Preventive home visits to elderly people by public health nurses aim to maintain or improve the functional status of elderly and reduce the use of institutional care services. A number of trials that investigated the effects of home visits show positive results, but others do not. The outcomes can depend on differences in characteristics of the intervention programme, but also on the selection of the target population. A risk group approach seems promising, but further evidence is needed. We decided to carry out a study to investigate the effects in a population of elderly with (perceived) poor health rather than the general population. Also, we test whether nurses who are qualified at a lower professional level  are able to obtain convincing effects. The results of this study will contribute to the discussion on effective public health strategies for the aged.The study is carried out as a parallel group randomised trial. To screen eligible participants, we sent a postal questionnaire to 4901 elderly people (70–84 years) living at home in a town in the south of the Netherlands. After applying inclusion criteria  and exclusion criteria , we selected 330 participants. They entered the randomisation procedure; 160 were allocated to the intervention group and 170 to the control group. The intervention consists of (at least) 8 systematic home visits over an 18 months period. Experienced home nurses from the local home care organisation carry out the visits. The control group receives usual care. Effects on health status are measured by means of postal questionnaires after 12 months, 18 months (the end of the intervention period) and after 24 months (the end of 6-months follow-up), and face-to-face interviews after 18 months. Data on mortality and service use are continuously registered during 24 months. A cost-benefit analysis is included.The design and setting of the study, the selection of eligible participants and the study interventions are described in this article. Other included items are: the primary and secondary outcome measures, the statistical analysis and the economic evaluation. The number of elderly people is increasing. Due to the ageing population, more demands are made on health care services . In the et al [et al [et al [In the past years 3 reviews were published on the effects of preventive home visits to elderly people living in the community -4. Theseet al  reviewedOne can argue about the differences in the approach of each review, but in general the results of the home visiting studies are heterogeneous with respect to the different outcome measures. Many factors can play a role in the effectiveness of the interventions, including the target population, characteristics of the intervention, the persons carrying out the visits and the compliance to the given advice.Research in the Netherlands showed that preventive home visits do not seem to be useful for the general population of elderly people . In thatThe probable usefulness of home visits for a high risk group was confirmed in five controlled studies -11. HoweThis study will investigate the effects of systematic home visits by home nurses to elderly people with (perceived) health problems in terms of their health status, the use of care services and the cost-effectiveness. We expect that the visits will improve the functional abilities, perceived health and quality of life of the participants. We also hypothesize that they will reduce specialist care, institutionalisation, especially hospital (re-) admissions, and total health care expenditures. Evidence regarding the usefulness of the proposed risk group approach is needed to decide on the future implementation of the visits. This article presents the design of this new trial.The study is carried out as a parallel group randomised trial. It is conducted in co-operation with a large home care organisation in the south of the Netherlands (Sittard and surroundings). The addresses we used to screen eligible persons for the study were drawn from the population register of the municipality. After the screening procedure we randomised 330 elderly. Effects of the intervention are measured by means of postal questionnaires after 12 months, 18 months (at the end of the intervention period) and after 24 months (at the end of a 6-months follow-up period) and by face-to-face interviews after 18 months. Mortality and data on the use of care services are continuously registered over the 24-months research period. A cost-benefit analysis is also included. The design of the study is shown in figure The study has obtained the approval by the Medical Ethical Committee of Maastricht University/Academic Hospital Maastricht.We sent a postal questionnaire to 4901 elderly people between the age of 70 and 84 years who were still living at home. These lived in 14 districts in the research area. We included districts with close proximity to the centre of town where the home care organisation is situated. In this way we limited the travelling time of the nurses to carry out the visits. Districts with large industrial areas were excluded.Reminders were sent after 2–3 weeks to 45% of the elderly. The response rate was 76% after about 6 weeks. The response rates per district fluctuated between 65% and 81%. The average time to fill out the questionnaire was about 30 minutes. The elderly could do this by themselves or, if they needed help, with the assistance of family, friends or volunteers. A list of names and addresses of volunteers was added to the questionnaire. Even if persons did not want to participate in the study, we kindly requested them to fill out the questionnaire and return it to us. A postage free envelope was included.The questionnaire was used as a screening instrument and also served as a baseline measurement for the participants of the trial. Among the respondents (n = 3,689, see figure After applying the in- and exclusion criteria, 492 persons were eligible to take part in the study. However, we excluded 162 more persons for the following reasons: their GP did not want to co-operate with the study (n = 139), respondents had too many missing values on the functional status scale (n = 11), the health insurance company was unknown (n = 1) or it was uncertain whether the health insurance company would be willing to co-operate (n = 11). The health insurance companies of 96% of the finally selected participants had already given consent to provide us with data on health service use. As we selected persons whose GP was willing to co-operate, relevant health care data from the GP practices are available for all participants. A flow diagram of the selection of participants is shown in figure We calculated the sample size from the data of our previous home visitation study in the Netherlands . ParticiBased on data of the selection criteria we estimated that about 10% of the screened population was eligible for the study (including informed consent). Therefore, we needed to mail a minimum of 3,500 questionnaires to persons aged 70–84 years living in the community. To account for unforeseen circumstances, we decided to send out about 5,000 questionnaires. After applying the inclusion and exclusion criteria, and taking into account the GPs' willingness to co-operate with the study, there were sufficient participants eligible for the study to raise the selected number to 330. This slightly increased the power of the study.The baseline measurements included questions on relevant prognostic factors related to the health status and service use. Before randomisation we divided the 330 participants into two groups: couples (n = 46) and those for whom this did not apply (n = 284). In this way we made sure that eligible persons who lived together, were always allocated to the same study group (in order to avoid contamination of the intervention). The 23 couples were divided into 3 strata on the basis of their (added) score on functional status (0–4, 5–7 or more than 7 out of 18 activities that cannot be carried out independently). The other 284 participants were stratified into 8 strata based on 3 prognostic factors – two health-variables and one service use-variable:1. functional status (0–2 or more than 2 activities that the elderly cannot carry out independently)2. changes in health during the 3 months prior to completion of the questionnaire (same/better or worse)3. contact with a medical specialist in the 3 months prior to completion of the questionnaire (contact yes or no).The participants in each of the 8 strata were then randomised into either a control or intervention group using a computer generated randomisation list with a block length of 4 . RandomlThe participants in the intervention group were assigned to one of the three home nurses. This depended on the location of their GP practice, as each nurse was assigned to a number of GP practices. We assumed that this would facilitate the co-operation with the GPs. Each nurse was responsible for 52–56 elderly during the intervention period.Our previous study showed that positive effects of the visits for people with a poor health status emerged already within 1.5 years. In the new trial the intervention period is restricted to this period. At the same time we increased the frequency of the visits. This enables the nurses to intervene more promptly on identified problems and risks, and to establish a position of trust in a shorter time period. Experienced home nurses therefore visit the intervention group at least 8 times over an 18 months period. If necessary, extra visits can be made. The duration of the visits can last between 60 and 90 minutes. Participants in the control group receive usual care. As before, they can use or apply for all available services in the area.Three nurses work half time for the trial. An experienced public health nurse supervises the visits on a weekly basis. All 3 home nurses are recruited from the co-operating home care organisation. Home nurses, as well as public health nurses, are well trained to conduct home visits. They are embedded in community care organisations that traditionally have preventive tasks. The home nurses are not part of a multidisciplinary team, but advice can be obtained from in-home specialists within the home care organisation, e.g., a dietician, a diabetes specialist and an occupational therapist. A nurse geriatric specialist from the local hospital can also be consulted, if necessary. At regular intervals, once every 6–8 weeks during the intervention period, he also advises the nurses on important geriatric issues.The home visits can be described as "systematic home visits to elderly people with health problems carried out by a home nurse". The 3 most important elements of the visits are (1) to detect problems or risks, (2) to give advice and (3) to refer to other professional or community services. This brief description is applicable to all home visiting studies that have been carried out so far. However, there are large differences in the protocols that have been used in earlier studies, ranging from an interview to collect information on social and health conditions  to a 'muThe visits are carried out in a systematic way according to a nursing model  that disOur starting-point is a client-centred approach. The elderly can indicate which problems they experience and which needs they have. The EasyCare Questionnaire ,21, an eAn individual plan for each elderly person is set up. The activities are planned in agreement with the elderly, as this will improve compliance. We only included elderly with a poor (perceived) health, hence a broad range of problems can come forward, including physical, mental as well as social problems. Guidelines on a number of geriatric topics are used for advice and referral regarding problems and risks that are identified. A Handbook of Nursing Diagnosis  is also The elderly are primarily themselves responsible to carry out the planned activities. The home nurse only supports the elderly. In order to improve compliance, the nurses contact the elderly by telephone 1 to 4 weeks after each visit, depending on the type of advice. They ask whether the advice has been followed, and if not, what the impediments are and if further assistance is necessary. The participants are offered consultation with the nurses by telephone each morning between 9.00 – 9.30 hours.The evaluation of each home visit takes place at the next visit. The cycle is then repeated and new or old, but not solved, problems can be dealt with.In the 3-months period before the start of the visits, the home nurses were actively involved in the development of the visiting protocol. They also received relevant training in communication skills and using assessment tools. They took courses on several subjects, e.g., relevant geriatric health topics, behaviour change and the usage of the Handbook of Nursing Diagnosis . SeveralCommunication between the nurses and the GPs is according to the 'normal' communication lines between nurses of the home care organisations and the GPs. Before the start of the study all GPs received a list of eligible participants registered at their practice, to screen very ill persons. After randomisation a definite list of participants was sent to them, but no reference was made to which treatment group they belong. The allocation of the participants to the 2 groups was disclosed after conclusion of the first 3 home visits. The GPs then received an overview of all treated problems for each participant in the intervention group, including the accompanying recommendations and the results of the interventions. The GPs were asked for their comments or suggestions and in this way they could become involved, if they wanted to. A similar overview will be sent to them for visits 4–6 and 7–8.All elements of the intervention are monitored as part of a process evaluation. This includes the registration of topics discussed at each visit, treated problems, advice given and referral to other services. The evaluation of each visit is registered at each next visit and includes the compliance with the given advice. Reasons for non-compliance are noted. The nurses' experiences with the visiting protocol, the role of the supervising public health nurse and the patient's experiences with the home visits will be assessed at the end of the intervention period by means of face-to-face-interviews.Other aspects of the intervention process assessed are: the time spent on the visits, including the travelling and preparation time and the time spent on telephone contacts. Elements of the telephone conversation after each visit, most importantly whether the elderly complied with the given advice, are registered.Detailed analyses of the intervention process and outcome data might help to identify which programme characteristics are related to possible favourable effects of the visits and may result in the development of more effective interventions. It might also provide additional information for the possible implementation of the visits in daily practice.The primary health related outcome measures are: self-rated health, functional status, quality of life and changes in self-reported problems. In addition, a variety of other health measures (secondary outcome measures) will be assessed. Information will be obtained, among other things, on health complaints, medication use, and loneliness and mental health. The municipality will supply mortality data (secondary outcome measure) over the entire research period.The use of services relates to the frequency and duration of care from the following services: domestic and community nursing care, GP, physiotherapy, day care in institutional care settings, hospital outpatient clinics, hospital, nursing home, home for the elderly, use of aids and modifications to the home. The primary outcomes for service use are specialist medical care and hospital (re-) admission. The health insurance companies will supply data on the use of services over the two-year research period. Additional data not covered by the health insurance companies, will be supplied by GPs, the hospital, the home care organisations, etc. Table The main analyses will be conducted according to the intention-to-treat principle. Analysis of primary and secondary endpoints will be performed using relevant significance tests . Regression techniques will be used, if necessary, to estimate the effects for the various outcome measures, adjusting for small differences between the groups at the start of the study. In addition, we will conduct per-protocol analyses; these are restricted to those participants who complied fully with the intervention protocol and outcome measurements. Preplanned subgroup analysis will be performed for the following subgroups: living alone/together; health deterioration over the previous 3 months; functional status and locus of control. Differences in approaches between the nurses will be investigated.A cost-effectiveness analysis will be carried out in which we consider costs from a societal perspective. The economic evaluation will measure and evaluate the 'real' costs. In this study we will include direct health care costs, i.e. costs made for the home visit programme and health care costs made by the participants. Costs of the intervention programme consist of costs for the screening procedure, salaries of the nurses, travel expenses, costs of training sessions for the nurses, etc. Health care costs include costs of inpatient and outpatient treatment, consultation by GPs and other medical practising specialists, physiotherapy, medication, professional home care, nursing home, meals on wheels, aids and appliances, etc. In order to estimate the costs, the quantity of each resource will be multiplied by its assigned unit cost of price.Direct non health care costs (e.g., the travel costs made by participants) are not included. These should preferably be gathered prospectively by means of a cost-diary . We consThe screening procedure was carried out in the fall of 2002. In January 2003 we sent a letter to the elderly notifying them that they were selected to participate in the study and whether they would receive home visits or not. In February the home visits started. They are carried out according to plan and will end in September 2004. The first effect evaluation, 12 months after the start of the intervention period, has taken place: 302 questionnaires were sent out in March 2004. The response rate was 95%. Since the beginning of the intervention period, a total of 24 participants died and 4 persons withdrew from the study. Three more effect evaluations will take place: two evaluations after the intervention period in October 2004  and one after the 6 months follow-up in March 2005 .The use of postal questionnaires turned out to be a good and inexpensive method to screen elderly people – there were more than sufficient eligible persons to participate in the research project. The response rate was high and less than one percent of the questionnaires were omitted due to too many missing values. For most of the variables, the percentage of missing values varied between 0 and 2 per cent. Media coverage shortly before sending out the questionnaires and accompanying letters from the municipality and the university may have contributed to the high response rate. It is not certain whether the results are comparable to other (larger) towns in the Netherlands. The response rate of the postal questionnaires used for the first effect evaluation (12 months after the start of the study) was 95%. Nearly all included elderly seemed to be motivated to participate.We selected elderly with a poor perceived health status, because we expected the home visits to be more beneficial for this group rather than for those who are still in good health . ResultsWe considered including a third group of elderly with poor health status to receive home visits from voluntary workers. This was, however, not feasible, mainly because the number of participants with a participating GP was too low. The frequency of the visits and the level of professionalism, nurses versus voluntary aids , could be a topic of study in another trial depending on the outcome of this study.The author(s) declare that they have no competing interests.Erik van Rossum and Paul Knipschild were responsible for the research question. They contributed to drafting of the study protocol, as did Ruud Kempen. Ans Nicolaides-Bouman made the first draft of this paper. The other 3 authors commented on it and approved the final version.The pre-publication history for this paper can be accessed here:


When Francis Collins and Craig Venter reported the draft sequence of the human genome in 2001, Collins described the so-called book of life as more of a life sciences encyclopedia. In it, we can find our evolutionary history written in the fossil record of our DNA, a parts manual listing the genes and proteins needed to build and operate a human being, and a medical text, gleaned from the genetic variants linked to human disease. Unfortunately, he added, the texts are written in a language “we don't entirely know how to read yet.” Since then, biologists have made great progress in extracting meaning from the human genome. Humans are 99.9% alike genetically, and that 0.1% makes all the difference in terms of appearance, personality, and susceptibility to disease. That 0.1% promises to shed light on the evolutionary forces that control genetic variation as well as the genetic origins of human disease.Very small genetic variations—including differences of a single DNA base, called single nucleotide polymorphisms, or SNPs—occur through random mutations. Individuals have two of each chromosome (one from the mother and one from the father), and the combination of SNPs found together on one chromosome can change through the random shuffling of genetic material between the two chromosomes when sperm and egg cells are produced during meiosis. By studying the location and frequency of this reassortment in the genome, biologists hope to understand how recombination affects the overall pattern of SNP variation and how these patterns relate to human disease. An international collaborative effort called the HapMap Project aims to identify the most common SNP associations within chromosomes, known as haplotypes, and then determine which haplotypes are associated with disease. This approach relies on what's known as “linkage disequilibrium”—the nonrandom association of alleles (gene variants) at different locations on a chromosome—to facilitate their search for candidate disease genes. Adjacent SNPs show strong linkage disequilibrium, which means that researchers can select a limited number of SNPs as markers for a haplotype and test their association with disease rather than testing each SNP.Patterns of linkage disequilibrium depend on the rate of recombination—higher recombination rates typically cause less linkage. Demographic factors and chance also affect levels of linkage disequilibrium; while both vary across populations, it has been thought that recombination rates do not. Recombination appears to favor specific genomic regions, termed hotspots, but the observation that the recombinant chromosomes are not passed down in equal proportions suggests that recombination hotspots may be short-lived, appearing as transient blips on the evolutionary radar. Exploring this possibility, Susan Ptak et al. compared a well-studied recombination hotspot in humans, called TAP2, with a similar region in our closest evolutionary cousins, chimpanzees, to see whether they are similarly endowed.Since recombination occurs relatively rarely, researchers have relied largely on indirect methods to determine regional recombination rates. Though recent advances have made sperm analysis in humans more practical , such techniques are less feasible with chimps because collecting large amounts of sperm from individual males might compromise their success in mating competition or reduce the genetic diversity of endangered chimp populations. Here, the researchers used an indirect approach to estimate recombination rates from the patterns of linkage distribution, which “reflect the rate and distribution of recombination events in the ancestors of the sample.” They focused on chimps from a single subspecies because the reported high level of genetic differentiation between subspecies could skew estimates of recombination rate variation. Analysis of the TAP2 region revealed 47 SNPs in the human and 57 in the chimp, with an overall lower level of linkage disequilibrium in humans: strong linkage was seen only in adjacent pairs of SNPs in humans, but was found in both adjacent and more distant pairs in the chimps. Using a statistical approach to characterize recombination rate variation between the two species, Ptak et al. found “extremely strong support” for rate variation in humans but found strong evidence against such variation in chimps.Humans and chimps diverged from a common ancestor five to six million years ago and differ at only 1.2% of base pairs on average. That the recombination hotspot does not exist in both species suggests that hotspots are not stable and can evolve fairly quickly. If recombination rates within a small genomic area—at the level of a few thousand bases—can change in such a short time frame between such closely related species, Ptak et al. reason, they may do so within species, too. Such a prospect has important implications for the HapMap Project and disease association studies that rely on linkage disequilibrium. While haplotypes offer a shortcut for identifying candidate disease genes based on typing a certain number of markers, the number of markers required depends on the strength of linkage disequilibrium. If recombination rates differ across human populations, as these results suggest, then the strength of linkage disequilibrium will too—which means that association studies might need to adjust the number of markers needed to flag candidate disease genes in different populations.


Genes expressed both in normal testis and in malignancies (Cancer/ Testis associated genes – CTA) have become the most extensively studied antigen group in the field of tumour immunology. Despite this, many fundamentally important questions remain unanswered: what is the connection between germ-cell specific genes and tumours? Is the expression of these genes yet another proof for the importance of genome destabilisation in the process of tumorigenesis?, or maybe activation of these genes is not quite random but instead related to some programme giving tumours a survival advantage?This review collates most of the recent information available about CTAs expression, function, and regulation. The data suggests a programme related to ontogenesis, mostly to gametogenesis. In the "brain-storming" part, facts in conflict with the hypothesis of random CTA gene activation are discussed. We propose a programme borrowed from organisms phylogenetically much older than humans, which existed before the differentiation of sexes. It is a programme that has served as a life cycle with prominent ploidy changes, and from which, as we know, the germ-cell ploidy cycle – meiosis – has evolved. Further work may show whether this hypothesis can lead to a novel anti-tumour strategy. Cancer/Testis (CT) antigens are a group of tumour antigens with gene expression restricted to male germ cells in the testis and to various malignancies. Their function in tumours is enigmatic and a common between testis genes (gametogenesis) and cancer remains elusive. When the causal link is not evident, it is tempting to believe the association is random, and assign it to general aspects of "genome instability in cancer". However, we believe a more direct link may exist. As suggested in this review, possible clues may be found in the common evolutionary pathway between ploidy cycles in meiosis and polyploidy in tumour cells. The latter, along with CT antigen expression, is a characteristic feature of well-progressed tumours. However, before discussing such possibilities, it is necessary to review the established literature.The search for tumour antigens began in the 1960's with two groups identifying first alpha-fetoprotein (AFP), a serum marker for hepatoma and germ-cell tumours . These aThe first CTA, MAGEA-1, was identified in 1991 by Boon and colleagues using T-cell epitope cloning, a very complicated and time-consuming method . In 1995By initial definition, expression of genes coding for CT antigens should be restricted in normal tissues to male germ cells in the testis and to malignancies of various histological types. However, the criteria proposed in the 90's are not true for all antigens of this group as seen today. Furthermore, for many of the recently discovered gene products with the described expression profile, no T-cell recognized epitopes have hitherto been identified. This is why CTA – "Cancer/Testis Associated" is a more appropriate name for this family of genes (and will be used in this context further in this review), because a lot of its members still need to be proven as possessing antigenic properties in cancer patients.Attributing genes to the CTA gene family is based on several characteristic features ,8:1. Predominant expression in germ cells of the testis and generally not in other normal tissues.2. Expression in a number of malignant tumours of various histological types.3. Mapping of the gene to the X-chromosome4. Membership of a multigene family.5. Antigenic properties in tumour-bearing patients.Some exceptions to these criteria for certain CTAs will be described and discussed later.To date, 89 individual CTA genes or isoforms have been described, which are organised in to 44 families see . From thThe expression pattern of CTAs during spermatogenesis is of special interest. Functional analysis of these genes during gametogenesis might well give some clues about their possible role in tumours. Their expression is restricted exclusively to spermatogenic germ cells with other tubular cells (e.g. Leydig and Sertoli) being negative. This fits well with the findings of Yuasa et al.  who demoDifferent CTAs are expressed during different stages of spermatogenesis Fig. ., so oneIn normal tissues, expression of NY-ESO1, MAGE-A3, -A4, and -A8 through -A11 as well as of several members of the XAGE gene family is found in the placenta. NY-ESO1 and several XAGEs are also expressed in the fetal ovary .The mechanisms involved in regulation of CTA expression have recently been comprehensively reviewed by Albert Zendman et al . Thus, oviz. 'Boris', is interesting. Boris is reported as being expressed in several types of malignancies, and normally plays a major role in regulating methylation processes during spermatogenesis – it removes imprinting from genes during the last mitotic division of spermatocytes (reviewed in [Mostly, methylation processes are responsible for the ectopic derepression of CTA genes. Using the demethylating agent, 5-aza-2-deoxycytidine (5DC), expression of several CTAs in cultured tumour cell lines can be induced/upregulated . 5DC entiewed in ). SeveraHistone deacetylase (HDAC) inhibitors, on their own or in combination with 5DC, can also induce CTA expression, including MAGE, SSX, and NY-ESO-1 family members . The CTAThere exist definite expression patterns (sets) of different CTAs in certain tumours. Marked heterogeneity of CTA expression is found in cells of some tumours, which cannot easily be explained by a global demethylation process -24. The Information regarding the function and cellular localization of CTAs is far less comprehensive. Often the proposed function is based purely on sequence homology with another protein of a known function. The only CTA proteins functionally established in gametogenesis are SCP-1, involved in chromosome pairing during meiosis , OY-TES-Suggestions for the functions of other CTAs mostly arose from studies of their homology with some well-known proteins and their domains, TSP50 being protease-like, CT17 phospholipase-like, and CT15 metalloproteinase-like . The CT1LDHC, the germ cell-specific member of the lactate dehydrogenase family, escapes from transcriptional repression, resulting in significant expression levels in virtually all tumour types tested. It might contribute to the constitutive activation of an anaerobic pathway in tumours, because its expression in tumours is not dependent on hypoxia .Suggestions as to the role of MAGEs with a CTA expression profile mainly depend on studies of their ubiquitously expressed family members (e.g. Necdin, MAGE-D1, NRAGE, Dlxin-1). In general, the data point to a role for MAGEs via transcriptional regulation in cell cycle control and apoptosis. F.ex, Necdin-related MAGE proteins differentially interact with the E2F1 transcription factor and the p75 neurotrophin receptor . The higThe products of the SSX genes belong to the family of highly homologous synovial sarcoma X (SSX) breakpoint proteins. These proteins may function as transcriptional repressors; SSX1, SSX2 and SSX4 genes have been involved in the t  translocation characteristically found in all synovial sarcomas -35. ThisThe HOM-TES-85 protein has structural peculiarities that are shared exclusively with the N-myc oncoprotein. However, functional studies are required for confirmation . BesidesBRDT is similar to the RING3 protein family. It possesses 2 bromodomain motifs and a PEST sequence (a proline, glutamic acid, serine, and threonine cluster) characteristic of proteins that undergo rapid intracellular degradation). The bromodomain is found in proteins that regulate transcription .PLU-1, a large multi-domain nuclear protein also has a strong transcriptional repression activity. It is a member of the ARID family of DNA-binding proteins. Plu-1 mRNA and PLU-1 protein are both highly expressed in the mitotic spermatogonia. The expression is reduced in the early prophase I stages (leptotene, zygotene), but reappears at pachytene, still being detectable in diplotene cells. It is located diffusely over the nucleus. PLU-1 might have a role in regulating meiotic transcription, restricted to certain meiotic stages .In situ hybridization of human testis sections showed SPAN-X mRNA expression in round and elongated spermatids [The protein encoded by the SPANX gene targets the nucleus and associates with nuclear vacuoles and the redundant nuclear envelope in sperm cells . In situermatids . These rermatids  and irraermatids .The protein encoded by IL13RA1 gene is a subunit of the interleukin-13 receptor. This subunit forms a receptor complex with IL-4 receptor alpha, a subunit shared by IL-13 and IL-4 receptors. This subunit serves as a primary IL-13-binding subunit of the IL-13 receptor, and may also be a component of IL-4 receptors. This protein binds tyrosine kinase TYK2, and thus may mediate the signalling processes that lead to the activation of JAK1, STAT3 and STAT6 induced by IL-13 and IL-4 .SGY-1 (soggy-1), a secreted protein related to the Dickkopf protein family, is involved in suppressing the Wnt signal-transduction pathway controlling transcription activation of genes such as c-myc, c-jun, Fra, and cyclin D1 by preventing the accumulation of beta-catenin ,47. Wnt NY-ESO-1 is one of the most immunogenic and therapeutically promising CTAs but functional studies on this gene are severely lagging behind its practical application. Unlike the majority of CTA genes, NY-ESO-1 stops its expression in well-progressed tumours, so it can be used as a marker to follow the early progression of testicular tumorigenesis .CAGE  and HAGETPX-1 is now seen as an integral protein of the outer dense fibres and the acrosome of spermatids in rats .In summary, we see that the functions of individual CTA, when known, are very diverse, including, for example, both activators and repressors of proliferation and transcription.H) mediated immune responses. Identification of these epitopes is one of the main goals of CTA research. Knowledge of epitopes recognized by the immune system allows the creation of the tumour-specific vaccines. In the vaccination process, T-cell epitopes are often administered together with different adjuvants or cytokines, or delivered using peptide pulsed autologous dendritic cells, all of which are aimed at enhancing the immune reaction [H responses, which are required to support the activity of CTLs, and provide a more "complete" immune response. However, vaccines designated to prime the immune system against tumours expressing various CTA have so far shown only partial clinical success [Immunogenicity in cancer patients is elicited only by short peptide sequences of CTA epitopes, which are presented on the tumour cell surface by HLA Class I molecules in the case of cytotoxic T lymphocyte (CTL) mediated immune responses and by HLA Class II molecules on the surface of APC (antigen presenting cells), in the case of T-helper cell  seems likely . Are CTADiversity of functions, including genes involved in ontogenesis suggest that CTAs are activated either as a result of the genome instability (however, why testicular genes?) or as part of a complex program. Also from this point, this looks like a program related to gametogenesis. The same idea was proposed by Old .If CTAs generally do not enhance tumour growth except by stimulating proliferation, like oncogenes, another possibility is that they could do it by stimulating DNA repair or inhibiting apoptosis . Indeed, some CTAs have relation to the DNA repair factors by homologous recombination. These are SPO11, SYCP1, helicase-like CAGE and HAGE acting in meiotic prophase of gametogenesis. In turn, homologous recombination in tumours was shown to act anti-apoptotically .Are these CTAs restricted only to prophase of meiosis, where recombination takes place? It appears not. For example, some are associated with the spermatogonial stage (Plu-1 mRNA and PLU-1), and some with maturation of the acrosome  one. Let us remember the experiments by Mintz and Illmeisee  who clonAnother question is whether CTA genes govern a life-cycle-like program with its key events similar to meiosis? In turn, to which process does meiosis provide a key – recombination and reduction division? Are tumours capable of these? We also have to consider why X-chromosomes are involved, and not Y-chromosomes, which are responsible for sex? Perhaps this is because the X-chromosome is better conserved evolutionary and appeared prior to sex discrimination.So, this looks like an ontogenetic program, which evolutionary preceded sexual (amphimictic) life-cycle, with the key in recombination and reduction division; but what is it? The answer is evident – ancient ploidy cycles ,79. In gThere may be reduction of ploidy 2n-n  or from nx to 2n DNA numbers, in the asexual ploidy cycles characteristic for a few protists, as in Amoebae and foraminiferans ,81 as weIn this context, expression of some CTAs by the placenta is of interest because trophoblast and decidua are the only mammalian tissue capable of endoreduplication creating enormously large ploidies . an abilNow let us return to DNA recombination repair in tumours as a means of survival, as already mentioned. Repair by homologous recombination can protect malignant tumour cells from apoptosis. In particular, as shown in our laboratory, endopolyploid cells employ this mechanism . LikewisIn turn, some giant tumour cells show the capability to segregate their genomes and return to mitosis ,88-91, s.A hypothesis is put forward that activation of at least some of the CTA genes in p53-deficient human tumours could be due to the genetic program running "relic" ploidy cycles in tumour cells. This hypothesis offers new opportunities for the design of novel tumour treatment strategies. In particular, passive therapy using CTAs to prime the host immune system against the tumour could be replaced with gene therapy aimed to block the function of CTA gene products or even their expression. This approach is promising, because normally only the germ cells in the testis express CTA genes and they are well protected by the blood-testis barrier. Thus, there should not be any problem with tumour-specific priming, and respectively we would predict there to be no side effects."Chromosomal localization, type of immune response, identification method and identification references for all known 44 CTA gene families".Click here for file" CTA Frequency (%) of expression in various tumour types".Click here for file


Alternative splicing contributes significantly to the complexity of the human transcriptome and proteome. Computational prediction of alternative splice isoforms are usually based on EST sequences that also allow to approximate the expression pattern of the related transcripts. However, the limited number of tissues represented in the EST data as well as the different cDNA construction protocols may influence the predictive capacity of ESTs to unravel tissue-specifically expressed transcripts.We predict tissue and tumor specific splice isoforms based on the genomic mapping (SpliceNest) of the EST consensus sequences and library annotation provided in the GeneNest database. We further ascertain the potentially rare tissue specific transcripts as the ones represented only by ESTs derived from normalized libraries. A subset of the predicted tissue and tumor specific isoforms are then validated via RT-PCR experiments over a spectrum of 40 tissue types.Our strategy revealed 427 genes with at least one tissue specific transcript as well as 1120 genes showing tumor specific isoforms. While our experimental evaluation of computationally predicted tissue-specific isoforms revealed a high success rate in confirming the expression of these isoforms in the respective tissue, the strategy frequently failed to detect the expected restricted expression pattern. The analysis of putative lowly expressed transcripts using normalized cDNA libraries suggests that our ability to detect tissue-specific isoforms strongly depends on the expression level of the respective transcript as well as on the sensitivity of the experimental methods. Especially splice isoforms predicted to be disease-specific tend to represent transcripts that are expressed in a set of healthy tissues rather than novel isoforms.We propose to combine the computational prediction of alternative splice isoforms with experimental validation for efficient delineation of an accurate set of tissue-specific transcripts. PDE1C [IRF-3 [The large difference between cells from different tissues is the consequence of a complex regulatory machinery guiding the tissue specific expression of genes and their transcripts. Several genes have been described to exhibit differential splicing patterns for different tissues (E.g. C [IRF-3 ) that reC [IRF-3 ,5.The resource mainly used to predict tissue-specific expression is the rapidly expanding repertoire of expressed sequence tags (ESTs) in the public databases representing a wide spectrum of tissue types. Unlike serial analysis of gene expression (SAGE) which mainly yields the tissue specific expression of genes , the ESTBesides the detection of the existence of alternative splice isoforms the tissue annotation of ESTs can be used for the computational prediction of the expression pattern of these transcripts where the tissue-wise count of transcript-specific ESTs with respect to a random background distribution defines an expression level -14. TranThe EST-based prediction of alternative splice iso-forms revealed 427 genes each contributing at least one potential tissue-specifically expressed variant. These variants show specificity for 28 different tissue types, where brain, testis and placenta account for approximately half of these transcripts see . Many ofOut of the 20 isoforms tested experimentally see . The remThe predicted expression of the isoforms in a single tissue could not be confirmed for half of the variants analyzed (standard conditions). However, the isoforms were always detected to be expressed in the tissue that was originally predicted by our software. The observed expression pattern of the 'unspecific' isoforms ranges from expression in only a few, sometimes related tissues (LMO7 : brain, The number of genes with transcripts exclusively expressed in tumors is relatively large 1120) as compared to the number of genes revealing tissue specific isoforms. Interestingly, 2 out of 4 such disease-related transcripts  or t or t24])The separate evaluation of EST data from non-normalized as well as from normalized cDNA libraries will help to categorize transcripts into highly and lowly abundant ones thus facilitating the integration of EST-based predictions with expression data from microarray experiments. We suggest that large-scale analysis of tissue-specific transcripts should be ideally based on a computational prediction of isoforms that ranks candidate transcripts, tightly coupled with experimental validation via RT-PCR or DNA microarray experiments . Such anThe basis of our work is the tissue/tumor annotation of ESTs is GeneNest database  and the The cDNA libraries of the GeneNest database were semi-automatically categorized into non-normalized, normalized/subtracted and PCR-based libraries by screening for the appropriate keywords in the original annotation of the respective EMBL database entries. All libraries for which none of the keywords were found were defined as being non-normalized. PCR-based libraries like those derived by ORESTES PCR were not used for the current analysis. Additionally, to avoid miscounting caused by PCR amplification, ESTs of the same library and with identical start/end positions in the alignment were treated as a single sequence. Since the level of normalization of different libraries may differ depending on the number of rounds of subtractive hybridizations performed, we also extracted the normalization level (measured as Cot or Rot: ) as far Alternative splice isoforms in the SpliceNest database are revealed by aligning EST consensus sequences (putative transcripts) related to one gene to the appropriate genomic sequence. Significant differences in the boundaries of the putative exons are interpreted as alternative splicing events. For all exon-exon-boundaries that define a certain splice iso-form the annotation of ESTs covering the respective boundary is evaluated. Isoforms overrepresented by ESTs from particular tissue are tagged as putative tissue/tumor specific splice isoforms. Several parameters (e.g. number of ESTs from a particular tissue, number of ESTs from other tissues, number of associated mRNA sequences etc.) are computed for these isoforms and finally stored in a relational database system. The refined set of tissue and tumor specific variants is then generated by setting the requirement of at least 3 ESTs in both alternative forms. Fig. A set of putative tissue specific (n = 16) and disease-related (n = 4) alternative splice events was arbitrarily selected for RT-PCR experiments. PCR primers were generated on the alternatively spliced exon as well as on either side of the event Fig.  using thFor the subsequent RT-PCR experiment, total RNA was prepared using the single-step guanidinum method according to the manufacturer's instructions (TRIZOL, Gibco BRL). First strand cDNA synthesis was carried out in 20 μl reaction using the Omniscript Reverse transcriptase (Qiagen) and the oligo(dT) primers with 2 μg of total RNA. RT-PCR was carried out in a 20 μl reaction in 1 × buffer [1.5 mM Mg2+, 0.2 mM dNTPs, 0,4 μM primers each, 1 Unit of Taq polymerase (Roche)] and 1 μl of cDNA. Amplification steps were as follows: 95°C for 90 sec; 9 cycles of 94°C for 20 sec, 64°C for 10 sec decreasing the annealing temp for 1°C with each cycle (touchdown), 72°C for 20 sec; followed by 30 cycles of 94°C for 20 sec, 55°C for 10 sec, 72°C for 20 sec, followed by an extension at 72°C for 10 min. For the refined PCR, the amplification step was repeated with identical PCR conditions but with 2 μl of PCR product instead of 1 μl of cDNA.All PCR products were resolved on 2% agarose gels run at 90 V/20 cm for 1.5 h in TAE buffer. Gels were then manually examined for exact size, genomic contamination and the tissues in which the bands are observed. As a control, a fraction of variants were sequenced using the ABI Prism BigDye Terminators and the ABI Prism 3100 sequencer (Applied Biosystems).SG wrote the prediction software as well as designed PCR primers for experimental analysis. SH and MV provided guidance for the computational work. DZ performed the RT-PCR experiments with the guidance of BK.List of tissues for which tissue specific transcripts are predicted. This is a text file with a listing of all tissues for which specific trascripts exist along with the number of ESTs related to individual tissues. Furthermore, the ESTs derived from normalized libraries and the specific variants predicted via such ESTs are also listed.Click here for fileDetailed description of RT-PCR experiments This is an excel file containing the primer sequences used for RT-PCR experiments along with detailed comments on the gel pictures subsequently obtained.Click here for fileRT-PCR picture (jpeg file) showing the expression pattern of gene PRAME This gene shows specific expression for several tumor types, along with testis as the only normal tissue.Click here for file


Assembling a complex structure like an automobile requires the tight coordination of hundreds of independent entities—parts must be shipped and arrive on time, workers with the right skills must be in the right place on the assembly line, four (not three, not five) wheels must be bolted into place just so. Overseeing the entire operation is a cadre of managers, whose ability to monitor and respond to changing conditions keeps the entire process moving forward on time and in step.Bacillus subtilis. In this issue, Richard Losick and colleagues show that spore formation in this bacterium is ultimately governed by the temporal interactions of five genes, which together coordinate the activity of almost 400 others.A living cell is orders of magnitude more complex, and yet has no omniscient manager at the helm. So how does a cell make anything happen on time, and equally important, keep everything from happening all at once? These central questions in developmental biology now have the outlines of an answer in one species, B. subtilis divides to form two different cell types: one is a resistant spore, and the other is a mother cell, which engulfs the spore and surrounds it with a protective coat. Building on the large literature addressing the genetic events underlying mother-cell development, Losick et al. performed a variety of experiments to determine exactly which genes turned on and off when, and which genes controlled which others. Over the five-hour process of mother-cell development, they determined that 383 individual genes were activated, representing 9% of the bacterium's 4,106 genes.When conditions are right, E). Sigma factors, such as the sigma-E protein, bind to RNA polymerase, and in so doing, increase its affinity for, and therefore its ability to activate, other genes. Thus, sigma factors preferentially activate a specific set of genes. Sigma-E turns on 262 genes (which together compose its “regulon”), kick-starting a variety of processes in the early development of the mother cell.The instigator of the entire process is a protein called sigmaE . By generating the DNA-binding protein GerE, this factor begins a second feed-forward loop, and together, sigma-K and GerE activate the final set of genes needed for mother-cell development. In outline, the system looks like this: sigma-E → SpoIIID/GerR → sigma-K → GerE.Among the joint targets for sigma-E and SpoIIID is another sigma factor, sigma-K (σThe consequence of all this activity is a series of transcriptional pulses, timed to supply proteins just as they are needed, and then turn off their production when the need passes. For instance, to form the multilayered coat around the spore, sigma-E turns on genes that form the bottom layer, or substratum; these are turned off by SpoIIID. Genes for outer layers, also turned on by sigma-E, are not turned off by SpoIIID, but instead by GerE. Sigma-K turns on genes which form the polysaccharide surface of the coat, which is needed later on.B. subtilis development, let alone development in more complex organisms. There is much still to be learned about how genes lower down in the hierarchy—the “middle managers”—do their jobs, and how the system is fine-tuned by environmental conditions. And while the general scheme of feed-forward loops and hierarchical control is likely to apply to multicellular, eukaryotic organisms, the details are certain to be different, and much more complex.The elucidation of this complex pattern of gene expression doesn't by any means answer every question about


Quantitative proteomic analyses have traditionally used two-dimensional gel electrophoresis (2DE) for separation and characterisation of complex protein mixtures. Among the difficulties associated with this approach is the solubilisation of protein mixtures for isoelectric focusing (IEF). To find the optimal formulation of the multi-component IEF rehydration buffer (RB) we applied the Taguchi method, a widely used approach for the robust optimisation of complex industrial processes, to determine optimal concentrations for the detergents, carrier ampholytes and reducing agents in RB for 2DE using commercially supplied immobilised pH gradient (IPG) gel strips.Our optimisation resulted in increased protein solubility, improved resolution and reproducibility of 2D gels, using a wide variety of samples. With the updated protocol we routinely detected approximately 4-fold more polypeptides on samples containing complex protein mixtures resolved on small format 2D gels. In addition the pI and size ranges over which proteins could be resolved was substantially improved. Moreover, with improved sample loading and resolution, analysis of individual spots by immunoblotting and mass spectrometry revealed previously uncharacterised posttranscriptional modifications in a variety of chromatin proteins.While the optimised RB (oRB) is specific to the gels and analysis approach we use, our use of the Taguchi method should be generally applicable to a broad range of electrophoresis and analysis systems. Xenopus eggs. The combination of 2D gel electrophoresis (2DE) and Mass Spectroscopy (MS) are powerful tools for this analysis.During cell cycle progression different functional protein complexes associate with and dissociate from chromosomal DNA ,2. We ha2DE is capable of resolving thousands of proteins in a single separation procedure . Develop4) separate reactions. Using Taguchi approach the same task can be accomplished in the experiment with just nine reactions.Good solubilisation of protein samples is critical for high performance 2D electrophoresis and there is a wide range of protein solubilisation cocktails reported in the literature. However we have not found any systematic studies reporting optimal concentrations of critical ingredients, possibly because conventional approaches to optimisations are very time consuming: varying all the possible components in turn and in combination is quite laborious. There are, however, methods for reducing the complexity of multi-parametric matrices. The Taguchi method, has been widely used for several decades in the development of industrial processes and recently found its way to the area of life sciences -10. The To find the optimal and most robust conditions for 2DE, we applied a modified Taguchi method  for the Rehydration buffers for IEF generally consist of chaotropes (urea, thiourea), detergent(s), reducing agent(s) and carrier ampholytes (reviewed in ). The stXenopus egg proteins were solubilised in each RB and separated by 2DE  respectively. IAA serves to alkylate reduced cysteine residues and prevent their modification during and after SDS-PAGE. There is evidence that this protocol is not very efficient as the SDS in equilibration solution interferes with IAA alkylation. Variable alkylation can cause a substantial number of artefactual spots on 2D gels ,19. One Xenopus egg extract containing 50 μg of protein were dissolved in rehydration buffers formulated according to the L9 orthogonal Taguchi array shown in Table SNR values for each level of a given component. The signal-to-noise ratio (SNR) function is a statistical measure of performance and takes into account both the mean and variability. In its simplest form, the SNR is the ratio of the mean  to the standard deviation (noise). While there are many different possible SNRs their simple interpretation is always the same: the larger the SNR the better. We used the Taguchi SNR function that is most applicable to the situation where the highest yield of optimised process is desirable .Aliquots of total SNR graphs from a representative optimization experiment. For two variables in this experiment, CHAPS and DTT, the SNR functions had a maximum within the analysed range and highest SNRs were achieved at 1.32% CHAPS and at 34 mM DTT % CHAPS, (43 ± 12) mM DTT, 0.25% ampholytes and 0.4% ASB14 (the last two are the lowest concentrations used in our optimisation experiments).SNR response for ASB14 suggests there may be two concentrations that increase the number of detected spots. While we chose detected spot number as a general reporter of RB performance, other factors such as spot circularity, streaking, spot intensity, etc are also critical. We noted that concentrations of ASB14 slightly higher than 2.0% induced significant streaking and spot shape changes leading to a reduced number of detectable spots  and with oRB (7 M urea, 2 M thiourea, 1.2% CHAPS, 0.4% ASB14, 0.25% ampholytes)  Figure . We obseTo extend our analysis we have evaluated the performance of oRB with a variety of different samples. Chromatin associated proteins are often enriched in lysine and arginine residues that mediate interactions with DNA. When we analysed a preparation of mitotic chromosome proteins  using sRAs a test of the use of an oRB, we characterised a series of well-known chromatin proteins whose function is regulated during the cell cycle. Our improved RB and revised alkylation procedure eliminated ambiguities in immunoblots of high molecular weigh proteins and revealed specific modifications that have not been described previously. XCAP-E (M.W. 140 kDa), a component of condensin, a protein complex involved in the assembly of mitotic chromosomes , was anaPhosphorylation is one of the post-transcriptional modifications that can change the pI of proteins. Previous analysis has not detected significant phosphorylation in XCAP-E and treatment of these samples with phosphatase produced no change in the 2DE pattern (data not shown). We are currently exploring the nature of this apparently specific modification. Regardless, an optimised 2DE methodology can reveal multiple forms of proteins, even relatively large ones that are traditionally poorly resolved.Xenopus chromatin from the end of mitosis and are released during S-phase [Xenopus chromatin at the beginning of S-phase in a control experiment  combinations of zwitterionic detergents, appropriately optimised, can provide improved solubilisation of proteins for 2DE; (2) the concentration of carrier ampholytes must be optimised; (3) the use of a technique like the Taguchi method can rapidly and relatively easily determine an optimal combination of RB components for 2DE. Whereas systematic evaluation of all possible combinations in a multi-component system is prohibitively costing and time consuming, the Taguchi method provides a systematic assessment of a systems behaviour that is reasonably straightforward to perform.Our results also extend the power of 2DE in the analysis of post-translational modifications. For subsequent MS identification of proteins and characterisation of post-translational modifications, difficulties with differential solubility and focussing must be resolved. The oRB addressed these issues and substantially improved the resolution of protein isoforms, including those larger than 100 kDa.We have successfully applied the Taguchi method to optimise the complex composition of a rehydration buffer used in 2DE. The strategy greatly reduced the number of experiments required compared to classical designs, reduced cost, and has produced a substantially improved 2DE RB.Immobiline DryStrip 7 cm gels and carrier ampholytes of different pH ranges were purchased from Amersham Pharmacia Biotech UK Ltd. The Protean IEF cell from BioRad (Bio-Rad Laboratory Ltd., Hemel Hempsted, UK) was used to run the isoelectric focusing separation. For the second dimension, the focused IPG strips were loaded on precast Novex ZOOM gels (Invitrogen, Ltd, Paisley, UK) and run with NuPage MOPS SDS buffer as instructed by the manufacturer.The zwitterionic detergent ASB14 was obtained from Calbiochem (Merck Biosciences, Ltd., Nottingham UK). Urea of AristaR grade and other general chemicals of AnalaR grade were purchased from BDH (Merck House, Poole, Dorset, UK). SYPRO Ruby (Molecular Probes, Leiden, The Netherlands) was used to stain 1D and 2D gels according to manufacture recommendations. The images of stained gels were acquired by a Fujiimager LAS1000 using the Dark Reader trans-illuminator . Spot detection, matching, quantification and analysis were carried out using Phoretix 2D Pro software (Nonlinear Dynamics Ltd, Newcastle, UK). The antibody against XCAP-E (R5-5) was kindly supplied by T. Hirano ,29.Xenopus egg extract preparation, chromatin reconstitution, and elution of chromosomal proteins was carried out as previously described [g in a micro-centrifuge and transferred to rehydration chambers. The dry IPG strips were allowed to re-swell in RBs over night before IEF separation. For samples that were prepared in sRB solubilisation was continued for 2 hours without alkylation before starting the IPG re-swelling.escribed ,28,20. FIsoelectric focusing was performed using a two phase protocol: (1) 250 V for 30 min and (2) 250 – 5500 V fast ramping voltage gradient to accumulate 33000 total volt-hours. The focused IPG strips were subjected to additional reduction and alkylation treatment before the second dimension SDS-PAGE. The strips were equilibrated for 20 min in 25 mM DTT dissolved in 6 M Urea/2% SDS/30% glycerol/50 mM Tris HCl pH8.8 and then alkylated by incubating with 360 mM acrylamide for 20 min in the same buffer. Equilibrated IPG strips were applied to precast Novex 4–12% Zoom gels and run at room temperature for 1 hour at 200 V. SYPRO Ruby stained gels were imaged and the total number of spots in individual gels was determined by Phoretix 2D Pro imaging software. The resulting data were visually inspected to remove background artefacts. The number of spots identified in each individual gel was used in the Taguchi calculations.SNR) for each component. The goal with 2DE is to maximise the numbers of detectable spots. For this aim G. Taguchi designed the following SNR function:In the Taguchi method, variables under optimisation are arranged into orthogonal array , where CHAPS was present at 1% level phosphine hydrochloride, DTT – dithiothreitol, IAA – iodoacetamide, IPG – immobilised pH gradient, MCM – mini-chromosome maintenance proteins, PTM – post-translational modifications.RB – rehydration buffer, sRB – standard rehydration buffer, oRB – optimised rehydration buffer, None declared.Xenopus licensing complex and human nucleolar proteins and drafted the manuscript. IMP carried out 2DE analysis of Xenopus condensin complex and mitochondrial extracts from mouse and shrimp cells. JJB participated in the design of the study and coordination. JRS conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.GAK designed and carried out optimisation experiments, 2DE analysis of Xenopus egg extract were dissolved in RBs containing 7 M urea, 2 M thiourea, 1.2% CHAPS, 43 DTT and variable amount of ASB14 and ampholytes as indicated on each picture. Spots detected: (A) 682, (B) 227, (C) 662, (D) 640.2DE response to variations of ASB14 and ampholytes concentrations. 50 μg aliquots of total Click here for file


Aedes and Anopheles mosquitoes, ribosomal protein RPS6 has an unusual C-terminal extension that resembles histone H1 proteins. To explore homology between a mosquito H1 histone and the RPS6 tail, we took advantage of the Anopheles gambiae genome database to clone a histone H1 gene from an Anopheles stephensi mosquito cell line.In Anopheles stephensi histone H1 corresponding to a conceptual An. gambiae protein, with 92% identity. Southern blots suggested that Anopheles stephensi histone H1 gene has multiple variants, as is also the case for histone H1 proteins in Chironomid flies.We designed specific primers based on RPS6 and histone H1 alignments to recover an Anopheles stephensi and Anopheles gambiae mosquitoes share 92% identity to each other, but only 50% identity to a Drosophila homolog. In a phylogenetic analysis, Anopheles, Chironomus and Drosophila histone H1 proteins cluster separately from the histone H1-like, C-terminal tails on RPS6 in Aedes and Anopheles mosquitoes. These observations suggest that the resemblance between histone H1 and the C-terminal extensions on mosquito RPS6 has been maintained by convergent evolution.Histone H1 proteins from  Ribosomal protein (RP) S6 is a phosphorylated protein that resides on the small subunit of eukaryotic ribosomes. Phosphorylation occurs on a cluster of five serine residues near the C-terminal end of the protein. Although details remain unclear, the phosphorylation state of RPS6 is believed to influence translational efficiency of some mRNAs , possiblAedes aegypti and Aedes albopictus mosquitoes, the RPS6 protein is ~17 kDa larger than its Drosophila homolog, and on polyacrylamide gels, it migrates as the largest protein from the small ribosomal subunit. Ae. aegypti and Ae. albopictus RPS6 cDNAs encode an approximately 100 amino acid extension at the C-terminal end of the protein. The extension is particularly rich in lysine, alanine and glutamic acid, and most closely resembles the sequence of histone H1 proteins from diverse sources [In  sources .Drosophila RPS6 proteins. We have recently shown that RPS6 protein isolated from ribosomal subunits retains its histone H1-like tail [Because RPS6 is thought to have regulatory function(s) in a variety of cell signaling pathways , we wereike tail . Thus, uike tail , the hisRpS6 cDNA from an Anopheles stephensi cell line encodes an approximately 170 amino acid histone H1-like C-terminal extension, and in silico analysis reveals a similar modification encoded by the rpS6 gene in Anopheles gambiae. In both Aedes and Anopheles mosquitoes, the C-terminal extension was completely encoded in Exon 3, directly contiguous with upstream open reading frame encoding the series of serines that may be phosphorylated [Aedes and Culex [Anopheles mosquitoes represents the ancestral state, and that the RPS6 tail has been lost in the higher Diptera, which include D. melanogaster.orylated . AnophelAedes and Anopheles mosquitoes was high, relative to the conventional portion of the RPS6 coding sequence. Because histone H1 is the most variable of the histone proteins, and functions as a linker, rather than as a component of the histone octamer, we set out to clone a cDNA encoding a bona fide histone H1 protein from an An. stephensi cell line. In a phylogenetic comparison, the An. stephensi histone H1 protein clusters with homologs from Drosophila and Chironomus, rather than with RPS6 histone H1-like tails from mosquitoes. These results indicate that the histone H1-like tails on mosquito RPS6 proteins are evolving independently of conspecific histone H1 proteins.Although mosquito RPS6 tails in general resemble histone H1 proteins, their divergence between Drosophila melanogaster histone H1 spans 1204 nucleotides, and encodes a 256 amino acid protein in a single exon [His1 allele in Drosophila [Drosophila histone H1 protein (Accession NM_058232) was compared to the Anopheles gambiae genome using the program BLAST [), we obtained 5 accessions with E values ranging from 3e-35 to 8e-43, distributed on mosquito chromosomes 2 and 3. Upon further examination, we noted that XP_314184 and XP_314186 (chromosome 2) corresponded to the same protein. Two additional histone H1 candidates (XP_311486 and XP_309451) were encoded on chromosome 3. These three conceptual Anopheles proteins shared 70–80% identity to one another, and about 50% identity to the Drosophila H1 protein sequence. In the EST-other database, we found a single uninformative match to an unidentified An. gambiae entry (dbEST id = 11236311), with the relatively modest E value of 0.055. Histone H1 sequences from Aedes mosquitoes are not yet in existing databases.The gene encoding gle exon . There iosophila , while mosophila -11. WhenDrosophila and Anopheles histone H1 proteins was relatively low, compared to approximately 80% amino acid identity between Drosophila and Anopheles RPS6, exclusive of the histone-H1-like tail in the mosquito protein. The Drosophila H1 histone was also ~50% identical to that from Chironomus thummi, a fly closely related to mosquitoes in the infraorder/superfamily Culicomorpha [The 50% identity between comorpha .rpS6, we aligned one of the An. gambiae H1 candidate proteins (XP_311486) to a histone H1 protein from C. thummi, and examined the alignment for precise matches  corresponded to residues AAKKPKA , with total RNA as the template. The absence of a poly(A) tail on histone mRNAs required an unconventional strategy to obtain the 3'-end of the coding sequence. First, we used HindIII-digested genomic DNA template, with a primer based entirely on the 3'-UTR of An. gambiae XP_314184, without success. When we designed a second primer  matched An. gambiae histone H1 candidates on chromosomes 2 and 3 with an E value of 0.0. In addition, 6 unmapped sites also had E values of 0.0. A final two sites had E values of 4e-170 and 3e-127. The deduced An. stephensi protein sequence was 92% identical to An. gambiae protein XP_314184 on chromosome 2  deduced Anopheles gambiae protein (not shown). Identity with An. gambiae XP-311486 was 79%. Based on these criteria, we have cloned the An. stephensi homolog of An. gambiae XP_314184.The e 2 Fig. . A similDrosophila and Anopheles (or Drosophila and Chironomus) histone H1 proteins was only 50%. This divergence undoubtedly reflects the ~250 million years [Aedes and Anopheles RPS6 histone H1-like tails, exclusive of the conventional RPS6 protein sequence, with histone H1 proteins from the nematode Caenorhabditis elegans (AAM44399), the closely-related flies Chironomus thummi (Q07134) and Chironomus tentans (AAB62239), Drosophila, and the Anopheles gambiae and Anopheles stephensi homologs  polymerase (PARP). In future studies, we plan to explore whether the histone H1-like tail undergoes posttranslational modification, and whether it plays a functional role in ribosome biogenesis, perhaps through the activity of PARP.Because RPS6 is considered an important functional component of the ribosome, it seems surprising that a histone H1-like tail occurs at the C-terminal end of this particular protein. However, histone H1-like tails have been reported at the N-terminus of and L23a . The An.Anopheles stephensi mosquito cell line [We used the ASE-IV ell line , which well line . This foCells grown as suspended vesicles for 4 to 5 days in twenty 60 mm plates were collected by centrifugation, and the cell pellet was washed twice with phosphate-buffered saline . The ceHindIII (Promega) at 37°C overnight. Enzyme was removed by phenol:choloroform extraction, and the DNA was recovered by precipitation with ethanol and dissolved in TE. Digested DNA (100 ng) was used as template for the PCR reaction, which contained 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM of each of the four dNTPs, 0.4 μM of primer F1 (5'CCG AAG AAG CCG AAG AAG CCC) and R1 (5'TGC TTT CGG CTT CTT GGC AGC) and 2.5 units of Taq DNA polymerase (Promega, Madison, WI). PCR was performed with an initial denaturation at 94°C for 2 minutes. The next 35 cycles included 94°C denaturation for 45 sec, 55°C annealing for 1 minute, and 72°C extension for 1 minute. The reaction was terminated by a final elongation cycle at 72°C for 2 minutes. The PCR product was recovered from a 0.9% agarose gel, purified using Ultra-Clean 15 (MO Bio Laboratories Inc., Solana Beach, CA) and cloned into PGEM T-Easy vector (Promega). The 3'-end of the gene was obtained in a similar manner, using primers R2  was digested with Total RNA was recovered from ASE-IV cells by guanidine isothiocyanate extraction and cesium chloride centrifugation as described by Davis et al. . The finC. elegans H1 protein designated as an outgroup. The An. stephensi histone H1 sequence has GenBank accession # AY672907.The analysis in Fig. YZ did the experimental work, AMF helped with experimental design and manuscript preparation. Both authors read and approved the final manuscript.


The pancreas is a rare but occasionally favored target for metastasis. Metastatic lesions in the pancreas have been described for various primary cancers, such as carcinomas of the lung, the breast, renal cell carcinoma and sarcomas.We report the case of a 60-year old female with a mass in the pancreatic head four years after partial gastrectomy for gastric adenocarcinoma. The patient underwent a pancreatoduodenectomy. Pathological examination revealed metastases of the primary gastric carcinoma within the pancreatic head and in regional lymph nodes.Pancreatic tumors in patients with a history of non-pancreatic malignancy should always be considered to be a putative metastatic lesion at an unusual site. If the pancreas can be identified as the only site of spread, radical resection may prolong survival. The pancreas is an uncommon location for solitary metastasis from other primary cancers . DespiteA 60-year-old woman presented with elevated blood levels of the tumor markers CEA  and CA 19-9 . Four years before being referred to our institution, the patient had undergone gastric resection (Billroth II gastrectomy) for an adenocarcinoma of the stomach. The tumor was located at the lesser curvature of the gastric antrum, measuring 3 cm in the largest diameter. Pathologic examination revealed a gastric carcinoma of low differentiation, which infiltrated the gastric wall into the subserosal layer without penetrating the serosa. Microscopically, the carcinoma was mainly composed of tubular formations of mitotically active, atypical epithelial cells Figure . The tumFour years after gastric resection, however, ultrasound examination, computed tomography and magnetic resonance imaging revealed an inhomogeneous mass of the pancreatic head, measuring 4 cm in largest diameter Figure . RadiogrThe patient was discharged from the hospital without any perioperative morbidity on the ninth postoperative day. The postoperative blood levels of the tumor markers declined to normal values (CEA 2.6 μg/l, CA 19-9 24 U/ml). Due to the complete surgical resection and the lack of risk factors for recurrence, the patient received no further adjuvant therapy. Under regular follow-up for one year with determination of the tumor markers and computed tomography, the patient revealed no signs or symptoms of local or systemic recurrence.Death from recurrence of gastric adenocarcinoma occurs in 70–75% of patients during the first two years after surgical intervention, however, reports of recurrences more than 10 years after primary diagnosis have been reported as well . The moset al [Adenocarcinomas of the pancreas and of other primary sites frequently display a large histomorphological and immunohistochemical overlap. Thus the differential diagnosis of primary pancreatic cancer versus solitary metastases of other adenocarcinomas may be very difficult – if not impossible – using common pathological and immunohistochemical techniques. According to Robbins et al , solitarPancreatic resections can nowadays be performed with low morbidity and mortality rates, in particular in high-volume centers ,12. ResuThe authors declare that they have no competing interests.MNW collated the information, searched the literature and wrote the manuscript.FB and PS contributed to the pathological aspects of the manuscript and helped in preparing the manuscript.BEF assisted in literature search and writing of the manuscript.MWB and HF managed the patient and helped in preparing the manuscript and edited the final version with PS.All authors read and approved the final version of the manuscript.


ChaB contains a conserved 60-residue region of unknown function found in other bacteria, archaeabacteria and a series of baculoviral proteins. As part of a structural genomics project, the structure of ChaB was elucidated by NMR spectroscopy.ChaB is a putative regulator of ChaA, a NaThe structure of ChaB is composed of 3 α-helices and a small sheet that pack tightly to form a fold that is found in the cyclin-box family of proteins.2+ and Ca2+ divalent metal ions.ChaB is distinguished from its putative DNA binding sequence homologues by a highly charged flexible loop region that has weak affinity to Mg Escherichia coli, among the membrane proteins that catalyze the exchange of ions across the cell membrane [+/H+ antiporters NhaA, NhaB and ChaA, which are involved in sodium ion extrusion. Within E. coli and other enteric bacteria, antiporters encompass the primary systems responsible for adaptation to growth in conditions of high Na+ concentrations and varying pH [+/H+ antiporters, ChaA is unique in that it also shows pH-independent Ca+/H+ antiporter activity. ChaA is also regulated by Mg2+, which inhibits both its Na+/H+ and Ca+/H+ antiporter activity [The regulation of cellular ion concentrations is an essential process in all organisms, necessary to sustain a multitude of physiological processes including pH balance and ion homeostasis. This process is accomplished mainly through membrane ion transporters. In membrane  are the rying pH -8. It isrying pH ,10. Of tactivity .chaA, chaB and chaC found at ~27 minutes on the E. coli chromosome [The Cha operon consists of 3 genes, romosome . Both Ch1H-15N HSQC spectrum of ChaB  were made for the 1H, 13C, and 15N side chain resonances. Resonance assignments have been deposited at BMRB (code 6117). Six signals in the 1H-15N HSQC spectrum originated from the 21 residue N-terminal His-tag 5). This de 1SIG, ) with a 1SIG, [RN, with lieoenzyme ,17. Althproteins , a classproteins .70 and σRN based on their structural similarity  indicates that the highly charged  and not likely to be physiologically significant. Given ChaB's proposed role as a regulatory protein, it is possible that the affinity for Ca2+ or Mg2+ is increased in the presence of ChaA or ChaC.Given the proposed function of ChaB as a regulator and the effect of magnesium as an inhibitor of the Caged Fig.  flexibleE. coli and Salmonella typhi) and archeabacteria (Methanosarcina mazei), while Group II contain ChaB related proteins that are found in Baculoviridae. Thus far, no ChaB domains have been identified in vertebrate and plant species. One major difference between the two classes of ChaB proteins is the presence of the charged loop , which contains an extended poly-linker region and an 8× N-terminal histidine tag with a modified thrombin cleavage site.The gene encoding full length ChaB (residues 1–76) was amplified from genomic E. coli BL21-Gold (DE3) cells (Stratagene) as an 8× His-tagged fusion protein. At an OD600 of 0.8, the cells were induced with 1 mM IPTG and grown for another 3 hours at 30°C. The protein was purified to homogeneity by absorption onto a Ni2+ charged chelating sepharose column (Amersham Biosciences) under native conditions. The recombinant protein used for NMR studies consists of the ChaB sequence with an extra 21 residues (MGSSHHHHHHHHSSGFNPRGS) at the N-terminus containing the 8× His-tag and a thrombin cleavage site. This tag replaced the first residue, a methionine, of the genomic sequence of ChaB. In the analysis vide ante ChaB refers to residues P22-S96 of our construct (i.e. the wild type ChaB sequence excluding the first Met residue). Thus, ChaB begins at P22 in our numbering scheme, corresponding to P2 in the native ChaB sequence. The mass of ChaB was confirmed by SDS-PAGE and electrospray mass spectroscopy.The ChaB construct (plasmid ID: pPI489) was expressed in 15N and/or 13C was achieved by growing the bacteria in M9 minimal medium supplemented with BME vitamins (SIGMA) and (15NH4)2SO4 and/or 13C6-glucose as the sole nitrogen and carbon sources at 37°C. ChaB was purified as described above. NMR samples were obtained by exchanging ChaB into an NMR buffer comprising 50 mM CaCl2 at pH 6.3 using a PD-10 column and subsequent concentration to ~200–300 μL using an Amicon Ultra-4 (5 KDa cutoff, Millipore). Typical protein concentrations ranged from 1.5–2.0 mM.Uniform enrichment of ChaB with 1H, 13C and 15N assignments were completed from CBCA(CO)NH, CBCANH and HBHA(CBCACO)NH spectra using a combination of NMRView [1H, 13C and 15N sidechain assignments were obtained by manual analysis of the H(CC)(CO)NH, C(C)(CO)NH and HCCH-TOCSY experiments using NMRView and in-house written scripts. 1H, 13C and 15N chemical shifts were referenced to DSS according to the IUPAC recommendation [NMR spectra for resonance assignments were recorded at 303 K on a Bruker Avance DRX 600 MHz spectrometer equipped with a triple-resonance CryoProbe and processed with NMRPipe . Backbon NMRView  and SMAR NMRView  packagesendation .13C/15N-edited NOESY experiment (τm= 120 ms) in 90% H2O/10% D2O, and 13C-edited NOESY (τm = 100 ms) and 13C-edited NOESY (aromatic region) (τm = 100 ms) experiments acquired in 99.9% D2O. The experiments in D2O were acquired at 800 MHz on a Varian INOVA spectrometer at NANUC). A 4D 13C-13C edited NOESY experiment (τm = 100 ms) was acquired at 600 MHz to resolve ambiguities involving methyl groups. For all experiments at 600 MHz, the minimal number of scans dictated by the phase cycle was used in combination extensive folding in 15N and 13C to reduce experimentation time. Additional restraints used in structure calculations were: dihedral restraints, derived from 3JHN-Cα coupling constants obtained from the HNHA experiment [1H-15N residual dipolar couplings extracted from comparison of IPAP-HSQC experiments recorded on ChaB with and without 11 mg/mL Pf1 phage [2 precipitated the Pf1 phage.Distance constraints were obtained from a simultaneous 3D periment  and 1H-1f1 phage . For the1H}-15N NOE spectra were acquired in an interleaved manner in which each individual FID was collected with and without presaturation and a recycle delay of 4 s [Steady state {y of 4 s . Saturat13C-13C NOESY experiment was important for manually assigning a number of ambiguous assignments. A total of 1794 unambiguous and 346 ambiguous NOE restraints were obtained from this method and used in combination with dihedral restraints to calculate an ensemble of ChaB structures using CNS [a = 13.7 and R = 0.325. Only residues exhibiting a heteronuclear NOE > 0.65 were included as residual dipolar couplings. Seventeen lowest energy structures with the fewest violations were selected to represent the ChaB structure. No NOE violations over 0.2Å were observed. Structural statistics for this ensemble as calculated by CNS [A set of unambiguous NOE constraints were extracted from the 3D-NOESY spectra and used in conjunction with dihedral angle restraints to generate a preliminary fold of ChaB using CNS1.1 . The ressing CNS . These ssing CNS  and optising CNS  and deted by CNS , PROCHECd by CNS  are summ2 or 2 M MgCl2 to 15N labeled ChaB. Prior to titration, metal impurities were removed by addition of EDTA to the ChaB sample followed by exchange into 20 mM Bis-Tris buffer, pH 6.3 using a PD-10 column. Aliquots of CaCl2 or MgCl2 were added up to a final concentration of 50 mM. Minimal changes in pH and volume were ensured throughout. Chemical shift perturbations were measured as a weighted vector sum of the 1H and 15N chemical deviations: {[(Δ1H ppm)2 + (Δ15N ppm × 0.2)2]0.5}.The effect of calcium on ChaB was determined from addition of aliquots of 5 M CaClNMR: nuclear magnetic resonance NOE: nuclear Overhauser enhancement HSQC: heteronuclear single quantum coherence PPM: parts per million RMSD: root mean squared deviation PDB: Protein Data BankE. coli proteins cloned as part of the structural genomics initiative. PI completed the initial cloning of chaB. NS expressed, purified and characterized ChaB by mass spectrometry. MJO drafted and NS contributed to the written manuscript. KG coordinated and provided financial support for this study.MJO expressed and purified isoptically enriched ChaB, collected all NMR spectra at 600 MHz, processed and analyzed NMR data, performed structural calculations and structural refinement. NS identified ChaB among a series of