Public satisfaction with policy process influences the legitimacy and acceptance of policies, and conditions the future political process, especially when contending ethical value judgments are involved. On the other hand, public involvement is required if effective policy is to be developed and accepted.Using the data from a large-scale national opinion survey, this study evaluates public appraisal of past government efforts to legalize organ transplant from brain-dead bodies in Japan, and examines the public's intent to participate in future policy.A relatively large percentage of people became aware of the issue when government actions were initiated, and many increasingly formed their own opinions on the policy in question. However, a significant number (43.3%) remained unaware of any legislative efforts, and only 26.3% of those who were aware provided positive appraisals of the policymaking process. Furthermore, a majority of respondents (61.8%) indicated unwillingness to participate in future policy discussions of bioethical issues. Multivariate analysis revealed the following factors are associated with positive appraisals of policy development: greater age; earlier opinion formation; and familiarity with donor cards. Factors associated with likelihood of future participation in policy discussion include younger age, earlier attention to the issue, and knowledge of past government efforts. Those unwilling to participate cited as their reasons that experts are more knowledgeable and that the issues are too complex.Results of an opinion survey in Japan were presented, and a set of factors statistically associated with them were discussed. Further efforts to improve policy making process on bioethical issues are desirable. In Japan, it was not until 1997 that a law was finally enacted to legalize organ transplant from a brain-dead body. Since 1968, when the first heart transplantation from a person declared brain dead was performed, there have been long-standing struggles in Japan for and against this procedure. In addition to many non-governmental institutions and individuals, the Japanese government – both the legislature and administrative bodies – engaged in a variety of efforts for this enactment. A number of factors have been suggested for the prolonged lack of policy in this area: deep public mistrust of the medical profession caused by the 1968 heart transplant; the Japanese culture which still holds traditional Japanese view of death and the body; and the lack of the broad public consensus required as a precondition for a policy [As elsewhere, public policy to resolve these social disputes was pursued . ObserveIn the policy process, which entails the introduction and implementation of a policy, public opinion is generally considered an important factor affecting its fate. Without favorable public opinion, a policy cannot be introduced and implemented effectively . Public An important aspect in policy making is the degree of public participation, which is defined as a set of measures to consult with, involve, and inform the public to encourage participation in policy development . Since eThis study examines public appraisal of past efforts of the Japanese government to legalize organ transplant from brain-dead bodies. Other goals are to quantify the public's intent to participate in future policy discussions and to identify possible factors affecting both appraisals and intentions to participate. A brief chronology of Japan's efforts since the 1960's toward legalization of organ transplant upon brain death is also presented. Implications of the study findings are discussed, as are suggestions for future research.A questionnaire includes sections on demographic characteristics (age, sex, education, occupation), health conditions , period of first issue attention (when did you first hear of the issue on brain death and organ transplant?), period of opinion formation (when did you arrive at the opinion you have now on this issue?), knowledge about donor card , knowledge of past government efforts at informing the public and inviting their opinions (asking number of measures employed by the government), appraisal of past government efforts (How do you rate past government efforts around the issue of brain death and organ transplant?), and intention to participate in future (bioethics-related) policy discussions. Those who indicated unwillingness to participate were asked to provide reasons for that decision. The final questions concerned important agenda items for future policy processes.Questionnaires were sent to 3000 people, selected from 15 cities and towns nationwide by the stratified random sampling method. They were requested to answer the questions on the sheets, and send them back by mail in a postage prepaid envelope. The study was conducted in January 2002, and the overall response rate was 34.5%.First, association of the questionnaire items both with a) appraisal of past government efforts and b) participation intent was examined using Mann-whitney tests (between two groups), Kruskal-Wallis tests (among multiple groups), and/or Spearman's rank correlations. Next, a multiple logistic regression model was applied to identify possible explanatory variables (entered and removed at the significance level of p = 0.05) to determine a set of variables that best predicts the dependent variables. Also, stepwise logistic regression analysis was conducted to determine factors affecting people's selection of important future agenda items.Extant opinion polls regarding public perceptions of brain death and organ transplants from brain-dead patients were studied to identify possible trends. When more than one poll was conducted in a year, results were averaged to avoid possible biases deriving from different survey designs. The following national polls were used to plot the trends in opinions: Yomiuri Shimbun (1982, 1984–95, 1997–99); Asahi Shumbun (1985, 1988, 1992, 1996–98); Mainichi Shimbun (1985, 1990–91, 1997); Office of the Prime Minister (1987, 1991, 1998); Nippon Hoso Kyokai (1991–92, 1996); and Jiji Tsushin (1992, 1994) [Descriptive statistics indicating basic attributes of study participants, as well as all other survey results, are shown in Table Figure Table Moreover, when people are older, they are more likely to have recognized the issue and formed their opinions earlier, but know less about donor cards and past governmental efforts. Knowledge of donor cards and of government efforts were positively correlated, as were period of first attention to the issue and period of opinion formation. There was no significant correlation between appraisal and participation intent.Table As shown in Table Those who were unwilling to participate in future policy discussion cited several reasons for their decision: Experts know better (50.1%); Issue is difficult (44.9%); Participation is ineffective (21.6%); Too busy (14.1%); and Not interested (7.9%).Using data obtained in an opinion survey, this study seeks to evaluate public appraisal of past government efforts to legalize organ transplant from brain-dead bodies in Japan. Even though public opinion is relatively unstable and sometimes an irrational response to surrounding symbols, it continues to be an important factor in policy advocacy. Public acceptance of a policy is considered important not only for the practical reason that adopted policy cannot be implemented effectively and efficiently without public consent, but also for the ideological democratic point of view that policy is to be based on the judgment of a rational, informed and willing public, or at least on conscious delegation of individual autonomy to expert authorities . The degThe chronology of the major events leading to enactment of the Organ Transplant Act and the successful implementation of the first several operations in Japan has been described elsewhere in detail . Here weAn act which enabled cornea transplant operations with the consent of the family was passed in 1957. In the same time period, kidney (1956-) and liver (1964-) transplants also commenced. The first heart transplant in Japan was performed at Sapporo Medical College in 1968. Since brain death had not been officially established, there was concern about a possible conflict of interest, and some believed that the extraction of a heart was murder. Extended efforts have taken place since then, to develop criteria for death, specifically medical and biological definition and diagnosis, their social use, and how to determine a human/ individual death in relation to the criteria.In the medical field, a committee of the EEG Society published standards for diagnosing brain death in 1974. The donor card system was sanctioned by the government shortly thereafter in 1977, without defining the criteria for death. In the meantime, organ transplant was sporadically performed with the consent of families, but without any official regulation of the process. Consequently, the transplant of cadaveric organs spread only gradually, since harvesting organs from brain-dead bodies continued to invoke public dispute and sometimes resulted in lawsuits. In 1984, for example, when the first multiple transplant (a combined kidney/pancreas transplant) was performed from a brain-dead body at Tsukuba University, the Patients' Rights Conference (PRC) soon filed a charge of murder in the case.The medical community began to advocate legislation governing brain death and organ transplantation more openly. In 1985, the MHW announced its diagnostic criteria for brain death, though it stated that a patient's death couldn't be judged by brain death. [Period 1]. In 1986, the Japan Medical Association formed the Bioethics Discussion Group, a study group of interdisciplinary nature, and in 1988 issued its Final Report, which encouraged brain death legislation to facilitate organ transplantation. With the goal of shaping public opinion, the Japan Organ Transplantation Society sponsored a series of open symposia in 1989. [Period 2]. A group of politicians from the major party started investigating the current situation in other countries, considering possible legislation. Activities of patients' groups reportedly helped shift public attention away from the brain-dead potential donor to the seriously ill person who needs a transplant ,11. At tFinally, in early 1990, more than 30 years after the first transplant, the office of the Prime Minister established a special commission, Provisional Commission for the Study of Brain Death and Organ Transplantation. To encourage public involvement, the Commission held a series of public hearings and town meetings, and issued newsletters. Its 1991 interim and 1992 final reports presented both a majority view and a minority view. The former stated that a social consensus on brain death had already been achieved, and the latter argued that such a consensus had not yet been achieved. Both groups approved organ transplant when the consent of the donor was definitely obtained. A number of scholars argued that it should be a personal decision whether or not one's death is to be determined by brain death criteria, making individual consent the basis of both brain death and organ donation . [PeriodA bill to legalize the transplantation of organs was presented to the Diet first in 1994. After several years of discussion, the modified bill finally became law in October 1997. Organ transplantation was thereby legalized where the donor has given written consent both to transplant and to the determination of brain death. Brain death was accepted only in such a case to enable organ transplants. In practice, the patient's family can still override the prior consent decision. [Period 4]. In 1999, two years after the law was passed, the first heart transplantation was successfully conducted, at Osaka University Hospital . That saAs was indicated in Figure Our results also indicate that as the number of people approving organ transplants from brain-dead bodies increased, the number of opponents increased in parallel, although in smaller numbers. This increase in political awareness or in political knowledge, as suggested elsewhere , led to As in other countries, the public debates on brain death and organ transplant, both in the private sphere and in the public sphere, were new attempts at governance of socio-technical innovation in the field of biomedicine. If social mobilization is fueled by the inability of the institutional system to respond adequately to public concerns, the issue status in Japan in the 1980s, when many individuals and institutions started to pay attention and get involved in the debate, might indicate insufficient mediation of the actors for conflict resolution (by the government) before and during that period. Generally in post-war Japan, a relatively small number of political and administrative elites have left the handling of many social conflicts to the workings of traditional social relations . SimilarIn the early 1990s when the Ad-hoc Council was set up, the Japanese government introduced a variety of measures to resolve social disputes by inviting the public into policy discussions. Our finding that many people recognized the issue and formed their opinions at times other than this period, however, suggests that these tactics were not very effective in terms of raising public awareness. In European countries, it was reported, public involvement measures served well as focusing devices, which helped attract attention and facilitate discussion among the various public . The difIt is remarkable that more than 40% of respondents were unaware of any past government policy, despite longstanding struggles around the issue and much media coverage. Of those who were aware, about half of them had favorable opinions of government efforts, while a slightly larger percentage had negative views. The fact that only 30% of respondents reported satisfaction indicates that there is much room to improve public awareness, acceptance, and appraisal in the policy process on bioethical issues.Multiple regression analysis disclosed that age, period of opinion formation, and knowledge of donor cards are independent factors affecting public appraisal of past governmental efforts. Individuals tended to give higher appraisal points when they were older. Indeed, age seems to be a major factor in opinion formation on several policy issues . PerhapsA national effort to incorporate ethical considerations into policy rests on an academic reservoir of technical experts, legal scholars and humanists, and on the public understanding of science and its social implications, as well . The proAccording to Taylor and Fiske , people In relation to this point, it should be noted that some changes in the policymaking process were regarded as important. A majority of respondents suggested that better information disclosure and more respect for both patients' and experts' opinions are desirable in the policy process. It follows that more effective involvement of the public, especially those stakeholders, in policymaking is warranted. Although an empirical assessment is not available, some procedures used in France and Germany might merit attention in modeling future policymaking for other countries. The National Consultative Bioethics Committee of France holds an annual public conference where, in addition to an activity report from the Committee, many ethical topics are discussed, with the participation of both experts and laypeople . The GerIn many countries, participation has gained momentum in a variety of policy domains . In healIn our study, a majority of people (61.8%) responded that they would not participate in future policy discussions, while the rest (38.2%) responded that they would. The absence of association between government appraisal and participation intent indicates that the latter is determined by factors other than the former. Multivariate analysis showed that younger age, earlier period of first attention, earlier period of opinion formation, and more knowledge of past governmental efforts are positively associated with the intent to participate in future policy discussions. This indicates that the more attentive members of the public, namely those long-term observers with their own opinions and knowledge of current policy, have more interest and consequently a greater intention to participate in policy discussions. This finding is consistent with past studies indicating that participation is facilitated by policy knowledge and/or political sophistication . PositivReasons cited for being unwilling to participate in the process indicate that many feel unqualified or unknowledgeable but not necessarily too busy or uninterested. Further analysis revealed that older people are more likely to cite "Experts better" and "Ineffective", and are less likely to choose "Busy"; that females are more likely to cite "Difficult", and that people tend to cite "Ineffective" when they are more knowledgeable about past government efforts, while choose "Busy" or "Uninterested" when they are less knowledgeable. These findings suggest that despite their latent interest in the issue, people are unwilling to commit themselves to policy discussions because of their perception of inadequacy stemming both from their lack of knowledge and sense of inefficacy, as judged from past experiences. The absence of an association between appraisal and participation suggests that people might be uncertain about their own competence and efficacy in policymaking. It can be inferred that people hope for a means of understanding the issue, so as to formulate their opinions for themselves.Political participation is facilitated by having a personal stake and perceived self-efficacy in policymaking. Conversely, it could be hampered by both indifference to the issue and a sense of powerlessness . More spIn the context of Japan, it is important to remember that public involvement measures thus far used were not very effective in raising public awareness and that people consider some changes desirable in the policymaking process. It is possible that more people could be inspired to participate by changing the design of public involvement measures, from a consultation type of involvement to a partnership model . EffortsMore innovative methods of public participation show promise and deserve consideration in improving policy process on medico-ethical issues and increasing public satisfaction with policy and politics. These include consensus conferences, citizens' juries, scenario workshops, deliberative opinion polls, among others . DistincAs is always the case with mass opinion surveys, this study cannot escape the possible bias introduced by the low response rates of polls . A samplAmong many topics to be considered for future research is the function of (mass) media vis-à-vis public opinion formation. The media should be examined critically as they influence both experts, policymakers, and the public. Mass media have a dual function in these processes: as a conduit of debates and negotiations as well as a source of influence . Also onGovernment decisions and their outcomes, namely the enactment and subsequent implementation of organ transplants, attracted public attention and helped formulate public opinion on the issue, more than did the processes leading to enactment. In the case of the concept of brain death and the legalization of organ transplant in Japan, many people still were unaware of past government efforts in policymaking, including the measures used for public involvement, despite past longstanding social debates. Only a small percentage of the public indicated satisfaction with the process. However, those who were attentive to the issue, knowledgeable of the past policy process as well as of the current policy, tended to rate the policy process more positively. Although people do not always manifest their intent to participate in future policy discussions, they might maintain sufficient interest in biomedical issues and have a latent wish to get involved in the policy process.The author(s) declare that they have no competing interests.All the authors (HS, AA, IK) fully participated in the planning, designing, and carrying out of the surveys for this study. HS performed the statistical analysis and drafted the article. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The issue of overpopulation has fallen out of favor among most contemporary demographers, economists, and epidemiologists. Discussing population control has become taboo. This taboo could be hazardous to public health The issue of human overpopulation has fallen out of favor among most contemporary demographers, economists, and epidemiologists. Discussing population control has become a taboo topic. Yet, this taboo has major implications for public health.The silence around overpopulation prevents the global health community from making the necessary link between the planet's limited ability to support its people (its carrying capacity—see sidebar on following page) and health and development crises. In this article, I describe how popular thinking on population control has been shaped over the last 200 years, and how our failure to address the population explosion may be one cause of recent epidemics and social unrest.The question of human overpopulation and its relationship to human carrying capacity has been controversial for over two centuries. In 1798 the Reverend Thomas Malthus put forward the hypothesis that population growth would exceed the growth of resources, leading to the periodic reduction of human numbers by either “positive checks”, such as disease, famine, and war, or “preventive checks”, by which (in the absence of contraception) Malthus meant restrictions on marriage. This “Malthusian view” was rapidly accepted by most politicians, demographers, and the general public, and remained popular until fairly recently.The Population BombMalthus's worst fears were not borne out through the century following his death in 1834—food production largely kept pace with the slowly growing global population. However, soon after 1934, the global population began to rise steeply as antibiotics, vaccines, and technology increased life expectancy. By the 1960s, concerns of a mismatch between global population and global food supply peaked—expressed in books such as Paul Ehrlich's 1968 In 1966, United States President Lyndon Johnson shipped wheat to India to avert a famine on the condition that the country accelerate its already vigorous family planning campaign But the 1970s surprised population watchers. Instead of being a period shadowed by calamitous famine, the new crop strains introduced by the “Green Revolution”  caused a dramatic increase in the global production of cereals, the main source of energy in the global diet. Among the development community, despair turned into cautious optimism. By the end of the decade, the public health community felt sufficiently empowered to proclaim “Health for All by the Year 2000”. Average life expectancy continued to zoom upwards almost everywhere—even in sub-Saharan Africa.The introduction of safe contraception contributed to a rapid fertility decline in many countries. But while the rate of global population growth declined from its peak in the late 1960s, the absolute increment of increase in annual global population continued to grow. Most population-related scientists, including food scientists and demographers, as well as US President Jimmy Carter, continued to be very concerned about global overpopulation. In 1970, the father of the Green Revolution, the agricultural scientist Norman Borlaug, was awarded the Nobel Peace Prize. In his Nobel lecture, Borlaug warned that the success of the Green Revolution would buy a breathing space for humankind of three decades, unless equivalent action was taken to reduce fertility rates With hindsight, the 1970s can be seen as the decade when widespread concern about overpopulation started to fade. The social and economic milieu of many developed countries, especially in the US, started to change. US foreign aid, as a percentage of the gross national product, declined from the late 1960s, perhaps in part because of the competing needs of the Vietnam War but also perhaps because of the apparent success of development in the Third World. The economic policies known as Keynesianism, which had been dominant since the end of World War II in many developed nations, came under sustained attack. These policies had placed a high value on full employment and social security. Keynesian policies restrained domestic inequality through high taxation and the promotion of social norms that censured conspicuous consumption . Shortly before his death, J. M. Keynes had also been crucially involved in the establishment of the World Bank. Keynes appears to have been personally committed to the advance of global justice, and to the reduction of inequality both within and between nations The world oil shock in 1973 contributed both to “stagflation”—a combination of rising unemployment with higher prices—and to increased economic power for the oil-producing countries of the Third World. Indeed, the term “Third World” came to be considered pejorative and was replaced by the “South”. Stagflation was interpreted as a failure of Keynesian policy. The demise of Keynesianism was accompanied by a further decline in concern for Third World development among elite economists and the general public.It is unlikely that the issue of global population policy figured into the election that put US President Ronald Reagan into office in 1980. Nevertheless, Reagan's policies were to cement a new orthodoxy about global overpopulation and development strategies. Unlike his republican predecessor, Richard Nixon, Reagan considered concerns about global population size to be “vastly exaggerated” As foreign aid budgets fell, the “Health for All” targets began to slip from reach. Instead, international agencies promoted structural adjustment programs, health charges for patients (“user fees”), and the “trickle down” effect as the best ways to promote development. It is plausible that a fraction of the public who remained concerned about Third World development thought that these new economic policies deserved a chance. Less charitably, the new economic policies also appeared to allow people already financially comfortable to abdicate concern for Third World development because the new orthodoxy asserted that market deregulation, rather than aid, was the royal road to development. The increased domestic inequality of recent decades in developed countries It is now clear that market deregulation and generally high birth rates have proven disastrous in many Third World countries. “Health for All”, if recalled at all, is now seen as absurdly optimistic. The failure of development is most obvious in many sub-Saharan countries, where life expectancy has fallen substantially. But life expectancy has also fallen in Haiti, Russia, North Korea, and a handful of other nations Causal theory is complex. Every cause has a cause, and, increasingly, causes are being considered as a part of causal chains, causal webs, and causal snowballs. Some theorists distinguish between identifiable “proximal” causes and deeper, underlying, or “distal”, causes Often, the carrying capacity of one region at one point in time is boosted by the appropriation of the carrying capacity from other people and even other generations. Such resources include oil, deep sea fish, and the stability of the global climate and ecological systems. But in Rwanda, the most densely populated country in Africa, the importation of such resources has long been limited. Unlike other densely populated countries such as Hong Kong and Holland, Rwanda's economy at the time of its most infamous genocide, in 1994, depended almost exclusively on its primary production Among the many different explanations for the horrific 1994 Rwandan genocide, the possibility of a Malthusian check  is scarcely mentioned ,19. A MaThere is even less scientific discussion that entertains the possibility that the sub-Saharan epidemic of HIV/ AIDS may also be a Malthusian check Maurice King refers to the silence on overpopulation as the “Hardinian Taboo”, named after the American ecologist Garett Hardin, who described the taboos that humans use to avoid confronting the need for population control Whatever the cause of the scarcity of modern academic analysis, the related issues of human carrying capacity and overpopulation deserve fresh consideration. The entrapment model has an explanatory power that is lacking in more superficial causal explanations. Of course, solving entrapment is very difficult, but as with most medical problems, a proper diagnosis will help identify the proper treatment.Human carrying capacity is the maximum population that can be supported at a given living standard by the interaction of any given human-ecological system. This apparently simple concept has many nuances and is rarely used by population scientists. However, in rejecting this term, purists risk making a terrible conceptual flaw, that of thinking that environmental and human resources are largely irrelevant to human population size.It is irrefutable that human ingenuity and cooperation can increase human carrying capacity Even culturally homogenous groups can outgrow their carrying capacity, as in the case of the Great Hunger in Ireland in the 1840s, when the population crashed because of famine, disease, and emigration. Indeed, Malthusian theory was used, in part, to justify the scanty aid provided to the Irish from Britain, a country that did not identify closely with the Irish.


Gangliocytic paraganglioma are rare neoplasms that predominantly arise in periampulary region. Though considered benign the disease can spread to regional lymphatics.A 49 year old woman presented with melena and was found to have a periampullary mass. Endoscopic evaluation and biopsy demonstrated a periampullary paraganglioma. The tumor was resected with pylorus-preserving pancreaticoduodenectomy and was found to represent a gangliocytic paraganglioma associated with nodal metastases. In a controversial decision, the patient was treated with adjuvant external beam radiation therapy. She is alive and well one year following resection. The authors have reviewed the current literature pertaining to this entity and have discussed the biologic behavior of the tumor as well as the rationale for treatment strategies employed.Paraganglioma is a rare tumor that typically resides in the gastrointestinal tract and demonstrates low malignant potential. Due to rarity of the disease there is no consensus on the adjuvant treatment even though nearly 5% of the lesions demonstrate the malignant potential. This entity was first discussed by Dahl  in 1962 . The pat in 1962 . Long teA 49-year-old woman presented with a 6-month history of melena. During the preliminary consultation, she also complained of right upper quadrant pain, which radiated to her right lower quadrant and upper back. She underwent upper gastrointestinal (GI) endoscopy which demonstrated a 3 cm, ulcerated, ampullary mass figure . Endoscoth postoperative day.The patient underwent pylorus-preserving pancreaticoduodenectomy and lymph node dissection. The postoperative course was uneventful except for delayed gastric emptying diagnosed by frequent vomiting during postoperative days 7 to 10, and confirmed by gastrograffin swallow. These symptoms eventually resolved with conservative management and the patient was discharged from the hospital on 15Gross pathological evaluation of the resected specimen included a portion of duodenum with ampulla, measuring 16 cm. in length, and a portion of pancreatic head measuring 5 cm. in length. There was a polypoid periampullary mass protruding into the duodenal lumen, measuring 1.4 × 1.2 × 0.7 cm. On cut section, the mass was moderately firm and pink-tan. It was circumscribed but unencapsulated, and appeared to be covered by normal appearing duodenal mucosa. There was no evidence of pancreatic invasion by the tumor. A total of seven lymph nodes were also removed, 5 peripancreatic (3.0 cm in greatest dimension) and 2 periduodenal (1.2 cm and 1.0 cm in greatest dimension, respectively).Histologically, the tumor consisted of a complex neoplastic proliferation that included a component resembling carcinoid or islet cell tumor figure , admixedImmunohistochemical analysis demonstrated that the tumor stained positively for S-100, chromogranin, synaptophysin, and cytokeratin AE1 and AE3. No reactivity was observed with MART-1 or HMB 45. Staining for c-kit (CD 117) was performed on sections of the primary tumor and of one of the lymph nodes with metastatic tumor. The carcinoid-like epithelial cells and the spindle-shaped neurofibrillar cells stained negatively for the cell marker c-Kit. The gangliocytic cells stained strongly positive for c-kit.Due to the evidence of regional lymph node metastasis the patient was counseled regarding adjuvant therapeutic options. The treating physicians queried recognized experts in the field of radiation therapy for gastrointestinal malignancies via email and a consensus developed that external beam radiation therapy might be reasonable, although it was admitted that no data are available regarding the use of this modality for this disease entity. Ultimately, a decision was made to administer external beam radiotherapy to the abdomen in an effort to eradicate any possible residual disease not removed during surgery and to reduce the risk of locally recurrent disease. No chemotherapy was advised due to the rarity of distant metastases and the lack of response of these neoplasms to conventional systemic therapy.The patient was treated with intensity-modulated radiotherapy in 28 fractions of 180 cGy/fraction over 37 elapsed days; 6 mv photon beam energy was used. The target was a postoperative tumor bed with a 5–10 mm margin. The total dose was 5,040 cGy. She tolerated treatment well and is now symptom free more than one year following resection. Surveillance CT scans and endoscopy have been performed both of which reveal no evidence of recurrent disease.et al reported that there seems to be a slight male predominance and an average age of 54 at presentation [Gangliocytic paraganglioma is a rare, typically benign tumor of the gastrointestinal tract most commonly located in the second portion of the duodenum, with a few cases having involved the jejunum and pylorus -7. Burkeentation . This leentation .Histologically, our patient's tumor demonstrated the characteristic tricellular pattern of gangliocytic paragangliomas. These tumors are typically composed of an admixture of ganglion cells, spindle cells and epithelial cells ,10-13. TImmunohistochemically these tumors stain positive for a variety of markers as was demonstrated in this report. Such markers include those mentioned above as well as neuron-specific enolase, pancreatic polypeptide, somatostatin, myelin basic protein and neurofilament proteins ,12,13,17There is no data in the literature to guide clinicians on the use of adjuvant therapy despite the fact that approximately 5% of cases demonstrate malignant features . Since tGangliocytic paraganglioma is a rare duodenal tumor that can present with non-specific symptoms. Positive diagnosis can be obtained histologically by observing three characteristic cell types. Although this tumor is considered benign, the possibility exists for regional lymph nodal spread. Due to the rarity of the disease, no clear adjuvant treatment strategy has been determined in cases that demonstrate regional or distant metastasis.The authors(s) declare that they have no competing interests.AW wrote the original manuscript.AM performed surgical resection and prepared requested revisions of the manuscript.JM performed histopathological evaluation of the lesion and prepared photomicrographs.CT administered radiation therapy to the patient, made editorial suggestions, and supervised AW.


While it is well known that bradykinin B2 agonists increase plasma protein extravasation (PPE) in brain tumors, the bradykinin B1 agonists tested thus far are unable to produce this effect. Here we examine the effect of the selective B1 agonist bradykinin (BK) Sar-[D-Phe8]des-Arg9BK (SAR), a compound resistant to enzymatic degradation with prolonged activity on PPE in the blood circulation in the C6 rat glioma model.SAR administration significantly enhanced PPE in C6 rat brain glioma compared to saline or BK (p < 0.01). Pre-administration of the bradykinin B1 antagonist [Leu8]-des-Arg (100 nmol/Kg) blocked the SAR-induced PPE in the tumor area.Our data suggest that the B1 receptor modulates PPE in the blood tumor barrier of C6 glioma. A possible role for the use of SAR in the chemotherapy of gliomas deserves further study. The b® RMP- have beemor area . B1 recemor area ,9. The Bmor area . Thus, imor area .p < 0.01). This effect was abolished, with a return to basal levels, by pre-treatment of the animals with the BK receptor B1 antagonist LEU (35.8 ± 8.6 μg EB/g dry tissue). The effect of SAR in increasing PPE in C6-inoculated right hemispheres (91.9 μg EB/g dry tissue ± 7.9) was also observed (t = 24.9, p < 0.001) when compared to the contralateral non-tumoral hemisphere (21.7 μg EB/g dry tissue ± 3.0, n = 6) . We determined PPE in experimental groups of animals treated with bradykinin (BK), the BK receptor B1 agonist (SAR) or the BK receptor B1 antagonist (LEU)  has been successfully employed to enhance BTB permeability to chemotherapeutic agents [It was recently described that gliomas may express both B1 and B2 BK receptors, with B2 being located closer to the periphery of the tumor cells while B1 was observed throughout the cell . In viewc agents ,20, but c agents .In general, B1 receptors are non-constitutive  and restBK or BK agonists have been previously used to increase BTB permeability but, due to their faster enzymatic degradation ,8, they Our data suggest that the B1 receptor modulates PPE in the blood tumor barrier of C6 glioma. A possible role for the systemic use of SAR in the chemotherapy of gliomas or other CNS neoplasms deserves further study.The C6 rat glioma cell line was cultured as previously described . Brieflyad libitum. Animals were treated according to the Guidelines for the Use of Animals of Universidade Federal de Santa Catarina. Before the procedures, the rats were anesthetized with sodium pentobarbital (50 mg/Kg body weight, i.p.) and then injected with 1 × 106 C6 cells prepared in 5 ml DMEM/FCS (vehicle) into the right cerebral hemisphere using the following coordinates: 4 mm lateral from right bregma and 4.5 mm deep from the dural surface, as previously described [Male Wistar rats  were used. The rats were maintained in a temperature- and light-controlled vivarium , with food and water available escribed .n = 10, C6-inoculated; n = 6, control) or in combination with 100 nmol/Kg bradykinin (RBI, USA) (n = 6, C6-inoculated) or with 100 nmol/kg of selective bradykynin B1 agonist Sar-[D-Phe8]des-Arg9BK  (SAR, a gift from Dr Domenico Regoli, Université de Sherbrooke, Sherbrooke, Québec, Canada). An additional group of C6-inoculated animals (n = 6) were treated intravenously with SAR (100 nm/Kg) 5 min after pre-treatment with 100 nmol/Kg of the B1 receptor antagonist [Leu8]-des-Arg9BK (LEU, Sigma). Previous pilot studies conduced in our laboratory (data not presented) showed that SAR had a maximum effect at the equivalent dose of 100 nmol/Kg. These assays also showed that LEU at the dose used was effective in blocking the effect of SAR effect on isolated brain parenchyma and dura-mater. Fifteen minutes after the treatments, rats were perfused with 4 ml/Kg 0.9% NaCl for 3 min [Seventeen days after C6 inoculation, BBB or BTB extravasations were assessed by the Evans blue (EB, Sigma) method . EB  or by the Student t-test for paired samples . Values were considered statistically significant when BBB-Blood brain barrierBK-BradykininBTB-Blood tumor barrierB1-Bradykinin receptor B1B2-Bradykinin receptor B2EB-Evans BlueLEU-[Leu8]-des-Arg9BKMR-Metabolic resistancePPE-Plasma protein extravasationSAR-Sar-[D-Phe8]des-Arg9BK metabolically resistant bradykinin B1 agonistBLS and RCC contributed equally to this work. BLS and RCC carried out the surgical procedures and cell culture. MMB, RW, and CGCJr participated in the preparation of the manuscript. MAS carried out the absorbance assays. AGT and MN conceived the study and participated in its design and coordination. All authors participated in the analysis of the data and discussion of the results. All authors read and approved the final manuscript.


A method for identifying short-live proteins using a GFP-fusion cDNA library for monitoring degradation kinetics is described. We have developed a screening technology for the identification of short-lived proteins. A green fluorescent protein (GFP)-fusion cDNA library was generated for monitoring degradation kinetics. Cells expressing a subset of the GFP-cDNA expression library were screened to recover those in which the fluorescence signal diminished rapidly when protein synthesis was inhibited. Thirty clones that met the screening criteria were characterized individually. Twenty-three (73%) proved to have a half-life of 4 hours or less. Cellular proteins differ widely in their lability, ranging from those that are completely stable to those with half-lives measured in minutes. Proteins with a short half-life are among the most critical to the cell. Regulated degradation of specific proteins contributes to the control of signal transduction pathways, cell-cycle control, transcription, apoptosis, antigen processing, biological clock control, differentiation and surface receptor desensitization ,2. RapidIn vitro assay of degradation is simpler than in vivo analysis, but an in vitro assay system may not fully mimic the degradation of proteins in the cells. Genome-wide functional screening and systemic characterization of cellular short-lived proteins has received little attention [Aequorea victoria, has been widely used to monitor gene expression and protein localization [Analysis of labile proteins has been time-consuming and labor-intensive. The most definitive form of analysis requires pulse-chase labeling cells and immunoprecipitation extracts. ttention . GFP, thlization . Recentllization , a labillization  and thatlization . These slization . Furtherlization .We developed a GFP-based, genome-wide screening method for short-lived proteins. We made a GFP fusion expression library of human cDNAs and introduced the library into mammalian cells. Transfected cells were FACS-fractionated into subpopulations of uniform fluorescence. Individual subpopulations were treated with cycloheximide (CHX) to inhibit protein synthesis and re-sorted after 2 hours of treatment. Sorting was gated to recover cells with a fluorescent signal that was diminished compared to the population mode. Repeated application of this process resulted in a high yield of clones that encode labile fusion proteins.6 cell transformants per microgram of DNA. In addition, we confirmed by PCR amplification that 95% of clones contained a cDNA insert larger than 800 base-pairs (bp) (data not shown). The libraries were thus deemed to be useful for screening short-lived proteins in mammalian cells. We used 293T cells as the recipient. These cells offer two advantages. First, they express the SV40 large T antigen. This allows the library plasmids, which contain an SV40 origin of replication, to be highly replicated. Plasmids can therefore be recovered easily. Second, 293T cells have high transfection efficiency.The selection scheme is shown in Figure 10 intervals of fluorescence , a protein synthesis inhibitor. We chose a short treatment time to avoid selecting cells that became dimmer as a result of secondary responses other than rapid turnover of the GFP tagged proteins. To enrich for cells that are susceptible to CHX treatment, we started with a cell population that has an approximately log-normal fluorescence histogram distribution, with a working range of 1.5 to 4.5 logs. We used FACS fractionation to divide this population into five subpopulations (R2, R3, R4, R5, R6) of ascending brightness, gating each on successive one-half log6 cells from the shifted population, the left shoulder of the population observed in the CHX-treated but not in the untreated R4 cells  is a stable protein, its fluorescence intensity would not be changed by treatment with CHX. We found that eight of the 12 groups showed a decrease of the fluorescence intensity peak by 30-50% (compared to untreated cells) after 2 hours of CHX treatment. In four out of 12 groups, no change in fluorescence intensity was detected.To isolate individual clones with the desired property, we randomly chose one of the eight CHX-responsive groups and characterized individual clones. We analyzed 30 clones from this group by individually transfecting them into 293T cells and determining the half-life by FACS-based analysis of CHX chase kinetics. We found out that 22 clones showed a decrease in fluorescence intensity ranging from 30 to 90% after treatment with CHX for 2 hours. Assuming first order kinetics of turnover, this single-time-point experiment implies that the proteins corresponding to these 22 clones have a range of half-lives ranging from about half an hour to 3-4 hours Table . The 22 As no data are available on the intracellular turnover kinetics of the 19 identifiable proteins, we picked three clones - splicing factor SRp30c, a guanine nucleotide-binding regulatory protein (G protein), and cervical cancer 1 proto-oncogene protein - and examined their turnover by CHX chase and western blot analysis. These three clones Table . The halSequencing analysis indicated that these three GFP fusion cDNAs do not contain a full-length coding sequence. SRp30c cDNA is missing 17 amino acids at its amino terminus, G protein 20 amino acids, and proto-oncogene p40 three amino acids. To exclude the possibility that the missing amino acids or the fused GFP domain contribute artifactually to protein liability, we amplified the full-length coding sequences of these three genes and expressed them as Myc fusion proteins. Their turnover was examined by CHX chase and western blot analysis with antibody to the Myc tag Figure . TurnoveThis technology is subject to two kinds of false-positive results. First, fusion to a detection tag such as GFP or Myc may affect the folding of tagged proteins, which could accelerate their turnover. Second, expression of the fusion proteins under the control of viral promoter elements could result in overexpression, with concomitant misfolding or failure to associate with endogenous interaction partners. To rule out these artifacts, we measured the degradation of native non-fusion endogenous counterparts of two of the proteins we identified, those for which antibodies were available. Turnover of the proteins associated with clone 19 and clone 25 was measured by CHX chase and western blot analysis. The results Figure  demonstrThe abundance of a given cellular protein is determined by the balance between its rate of synthesis and degradation. The two are of equal importance in their effect on the steady-state level. Furthermore, degradation determines the rate at which a new steady state is reached when protein synthesis changes . DespiteWe have developed an efficient and rather specific screen by combining GFP fluorescence, as a high-throughput measure of protein abundance, with pharmacologic shutoff of protein synthesis. Of 30 clones that were recovered from the screen Figure  and indiFor some of the proteins uncovered in this survey, rapid turnover can be rationalized as intrinsic to their cellular function. SRp30c factor (accession number U87279) is responsible for pre-mRNA splicing. Alterative splicing is a commonly used mechanism to create protein isoforms. It has been proposed that organisms regulate alternative splice site selection by changing the concentration and activity of splicing regulatory proteins such as SRp30c in response to external stimuli . The finThe G proteins are a ubiquitous family of proteins that transduce information across the plasma membrane, coupling receptors to various effectors ,17. AbouIn mammalian cells, proteasomes have the predominant role in the degradation of short lived proteins, whereas lysosomal degradation appears to be quantitatively less important . DetermiIn this study we have developed an innovative technology to identify labile proteins using GFP-fusion expression libraries. Using this technology we have discovered short-lived proteins in a high-throughput format. This technology will greatly facilitate the discovery and study of short-lived proteins and their cellular regulation.XhoI restriction site and with StrataScript reverse transcriptase. Synthesis was performed in the presence of 5-methyl dCTP, resulting in hemimethylated cDNA, which prevents endogenous cutting within the cDNA during cloning. Second-strand cDNA was synthesized using E. coli DNA polymerase and RNase H. Adaptors containing EcoRI cohesive ends were introduced into the double-stranded cDNA, which were then digested with XhoI. The cDNAs contained two different sticky ends: 5' EcoRI and 3' XhoI. The cDNAs were separated on a 1% SeaPlaque GTG agarose gel in order to collect those larger than 800 bp. After extracting cDNAs from the agarose gel with AgarACE-agarose-digesting enzyme followed by ethanol precipitation, the cDNAs were directionally cloned into EGFP-C1/2/3 expression vectors with three open reading frames (ORFs) (Clontech). The vectors were modified within the multiple cloning sites in order to be compatible with the cDNA orientation. By this means, cDNA ORFs were aligned to the carboxy terminus of EGFP. The host cell used for plasmid transfection and expression, 293T, expresses the SV40 large T antigen. Therefore, the cDNA EGFP-C1/2/3 vector containing the SV40 origin of replication can replicate independently from chromosome DNA in the host cells, which facilitates the recovery of plasmid DNAs from the host cells.Messenger RNAs from brain, liver, and the HeLa cell line (Clontech) were used as templates for cDNA synthesis, using a cDNA synthesis kit from Stratagene according to the manufacturer's recommendation, with some modifications. First-strand cDNA was synthesized using an oligo(dT) primer-linker containing an 293T cells were cultured at 37°C in DMEM (Invitrogen) supplemented with 10% FBS, 1% nonessential amino acids and 100 U/ml penicillin, 0.1 mg/ml streptomycin. One day before transfection, cells were seeded in 10-cm plate in 10 ml growth medium without antibiotics. Transfection was performed using Lipofectamine 2000 reagent according to the manufacturer's instructions. Samples (25 μg) of a cDNA library were diluted in 1.5 ml Opti-MEM (Invitrogen). Lipofectamine 2000 was diluted in 1.5 ml Opti-MEM and mixed with diluted DNA. After 20 min incubation, the DNA-Lipofectamine 2000 complex was added to the cells. The cells were incubated for 16 h before analysis.6 cells were collected in 12 × 75 mm glass tubes containing 200 μl serum to enhance the cell survival rate. For short-lived protein screening, sorted cells were recultured in a 12-well plate and treated with or without 100 μg/ml CHX for 2 h. The cells then were collected and subjected to FACS analysis and sorting. The cells showing a decrease in fluorescence intensity with CHX treatment were collected for further analysis.Cells were harvested by trypsinization, washed, and resuspended in DMEM. Cytometric analysis and sorting were performed using a hybrid cell sorter combining a Becton Dickinson FACStarPLUS optical bench with Cytomation Moflo electronics (Stanford Beckman Center shared facility). Green fluorescence was measured using a 525/50 band pass filter. Gates were set to exclude cellular debris and the fluorescence intensity of events within the gated regions was quantified. Fluorescence-activated cell sorting was performed with a lower forward scatter threshold to detect transfected cells while ensuring that debris and electronic noise were not captured as legitimate events. Transfection efficiency was so high that normal voltages for detecting GFP were reduced. For fractionation, the cell population was gated on the basis of the fluorescence intensity. Cells were sorted at a rate of 8,000 events/sec. 10E. coli (Invitrogen). Bacterial colonies were transferred to 96-well plates containing LB with 50 μg/ml kanamycin and 30% glycerol. After overnight growth at 37°C, the colonies are stored at -80°C. Plasmid DNAs were prepared from individual clones, sequenced and BLAST searches performed against the NCBI database.Plasmid DNA was extracted from sorted cells using a Qiagen mini-plasmid preparation kit. Plasmid DNAs were eluted in water and transformed into electro-competent DH10B To obtain full-length coding sequence of the genes, we amplified them with a human full-length cDNA kit (Panomics) according to the manufacturer's instructions. The full-length coding sequences of cDNAs were then cloned into the pCMV-Myc vector (Clontech) for expression in 293T cells.The plasmid DNAs of individual clones were prepared and transfected into 293T cells. The transfected cells, with or without CHX treatment, were collected in PBS and cell lysates were prepared by sonication. Proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to a membrane. Fusion proteins were detected using a polyclonal antibody against GFP (Clontech), a monoclonal antibody against the Myc epitope (Sigma), a polyclonal antibody against G protein (Santa Cruz) or an antibody against Hsp70 (Santa Cruz). Bands were visualized with SuperSignal West Pico kit (Pierce).Additional data file The original data used to perform this analysisClick here for additional data file


MgSO4*7H2O was administered daily for two weeks. Animals were examined for the contractile properties and for the number of motor units of extensor digitorum longus and soleus muscles at three postnatal stages and adulthood. Four experimental groups were included in this study: i) controls, ii) axotomized rats, iii) magnesium treated controls and iv) axotomized and Mg2+-treated rats.The nerve crush was performed on the 22+ were evident immediately after the Mg2+-treatment. Immature EDL and soleus muscles were slow and fatigueable. Soleus gradually became fatigue resistant, whereas, after axotomy, soleus remained fatigueable up to adulthood. EDL gradually became fastcontracting. Tetanic contraction in axotomized EDL was just 3,3% of the control side, compared to 15,2% in Mg2+-treated adult rats. The same parameter for axotomized soleus was 12% compared to 97% in Mg2+-treated adult rats.Axotomy resulted in 20% MU survival in EDL and 50% in soleus. In contrast, magnesium treatment resulted in a significant motor unit survival . The neuroprotective effects of Mg2+ treatment in vivo.These results demonstrate that motoneuron death occurs mostly within two weeks of axotomy. Magnesium administration rescues motoneurons and increases the number of motor units surviving into adulthood. Fast and slow muscles respond differently to axotomy and to subsequent Mg In the rat the first 3 weeks of life constitute a critical period of neuromuscular plasticity. Contractile properties of muscles are not inherent, but are determined by the motor nerve, that supplies the muscle. This was shown by experiments of crossinnervation between fast and slow muscles .Axotomy of peripheral nerves in neonatal rats, leads to loss of the bigger immature motoneurons through an excitotoxic process, with bigger neurons firing at a greater frequency. Consequently there is loss of the fast-contracting muscle fibers being innervated by bigger nerve cells .2+ influx into the cell and subsequent activation of a cell death cascade (activation of proteases, lipases and other enzymes leading to cell lysis).There is much evidence that overactivation of glutamate receptors plays a significant role in this process (glutamate excitotoxicity) . GlutamaFollowing neonatal sciatic nerve injury, the surviving motoneurons, take at least 8 days to grow back to the hind limb muscles, whereas most of the motoneurons that die, do so by apoptosis within the first two days . Howeverth postnatal day (P5) does not cause significant motoneuron loss, since damaged axons reinnervate muscle fibers successfully [Target deprivation from muscle after the 5essfully ,7,9.nd postnatal day. It could be assumed that in our model, magnesium ions penetrate the blood brain barrier, as magnesium has been shown to concentrate in the cerebrospinal fluid after intraperitoneal injection and increase the electrical threshold required to control seizures, in a rat model, by other investigators [In this study we examine the possible neuroprotective effects of systemic administration of magnesium ions, after axotomy in neonatal rats on the 2tigators .We also investigated the time course of motor unit loss and the alterations in the contractile properties of a fast- (EDL) and slow-contracting (soleus) muscle.nd postnatal day the sciatic nerve of the left hindlimb was crushed, in order to perform axotomy. Four experimental groups were included in this study: i) control, ii) rats whose sciatic nerve was crushed (axotomy), iii) magnesium treated controls and iv) rats whose sciatic nerve was crushed and were magnesium treated.Wistar Albino rats of both sexes were used in these experiments. All efforts were made in order to minimize the number of animals used and their suffering. The research project complies with the guidelines for animal use, established by the American Physiological Society and was approved by the local ethical committee in accordance with EEC Council Directive 86/609. The day of birth was taken as P0 (zero). At the 22+-treated and non-treated rats at: a) Postnatal day 14 (P14), b) Postnatal day 21 (P21), c) Postnatal day 28 (P28) and d) Adult rats (older than 2 months). In our study six successfully tested Mg2+-treated and six non-treated rats of each age-group (48 rats) were included.The rats were examined for the contractile properties and the motor unit number of two hindlimb muscles (EDL and soleus) at several stages of postnatal development. Tension recordings were held in both hindlimbs (operated and control) in groups of Mgnd postnatal day and were operated under sterile conditions. Surgery was performed under an operating stereoscope (10× magnification). The sciatic nerve was identified and crushed at the mid-thigh, just proximal to its division to the tibial and common peroneal nerves. Care was taken not to damage the blood supply to the surrounding tissues. Crush was performed using a fine forceps for 30 seconds. Then the nerve was examined to ensure that the epineural sheath was intact but translucent (axotomy). The wound was closed with fine sutures. All surgical procedures were carried out by the same surgeon. Three hours after wound suturing the neonates were placed back with their mothers.The animals were anesthetized with ether at the 2Specific tests were performed daily in order to confirm the efficacy of axotomy:a) The plantar- and the dorsi-flexion reflexes were checked: The examiner, placing his index finger on the animals foot, forces it to plantar- and dorsi-flex the ankle. The animal reflects actively, doing the opposite movement, if able to dorsi- and plantar-flex, respectively. No reactive movements are evident after successful denervation.b) Animals were suspended by their tail and the inability of normal movement of the left hindlimb, indicating successful nerve axotomy, was assessed for the following :_ Hip and knee in extension_ Ankle in the operated side in plantar flexion_ Adduction of the whole limb_ Weakness of digit extension in operated sideThe first behavioral signs of reinnervation after axotomy, should be evident at about 10–12 days after injury, according to other investigators [Only those animals, whose successful axotomy was verified, were included in our study.2+ was injected subcutaneously as a solution of MgSO47H2O (0,05 ml of 1 M solution / 10 g body weight). This was the highest tolerable dose. Mortality in higher doses exceeded 80%. The treatment continued until the rats were 14 days old. Control groups were injected daily with the same volume of normal saline (0,05 ml / 10 g body weight). In Mg2+ treated animals weight gain slowed down during the first month of their life. Treated and control adult rats, however, did not show statistically significant differences in their body weight. Axotomized rats, presented with motion deficit on the denervated limb, as described above, for about 2 weeks. After Mg2+ treatment normal kinetic behaviour and reflexes returned on the 7th to 9th postnatal day.The rats of some litters were treated daily with magnesium ions. Mg3 25 mM, glucose 5,55 mM and CaCl2 1,89 mM) [Animals were anesthetized with Chloral Hydrate (4,5% 1 ml / 100 g of body weight). The EDL and soleus muscles of both the axotomized and the contralateral control leg were prepared. The distal tendons were dissected free and attached to a strain gauge transducer (Dynamometer UFI, Devices) by a short silk suture and the exposed parts of the muscles were kept moist with warm (37°C) Krebs solution (NaCl 118,08 mM, NaHCO1,89 mM) . The sciThe tendons of EDL and soleus muscles were connected to a strain gauge and the tension elicited by sciatic nerve stimulation (Digitimer DS9A stimulator) was displayed on the screen of an oscilloscope (Fluke PM 3380 A). Muscle length was adjusted so as to produce maximal single twitch tension , through a micromanipulator allowing motion on the 3 axes (Prior, England). Then stimulus intensity was adjusted in order to elicit maximal tension, using supramaximal (3–9 volts) square pulses of 0,5 msec duration. The signal from the transducer was amplified by a DC transducer amplifier (Neurolog NL 107). Time To Peak (TTP) and Half Relaxation Time (HRT) of the Single Twitch recording were measured. Tetanic contractions were then elicited by stimulating the nerve at 10, 20, 40, 80 and 100 Hz. The duration of stimulus was 250 msec. All devices during the tension-recording procedure were controlled by a pulse programmer (Digitimer D4030).The number of motor units (MU) was estimated by the incremental method (the number of different single twitch tensions produced by stepwise increments of stimulus intensity). The fatigueability of the muscles was tested by stimulating them at 40 Hz for 250 msec every second. The decrease in tension at 3 minutes of such stimulation was measured and the % Fatigue Index (FI) was calculated:F.I. =  / Initial Tension.After tension recordings were completed, muscles were excised and weighed. The animals were killed by cervical dislocation.Stiatical analysis of the results was performed using SPSS 10.0 software for Windows. Nonparametric tests  were used in order to compare data, of different groups. Criterion of statistical significance was set at p < 0.05.2+ does not affect the number of motor units of control muscles. Axotomy at P2 results in statistically significant (p < 0.05) motor unit loss in both EDL and soleus in all age groups. This means that motoneuron death is already established at P14. Treatment with Mg2+ results in a statistically significant difference in the survival of motor units compared to non-treated axotomized rats. It is obvious that the neuroprotective effect of Mg2+ is already established at P14 immediately after the period of treatment  muscles, however, are not yet differentiated into fast- or slow-contracting. In adult rats TTP and HRT are respectively a) 36 ± 4.4 msec and 28 ± 2.28 msec in the EDL and b) 56.2 ± 3.37 msec and 59.5 ± 3.78 in the soleus . It caused axotomized EDL to become fast-contracting in adult rats (TTP: 38 ± 7.53 and HRT: 43 ± 4.13), as it is predicted for control muscles, whereas soleus' contractility was not affected.Mg2+ administration, with a fatigue index of about 65% (Table 2+ treatment not affecting this state. Axotomy however, causes EDL to become fatigue resistant in adult rats (F.I = 15.6). Values of operated side compared to those of control muscles, are statistically significantly different even at P21. Mg2+ administration after axotomy does not induce conversion of EDL to a fatigue resistant muscle.Soleus is a fatigue resistant muscle in adult rats, whereas EDL is not. However, both immature EDL and soleus muscles show properties of fatigueable muscles. EDL (control or operated) at P14 is not fatigue resistant, irrespective of Mg2+ treatment, the development of soleus into a fatigue resistant muscle is not hindered.While soleus is not fatigue resistant at P14 F.I.= 55.6%), it gradually becomes fatigue resistant during normal development , an NMDA antagonist, has been used in animal models  axotomy ,16. Howe2+ site within the pore of the ion channel [Magnesium is a non-competitive, voltage dependent, NMDA-receptor antagonist, acting by coupling with the specific Mg channel -19. Its  channel .Moreover, axotomy in early postnatal period does not only reduce the number of surviving motoneurons and motor units, but also provokes changes in the contractile properties of limb muscles . ImmaturIt has been shown that the number of motor units remains constant after birth ,23. ThisIt should be mentioned that soleus contractile properties are not significantly altered throughout early postnatal life. Soleus is a slow, non-fatigue resistant muscle at P14 that progressively becomes fatigue resistant. The process of soleus' development into a fatigue resistance muscle is stopped, after sciatic nerve axotomy. Axotomized soleus becomes less fatigue resistant in adult rats, compared to control muscles. However the process of muscle necrosis, as proposed by other authors, could contribute to this result, as well, rather than the loss of motor units alone .Denervated soleus muscle at birth is less fatigue resistant than control muscles in adult rats. This state is already established at P28, with this process progressing further on to adulthood.As mentioned already, it is noticeable that force outcome even by this 'conservative' muscle is largely affected by axotomy as the animal grows up, after the first month of life. Our data show that even when no marked reduction in the number of motor units occurs, muscle weight does not improve from P28 to adulthood neither does single twitch or maximal tetanic tension. The same phenomenon appears in the EDL as well. The discrepancy seen during the P28 to adulthood interval, both in EDL and soleus, between the reduction in the number of motor units on the one hand, and force outcome (single twitch and tetanic twitch tension) and muscle weight after axotomy on the other, can be explained as a consequence of marked muscle atrophy and necrosis.As shown by other workers , all immBoth immature EDL and soleus muscles are slow contracting at P14. During normal development EDL gradually converts into fast contracting, remaining not fatigue resistant throughout life. We found that EDL has already gained characteristics of a fast muscle at P21. Axotomy causing death of the bigger motoneurons, thus destroying the large motor units, converts the muscle into slow-contracting. It was also shown that increase in speed of both EDL and soleus was much reduced after denervation .Our data show that EDL was affected by axotomy much more than soleus. If connection between muscle and nerve is disrupted, as is the case after axotomy, fastcontracting muscles are mostly impaired ,21,27. IHistological findings  and studPrevious studies  have shoMagnesium administration by other workers  has beenOur findings strongly support the findings of previous work , that haLocal application of magnesium ions to the muscle, by implants affecting achetylcholine release in the neuromuscular junction , is suggMagnesium administration did not cause any statistically significant influence on the contractile properties of the control muscles in the right unoperated leg, in any age group. Neuroprotection by magnesium reversed the effects of excitotoxicity, predominately in the fast-contracting EDL.2+ administration rescues motoneurons and increases the number of motor units surviving into adulthood. Furthermore, fast and slow muscles respond differently to axotomy, as well as, to subsequent in vivo treatment with Mg2+.In conclusion, our results show that motoneuron death occurs mostly within two weeks of axotomy, while systemic MgThe authors declare that they have no competing interests.AMPA: a-amino-3-hydro-5-methyl-4-isoxazolo-propionic acidCNS: Central Nervous SystemEDL: Extensor Digitorum Longus muscleFI: Fatigue IndexHRT: Half Relaxation TimeMg: MagnesiumMU: Motor UnitsNMDA: N-methyl-D-aspartateP: PostnatalSD: Standard DeviationTTP: Time to PeakNG carried out the experiments, participated in the sequence alignment as well as in the design of the study and drafted the manuscript.AH participated in the experiments and performed the statistical analysisDK participated in the experimentsMA conceived of the study, and participated in its design and coordinationThe pre-publication history for this paper can be accessed here:


Following publication of this work , the aut


In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44), kinases (EGFR-cytoplasmic domain, CDK2 and 4), proteases (MMP1, CASP2), signal transduction proteins (GRB2, RAF1, HRAS) and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX). Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study.Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY), or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx) and maltose binding protein (MBP) were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions.E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases proteins may be truncated to minimise molecular weight and the numbers of contiguous hydrophobic amino acids and low complexity regions to aid soluble expression in E. coli.By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in  To achieve this on a whole proteome scale a generic approach must be taken to the expression of protein families, unlike the traditional approach of protein chemistry in optimising the isolation of individual proteins on a case by case basis. E. coli has been the expression system of choice for the majority of laboratories engaged in high-throughput, multi-plexed cloning, expression and purification of proteins for structural genomics [E. coli as an expression host include well studied physiology, genetics and availability of advanced genetic tools [C. elegans full-length genes were cloned and expressed in E. coli only 11 % yielded soluble protein [in vitro refolding has been used in a number of cases, but refolding conditions are highly protein specific and so unlikely to be useful for high-throughput protein expression.The production of purified proteins is important for several experimental approaches aimed to assign gene function including antibody generation for immunocytochemistry and immunoprecipitation studies -3, in viractions ,5 and stractions . The avagenomics . The advic tools -10, rapiic tools  and protic tools . There a protein . Similar protein . With th protein ,16 or en protein  assays. E. coli one of which is to truncate long multi-domain proteins into separate domains, as has been performed for the Ephb2 receptor [There are several fall-back strategies for expression of correctly folded eukaryotic proteins in receptor ,18,19. Rreceptor  or inducreceptor . Alternareceptor ) or chapreceptor ; the indreceptor  or chemireceptor ; or the receptor ,26, overreceptor  or lipidreceptor . Perhapsreceptor -31.The aim of this work was to ask if it is possible to derive some general conclusions regarding which expression strategy would most likely result in the expression of soluble, functionally active mammalian protein on a family-by-family or domain-by-domain basis. A deep-mining approach was taken to maximise the chances of successful expression by examining the soluble expression of 30 different proteins using 14 different expression vectors. This study allowed us to make several conclusions regarding the best strategies to adopt for the soluble expression of different mammalian proteins in bacteria. The conclusions were tested by the expression of an additional 95 mammalian proteins. and sequence confirmed. A recombinational cloning strategy was employed termed "GATEWAY" cloning [The 30 proteins chosen for this expression study are listed in Table  cloning ,36 based cloning . Primers cloning  and openFor this study a set of destination vectors were constructed by modifying pET-DEST42 . The T7 promoter was chosen over other promoters commonly used for bacterial expression because of the high specificity and processivity of T7 RNA polymerase and the wide choice of expression strains currently available. Briefly, multicloning sites were created either 5' of the attR1 or 3' of the attR2 recombination sites for insertion of DNA inserts encoding N or C-terminal tags respectively. The expression vectors contained a T7lac promoter  for imprE. coli BL21(DE3), an expression strain containing chromosomally integrated T7 RNA polymerase gene (λDE3 lysogen) under the control of the lacUV5 promoter. To handle a large number of expression experiments  and associated manipulations to screen for total and soluble expression in E. coli, the recombinational cloning, transformation, growth of expression cultures and cell lysis and filtration separation of insoluble protein were performed in 96-well plate format. Figure Expression plasmids generated by recombination reactions were used to transform eriments 0 total aLooking first at the results for total (soluble and insoluble) expression, no clear patterns emerge for the various expression vectors used. With the exception of CASP2, CDKN2A, Trp53, EGFR(TK), FOS and CD44 most proteins expressed well across all expression vectors. Interesting differences are apparent however when one looks at the production of soluble protein. Using decahistidine green fluorescent protein (H10-GFP) or decahistidine glutathione-S-transferase (H10-GST) as fusion partners at the N-terminus gave poor yields of soluble intact product. This may not be because they were poor at promoting soluble expression but because they were prone to proteolysis during cell lysis reducing the yield of full-length soluble protein. A set of proteins (GFP, RAF1(Ras-bd), HRAS, mdm2(p53-bd), Ephb2(TK) and CCND2) gave high soluble expression levels in the baseline N-terminal decahistidine vector, which was not improved when expressed as decahistidine thioredoxin (H10-Trx) or decahistidine maltose binding protein (H10-MBP) fusions. The molecular weight of these proteins ranged from 9 – 35 Kda and averaged 22.8 Kda. These proteins are all expressed in the cytoplasm, have an average of 1 low-complexity region, 3.8 contiguous hydrophobic amino acids (hp_aa), pI of 6.6, grand average of hydropathicity index  was dramatically poorer at expressing soluble protein compared with the vector pN110 (which encodes a H10 tag), with average soluble expression yields of 0.8 and 3.3 mg per litre of culture respectively. Both vectors contain T7 RNA polymerase promoters, but pN110 also contains a lac operator (lacO) downstream of the promoter and the gene encoding the lac repressor (lacI) for tighter control of gene expression. This may result in a faster rate of transcript synthesis, after induction with IPTG, and hence translation rates (due to an increased concentration of mRNA) for pDEST17 compared with pN110. If translation rate exceeds the rate of protein folding, then increased production of insoluble protein would occur.A similar study was performed where the 30 ORFs were cloned into 8 different C-terminal tag expression vectors shown in Figure MBP-H10 was the most effective tag at the C-terminus to promote protein solubility with an average construct full-length soluble yield of 5.0 mg/l, which compares well with an average of 5.8 mg/l when this tag is fused at the N-terminus. The order of C-terminal tags to promote soluble expression was similar for total expression: MBP-H10 > GST-H10 > V5-H6 > Dhfr-H10 ~ GFP-H10 ~ Trx-H10 > H10 ~ DHFR-H10. Thioredoxin was not as effective a solubility enhancing tag when fused at the C-terminus with an average soluble yield of only 0.7 mg/l compared with 6.0 mg/l when fused to the N-terminus.Several correlations with protein features are seen when one groups the MPB fusions according to soluble protein expression levels. For the first group, where soluble expression levels were in the range of 5 – 50 mg/l, the average molecular weight, pI and GRAVY score were 20.6 KDa, 5.9 and -0.58 respectively. The average numbers of contiguous hydrophobic amino acids, low complexity and coiled-coil regions were 3.1, 0.56 and 0.22 respectively. The second group displayed soluble expression levels between 1 – 5 mg/l. Here, the average molecular weight, pI and GRAVY score were 25.1 KDa, 7.9 and -0.39 respectively and the average numbers of contiguous hydrophobic amino acids, low complexity and coiled-coil regions were 4.3, 0.71 and 0 respectively. The last group displayed soluble expression levels between 0 – 1 mg / l. Here the average molecular weight, pI and GRAVY score were 41.1 KDa, 6.2 and -0.51 respectively and the average numbers of contiguous hydrophobic amino acids, low complexity and coiled-coil regions were 5, 2.43 and 0.21 respectively. There were representatives of nuclear, cytoplasmic and extra-cellular proteins in all three groupings.E. coli previously (Nfkb1), to those that contain no PfamA domains and have not been expressed in E. coli previously (Maat1, BC031407, Ttyhl, 1500001H12RIKEXT2, Ext2, KIAA1136, G2 and KIAA1549). They included 24 proteins not annotated as PfamA domains, with unknown function. All cDNAs were amplified from a primary cDNA library, cloned into pDONR221 and sequence confirmed prior to transfer to pDEST-N112-MBP  , sub-celet al.  tested tet al. found a similar relationship with their set of 32 human proteins with 4 different N-terminal fusions [There was a strong correlation between successful soluble expression and molecular weight of the protein. Small proteins with an average molecular weight of 22.8 KDa did not require to be fused with solubility enhancing proteins for soluble expression whereas proteins that required to be fused with N-terminal MBP or Trx for soluble expression had an average molecular weight of 40.4 KDa and those where the addition of a N-terminal fusion could not rescue soluble expression had an average size of 51.4 KDa. The same pattern also emerged in the C-terminal fusion study. The decreasing probability of successful soluble expression of mammalian proteins with increasing molecular weight is likely due to increasing protein complexity, perhaps requiring specialised eukaryotic chaperones for folding or stabilising binding partners. The majority of proteins solubly expressed in this study contained single domains and as fusion proteins were either capable of self-folding or were folded with the aid of prokaryotic chaperones. Braun  fusions .in vivo.A correlation in this study was observed between increasing numbers of contiguous hydrophobic amino (hp_aa) acids (AILFWV) and soluble expression. This ranged from an average of 3.8 hp_aa for those proteins not requiring a N-terminal fusion for high level soluble expression to 5 hp_aa for proteins requiring a N-terminal fusion for successful expression and 5.6 hp_aa where expression failed under the conditions described here. This pattern was also repeated in the C-terminal fusion study where good expression proteins had an average of 3.1 hp_aa whereas poor expression proteins had an average of 5 hp_aa. In a study of the sequences of 2753 non-membrane proteins it was found that the sequences of three or more consecutive hydrophobic residues are suppressed in globular proteins . Low comSome interesting conclusions were drawn when soluble expression was measured for an additional set of 95 mammalian proteins expressed as H10-MBP fusions Table . In seveet al. [There have been three comparative studies recently where sets of proteins were cloned into several expression vectors and the effects of the fusion partner on total and soluble expression yield were examined. Hammarstrom et al.  cloned 2et al.  Here GSTet al. . HoweverIn this study it was found that, on average, N-terminal fusion partners are preferable for optimal protein expression. When proteins are expressed with their native N-terminus, as in our C-terminal fusion proteins, total expression levels can be more variable than when expressed with a constant N-terminal tag. This may be because of variable RNA secondary structures in the region around the start codon which could interfere with ribosome binding. An additional explanation is that during translation the expressed protein emerges from the ribosome first and initiates an incorrect, irreversible, folding pathway before the soluble fusion partner has been translated and folded. The mis-folded protein would be ubiquitin labelled and targeted to the proteasome for degradation resulting in lower total expression levels. This scenario is more likely when expressing mammalian proteins in a bacterial system which lacks specific eukaryotic chaperone proteins. It has been shown previously that proteins prone to mis-folding and aggregation can arrest GFP folding when fused at the C-terminus . HoweverIt was found that Trx and MBP were the best N-terminal protein fusions to promote protein solubility. The best C-terminal fusion to promote protein solubility was MBP and this may be acting as a true intra-molecular chaperone , able toIt must be stressed here that although protein solubility is a useful indicator of correct folding, additional measurements need to be performed to give supporting evidence for correct folding. These may include removing the protein fusion with a protease and analysis of the cleaved protein of interest by a variety of biophysical and functional assays such as analysis of monodispersity by light scattering , NMR 5656,57, CDGFP did not significantly enhance soluble protein expression when fused to the C-terminus of the proteins in this study, supporting the use of this tag as an indicator of soluble protein expression of fused ORFs.,41 The oE. coli? If the protein has a molecular weight of less than 30 KDa and contains 1 or less low complexity regions and less than 4 contiguous hydrophobic amino acids expression of the full-length protein in E. coli should give good levels of soluble protein. As a generic strategy we would recommend expressing the protein with a fusion partner and found MBP and Trx to be the best fusions to enhance protein solubility as N-terminal tags with MBP being superior as a C-terminal fusion. C-terminal fusions are desirable for proteins such as the P450s where N-terminal tags can inhibit functional activity. When fused to an optimal fusion partner, nuclear, cytoplasmic and extra-cellular domains were equally likely to be expressed solubly. For larger proteins over 50 KDa, truncations should be considered to express specific protein domains and to minimise the molecular weight, number of low complexity regions and contiguous hydrophobic amino acids. In conclusion, this study will help enable a systematic expansion in the number mammalian proteins and domains that can be successfully expressed in E. coli as soluble product, and also predict which are best targeted for a eukaryotic expression system.What guidelines have emerged from this study in developing a strategy for the production of soluble mammalian proteins in E. coli DB3.1 and BL21(DE3)Star pLysS, Gateway BP and LR clonase enzyme mix, pre-cast 4–12 % NuPAGE Bis-Tris gels and PVDF membranes (0.45 μm pore size) were all from Invitrogen (Paisley, UK). Entry plasmids in both open (minus stop codon) or closed format (plus stop codon) containing the full-length genes for GRB2, HRAS, JUN, FOS, MAD, MAX, CDK2, CDK4, CDKN1B, CASP2, MMP1, CDKN2A and CD44 were provided by Pascal Braun and Josh LaBaer (Harvard Institute of Proteomics, Cambridge, USA). A full length clone containing the full-length human EGFR ORF was provided by the RIKEN BioResource Center (Tsukuba, Japan) and Efna1 from the Mammalian Gene Collection (MGC) archived at the Wellcome Trust Sanger Institute (Hinxton, UK). First strand synthesis human and mouse cDNA was from BD Biosciences (Oxford, UK). Plasmid, gel extraction and PCR purification kits and 6xHis protein ladder were purchased from Qiagen (Crawley, UK). The expression strain BL21(DE3), BugBuster protein extraction reagent and His tag monoclonal antibody was from Merck Biosciences (Nottingham, UK). The 96-well multiscreen-DV durapore filter plate with 0.65 μm pore size was from Millipore (Watford, UK) and Cy5-labelled goat anti-mouse IgG from Amersham Biosciences . Europium labelled antibodies and DELFIA reagents were from Perkin Elmer (Beaconsfield, UK) and all other chemicals unless otherwise stated were from Sigma-Aldrich (Gillingham, UK).Oligonucleotides were synthesised by Qiagen-Operon (Cologne, Germany) or Sigma-Genosys (Haverhill, UK). All restriction enzymes were from New England Biolabs (Hitchin, UK). The vectors pET-DEST42, pDEST17 and pDONR201 and 4, and 0.5 unit KOD hot start DNA polymerase (Novagen). PCR cycling conditions were: 94°C – 2 mins followed by 15 cycles of 94°C – 15 s, 59°C – 30 s, 68°C – 9 mins. The 7468 bp PCR product was purified using a PCR purification spin column (Qiagen) and eluted with 30 μl of 10 mM Tris-HCl (pH8.5), digested with 20 units of DpnI enzyme at 37°C for 4 hrs, to remove methylated plasmid DNA, purified by spin column and an intramolecular ligation reaction performed using 16 ng of linear PCR product and 5 units T4 DNA ligase and the buffers from the rapid ligation kit (Roche). The ligated PCR product was used to transform E. coli DB3.1 and the resultant pET-DEST42-MCS plasmid DNA prepared and sequence confirmed. Insert 1, encoding a decahistidine tag with a 5'-NdeI site and blunt 3' end, was prepared by PCR with primer pairs 22 (5' GGAATTCCATATGAAUCAC 3') and 24 (5' pGTGATGGTGATGGTGATGGTGATGGTGATTCATATGGAATTCC) and insert 2 encoding a decahistidine tag flanked by a 5'-NdeI site and 3'-KpnI site was prepared with primer pairs 22 and 26 (5' CGGGGTACCATGGTGATGGTGATGGTGATGGTGATGGTGATTCATATGGAATTCC 3'). PCR reactions were as above except the annealing temperature dropped to 44°C, extension time to 10 s and 12 cycles employed. Insert size was checked by 10 % TBE-PAGE and purified by a nucleotide removal kit (Qiagen). Expression vectors (b) pDEST-N110 and pDEST-N112 , pET41a or pET32 (Novagen) respectively The primer pairs for MBP were 78 (5' TTATTACACGAAGTGAAAATCGAAGAAGGTAAACTGGTAATC 3') and 79 (5' pGTTCGAGCTCGAATTAGTCTGCGCGTCTTTC), for GFP 84 (5' TTATTACACGAAGTGGCTAGCAAAGGAGAAGAACTTTTCACTGGAG 3') and 85 (5' pTTTGTAGAGCTCATCCATGCCATGTGTAATC 3'), for GST 86 (5' TTATTACACGAAGTGTCCCCTATACTAGGTTATTGGAAAATTAAGGG 3') and 87 (5' pATCCGATTTTGGAGGATGGTCGCCACC 3') and for Trx 88 (5' TTATTACACGAAGTGAGCGATAAAATTATTCACCTGACTGAC 3') and 89 (5' p CAGGTTAGCGTCGAGGAACTCTTTC 3'). The inserts were digested with DraIII and ligated with DraIII, BfrBI cut pDEST-N112 vector to create the GATEWAY destination vectors pDEST-112-MBP, pDEST-112-GFP, pDEST-112-GST, pDEST-112-Trx.To prepare pET-DEST42-MCS, a multi-cloning site was inserted into pET-DEST42 (Invitrogen) at nt396, between the shine-dalgarno sequence and the attR1 recombination site, encoding the recognition sequences for NdeI, KpnI, DraIII and BfrBI. Inverse or whole plasmid PCR was performed on pET-DEST42 with 5'-phosphorylated PAGE purified primer pairs 20 (5' TACCCACGAAGTGATGCATACAAGTTTGTACAAAAAAGCTGAACG 3') and 21 (5' CCCATATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAG 3') in a 20 μl reaction containing 10 ng pET-DEST42, 0.3 μM primers 20 and 21, 20 mM Tris-HCl (pH 7.5), 0.5 mM DTT, 200 μM each of dATP, dCTP, dGTP and dTTP, 1 mM MgSOpDEST-C101 was designed to insert a decahistidine encoded sequence between the attR2 recombination site and T7 transcription termination region. pDEST-C102 is as C101 except a DraIII, BfrBI site was inserted downstream of the attR2 recombination site. Inverse PCR was performed as described above with primer pairs 1 (5' pCACCATCACCATCATCACCATCACCATTGAGTTTGATCCGGC) and 2 (5' pATGCACCACTTTGTACAAGAAAGCTGAAC) to generate pDEST-C101 and primer pairs 1 and 3 (5' pATGCATACCACTCACTTCGTGCACCACTTTGTACAAGAAAGCTGAAC) to prepare pDEST-C102. Murine and human dihydrofolate reductase (Dhfr and DHFR respectively) inserts flanked by a 5' DraIII site and blunt end at the 3' were amplified from MGC clones using the primer pairs 82 (5' TTATTACACGAAGTGCGACCATTGAACTGCATCGTCGCCGTG) and 83 (5' pGTCTTTCTTCTCGTAGACTTCAAACTTATAC 3') for Dhfr and 80 (5' TTATTACACGAAGTGGGTTCGCTAAACTGCATCGTCGCTGTG) and 81 (5' pATCATTCTTCTCATATACTTCAAATTTG) for DHFR. The DraIII digested inserts were ligated with DraIII, BfrBI digested pDEST-C102 vector to create pDEST-C102-MBP, GFP, GST, Trx, Dhfr and DHFR as shown in Figure et al. [4, and 0.5 unit KOD hot start DNA polymerase (Novagen) in a total volume of 20 μl. The PCR reaction consisted of 94°C – 2 mins, and 30 cycles of 94°C – 15 s, 55°C – 30 s, 68°C – 2.5 mins followed by 68°C – 5 mins. A 50-fold dilution of the PCR 1 reaction was made for the second 30 cycle PCR containing the ORF specific primers. Linkers were added to these primers encoding half the attB1 and attB2 sites for forward and reverse primers respectively. For entry clone generation to be transferred to N-terminal tag expression vectors the 5'-linkers for the forward and reverse primers were 5' AAAAAGCAGGCTCT 3' and 5' AGAAAGCTGGGTTCTA 3' respectively with the reverse primer adding a stop codon. For inserts destined to the C-terminal tag expression vectors the forward and reverse primers were 5' AAAAAGCAGGCTTCGAAGGAGATAGAACCATGG 3' and 5' AGAAAGCTGGGTT 3' respectively with the forward primer encoding the shine-dalgarno and kozak sequences and start codon. PCR 2 products were analysed by 1 % TBE-agarose electrophoresis[A nested PCR strategy was used to isolate protein encoding ORFs directly from cDNA adapted for GATEWAY cloning from the method described by J. E. Collins et al. . Brieflyophoresis and corrophoresis except tophoresis were use600 had reached 1 (3 hrs for BL21(DE3)), 60 μl was transferred to 1.2 ml 2xYT media in a 96-deep-well block containing the appropriate antibiotics, placed on a shaking incubator at 37°C and when the OD600 reached 0.5 (2 hrs for BL21(DE3)) IPTG added to a final concentration of 1 mM and shaking continued at 25°C for 12 hours. Total protein was analysed by transferring a 20 μl aliquot of the induced culture to a 96-well PCR plate containing 20 μl of 2 × NuPage LDS loading buffer (Invitrogen), 0.1 M DTT, heated to 95°C for 10 mins and cooled on ice prior to loading 10 μl on a 17-well 4–12 % NuPAGE Bis-Tris gels with a multi-channel gel loading syringe (Hamilton). Soluble protein was extracted by transferring 290 μl of induced culture to a shallow well plate, centrifugation at 3000 g for 5 mins, supernatant removed and cells were resuspended in 58 μl BugBuster containing 1.4 units of benzonase and 58 units of recombinant lysozyme (Novagen). For the C-terminal tag and expression strain comparison this buffer was also supplemented with 0.58 μl protease inhibitor cocktail set III 10-fold diluted in DMSO (Novagen). The cell-pellets were resuspended with a multi-channel pipette and incubated with slow shaking for 20 mins at room temperature prior to transfer to 96-well multiscreen-DV durapore filter plates with 0.65 μm pore size (Millipore). The filter plate was placed on top of a shallow 96-well plate and centrifuged at 1000 g for 2 mins. 4 μl of the filtrate was then added to a 96-well plate containing 5 μl of 4 × NuPage LDS loading buffer (Invitrogen), 11 μl of 182 mM DTT, the plate heated at 95°C for 5 mins and loaded onto a 17-well 4–12 % NuPAGE Bis-Tris gel. A His-tagged molecular weight ladder (Qiagen) was also loaded onto each gel. Gel electrophoresis and electro-transfer to PVDF membrane was as described.[All BL21(DE3) transformants were selected and propagated in the presence of 100 μg/ml ampicillin. A single antibiotic resistant colony was used to inoculate 0.5 ml 2xYT media in a 96-deep well block containing the appropriate antibiotics and shaken at 210 rpm at 37°C. When the average ODMRD performed the molecular biology, participated in the bioinformatics, expression screening, quantitation, experimental design and drafted the manuscript. SPS and RLP participated in the expression screening and quantitation. KJV helped with the bioinformatics (database searching, protein domain annotation and primer design). JM participated in the experimental design, coordination and helped to draft the manuscript. All authors approved the final manuscript.