dealing with the issues at hand and most of the issues could have been alleviated had they taken proper care in the construction of the failed tank.

The known effects of the introduction of the diesel fuel into the environment include the death of fish, birds and the contamination of dozens of miles of shoreline. Studies conducted after the crisis do not account for the emotional anguish of citizens, for personal inconveniences, unknown health risks or for the economic losses on commercial activities. Aside from legal responsibility, the ethical liabilities remain as long as the relationship exists between the spill, environment and the community.

Rawls' basic idea is that justice should involve developing social institutions that do not lead to unfair advantages for some at the expense of others. In particular he emphasizes on the individual's freedom of choice and, one could argue, sense of duty to make decisions which will act in the interests of all, rather than just oneself. Furthermore, Rawls argued that present generations bear a heavy burden for the sake of future generations because it is unjust for present generations to leave nothing for future generations. Each generation should put themselves in the "original position and should; Ask what is reasonable for members of adjacent generations to expect of one another at each level of advance. They should try to piece together a just savings schedule by balancing how much at each stage they would be willing to save for their immediate decedents against what they would feel entitled to claim of the immediate predecessors.

Thus, imagining themselves to be parents, say, they are to ascertain how much they would set aside for their children by noting what they would believe themselves entitled to claim of their own parents. With this said, based upon Rawls' theory that we should not hand the next generation a situation worse than we received from the generation preceding us, Ashland Oil had a great responsibility to ensure care for the surrounding community and the environment.

The failure by Ashland to find the flaw located near the top edge of a steel plate in the oil tank was a serious departure from sound practice and code compliance by the company. Ashland, its employees and some contractors displayed a dangerous pattern of negligence and ignorance in selecting, assigning, constructing, supervising and inspecting the reconstruction project. Reconstruction of the tank failed to conform to industry standards or the terms of the contract for the project. Ashland Oil's response to the incident was adequate but slow. The company's response indicated that it had governmental guidelines to respond to an incident of this nature, but none of its own standard operating procedures for crisis response. This crisis highlights the company's lack of internal conduct and safety operating procedures for checks and balances of its industries. Furthermore, this incident showed a lack of policy and involvement by government agencies. These agencies should have taken active roles through continual inspections and verifications of the plants operations.

A more rapid establishment of a central command post would have enhanced response coordination. In addition, the delay of the incident-specific Regional Response Team activation until two days after the incident may have decreased opportunities for valuable assistance to the responding agencies. Communication problems and lack of available containment and monitoring equipment also hindered response efforts. Inventories of locally available equipment be prepared to assist emergency responders in quickly locating needed equipment. The collapse could have been and should have been averted. Both the existence of the flaw and the tendency of the tank steel to react brittle under normal regional climatic and service conditions were discoverable through the application of good engineering, construction and inspection practices and by compliance with applicable industry and governmental standards.

4. Restatement of the scope of the problem and proposed options/alternatives

(TIME) Better testing mechanisms - Ashland Oil made the choice to reuse 40 year old steel that had previously been cut up which could have possibly decreased its holding strength. They also decided to not use the standard safety practices for testing the holding capacity of the tank by not filling it with water before introducing oil. Both of these actions should have had priority due to the overall condition of the steel and the winter conditions being experienced at that time.

(TIME/EPA)Adequate secondary safety mechanisms- After investigations were conducted on the failure of the dikes, which have the purpose of containing accidental spills, it was found that they were of adequate size by EPA standards. Seeing how dikes that dikes that meet the EPA standards may have very little or no affect on containing an accidental spill, it is in the best interest of the EPA to review these standards and make revisions. The revisions should address the possibility of tanks being filled at settings that may not have adequate dikes that can help counter accidental spills.

(EPA) Better location for refilling oil tanks- How the oil spill got access into the Monongahela River and subsequently into the Ohio River was traced by an uncapped storm drain. There should have been better placement or monitoring of this particular storm drain due to its proximity to possible toxic material. Not only was this not paid any attention but it was left uncovered. If adequate focus was used than there is a possibility that the oil would not have spread so quickly and contaminated so much.

(EPA) Establish a chain of command for multiple agencies to use-There needs to be a better and more structured network for events that involve different agencies from different areas. Not establishing such a structured network resulted in delayed response in coordinating efforts for the clean up and may have resulted in worse damage by the oil spill. Also, the lack of structured communication resulted in the Regional Response Team to be activated two days after the initial spill. This delayed response time may have resulted in decreased opportunities to provide assistance to those assisting in the cleanup effort.

5. Choice and Justification of a Plan to Address the Problem

Ashland Oil being the largest oil refiner in the United States when this spill occurred, it meant that the company has sufficient benefit and financial stability to act appropriately. Therefore, the company officers should have met the American Petroleum Industry standards. These standards focused in building and testing of the oil tanks. Methods had not been successful in detecting and characterizing the lick in the tank, however, the approach to control the oil spill in order not to expand water contamination.

We propose that any oil companies follow the American Petroleum Industry standards. In addition, the officers should be trained on the EPA regulation standards. Regardless the proximity or not from drinking water sources, these trainings must be provided. These trainings must first start with the Safe Water Drinking Act and the Clean Water Act. Then, add additional training such as emergency planning strategies, not only to supervisors, but also to all workers in order to make all staff proactive in any future spill. Furthermore, the company should post around the tanks standards protocol and chain of command so that any employer can refer to in case of emergency.

View the impact of this type of catastrophe in community and wild life, effort should be devoted in getting some emergency planners that can time to time simulate an oil spill in order to test not only the employers’ readiness and community awareness but also the company’s equipment and automatism.

This is Preview only. If you need the solution of this assignment, please send us email with the complete assignment title: ProfessorKamranA@gmail.com

Structure Organization And Function Of The Human Body Biology Essay

Biology » Structure Organization And Function Of The Human Body Biology Essay

Cell are the structural and functional units of all living organisms. Some organisms, such as bacteria, are unicellular, consisting of a single cell. Other organisms, such as humans, are multicellular, or have many cells—an estimated 100,000,000,000,000 cells! Each cell is an amazing world unto itself: it can take in nutrients, convert these nutrients into energy, carry out specialized functions, and reproduce as necessary. Even more amazing is that each cell stores its own set of instructions for carrying out each of these activities.

Prokaryotic Cells - organisms that are lack of nuclear membrane, the membrane that surrounds the nucleus of a cell. Bacteria are the best known and most studied form of prokaryotic organisms, although the recent discovery of a second group of prokaryotes, called archaea, has provided evidence of a third cellular domain of life and new insights into the origin of life itself.

- prokaryotes are unicellular organisms that do not develop or differentiate into multicellular forms.

- are capable of inhabiting almost every place on the earth, from the deep ocean, to the edges of hot springs, to just about every surface of our bodies.

Prokaryotes are distinguished from eukaryotes on the basis of nuclear organization, specifically their lack of a nuclear membrane. Prokaryotes also lack any of the intracellular organelles and structures that are characteristic of eukaryotic cells. Most of the functions of organelles, such as mitochondria, chloroplasts, and the Golgi apparatus, are taken over by the prokaryotic plasma membrane. Prokaryotic cells have three architectural regions: appendages called flagella and pili—proteins attached to the cell surface; a cell envelope consisting of a capsule, a cell wall, and a plasma membrane; and a cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of inclusions.

Eukaryotes include fungi, animals, and plants as well as some unicellular organisms. Eukaryotic cells are about 10 times the size of a prokaryote and can be as much as 1000 times greater in volume. The major and extremely significant difference between prokaryotes and eukaryotes is that eukaryotic cells contain membrane-bound compartments in which specific metabolic activities take place. Most important among these is the presence of a nucleus, a membrane-delineated compartment that houses the eukaryotic cell’s DNA. It is this nucleus that gives the eukaryote—literally, true nucleus—its name.

The outer lining of a eukaryotic cell is called the plasma membrane. This membrane serves to separate and protect a cell from its surrounding environment and is made mostly from a double layer of proteins and lipids, fat-like molecules. Embedded within this membrane are a variety of other molecules that act as channels and pumps, moving different molecules into and out of the cell. A form of plasma membrane is also found in prokaryotes, but in this organism it is usually referred to as the cell membrane.

The cytoskeleton is an important, complex, and dynamic cell component. It acts to organize and maintain the cell's shape; anchors organelles in place; helps during endocytosis, the uptake of external materials by a cell; and moves parts of the cell in processes of growth and motility. There are a great number of proteins associated with the cytoskeleton, each controlling a cell’s structure by directing, bundling, and aligning filaments.

Inside the cell there is a large fluid-filled space called the cytoplasm, sometimes called the cytosol. In prokaryotes, this space is relatively free of compartments. In eukaryotes, the cytosol is the "soup" within which all of the cell's organelles reside. It is also the home of the cytoskeleton. The cytosol contains dissolved nutrients, helps break down waste products, and moves material around the cell through a process called cytoplasmic streaming. The nucleus often flows with the cytoplasm changing its shape as it moves. The cytoplasm also contains many salts and is an excellent conductor of electricity, creating the perfect environment for the mechanics of the cell. The function of the cytoplasm, and the organelles which reside in it, are critical for a cell's survival.

Two different kinds of genetic material exist: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Most organisms are made of DNA, but a few viruses have RNA as their genetic material. The biological information contained in an organism is encoded in its DNA or RNA sequence.

Prokaryotic genetic material is organized in a simple circular structure that rests in the cytoplasm. Eukaryotic genetic material is more complex and is divided into discrete units called genes. Human genetic material is made up of two distinct components: the nuclear genome and the mitochondrial genome. The nuclear genome is divided into 24 linear DNA molecules, each contained in a different chromosome. The mitochondrial genome is a circular DNA molecule separate from the nuclear DNA. Although the mitochondrial genome is very small, it codes for some very important proteins.

The human body contains many different organs, such as the heart, lung, and kidney, with each organ performing a different function. Cells also have a set of "little organs", called organelles, that are adapted and/or specialized for carrying out one or more vital functions. Organelles are found only in eukaryotes and are always surrounded by a protective membrane. It is important to know some basic facts about the following organelles.

The nucleus is the most conspicuous organelle found in a eukaryotic cell. It houses the cell's chromosomes and is the place where almost all DNA replication and RNA synthesis occur. The nucleus is spheroid in shape and separated from the cytoplasm by a membrane called the nuclear envelope. The nuclear envelope isolates and protects a cell's DNA from various molecules that could accidentally damage its structure or interfere with its processing. During processing, DNA is transcribed, or synthesized, into a special RNA, called mRNA. This mRNA is then transported out of the nucleus, where it is translated into a specific protein molecule. In prokaryotes, DNA processing takes place in the cytoplasm.

Ribosomes are found in both prokaryotes and eukaryotes. The ribosome is a large complex composed of many molecules, including RNAs and proteins, and is responsible for processing the genetic instructions carried by an mRNA. The process of converting an mRNA's genetic code into the exact sequence of amino acids that make up a protein is called translation. Protein synthesis is extremely important to all cells, and therefore a large number of ribosomes—sometimes hundreds or even thousands—can be found throughout a cell.

Ribosomes float freely in the cytoplasm or sometimes bind to another organelle called the endoplasmic reticulum. Ribosomes are composed of one large and one small subunit, each having a different function during protein synthesis.

2. Describe and distinguish between the cell and tissue organizations and systems.

Tissues are the collection of similar cells that group together to perform a specialized function. The four primary tissue types in the human body: epithelial tissue, connective tissue, muscle tissue and nerve tissue.

Epithelial Tissue - The cells are pack tightly together and form continuous sheets that serve as linings in different parts of the body.  It serves as membranes lining organs and helping to keep the body's organs separate, in place and protected.  Some examples of epithelial tissue are the outer layer of the skin, the inside of the mouth and stomach, and the tissue surrounding the body's organs.

Connective Tissue - There are many types of connective tissue in the body.  It adds support and structure to the body.  Most types of connective tissue contain fibrous strands of the protein collagen that add strength to connective tissue.  Some examples of connective tissue include the inner layers of skin, tendons, ligaments, cartilage, bone and fat tissue.  In addition to these more recognizable forms of connective tissue, blood is also considered a form of connective tissue.

Muscle Tissue - Muscle tissue is a specialized tissue that can contract.  Muscle tissue contains the specialized proteins actin and myosin that slide past one another and allow movement.  Examples of muscle tissue are contained in the muscles throughout your body.

Nerve Tissue - Nerve tissue contains two types of cells: neurons and glial cells.  Nerve tissue has the ability to generate and conduct electrical signals in the body.  These electrical messages are managed by nerve tissue in the brain and transmitted down the spinal cord to the body.

This is Preview only. If you need the solution of this assignment, please send us email with the complete assignment title: ProfessorKamranA@gmail.com

Subcloning Experiments And Procedures Biology Essay

 

Subcloning is a technique used to produce recombinant DNA. A fragment of DNA containing a gene of interest is inserted into a vector/ plasmid DNA that replicates independently of chromosomal DNA to produce recombined DNA. In order for DNA to recombine, isolation, purification, quantification, digestion, electrophoresis, ligation, transformation, and screening must for all intents and purposes be performed. First alkaline lysis is used to isolate the vector and insert of choice, from bacterial cultures, by opening up the bacterial cell wall and releasing plasmid DNA. Purification removes RNA and protein that may contaminate insert and vector DNA. Quantification reveals the amount of DNA that was isolated (Schramm, Molecular & Cellular Biology Laboratory.). Digesting the DNA sequences with restriction enzymes allows for the extraction of insert and vector DNA at proper sites, which will determine the purity of the DNA samples by using agarose gel electrophoresis (Schramm, Molecular & Cellular Biology Laboratory.). Ligation of the insert to the vector is performed and then transformed into competent cells and grown on plates containing a selectable marker. To determine if the preceding procedures mentioned were successful, the DNA is isolated by doing a restriction digest and the recombinant gene is screened (Schramm, Molecular & Cellular Biology Laboratory). In this particular experiment, if the procedures are carried out successfully the vector will take in the insert gene containing ampicillin resistance and as a result bacterial growth should be seen in the presence of ampicillin and isolation of this recombinant DNA should be possible.

Isolation and purification of DNA from a bacterial culture of E. coli with a vector/plasmid DNA sequence from another bacterial culture of E. coli containing ampicillin resistance, as an insert. An alkaline lysis, with SDS detergent, was used to isolate the DNA from the E. coli cells along with COLD lysis solution. The lysate was incubated at room temperature for 3 minutes and then poured into a spin column and centrifuged, then washed with wash buffer. The DNA was eluted with water, and then underwent centrifugation twice while decanting the flow through, which was then used for quantification while the spin column was discarded. Two vectors and two inserts were used to increase the chances of obtaining purified vector and insert. For quantification, 5 tubes were used, 1 blank (water), 2 separate tubes each containing inserts, and 2 separate tubes containing vector. The blank contained 1000ul of water, each of the 4 tubes contained 5ul of vector DNA and insert DNA and 995ul of water. A spectrophotometer was used to determine the concentration of DNA, and the absorbance. Calculation of the volume of vector and insert was done to obtain 5ug of DNA to be digested by restriction endonucleases Xba1 and BamHI.

Subsequently, 1% agarose gel was prepared with dissolved TAE buffer and ethidium bromide and then solidified. The vector and insert DNA was loaded into the gel. Electrophoresis was done by running the gel for an hour at 120V. Vector1 and insert1 were excised out of the gel as they were highest in concentration of DNA, and then weighed. Three volumes of binding buffer was added to every volume of gel slice and incubated at 50oC for 15 minutes. The original volume of vector and insert of isopropanol was added and mixed by inversion to the DNA samples. Wash buffer was put in and then centrifuged and the filtrate were removed. Centrifugation, elution with 20ul of water, and another centrifugation were done. Next, ligation of the insert to the vector was performed by joining 100ng of vector with no insert (a1:0 ratio), with an equal molar concentration of insert (a 1:1 ratio), and then 3 times the concentration of insert with the vector (a 1:3 ratio). The molar ratio of insert to vector was determined to calculate the volume of the vector and inserts. These volumes were adjusted to 10ul of deionized water. Ligation buffer was added and then mixed. T4 DNA ligase was added and then mixed and centrifuged briefly, then incubated at room temperature for 15 minutes. Later, 5ul of the ligation mixture was transferred into a microfuge tube. Competent cells were added to the DNA and gently mixed by pipeting up and down. The mixture was incubated on ice for 30 minutes and heat shocked for 2 minutes at 37oC, and then cooled on ice for 5 minutes. Luria broth was added, followed by incubation of the cells for 1 hour, 37oC. These mixtures were added to and spread on the appropriate plates. The plates were then incubated at 37oC overnight.

Screening and purification of the recombinant DNA were carried out. Bacterial culture was pelleted, followed by removal of supernatant. This step was repeated with COLD lysis buffer, followed by constant vortexing. The lysate was incubated at room temperature for 3 minutes, and then centrifuged. Wash buffer was added to the tube, followed by centrifugation, removal of the filtrate, decant, and then centrifuged again. The sample was eluted with 30ul of water and centrifuged. Then, DNA quantification was performed. Restriction digest, with the enzyme HindIII, of the quantified recombinant DNA, with a master mix of BSA, Restriction buffer enzyme buffer, and restriction enzyme A, in one microfuge tube was performed. Four more tubes were filled with 3ul of DNA each, and 4ul of H2O, and 3ul of master mix into each of the 4 tubes. These tubes were spun for 1 minute, and incubated at 37oC for 30 minutes. Lastly, 2ul of 5xdyes was added to each tube, so they can be used in electrophoresis, in 1% agarose gel.

This is Preview only. If you need the solution of this assignment, please send us email with the complete assignment title: ProfessorKamranA@gmail.com

Studying Cells With Super Resolution Microscopies Biology Essay

With a growing interest in biology and the composition of living biological entities as well as a good understanding about the fact that biological entities were composed of extremely small complexes, it was essential to come up with an instrument which would help in viewing objects that could not be seen with an unaided eye. The earliest development of the microscope can be traced back to the use of a magnifying glass even though it wasn’t until the 16th century when the earliest simple microscope was developed by inverting a telescope and this was further modified and improved in the 17th century. Ever since then, new techniques have been developed in order to gain a better understanding of biological entities and presently, the world has reached an era of ‘super-resolution microscopy’ which helps I surpassing ‘Abbe’s resolution limit’. These techniques have helped in imaging nanoscopic molecules that play an essential role in different biological processes and has improved the understanding of the structural and functional properties of subcellular components. Although these techniques have been developed to provide a wide range of properties like 3-dimensional imaging and live imaging, each of them still has its advantages and pitfalls and this essay discussed a few of these techniques in detail.

Introduction

The history of discoveries in cell biology and its related fields is mirrored with the advancements made with the microscope over the past five centuries. Although the simplest microscope was first known to be made and used by Robert Hooke, It was Antonie van Leeuwenhoek who earned the title of “Father of the Microscope” for building the first microscope  in 1674, and pioneering discoveries concerning bacterial cells and erythrocytes. The nineteenth century was marked with improvements in microscopes and staining methods, which further led to scientists establishing the cell theory and viewing the key cell components, understanding cell division and differentiation and the discovery of mitochondria. With the breakthrough of research in the biological field, Ernst Abbe, a mathematician formulated the “Abbe Sine Condition” which enabled calculations that allowed the maximum resolution in microscopes possible  . However, this also meant that that the resolution of optical microscopes was limited by diffraction, which would reach a peak and limit the ability of seeing molecules closely located to one another.

Cell biology revolutionised in the mid-twentieth century with the advances in fluorescent-labelling techniques, which proved to be important tools in biological research, and advances in microscope design and technology. Since then, more specifically in the past decade, there has been an outbreak in the practical implementation of microscopic techniques, with the emergence of super-resolution microscopy that can overcome Abbe’s limit of resolution  , hence converting fluorescence microscopy into an effective 3D visualization tool  . This enables scientists to view single nanoscopic molecules of 10-20nm, not only in all three dimensions, but also trace these molecules in cellular processes. These techniques, as seen in fig.1, follow one of the two approaches; the first is based on spatial patterning of excited light (illumination-based) and this is used in stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM). The other approach is based on the localization of single molecules (probe-based) and this is used in (fluorescence) photoactivation localization microscopy/Stochastic optical reconstruction microscopy [(f) PALM/STORM]  .

STED

SSIM

TIRFM

PALM/STORM

Best resolution (nm)

~20(lateral)

~50 (spatial)

~50 (lateral)

~90 (spatial)

~230(lateral) ~100 (spatial)

~ 20-30 (lateral)

~60-70 (spatial)

Principle approach

Patterning of excited light using two laser beams

multiple interfering light beams to form moiré patterns

Evanescent field produced by total internal reflection of light

single-molecule localization of photoswitchable fluorophores

Limitations

Photobleaching can occur due to limited light wavelengths

Technical faults

High-intensity pulsed lasers can cause damage to the sample

Photobleaching can occur

Very sensitive to even the smallest changes in sample position

For 3D SSIM, a large number of images need to be taken per wavelength; this takes a long time.

Only 1 plane can be imaged

3D imaging is not possible unless TIRFM is combines with another technique  

Require large number of raw images  

Data acquisition speed is very slow, bearing a direct effect on imaging live samples

Live imaging

TIRFM-SSIM

3D imaging

TIRFM-SSIM

Multicolour imaging

Table 1: Comparison of the distinct features of the different super-resolution techniques.

Two different approaches to breaking the diffraction limit. A. STED microscopy uses two different lasers- an excitation laser (left) and a doughnut-shaped STED laser (middle- this laser deactivates the fluorophores molecules). Using these 2 lasers, the effective excitation area is limited to a small central zone (right). B. Single molecule localization microscopy methods such as PALM and STORM use photoactivatable fluorophores which can switch between their excited state and ground state to successively image the localization of a small number of molecules at a time at high precision by finding the molecule’s centroid. The many ‘raw’ images are then reconstructed to generate the final super-resolution image.

The emergence of super-resolution microscopy has opened many doors in the field of modern biology and medicine, giving an insight on processes that were unable to be followed using conventional microscopy. It is important to understand that every protein found in living cells has a specific function and is a part of a much larger molecular network. In order to understand the functioning of these large networks, it is important to track the movement and interactions of these proteins within the cell  . Super-resolution microscopy, aids in visualising the 3D-structure and accurate location of single protein molecules  on distinct organelles and on structures like lysosomes and microtubules, helping in understanding protein interactions and providing a better understanding of the molecular-scale architecture of cells  .

Three dimensional STORM image of the mitochondria network in a mammalian BS-C-1 cell. The z-position is colour-coded according to the colour scale bar.

In the past decade, super-resolution microscopy has been used to map the 3D-organization of distinct components of the nuclear pore complex; the polygonal network that makes up the endoplasmic reticulum in cells was imaged, as seen in fig.3, in living PtK2-cells of the kidney; the movement of synaptic vesicles have been traced inside living neurons by tagging the vesicle protein synaptotagmin with antibodies  and these techniques have been used to study the co-localization of two mitochondrial proteins by labelling them with different fluorophores. These studies would not have been possible without nanoscale-resolution provided by these techniques since all these structures are extremely small in size  .

Super-resolution imaging of the endoplasmic reticulum in living PtK2-cells of the kidney cell. (A) Shows the confocal image and (B) shows the simultaneously recorded STED (x, y) images from the ER marked by the fluorescent protein Citrine targeted to the ER. The arrows point out rings formed by the tubular network of the ER, which are clearly visible only in the STED image (B). 

The emergence of super-resolution microscopy has put light on important details of cell biology, holding great importance for research in the future. This essay discussed the different techniques of super-resolution microscopy, its application in cell biology, and its limitations as an instrument.

The first super-resolution microscopy technique, STED microscopy’s concept was introduced a decade ago and has advanced within the past few years. It is based on patterning the excited light in such a way that the volume of light in the excited-state is extremely small, hence maintaining the amount of light that emits fluorescence to small volumes  .

This is achieved by using two pulsed laser beams of different wavelengths; the wavelength of light from the first laser beam excites the fluorescent marker and the second laser beam illuminates the sample with a doughnut-shaped beam (called the STED-beam)  as seen in fig.4. The wavelength of light from the STED-beam is such that it causes the excited fluorescent molecules to de-excite, bringing them back to the ground-state via stimulated emission. The doughnut-shaped beam from the second laser ensures that the molecules of the centre-most part of the labelled sample are in the excited state, and fluorescence is detectable  .

Schematic diagram showing the use of the excitation and deexcitation (STED) beams for 3D-STED imaging inside a living cell. (A) An objective lens focuses the excitation (blue) beam and deexcitation (orange) beam into the ER while also collecting the resulting beam from the fluorescence photons. (B) xy-axes imaging: excitation spot (blue) and doughnut-shaped focal spot (orange) for stimulated emission (C) xz-axes imaging: excitation spot (blue) and STED spot composition consisting of a spot featuring a maximum above and below the focal plane along the z- axis, referred to as STEDz, and an enlarged doughnut-shaped beam called STEDr.

The lateral resolution of STED microscopy has been pushed to below 20nm and has been successful in imaging the synaptic vesicle movement in live neurons after neurotransmitter release during an impulse. In the past, synaptic vesicle exocytosis was suggested and confirmed by using electron microscopy, where ‘pockets’ in the pre-synaptic membrane terminals of chemically-fixed nerve cells were seen, hinting on exocytosis as the process of neurotransmitter release  . Further, fluorescence microscopy was used to study the vesicular movement after neurotransmitter release by using FM-dyes  . Even though it was known that vesicles are recycled via endocytosis, the fate of its components after fusion with the membrane was still unclear since the vesicles were too small to be resolved by available microscopes.

To solve this problem, STED microscopy was used to determine the entire process of vesicle endocytosis. Monoclonal antibodies against the intravesicular membrane protein synaptotagmin was used for imaging purposes; these antibodies only bound to those protein molecules that were exposed during vesicle exocytosis and were internalised when the vesicle was endocytosed. Fluorescent-labelled secondary antibodies were attached after membrane fixation and permeabilization and were used for visualisation of these vesicles. Images showed synaptotagmin molecules clustered on the pre-synaptic membrane, suggesting that vesicle components remain together on the pre-synaptic membrane during recycling by endocytosis. Each synaptic vesicle is 40-50nm in size and they usually occur in groups of 100-300 vesicles. Therefore, fig.5 shows that using STED microscopy was essential for localising individual vesicles, and contrary to previous beliefs that vesicles hardly move, STED revolutionised the understanding of vesicle-recycling by showing that vesicles constantly move rapidly and randomly  .

Comparison of confocal (left) and STED (right) counterpart images of a small region of a neuron terminal labelled with an anti-synaptotagmin antibody, ?xed, permeabilized and visualized using Atto532-labelled secondary antibodies. The STED image reveals a marked increase in resolution and also shows the accurate location of individual vesicle components on the neuron membrane.

However, STED microscopy is limited by wavelength. The absence of sufficient tuneable pulsed light sources in the visible range of light which de-excite the already excited fluorescent-labelled molecules has limited STED microscopy to only a small fraction of fluorophores, which causes bleaching and phototoxicity  . STED also requires the use of high intensity pulsed lasers which can cause significant damage to the samples. Furthermore, there are technical limitations set by the laser power required  for this technique and the very often, mechanical drift of the optical instruments causes imperfection of the doughnut-shaped beam around the sample, limiting the spatial resolution.

Another example of an illumination-based technique, SSIM follows the approach of illuminating the sample with multiple interfering light beams in order to break the resolution barrier  . When multiple beams of mutually coherent light are allowed to interfere, they form a structured pattern, like that of Moiré fringes seen in fig.6. When focussed on the labelled sample, the illumination pattern further interacts with the fluorescent probes. The emitted light contains image details of higher resolution, including details that cannot be resolved using a normal microscope. The illumination patterns are modulated by changing the orientation of light on the sample and high-resolution images are captured within the illumination from different patterns.

The approach of resolution enhancement followed by structured illumination. (a) If two line patterns are superposed in each other, moiré fringes will be formed as a product (seen here as the apparent vertical stripes in the overlap region). (b) A conventional microscope is limited by diffraction to a circular ‘observable region’ of reciprocal space. (c) A sinusoidally striped illumination pattern-the possible positions of the two side components (light beams) are limited by the same circle that defines the observable region (dashed). If the sample is illuminated with structured light, moiré fringes which represent information that has changed position in reciprocal space will appear. The observable region will contain normal information and moved information that originates in two offset regions (d). From a series of images with different orientation and pattern phase, it is possible to recover information from a region that is twice the size of the normally observable region can be obtained, corresponding to twice the normal resolution (e).

The images are collected and reconstructed using computer software which extracts the details from the moiré images, reconstructing them into 3-dimensional images with doubled resolution. The original 2D-SIM involved using two beams of light which interacted with the sample probe to increase its resolution and form 2D images. However, this technology was extended by using 3 light beams, generating resolved images with finer details of the sample in the axial and lateral directions, resulting in a 3-dimensional image of the sample.

Using 3D-SIM in comparison with conventional wide-field epifluorescence-microscopy, experiments to better the understanding of higher order chromatin and to study the accurate localizations of other nuclear components like the nuclear pore complexes (NPCs) and nuclear lamina were performed. The chromatin of formaldehyde-preserved mouse C2C12-myoblast cells were stained with 4',6-diamidino-2-phenylindole (DAPI) and they were observed using 3D-SSIM. The Images obtained from this technique showed a large number of ‘holes’ within the area of the stained chromatin as in fig.7, a feature that could not be observed in the images obtained by wide-field epifluorescence-microscopy.

Comparison of 3D-images obtained from conventional wide-field microscopy (left) and 3D-SIM (right) used in order to resolve interphase chromatin structure of the same DAPI-stained C2C12 cell nucleus. Deconvolution was applied to the wide-field data set (middle). (A) Mid cross-section shows brightly stained clusters of centromeric heterochromatin. Inset shows higher-detail information of chromatin substructures when recorded with 3D-SIM. Arrow in 3D-SIM inset points to a less-bright chromatin structure that has been spuriously eroded by the deconvolution procedure. (B) Apical sections (corresponding to a thickness of 0.5 µm) taken from the surface of the nuclear envelope closest to the coverslip. The raw image shows diffuse DAPI-staining, the deconvolved image shows more pronounced variations in fluorescence intensities and the image obtained from 3D-SIM shows extended resolution and reveals a punctuated pattern of regions without DAPI-staining.

Further taking advantage of SSIM’s multicolour and 3D-imaging properties, the same cells were immunostained with antibodies specific to the nuclear pore complexes (NPC), which detect the NPC proteins; and antibodies against lamin-B, a major component of the nuclear lamina (intermediate filament protein). Hence, the 3D-SSIM images showed the chromatin on the nucleoplasmic side, followed by nuclear lamina and then the nuclear pore complexes on the cytoplasmic side forming a triple-layered organization as in fig.8. Not only was the heterochromatin distinguished from the euchromatin, but at every ‘hole’ where DAPI-labelled chromatin was absent, some amount of NPC-staining was present, suggesting that chromatin was absent within close proximity of the NPCs. Even though all three sub-nuclear structures were obtained using conventional fluorescence microscopy, the spatial organization of these structures was obtained only by using 3D-SSIM.

multicolour imaging of DNA, nuclear lamina, and NPC structures in C2C12 cells by 3D-SIM. The cells are immunostained with antibodies against lamin B (green) and antibodies that recognize different NPC epitopes (red). DNA is counterstained with DAPI (blue). The image on the top left shows the same sample imaged using confocal laser scanning microscopy (CLSM) and the image on the top right shows the images obtained using 3D-SSIM which are better resolved and more clearer. The bottom picture clearly shows the triple layered organization of the three structures.

Therefore, 3D-SSIM has proved to be essential in understanding the spatial organization and interactions of sub-cellular structures that were unable to be studied before. Even though some of the initial limitations of SSIM like the time required to reconstruct and analyse the images have been overcome, SSIM is still restricted by the photostability of the fluorophores used since photobleaching leads to a less intensive image.

Even though total internal reflection fluorescent microscopy (TIRFM) was first used in 1981, it’s still a very important technique and has been used extensively since it allows selective excitation of labelled molecules in a cellular/aqueous environment which are near the surface only. This is not only beneficial because of its ability to view labelled molecules, but also because the region of interest is thin enough to obtain the highest frame-rates. TIRFM combined with structured illumination microscopy (SIM) has developed into a super-resolution technique which can break the resolution barrier and improve resolution of the region of interest.

The conventional-TIRFM is based on the diffraction properties of a light beam when incident onto a surface separating two media with different (high and low) refractive indexes. At a high incident angle (greater that the critical-angle), all the incident light is ‘totally reflected’ as long as it is coming from the medium with a high refractive index through the medium with a low refractive index  . At this surface, an ‘evanescent field’ is produced. This field is considered to be an electromagnetic field capable of exciting fluorophore molecules present on the surface. This field rises from the surface into the medium of lower refractive index  . The depth of fluorophore excitation is minimised in this phenomenon because as the evanescent field rises parallel to the surface and the distance between the field and surface increases, its strength decreases exponentially, limiting the fluorescent region. TIRFM not only provides a very thin, sectioned layer of excited fluorophores which helps in minimising the background noise caused by water molecules, it also omits unwanted fluorescence of molecules that are out of focus. However, the major drawback of this technique is that only one plane (z-plane) can be imaged, limiting its use to study cell surface events. Therefore, to obtain limit-breaking resolutions and to view multiple planes of the sample region, TIRFM is used in combination with SIM.

The TIRFM-based SIM was used to image EGFP-labelled a-tubulin of living S2-cells of Drosophila. a-tubulin is a protein present in microtubules. Comparing the images of the same sample region obtained by using conventional-TIRFM and TIRFM-SIM, the latter showed a significant improvement in the resolution of the image as seen in fig.9(a,b).

Comparison of conventional TIRF (a) and TIRF-SIM (b) images of the microtubule cytoskeleton in a single S2 cell. The image obtained after combining TIRF and SIM shows better resolution hence giving a clearer image.

Live imaging using TIRFM-SIM was applied to image polymerisation-depolymerisation of microtubules located near the centrosome of a Drosophila S2-cell which was in its mitotic state. Since the length of microtubules was constantly changing due to its polymerisation and depolymerisation, kymographs were used to process images and to determine the spatial-position of the microtubules over time by determining the difference in GFP-labelling density along the microtubule length at different times. Combing SIM with TIRFM helped in imaging the GFP-labelled a-tubulin with enhanced clarity and allowed accurate localization of the end of the microtubule, hence being able to follow it through the process. The images obtained from live-SSIM showed distinct transformation between the microtubule’s polymerisation state, depolymerisation state and its steady state, hence being able to track the dynamics of the microtubules (fig.10), a phenomenon which was not possible to understand properly using conventional-TIRFM.

TIRF-SIM images at different time frames of EGFP-a-tubulin in a S2 cell. (a) 95th image from a 180-frame sequence. Each frame was acquired in 270ms. (b) The green-boxed area of (a) shown at selected times as indicated on the individual images, using conventional TIRF (left) and TIRF-SIM (right). Green arrows follow the end of a single microtubule, which can be seen elongating until approximately the 100 s time point, and then rapidly shrinking. These changes are much easier to follow in the TIRFM-SIM images which are much clearer compared to the TIRFM images obtained.

In contrast to STED and SIM-microscopy (based on the spatial patterning of excited light), STORM and photoactivated localization microscopy (PALM) are probe-based methods principled on single-molecule localization and were developed recently in 2006. These techniques combine 3D and multicolour-imaging and obtain images with a spatial-resolution of 20-30nm and an axial and lateral-resolution of 60nm and 70nm respectively  . Keeping in mind that single molecule localization is made difficult in fluorescently-labelled biological samples because it contains millions of fluorophore molecules in a large density  , PALM/STORM use photoswitchable probes which can be switched between its visible (fluorescent, excited) and invisible (nonfluorescent, de-excited) state by using light of different wavelengths. Therefore, this approach consists of repeated cycles of sample imaging. In each cycle, different fluorophore-molecules within a diffraction-limited region are excited, such that each excited molecule can be individually imaged without overlapping (due to the images of closely located fluorophore molecules which are invisible in this cycle) and subsequently deactivated to the ground-state  as seen in fig.11. In following cycles, a stochastically different set of fluorophore-molecules are excited, determining the accurate coordinates of different molecules in each image. Using these individual images, an overall image is constructed and the position of each molecule in the sample is determined. The PALM/STORM techniques and based on the same concept of single-molecule localization, the only difference being the fluorescent probes that each of them uses. While PALM originally used photoactivable fluorescent proteins that are attached to sub-cellular structures, STORM used synthetic photoswitchable cyanin dyes that carried out the same function.

Schematic diagram showing the basic principle followed by STORM imaging. (a) Shows the microtubules within a cell. (b) shows a distinct set of fluorophore molecules in its excited state. (c), (d) and (e) show different set of fluorophore molecules that are excited while the other closely situated molecules are in the ground-state by their photoswitchable property. (f) shows the complete reconstructed image formed by compiling all the raw images into one image.

Further, STORM is developed to provide multi-coloured imaging by using combinatorial pairs of “reporter” dyes which cause the fluorescence and “activator” dyes which can reactivate the ‘switched-off’ reporter dyes when placed in close proximity to the reporter. Thus, each pair has a different colour of emitted light, determined by the reporter dye and a different colour light that activates the reporter, determined by the activator dye  . This technique, therefore, allows the study of molecular interactions between different sub-cellular structures by co-localizing them within a cell.

Comparison between images of microtubules in a mammalian cell obtained from conventional microscopy and 3D-STORM (A) Conventional immunofluorescence image of microtubules in an area of a BS-C-1 cell. (B) The 3D-STORM image of the same area with the z-position of the microtubules colour-coded according to the colored scale bar. (C-E) Show the x-y, x-z and y-z cross-sections of a small region of the BS-C-1cell outlined by the white box in (B), showing 5 microtubule filaments.

To understand the interaction and spatial relation between mitochondria and microtubules within a cell, a two-colour 3D-STORM was performed which proved to be fundamental towards the understanding of mitochondrial-microtubule interactions. It is certain that mitochondria are the “power houses” of a cell and hence, to maintain its dynamic morphology  , these organelles are constantly moving about a cell with help from motor-proteins which attach particularly to microtubules within a cell. For this experiment, fixed monkey kidney BSC-1-cells were used and two different sets of reporter-activator dyes were used to stain Tom20, part of the translocase outer mitochondrial membrane complex (used as an outer membrane marker for mitochondria) and ß-tubulin, a protein present in microtubules; the reporter dyes were attached to secondary antibodies.

Comparison of images of microtubule-mitochondrial interactions in mammalian cells as obtained from conventional and 3D-STORM microscopy. (a) A conventional fluorescence image of mitochondria (magenta) and microtubules (green). The image is slightly blurred and the distance between the mitochondria and microtubules, if any, is not visible since a single mitochondrion is seen to touch multiple microtubules. (b) STORM image of the same area with all localizations at different z positions stacked. The image is acquired in aqueous media and reconstructed from 500,000 localization points. This image, contrary to the conventional image (a), clearly shows a 150nm separation between the mitochondrion and one microtubule, whereas the same mitochondrion was in much closer proximity to another microtubule.

The STORM-image provided a clear picture of mitochondria and microtubules, allowing a better understanding of the spatial relation between them as compared in fig.13. STORM is not limited to imaging the interactions of only two sub-cellular structures and can be used to image multiple structures by differentially labelling them. STORM can be extended to imaging motor-proteins, the main complexes which facilitate the mitochondrial movement along microtubules, further illustrating their interactions and providing a better understanding of the regulation of morphology of these “power houses” within a cell, thereby having much potential for future nanoscale-research.

However, STORM requires large numbers of raw images of localised-molecules to be taken from different imaging-frames so that the entire super-resolution image can be constructed, and this limits the speed of this technique and the acquisition time required to construct the highest-resolution image requires a few minutes  .

Conclusion

It is safe to conclude by saying that microscopy has come a long way since its first discovery in the late 16th century and reached an era when the diffraction limit is being surpassed so that individual nanoscale molecules can be observed. In the past decade, super-resolution microscopy has taken a big jump and techniques like STED, SSIM, TIRF-SSIM and PALM/STORM have been developed. Even though each of these techniques accommodates features like greatly improved image-resolution, 3-dimensional imaging, live-sample imaging and multi-coloured imaging, each of these has its own limitations. In the ideal world, STED microscopy would be expected to work independent of light-wavelength and unaffected by the high-intensity lasers. Similarly, PALM/STORM would be expected to be faster techniques requiring lesser raw-images and SSIM would be expected to be unaffected by photobleaching and sample-positioning. SIM can be applied for live-imaging, 3D-imaging and multicolour-imaging; however, its resolution is still not as good as that provided by STED microscopy (live-imaging, 3D-imaging) and PALM/STORM (3D-imaging, multicolour-imaging). Therefore, at this point, it is hard to tell as to which of the above explained techniques is the best since each of them have their advantages and pitfalls and each has significant potential in different areas of biological research. As of now, considerable progress has been made in microscopy, hence opening many doors in cell biology and it is safe to say that in the future, technology will improve, and new imaging techniques will be developed

This is Preview only. If you need the solution of this assignment, please send us email with the complete assignment title: ProfessorKamranA@gmail.com

Structure Function Studies Of Helicobacter Pylori Urease Biology Essay

Biology » Structure Function Studies Of Helicobacter Pylori Urease Biology Essay

Urease catalyzes the hydrolysis of urea into ammonia and carbon dioxide. The release ammonia neutralizes the gastric acid, and allows the colonization of Helicobacter pylori in human stomach. The apo-urease undergoes a post-translation carbamylation of an active-site lysine residue, followed by insertion of two nickel ions essential for metal catalysis to the active site. In H. pylori, four urease accessory proteins, UreE, UreF, UreG, and UreH, are essential to the maturation process of urease. It is postulated that the apo-urease either bind a pre-formed UreG/UreF/UreH complex, or the individual urease accessory proteins sequentially to form a pre-activation complex. The Ni-binding protein UreE then interacts with the UreG of the complex, and triggers the GTP-dependent activation of urease.

How these urease accessory proteins interact with each other and with the urease to form the activation complex is poorly understood, partly because of the lack of high-resolution structures available for these urease accessory proteins. Until recently, the only urease accessory protein whose structure is available is UreE. We have recently determined the crystal structure of UreF, and have obtained a preliminary structure of the UreF/UreH complex. The novel structural information allows us to use the protein engineering approach to address the following questions: (1) Is the dimerization of H. pylori UreF/UreH complex essential to the maturation of urease? (2) Is the interaction between UreF and UreH essential to the maturation of urease? (3) Where is the interacting surface on the UreF/UreH complex that are responsible for binding UreG and the urease? As we have already obtained crystals of the UreG/UreF/UreH complex that diffract to good resolution, we propose to determine the crystal structure of this ternary complex. Through this work, the structures of all urease accessory proteins involved in urease maturation will be available, and together with the mutagenesis data, we will have a better understanding of how the urease accessory proteins associate with the urease to form the pre-activation complex for the maturation of urease.

Infection of Helicobacter pylori induces inflammation in the human stomach, and causes gastric or duodenal ulcers. High activity of urease is one of the factors that facilitate the colonization of H. pylori in the stomach. Urease catalyzes the hydrolysis of urea into ammonia, which neutralizes the gastric acid and allows the pathogen to survive in the human stomach.

In the active site of urease, there is a carbamylated lysine residue that are involved in binding two nickel ions essential to the metal catalysis of the enzyme. In H. pylori, the maturation of urease (i.e. the carbamylation of the active site lysine residue and the insertion of nickel ions) is assisted by four urease accessory proteins, namely, UreE, UreF, UreG, and UreH (UreH is the H. pylori ortholog of UreD found in other species). The current model for urease maturation suggests that the urease binds UreF, UreG and UreH to form a pre-activation complex, which then interacts with the Ni-binding protein UreE to trigger the GTP-dependent activation of urease.

How the urease accessory proteins interact with each other and with the urease for the maturation process is poorly understood. This proposal aims to address a number of important questions concerning the structure-function of urease accessory proteins UreF and UreH:

(1) Is the dimerization of H. pylori UreF/UreH complex essential to the maturation of urease?

We have determined the crystal structure of H. pylori UreF and UreF/UreH complex. Both of them exist as dimers in the crystal structure. Moreover, we showed that H. pylori UreF/UreH complex exists as a 2:2 dimer in solution. Based on the crystal structures, we will introduce mutations in the dimeric interface to break the dimerization of H. pylori UreF/UreH complex, and test if these mutations affect the in vivo maturation of urease.

(2) Is the interaction between UreF and UreH essential to the maturation of urease?

Our preliminary data showed that the truncation of the C-terminal residues of UreF breaks the UreF/UreH complex, and abolishes the activation of urease. To further investigate the functional importance of UreF/UreH interaction in urease maturation, we will introduce mutations in the interface of the UreF/UreH complex, and test if these mutations affect the formation of UreF/UreH complex, and in vivo maturation of urease.

(3) How does the UreF/UreH complex interact with UreG and the urease?

The UreF/UreH is also known to form bigger complexes with UreG, and with the urease. How UreF/UreH complex associates with UreG and the urease to form the activation complex for the maturation of urease is poorly understood. Based on the crystal structure of the UreF/UreH complex we solved, we propose to perform scanning mutagenesis to map the surface on UreF and UreH for interaction with UreG and with the urease. The mutants’ ability to form complex with UreG and the urease will be correlated with their ability to activate urease in vivo.

(4) What is the structure of UreG/UreF/UreH complex?

We have already obtained crystals of UreG/UreF/UreH complex. We propose to solve the high-resolution structure of the complex by X-ray crystallography. The structure will provide the first high-resolution structure of how these urease accessory proteins interact with each other.

Through this work, we will determine the crystal structure of UreF/UreH/UreG complex. Together with the mutagenesis data and the in vivo urease activation assay, our proposed work will contribute a significant step towards a better understanding on the structure-function relationship of these urease accessory proteins.

Objectives

To test whether the dimerization of the H. pylori UreF/UreH complex is essential to the maturation of urease

To investigate the functional interaction between UreF and UreH by mutagenesis.

To map the interaction surface on the UreF/UreH complex for binding UreG and the urease by scanning mutagenesis.

To determine the structure of UreG/UreF/UreH complex by X-ray crystallography

Activity of urease is one of the factors that facilitate colonization of Helicobacter pylori in the human stomach. Urease is a nickel-containing enzyme that hydrolyzes urea into ammonia and carbamic acid, which decomposes spontaneously into carbonic acid and ammonia [1].

NH2-CO-NH2 + H2O ? NH3 + NH2-COOH

NH2-COOH + H2O ? NH3 + H2CO3

The ammonia neutralizes the gastric acid, and allows the pathogen to survive in the human stomach.

The structure of urease from various species have been determined [2-5]. Urease is composed of ?, ? and ? subunits. In H. pylori, the ureA gene encodes the b and g subunits as a fusion protein, and the ureB gene encodes the a-subunit. In the active site of urease, a carbamylated lysine residue is involved in binding two nickel ions, which are essential to the catalysis of urea hydrolysis. The maturation of urease involves the carbamylation of the lysine residue and the insertion of nickel ions to the active site. In H. pylori, the urease accessory proteins that are involved in urease maturation are: UreE, UreF, UreG and UreH [1]. UreH is the H. pylori ortholog of UreD found in other species. In this proposal, we use the notation of ‘UreH(D)’ when we refer in general to the homologous UreH or UreD proteins, and use ‘UreH’ when we refer specifically to the protein UreH in H. pylori.

UreF and UreH(D) play pivotal roles in the formation of activation complex for the maturation of urease. UreF was reported to form complex with UreH(D) [6-9], and the two proteins interact with UreG to form the heterotrimeric complex UreG/UreF/UreH(D) [9, 10]. UreG is a SIMIBI class GTPase, which is homologous to the hydrogenase maturation factor HypB [11]. The apo-urease can form complex with UreG/UreF/UreH(D), or its components of UreH(D) and UreF/UreH(D) [9, 10, 12, 13]. It has been shown that apo-urease can be activated in vitro by just adding excess amount of carbon dioxide and nickel ion [14]. The in vitro activation of urease is increased when in complex with UreF/UreH(D) and UreG/UreF/UreH(D) [13, 15]. Chemical cross-linking experiments suggest that binding of UreF/UreH(D) may induce conformational changes of the urease [16], which may allow the diffusion of nickel ion and carbon dioxide into the active site to promote activation of urease [17].

The current model for in vivo urease maturation proposed by Hausinger’s group is illustrated in Fig. 1 [1]. The apo-urease interacts with UreG, UreF and UreH(D) to form a pre-activation complex. UreE, a dimeric nickel-binding protein, then interacts with UreG of the complex, and triggers the GTP-dependent activation of urease [15, 18, 19].

The formation of activation complex for the maturation of urease involves protein-protein interaction among the urease accessory proteins and the urease. However, structure-function studies of how these urease accessory proteins interact with each other was only poorly understood. One obstacle was that expression of UreH(D) alone in E. coli resulted in the formation of inclusion bodies. Recently, Hausinger’s group has successfully expressed soluble K. aerogenes UreD in fusion with the maltose binding protein (MBP-UreD), which allows for the first time in vitro characterization of UreH(D). They showed that UreH(D) can interact with UreF in ~ 1:1 binding ratio, but only weakly with UreG [9].

Until recently, the only urease accessory protein whose structure is available is UreE [18, 20-22]. The structure of UreF was recently determined by Chirgadze’s group [23], and in parallel, by our group [24]. The work proposed here will fill the much-needed gap of knowledge on the structure-function studies of urease accessory proteins.

1. We have determined the crystal structure of H. pylori UreF. Our group has determined the crystal structure of H. pylori UreF using the MAD method with Se-Met labeled protein [24]. The structure of the native UreF, refined to 1.85 Å resolution by us, is similar to the structure of Se-Met derivative reported independently by Lam et al. [23]. UreF is an all-alpha protein consisting of 10 helices. It forms dimers in the crystal structure (Fig. 2). The dimeric interface is formed by docking of helix-1 to the helix-8 and helix-9 of the opposite UreF molecule.

2. We have established an efficient protocol to express and purify UreF/UreH complex. As mentioned above, one obstacle for the structure-function studies of the UreF/UreH complex was that expression of UreH alone resulted in insoluble inclusion bodies (Fig. 3). We have successfully solved this problem by co-expressing UreH with GST-UreF in E. coli (Fig. 3). After affinity chromatography purification and removal of the GST-fusion tag, the UreF/UreH complex can be purified in large quantity (~10 mg per liter of bacterial culture).

3. We have established assays to correlate in vitro protein-protein interactions with in vivo maturation of urease. We showed that when co-expressed together, UreF and UreH form a soluble complex that can be pull-down by GST affinity column (Fig. 4A, lane 2). We noticed that the C-terminal residues of UreF were protected from degradation upon complex formation with UreH (Fig. 4A, lane 1 & 2). We showed that truncation of the C-terminal residues of UreF (UreF-DC20) disrupted the formation of a soluble UreF/UreH complex (Fig. 4A, lane 3). We have also established an assay to test the in vivo maturation of urease (Fig. 5), and showed that the mutation (UreF-DC20) that disrupted the interaction between UreF and UreH also abolished in vivo maturation of urease. By GST pull-down, we demonstrated that the UreF/UreH complex interacts with UreG (Fig. 4B, lane 4), and with the urease (Fig. 4B, lane 4). These preliminary data demonstrated that feasibility of the proposed structure-function studies.

4. We have obtained the preliminary crystal structure of H. pylori UreF/UreH complex. With the purified UreF/UreH complex, we were lucky to obtain crystals of the complex that diffract to high resolution (Fig. 6A). Diffraction data was collected to 2.5Å resolution. We phased the structure by molecular replacement using the structure of UreF as a search template. Our preliminary structure of UreF/UreH complex showed that the UreF/UreH complex forms a 2:2 dimer in the crystal structure (Fig. 6B). We anticipate that the refinement of the UreF/UreH complex structure will be finished very shortly, and the structure will provide a rational based for the mutagenesis studies proposed in this study.

5. We have showed that the UreF/UreH complex form dimers in solution. To test if the UreF and UreF/UreH form dimers in solution, we have loaded purified samples of UreF and UreF/UreH to an analytical size-exclusion-chromatography column coupled to a static light scattering detector (Fig. 7). The apparent M.W. for UreF was 43 kDa, which is in between the theoretical M.W. of a monomeric (28 kDa) and a dimeric (56 kDa) form of UreF. The results suggest that UreF alone does have a tendency to form dimers, and the dimeric form of UreF is in exchange with the monomeric form in solution. On the other hand, the formation of dimer is more-or-less complete in the UreF/UreH complex. The apparent M.W. measured for UreF/UreH complex was 116 kDa, which is consistent with the theoretical M.W. of 116 kDa for a 2:2 dimer of UreF/UreH complex.

6. We have established an efficient protocol to express and purify UreG/UreF/UreH complex, and obtained crystals of the complex. We have found that the most efficient way to obtain the H. pylori UreG/UreF/UreH complex is to co-express UreG, GST-UreF and UreH together in E. coli. The ternary complex can be easily purified by affinity chromatography followed by removal of GST-fusion tag by protease digestion. In our hand, the yield of UreG/UreF/UreH complex is ~5mg per liter of bacterial culture. More encouraging is that we have successfully obtained crystals of UreG/UreF/UreH that diffracted to a reasonable resolution of ~3Å (Fig. 8). These preliminary data strongly suggest that the proposed structure determination of the ternary complex of UreG/UreF/UreH by X-ray crystallography is highly feasible.

Track Record of PI

The PI has extensive experience on structure determination by both NMR and X-ray crystallography, and using protein engineering to probe the structure-function of proteins. In addition to the structure determination of UreF and UreF/UreH complex discussed above, he has solved the solution structure of barstar, an inhibitor of barnase, and studied its dynamics behavior by NMR spectroscopy [25, 26]. He also studied the effect of mutations on the stability and structural perturbation on the DNA-binding domain of the tumor suppressor p53 by NMR spectroscopy [27, 28]. He has used an approach that combines evidence from NMR experiments and molecular dynamics simulation to study the folding pathway and the denatured states of barnase and chymotrypsin inhibitor-2 [29-31]. Supported by previous GRF grants, he solved the solution [32] and crystal [33] structure of ribosomal protein L30e from Thermococcus celer, the crystal structures of a thermophilic acylphosphatase from Pyrococcus horikoshii to 1.5Å [34], and human acylphosphatase to 1.45Å [35], an orange fluorescent protein from Cnidaria tube anemone to 2.0Å [36], seabream antiquitin to 2.8Å [37], the crystal structure of trichosanthin in complex with the C-terminal residues of ribosomal stalk protein P2 to 2.2Å [38], and the solution structure of the N-terminal dimerization of P2 [39]. We believe that, with our strong background in structural biology and the solid preliminary data, we are in a leading position to determine the structure of the UreG/UreF/UreH ternary complex, and to study the how the urease accessory proteins interact with each other for the maturation of urease.

Our preliminary data suggest that the H. pylori UreF/UreH complex forms a 2:2 dimer in solution. Both the crystal structure of H. pylori UreF, and the preliminary structure of UreF/UreH complex suggest that the dimerization is likely to be mediated by UreF. It is presently not known whether the dimerization is a unique property of H. pylori UreF - for example H. pylori and K. aerogenes UreF only share 19% sequence identity. Interestingly, the quaternary structure of H. pylori urease is different from ureases from other bacterial species. Unlike the urease (UreABC) from K. aerogenes that forms a trimeric complex (UreABC)3, the H. pylori urease (UreAB) forms a tetramer of trimers ((UreAB)3)4. Nevertheless, that H. pylori UreF/UreH complex exists as a dimer in solution and in crystal structure raises an interesting question - is the dimerization of H. pylori UreF/UreH complex essential to the maturation of urease?

To address this question, we will introduce mutations that are designed to break the dimerization of UreF and UreF/UreH complex. As shown in Fig. 2, the dimeric interface is formed by docking of helix-1 to the helix-8 and helix-9 of the opposite UreF molecule. A closer look at the dimeric interface of the crystal structure of UreF reveals a number of interactions that may be importance to the dimerization of UreF (Fig. 9). For example, to break the hydrogen bonding network among Q37, Q205 and Q212, we will replace the Gln residue with either alanine or asparagine to create triple mutants of Q37A/Q205A/Q212A and Q37N/Q205N/Q212N. We anticipate that both truncation of and shortening of the amide chain should break the hydrogen bond network. To disrupt the hydrophobic interaction around F33, we will substitute the Phe residue with alanine (F33A) or with a polar residue (e.g. F33R). Substitution of polar residue like arginine at Phe-33 should highly disfavor dimerization because the high desolvation penalty will prevent the polar residue to be buried upon dimerization. If necessary, we will create quadruple mutants (e.g. Q37A/Q205A/Q212A/F33A) to ensure disruption of UreF dimerization.

3.1.1 GST pull-down assay for UreF/UreH interaction. First, we test if these mutants will affect the formation of soluble UreF/UreH complex by GST pull-down assay (Fig. 4A). UreH will be co-expressed with mutants of UreF fused with GST-tag, and the bacterial lysate will be loaded to a GSTrap column (GE Healthcare). After extensive washing with binding buffer (20 mM Tris pH7.5, 0.2M NaCl, 5mM DTT), the proteins will be eluted with 10mM glutathione.

As these mutations are located at the dimerization interface, which are far away from the UreF/UreH interface, we anticipate that they will not affect UreF/UreH interaction.

3.1.2 Size-exclusion-chromatography/static-light-scattering (SEC/LS). We will test if these mutants affect dimerization of UreF by SEC/LS. Purified samples of UreF mutants and its complex with UreH complex will be loaded to an analytical Superdex 200 column connected to an online miniDawn light scattering detector and an Optilab DSP refractometer (Wyatt Technologies). The light scattering data will be analyzed using the ASTRA software provided by the manufacturer to obtain the molecular weight of the protein samples.

If the mutations break the dimerization, we anticipate that the measured molecular weight will be 28 kDa for UreF, and 58 kDa for UreF/UreH complex.

3.1.3 In vivo maturation of urease. We will test if test if these mutants affect in vivo maturation of urease. We have established an assay for in vivo maturation of urease (Fig. 5). We have cloned the H. pylori urease operon, ureABIEFGH, into the pRSETA vector to create the pHpA2H vector. We will introduce the mutations into the ureF gene in the pHpA2H vector. E. coli will be transformed with wild-type and mutant pHpA2H vectors, or the negative control plasmids (pHpAB and the empty vectors). The bacterial cells will be grown in the presence of 0.5 mM nickel sulfate, and were induced overnight with 0.4 mM IPTG. After cell lysis by sonication, urease activity of the bacterial lysate will be assayed in 50 mM HEPES buffer at pH 7.5 with 50 mM urea substrate, and will be measured by the amount of ammonia released using the method described in ref. [40].

If the dimerization of UreF and UreF/UreH is essential to the maturation of urease, the mutations that break the dimerization will also abolish the maturation of urease. On the other hand, if the maturation of urease is not affected by these mutations, it is likely that the dimerization is not essential to the urease maturation.

Our preliminary data showed that removal of the C-terminal residues of UreF breaks the UreF/UreH complex, and abolishes the maturation of urease. The availability of a preliminary structure of UreF/UreH complex allows us to introduce site-directed mutations that are designed to break the UreF/UreH interaction, and to further investigate the functional importance of UreF/UreH interaction. Our structure showed that upon complex formation, the C-terminal residues of UreF become structured and form an extra helix (helix-11) that dock to a binding cavity of UreH. Three hydrophobic residues V235, I239, and M242 on helix-11 are buried to a hydrophobic pocket of UreH.

To further investigate the functional importance of UreF/UreH interaction in urease maturation, we will create alanine and hydrophobic-to-polar (e.g. V?N) substitutions at V235, I239 and M242, which are designed to break the UreF/UreH complex formation. We will test if these mutations affect the formation of the UreF/UreH complex by the GST pull-down assay described in 3.1.1, and if they affect maturation of urease as described in 3.1.3. If the interaction between UreF and UreH is essential to the maturation of urease, we anticipate the mutations that break the UreF/UreH interaction will also abolish the maturation of urease.

The UreF/UreH is also known to form bigger complexes with UreG, and with the urease (UreA/UreB). How UreF/UreH complex associates with UreG, and the urease to form the pre-activation complex (UreA/UreB-UreG/UreF/UreH) for the maturation of urease is poorly understood. It has been reported that UreG does not interact directly to the urease, suggesting the UreF/UreH complex serves as a bridge that recruits UreG to the activation complex.

Our group has recently collected 2.5Å diffraction data for the H. pylori UreF/UreH, and has obtained a preliminary structure of the complex, which allows us to identify surface residues of UreF and UreH. To map the interacting surface of UreF/UreH complex for binding of UreG and the urease (UreA/UreB), we propose to perform alanine-scanning mutagenesis of surface residues on UreF and UreH. We will first focus on relatively more conserved surface residues (For UreF: P44, I45, Y48, S51, E55, Y72, E119, R121, Y183, K195, Q201, Q205, H244, E245, R250, L251, S254. For UreH: D60, G61, T78, K84, P111, I115, F177, E140, R146, E151, R213). We will also introduce multiple substitutions at these residue positions, if they are close in space according to the preliminary structure of UreF/UreH complex. We will first test if these mutations affect UreF/UreH interaction as described in 3.1.1. If so, we will exclude those mutants from the library.

After we have created the mutant library of UreF/UreH complex, we will test the mutants' ability to form complex with UreG, and with the urease (UreA/UreB). In brief, mutants of the GST-UreF/UreH complex will be co-expressed in E. coli. Our preliminary data suggest that the GST fusion tag will not interfere with binding of UreG or UreA/UreB (Fig. 4B). The bacterial lysate of GST-UreF/UreH (or its mutants) will be mixed with bacterial lysate expressing UreG or UreA/UreB, and then loaded to a GSTrap column for the pull-down assay. For those mutations that break the interaction, we will also perform the reciprocal pull-down in which the GST-tag is fused to the UreH, UreG, UreA or UreB. This is to confirm that the breakage of interaction is due to the mutations, but not due to a nearby GST-tag.

To address the question if the interaction between UreF/UreH and UreF (or UreA/UreB) is essential to maturation of urease, we will test the ability of the UreF/UreH mutants to activation urease in vivo as described in 3.1.3. If the interaction is essential to urease maturation, we anticipate the mutations that break the interaction will also abolish the urease maturation.

3.4.1 Expression and purification of UreG/UreF/UreH complex - We have established an efficient expression purification protocols for the ternary UreG/UreF/UreH complex. His-GST-tagged UreF, UreG and UreH will be co-expressed together in E. coli BL21(DE3) strain using the expression plasmids pET-Duet-HisGST-UreF/UreG and pRSF-UreH. After affinity chromatography purification, the His-GST fusion tag will be removed by the PreScission Protease (GE Healthcare). The protein complex will be further purified by gel filtration. Typical yield of the UreG/UreF/UreH complex is ~ 5mg per liter of bacterial culture.

3.4.2 Optimization of crystallization conditions - Preliminary screening of crystallization conditions was performed. We have already obtained crystals of the UreG/UreF/UreH that diffract to ~3Å (Fig. 8). We will further optimize the crystallization condition by grid-searching the pH and precipitant concentrations, and addition of additives or detergents. Quality of the diffraction data will be used to guide optimization of the crystallization conditions. We will also optimize the cryo-protection procedures (e.g. the choice of cryo-protectants and their concentration) to improve the quality of diffraction data collected. When necessary, we have access to synchrontron beam line at Diamond Light Source, Oxford, through collaboration with Dr. Yu-Wai Chen (King's College London).

3.4.3 Phase determination - We will first attempt to phase the structure by molecular replacement. At the time of writing this proposal, we are refining the structure of H. pylori UreF/UreH complex. We will use the UreF/UreH complex structure as a search template to solve the phase of the UreG/UreF/UreH complex by molecular replacement. In parallel, we will also prepare selenium-methionine labeled sample of UreG/UreF/UreH for multi-wavelength anomalous diffraction (MAD) phasing by expressing the protein complex in minimal medium containing Se-Met as described in Doublie [41]. The H. pylori UreG, UreF, and UreH proteins contain 9, 10, and 8 methionine out of 199, 254, and 265 residues, which should provide enough phasing power for MAD phasing. The PI's group has previously established the expression protocols for Se-Met labeling for H. pylori UreF, and determined its structure by MAD phasing. We have access to synchrontron beam line at Diamond Light Source for collection of MAD data.

3.4.4 Model building and refinement - Models will be built interactively by the program COOT [42], and refined using PHENIX [43]. The progress of refinement will be monitored by Rfree- and R-factors. Quality of the crystal structure will be validated by the program MOLPROBITY [44].

This is Preview only. If you need the solution of this assignment, please send us email with the complete assignment title: ProfessorKamranA@gmail.com