Endothelial nitric oxide synthase (eNOS) has previously been detected in the glandular part of the human gastric mucosa. Furthermore, nitric oxide (NO) has been shown to influence gastric secretion in various animal models. The present study was conducted to investigate the influence of exogenously and endogenously derived NO on histamine- and cAMP-stimulated gastric acid secretion in isolated human oxyntic glands.14C]aminopyrine.Oxyntic glands were isolated from human gastric biopsies and were subsequently pre-treated with NO donors and nitric oxide synthase inhibitors and then exposed to histamine or dibutyryl-cAMP (db-cAMP). The secretory response of the glands was determined as accumulation of [G-nitro-L-arginine methyl ester (L-NAME) or NG-nitro-L-arginine (L-NNA) enhanced the secretory response.The histamine- or db-cAMP-induced acid secretion was attenuated by L-arginine, a known source of endogenous NO, and also by the NO-donors sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP). Pre-treatment with either of the NOS inhibitors NOur results show that NO inhibits gastric acid secretion in isolated human gastric glands, and that there is endogenous formation of NO within the glandular epithelium in the vicinity of the parietal cells. Nitric oxide (NO) is produced from L-arginine in a reaction catalyzed by the enzyme nitric oxide synthase (NOS),2. NO isAnimal experiments have provided conflicting information about the interaction between NO and gastric acid secretion. For instance, studies in vitro have shown that NO stimulates secretion of gastric acid in the mouse,18 and bIn an earlier study, we found morphological support that endogenous NO plays a role in regulation of parietal cell function . Also, tTwenty-four healthy men ranging in age from 22 to 31 years were recruited as paid volunteers. The selection criteria stipulated that the subjects had to be free from disease and should not have taken any medications or imbibed alcohol for at least one week prior to examination. The men fasted for at least six hours before examination.®, AstraZeneca, Södertälje, Sweden), after which routine gastroscopy was performed using an Olympus GIF-100 endoscope. Pinch biopsy forceps (Olympus FB 24K-1) were used to take tissue specimens from the greater curvature, immediately distal to the fundus. In all subjects, the gastric mucosa appeared to be normal, both macroscopically and histologically. All subjects tested negative for Helicobacter pylori infection in the urease breath test .Pharyngeal anaesthesia was induced with lidocaine spray , and all subjects gave informed consent.The current experiments were based on a technique that was first described in 1976 for use in rabbits in vitro  and is n2) collagenase enzyme solution -tosyl-L-lysine chloromethyl ketone [TLCK], 10 μM indomethacin, 10 mM glucose, 2 mg/ml human serum albumin , and 1 mg/ml collagenase type IA [Sigma]). The mixture was placed in a 37°C water bath and was gently stirred for 120 minutes, after which most of the treated specimen had disintegrated, leaving mainly isolated gastric glands. The mixture was subsequently filtered through a 200 μm mesh. The isolated glands were washed and re-suspended in pre-warmed (37°C) respiratory medium . The glands were then transferred to vials containing fresh respiratory medium to which we added one of the following: the NOS inhibitor NG-nitro-L-arginine methyl ester  or equivalent amounts of its biologically inactive enantiomer NG-nitro-D-arginine methyl ester (D-NAME); the NOS inhibitor NG-nitro-L-arginine ; either of the two NO donors sodium nitroprusside  and S-nitroso-N-acetyl-penicillamine ; the substrate for endogenous NO production, L-arginine (0.1 mmol/L) [The human oxyntic mucosal biopsies used in our study were washed and stored no longer than 15 minutes in ice-cold oxygenated phosphate-buffered saline (PBS). The tissue specimens were cut into smaller pieces with a pair of scissors and transferred to oxygenated (100% O14C-labeled aminopyrine (AP) in the glands themselves and in the supernatant after centrifugation and then calculate the ratio between those two values  [14C]labeled aminopyrine was added to the vials, which were then further incubated at 37°C for 90 minutes. Thereafter, the gland suspension was transferred to previously dried and weighed tubes, which were centrifuged at 4,000 rpm for two minutes. The supernatant was removed and transferred to scintillation vials. The pellets (glands) were dried at 100°C, and the dry weight was determined, and the glands were subsequently re-suspended in 0.5 mol/L NaOH at 60°C and transferred to scintillation vials. The radioactivity of the glands and the supernatant was determined in a liquid scintillation counter , and the AP ratio was calculated using the following formula[A well-established method used to indirectly measure acid secretion by isolated gastric glands is to determine accumulation of P ratio) . In shorg formula:where IGW is intraglandular water volume (= 2 × the dry weight of glands in mg).Background accumulation of AP is included in the values representing the secretory response. The AP ratios for background and histamine-and db-cAMP-stimulated conditions differed between the individuals. Therefore, for each subject, we determined the AP ratio for stimulated glands and considered that value to be 100% and used it as an individual reference value. All values are based on single analyses.Data were analysed by one-sample sign tests comparing median values using MINITAB™ Statistical Software. P values less than 0.05 were considered significant.® Avidin (Vector Laboratories) for 1 h. Following that treatment, the slides were washed and coverslipped using Vectashield® mounting medium. A Nikon Eclipse® E800 fluorescence microscope with a VFM EPI-fluorescence attachment was used to examine and evaluate the slides. A band-pass filter with a wavelength range of 520–560 nm and a long-pass filter with cut-on wavelength at 590 nm (for emitted light) were employed to visualize the Texas Red® molecules.Isolated gastric glands from five test subjects were placed on charged Super Frost*/Plus glass slides  and then washed with PBS and permeabilized with 100% ethanol at -70°C for 5 min. Thereafter, the slides were incubated at room temperature overnight with rabbit anti-NOS3 antibody  and then washed thoroughly in PBS and incubated for 1 h with biotinylated goat anti-rabbit secondary antibody. Slides where primary antibody had been left out served as negative controls. The slides were subsequently washed again, and biotinylated antibody was detected by exposure to 20 μg/ml Texas RedFor morphological evaluation, glands were fixed on glass slides and stained with Harris hematoxylin for five minutes and 0.5% eosin Y for two minutes. Each step was followed by a rinse in tap water.We studied the effects of NO on acid secretion induced by various stimulants in gastric glands isolated from stomach biopsies from human. Morphological examination of the hematoxylin-eosin-stained slides revealed that the isolation procedure had successfully yielded whole-gland preparations and that parietal cells were present in the gastric glands. Immunohistochemical analysis showed that the isolated glands contained eNOS-immunoreactive cells Fig. , which aBackground AP accumulation was observed in the isolated glands, with a median AP ratio of 8.6 . After administration of 50 μmol/L of histamine or 1 mmol/L of db-cAMP, the median AP ratios were 24.7  and 38.2  respectively. The response to both histamine and db-cAMP exceeded the background by a factor of about 2–4 in all preparations  reduced the histamine response to a median of 48% (n = 7) of the response seen in non-pre-treated glands (100%). In four out of five gland preparations, the substrate for endogenous NO formation, L-arginine (0.1 mmol/L), decreased the AP ratio. This effect however was not statistically significant Fig. . When isExposing the isolated glands to db-cAMP increased the secretion of gastric acid compared to the background level. Compared to untreated glands, those that were pre-treated with SNP (1 mmol/L) or SNAP (0.1 mmol/L) accumulated less AP after stimulation with db-cAMP; 63%(n = 9) and 81%(n = 8) respectively Fig. . MoreoveThe method of using isolated gastric glands in vitro is well suited for studying the interaction between gastric acid secretion and various endocrine signals. This technique has been thoroughly evaluated, chiefly in experiments on animals, and it has been reported to offer good reproducibility . FurtherIn a previous study conducted by our research group , examinaThe exact mechanisms behind this paracrine regulation of gastric acid secretion is yet to be elucidated. There are several different pathways within the parietal cell that might be affected by NO. It can induce ADP ribosylation of G-actin , therebySince it is known that guanylate cyclase is a general target of NO in many cell systems, some investigators have suggested that NO exerts its effects via cyclic guanosine 3',5'-monophosphate (cGMP) in both rat and rabbit parietal cells ,15. An oFurther investigations are needed to clarify the role of NO in parietal cell function.The findings of the present study suggest that NO produced endogenously in the human oxyntic mucosa can reduce the stimulatory effects of histamine or db-cAMP on gastric acid secretion. We obtained uniform results for gastric glands isolated from different healthy human subjects, which implies that NO released from specific cells within the secretory mucosa plays an important physiological role in the regulation of gastric acid secretion.None declared.AB participated in the design and coordination of the study, performed the secretory studies, carried out the immunohistochemical procedures, and drafted the manuscript. SR was the surgeon in charge and carried out all gastroscopic procedures. ACE and SES were involved in the design of the study and in drafting of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The study investigated the extent to which approaches to work, workplace climate, stress, burnout and satisfaction with medicine as a career in doctors aged about thirty are predicted by measures of learning style and personality measured five to twelve years earlier when the doctors were applicants to medical school or were medical students.Medical Register. The current questionnaire included measures of Approaches to Work, Workplace Climate, stress , burnout (Maslach Burnout Inventory), and satisfaction with medicine as a career and personality (Big Five). Previous questionnaires had included measures of learning style (Study Process Questionnaire) and personality.Prospective study of a large cohort of doctors. The participants were first studied when they applied to any of five UK medical schools in 1990. Postal questionnaires were sent to all doctors with a traceable address on the current or a previous Doctors' approaches to work were predicted by study habits and learning styles, both at application to medical school and in the final year. How doctors perceive their workplace climate and workload is predicted both by approaches to work and by measures of stress, burnout and satisfaction with medicine. These characteristics are partially predicted by trait measures of personality taken five years earlier. Stress, burnout and satisfaction also correlate with trait measures of personality taken five years earlier.Differences in approach to work and perceived workplace climate seem mainly to reflect stable, long-term individual differences in doctors themselves, reflected in measures of personality and learning style. Sir William Osler (1849–1919), one of the most distinguished physicians of the nineteenth and early twentieth century, recognised that only some doctors are happy in their professional lives:"To each one of you the practice of medicine will be very much as you make it – to one a worry, a care, a perpetual annoyance; to another, a daily joy and a life of as much happiness and usefulness as can well fall to the lot of man."The modern medical workplace is a complex environment, and doctors respond differently to it, some finding it stimulating and exciting, whereas others become stressed and burned out from the heavy workload. The medical workplace also provides an environment where new skills are continually being learned, both as a result of medical knowledge evolving and because a doctor's work changes, in part due to career development and progression through different jobs.et al . Theth March 2003, a few questionnaires were returned up until the end of August 2003.Questionnaires, along with a postage-paid return envelope, were posted at the beginning of December 2002. Two reminders were sent to non-respondents. Although the official closing date was 25Statistical analysis used SPSS version 10.5, and structural equation modelling used LISREL 8.52.Supplementary Information see The tracing exercise looked for 2,912 individuals thought to have completed basic medical sciences and entered a clinical course. Eighty-nine had never been on the UK Medical Register, and either had failed finals, had never registered, or had emigrated. Of 2,823 individuals who were traced, 2,754 doctors were on the 2002 Register, 7 returned to the Register during 2002, and 64 were on an earlier Register. Of 2,823 questionnaires sent, 176 were returned by the Post Office as undeliverable, 10 doctors were travelling and hence uncontactable, and2 had died. Of the remaining 2,635 doctors, 1,668 returned questionnaires, giving a response rate of 63.3%. There was no evidence of response bias . There was substantial variation in the scores on the aAWQ and the aWCQ, and the factor structures of the aAWQ and aWCQ were similar to those reported elsewhere [Supplementary Information see The mean age of respondents on 1lsewhere  (see Supet al study, the highest correlations were for a surface-disorganised approach correlating with high workload, and a deep approach correlating with a supportive-receptive environment and with choice-independence (table Approaches to work and learning were correlated with climate in the workplace, and as in the Delva ce table .Table Table Table Supplementary Information see Tables A surface-disorganised approach to work is predicted by surface learning in medical school and by higher neuroticism scores and lower conscientiousness (see tables A workplace climate dominated by a high workload is predicted by higher stress and emotional exhaustion measures five years earlier, and by lower openness to experience (see tables Supplementary Information see Although in the previous analyses, stress and burnout have been used as predictors of approaches to work and workplace climate, they are also important outcome measures in their own right. Table Supplementary Information (see The complex relationships described by the various correlations are best analysed and described by means of path analysis or causal modelling , which ation see . AlthougSupplementary Information (see Although the path diagram in figure tion see .Stress in our model is caused by personality differences, being greatest in those having high neuroticism scores, low extraversion scores, and low conscientiousness scores. It is unrelated to learning style.Learning styles at medical school relate to different personality measures, in particular showing no relationship to neuroticism. Deep learning is highest in extraverts who are open to experience, whereas strategic learning is highest in highly conscientious individuals with low openness to experience. Surface learning style is higher in introverts who are low in openness to experience. These findings are similar to those of others .Approaches to work are mainly but not entirely driven by learning styles. A deep approach to work occurs in extraverts who are open to experience and have a deep learning style. The surface-rational and surface-disorganised approaches to work are both greater in those with a surface learning style. However, a surface-disorganised approach occurs in individuals with higher neuroticism scores, in those with lower conscientiousness scores, and in those who have been stressed, whereas the surface-rational approach to work occurs in strategic learners and in those who are low in openness to experience.Workplace climate has a range of influences. High perceived workload occurs in those with a surface-disorganised approach to work, who have been stressed and are more neurotic. In contrast, choice-independence and a supportive-receptive environment both occur in individuals who have not previously been stressed, the choice-independence approach occurring in those with a deep approach to work, whereas the supportive-receptive approach occurs in those who have higher scores on the personality trait of agreeableness.Many doctors at the age of 30 are unhappy in their jobs, and a fifth of our sample reached the conventional GHQ criterion of psychiatric 'caseness'. In contrast, many doctors reported high levels of personal accomplishment, choice and independence in their work environment, satisfaction with medicine as a career, and intellectual and emotional satisfaction from their work. That is not new; Sir William Osler in 1905 contrasted doctors "whose stability of character and devotion to duty make one proud of our profession" with those who find it difficult to keep "the flame alive, smothered as it is apt to be by the dust and ashes of the daily routine" [BMJ Careers devoted to "Doctors' Wellbeing", that excessive workload and absence of support are directly caused by poor working conditions: "the way in which the NHS is run generates stress for members of the workforce every day" [with stress and burnout reported five or six years earlier, when the doctors were PRHOs and carrying out entirely different jobs. High perceived workload and poor support are therefore determined as much by doctors themselves as by specific working conditions. That view was expressed in another article in the special edition of BMJ Careers: "A critical element contributing to the stress that many conscientious doctors experience is internal..." [In 2001, Richard Smith asked "Why are doctors so unhappy?" and concluded that "The most obvious cause of doctors' unhappiness is that they feel overworked and undersupported" . Certainery day" . Howeverery day" . It is prnal..." . A similrnal..." .et al [surface-disorganised approach to work, which in turn is correlated with being a surface learner at application to medical school, a dozen years previously. Surface-disorganised doctors are also high on the personality trait of neuroticism and low on the trait of conscientiousness; and again those correlations are with measures taken six years earlier when the doctors were PRHOs. Doctors reporting a work climate low in support were lower on the personality scale of agreeableness in the measures collected when they were PRHOs.If differences in reported workload are partly explained by differences among doctors, what in turn explains those differences? Doctors reporting a high workload also have what Delva et al  describeSome doctors may be stressed and burned out, but what predicts those others who are happy in their work? Doctors reporting high satisfaction with medicine as a career have a deep approach to work, and that approach is more common in those who also had a deep learning style when they applied to medical school. Satisfaction with medicine also relates directly to the personality traits of greater extraversion and lower neuroticism, and the deep approach to work correlates with greater extraversion and more openness to experience. Doctors who describe their colleagues as receptive and supportive score more highly on the personality trait of agreeableness; and as in many other correlations reported here, that correlation is stable across time – those who are more agreeable at the age of 24 have a more receptive and supportive work environment when aged 30.An overview of our findings is that approaches to work are predicted by earlier measures of study habits and learning styles, whereas perceived work climate, and its pathologies such as stress and burnout, are predicted mainly by personality. Although unfortunately our study did not measure personality during selection, the high stability of the Big Five measures across the life-span -29 (and If, as William Wordsworth said, "the child is father to the man", then the seeds of subsequent job satisfaction and dissatisfaction in doctors may be visible in the personality, motivations and learning styles of medical school applicants. This argument may provide some justification for using such measures in selection, particularly given the general association of job performance and satisfaction with personality  and motiHowever, just as genes are not destiny, so neither personality nor learning style is destiny. Nurture interacts with nature , the envFormal education, particularly effective formal education , can alscorrelates of approaches to work, workplace climate, stress, burnout and satisfaction with a medical career, but are causes, events later in time being predicted by events earlier in time [Longitudinal data suggest that personality and learning style are not merely  in time . DoctorsThese results imply that differences in approach to work and workplace climate in our study result from differences among doctors themselves, as much as they do from differences in working conditions.None declared.The cohort study was designed by ICMcM. The present follow-up was designed by ICMcM and EP, who also prepared the questionnaire. AK was responsible for day-to-day running of the study, and for data entry and cleaning. ICMcM was primarily responsible for data analysis and for writing the first draft of the paper. ICMcM, EP and AK were all involved in preparing the final draft of the paper.The pre-publication history for this paper can be accessed here:Click here for file


The European Union recently awarded 12 million Euros to the ZF-MODELS research consortium to study zebrafish models for human development and disease Danio rerio) in 1822, would be astounded to see the scientific attention now afforded to this two-inch-long native of Indian rivers. A fish with no economic worth was how he described this little creature. Yet recently, the European Union awarded 12 million Euros to the ZF-MODELS research consortium to study zebrafish models for human development and disease. When and why did zebrafish swim from home aquaria into research labs, and what can we learn about our biology from this surprising source?Francis Hamilton, the Briton who first described zebrafish , ‘who laid the groundwork for the use of zebrafish as a developmental model’.Xenopus neural development but soon became attracted to zebrafish as a model organism. By the early 1980s, she explains, Streisinger had worked out many of the genetic tricks needed to tackle zebrafish development. What's more, the fish had ‘wonderful embryology’. The embryo, which develops outside its mother, is transparent. ‘You can see different cell types, watch individual cells develop, do transplantation experiments’, Eisen enthuses, ‘and development is quick but not too quick’. Being able to watch individual neurons developing in real time opened up whole new avenues of research for Eisen and other neurobiologists.Eisen, who now heads her own research group, went to Oregon in 1983 to work on The properties of zebrafish that attracted Eisen soon attracted people interested in other aspects of vertebrate development to the stripy tiddler . As EiseDevelopment was dedicated to the mutants that had been isolated and characterised. These screens, says Ralf Dahm , project manager of the ZF-MODELS consortium, ‘were the first major zebrafish projects, and they showed that zebrafish was a model organism to be reckoned with’.The speedy expansion was driven in great part by two genetic screens initiated in 1992–1993 by Christiane Nüsslein-Volhard in Tübingen, Germany, and Wolfgang Driever and Mark Fishman in Boston, Massachusetts. The aim of both screens was to identify genes with unique and essential functions in zebrafish development, and in 1996 an issue of the journal ‘That was a fantastic time’, says Derek Stemple, then a postdoc with Driever but now a group leader at the Wellcome Trust Sanger Institute  and a ZF-MODELS participant. ‘From Wolfgang's lab, I was able to take the mutations that affected notochord development, and have been studying them ever since’. The notochord is an embryonic structure that forms the primitive axial skeleton of the developing embryo, and because mutations affecting notochord development result in shortened embryos, seven of the affected genes have been named after the dwarves in Snow White—zebrafish, like some other developmental models, have many imaginatively named mutants. Stemple now knows the identity of six of these mutated genes, all of which lead to disruption of basement membrane around the notochord.Many mutants from those first two screens are still used by developmental biologists, but another set of mutants has recently been isolated by Nancy Hopkins, Amgen Professor of Biology at the Massachusetts Institute of Technology . About ten years ago, Hopkins started to develop insertional mutagenesis in zebrafish . In thisThe Tübingen researchers did another chemical mutagenesis screen between 2000 and 2001, and are now starting a third screen of 6,000 genomes as part of the ZF-MODELS project. ‘Each of our screens has built on the previous one by including more specific assays’, explains Dahm. Mutagenesis for the third screen is underway, but the assays, which include looking for defects that specifically affect adults, are still at the pilot stage; this autumn, the project's executive committee, which is headed by Nüsslein-Volhard, will decide which assays to use in the full-scale screen. ‘Just over half the 17 partners in the consortium will come to Tübingen to do screens’, predicts Dahm. ‘By bringing in expertise in different systems in this way we should greatly increase the efficiency of the screen’.The ZF-MODELS consortium, which is funded under the European Union's Sixth Framework Programme, aims to establish zebrafish models for human diseases, discover genes that will lead to the identification of new drug targets, and gain fundamental insights into human development. ‘We will mainly focus on using advanced technologies that have recently become available’, says scientific coordinator Robert Geisler . For example, Geisler's lab will use DNA chip technology to investigate gene expression patterns in zebrafish mutants and so provide increased knowledge of the regulatory pathways that act in zebrafish development.Consortium members will also use ‘reverse genetics’ to investigate these pathways. In reverse genetics, researchers start with a gene of interest and investigate the phenotypic effect of altering its activity; by contrast, in ‘forward genetics’ the starting point is to look for a particular phenotype and then hunt out the altered gene that is causing it. Two reverse genetics approaches will be used by the consortium. Gene expression will be transiently knocked down with morpholinos, short segments of the gene that block its function. In addition, a recently developed technique known as TILLING  will be used to knock out gene activity permanently.The first step in TILLING is to mutagenise male zebrafish and mate them with untreated female fish, explains Stemple, whose group is one of three ZF-MODELS partners who will use this approach. Offspring are raised to adulthood, and the DNA of each individual is then genotyped for the exon of interest. The consortium already has a collection of 6,000 such individuals, and once a fish carrying a mutation in the gene of interest has been identified, it will be outcrossed to produce offspring, half of which will carry the desired mutation on one of their chromosomes. ‘It is then a matter of identifying these heterozygote fish and incrossing them to get homozygous fish in which you can see the phenotype that correlates with that mutation’, says Stemple.As well as helping to produce knock-outs for other researchers, Stemple is also using the TILLING technique to develop zebrafish models for muscular dystrophy. Among the genes that are important in notochord development are those that encode laminins. This led Stemple into studying muscular dystrophy because laminins are involved in the human disease. ‘When we used morpholinos to disrupt [the production of] dystroglycan, a laminin receptor, we got a good model for muscular dystrophy’, he explains. Now, he plans to use TILLING to disrupt up to 30 other genes known to be involved in human muscular dystrophy. ‘In particular, we will look for hypomorphic mutants, fish that are viable but on the edge of falling apart’. These mutants can be used to identify small molecules that push the fish into muscular dystrophy. Finding molecules that can cause a disease in this way ‘might give us a handle on something to fix the disease’, says Stemple.In another strand of the ZF-MODELS project, zebrafish expressing green fluorescent protein (GFP) in specific cells or tissues will be generated and characterised . In suchWilson's own interest is in neuroanatomy. Together with Jon Clarke, another developmental neurobiology group leader at University College London, he plans to analyse GFP lines in which small groups of neurons or particular parts of neurons are labelled, and in this way start to build a detailed reconstruction of early brain neuroanatomy. This, in combination with other work on zebrafish carrying mutations affecting neural development, will give the team ‘a better picture of how a vertebrate brain is built’.A final, important aspect of the ZF-MODELS project, adds Dahm, is database construction. ‘We will be developing a set of databases that will integrate all of the project data’, he explains. ‘In addition, we hope to integrate our data with that of ZFIN in the United States to make one central zebrafish resource’.The researchers of the ZF-MODELS consortium are understandably excited about participating in what will, says Geisler, bring an already strong European zebrafish community closer together. But zebrafish researchers in the United States are also excited by the ZF-MODELS project. ‘We need big lab models like ZF-MODELS in developmental biology’, says Hopkins, noting that the days of small groups working in isolation are long gone. This consortium, adds Howard Hughes Medical Institute Investigator Leonard Zon , ‘will not only have an effect on European zebrafish science but also on how it is done in the United States’.But how much can zebrafish tell us about human development and disease? A lot, say zebrafish researchers. ‘Fish really are just little people with fins’, says Hopkins. ‘Of course, there are developmental differences between people and fish, and no one pretends that we can answer every question about human development in zebrafish’. Nevertheless, zebrafish studies can provide valuable clues to the genes involved in human diseases and to potential targets for therapeutic interventions. Hopkins provides the following illustration: ‘We have been doing “shelf screens”, in which we go back to our collection of mutants to find all those that affect the development of a single organ. When Zhaoxia Sun, a postdoc in my lab who now has an independent position at Yale Medical School, screened three-day-old fish for cystic kidney disease, she found 12 different genes. Two were known to cause human cystic kidney disease, so we knew we were in the human disease pathway somewhere, but we had no idea what the other genes were’. Hopkins and Sun have since identified the remaining genes, and these point to a single pathway being involved in the human disease.Developmental geneticist Didier Stainier  is also using zebrafish to study organ development, in particular, heart development. The zebrafish heart is like the early human heart—a tube with an atrium, ventricle, and valves. ‘Everything we have found in the fish is relevant to the human heart’, says Stainier. ‘Obviously, there are additional processes involved in humans, but the basic outline of heart development in fish and people is largely similar’. Stainier has a collection of zebrafish in which valve formation is faulty. ‘Some of the genes we have found will be involved in human congenital valve defects’, he predicts. Knowing the identity of these genes will be useful diagnostically, but, in addition, zebrafish studies can reveal exactly what has gone wrong at a cellular level. The ability to follow individual cells as organs develop is key to this, says Stainier, who reported in March that fibronectin is required for heart development because, by regulating the polarisation of epithelial cells, fibronectin ensures the correct migration of myocardial cells. And in this issue of PLoS Biology, Stainier's lab have identified another zebrafish gene that is involved in heart development—cardiofunk, which encodes a special type of muscle protein.Many researchers are now recognising the value of zebrafish models of human disease. Over the past three to four years, says Zon, this area of research has become a growth industry. The interest in disease models has grown hand-in-hand with the development of morpholinos to knock out specific genes, and the advent of TILLING, says Zon, ‘has set off a whole new fury. There are now large numbers of investigators who will try to knock out their favourite gene and come up with a model’.Zon has worked on disease models for blood , blood vBigger and bigger seems to be the consensus. Chemical screens like Zon's for anticancer drugs can be set up for other human diseases such as muscular dystrophy. Work like Stainier's on organ development may have applications in tissue engineering. ‘If we can find out what drives differentiation in zebrafish’, he suggests, ‘we might be able to do the same for human cells’, making human tissue replacement therapy a practical possibility. And while many zebrafish researchers will continue to study development, others are now moving into the realms of physiology and behavioural studies.Geisler sums up zebrafish developmental research past, present, and future as follows: ‘No other [vertebrate] organism offers the same combination of transparent and accessible embryos, cost-effective mutagenesis screening, and, more recently, a sequenced genome, [DNA] chip, GFP, and knockout technology’. Add to that the potential of zebrafish embryos as a screening platform for small molecule libraries and the new technologies that allow forward and reverse genetics, and it is clear that zebrafish are not about to revert to being pretty pets swimming in small tanks in the corner of the living room.ZF-MODELShttp://www.zf-models.orgMore details of the work included in this European Union Integrated Project can be found at ZFINhttp://z.n.org/ZFIN, provides an extensive database for the zebrafish community including genetic, genomic, and developmental information; search engines for zebrafish researchers and laboratories; listings of meetings; and links to many other zebrafish sites, including sites with movies of zebrafish development.The ZFIN Web site, at Development  on the first two mutagenesis screens contains 37 research articles and can be freely accessed at http://dev.biologists.org/content/vol123/issue1/index.shtmlThe special issue of


Epidemiological studies have shown that individuals who regularly consume NSAIDs have lower rates of mortality associated with colorectal cancer. Because COX-2 inhibitors prevent tumor growth through some mechanisms, we assessed the effect of JTE-522, a selective COX-2 inhibitor, on pulmonary metastases of colon cancer in a rat model.6 RCN-9 (rat colon cancer cells) was injected into the tail vein of 24 anesthetized male F344/DuCrj rats. Oral JTE-522  was administered from the day before RCN-9 injection until the end of the study. Twenty-four days later, the lungs were removed from sacrificed rats and weighed. Pulmonary metastatic tumors were microscopically evaluated in the largest cross sections. We also performed immunohistochemical staining for both COX-2 and VEGF.A suspension of 5 × 10JTE-522 dose-dependently decreased lung weight (p = 0.001) and the size of pulmonary metastatic tumors (p = 0.0002). However, the differences in the number of metastatic tumors among 4 groups were insignificant. Significant adverse effects of JTE-522 were undetectable. Immunohistochemical staining showed high levels of both COX-2 and VEGF in pulmonary metastatic tumors.JTE-522 dose-dependently decreased the size, but not the number of pulmonary metastases. COX-2 inhibitors might block metastatic tumor growth, but not actual metastasis. Selective COX-2 inhibitors might be useful as therapeutic agents that inhibit the growth of metastatic tumors, as well as the tumorigenesis of colorectal cancer. They fo in 1991 ,6. Many  in 1991 . Oshima out mice . In addiat model , and anoat model . We repoat model . Tomozawl cancer .It is well known that colorectal metastasis occurs primarily in the liver and secondarily in the lung . A laparThe selective COX-2 inhibitor, JTE-522 , was susTwenty-four 4-week-old male F344/DuCrj rats, weighing 50 to 70 g purchased from SLC  were acclimatized for one week before use in this study. The rats were maintained in an environment with a controlled temperature (22 to 24°C), humidity (40 to 50%), and lighting (12 hours light-dark cycle) with free access to tap water and standard rodent chow .et al. after inbred F344/DuCrj rats were subcutaneously injected with neutralized 1, 2- dimethylhydrazine (DMH) [The RIKEN CELL BANK donated the rat colon cancer cell line, RCN-9, which was established by Inoue ne (DMH) . The tum6 RCN-9 cells in 0.4 ml of PBS was injected into the tail vein.F344 male rats were anesthetized with ether and then a tumor suspension containing 5 × 10Twenty-four rats were divided into four groups. Group 1 (control) was not given JTE-522. The other three groups all orally received daily doses of 3, 10 and 30 mg/kg JTE-522  by gavage from the day before RCN-9 injection until the end of the study. The rats were sacrificed 24 days after injection and the lungs were removed and weighed. After 3-day formalin fixation, lungs were embedded in paraffin. Paraffin-embedded sections were cut into 3-μm serial sections, which stained with hematoxylin and eosin to evaluate the pharmacological effects of JTE-522 on pulmonary metastases. The number and the largest diameter of metastatic tumors were examined microscopically in the largest sagittal cross section. The largest metastatic tumor was selected after measuring the diameter of 10 tumors in each slide.2O2 in methanol for 20 minutes. After a short rinse in phosphate-buffered saline (PBS), non-specific binding was blocked by incubating the sections in 10% normal goat serum for 10 minutes at room temperature. Thereafter, the slides were incubated with primary antibodies directed against COX-2  for 2 hours or vascular endothelial growth factor (VEGF)  for 1 hour at room temperature. The slides were washed in PBS (3 × 5 minutes) and incubated with secondary antibody  for 30 minutes at room temperature. After washing in PBS, color was developed using 3, 3'-diaminobenzidine tetrahydrochloride (Sigma) in 50 mmol Tris-HCl (pH 7.5) for 5 minutes and counterstaining with 1% Mayer's hematoxylin. Sections known to show intense immunostaining for COX-2 and VEGF were included in each experiment, having been incubated with either primary antibody or PBS as positive and negative controls, respectively. The positive controls were always obviously stained and negative controls were never stained.Slides were immunohistochemically examined using the Universal Immuno-enzyme Polymer (UIP). Sections of 3 μm thick were cut from formalin-fixed and paraffin-embedded blocks of rat tissues. The slides were deparaffinized in xylene, rehydrated through a graded series of ethanols and washed with distilled deionized water. Antigen was retrieved by incubating the sections in 10 mmol/L citrate buffer (pH 6.0) in an autoclave (120°C) for 5 minutes. Thereafter, slides were cooled to room temperature and then washed with distilled deionized water. To quench endogenous peroxidase activity, slides were immersed in 3% Het al [The specimens immunostained for COX-2 and VEGF were independently scored by two blinded investigators. Both the distribution (the percentage of positive cells) and the intensity of staining were assessed in a semiquantitative fashion. Sections were scored, according to the method of Yukawa et al . Both thThe effects of JTE-522 on pulmonary metastasis were analyzed by the Kruskal-Wallis test and are expressed as means ± standard deviation. A correlation between COX-2 and VEGF expression was analyzed by Spearman's rank test. In all instances, statistical significance was established at p < 0.05.Each group consisted of 6 rats. JTE-522 significantly and dose-dependently decreased the weight of the lungs  supplied from the date of implantation [et al. evaluated the pharmacological effects of celecoxib on lung metastasis by counting the metastatic tumors using a stereomicroscope and by histochemical analyses of consecutive lung sections. Tomozawa et al. investigated the effect of JTE-522 on lung metastasis of colon cancer in mice [JTE-522 dose-dependently reduced the weight of the lung with metastases and the size of metastatic tumors. These observations are consistent with those of other studies of selective COX-2 inhibitors. In the present study, however, the number of metastatic lesions determined microscopically was not reduced by JTE-522, which conflicts with the earlier findings. Masferrer antation . Celecox in mice . Their sxib 160–30 ppm supet al. identified a significant correlation between COX-2 and VEGF, the latter of which might be one of the most important mediators of the COX-2 angiogenic pathway [et al. reported that hypoxic animals have significantly higher VEGF concentrations compared with normoxic controls [Here, we immunohistochemically stained COX-2 and VEGF because some reports indicate that prostaglandins regulate VEGF expression ,22 and t pathway . Immunoh pathway . Our res pathway . VEGF wacontrols . On the We reported that COX-2 is related to human colorectal tumorigenesis. COX-2 mRNA expression is obviously increased in sporadic adenomas compared with normal colorectal mucosa and is significantly greater in adenomas with larger diameters . Additioet al. demonstrated that rofecoxib decreased the growth and metastatic potential of colorectal cancer in mice through multiple mechanisms [et al. reported that refecoxib negatively regulated angiogenesis in human colorectal cancer liver metastases [Recent reports have shown the inhibitory effect of rofecoxib on liver metastases. Yao chanisms . Fenwicktastases . It is cIn conclusion, JTE-522, a selective COX-2 inhibitor, dose-dependently inhibited the growth of pulmonary metastasis from colorectal cancer in a rat model. Selective COX-2 inhibitors may constitute useful therapeutic agents that can interfere with the growth of hematogenous metastasis, as well as the tumorigenesis of colorectal cancer.The author(s) declare that they have no competing interests.HK carried out the animal experiment and drafted the manuscript. HK and TH performed immunostaining and its evaluation. HU performed the statistical analysis. ME and KS participated in the design of the study and its coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


ASPM, which encodes the human homologue of a fly protein essential for spindle function, are the most common known cause of MCPH. Here we have isolated large genomic clones containing the complete ASPM gene, including promoter regions and introns, from chimpanzee, gorilla, orangutan, and rhesus macaque by transformation-associated recombination cloning in yeast. We have sequenced these clones and show that whereas much of the sequence of ASPM is substantially conserved among primates, specific segments are subject to high Ka/Ks ratios (nonsynonymous/synonymous DNA changes) consistent with strong positive selection for evolutionary change. The ASPM gene sequence shows accelerated evolution in the African hominoid clade, and this precedes hominid brain expansion by several million years. Gorilla and human lineages show particularly accelerated evolution in the IQ domain of ASPM. Moreover, ASPM regions under positive selection in primates are also the most highly diverged regions between primates and nonprimate mammals. We report the first direct application of TAR cloning technology to the study of human evolution. Our data suggest that evolutionary selection of specific segments of the ASPM sequence strongly relates to differences in cerebral cortical size.Primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by global reduction in cerebral cortical volume. The microcephalic brain has a volume comparable to that of early hominids, raising the possibility that some MCPH genes may have been evolutionary targets in the expansion of the cerebral cortex in mammals and especially primates. Mutations in  ASPM gene is associated with abnormally small brain size. Comparison of the ASPM gene from several primate species suggests it as a target of evolutionary selection for increased brain sizeMutation of the  ASPM gene  is an autosomal recessive neurodevelopmental disorder in which the brain fails to achieve normal growth. The affected individuals have severe reduction in brain size; however, the gyral pattern is relatively well preserved, with no major abnormality in cortical architecture . MoreoveSPM gene .ASPM gene encodes a 10,434-bp-long coding sequence (CDS) with 28 exons, and spans 65 kb of genomic DNA at 1q31. ASPM contains four distinguishable regions: a putative N-terminal microtubule-binding domain, a calponin-homology domain, an IQ repeat domain containing multiple IQ repeats , and a C-terminal region , is localized in the mitotic centrosome and is known to be essential for both the organization of the microtubules at the spindle poles and the formation of the central mitotic spindle during mitosis and meiosis. Mutations in asp cause dividing neuroblasts to arrest in metaphase, resulting in reduced central nervous system development ((Mus musculus) brain, the Aspm gene is expressed specifically in the sites of active neurogenesis. Expression in the embryonic brain was found to be greatest in the ventricular zone, which is the site of cerebral cortical neurogenesis , gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and rhesus macaque (Macaca mulatta) were isolated by transformation-associated recombination (TAR) cloning in yeast (Saccharomyces cerevisiae), the technique allowing direct isolation of a desirable chromosomal region or gene from a complex genome without constructing its genomic library  microsatellite expansions, three Alu insertions, including retroposition of AluYi9 in the orangutan–gorilla–chimpanzee–human clade, and retroposition of a new macaque-specific AluY subfamily similar to human AluYd2. Analysis of eight different macaque individuals showed that this particular insertion is polymorphic in the macaque population (data not shown), and thus the insertion appears to be very recent. One macaque-specific 245-bp-long insertion is linked to expansion of a 49-bp-long, minisatellitelike array. The remaining macaque-specific insertion (50 bp) is nonrepetitive. A closer analysis suggests that the insert is not a processed pseudogene of known genes (data not shown).We have compared complete gene sequences from primate species with a 65-kb, full-size human rst exon . ASPM seervation H, and paervation . Multiplervation B. Seven Alu–Alu recombination . Except for orangutan and rhesus macaque, two or more ASPM CDSs were used for analysis. ASPM proteins showed the same overall length and domain structure  are more variable. Indeed, nonsynonymous substitutions in hominoid primates . Also the model that the complete gorilla–chimpanzee–human clade is evolving at one ω rate different from that in the rest of the tree is well supported (p < 0.01). Because ASPM consists of regions with different degrees of sequence conservation  revealed an elevated value in the human branch A. Accordtion see , we sepation see B the contion see C. The lition see D. As seetion see D. It is tion see . TherefoASPM polymorphism in those species . Chimpanzee sequences differed in five synonymous and 11 nonsynonymous sites. In order to interpret this seemingly high level of observed polymorphism, intraspecific diversity was compared to interspecific diversity using the McDonald and Kreitman test (p < 0.001) to conclude that the pattern of gorilla polymorphism is therefore different from the divergence pattern. Indeed gorilla polymorphism is less than variation resulting from divergence: within species, the ω ratio is 1.43 for gorillas compared to 2.2 for the divergence between the gorilla common ancestor and the human–chimpanzee common ancestor. Intraspecific variation, although seemingly unusual in showing so many replacement substitutions in both chimpanzee and gorilla, is less than or in line with what we have observed for ASPM divergence between species. Therefore, relaxation of selection cannot explain the high nonsynonymous/synonymous substitution ratios among African hominoids, further supporting the idea that adaptation has occurred in ASPM.Sequencing of two CDSs in African green monkey, three in gorilla, and three in chimpanzee allowed us to look for cies see E. Human man test . In the ASPM gene, which is involved in determining the size of the human brain and in which mutations lead to MCPH. The ASPM homologue in the fruit fly is essential for spindle function, suggesting a role for this gene in normal mitotic divisions of embryonic neuroblasts. Complete gene homologues from five primate species were isolated and sequenced. In agreement with the predicted critical role of ASPM in brain development, both coding and noncoding regions of ASPM homologues showed a marked degree of conservation in humans, other hominoids, and Old World monkeys. The differences found in noncoding regions were small insertions/deletions and lineage-specific insertions of evolutionarily young Alu elements into introns.In this study, we applied TAR cloning technology to investigate molecular evolution of the ASPM begins well before brain expansion suggests that the molecular evolution of ASPM in hominoids may indeed be an example of a molecular “exaptation”  selection in human and African ape lineages. Such “mosaic” selection has been previously described for other proteins . When ouptation” , in thatASPM, rapidly evolving residues are mainly concentrated in the IQ repeat domain containing multiple IQ motifs, which are calmodulin-binding consensus sequences. While there is no direct evidence yet, it is likely that the function of human ASPM is modulated through calmodulin or calmodulinlike protein(s). Previous interspecies comparisons of ASPM proteins have shown a consistent correlation of greater protein size with larger brain size mainly because of the number of IQ repeats  and possibly even earlier in primate evolution. IQ motifs are seen in a wide variety of proteins, but the ASPM proteins in primates are unique, because they have the largest known number of IQ repeats. Given the proposed role of ASPM in regulating divisions of neuronal progenitors, both the number of repeats and the particular amino acid substitutions in the IQ repeats may be strongly related to brain evolution.In the case of  repeats . For exan humans . ASPM hoASPM gene mutations which lead to MCPH provide a direct link between genotype and phenotype. ASPM is yet another example on the growing list of positively selected genes that show both accelerated evolution along the human lineage and involvement in simple Mendelian disorders  is barely different from that in gorillas  and common chimpanzees   , yet accears ago . TherefoHow might evolutionary changes in the ASPM protein affect cerebral cortical size? One potential mechanism might be that changes in ASPM induce changes in the orientation of the mitotic spindle of neuroblasts. Normally, neural precursor cells can have mitotic spindles oriented parallel to the ventricle or perpendicular to the ventricle. Mitoses in which daughter cells are oriented next to one another at the ventricular zone are typically “symmetric” in that a single progenitor cell generates two progenitor cells, causing exponential expansion of the progenitor pool. In contrast, mitoses that generate daughter cells that are vertically arranged are typically “asymmetric” so that one daughter cell becomes a postmitotic neuron, whereas the other daughter cell remains as a progenitor, causing only a linear increase in cell number. Control of this proliferative symmetry can cause dramatic alterations in cerebral cortical size , and so ASPM gene evolution in hominoids. Here, we have demonstrated the utility of TAR cloning for evolutionary sequence comparisons among humans and other primates. In addition, the ASPM TAR clones isolated in these studies could provide valuable reagents for studying ASPM gene regulation in its natural sequence context. Overall, we anticipate this technology will be extremely useful in studying the evolution of other genes that may be responsible for uniquely human traits.Functional genomics studies are clearly needed to elucidate the exact nature of the molecular mechanisms affected by Human Molecular Genetics shortly after this paper was submitted.The related paper by ASPM gene from the human (Homo sapiens), chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and rhesus macaque (Macaca mulatta) genomes, a TAR vector containing two unique hooks was constructed. Two targeting sequences were designed, 131 bp 5′ and 151 bp 3′, from the available human genomic sequence of ASPM . The targeting sequences were PCR amplified from the genomic DNA using two specific primers  in agarose plugs. Spheroplast transformation experiments were carried out as previously described in ASPM, yeast transformants were examined by PCR using diagnostic primers specific for exon 2 and exon 27 of ASPM  by homologous recombination in yeast using a BAC/NeoR retrofitting vector, BRV1, and then transformed into a recA DH10B E. coli strain  and exon 18 of the red-chested mustached tamarin (Saguinus labiatus) and black-handed spider monkey (Ateles geoffroyi) (approximately 4.7 kb) were PCR amplified using a pair of specific primers  using TRIzol reagent . Reverse transcription and 5′- and 3′-RACE reactions were performed using SMART RACE cDNA Amplification Kit . RNAs were extracted from primate cell lines . Primer pairs for cDNA sequencing were designed based on the human ASPM mRNA sequence. Primer sequences are available upon request. All sequences were named and numbered according to the clone/accession identifier.Chimpanzee, gorilla, orangutan, and rhesus macaque TAR clones containing full-size BAC DNAs . Forwardhttp://baboon.math.berkeley.edu/mavid/)  (http://pbil.univ-lyon1.fr/software/seaview.html) (http://www.hgmp.mrc.ac.uk/Software/EMBOSS/) for sequence analysis. Short nucleotide patterns associated with genome rearrangements were searched using FUZZNUC (EMBOSS). We searched for the following recombinogenic motifs: chi-like octamer (GCWGGWGG), immunoglobulin heptamer (GATAGTG), translin GCCCWSW and GCNCWSCTNGCCCWSSW), topoisomerase II (RNYNNCNNGYNGKNYNY), topoisomerase IId (GTNWAYATTNATNNR), topoisomerase IIi (YYCNTASYGGKYYTNNC), and V(D)J recombinase (CACAGTGN(12/23)ACAAAAACC). For short or highly ambiguous patterns (topo-isomerase II), no mismatches were allowed; for longer motifs J recombinase) up to two mismatches were permitted. Prediction of CpG islands was performed by CPGPLOT (EMBOSS) with default parameters . CENSOR (http://www.girinst.org/Censor_Server-Data_Entry_Forms.html)  were used for identification of repetitive elements. Minisatellites were detected by TANDEM REPEAT FINDER   ASPM sequence. Primate CDSs were deduced from the ASPM gene alignment with human sequences. Synonymous and nonsynonymous substitutions were detected by SNAP (http://www.hiv.lanl.gov/content/hiv-db/SNAP/WEBSNAP/SNAP.html). Codon maximum likelihood (ML) in CODEML in PAML v. 3.13 (http://abacus.gene.ucl.ac.uk/software/paml.html) (http://pbil.univ-lyon1.fr/software/phylowin.html) (http://paup.csit.fsu.edu/index.html) and corrected for multiple substitutions using the Tamura-Nei algorithm.Genomic sequences were aligned using MAVID (/mavid/) ; proteinialign/) . Alignmeew.html) . We havems.html)  and REPET FINDER . ASPM se/hgBlat) . First, ml.html)  has beenml.html) . For larin.html) . SegmentCommentaryASPM is an AT-rich gene (GC content 36.4%–37%) and, as expected . On the other hand, the 3′ breakpoint sequences are diverged from the chi consensus (second box). Both Alu elements in the alignment are shown from the first position and end at the same position, and thus positions in one element correspond to positions in the other Alu copy. As can be seen, the breakpoint position in the first AluSp repeat exactly corresponds to the breakpoint position within the second AluSz element, suggesting homologous recombination between the two repeats.Both orangutan breakpoints are located within 5′ portions of two (163 KB PDF).Click here for additional data file.Figure S2ASPM segment (percent same), and a schematic alignment of segmental duplications. The ASPM segment (black) shares similarity with 24 segmental duplications that contain additional sequences and are present on several human chromosomes. The ASPM copy and three duplications on Chromosome 7 share the same L1P4 terminal insertion, which is absent from all other duplications. The tree on the left shows evolutionary relationships among the duplications estimated by the neighbor-joining method.From left to right: phylogeny, chromosomal position, band name, identity to (169 KB PDF).Click here for additional data file.Figure S3The amino acid identity in the conserved regions is 85.44%, 49.39%, and 68.74% for exon 3, exon 4, and the IQ domain, respectively. In addition, while the alignment of conserved regions is completely gap-free, the variable domains exhibit several gaps including a large deletion in the mouse IQ domain (human positions 2655–2943).(97 KB PDF).Click here for additional data file.Table S1ASPM and lower case letters indicate cloning sites.Upper case letters indicate sequences homologous to (118 KB PDF).Click here for additional data file.Table S2p-value is in parentheses. The section over the diagonal is calculated using the Pearson (linear) correlation coefficient; under the diagonal are correlations obtained using the Spearman's rank coefficient—nonparametric). Nontrivial or interesting significant correlations are shown in bold and italics. The CAI represents the mean for all species . The ω ratio, Ka, and Ks  correspond to all branches of the phylogenetic tree. They were obtained using a ML model with one fixed ω ratio for all branches.All correlations were obtained for the same 100-amino-acid-/300-nucleotide-long, nonoverlapping windows. The first value shows the correlation coefficient; Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/) accession number for the human ASPM mRNA sequence used in this study is NM_018136. The sequence data from chimpanzee, gorilla, orangutan, and rhesus macaque full-length ASPM have been submitted to GenBank under accession numbers AY497016, AY497015, AY497014, and AY497013. The sequence data from chimpanzee, gorilla, and African green monkey ASPM cDNA have been submitted to GenBank under accession numbers AY508452, AY508451, and AY486114. The sequence data from spider monkey and tamarin exon 18 have been submitted to GenBank under accession numbers AY497017 and AY497018.The GenBank (


Results obtained from brain imaging studies indicate that serotonin transporter (SERT) and dopamine transporter (DAT) densities are altered in major depression. However, no such studies have been published on current mania or hypomania.123I]nor-β-CIT we present a case with simultaneous symptoms of major depression and hypomania. She had an elevated serotonin transporter availability (SERT) in the midbrain and elevated dopamine transporter availability (DAT) in the striatum, which normalised in a one-year follow-up period during which she received eight months of psychodynamic psychotherapy.In this single photon emission computed tomography (SPECT) study with [To our knowledge, this is the first report on SERT and DAT associated with mania. In our case the availability of both SERT in the midbrain and DAT in the striatum were elevated at baseline and declined during psychotherapy, while the SERT and DAT of the depressed controls increased during psychotherapy. Symptoms of hypomania in the case were alleviated during psychotherapy. Clinical recovery was also reflected in the Hamilton Depression Rating Scale (HDRS) scores. Decreased serotonin transporter densities (SERT) have been recorded from the brains of depressed patients ,2, whichIn this paper we present a case with simultaneous symptoms of major depression and hypomania. The structural diagnostic classifications DSM-IV and ICD-10 are unable to diagnose this kind of mixed state, even though similar cases have been presented earlier in the literature .The index subject was a 25-year-old female with a history of two periods of major depression according to the DSM-IV-R criteria. She had also had hypomanic periods that did not completely fulfil the criteria of mania, and at baseline she fulfilled the criteria of hypomania, moderate depression and dysthymia . She was overactive, restless and irritated and coped with a reduced amount of sleep. She had difficulty in concentrating on one thing at a time, and she appeared very lively and talkative. She was therefore diagnosed with bipolar mood disorder type II. In other words, our index patient had a mixed mania without fulfilling the criteria of full-blown mania during the baseline scan.The 6 female depressed controls were diagnosed to have severe or moderate depression . Three of them also fulfilled the criteria for dysthymia (300.40) and three had previously suffered periods of major depression. They were participants of our larger study on the effects of psychotherapy on SERT and DAT densities to be reported in full elsewhere.The group of 10 healthy controls consisted mainly of employees of Kuopio University Hospital and medical students. Depression was an exclusion criterion for the control group. Controls had previously received no psychotropic medication or other psychiatric treatment and were physically healthy.Psychiatric diagnosis was based on clinical assessment and verified for all study subjects by a trained independent psychiatrist using the Structured Clinical Interview for DSM-IV-R (SCID-I) . The sevThe depressed controls (see Table for sociodemographic characteristics) received psychotherapy for 12 months in an outpatient clinic of the Department of Psychiatry of Kuopio University Hospital. The case discontinued the therapy after eight months because she felt it was no longer useful to her. The case and the depressed controls had received no psychiatric treatment or medication prior the SPECT imaging and they were treated without medication. Psychotherapy sessions occurred twice a week, 80 sessions per year. The psychotherapists had formal postgraduate professional training in psychodynamic psychotherapy. The study design was approved by the ethics committee of Kuopio University Hospital.Participants of the study were right-handed and all were female. The depressed and the healthy controls were age-matched with the case (Table). The healthy controls were not followed up.123I] nor-β-CIT  was diluted in a volume of 10 ml physiological saline. The specific activity was higher than 1.8 × 1011 Bq/μmol [SPECT imaging was performed on the Wednesday after the menstrual bleeding that preceded the psychotherapy and on 12-month follow-up. A dose of 185 MBq of [The SPECT scans were decay-corrected and reconstructed with Butterworth-filtered back-projection in a 128 × 128 matrix with a pixel size of 3 × 3 mm, and were attenuation-corrected with Chang's algorithm ,11. The D - 1. The striatal uptake was pooled.Region of interest placement was based on a Siemens semi-automatic brain quantification program. The lower threshold of 60% of the maximum count was used to reduce the volume averaging and partial volume errors. Regions of interest were the cerebellum, striatum and the midbrain. It was assumed that the cerebellum (reference region) corresponds to a two-compartment model  . The speFunctional neuroanatomy by means of SPECT was confirmed using magnetic resonance imaging (MRI) within two weeks of the SPECT imaging. If any cerebral focal abnormalities or organic brain diseases were detected by MRI scan, the patient was excluded from our study. The participants of our study thus had normal findings.The reproducibility of the SPECT scan had been previously studied with eleven healthy subjects . SPECT studies were performed twice with a 12-month interval. The correspondence between the studies was good, with the mean difference being 0.00 ± 0.08 (SD = standard deviation) for SERT  and 0.04 ± 0.18 for DAT . The intraclass correlation coefficient was 0.82 (p < 0.01) and 0.79 (p < 0.01), respectively (unpublished data).The Student's t-test was used to compare the depressed controls and the healthy controls and paired samples t-test to compare SERT densities of the depressed controls at baseline and on follow-up. A p-value of less than 0.05 was considered as the criterion for statistical significance.The background characteristics of the case and the controls are presented in the table. The case and the depressed controls had HDRS scores of 14 or more.The midbrain SERT availability did not correlate with HDRS scores in depressive controls either at baseline or on follow-up. Neither did the change in the HDRS score correlate with changes in SERT or DAT capacities under therapy.The case had an elevated SERT availability in the midbrain at baseline, while the depressed controls had decreased levels compared to the healthy controls . The noradrenergic cell-body rich nucleus ceruleus is close to our target region. This is assumed to contain the nucleus raphe, which is mostly comprised of serotonergic cell-bodies. The dopamine cell-body rich substantia nigra is also located close to our region of interest. However, nor-β-cit is considered to be more serotonin-specific than previous radioligands .Three studies have been published on the association between SERT availability and the serotonin transporter genotype. Two of these were performed on healthy subjects ,23, whilWe have found no previous imaging studies showing that serotonin plays a role in mania. Further research is needed to determine whether our findings could be generalised to all manic states.None declared.TT planned the study and analysed and interpreted the data. MJ also interpreted the data. PIS initiated and planned the study and organised the psychotherapy setting. HM took care of the imaging procedures. PA organised the psychotherapy setting. RV was responsible for the MRI scans and the interpretation of the scans. JK performed SPECT analyses. JT participated in the design of the study. JL initiated and planned the study project. All authors read, critically revised and approved the final manuscript.DAT: Dopamine transporterHDRS: Hamilton Depression Rating ScaleSD: Standard deviationSERT: Serotonin transporterSPECT: Single photon emission computed tomographyThe pre-publication history for this paper can be accessed here:


The role of inflammation in Alzheimer's disease (AD) has been controversial since its first consideration. As with most instances of neuroinflammation, the possibility must be considered that activation of glia and cytokine networks in AD arises merely as a reaction to neurodegeneration. Active, healthy neurons produce signals that suppress inflammatory events, and dying neurons activate phagocytic responses in microglia at the very least. But simultaneous with the arrival of a more complex view of microglia, evidence that inflammation plays a causal or exacerbating role in AD etiology has been boosted by genetic, physiological, and epidemiological studies. In the end, it may be that the semantics of "inflammation" and glial "activation" must be regarded as too simplistic for the advancement of our understanding in this regard. It is clear that elaboration of the entire repertoire of activated microglia – a phenomenon that may be termed "malactivation" – must be prevented for healthy brain structure and function. Nevertheless, recent studies have suggested that phagocytosis of Aβ by microglia plays an important role in clearance of amyloid plaques, a process boosted by immunization paradigms. To the extent that this clearance might produce clinical improvements (still an open question), this relationship thus obligates a more nuanced consideration of the factors that indicate and control the various activities of microglia and other components of neuroinflammation. Alzheimer's disease (AD) is a progressive degeneration of neural structure and function that arises in the cerebral cortex. Behaviorally, affected individuals usually present with semantic difficulty, followed by a deficiency in episodic memory, spatial disorientation, sleep disturbances, depression, agitation, loss of longer memories, general difficulty with the activities of daily living, and eventually, death. Neuropathological findings include a relatively high number of extracellular deposits of the amyloid β-peptide (Aβ), argyrophillic cytoskeletal aggregates in neurons, accumulation of α-synuclein, loss of synapses, loss of cholinergic and adrenergic fibers, loss of pyramidal neurons, and cerebral amyloid angiopathy (CAA) – deposition of Aβ around blood vessels.Most of the AD correlates above have been connected in some way to inflammation. For instance, the plaques – comprised primarily of aggregated amyloid β-peptide (Aβ) – are inundated with microglia that show profiles of morphology and gene expression consistent with inflammation. Indeed, if one characterizes any activity by microglia as a sign of "neuroinflammation," it can be said that inflammatory responses have been evident in AD for at least 40 years . But, itDespite these indications, there are reasons to believe that the changes observed in glia and inflammatory cytokines constitute a compensatory response in AD. Indeed, some investigators have been reluctant to apply the term "inflammation" to the constellation of events related to AD pathology. Some of the cytokines and other gene products expressed in peripheral sites of inflammation are present in the AD brain, but there is no apparent vasodilation or extravasation of neutrophils. In general, there seems to be less of the molecular and cellular behavior that is responsible for bystander tissue damage in peripheral inflammation. This journal was founded partially out of recognition that "neuroinflammation" is distinct. In essence, the concept reflects a compromise befitting the difficult line that must be maintained between effective cell-mediated immune responses and damage to the precious components of the CNS. Microglia elevate their expression of neurotrophic factors under many of the same conditions in which they show inflammation-related responses such as phagocytosis, retraction of processes, release of excitotoxins, and production of IL-1β and IL-6 and tumor necrosis factor ; in factRecent experiments with anti-Aβ immunization have highlighted another beneficial effect of "activated" microglia: removal of Aβ. It has long been recognized that microglia can efficiently phagocytose and at least partially degrade Aβ both in vitro and in vivo. But the persistence of amyloid plaques suggests that microglia are stymied in this regard during the development of AD or in the deposition of Aβ in mice transgenically engineered to produce large amounts of the peptide. Introduction of antibodies recognizing Aβ, either by active vaccination or by passive immunization , results in removal of some Aβ deposits and/or prevention of their formation. Although the phenomenon has been studied most rigorously in the transgenic mouse models, similar clearance of parenchymal plaques seems to have occurred in two human subjects that participated in an Aβ-vaccine trial ,15. And et al. [+ cells presumed to be microglia. The experimental paradigm was one of passive immunization of transgenic mice at nearly two years of age, old enough to have accumulated substantial Aβ deposits. Consistent with expectations, injection of anti-Aβ antibody diminished deposits in the parenchyma, even those that were mature enough to stain with Congo red. However, vascular deposition of Congo-red staining was elevated by approximately four-fold in the anti-Aβ-treated animals. Pfeiffer et al. found similar results in another transgenic line [et al. now show that the regional accumulation of vascular amyloid was accompanied by an elevated index of hemorrhages and a congregation of CD45+ cells, presumed to be microglia [+ cells observed by Wilcock and coworkers are microglia or another cell type. It is also unclear whether the accumulation of amyloid and inflammatory cells at the blood vessels represents an arrested state in Aβ clearance or simply a bottleneck in the transport, one that would eventually yield to complete removal of the peptide. However, the appearance of CAA in the human subjects that suffered from acute encephalitis suggests that the vascular accumulation is an untoward event, created or facilitated by inflammation. Another vascular irregularity caused by Aβ has been linked to inflammatory events in both transgenic mice and isolated human blood vessels [Wilcock et al.  have nowet al.  – and a nic line . Furthericroglia . Given ticroglia , one inticroglia . In thos vessels .malactivation" will be applied here to microglial activation which produces neurodegeneration. One obvious question is whether there might be a mode of "activation" that permits phagocytosis while limiting malactivation. In fact, stimulation of Fc receptors – the antibody receptors that initiate a good deal of antibody-triggered phagocytosis – can inhibit cytotoxicity in macrophages [The apparent contributions of inflammatory mechanisms to both Aβ clearance and vascular pathology illustrate a somewhat unique example of microglial ambivalence. While many had argued that microglial "activation" by Aβ was at least partially responsible for AD-associated degeneration, others had pointed to microglial phagocytosis as a desirable consequence of activation. For the purposes of discussion, the term "rophages . Similarrophages . Evidencrophages . Althougrophages . Interesrophages .While some have argued that CAA is of little consequence in AD , the elaAD: Alzheimer's diseaseAβ: amyloid β-peptideCAA: cerebral amyloid angiopathyIL-1, -6, -8: interleukin-1, -6, -8NSAID: nonsteroidal antiinflammatory drugTGFβ: transforming growth factor βThe author(s) declare that they have no competing interests.


Our recent study has shown that HIV-1 nef-derived miRNAs are produced in HIV-1 persistently infected cells. Furthermore, nef short hairpin RNA (shRNA) that corresponded to a predicted nef miRNA  can block HIV-1 Nef expression in vitro and the suppression by shRNA/miR-N367 would be related with low viremia in an LTNP (15-2-2). In the 15-2-2 model mice, the weight loss, which may be rendered by nef was also inhibited by shRNA/miR-N367 corresponding to suppression of nef expression in vivo.Here, we show the possibility that nef/U3 miRNAs produced in HIV-1-infected cells may suppress both Nef function and HIV-1 virulence through the RNAi pathway.These data suggest that  The human immunodeficiency virus (HIV), which infect humans cause acquired immunodeficiency syndrome (AIDS), which has reached pandemic levels in some societies, especially those in Southern Africa and Southeast Asia . Given tC. elegans germline [Drosophila germline, and the response against invading viruses in plants [et al., 2002), fragile X mental retardation [RNAi is a defense mechanism against aberrant transcripts that may be produced during viral infection and mobilization of transposons ,4. The Rgermline ,6, silenn plants . Post-trn plants . In contn plants . The matn plants ,11. At tardation ,14 and cardation  have beenef, is located at the 3' end of the viral genome and partially overlaps the 3'-long terminal repeat (LTR). The nef gene is uniquely conserved in HIV-1, HIV-2 and simian immunodeficiency virus (SIV) and is not essential but important for viral replication in vivo [nef gene is expressed during HIV infection and often accounts for up to 80% of HIV-1 specific RNA transcripts during the early stages of viral replication [nef dsRNA containing the 3'-LTR regions, obtained from long-term non-progressor (LTNP) AIDS patients, actually inhibited the transcription of HIV-1 [cis-expression of mutated F12-HIV-1 nef inhibits replication of highly productive NL43-HIV-1 strain, which is not related to down-regulation of CD4 [nef gene cloned from the provirus of naturally occurring HUT-78 T cells infected with the supernatant of the peripheral blood mononuclear cells (PBMCs) of an HIV-seropositive non-producer patient, induces a block of viral replication [nef RNAs may be a cis-regulatory factor for HIV-1 replication [One of the human immunodeficiency virus type 1 (HIV-1) coding accessory genes,  in vivo . The nefof HIV-1 . Furthern of CD4 ,19. It hlication .nef-derived miRNAs are produced in HIV-1-infected cells. The results presented here show that nef short hairpin RNAs (shRNAs) corresponding to the nef miRNAs efficiently block RNA stability or mRNA translation, perhaps an indication that HIV-1 regulates its own replication by using nef miRNAs.In the current study, we have established the link between miRNAs and HIV infections by demonstrating that nef miRNAs and HIV-1-infected cells. To achieve this purpose, we extracted total RNA from HIV-1 IIIB strain persistently infected MT-4 T cells and northern blot analysis was performed using eight probes against the nef coding region, as shown in Figure nef region. However HIV-1 major transcripts were not detected (data not shown), indicating that the structure of the small RNA molecules could be double-stranded RNAs (miRNAs). Some variability was observed when the quantity of the miRNAs was compared with the total of the major transcripts. A maximum of 3.2% of miRNAs was detected by using #367 probe when compared with total HIV-1 transcripts, and the minimum of 0.3% was detected by using probe# 90 -encoded miRNAs were identified. Thus, during the preliminary stages of this study, our curiosity was fixed on the need to find out if indeed there was any relationship between  90 Fig. .nef miRNA, ~25-nt RNAs were gel purified, cloned and sequenced. The sequences from the nef miRNA clones were 5'-acugaccuuuggauggugcuucaa-3' or similar ones, corresponded to the nucleotides approximately 420 to 443 conserved region of nef (miR-N367). The most notable feature of this analysis is that it has proven beyond reasonable doubts that nef-derived miRNAs are produced in HIV-1 infected cells.To randomly clone the nef expression by the nef miRNA, we constructed eight shRNAs homologous to the native miRNA or probes used in Figure nef gene expression or not  as a positive or negative control. All the shRNA-expressing plasmids including the controls were co-transfected into Jurkat T cells with pYM2.2 and cell fluorescence resulting from the expression of EGFP reporter gene was quantified by flow cytometry. The sinef176, 190, 367/miR-N367 and control siegfp all showed efficient reduction, but the sinef 007, 084, 299, 468 and 580 constructs gave only modest reductions, and no suppression was observed with si(-) and luc -based live vector. The PFV vector expressed HIV-1 SF2 nef gene as a reporter and the STYLE vector expressed shRNA as effectors. The full-length nef gene was inserted into the bel-2 portion of a PFV clone (22) in frame to obtain pPFV/nef , was collected at 72 h after transfection. For preparation of the STYLE vectors to deliver the shRNAs, the env gene portion of pSKY3.0 was replaced with the shRNA expression cassette under the control of the H1 promoter. The pSTYLE was produced -PCR. The integration of the DNAs into the genome of Jurkat T cells was also confirmed by PCR of the LTR, gag and/or nef regions . The most efficient sinef176, 190 and 367/miR-N367 vectors for reduction in nef expression  suppressed Luc activity when compared to controls .Since different host gene products are required for siRNA-mediated RNAi and miRNA-mediated translational repression with  in vivo . To testBecause extracellular Nef is internalized into human and mouse lymphocytes and macrophages -27, we ein vitro [PPARγ mRNA and body weights of mice. Significant PPARγ mRNA expression in intestinal adipose tissue of group 3, but not group 1 and 2, was detected on day 2 , our results show that nef-derived miRNAs are produced in HIV-1 infected cells, and support the possibility that miRNA and siRNA may be functionally identical, at least in a retrotransposon such as HIV. Recent studies have revealed that miRNAs and siRNAs could block mRNA expression by similar mechanisms [nef miR-N367 could regulate nef expression even in vivo. In our unpublished data, HIV-1 LTR promoter activity was inhibited by miR-N367  expression, of which activity was dependent on negative responsive element (NRE) of U3 region (our unpublished data). Although no mismatch shRNA against region #367 was active, the miR-N367 from HIV-1 genome may have some mismatches and effectively inhibit HIV-1 transcription. Further the effects of siRNAs of Tc1, in particular those to the terminal inverted repeats derived from read-through transcription of entire transposable elements, were presented for silencing transposase gene expression by RNAi machinery in germ lines of C. elegans, [in vivo, however the inserted nef gene in the foamy retrotransposon may represent miRNAs which could inhibit nef mRNA expression by presumably an identical mechanism to that observed of siRNAs. Thus, RNAi might serve as a new sequence-specific therapeutic arsenal in AIDS prevention and possibly treatment.In this study, whereas siRNA has been reported to inhibit hepatitis B virus replication chanisms  and thatchanisms  and EBV-chanisms . Our resin vivo.Overall, our results indicate that nef shRNA transduced into T cell line inhibited HIV-1 transcription. Further, nef miRNAs could be produced from infected T cells and can block the trans-activity of Nef as well as HIV-1 replication on its own via the cis-action of nef. These functions of nef via RNAi pathways may allow persistently low pathogenic or latent infection as observed in HIV-infected non-progressors. Cumulatively, these data suggest that Nef may be involved in both viral replication and the disease progression, the findings, which may facilitate new strategies for HIV control nef sequence, because of the inclusion of viruses, which were also subtype B. The SF2 contained full-length nef reading frame as indicated in Table Patient selection is showed in Table 6 cells per ml for 3 days.HeLa and BHK cells were grown in Dulbecco's modified Eagle Medium (DMEM)  supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. CRFK cells were grown in Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO) with 10% FBS and antibiotics. Jurkat T cells and MT-4 T cells persistently infected with HIV-1 IIIB strain were cultured in RPMI-1640 medium (GIBCO) supplemented with 10% FBS and antibiotics. The packaging cells (CRFKsugi) were made by transfecting CRFK cells with 10 μg of the pFFenv with Lipofectin Reagent (Invitrogen) and selecting transformants after culture for 14 days with 25 μg/ml of hygromycine B (Invitrogen). After 14 days, FFV Env protein expression was measured by flow cytometry and immunoblot analyses with FFV-infected cat B serum . The pPFThe details of plasmid constructs and the primer sequences used in cloning strategies are shown in supplementary file see .Flow cytometry was performed with a FACS Calibur  as described previously .Firefly Luc assay was performed using the Luciferase Assay System (Promega) as described previously . ImmunobThe concentration of p24 supernatant was determined by an antigen capture assay  according to the manufacturer's instructions.Confocal laser microscopy analysis was performed as described previously .SalI and cloned into SalI site of pBluescript SK(-), followed by sequence analysis. The secondary structures of RNAs were predicted by GENETYX-MAC program .Total RNAs were extracted from HIV-1 IIIB or SF2 persistently infected or uninfected MT-4 T cells using TRIzol reagent (Invitrogen). Approximately 40 μg of total RNA was treated with RNase A and T1  as described previously , subjectgag, VIII (5'-tgtggtggaatgccactag-3') and IX (5'-attgtcatggaattttgta-3') for PFV LTR, XI (5'-tcttacagaccagtaacaa-3') and XII (5'-gtcaatcattacatctgca-3') for PFV gag, XIII (5'-aactactagtacccttcagg-3') and XIV (5'-aaaactcttgctttatggcc-3') for HIV-1 gag, XV (5'-atgggtggcaagtcaaaacg-3') and XVI (5'-tcagcagtctttgtagtactccg-3') for HIV-1 nef, XVII (5'-gttatgggtgaaactctgggagat-3') and XVIII (5'-atgttcctgaacataatcgtc-3') for PPARγ, XIX (5'-gacaacggctccggcatgtgcaaag-3') and XX (5'-ttcacggttggccttagggttcag-3') for β-actin, respectively. The nested PCR followed RT reaction was performed as described previously [nef or gag bands/integrating number of β-actin X 100.Semi-quantitative RT-PCR analysis was performed using the ThermoScript RT-PCR System  according to the manufacturer's protocol with the following primers: III (5'-atcatgggccaaagagaattc-3') and IV (5'-aaatttcactcaatcgagcc-3') for FFV LTR, VI (5'-aggacctgaaaggcatg-3') and VII (5'-ttgttgagatcgtcccg-3') for FFV eviously . PCR pro5 IFU of PFV/nef and STYLE-367 by intravenous (i.v.) injection. RT-PCR analyses were performed 2 days after infection. For histological analysis, cryostat sections were prepared from both human and mouse tissues. The fixed sections were rinsed with PBS and incubated with 5% BSA for at least 1 hr to inhibit nonspecific binding of antibodies. Sections were incubated overnight at 4°C with anti-Nef rabbit serum, F3 or 305 mAb, and incubated with peroxidase or FITC conjugated secondary antibodies. The washed sections were incubated in 0.03% 3,3'diaminobenzidine (Sigma) solution in 0.05 M Tris buffer with 0.01% H2O2 for development of peroxidase activity. After counterstaining with hematoxylin or methylgreen, the sections were dehydrated and mounted.Balb/c and C3H/Hej mice were raised under specific pathogen-free (SPF) conditions. Mice were infected with 1 ml of 10Data were analysed using a one-way ANOVA analysis with a post-hoc Fisher's test. P values of 0.05 or more were determined for that of cut off.HIV-1 human immunodeficiency virus type 1miRNA microRNAnt, nucleotidesLTNP long-term non-progressorsshRNA short hairpin RNAAIDS acquired immunodeficiency syndromeRNAi RNA interferencesiRNA small interfering RNALTR long terminal repeatSIV simian immunodeficiency virusPBMCs peripheral blood mononuclear cells;EBV Epstein-Barr virus;EGFP enhanced green fluorescence proteinPFV prototype foamy virusTA trichostatinA IFU infectious unitsFFV feline foamy virusRT reverse transcriptionm.o.i., multiplicity of infectionmAb monoclonal antibody;Ner Nef receptor molecule;NRE negative responsive element.The authors declare that they have no competing interests.in vivo studies and tissue analyses. Y.I. and H.O. participated in data validation and overall experimental design. E.A.B. and N.K.S. carried out the clinical, sequencing, and virological studies and the writing of the manuscript. Y.R.F. participated in the design of the study and coordinated it. All authors read and approved the final manuscript.S.O. carried out northern analyses, immunoblot analyses, RNAi assays and was involved in the construction of plasmids. M.I. and Y.T. participated in Click here for file