Severe acute respiratory syndrome (SARS) is a new infectious disease caused by a novel coronavirus that leads to deleterious pulmonary pathological features. Due to its high morbidity and mortality and widespread occurrence, SARS has evolved as an important respiratory disease which may be encountered everywhere in the world. The virus was identified as the causative agent of SARS due to the efforts of a WHO-led laboratory network. The potential mutability of the SARS-CoV genome may lead to new SARS outbreaks and several regions of the viral genomes open reading frames have been identified which may contribute to the severe virulence of the virus. With regard to the pathogenesis of SARS, several mechanisms involving both direct effects on target cells and indirect effects via the immune system may exist. Vaccination would offer the most attractive approach to prevent new epidemics of SARS, but the development of vaccines is difficult due to missing data on the role of immune system-virus interactions and the potential mutability of the virus. Even in a situation of no new infections, SARS remains a major health hazard, as new epidemics may arise. Therefore, further experimental and clinical research is required to control the disease. Severe acute respiratory syndrome (SARS) is the first new infectious disease of this millennium. SARS has originated from Southern China at the end of 2002 and has a high mortality and morbidity. Within a period of six months beginning at the end of 2002, the disease has affected more than 8,000 people and killed nearly 800 . The disSARS is caused by a novel, SARS-associated coronavirus (SARS-CoV) -6 which Shortly after the initial global alert, the WHO initiated a collaborative multi-center research project on SARS diagnosis, led by eleven principal laboratories in nine countries . Using mParallel to the progress made in the epidemiology and clinical diagnosis which has recently been demonstrated by numerous case reports, clinical studies and definitions , scientiWhile other reviews have focused on the epidemiology, clinical presentation and potential treatment of SARS, the present overview aims to analyze and present the currently available data on molecular mechanisms of SARS. In this respect, it is important to underline that in the present state of no specific drug or vaccine being available, research on molecular mechanism is crucial to identify potential treatment targets.Prior to the development of therapeutic regimes based on molecular mechanisms of the disease, the causative agent had to be isolated and analysed. Soon after the fast establishment of the international WHO laboratory network, rapid progress was made in the identification process of the causative agent, and it was reported that SARS is most probably caused by a novel strain of the family of coronaviruses -6. TheseSoon after the identification of a new coronavirus as the causative agent of SARS and of a southern Chinese province as the first area of occurrence, animal species of this area have been speculated to be the origin of the SARS-CoV. As analysis of the SARS-CoV genetic sequence revealed large differences to any other currently known coronaviruses in humans or domestic animals ,17, it wUntil the identification of the new SARS-CoV, the coronaviruses have been divided into three subgroups, which differ with respect to their genome . The firDespite the fact that this new virus most likely jumped to humans from wild animal species, it has remarkably well adapted to the human organism as shown by its high person-to-person transmissibility.The structure of the SARS viral RNA is organized in 13–15 open reading frames (ORF) and contains a total of approximately 30,000 nucleotides ,16,17.Recently, 61 SARS-CoV sequences derived from the early, middle, and late phases of the SARS epidemic together with two viral sequences from palm civets were analyzed . GenotypOnly ORFs exceeding fifty amino acids in translational capacity are considered relevant as they contain the sequences for the structural and functional properties of the virus and are therefore of potential interest for the development for future therapeutic strategies. The comparison of the different SARS-CoV ORFs with those of other coronaviruses reveals a familiar pattern of structural gene arrangement with replicase and protease genes (gene 1a-1b) and the spike (S), envelope (E), membrane (M) and nucleocapsid (N) genes in a typical 5'- to 3' order of appearance . The propro, one of the two papain-like proteinases operating on cleavage sites at the N-terminus of the polyproteins , also called 3C-like protease (3CLpro), is responsible for the cleavage of all the remaining proteins encoded by gene 1a-1b [Two-thirds of the SARS RNA is organized in the gene 1a-1b. The sequence of this gene is highly conserved among all coronaviruses . ORFs 1as Figure . The maine 1a-1b ,30.Apart from gene 1, coronavirus genes are known to be usually expressed from subgenomic mRNAs. They share a common leader sequence at the 5'-end and initiate at different places in the genome extending toward the 3'-end of the virus genome . Some ORpro and Mpro) were addressed as they are involved in replication, transcription or post-translational polyprotein processing [So far, five to eight subgenomic mRNAs were found in SARS-CoV-infected cells ,27. Thieocessing .In conclusion, research in the area of coronavirus gene expression is important to delineate components which directly affect SARS-CoV virulence.The structural proteins of the new SARS-CoV are potential targets for new treatment options. The new SARS-CoV only contains the three envelope proteins, spike (S), envelope (E), and membrane (M) but not the hemagglutinin-esterase (HE) protein, which is present in some coronaviruses of the second group.The spike glycoprotein is responsible for the characteristic spikes of the SARS-CoV Figure . Intra- The SARS-CoV has also been reported to cause the formation of syncytia in vivo, but so far only under the condition of cultured Vero cells . The SARAn interesting feature of the SARS-CoV and other coronaviruses is the resistance against the gastrointestinal fluids despite the lipid composition of their envelope. It has been reported that the SARS-CoV can survive in diarrheal stool for four days and also, patients with SARS often suffer from gastrointestinal symptoms with the virus to be detected in the stool . As the -4 for RNA-dependent RNA polymerases [It is unclear when and how novel pathogens such as the SARS-CoV cross the barriers between their natural reservoirs and human populations, leading to the epidemic spread of novel infectious diseases . As withymerases , RNA virymerases . This obymerases .Next to mutations, a further threat of the SARS-CoV is based on the ability of coronaviruses to undergo RNA recombination at a high frequency . For a vAlthough human coronaviruses are characteristically causing self-limiting short diseases, the question of potential chronic SARS infections is of major importance for a future disease control. If the SARS-CoV is able to cause a chronic persistent infection, chronic carriers may serve as sources for new SARS outbreaks. However, the detection of SARS-CoV in stool of patients for longer periods than 6 weeks after hospital discharge has not been reported so far. Therefore, the danger of chronic carriers may not be relevant. In contrast to common human coronavirus infections with short durations, most animal coronaviruses cause persistent infections. As an example, the feline coronavirus FIPV infects animals which then continue to shed virus for periods reaching up to seven months after infection without carrying disease symptoms . Also, TThe mean incubation period of SARS was estimated to be 6.4 days . The mean reported time from the onset of clinical symptoms to the hospital admission varied between three and five days .Main clinical features of the disease are in the initial period common symptoms such as persistent fever, myalgia, chills, dry cough, dizziness, and headache. Further, although less common symptoms are sore throat, sputum production, coryza, vomiting or nausea, and diarrhea ,51. SpecThe clinical course of the disease seems to follow a bi- or triphasic pattern. In the first phase viral replication and an increasing viral load, fever, myalgia, and other systemic symptoms can be found. These symptoms generally improve after a few days. In the second phase representing an immunopathologic imbalance, major clinical findings are oxygen desaturation, a recurrence of fever, and clinical and radiological progression of acute pneumonia. This second phase is concomitant with a fall in the viral load. The majority of patients is known to respond in the second phase to treatment. However, about 20% of patients may progress to the third and critical phase. This phase is characterized by the development of an acute respiratory distress syndrome (ARDS) commonly necessitating mechanical ventilation.Rapid progress has been made in understanding the clinical presentation of SARS in adults and children -56. In cThe course of the disease in teenagers more likely resembles adults in concerning clinical presentation and disease progression . SARS maCoronaviruses are known to exert their effects by cytocidal and immune-mediated mechanisms. In vitro studies using cell culture assays have shown that coronavirus infection commonly results in cytopathic effects such as cellular lysis or apoptosis . Also, tNext to cytocidal effects, also immune-mediated mechanisms of both the innate and adaptive immune system seem to contribute to the pathogenesis of SARS-CoV infections. In this respect, it has been shown that in MHV infection, T cells and cytokines play an important role in development of the disease . Also, hFor SARS-CoV infections, it has been reported that there seems to be an inflammatory cell influx consisting in particular of macrophages in the airways, and a massive release of cytokines during the peak of the infection ,68. It iThe pharmacotherapy for SARS with anti-inflammatory steroids is controversial and largely anecdotal . It was The most obvious gene which is likely to be a key modifier of SARS pathomechanisms is the spike (S) protein gene. As known for other coronaviruses, it does not only affect viral pathogenesis by determining the target cell specificity but also by other mechanisms. In this respect, a single mutation in the S gene of MHV has significant effects on the viral virulence and tissue tropism . Also, mThe primary target cells of SARS-CoV infection are respiratory epithelial cells. As the virus can also be detected in stool specimen and patients with SARS often also have gastrointestinal symptoms, epithelial cells of the gastrointestinal tract also seem to be major target cells. Next to these epithelial cells, the SARS-CoV has also been found in macrophages and many other cells as it has been detected in not only in the respiratory tract and stool specimen but also in the blood, liver, kidney and urine . In thisThe SARS-CoV target cell specificity is determined by the spike protein affinity to cellular receptors. In contrast to the all group III coronaviruses and the SARS-CoV for which the receptors have not been finally analyzed, it is known that group I coronaviruses bind to aminopeptidase N (CD13) as receptors , while gRecently, it was shown that a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), efficiently binds the S1 domain of the SARS-CoV S protein. SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Also, anti-ACE2 but not anti-ACE1 antibodies blocked viral replication on Vero E6 cells, indicating that ACE2 is a functional receptor for SARS-CoV  which waRecently, the C-type lectin CD209L  was discovered to be a further human cellular glycoprotein that can serve as an alternative receptor for SARS-CoV . The intAfter binding to the receptor, the next molecular step of potential use for the development of anti-SARS drugs is the virus entry into the cells. While most coronaviruses enter their target cells via plasma membrane fusion, a further entry mechanism may be acidic pH-dependent endocytosis . FocusinAfter the binding to a host cell receptor and entry into the cells, the molecular steps of transcription, translation and protein processing display further potential targets for new therapeutic strategies. In this respect, the RNA-dependent RNA polymerases (SARS-CoV RdRp) may be a potential target for a future anti-SARS therapy. A recent study located its conserved motifs and built a three-dimensional model of the catalytic domain . The autpro) or the 3C-like proteinase (3CLpro), and two auxiliary, papain-like proteinases (PL1pro and PL2pro). The latter two are responsible for the cleavage of the viral polyproteins, pp1a and pp1ab, at three sites near the amino-terminus, while the Mpro processes these proteins at as many as 11 additional sites. Interestingly, SARS-CoV lacks the PL1pro [pro [pro on PL1pro cleavage sites has been shown in IBV and HCoV [pro gene in other coronavirus genomes, SARS-CoV displays a domain within ORF1a that lacks any detectable sequence homology and has therefore been named the SARS-unique domain (SUD) [Also, protease activity is crucial for SARS-CoV RNA replication and protein processing ,85, and e PL1pro ,17, but pro [pro . This isand HCoV . Roughlyin (SUD) . It is npros of TGEV [pro in the crystalline state. The X-ray structure of the complex revealed binding of the P1 glutamine, P2 leucine, and P4 threonine side chains of this compound to the respective subsites in the substrate-binding cleft, in agreement with the pronounced specificity for cleavage by the Mpro after the substrate sequence -Xaa-Leu-Gln. The structure also showed the expected covalent attachment of the methyl ketone group at P1 of the inhibitor to the catalytic cysteine of the enzyme.Crystal structures have been determined for the M of TGEV , HCoV 22 of TGEV , and, mo of TGEV . They al of TGEV ,87,88. T of TGEV  have synpros of HCoV 229E and TGEV, the crystal structure of the SARS-CoV Mpro revealed some surprises [pro from the viral polyprotein, which probably involves a pH-dependent step.In spite of 40% and 44% sequence identity, respectively, to the Murprises . Within pro was employed by Yang et al. [The same hexapeptidyl chloromethylketone inhibitor used by Anand et al.  in theirg et al.  to charapro resembled that of AG7088 in complex with its target, the 3C proteinase of human rhinovirus [On the basis of their crystallographic work, Anand et al.  found thinovirus , even thinovirus  thereforinovirus .pro and screened 29 approved and experimental drugs against a model of the SARS CoV proteinase as well as the experimental structure of the transmissible gastroenteritis virus (TGEV) proteinase [Other studies used molecular dynamics simulations of the Moteinase . It was oteinase . HoweverFurther potential targets are the E and M proteins Figure  as they With regard to the multitude of potential epithelial target cells, specific endogenous drug delivery systems may also be of relevance. In this respect, the family of peptide transporters consisting of PEPT1 and PEPT2 which are differentially expressed in potentially infected cells of the respiratory tract ,95, smalAs most patients develop an immunity against the SARS-CoV and survive the infection, the possibility of creating an effective and safe vaccine seems to exist . There aLive-attenuated coronavirus vaccines can be generated by deletions in "group-specific genes". The deletions of these genes do not change replication properties but attenuate the virus . ExampleWhole killed vaccines are generally safe and easy to generate. In fact, this technique has been applied in veterinary medicine to generate vaccines for BoCV and IBV . Also, aUsing molecular biology techniques to generate large quantities of recombinant viral proteins, recombinant subunit vaccines, e.g. against the spike protein, are expected to be created relatively easy as shown by two recent studies ,117. EigAn approach using recombinant vectored vaccines with DNA or a viral vector could be a promising target. The DNA prime and adenovirus or MVA boost approach which is currently analysed for a potential use in the development of HIV vaccines, may also offer a strategy to prevent SARS infections. In this respect, a multi-valent approach which induces both humoral and T cell-mediated host responses seems to be the most attractive strategy.From the field of veterinary medicine, data on this approach are already available: A recombinant fowlpox with the S1 gene of IBV was demonstrated to be relatively protective against IBV . Also, aA further strategy is based on the use of epitopes which can be delivered using viral or DNA vectors. Such an epitope-based strategy for coronavirus vaccination has already been reported  and the In summary, the important development of SARS vaccines can be approached using several techniques which should ideally encompass the induction of both humoral and cell-mediated mechanisms. As coronavirus vaccines in animals have partly been reported to cause an enhancement of viral infections , a cautiIn summary, the onset of the SARS epidemic in different continents has led to the formation of a successful laboratory network to identify the molecular mechanisms underlying the SARS infection. Next to the development of early diagnostic tests and effective treatment strategies, it is most important to orchestrate research activities which lead to the development of vaccines and antiviral agents, as there is no established therapy to date. Even now in a situation of only a handful of new cases, SARS remains a major global health hazard which may reappear.


Tula hantavirus carrying recombinant S RNA segment (recTULV) grew in a cell culture to the same titers as the original cell adapted variant but presented no real match to the parental virus. Our data showed that the lower competitiveness of recTULV could not be increased by pre-passaging in the cell culture. Nevertheless, the recombinant virus was able to survive in the presence of the parental virus during five consecutive passages. The observed survival time seems to be sufficient for transmission of newly formed recombinant hantaviruses in nature. Recombination in RNA viruses serves two main purposes: (i) it generates and spreads advantageous genetic combinations; and (ii) it counters the deleterious effect of mutations that, due to the low fidelity of viral RNA polymerases and lack of proofreading, occur with high frequency . The purHantavirus, family Bunyaviridae) have a tripartite genome comprising the L segment encoding the RNA-polymerase, the M segment encoding two external glycoproteins, and the S segment encoding the nucleocapsid (N) protein [et al [Hantaviruses . These data confirmed our earlier observation [in situ formed recombinant S RNA disappeared from the mixture after four passages [First, we designed RT-PCR primers able to discriminate between non-recombinant (V-type) and recombinant (REC-type) types of TULV S RNA. The resullts presented in Fig. ervation  that thepassages , one shoin vivo situation, from one rodent to another. If transmission is performed in a sampling-like fashion – and this seems to be the case for hantaviruses [Although relatively short, the observed survival time of the recTULV in the presence of the original variant TUL02 seems to be sufficient for transmission of a recombinant virus, in a hypothetical aviruses  – the reaviruses  supportsaviruses ,21.The genetic swarm of S RNA molecules from the recTULV is represented almost exclusively by the variant with a single break point located between nt332 and nt368. The proportion of the dominant variant is larger in the passaged recTULV  than in the freshly formed mixture of recS RNAs (12 of 20 cDNA clones, or 60%) . Thus, rThe data presented in this paper show that the recTULV presents no real match to the original cell adapted variant and that the lower fitness of the recombinant virus can not be increased by pre-passaging in cell culture. The observed survival time of the recTULV in the presence of the parental virus seems to be sufficient for transmission of newly formed recombinant hantaviruses in nature.3 – 104 FFU/ml.RecTULV (clone 5) was purified from the mixture it formed with the original variant, TULV02, using two consequent passages under terminal dilutions . After t6 cells) were infected with the 1:1 mixture of recTULV and TULV02, approximately 104 FFU altogether. After 7–12 days the supernatant (~20 ml) was collected and RNA was extracted from the cells with TriPure™ isolation reagent, Boehringer Mannheim. Aliquots (2 ml) of the supernatant were used to infect fresh cells; the rest was kept at -70°C. The following nine passages were performed in the same way.Vero E6 cells (5 × 10RT was performed with MuLV reverse transcriptase (New England Biolabs); for PCR, AmpliTaq DNA polymerase (Perkin Elmer, Roche Molecular Systems) was used. To monitor the presence of TULV S RNA on passages, RT-PCR was performed with primers VF738  and VR855 . To monitor the presence of recTULV S RNA, RT-PCR was performed with primers RECF738  and RECR855 . All four primers were perfect matches to the homologous sequences; to the heterologous sequences, the forward primers have five mismatches while the reverse primers have six. Alternatively, complete S segment sequences of both variants of TULV were amplified using a single universal primer  and thenThe author(s) declare that they have no competing interests.AngP participated in the design of the study, carried out the experiments and helped to draft the manuscript. AlexP participated in the design of the study and drafted the manuscript. Both authors read and approved the final manuscript.


Do you want to know about evolution? Brian and Deborah Charlesworth provide an excellent and concise account of the core issues for a broad range of readers Evolution: A Very Short Introduction, is no mean feat.Their book is one of a series of short introductions, published by Oxford University Press, covering an eclectic array of subjects that aim to provide an accessible yet stimulating read for anyone wanting a thorough introduction to a topic. In this small volume, the Charlesworths have succeeded on both fronts and provide an excellent account of the core issues for a broad range of readers. One of the reasons for the book's appeal is that the authors draw on a range of carefully chosen human traits to illustrate their points. By contrast, most evolutionary textbooks (other than those purely on human evolution) tend to focus on nonhuman organisms. As with traits in every other organism, many human and human-related characteristics have evolved via genetic drift and natural selection, and they provide an effective means of convincing readers of the reality and relevance of evolution. For example, to explain how mutation can cause the loss of a function, the authors discuss the relatively poor sense of smell in humans, as compared with many other mammals, using an example of a vestigial ‘pseudogene’ of a human olfactory receptor gene. They also discuss tooth decay, enzyme aesthetics, heritable differences, cancer and other diseases, and the ability to taste and so on.Although the topics the Charlesworths choose to focus on are certainly appropriate, they provide only a brief mention of one important process—development. Evolutionary developmental biology is a burgeoning field that can provide interesting and important insights into our understanding of the mechanisms of evolution. For example, the absence of eyes in cavefish, rather than being the result of a degenerative process, might be the result of selection on genes that govern feeding morphology, a selection process that has included suppression of eye development . Such deIn Chapter 7, the authors discuss five topics that have traditionally been hard to understand from an evolutionary point of view. These ‘difficult problems’ are ageing, altruism, human consciousness, complex adaptations, and the origin of living cells. Difficult problems can be interpreted in two ways: those that, although hard to solve, have either been explained or will eventually be explained by modern evolutionary theories, and those problems that cannot be fully resolved with our current understanding but leave room for learning about additional mechanisms or factors. The Charlesworths generally consider only those problems of the former type—the explained ones. However, I think that some of the more intractable problems should be described in more detail. For instance, complex adaptation might be fully explained by mutations and natural selection, but additional unknown mechanisms might be essential for the evolution of the complex traits. I realize that opponents of modern evolutionary theory, such as creationists, have often cited these traditional problems to support their conclusion that modern evolutionary theory is wrong; but progress always depends on the consideration of new ideas, and there might be important mechanisms still to be discovered that play a key role in evolution. Describing potentially intractable problems might also spur on young readers who are thinking of studying evolutionary biology with the hope that there are still some theoretical battles to be conquered.The Blind Watchmaker by Richard Who is the target audience of this book? For many books, the topics chosen and the writing style can perhaps provide clues to the nature of the readers. For instance, In Japan, there seem to be few people who deny the facts of evolution, although there are many ideologically motivated books opposing natural selection and Darwinism. To convince creationists of evolution is usually extremely difficult, if not impossible, because they will never doubt their assumption that God created humankind. Education of young and curious people, however, can make a difference. This is where I think the book will be most successful, but this book should not just be limited to young people—I can recommend it to anyone who wants to know about evolution. Moreover, I can recommend it to Japanese students not only as an introduction to evolution, but also as an exercise in reading a well-written and engaging English text.


Immunotherapy in patients with Alzheimer's disease (AD) is rapidly becoming a hot topic of modern geriatric and clinical gerontology. Current views see immunization with Aβ peptide, the amyloidogenic protein found in senile plaque of AD patient's brains, or the infusion of preformed antibody specific for human Aβ, as possible therapeutic approaches to improve the cognitive status in the disease. Animal models of the disease have provided positive results from both approaches. Thus, an initial clinical trial using immunization with human Aβ in AD patients was started, but then shortly halted because of an unusually high incidence (6%) of meningoencephalitis. A long and currently ongoing debate in the scientific community about the pro or contra of vaccination or passive immunization with Aβ in AD is thereafter started. Here, the authors would like to stress few points of concern regarding these approaches in clinical practice. Immunotherapy in patients with Alzheimer's disease (AD) is rapidly becoming a hot topic of modern geriatric and clinical gerontology. M.E. Weksler  in the aCurrent views see immunization with Aβ peptide, the amyloidogenic protein found in senile plaque of AD patient's brains, or the infusion of preformed antibody specific for human Aβ, as possible therapeutic approaches to improve the cognitive status in the disease.Animal models of the disease have provided positive results from both approaches, since either vaccination with human Aβ or infusion of preformed antibodies specific for Aβ, have resulted in a decrease of amyloid plaques density in the brain of amyloid precursor protein (APP) transgenic mice ,3. ImproAn initial clinical trial using immunization with human Aβ in AD patients was started and then shortly halted because of an unusually high incidence (6%) of meningoencephalitis (see the Weksler article ). A longHere, we would like to stress few points of concern regarding these approaches in clinical practice.1) The claimed animal model for AD is unfortunately incomplete even if useful model for the human disease.2) Vaccination with human Aβ in mice induces an immune response against a foreign protein, i.e. human Aβ, and the mouse Aβ homolog does not appear to be involved. On the contrary, Aβ vaccination in man may potentially induce an autoimmune like disease in the brain and other peripheral tissues of susceptible patients. At the moment we do not have the ability to predict which patients will suffer destructive immune responses.3) The effects of both vaccination or passive immune therapy in AD brains might be non-specific, as already suggested by a recent report  and by t4) Vaccine therapy is not always effective in the elderly, since immune defects of variable degree are often present in old persons . In old 5) Historically vaccination has been successful in the prevention of the diseases much and much less in the therapy of ongoing diseases. A significant gain would be achieved by Aβ vaccination or passive immune therapy, if these manipulations will work in the very early stages of the disease. At the moment, no clinical data on this topic are available and those from animal models have not extensively addressed this topic.6) A general consideration on AD is also mandatory. In fact, clinical signs of dementia show up after extensive synapse and neuron loss have already occurred in the brain. How can an immune response restore an already compromised nervous circuit or revive dead neurons? The very modest decrement of cognitive deterioration rate claimed in a proportion of AD patients receiving the vaccine is a marginal therapeutic goal. In fact, the disease has not been cured, and the modest clinical slow down in the AD progression takes big prices, i.e. patients and care givers will continue to suffer the catastrophic effects of the disease for a longer time.We feel that Aβ vaccination and in a less extent passive immune therapy are now exciting experimental protocols for animal research and experimental neurology investigations, probably premature for clinical applications.


The gefitinib compassionate-use programme has enabled >39,000 patients worldwide to receive gefitinib ('Iressa', ZD1839) treatment. This paper reports the outcome of gefitinib treatment in Chinese patients who enrolled into the 'Iressa' Expanded Access Programme (EAP) at the Peking Union Medical College Hospital.Thirty-one patients with advanced or metastatic non-small-cell lung cancer (NSCLC) that had progressed after prior systemic chemotherapy were eligible to receive oral gefitinib 250 mg/day as part of the EAP. Treatment was continued until disease progression or unacceptable toxicity occurred. The impact of treatment on disease-related symptoms and quality of life (QoL) was evaluated with the Chinese versions of European Organization for Research and Treatment of Cancer Quality of Life Questionnaires (EORTC QLQ-C30 and QLQ-LC13).Gefitinib was well tolerated. Adverse events (AEs) were generally mild (grade1 and 2) and reversible. The most frequent AEs were acneform rash and diarrhoea. Only one patient withdrew from the study due to a drug-related AE. The objective tumour response rate was 35.5% ; median progression-free survival was 5.5 months (95% CI, 1.6 to 9.4); median overall survival was 11.5 months (95% CI, 5.6 to 17.3). The QoL response rates for five functioning scales and global QoL varied from 56–88%. The main symptom response rates varied from 44–84%. QoL and symptom response were correlated with objective tumour response.Gefitinib demonstrated safety and efficacy as monotherapy in this series of Chinese patients with advanced NSCLC and was also associated with remarkable symptom relief and improvement in QoL. Although clinical trials are needed to confirm these positive findings, the data suggest that treatment with gefitinib may be beneficial for some Chinese patients who do not respond to chemotherapy and have poor prognosis. Platinum-based combination chemotherapy is the standard first-line treatment for patients with advanced non-small-cell lung cancer (NSCLC). Although meta-analysis of clinical trials proved that combination chemotherapy for advanced NSCLC is superior to best supportive care  and new Many solid tumours express or highly express the epidermal growth factor receptor (EGFR). In lung cancer, deregulation of EGFR is seen mainly in NSCLC: EGFR is highly expressed in NSCLC at levels varying from 32–80% -6. AbnorGefitinib is an orally active small molecule EGFR tyrosine kinase inhibitor (EGFR-TKI). The tolerability and efficacy of gefitinib have been explored extensively in the West and Japan. In a Phase I trial, Nakagawa et al compared the safety profile, pharmacokinetic parameters and antitumour activity of gefitinib in patients with solid malignant tumours in Japan, the USA and Europe, and no significant difference was found for ethnicity . HoweverPatients with advanced NSCLC who had no alternative therapeutic options have been able to receive gefitinib treatment in a worldwide compassionate-use programme. Here we report the outcome of treatment with oral gefitinib 250 mg/day in Chinese patients with advanced NSCLC participating in the compassionate-use programme at Peking Union Medical College Hospital.9/L, absolute neutrophil count >1.5 × 109 cells/L, platelet count >100 × 109/L); liver function ; renal function (serum creatinine <150 μmol/L, blood urea nitrogen <10.7 mmol/L); PaO2 >60 mmHg.Patients aged ≥18 years with histologically or cytologically confirmed advanced or metastatic NSCLC were eligible for enrolment into the compassionate-use programme after providing written, informed consent. They were required to have failed prior chemotherapy, and had no other treatment options available. Patients who had received chemotherapy were included if the treatment ended >28 days prior to the study. Other eligibility criteria included: adequate bone marrow function , pregnancy and lactation.One oral gefitinib tablet (250 mg) was taken at about the same time each day. Gefitinib was administered every day without interruption unless disease progression or unacceptable toxicity occurred.2 during therapy. Patients were evaluated after the first and third cycles of therapy, then every three cycles. Tumour response was evaluated according to Response Evaluation Criteria In Solid Tumors [In this study, 28 days of treatment was defined as one treatment cycle. Baseline evaluation included medical history and physical examination, electrocardiogram, chest X-ray, thorax computed-tomography scan and ultrasonography of the upper abdomen. Laboratory investigations included complete blood counts, urinalysis, and renal and liver function tests. Performance status (PS) was evaluated according to Eastern Cooperative Oncology Group (ECOG) criteria. Brain magnetic resonance imaging and radionuclide bone scans were only performed if metastatic disease was suspected according to the clinical manifestations of each patient. Regarding interstitial lung disease, we paid attention to clinical respiratory symptoms (e.g. dyspnea, cough) and radiographic findings of patients, and monitored PaOd Tumors . In lineQoL was assessed using the Chinese version of the European Organization for Research and Treatment of Cancer (EORTC) core questionnaire, the Quality of Life Questionnaire (QLQ)-C30 (version 3.0) and the supplemental lung-cancer-specific module QLQ-LC13 . FollowiLogistical regression test models were used to identify baseline factors (gender, PS, histology, TNM stage and prior chemotherapy) that might independently predict tumour response. Median progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan-Meier method and a log-rank test was used to detect differences OS between strata.2 test or Fisher's exact test.Changes in symptoms and QoL were assessed in two different ways. Firstly, the mean scores of QoL and disease-related symptoms at baseline were compared with those at the end of the second cycle of treatment using a paired-sample t test. Secondly, the response rates of symptoms and QoL were calculated. For the assessment of symptoms and QoL, the classification system suggested by Stephens et al was used . The resThirty-one eligible patients were enrolled into the EAP at Peking Union Medical College Hospital between October 2002 and October 2003. Patient characteristics are listed in Table All patients were assessed for toxicity. Twenty-three (74%) patients had at least one AE. Treatment-related toxicities are listed in Table The assessment of patient response is listed in Table vs. 4.4 months).Median PFS was 5.5 months (95% CI, 1.6 to 9.4); median OS was 11.5 months (95% CI, 5.6 to 17.3). The tumor response was associated with improved OS. Median OS of patients with response was significantly higher than that of those with no response (log-rank test p = 0.0058) (17 At baseline, 25 (81% [25/31]) patients returned QoL questionnaires. All 25 patients returned the questionnaires at the first cycle of therapy, 23  at the second cycle of therapy, and 18  at the third cycle. The most frequently reported general symptoms were fatigue (100%) and appetite loss (68%), while dyspnoea (100%) and coughing (84%) were the most frequently reported respiratory symptoms. The changes in the mean scores for symptom scales, functioning and global QoL scales are presented in Table Patient responses for functioning, global QoL and main symptoms are shown in figure Haemoptysis had the highest response rate (84%), the rate of improvement (63%) being just lower than that of appetite loss among the five symptoms. The response rates for dyspnoea and coughing were 56% and 68%, respectively. For fatigue and appetite, the response rates were 44% and 80%, respectively.Differences in response rates for symptoms and QoL between patients with or without objective tumour response are shown in Table A daily oral 250 mg dose of gefitinib was well tolerated in this series of Chinese patients with advanced NSCLC, who received gefitinib as part of a compassionate-use programme. AEs were mild and reversible and different to those associated with conventional chemotherapy, such as neutropenia, thrombocytopenia and neuropathy. No haematological or neural toxicity was observed in the study. As with prior clinical trials evaluating the toxicity of gefitinib, the most frequently reported AEs were acneform rash and diarrhoea (grade 1 or 2). Four patients developed grade 1 or 2 stomatitis during treatment, which has previously been associated with gefitinib treatment in 7.8% of patients in IDEAL 1 but was not reported in IDEAL 2 .Severe acute interstitial pneumonia is the most serious AE that has been linked with gefitinib therapy. Inoue et al recently reported that 4 of 18 patients receiving gefitinib in their clinic developed severe acute interstitial pneumonia . The worThe response rate (35.5%) was higher than that of recently presented Phase II trials (18.4% and 11.8% in patients receiving gefitinib 250 mg/day in IDEAL 1 and 2, respectively). The DCRs were 54.4% and 42% for the 250 mg/day dose in IDEAL 1 and 2, respectively, in contrast to 58.1% in our study. Unlike IDEAL 2, which was performed in the USA, IDEAL 1 was a global trial that recruited patients from 43 centres across Europe, Australia, South Africa and Japan and included a total of 102 Japanese patients with advanced NSCLC. The response rate for Japanese patients was higher than that of non-Japanese patients . However, the difference in response rate for ethnicity was not verified using multivariate logistical regression analysis. The difference in response rate between Japanese and non-Japanese patients was attributed to bias of baseline predictive factors of patients (gender, PS and histology) ,10.QoL is an important endpoint for assessment of gefitinib treatment and has been included in some phase I and phase II trials. In these trials, the Functional Assessment of Cancer Therapy – Lung questionnaire was used to assess QoL and symptoms. Its validation and sensitivity were verified. Significant improvements in symptoms and QoL were observed in IDEAL 1 and 2. In IDEAL 1, symptom and QoL improvement with a dose of 250 mg/day were 40.3% and 23.9%, respectively, and in IDEAL 2 they were 43.1% and 34.3%, respectively. In both trials, improvements in symptoms and QoL correlated well with tumour response .In our patient series, the EORTC QLQ-C30 and its supplement module QLQ-LC13 were used for assessment of QoL and symptoms. Together, they are thought to be one of the best developed instruments for lung cancer assessment . The queIn conclusion, the data from this patient series show that an oral dose of gefitinib 250 mg/day is well tolerated and has significant antitumour activity in Chinese patients with advanced NSCLC who had failed previous chemotherapy and for whom no other treatment options were available. Although clinical trials are needed to confirm these positive findings, the data suggest that treatment with gefitinib may be beneficial for some Chinese patients who do not respond to chemotherapy and have poor prognosis.'Iressa' is a trademark of the AstraZeneca group of companiesNone declared.XLM and LYL designed the experiments and wrote the manuscript. XTZ, SLW and MZW participated in patient's follow-up and tumour response evaluation.The pre-publication history for this paper can be accessed here:


Phenotypic analyses of animals with spontaneous mutations in DMT1 indicate that it functions at two distinct sites, transporting dietary iron across the apical membrane of intestinal absorptive cells, and transporting endosomal iron released from transferrin into the cytoplasm of erythroid precursors. DMT1 also acts as a proton-dependent transporter for other heavy metal ions including Mn2+, Co2+, and Cu2, but not for Mg2+ or Ca2+. A unique mutation in DMT1, G185R, has occurred spontaneously on two occasions in microcytic (mk) mice and once in Belgrade (b) rats. This mutation severely impairs the iron transport capability of DMT1, leading to systemic iron deficiency and anemia. The repeated occurrence of the G185R mutation cannot readily be explained by hypermutability of the gene. Here we show that G185R mutant DMT1 exhibits a new, constitutive Ca2+ permeability, suggesting a gain of function that contributes to remutation and the mk and b phenotypes.Divalent metal transporter-1 (DMT1/DCT1/Nramp2) is the major Fe David Clapham and colleagues offer new evidence that blurs the line between ion transporters and channels Based ondosomes . It is i2+, Mn2+, Cu2+, Zn2+, and Co2+ are required cofactors for many essential cellular enzymes. They cannot cross the plasma membrane through simple diffusion, and active uptake requires specific transporters. DMT1 is the only molecule known to mediate cellular iron uptake in higher eukaryotes. It is structurally unrelated to known Zn2+ and Cu2+ transporters, but DMT1 can transport those and other divalent metal ions  induced large inward currents in DMT1-expressing cells (42– to replace most of the Cl– ([Cl–]o = 5 mM) in low-pH bath solutions. With elimination of the background Cl– current, the proton-evoked current was inwardly rectifying (hyperbolic) (We studied wild-type DMT1 and the G185R mutant proteins by whole-cell patch–clamp in transiently expressing CHO-K1 and HEK-293T cells and in doxycycline-inducible DMT1-HEK-On and G185R-HEK-On cells. Consistent with previous studies, DMT1 expression significantly increased cellular t low pH B. As rep oocytes , reducting cells C and 1D.ng cells , as wellng cells . Becauseng cells , we usederbolic) E.2+ (data not shown) or Mn2+ induced an additional current with less pronounced slow inactivation , and Fe3+ is not transported by DMT1  was recorded (2+-induced current was dependent on [Mn2+]o, with a measurable response at [Mn2+]o < 1 μM (pH 4.2). In the presence of 100 μM Mn2+ (pH 4.2), the additional Mn2+-induced current was typically half the amplitude of the proton-induced current. Addition of Mn2+ alone (100 μM) at pH 7.4 did not induce any additional current. Since H+ or H+/Mn2+ induced two currents with distinct kinetics in DMT1-expressing cells, the underlying charge-carrying ion species and their relative contributions to the macroscopic currents were investigated. We monitored the reversal potential (Erev) and the current amplitude in ion-substitution experiments. Replacement of Na+ with N-methyl-D-glucamine (NMDG+) did not significantly change the Erev of H+ or H+/Mn2+-induced currents, although the net current amplitude was slightly increased  (Mn/PH can be estimated from the slope of Erev versus pH based on an extended Goldman–Hodgkin–Katz equation (+ and Mn2+). At pH 4.2, the slope of Erev versus pH did not differ significantly with or without Mn2+(see + to IDMT1 is much larger than that of Fe2+/Mn2+ (PMn/PH < 0.01), in contrast to the 1:1 stoichiometry proposed previously (2+ permeability was observed, even in isotonic (105 mM) Ca2+ solution (see In contrast, when Mnrode see F. Replaconse see . Our intonse see  or has ae Mn/PH) . PMn/PH equation  with two Mn2+see F. Therefeviously . Importation see C.2+ and 140 mM Na+) at pH 7.4 (G185R) was stable over minutes with no slow inactivation (see G185R in more than 85% of enhanced green fluorescent protein (EGFP)-positive cells transfected with the pTracer-G185R construct and in stable, doxycycline-induced G185R-HEK-On cells, but never in cells transfected with wild-type DMT1 . The inwardly rectifying current was cationic, since Ca2+ and Na+ substitution by NMDG+ completely abrogated the current  compared to wild-type DMT1 at similar protein expression levels , IG185R was 64 ± 7 pA/pF at −140 mV .Io 105 mM E, strengove 0 mV D, whereaG185R. The amplitude of IG185R was strongly dependent on [Ca2+]o (2+ in the bath, replacement of 140 mM NMDG+ by 140 mM Na+ only slightly (by approximately 15%) increased the current, indicating that Ca2+ permeated the plasma membrane of G185R-transfected cells much more readily than Na+. As shown in 2+]o not only augmented the current amplitude but also shifted Erev toward depolarized potentials. The slope of this shift (25 mV per decade) was close to the slope of 29 mV per decade predicted by the Nernst equation for a Ca2+-selective electrode . We determined G185R-specific currents by measuring the reversal potentials of the currents subtracted from two holding potentials  (G185R was less Ca2+-selective (PCa/PNa of approximately 10) than VGCCs (PCa/PNa of approximately 1,000). Single iG185R channels were not observed in cell-attached patches. Analysis of membrane current noise at −100 mV predicted a single-channel chord conductance of 0.4 ± 0.1 pS , too small to be observed under most patch–clamp conditions.We next investigated the cation selectivity of I [Ca2+]o F. With 1lectrode C. The reials see A and 4B lculated  and illuand Ba2+ D. While ransient D, the siransient , yieldinK+ > Cs+ E. Under sequence E of IG18 (VGCCs) , but IG12+ indicator dye Fura-2, we demonstrated G185R-mediated Ca2+ influx by monitoring intracellular Ca2+ levels in response to an elevation of [Ca2+]o (2+-influx and/or store release, we bathed cells in the presence of 50 μM SKF96365 and 50 μM 2-APB. Upon raising [Ca2+]o, [Ca2+]i rose from 105 nM to 240 nM in doxycycline-induced G185R-HEK-On cells, significantly higher than in control HEK-On cells treated with doxycycline. Thus, the permeability of G185R to Ca2+ is capable of increasing [Ca2+]i.Using the Ca [Ca2+]o F. To minG185R displayed hyperpolarization-induced inhibition (1/2) were −33 mV and −10 mV for control and isotonic Ca2+ solutions, respectively , nor was it blocked by antagonists of known Ca2+-permeant channels. In particular, the current was not blocked by ruthenium red (RR), Ca2+-release activated Ca2+ channel (CRAC) blockers SKF96365 and 2-APB (2+, Ni2+, Co2+), inhibited IG185R. La3+  blocked IG185R in a similar voltage-dependent manner , and CRAC currents, based on its current–voltage (I-V) relation, kinetics, permeation properties, and pharmacological sensitivity.Ihibition A and 4B hibition . The halectively B. The vopendence B. IG185Rnd 2-APB   and in mk enterocytes. A putative associated protein, if present in these different cell types, would have to be activated in a G185R-dependent manner. We did not find evidence of an associated protein when we immunoprecipitated wild-type or G185R DMT1 from transfected CHO-K1 cells (unpublished data). Furthermore, a distinct DMT1 mutant, G185K, also displayed Ca2+ permeability, but this mutant was less selective for Ca2+ over Na+ (unpublished data).We conclude that expression of G185R in transfected cells and in vivo in DMT1 mutants with impaired iron transport or more likely to be noticed by those managing the animal colonies. In parallel with these studies, we have generated knockout mice homozygous for a null DMT1 allele . Although detailed phenotypic characterization has not yet been completed, we have noted that –/–Dmt1 mice invariably die by the end of the first week of life, in contrast to mk/mk mice, which are poorly viable but can survive for more than a year . This suggests that the small amount of residual function of G185R DMT1, perhaps in combination with its gain-of-function Ca2+ conductance, contributes to viability.G185R mutations have occurred at least three times in rodents, which suggests that G185R not only inactivates DMT1, but may confer an unknown selective advantage. Because it has arisen in inbred colonies, the postulated selective advantage must either make the animals more viable than other 2+] has been reported to increase nontransferrin-bound iron uptake through an undefined transport system that has characteristics distinct from DMT1  in a 12-TM transporter protein conferred new Calocation . The “ch2+ entry gain of function helps to account for this remarkable pattern of remutation. Further investigation of this hypothesis will require direct and detailed comparison of DMT1-null and mk mice.We initiated these studies to investigate why a unique DMT1 mutation, G185R, has occurred independently at least twice in mice and once in rats ,1998. ThDMT1 cDNA used in this study was derived from one of four alternatively-spliced DMT1 gene transcripts. The G185R mutation was generated by using M13 phage and the oligonucleotide GTCCCCCTGTGGGGCCGAGTCCTCATCACCA. Wild-type DMT1 and the G185R mutant were tagged with a C-terminal FLAG epitope and subcloned into pTracer-CMV2 . CHO-K1 or HEK293T cells transiently transfected with DMT1 and G185R were used for the 55Fe uptake assay and Western blot analysis. To obtain a stable G185R-expressing cell line, the G185R-encoding DMT1 gene was subcloned into pRevTRE , a retroviral vector that drives expression from a Tet-responsive element. All constructs were confirmed by sequencing. DMT1 Western blot analyses were performed with an anti-FLAG M2 monoclonal antibody (Sigma, St. Louis, Missouri, United States) and, in some cases, with a goat polyclonal antibody raised against human DMT1 .The TM, or HEK-On) was generated, and expression was induced by adding 1–10 μg/ml doxycycline into the culture medium. Unless otherwise stated, the pipette solution contained 147 mM cesium, 120 mM methane-sulfonate, 8 mM NaCl, 10 mM EGTA, 2 mM Mg-ATP, 20 mM HEPES (pH 7.4). Bath solution contained 140 mM NaCl, 10 mM CaCl2, 10 mM HEPES, 10 mM MES, 10 mM glucose (pH 7.4). Unless otherwise stated, the low pH solutions contained only nominal free Ca2+ (1–10 μM). Data were collected using an Axopatch 2A patch–clamp amplifier, Digidata 1320, and pClamp 8.0 software . Whole-cell currents were digitized at 10 kHz and filtered at 2 kHz.Wild-type and G185R mutant DMT1 were subcloned into an EGFP-containing vector (pTracer-CMV2, Invitrogen) for transient expression in CHO-K1 and HEK293T cells. Cells were transfected using Lipofectamine 2000 (Invitrogen). Transfected cells, cultured at 37°C, were plated onto glass coverslips and recorded 24 (DMT1) or 30 (G185R) hrs after transfection. A stable cell line (HEK293 Tet-OnNa) was estimated according to rev upon replacing [Na+]o in nominally Ca2+-free bath solution (150 mM XCl, 20 mM HEPES, 10 mM glucose [pH 7.4]]), where X+ was Na+, K+, Cs+, or Li+. For the permeability to divalent cations (relative to PNa), bi-ionic conditions were used; Y2+ was Ca2+, Ba2+, or Sr2+ . The external solution was 140 mM NMDG-Cl, 10 mM Y2+Cl2, 20 mM HEPES (pH 7.4 adjusted with HCl). The permeability ratios of cations were estimated from the following equations  and 50 mM nitrilotriacetic acid. About 30 h after transient transfection, CHO-K1 or HEK293T cells were washed and harvested with PBS (for CHO-K1 cells, trypsin treatment was required). Cells were resuspended in glass test tubes at 0.5–1 million/ml in 490 μl assay buffer at 30°C. The reaction was started by adding 10 μl of 55Fe stock and stopped at 4, 8, and 16 min by quickly filtering the reaction mix on a nitrocellulose filter . Filters were washed twice with 2 ml of assay buffer, dried, and radioactivity counted by liquid scintillation spectrometry.The assay buffer contained 25 mM Tris, 25 mM MES, 140 mM NaCl, 5.4 mM KCl, 5 mM glucose, 1.8 mM CaCl2+] was constructed according to Cells were loaded with 2 μM Fura-2 AM in culture medium at 37°C for 30 min. Low levels of G185R protein were expressed in the absence of doxycycline in G185R HEK-On cells . Therefore, doxycycline-treated HEK-On cells not expressing DMT1 were used as controls in imaging experiments. We recorded Fura-2 ratios (F340/F380) on an UltraVIEW imaging system (Olympus, Tokyo, Japan). A standard curve for Fura-2 ratio versus [Camk mice (2SO4, 44 mM K2HPO4, 9 mM NaHCO3, 15 mM Na3Citrate, 10 mM HEPES, and 180 mM glucose (pH 7.4). The tissue was then incubated with gentle shaking for 3 min in a similar solution containing 7 mM K2SO4, 44 mM K2HPO4, 9 mM NaHCO3, 10 mM HEPES, 180 mM glucose, 1 mM DTT, and 0.2 mM EDTA (pH 7.4). The mucosal cells were gently squeezed from the duodenum with forceps into 5 ml of ice-cold DMEM/F12 medium, pelleted at 800 × g for 4 min, resuspended in 5 ml of prewarmed DMEM/F12 with 0.5 mg/ml collagenase type 1A, and incubated at 37°C for 10 min. Cells isolated by this procedure have been shown previously to be primarily of villus origin and hence are mature enterocytes. We confirmed this by alkaline phosphatase staining. Diluted cells were filtered through a 40-μm nylon cell mesh . The cells were then washed with DMEM/F12, resuspended in 20 ml of ice-cold DMEM and kept at 4°C. They were plated on coverslips coated with Cell-TakTM (BD Biosciences) and maintained on ice before patch–clamp recording at room temperature.Homozygous mk mice  were houmk mice . In briet-test with Bonferroni correction. A two-tailed value of p < 0.05 was taken to be statistically significant.Group data are presented as mean ± SEM. Statistical comparisons were made using analysis of variance and the Figure S1(A) Anion dependence of pH-induced response in a DMT1-expressing cell. Outward current usually appears later than the inward current.(B) Currents generated in response to a voltage ramp.. We attributed the outward currents shown in (A) and (B) to this endogenous Cl– current. Therefore, for our recordings on DMT1, SO42\– was usually used to replace most of the Cl– ([Cl–]o = 5 mM) for all low-pH bath solutions. (C) pH-induced outwardly rectifying current in a nontransfected CHO-K1 cell. A similar current was seen also in HEK293T and HEK-On cells, with properties similar to the cloned ClC-7 channel . This cu(718 KB PDF).Click here for additional data file.Figure S2Whole-cell currents were generated by voltage steps from −140 to +80 mV in 20 mV steps, 400 ms. The interval between steps was 1,000 ms. (1 MB PDF).Click here for additional data file.Figure S3+ by NMDG+ slightly increased the proton current (approximately 20%) and this was further augmented by adding 300 μM Mn2+. The concentrations used were Na+ and NMDG+, 140 mM, (pH 4.2); Mn2+, 300 μM. (141 KB PDF).Replacement of extracellular NaClick here for additional data file.www.ncbi.nlm.nih.gov/GenBank/) accession number for DMT1 is AF029758.The GenBank (


Trigonella are important from medical and culinary aspect. Among these, Trigonella foenum-graecum is commonly grown as a vegetable. This anti-diabetic herb can lower blood glucose and cholesterol levels. Another species, Trigonella caerulea is used as food in the form of young seedlings. This herb is also used in cheese making. However, little is known about the genetic variation present in these species. In this report we describe the use of ISSR and RAPD markers to study genetic diversity in both, Trigonella foenum-graecum and Trigonella caerulea.Various species of genus Trigonella foenum-graecum and nine accessions of Trigonella caerulea representing various countries were analyzed using ISSR and RAPD markers. Genetic diversity parameters  were calculated for ISSR, RAPD and ISSR+RAPD approaches in both the species. Dendrograms were constructed using UPGMA algorithm based on the similarity index values for both Trigonella foenum-graecum and Trigonella caerulea. The UPGMA analysis showed that plants from different geographical regions were distributed in different groups in both the species. In Trigonella foenum-graecum accessions from Pakistan and Afghanistan were grouped together in one cluster but accessions from India and Nepal were grouped together in another cluster. However, in both the species accessions from Turkey did not group together and fell in different clusters.Seventeen accessions of Trigonella caerulea as compared to Trigonella foenum-graecum. The genetic similarity matrices generated by ISSR and RAPD markers in both species were highly correlated (r = 0.78 at p = 0.001 for Trigonella foenum-graecum and r = 0.98 at p = 0.001 for Trigonella caerulea) indicating congruence between these two systems. Implications of these observations in the analysis of genetic diversity and in supporting the possible Center of Origin and/or Diversity for Trigonella are discussed.Based on genetic similarity indices, higher diversity was observed in  The family Fabaceae includes many crops useful for food, forage, fiber, wood and ornamental purposes. In this family, a few legumes such as chickpea, soybean, fababean, fenugreek, lentil, pea etc. are consumed as grain legumes. The grain legumes are plants used as food in the form of unripe pods, mature seeds or immature dry seeds, directly or indirectly . The graTrigonella is one of the largest genera of the tribe Trifoliatae in the family Fabaceae and sub-family Papilionaceae [Trigonella species, Trigonella foenum-graecum (commonly known as fenugreek) is a flowering annual, with autogamous white flowers occasionally visited by insects. Indigenous to countries on the eastern shores of Mediterranean, fenugreek is widely cultivated in India, Egypt, Ethiopia, Morocco and occasionally in England [Trigonella foenum-graecum is extensively grown in the tropical and subtropical regions of India. Different parts of the plant such as leaves and seeds are consumed in India. It is also used for medicinal purpose. According to ancient medicinal system, the Ayurveda, it is a herbal drugs that is bitter or pungent in taste. It is effective against anorexia and is a gastric stimulant [The genus ionaceae . Among T England . Trigonetimulant . Fenugretimulant . Researctimulant .T. caerulae, from the same family and commonly known as the Blue fenugreek, on the other hand, is a less commonly grown herb. This flowering annual with autogamous blue flower is found in Alps and in the mountains of eastern and south eastern Europe. Terminal leaves are mainly used for cooking while young seedlings are eaten with oil and salt. Dried and powdered leaves as well as flowers are used for flavoring and coloring bread, cheese, etc in China and Germany. They are also used as a condiment in soups and potato dishes and a decoction of leaves is used as aromatic tea [atic tea .T. foenum-graecum and T. caerulea although important in food and medicine are rarely grown outside their native habitat. Across the world only known and well-defined cultivars are grown in specific areas. Gene banks also harbor scanty germplasm collection of Trigonella species [T. foenum-graecum and T. caerulea representing various countries where they are grown using molecular marker technology.Grain legumes like  species . The negT. foenum-graecum of which 40 gave amplification. However, only 14 ISSR primers detected intraspecific variation in I7 accessions of T. foenum-graecum generating clear reproducible patterns and revealing 100 bands in the range of 500 bp to 2 kb. Among these seventy-two bands were polymorphic amounting to 72% polymorphism [Table T. caerulea, of the 100 ISSR primers used for initial screening, 47 gave amplification. Of these, 18 primers detected intraspecific variation in 9 accessions of T. caerulea showing 93.64% polymorphism [Table A set of 100 ISSR primers was used for initial screening of 7 accessions of sm Table . FurtherT. foenum-graecum of which 22 primers generated polymorphic patterns revealing 70.12% polymorphism [Table T. caerulea of the 40 primers used for initial screening, 10 primers produced polymorphic pattern giving 95.83% polymorphism [Table In case of RAPD analysis, 100 RAPD primers were used for initial screening in sm Table . Eight uT. foenum-graecum considering ISSR and RAPD approaches individually as well as together. Based on ISSR markers alone, the similarity index values ranged from 0.69 to 0.92. These values were used to construct a dendrogram using Unweighted Pair Group Method with Arithmetic averages (UPGMA). In the ISSR based dendrogram T. foenum-graecum genotypes formed 4 clusters [Figure not shown]. The first cluster grouped together accessions from Afghanistan (8707), Canada (1065), Pakistan (8717,8718), Iran (8675), and Turkey (8690). The second cluster contained accessions from India (8686,8689,8675). Remaining one accession from India (8687) and one from Nepal (8706) formed the third cluster. The fourth cluster contained accessions from Egypt (8698,8679) and Turkey (8692). Accessions from Ethiopia (8696), Turkey (8691) and Yemen (8714) out grouped from the main clusters. Three accessions from Turkey analyzed in the present study fell into different clusters.Genetic similarity was calculated from the Nei's similarity index value for all the 17 accessions of T. foenum-graecum genotypes formed 2 main clusters [Figure not shown]. The first cluster had two subgroups, the first subgroup contained accessions from Afghanistan (8707), India (8686,8687,8675), Turkey (8690) and Egypt (8698) while the second subgroup contained accessions from Pakistan (8717,8718), Turkey (8692), and Egypt (8679). The accession from Canada associated with these clusters. The second cluster contained accession from Iran (8685) and Nepal (8706) and the accession from India associated with this cluster. Accessions from Ethiopia (8696), Turkey (8691) and Yemen (8714) out grouped from these two clusters. In the RAPD based dendrogram also accessions from Turkey fell into different clusters.Based on RAPD markers alone, the similarity index values ranged from 0.71 to 0.91. In the RAPD based dendrogram, T. foenum-graecum accessions from Egypt (8698,8679) were grouped together. Accessions from Pakistan (8717, 8718) and Afghanistan (8707) were grouped together in one cluster. Accessions from India (8686,8689,8675,8687), Nepal (8706) and Iran (8685) were grouped together. However, all the three accessions from Turkey fell in different clusters and did not group among themselves. Bootstrapping was done using the WinBoot program to estimate the relative support at clades. The robustness of the cluster was not very strong in T. foenum-graecum (50–70%).Based on both the marker systems together the similarity index values ranged from 0.65 to 0.89 Fig. . Here thT. caerulea, genetic similarity was calculated from the Nei's similarity index value considering ISSR and RAPD approaches individually as well as together. Based on ISSR marker system, the similarity index values ranged from 0.41 to 0.92 while that on the basis of RAPD markers ranged from 0.34 to 0.93. Similarity indices values based on both the marker systems together ranged from 0.38 to 0.92 indicating more diversity in case of T. caerulea. The dendrograms based on ISSR and RAPD markers showed similar clustering pattern when used individually [Figures not shown] as well as together [Fig. T. foenum-graecum. The bootstrap values obtained using the WinBoot program in T. caerulea were very high (90–100%).In T. foenum-graecum and in Table T. caerulea. In T. foenum-graecum the Hav values calculated for ISSR, RAPD and ISSR+RAPD were 0.214, 0.203 and 0.211, respectively. In case of T. caerulea the Hav values for the same were 0.330, 0.346 and 0.338, respectively. For both, T. foenum-graecum and T. caerulea, the Hav values for ISSR and RAPD systems did not differ significantly. The marker index (MI) values calculated for ISSR and RAPD for T. foenum-graecum were 1.53 and 1.51, respectively while in T. caerulea they were 3.17 and 3.35, respectively. Thus MI values in both the species did not differ significantly for ISSR and RAPD systems.Heterozygosity was calculated using ISSR and RAPD marker systems individually as well as together as detailed in Table T. foenum-graecum, when the similarity matrices generated using ISSR and RAPD markers were compared, a value of r = 0.78, at P = 0.001 indicated a good correlation between data generated by both the systems [Fig. T. caerulea, a value of r = 0.98 indicated a very good correlation between the two marker systems [Fig. In ems Fig. . Similarems Fig. .Brassica napi [rapeseed, [Allium sect. Sacculiferum [Alliaceae, [Asimina triloba [pawpaw, [Cicer [[Cajanus [[Trigonella. In the present study, an attempt has been made to examine the level of genetic variation within T. foenum-graecum and T. caerulea accessions obtained from germplasm collection centers at Saskatoon (Plant Gene Resources of Canada) and Pullman (USDA-ARS Plant Introduction Station, Washington). The T. foenum-graecum accessions were selected in order to represent most of the countries where it is grown. In case of T. caerulea all the nine accessions available at Saskatoon and Pullman were used in present study.The two marker systems, ISSR and RAPD used in the present study have also been used as effective tools to evaluate genetic diversity and to throw light on the phylogenetic relationships in apeseed, ], Alliumliaceae, ] and Asi[pawpaw,  and 13]Brassica  [Cicer [ and 15]Brassica Cajanus [ and 18]Brassica T. foenum-graecum as well as in T. caerulea the Hav and the marker index (MI) values for ISSR and RAPD markers [Table T. foenum-graecum and T. caerulea [Fig. T. caerulea (0.33) as compared to T. foenum-graecum (0.21). Based on allozyme diversity, the estimated mean heterozygosity values have been reported for self-pollinating species, Vigna unguiculata, Hav = 0.027 [Vicia tetrasperma, Hav = 0.342 [Vigna unguiculata was lower while that for Vicia tetrasperma was higher as compared to T. foenum-graecum and T. caerulea. Based on ISSR markers, the estimates of genetic diversity, Hav = 0.358 reported in cultivated pawpaw (Asimia triloba), was higher as compared to T. foenum-graecum and T. caerulea [Polymorphism in a given population is often due to existence of genetic variants represented by the number of alleles at a locus and their frequency of distribution in a population. Heterozygosity corresponds to a probability that two alleles taken at random from a population can be distinguished using the marker in question. Thus a convenient quantitative estimate of marker utility and the polymorphism detected can be given in terms of the mean heterozygosity and the marker index . In T. frs Table  and 4, rlea Fig.  and 4. G = 0.027  and Vici = 0.342 . The hetcaerulea .T. foenum-graecum and T. caerulea. The accessions could be a mixture of different genotypes. Therefore, to have a complete representation of a specific accession, DNAs from fifteen plants were mixed in equal proportion. Thus within accession diversity was eliminated and a complete banding profile of the accession was used for the analysis.Data collected with ISSR and RAPD marker systems were used to compare genetic similarity between various accessions of T. foenum-graecum, ISSR and RAPD could detect almost similar level of polymorphism (72% with ISSR and 70.12% with RAPD). In the UPGMA analysis, T. foenum-graecum accessions from one country and the nearby region grouped together in some cases while they were placed in different clusters in certain cases. Accessions from Pakistan and Afghanistan grouped together in one cluster while accessions from India and Nepal grouped in another cluster. Moreover, the three accessions from Turkey fell in different clusters inspite of being geographically very close to each other. Thus, there was no clear clustering pattern of geographically closer accessions in the present study indicating that the association between genetic similarity and geographical distance was less significant. However, it is necessary to use more number of accessions from each geographical location to confirm the available pattern.In T. caerulea also, ISSR (93.64%) and RAPDs (94.83%) detected almost equal level of polymorphism. T. caerulea showed more polymorphism as compared to T. foenum-graecum. In case of T. caerulea also, the UPGMA analysis showed that plants from different geographical regions were distributed in different groups. Here again, the accessions from Turkey were not grouped together. Two accessions from Turkey out grouped from the main cluster while one was grouped in the first cluster with Australia. In T. caerulea we could obtain three accessions from Turkey and only one accession from other countries. Therefore, it would be premature to comment precisely about the correlation of geographic distance and genetic diversity in this case. To confirm the available pattern it is necessary to use more number of accessions from each geographical location.In The place of origin of a species as explained by Vavilov is an area that contains a large amount of genetic variability of that species. According to him variation is a function of time, hence the region containing the greatest variation in a species would have supported and sustained that species for a longer time than the other regions suggesting that region to be the Center of Origin and/or Diversity. He set up eight geographic centers, two of which namely the Near Eastern and Mediterranean Centers extent into Turkey .Trigonella, Trifolium and Medicago species [T. foenum-graecum and T. caerulea accessions from Turkey exhibited more diversity. These results support Vavilov's hypothesis. The Indian accessions of T. foenum-graecum i.e. accession number 8686 (Khandwa), 8685(Mumbai), and 8687  separated by an aerial distance of 52 km, 104 km, and 135 km, respectively from each other, were genetically more similar (similarity index 0.893) and clustered together [Fig. T. foenum-graecum from Turkey i.e. accession number 8692  and 8691 (Elbistan) separated by a distance of 100 km  were out grouped and were genetically more distant from each other although morphologically they were similar to each other as well to the accessions from other countries.It has been postulated by Vavilov that the Near East region extending from Israel through Syria, Southern Turkey into Iran and Iraq and the Mediterranean Center including Spain, Morocco and Turkey is the Center of Origin of  species . In the Vicia, Medicago, Trifolium, Lathyrus, Onbrychis, Trigonella, Pisum, Cicer, Lens, etc [Cicer, Lens, Pisum, Amygladus, Prunus, Triticum, etc have their Center of Origin and/or Diversity in this country [T. foenum-graecum the large seeded cultivars are abounded around the Mediterranean region whereas the small seeded cultivars are predominated eastwards. Thus, Turkey may also be the primary Center of Origin of T. foenum-graecum and T. caerulea. However, this hypothesis needs to be confirmed by considering more accessions distributed over a wide geographic range especially from the Near East and the Central Mediterranean region.Turkey is one of the significant and unique countries in the world from the point of view of plant genetic resources and plant diversity. The country has more than 3,000 endemic plants and immense diversity has been reported in many legumes such as ens, etc . Many ge country . Vavilov country . SimilarT. foenum-graecum and T. caerulea and simultaneously revealed molecular based genetic relationship. In the UPGMA analysis, no significant correlation was observed between geographic distance and genetic diversity. Our data further supported the hypothesis of the Near East and the Central Mediterranean to be the Center of Origin and/or Diversity for Trigonella as put forth by Vavilov.In conclusion, molecular markers allowed us to estimate the overall genetic diversity in T. foenum-graecum accessions were obtained from Plant Gene Resources of Canada (PGRC), Saskatoon, Canada. These accessions along with the TMP numbers and the country from where they have been collected are outlined in Table T. caerulea accessions were obtained from PGRC, Saskatoon, Canada and USDA-ARS Plant Introduction Station at Pullman, Washington (W-6), and are detailed in Table Seeds of A set of 100 anchored micro satellite primers was procured from University of British Columbia, Canada. PCR amplification of 20 ng of DNA was performed in 10 mM Tris-HCI pH 7.5, 50 mM KCI, 1.5 mM MgCl2, 0.5 mM spermidine, 2% formamide, 0.1 mM dNTPs, 0.3 uM primer and 0.8 U of Taq DNA polymerase (Ampli-Taq DNA polymerase, Perkin Elemer, USA) in a 25 ul reaction using Perkin Elmer 9700 thermocycler. After initial denaturation at 94°C for 5 minutes, each cycle consisted of 30 seconds denaturation at 94°C, 45 seconds of annealing at 50°C, 2 minutes extension at 72°C along with 5 minutes extension at 72°C at the end of 45 cycles.RAPD analysis was performed using arbitary decamer primers procured from University of British Columbia, Canada. The reaction mixture (25 ul) contained 10 mM Tris-HCI pH 7.5, 50 mM KCI, 1.5 mM MgCl2, 0.5 mM spermidine, 0.1 mM dNTPs, 15 pmoles of primer, 20 ng genomic DNA, and 0.8 U of Taq DNA polymerase (Ampli-Taq DNA polymerase, Perkin Elmer, USA). Amplification was carried out using Perkin Elmer 9700 thermocycle for 40 cycles, each consisting of a denaturing step of 1 minute at 94°C, followed by annealing step of 1 minute at 36°C and an extension step of 2 minutes at 72°C. The last cycle was followed by 5 minutes of extension at 72°C. Amplified products were electrophoresed on 2% agarose gel using 0.5× TAE buffer (10 mM Tris HCl and 1 mM EDTA pH. 8.0) and visualized by ethidium bromide staining. The patterns were photographed and stored as digital pictures in gel documentation system. The reproducibility of the amplification was confirmed by repeating each experiment three times.Amplified products were electrophoresed on 2% agarose gel using 0.5x TAE buffer (10mM Tris HCl and 1mM EDTA pH. 8.0) and visualized by ethidium bromide staining. The patterns were photographed and stored as digital pictures in gel documentation system. The reproducibility of the amplification was confirmed by repeating each experiment three times.Unequivocally reproducible bands were scored and entered into a binary character matrix (1 for presence and 0 for absence). The genetic similarity among accessions was determined by Nei’s genetic distance [26]. A dendrogram was constructed based on the matrix of distance using Unweighted Pair Group Method with Arithmetic averages. Scores entered in matrix were analyzed using TAXAN version 4.0 software based on the degree of bands sharing. Similarity matrix was generated using Dice coefficient as SI = 2Nab/Na+Nb where Na = total number of bands present in lane a, Nb = total of bands in lane b, Nab number of bands common to lanes a and b [27]. The dice values were then used to perform the UPGMA analysis. To evaluate the robustness of the grouping formed the binary data matrix was subjected to bootstrapping using WinBoot program [28]. The phenogram was reconstructed 1000 times by repeating sampling with replacement and the frequency with which the groups were formed was used to indicate the strength of the group. Correlation co-efficient for the similarity matrices generated by ISSR and RAPD data in both, T.foenum-graecum and T.caerulea, were calculated by method of Smouse et al [29].   The expected heterozygosity, Hn for a genetic marker was calculated as: Hn = 1 - pi2, where pi is the allele frequency of the ith allele [26]. The arithmetic mean heterozygosity Hav for each marker class was calculated as Hav = Hn/n, where n = number of markers or loci analyzed [18]. The average heterozygosity for polymorphic marker (Hav)p was further derived as; (Hav)p = Hn/np; where np is the no. of polymorphic markers or loci [18]. Marker index (Ml) for each marker system was also calculated as, MI = E (Hav)p; where E = effective multiplex ratio [E = nβ where β is the fraction of polymorphic marker or loci,18].RD carried out the PCR and molecular genetic study, participated in the design of the study and performed statistical analysis. ML participated in procuring the seeds from different sources and monitoring experiments and result. LC a taxonomist participated in procuring, growing and analyzing the seeds obtained from different sources. PR and VG conceived of the study, participated in its design and co-ordination and monitored the experiments and the results.All authors read and approved the final manuscript.


Furthermore, hypotension (systolic BP < 80 mm Hg) was present in 44% of children with severe acidosis (base deficit >15). These findings also indicate important baseline differences in two cohorts of children studied. We agree that reconsideration of guidelines for acute fluid management is warranted, particularly when current recommendations await an adequate evidence base. Nevertheless, conflicting opinions on the question of volume status in children with severe malaria can be satisfactorily resolved only through prospective randomized trials that include both fluid resuscitation and control groups. While the design and conduct of such trials will involve considerable challenges, optimal fluid management will never be resolved on the basis of theoretical consideration alone.The study by Planche et al.