MEN1 locus on chromosome 11q13 and somatic mutation of the MEN1 gene have been frequently associated with the development of MEN 1-type sporadic endocrine lesions. To further investigate the role of the MEN1 gene in sporadic endocrine tumorigenesis, we analysed DNA from 14 primary parathyroid lesions, 8 anterior pituitary tumours and 3 pancreatic tumours for the presence of somatic MEN1 gene mutations and LOH of seven microsatellite markers flanking the MEN1 locus. In addition, we similarly analysed 8 secondary parathyroid lesions which arose in patients with chronic renal failure. None of the patients studied had a family history of MEN 1. Three primary parathyroid lesions and one pancreatic tumour (glucagonoma) were found to have lost one allele at the MEN1 locus. Somatic mutations were identified by SSCP and sequence analysis in one of these parathyroid lesions (P320L) and in the glucagonoma (E179V). These results support previous findings that inactivation of the MEN1 tumour suppressor gene contributes to the development of sporadic MEN 1-type endocrine lesions but is not associated with the development of parathyroid hyperplasia seen in some renal failure patients. © 2000 Cancer Research CampaignEndocrine tumours of the pancreas, anterior pituitary or parathyroids arise either sporadically in the general population, or as a part of inherited syndromes such as multiple endocrine neoplasia type 1 (MEN 1). The mechanisms responsible for the development of sporadic endocrine lesions are not well understood, although loss of heterozygosity (LOH) of the


The hypoxic toxicity and binding of misonidazole (MISO) requires metabolic reduction. The influence of glucose on the toxicity and binding of MISO was studied because glucose is a major substrate for the supply of NADPH through the hexose monophosphate pathway (HMP). Hypoxic EMT6/Ro cells (10(6) cells ml-1) were incubated with varying concentrations of glucose (0.015 mM to 5 mM). The initial rate of glucose transport was found to increase linearly with the extracellular glucose concentration up to 5 mM (0.038 nmol glucose 10(-6) cells sec-1). About 1.5 percent of the total glucose consumed went through the HMP for hypoxic cells in 5 mM glucose. The rate of HMP progressively decreased as the glucose concentration was lowered. When exposed to 5 mM MISO, the HMP was stimulated. This stimulation declined from 3.2 times in 5 mM glucose to barely detectable below 1 mM glucose. Both the hypoxic toxicity and binding of 5 mM MISO to the acid-insoluble fraction were decreased as the concentration of glucose was lowered. Below 0.5 mM glucose, no significant toxicity due to MISO was observed. There was an initial burden of 2.5 nmol MISO 10(-6) cells bound with little toxicity. After this initial burden, the terminal slope was 1.8 mol MISO bound 10(-6) cells (63 percent decrease in the surviving fraction). These results indicate that glucose concentrations lower than 5 mM can decrease the HMP rate and the toxicity and binding of MISO to hypoxic cells, and imply that calibration curves with normal and low glucose concentrations should be used to estimate the possible hypoxic fraction when MISO is used as a hypoxic probe in vivo.


HNF1A gene since rare, penetrant mutations in these exons (which are only transcribed in selected HNF1A isoforms) are associated with a later age of diagnosis of Maturity onset diabetes of the young (MODY) than mutations in exons 1–7. The age of diagnosis in the subgroup of HNF1A-MODY individuals with exon 8–10 mutations overlaps with that of early multifactorial T2D, and we set out to test the hypothesis that these exons might also harbour low-frequency coding variants of intermediate penetrance that contribute to risk of multifactorial T2D.There is considerable interest in the hypothesis that low frequency, intermediate penetrance variants contribute to the proportion of Type 2 Diabetes (T2D) susceptibility not attributable to the common variants uncovered through genome-wide association approaches. Genes previously implicated in monogenic and multifactorial forms of diabetes are obvious candidates in this respect. In this study, we focussed on exons 8–10 of the HNF1A exons 8–10 in 591 European T2D subjects enriched for genetic aetiology on the basis of an early age of diagnosis (≤45 years) and/or family history of T2D (≥1 affected sibling). PCR products were sequenced and compared to the published HNF1A sequence. We identified several variants (rs735396 [IVS9−24T>C], rs1169304 [IVS8+29T>C], c.1768+44C>T [IVS9+44C>T] and rs61953349  but no novel non-synonymous coding variants were detected.We performed targeted capillary resequencing of HNF1A are unlikely to contribute to T2D-susceptibility in European samples. Nevertheless, the rationale for seeking low-frequency causal variants in genes known to contain rare, penetrant mutations remains strong and should motivate efforts to screen other genes in a similar fashion.We conclude that low frequency, nonsynonymous coding variants in the terminal exons of  HNF1A encodes the transcription factor hepatocyte nuclear factor 1 alpha and is the gene most commonly implicated in the pathogenesis of symptomatic Maturity-onset diabetes of the young (MODY) HNF1A encodes 3 different isoforms, termed A (encoded by exons 1–10), B (encoded by exons 1–7) and C (encoded by exons 1–6) which arise by alternative splicing and polyadenylation Despite the great success of the genome wide association (GWA) approach in identifying common genetic variants which predispose to T2D, the familial aggregation seen in this condition is far from fully explained HNF1A for example, there is evidence that a private common variant  is a major contributor to T2D pathogenesis in the Oji-Cree population (in whom the MAF is 8.7%) HNF1A variants across the allele frequency spectrum play a role in T2D-susceptibility derives from evidence that common variants in this gene are associated with multifactorial T2D Genes already known to play a role in the pathogenesis of monogenic or multifactorial diabetes are logical candidates in which to initiate the search for low frequency causal variants in T2D. In the case of HNF1A were particularly auspicious candidates in terms of harbouring low frequency, medium-penetrance variants involved in multifactorial diabetes. We set out to test this hypothesis by performing deep resequencing of these exons in a large sample of cases likely to be enriched for such variants, namely those with a strong family history of T2D and/or an early age at diagnosis.In the light of this evidence, we reasoned that the terminal isoform-specific exons of All subjects gave written informed consent and all protocols were approved by the local ethics committees. The Warren 2 Sibpair collection was approved by multiple local ethics committees in the UK including St Mary's Local Ethics Committee (EC3231). The Young Diabetes in Oxford Cohort was approved by the Oxfordshire Research Ethics Committee A (04/Q1604/97).To maximize the likelihood of identifying medium penetrance genetic variants influencing T2D-risk, a total of 591 individuals of British ancestry, ascertained for early-adult onset , and/or family history of T2D  were included .Sample 1 (the “Young Diabetes in Oxford Cohort”) includes subjects diagnosed with T2D between 18–45 years of age, recruited from Oxfordshire GP practices. We excluded those with type 1 diabetes (T1D) by requiring that all subjects had no permanent requirement for insulin therapy within 12 months of diagnosis and no evidence of islet autoimmunity (glutamic acid decarboxylase (GAD) antibody levels <14 WHO units/ml) Sample 2 consists of probands from the Warren 2 sibpair collection (W2SP): these have been described previously To establish the background allele frequency of variants identified in this study in a UK control population, we utilised a subset of the British Birth Cohort of 1958  HNF1A (including intron-exon boundaries) was performed on the ABI3700 platform  using standard protocols (primer sequences are available on request). Sequences were compared to the reference sequence (NM_000545.3) using the unidirectional analysis mode of Mutation surveyor V3.2 . This software package has been shown to have a sensitivity of >99% in the unidirectional analysis modeTargeted capillary resequencing of exons 8–10 of The background allele frequency of variants identified in this study was established in a UK control population using custom TaqMan assays  in resequencing the terminal 3 exons of ividuals . Howeverividuals . The onlividuals .HNF1A were noted to have been diagnosed as late as 38 years, and the median age of diagnosis was ∼27 years In a recent study of MODY families showing classical Mendelian segregation, individuals with mutations in exons 8–10 of In our study population, 60 (10%) individuals were diagnosed at 38.5 years or younger and the median age of diagnosis was 55 . Almost all cases had close relatives with T2D, a feature which is likely to reflect enrichment for medium penetrance variants.HNF1A and can be confident that there are no such variants with case allele frequencies exceeding 1.0%. Of course, we cannot exclude the possibility that additional low-frequency susceptibility variants will be found in other regions of this gene, nor in other genes known to be causal for monogenic forms of diabetes. Recent advances in high throughput “next-generation” resequencing technologies Despite these measures, we failed to detect any novel coding variants in the terminal exons of Table S1Assay by design sequences for genotyping of variants detected in exons 8–10 of HNF1A(0.03 MB DOC)Click here for additional data file.


Most teleost species, especially freshwater groups such as the Esocidae which are the closest relatives of salmonids, have a karyotype comprising 25 pairs of acrocentric chromosomes and 48–52 chromosome arms. After the common ancestor of salmonids underwent a whole genome duplication, its karyotype would have 100 chromosome arms, and this is reflected in the modal range of 96–104 seen in extant salmonids . The Atlantic salmon is an exception among the salmonids as it has 72–74 chromosome arms and its karyotype includes 12 pairs of large acrocentric chromosomes, which appear to be the result of tandem fusions. The purpose of this study was to integrate the Atlantic salmon's linkage map and karyotype and to compare the chromosome map with that of rainbow trout.in situ hybridization with BAC probes containing genetic markers mapped to each linkage group. The genetic linkage groups were larger for metacentric chromosomes compared to acrocentric chromosomes of similar size. Comparison of the Atlantic salmon chromosome map with that of rainbow trout provides strong evidence for conservation of large syntenic blocks in these species, corresponding to entire chromosome arms in the rainbow trout.The Atlantic salmon genetic linkage groups were assigned to specific chromosomes in the European subspecies using fluorescence It had been suggested that some of the large acrocentric chromosomes in Atlantic salmon are the result of tandem fusions, and that the small blocks of repetitive DNA in the middle of the arms represent the sites of chromosome fusions. The finding that the chromosomal regions on either side of the blocks of repetitive DNA within the larger acrocentric chromosomes correspond to different rainbow trout chromosome arms provides support for this hypothesis. The common ancestor of salmonid fishes, which include Atlantic salmon and rainbow trout, underwent a whole genome duplication sometime within the early Tertiary to the late Cretaceous periods . The eviThe karyotype of Atlantic salmon is the exception among the salmonids as it has an NF of 72–74 and includes 12 large acrocentric chromosome pairs, which appear to be the result of tandem fusions . The sma). Cytogenetic mapping utilizing fluorescence in situ hybridization (FISH) with BAC probes containing microsatellite loci that are linked to the male phenotype in Atlantic salmon identified the sex chromosome pair as chromosome 2 [Genetic linkage maps have been constructed for European Atlantic salmon using microsatellites , AFLPs  and SNPsmosome 2 . This almosome 2 .Hybridization experiments were carried out with BAC clones containing genetic markers mapped to each linkage group. It should be noted that there are 29 linkage groups in the Atlantic salmon genetic map. The nomenclature for the linkage groups is historical, and linkage groups 26, 27, 29 and 30 no longer exist . With the exception of two very small chromosome pairs, multiple BAC clones were assigned to each chromosome pair. Dual hybridizations were done to confirm assignment of probes from several pairs of duplicate genes . Figure The BAC clones used to assign the genetic linkage groups to chromosomes are given in Additional File Table Table Oncorhynchus species. When the rainbow trout chromosomes are arranged according to size, the smallest metacentric chromosome pair (LG 5) falls between the largest and second largest acrocentrics, a result which supports the inversion hypothesis for this chromosome. In addition, rainbow trout LG 5 corresponds to a single chromosome arm in Atlantic salmon and in the ancestral vertebrates [Oncorhynchus species. When the location of genetic loci mapped in rainbow trout are indicated on the Atlantic salmon merged female genetic map of the Br5 and Br6 SALMAP families  currently have unknown homologies to complete arm segments [A total of 207,869 BAC-end sequences with an average length of 666 bp were screened for microsatellites using a Perl script created in the Davidson Lab, and 20,606 microsatellites were identified. As of December 2008, 697 of these microsatellite markers have been placed on the Atlantic salmon genetic map using the SALMAP mapping families Br5 and Br6 ,19 and t from the flanking regions surrounding microsatellite markers identified as anchor loci on the Atlantic salmon genetic map. The resulting sequences were subsequently screened for suspected and known repetitive elements in salmonids using a salmonid-specific repeat masker . An additional probe (5'-GTTGCCAAATTCCGAGATCTTGGCGACGAAGCCACATGAT-3') was used to hybridize to the C. briggsae control anchor spots on the CHORI-214 BAC filters. Oligonucleotide probes were radioactively labeled at the 5' end with 32Pγ-ATP and T4 polynucleotide kinase (Invitrogen) in a 10 μL volume reaction at 37°C for 30 minutes. The labeling reaction included 1 μL of 10 μM probe, 10 U of T4 polynucleotide kinase, 2 μL of 5× forward reaction buffer, 2 μL of 32Pγ-ATP (3000 Ci/mmol) and 4 μL of water. Each of the CHORI-214 high density BAC filters was pre-hybridized for 3 hours in 50 or 100 ml of pre-hybridization solution  at 65°C. To each pre-hybridized filter 1.6 μL of radioactively labeled oligonucleotide probe and 1.6 μL of radioactively labeled C. briggsae overgo reference probe were added and hybridization was carried out at 65°C overnight (~18 hours). Three 1 hour washes consisting of 1× SSC and 0.1% SDS were performed at 50°C to remove unhybridized probe. Filters then were wrapped in Saran™ wrap and exposed on storage phosphor screens for 18 hours. Hybridization signals were detected using a Typhoon 9410 Phosphor Imager (Amersham Biosciences).Oligonucleotide probes (35–40 mer), which also acted as forward primers for PCR, and 20 mer reverse complement PCR primers were designed using primer3 software . These BAC clones were picked from the CHORI-214 Atlantic salmon BAC library and grown in 5 ml of LB medium containing 20 μg/mL chloramphenicol for 14–16 hours at 37°C with shaking at 250–300 rpm. Glycerol stocks were made by placing 700 μL of each culture into 1.5 mL Eppendorf™ tubes containing 300 μL of 50% glycerol. BAC DNA templates for PCR were prepared from selected clones by diluting the glycerol stock 1/40 in dH2O. Colony PCR of the selected clones positive by hybridization for a particular microsatellite's flanking region probe was performed in a T3 or T3000 Thermalcycler (Biometra) using appropriate primers and annealing temperature in 25 μL using the following procedure: initial denaturation step of 95°C for 5 min; 35 cycles with a denaturation step of 95°C for 45 sec, 45 sec at specific annealing temperature, extension at 72°C for 45 sec followed by a final extension at 72°C for 10 minutes. The PCR contained 0.05 U Taq DNA Polymerase (Qiagen), 12.5 pmoles of each specific primer, 2.5 μL 10× PCR buffer containing 1.5 mM MgCl2, 50 μM dNTP and 5 μL of BAC DNA template. PCR products were separated on a 1.5% agarose gel containing 1× TBE and 0.5 μg/ml ethidium bromide. The DNA fragments were visualized using a UV trans-illuminator (Ultra-Violet Products). Only those BAC clones that yielded clean single amplification products of the expected size were used for FISH analysis.BAC clones, positive by hybridization for a specific microsatellite or identified as containing a BAC-end microsatellite, were identified in the Atlantic salmon physical map Chromosome preparations were obtained from blood of the European strain of Atlantic salmon (2N = 58) by methods described previously . BrieflyWe examined several genetic maps for rainbow trout ,22-24 foKPL and RGD constructed the linkage map and integrated the markers with the BAC-based physical map. KAK, MRM, ABV and RBP carried out the FISH and chromosomal analyses. RGD and RBP correlated the Atlantic salmon and rainbow trout linkage maps and homologous chromosome arms. WSD, RBP, RGD and BFP conceived of the study. WSD, RBP and RGD drafted and prepared the final manuscript. All authors read and approved the final manuscript.Table Assignment of Atlantic salmon genetic linkage groups to chromosomes. This table shows the relationships of the genetic markers from each of the linkage groups, the BACs that contain the specific markers and the chromosome arm that each of the BACs was assigned to using FISH analysis.Click here for fileFigure Atlantic salmon consensus female genetic map from Br5 and Br6 families. This figure shows the female genetic map that was constructed based on the SALMAP Atlantic salmon mapping families, Br5 and Br6.Click here for fileFigure Atlantic salmon male genetic map from the Br5 family. This figure shows the male genetic map that was constructed based on the SALMAP Atlantic salmon Br5 mapping family.Click here for fileFigure Atlantic salmon male genetic map from the Br6 family. This figure shows the male genetic map that was constructed based on the SALMAP Atlantic salmon Br6 mapping family.Click here for fileTable Location of shared markers on Atlantic salmon and rainbow trout chromosome arms. This table shows the locations of the genetic markers that are shared in the Atlantic salmon and rainbow trout linkage maps and indicates their chromosomal positions in these species.Click here for file


Western analysis using a monoclonal antibody to cyclin D1 identified a 36 kDa protein in homogenates from seven tumours displaying cytoplasmic only and one tumour demonstrating both nuclear and cytoplasmic immunostaining. Using restriction fragment length polymorphism polymerase chain reaction and PCR-multiplex analysis, amplification of the cyclin D1 gene (CCNDI) was detected in 1/29 of the tumours demonstrating overexpression of cyclin D1 protein. We conclude that deregulation of CCND1 expression leading to both cytoplasmic and nuclear protein localization is a frequent event in ovarian cancer and occurs mainly in the absence of gene amplification. © 1999 Cancer Research CampaignThe expression of cyclin D1 protein in tumour sections from 81 patients with epithelial ovarian cancer was analysed using immunohistochemistry. The tumours that overexpressed cyclin D1 in more than 10% of neoplastic cells were considered positive. Thus overexpression of cyclin D1 was observed in 72/81 (89%) of the cases examined. Protein was detected in both the nucleus and the cytoplasm in 24/81 (30%) and localized exclusively in the cytoplasm in 48/81 (59%) of the tumours. Cyclin D1 was overexpressed in both borderline and invasive tumours. There was no association between protein overexpression and tumour stage and differentiation. Furthermore, no correlation between cyclin D1 expression and clinical outcome was observed. However, in tumours overexpressing cyclin D1 (


We analyzed national Haemophilus influenzae (Hi) surveillance data from 1994 and 1995 to describe the epidemiology of Hi invasive disease among persons of all ages. Serotype data were available for 376 (56%) of 669 reported Hi cases among children aged 4 years or younger; 184 (49%) were H. influenzae type b (Hib). Among children aged 4 or younger, incidence  of all Hi invasive disease was 1.8 in 1994 and 1.6 (p < 0.05) in 1995. Children aged 5 months or younger had the highest average annual incidence rate of Hib invasive disease ; children aged 6 to 11 months had the next highest rate (p < 0.05). Of 181 children with Hib invasive disease whose age in months was known, 85 (47%) were too young (aged 5 months or younger) to have completed a primary series with an Hib-containing vaccine. Of the 83 children with known vaccination status who were eligible to receive a primary series (aged 6 months or older), 52 (63%) were undervaccinated, and the remaining 31 (37%) had completed a primary series in which vaccine failed. Among persons aged 5 years or older with Hi invasive disease, the lowest average annual incidence was among those 20 to 39 years of age , and the highest was among those aged 80 years or older . Among persons aged 5 years or older, serotype data were available for 1,372 (71%) of the 1,940 Hi invasive disease cases; 159 (28%) of the 568 Hi cases with known serotype were due to Hib.


The aim ofthis study is the analysis ofthe results in 62 patients over 70 years ofage with acute cholecystitis treated in our Department from 1970 to 1990. The clinical picture in 47 patients was mild and in 15 severe. In 14 cases  the acute cholecystitis subsided with antibiotics (Group A). In 48more cases  following 1-3 days conservative treatment, operation was undertaken.Besides acute cholystitis there was gangrene of gallbladder in 10, choledocholithiasis in 7 andcholoperitoneum without perforation in 7 cases. Cholecystostomy in 25, cholecystectomy in 15 andcholecystectomy with exploration ofthe bill duct in 8 cases was performed (Group B). There was one deathin group A and 3 deaths .in group B. The hospital stay was 20 days. In conclusion the clinical findings inacute cholecystitis in the aged are usually mild. In the case of failure of medical treatment, after 2-3 daysemergency surgery should be performed.


The human TTC4 protein is a TPR (tetratricopeptide repeat) motif-containing protein. The gene was originally identified as being localized in a genomic region linked to breast cancer and subsequent studies on melanoma cell lines revealed point mutations in the TTC4 protein that may be associated with the progression of malignant melanoma.Here we show that TTC4 is a nucleoplasmic protein which interacts with HSP90 and HSP70, and also with the replication protein CDC6. It has significant structural and functional similarities with a previously characterised Drosophila protein Dpit47. We show that TTC4 protein levels are raised in malignant melanoma cell lines compared to melanocytes. We also see increased TTC4 expression in a variety of tumour lines derived from other tissues. In addition we show that TTC4 proteins bearing some of the mutations previously identified from patient samples lose their interaction with the CDC6 protein.Based on these results and our previous work with the Drosophila Dpit47 protein we suggest that TTC4 is an HSP90 co-chaperone protein which forms a link between HSP90 chaperone activity and DNA replication. We further suggest that the loss of the interaction with CDC6 or with additional client proteins could provide one route through which TTC4 could influence malignant development of cells. HSP90 and HSP70 both play a part in the cellular response to stress. However recently it has become apparent that they also co-operate to chaperone a variety of proteins (clients) in multiple cellular pathways in cells which are not stressed. They often play a vital role in the cellular functioning of the client The HSP90 co-chaperones are a group of diverse proteins which help HSP90/70 in their action. This group includes other heat shock proteins, TPR containing proteins, cyclophilins and others. The TPR domain is a 34 amino acid protein motif thought to be involved in protein:protein interactions. TPR motif containing proteins are widely distributed across multiple classes of proteins involved in a variety of cellular functions We have previously reported the identification of a protein from Drosophila (Dpit47) which shows properties consistent with it being a TPR co-chaperone The TTC4 gene was originally identified through its localisation in a genomic region linked to breast cancer In this paper we show that TTC4 is the human orthologue of the Drosophila Dpit47 protein. The two proteins are highly related in sequence, share a common cellular location, and show analogous interactions with both heat shock and the replication initiation protein CDC6. We show that loss of interaction with CDC6 occurs when some of the point mutations identified in melanoma samples are introduced into the TTC4 gene. This suggests a possible route through which TTC4 could affect the development of malignant melanoma. We also present evidence which suggests that TTC4 has a general role in the progression of cancers in addition to melanoma, since the levels of the protein are raised in a variety of tumour cell lines.Identification of the TTC4 gene was carried out using the Blastp programme at the NCBI. The full length gene in pCR2.1 (Invitrogen) vector was a kind gift of J. Cowell (Roswell Park Cancer Institute). All subsequent cloning steps were carried out by amplifying the TTC4 insert using a high fidelity polymerase (Expand–Roche) to introduce appropriate restriction enzyme sites. The gene sequence and cloning sites were subsequently checked by DNA sequencing (Lark Technologies).http://www.es.embnet.org/Doc/phylodendron/clustal-form.html)The phylogenetic tree was assembled by entering the sequences of the relevant genes into the EMBNET clustal W /phylodendron programmes on line (Human full length cDNA clones for HSP90 protein 1 beta (HSPCB) and HSP70 protein 8 (HSPA8) were obtained form Origene Technologies, Inc. HuCDC6 was obtained from A. Dutta (Dept of Pathology Boston).TTC4 was cloned in frame in the EcoR1 and Xho sites of the pJG4.5 vector.Hu CDC6 was cloned in frame in the BamH1 and Not1 sites of the pEG202 vector.HuHSP90 was cloned in frame in the Not1 and Xho1 sites of the pEG202 vector.HuHSP70 was cloned in frame in the BamH1 and Xho1 sites of the pEG202 vector.The sequences of all clones were verified by sequencing (Lark Technologies).http://proteome.wayne.edu/THPL.html)Yeast manipulations were carried out using standard two hybrid protocols . Quantitative analyses were performed using liquid beta-galactosidase assays as described (Full length TTC4 was subcloned in the pRSETa vector (Invitrogen). The 6Xhis tagged protein was purified under denaturing conditions and the resultant proteins sent to NeoMPS (Strasbourg) for antibody manufacture. Two different rabbit antisera were obtained. The specificity of the antibody was checked by affinity purification of the antibody against 6XhisTTC4 blotted onto Nitrocellulose membrane and competition with GST TTC4. For this GST TTC4 was bound to glutathione sepharose. Undiluted anti TTC4 serum was incubated for 1 hour at room temperature and then briefly spun down to pellet the beads associated with the specific antibodies. The depleted serum was used at a 1/1000 dilution in western blot. For all experiments both antibodies gave the same result.Anti HSP90 (ab13495), anti HSP70 (ab6535) and anti HuCDC6 (ab3220) were from Abcam. Alexa fluor 488 anti mouse and Alexa fluor 594 anti rabbit and Toto3 iodide were from Molecular Probes. Anti alpha-tubulin was from Sigma (clone DM 1A).Extracts for immunoprecipitation were prepared from sub confluent Hela cells. After treatment with trypsin, cells were washed twice with PBS. They were lysed in one volume of PBS, 1% triton X100 and Complete EDTA free protease inhibitors (Roche). After incubation for 10 minutes on ice the lysate was cleared by centrifugation for 5 minutes in a microfuge at 10 000 g at 4°C.Coupling of antibodies to Protein A sepharose beads and immunoprecipitation was carried out as described in 10 µg of GST-TTC4 (wild type or mutant) bound to glutathione beads was incubated with 6 µg of HSP90 in 10 mM Tris pH 7.5, 150 mM NaCl, 0.1% triton X100 and protease inhibitors . After 1 hour at 4°C the pellets were washed in the same buffer, resuspended in SDS PAGE loading buffer and analysed on a 12% PAGE SDS.2. Normal human melanocytes , and grown in RPMI 1640 medium with antibiotics, glutamine, 10% foetal calf serum and 10% COCells were trypsinised and then deposited on poly lysine treated coverslips. Fixation was carried out using 4 % paraformaldehyde diluted in cytoskeleton buffer . The cells were then treated with permeabilisation buffer . The coverslips were incubated with primary antibodies , either for 2 h at room temperature or 4°C overnight. They were washed with permeabilisation buffer and then incubated with secondary antibodies as described above. The secondary antibodies used were Alexa fluor 488 anti mouse and Alexa fluor 594 anti rabbit. The DNA was counterstained with Toto3 iodide. Prior to microscopy the coverslips were mounted in mounting medium Vectashield (Vector).This was carried out basically as described Cell pellets were washed twice in PBS and then resuspended in SDS-PAGE loading buffer at a concentration of 50,000 cells/µl. The equivalent of 150,000 cells was loaded in each lane. Cell debris was removed by centrifugation before separation by SDS –PAGE.Point mutations in TTC4 (pJG4.5) were introduced using the Quickchange site directed mutagenesis kit (Stratagene) as described in the manufacturers instructions.The Drosophila Pit47 protein was used to query the NCBI databases in order to find related proteins from other species. The closest homologue from any species was identified as the human TTC4 protein (29% identity). The degree of homology between the two proteins is shown in Our previous studies had shown that the Drosophila Pit47 protein interacts with HSP90 and HSP70. These interactions are very strong and easily detected using two-hybrid analysis. We therefore used similar methodology to determine whether TTC4 showed analogous interactions. The TTC4, human HSP90 and human HSP70 genes were cloned into two hybrid bait and/or reporter plasmids as described in the To confirm these interactions we performed immunoprecipitation experiments using anti-TTC4 antibodies. Full length TTC4 protein was overexpressed and used to raise two rabbit antibodies see . Both anBoth HSP90 and HSP70 were detected in the anti-TTC4 pellet. Staining of an SDS-PAGE of the immune pellet with colloidal coomassie blue showed that HSP90 was present in near stoichiometric amounts and HSP70 was present at a level of approximately 20% (data not shown). This is similar to what we previously observed for the Drosophila TTC4 homologue A comparison of the TPR sequences of HSP90-binding TPR co-chaperones has suggested that 2 basic residues (Lys-152 and Arg-156 in TTC4) are highly conserved and are involved in the interaction with HSP90 If TTC4 is a true orthologue of Pit47 we would expect that it would show interactions with proteins involved in DNA replication. The Drosophila protein was isolated through a two-hybrid interaction with the large subunit of the DNA polymerase alpha, however when we tried the analogous experiment with TTC4 and the human DNA polymerase alpha we failed to detect an interaction  and 5. WThis interaction was also confirmed by co-immunoprecipitation  of CDC6 Dpit47 is a nucleoplasmic protein at all stages of the cell cycle. We therefore analysed the subcellular location of TTC4 to determine if it was comparable. The antibodies were used to check the subcellular localisation of the TTC4 protein using indirect immunofluoresence in the melanocyte lines Nohm 1 and Hermes 3a /b. In boTo determine whether TTC4 was tightly bound to chromatin, Hela cells were fractionated into cytoplasmic, nucleoplasmic and pellet fractions (the latter consisting mostly of chromatin and nuclear matrix) as described in Analysis of the expression of the Drosophila protein at various stages in the life cycle of the fly suggested that the protein was more abundant at those stages where proliferation was taking place. A comparison of the levels of the TTC4 protein in first trimester placental tissue (contains many dividing cells) vs heart (non dividing)  showed tA comparison was also made of the level of the TTC4 protein in normal (MRC5 fibroblasts) versus tumour derived cell lines. Previous studies have reported a number of abnormalities in the TTC4 gene in melanoma tissues isolated from patients In this paper we have presented a number of lines of evidence which suggest that TTC4 is the human orthologue of the Drosophila pit47 protein: 1) The protein sequences of the 2 proteins show high levels of similarity, and this homology is not confined to the region of the TPR motifs; 2) Both proteins are contained in the nucleus and are most likely found mainly in the nucleoplasmic compartment. In each case several putative bipartite like NLS motifs Data presented in this paper also suggests that TTC4 is involved in the development or progression of cancer. A role for TTC4 in melanoma had already been suggested, since mutations seen in the gene in patient-derived samples and cell lines, seemed to correlate with increased invasiveness). Our data suggests that in addition to structural changes in the protein, melanoma lines contain more TTC4 protein than melanocytes. Furthermore increases in TTC4 protein levels are also observed in a number of other tumour derived cell lines compared to normal cells and tissues. It is therefore possible to suggest that an increased level of TTC4 contributes to the development of a variety of different tumours. An increased level of Dpit47 has also been reported in brain tumours in flies Although the precise role for TTC4 in tumour development is not clear, it may be partly related to a loss of protein interactions. Two of the naturally occurring TTC4 point mutations cause the protein to lose its interaction with hCDC6. Only one of these amino acids is fully conserved in the Drosophila protein but we have observed (GC and SC unpublished results) that mutation of this amino acid in Pit47 destroys its interaction with the Drosophila CDC6. We cannot say that the loss of the interaction with CDC6 is the precise cause of the defect, since the region into which the mutations map is likely to be involved in interactions with a number of proteins. (For instance the same region of pit47 is needed for interaction with DNA polymerase (David Loebel and SC unpublished observation)). In addition interaction of TTC4 with other cellular proteins has also been reported (e.g. TFIIIh


Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA–DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments  using newly designed specific PCR primer sets. Fluorescence  A limiting step in understanding any microbial ecosystem resides in our ability to inventory the microorganisms inhabiting the ecosystem, and to assess their metabolic potential, the interactions between them and their biotope. A partial answer to this challenge was (i) culture-independent studies based on the development of molecular microbial diversity analyses using the 16S rDNA gene as a phylogenetic marker , and  experiments helped to provide information about morphology and localization of the WWE3 bacteria within a microbial anaerobic sludge sample. Furthermore, the absence of the H17 helix in the WWE3 16S rDNA secondary structures is unprecedented and seems to be a characteristic of the bacterial candidate division WWE3 and its closest relatives.In the present study, we report that in the course of analysing a metagenomic fosmid library constructed from an anaerobic digester, we detected an unusual group of 16S rDNA bacterial gene sequences. These sequences, which presented many mismatches with the 16S rDNA universal primers, cannot be obtained by the classical polymerase chain reaction (PCR)-based amplification methods. Specific 16S rDNA PCR primers were developed for this group of sequences, named WWE3. Phylogenetic analyses show that this group constitutes a new bacterial candidate division. Fluorescence In order to analyse the microbial diversity and the metabolic potential of a mesophilic anaerobic digester, a large fosmid library was constructed using DNA extracted from the sludge digester of the WWTP of Evry, France. A part of the library (27 648 fosmid clones) was screened by hybridization with 16S rDNA gene targeted-hybridization probes. The 16S rDNA genes of 570 positive clones were directly sequenced using internal primers. While for 541 of these positive clones, the 16S rDNA gene sequences were obtained and affiliated to known bacterial or archaeal phyla, we were unable to obtain a 16S rDNA sequence for 29 clones. Analysis of HindIII fingerprints of these clones showed that their profiles were very similar. Southern blot hybridization using 16S rDNA-targeting probes revealed that 27 out of the 29 clones showed a common 1.6 kb HindIII positive fragment while the remaining two clones possess a positive 1.65 kb fragment. Shotgun sequencing of one of these 29 fosmid clones (DIGA11YD11) revealed that it does contain a complete 16S rDNA gene sequence which affiliates (88% identity) with a single sequence (AY953190) in public databases. The 16S rDNA sequences of the remaining 28 fosmids were determined by direct sequencing with specific primers derived from the DIGA11YD11 16S rDNA . All theExperimental procedures) were tested using the four DIGA11YD11-specific primer sets 1–4  16S rDNA libraries were constructed from DNA extracted from seven anaerobic digesters, using the DIGA11YD11-specific primers or a combination of specific and universal primers, (ii) sequence analysis of these 16S rDNA confirmed their inclusion in the WWE3 phylogenetic group and allowed us to design degenerate primers with a broader specificity. The presence of WWE3 bacteria was further investigated and confirmed in other anaerobic or anoxic environments such as swine lagoon slurries (6/10 samples) and freshwater biofilms (6/6 samples). The presence of WWE3 bacteria was tested by PCR on DNA sampled from three digesters over a 6-year period (2000–2006). During this period, WWE3 bacteria were detected in 16 out of 23 Evry sludge samples, three out of six from Corbeil and in all three sludge samples from Creil, showing that the WWE3 population size is subject to large variations in these anaerobic digesters.A total of 21 16S rDNA libraries were constructed from 12 DNA samples extracted from eight anaerobic digesters , one swiAn additional 61 bp insertion (type II) was found in the OTU-4 sequences. The two insertions (I and II) are located within the same region but exhibit completely different sequences. The overall intradivergence between WWE3 16S rDNA gene sequences reached 20% and is in the same order of magnitude as that for other uncultured bacterial candidate divisions . The retained sequences were then imported into the ARB database (http://www.arb-home.de) for phylogenetic analyses. An automatic alignment was performed which was manually checked and corrected.The 16S rDNA sequences obtained were edited and assembled with Phrap . Twenty-five 16S rDNA sequences representative of WWE3-defined OTUs, as well as representatives of OP11, WS6, OD1 and TM7 candidate divisions were used for tree construction. A modified version of the ‘Lane mask’ was used to choose homologous positions for tree construction . Phylogetest 3.0  to identtest 3.0  nucleotiradA annotated gene from DIGA11YD11 fosmid was aligned along with bacterial (sequences subset extracted from family HBG000623) and archaeal and eukaryotic (family HBG049531) radA homologues obtained from HOGENOM (http://pbil.univ-lyon1.fr). Phylogenetic analysis were performed using PhyML , six samples from Corbeil and three from Creil. The other digester samples are described in in situ hybridization experiments were performed as previously described  using the https://www.genoscope.cns.fr/agc/mage/wwwE3scope/.Gene prediction was conducted using the AMIGene software (Sequences determined in this study were deposited in the EMBL database under Accession No. CU367853 to CU367881 for sequences obtained from fosmid clones and CU392752 to CU392838 for sequences obtained from 16S rDNA clone libraries. DIGA11YD11 complete fosmid sequence was submitted under Accession No. CU367853.


DGAT1) gene by using data sets including sires with accumulated phenotypic observations of daughters . However, these data sets prevented analyses with respect to dominance or parent-of-origin effects, although an increasing number of reports in the literature outlined the relevance of non-additive gene effects on quantitative traits.Substantial gene substitution effects on milk production traits have formerly been reported for alleles at the K232A and the promoter VNTR loci in the bovine acylCoA-diacylglycerol-acyltransferase 1 (DGAT1 promoter VNTR alleles with milk production traits. We detected a dominant mode of effects for the DGAT1 K232A and promoter VNTR alleles. Namely, the contrasts between the effects of heterozygous individuals at the DGAT1 loci differed significantly from the midpoint between the effects for the two homozygous genotypes for several milk production traits, thus indicating the presence of dominance. Furthermore, we identified differences in the magnitude of effects between paternally and maternally inherited DGAT1 promoter VNTR – K232A haplotypes indicating parent-of-origin effects on milk production traits.Based on a data set comprising German Holstein cows with direct trait measurements, we first confirmed the previously reported association of DGAT1 locus have to be accounted for in more specific QTL detection models as well as in marker assisted selection schemes. The DGAT1 alleles in cattle will be a useful model for further investigations on the biological background of non-additive effects in mammals due to the magnitude and consistency of their effects on milk production traits.Non-additive effects like those identified at the bovine  DGAT1) gene is presumably the putative causal mutation for this QTL [DGAT1 K232A alleles have been confirmed in several breeds and populations [DGAT1 K232A locus representing the causal mutation for the QTL was achieved by functional in-vitro studies showing a higher efficiency in triglyceride synthesis of the allele DGAT1 232K compared to the allele DGAT1 232A [DGAT1 locus affecting milk production traits in cattle [DGAT1 promoter containing a SP1 transcription factor binding site motif (CCCGCC) was targeted as functional background for these additional alleles due to the functional relevance of juxtaposed SP1 binding sites for the regulation of gene expression in eukaryotes [DGAT1 promoter VNTR alleles consisting of a variable number of putative SP1 transcription factor binding sites showed an association with daughter yield deviations for milk production traits in a data set comprising bulls with homozygous genotype DGAT1 232A/232A [DGAT1 promoter VNTR alleles is underlined by in-vitro studies providing evidence for SP1 binding to the CCCGCC motif in the DGAT1 promoter and for induction of gene expression by the DGAT1 promoter VNTR alleles [Several mapping studies revealed a QTL for milk production traits in the centromeric part of cattle chromosome 14 (BTA14) [this QTL ,3. The pulations . FurtherAT1 232A . Howevern cattle -10. A VNkaryotes ,12. The 32A/232A . This re alleles .per se powerful granddaughter designs [DGAT1 polymorphisms in a data set comprising direct trait measurement in cows, however, demonstrate that the DGAT1 K232A and the promoter VNTR alleles display a dominant-recessive pattern of effects on milk production traits. This pattern of effects was previously unnoticed in data sets using breeding values or daughter yield deviations. Additionally, we demonstrate differences in the effects of maternally and paternally inherited DGAT1 promoter VNTR – K232A haplotypes on milk production traits, which indicate a parent-of-origin effect at the DGAT1 locus in cattle.There are several confirmed examples for non-additive QTL effects or QTL acting dependent of parental origin ,15 in a  designs , it is i designs . Our stuDGAT1 K232A and promoter VNTR alleles as well as of the DGAT1 promoter VNTR – K232A haplotypes based on the maternally inherited alleles/haplotypes are listed in Table DGAT1 232A and the promoter VNTR allele 3, respectively. All possible allele combinations were detected in the maternally inherited DGAT1 haplotypes with the maternally inherited promoter VNTR – K232A haplotype 3 – 232K showing the highest frequency. Strong linkage disequilibrium was observed between both DGAT1 loci, because DGAT1 232K was almost exclusively detected in linkage phase with the promoter VNTR allele 3. Due to their extremely low frequency, the DGAT1 haplotypes 1 – 232K, 2 – 232K, 4 – 232K, and 5 – 232K could not be included in the analysis of haplotype effects.Frequencies of the DGAT1 promoter VNTR allele 5 compared to all other promoter VNTR alleles are listed in Table 232A/232A homozygous. The DGAT1 promoter VNTR allele 5 showed a significantly superior effect on milk fat content and milk protein content, while milk yield was decreased compared to all other promoter VNTR alleles. The differences for effects on milk fat yield and on milk protein yield were not significant.To enable a direct comparison of this study with results of a previous study in German Holstein sires , the genDGAT1 K232A genotypes solitary showed highly significant effects on all milk production traits in a reduced model . Similar results were observed with the full model including the DGAT1 K232A genotypes as well as the promoter VNTR genotypes. The only exception was for milk protein yield, where no significant effect for the DGAT1 K232A locus was obtained in the full model. In contrast to the lack of association between DGAT1 K232A genotypes and milk protein yield, the DGAT1 promoter VNTR genotype had significant effects on all milk production traits including milk protein yield in the full model.The DGAT1 K232A genotype effects revealed that for all milk production traits the difference between 232A/232K heterozygotes and the 232A/232A homozygotes was larger than the difference between the 232A/232K heterozygotes and the 232K/232K homozygotes. This was most prominent for milk fat percentage in both models. Similarly, for milk yield and milk protein yield the difference between the DGAT1 promoter VNTR 4/4 homozygotes and the */4 heterozygotes  was larger than the difference between the */4 heterozygotes and the */* homozygotes. The calculated dominance values showed significant dominance effects on milk yield, milk fat yield and milk protein yield , milk fat content (DGAT1 232K) and milk protein content  (Table 4 had a lower milk yield and as a consequence a lower milk fat yield and a lower protein yield compared to the mean of the homozygotes with zero (*/*) or two copies (4/4) of the allele. Heterozygotes for the VNTR allele 5 had a higher milk protein content and heterozygotes for the DGAT1 232K showed a significantly higher milk fat content and a tendency towards a higher milk protein content and to a reduced milk yield.The analysis of the contrasts between 3 – 232K haplotype resulted in significantly higher milk yield and milk protein yield and lower milk fat content when inherited paternally. In contrast to that, the 1 – 232A haplotype resulted in significantly lower milk yield and higher milk fat content when passed by the sire to the offspring. For all other VNTR – K232A haplotypes, there is no influence of the parent-of-origin detectable at a significant level, but in the case of the 3 – 232A haplotype, there were tendencies to a lower milk fat content and lower milk protein content for the paternally inherited haplotype. In a further analysis, we tested if those differences between paternally and maternally inherited DGAT1 haplotypes could also be detected when investigating the DGAT1 K232A alleles only. As indicated in Table DGAT1 K232A alleles obtained results similar to those for the DGAT1 promoter VNTR – K232A haplotypes.The simultaneous test for a parent-of-origin effect over all haplotypes revealed a significant influence for the traits milk yield, milk fat content and a detectable, but not significant influence on milk protein yield Table . More spDGAT1 promoter VNTR genotypes showed significant effects on all milk production traits in a model also including the DGAT1 K232A genotypes. Our results provide an independent confirmation of previous findings [DGAT1 K232A mutation does not account for the entire QTL variation on milk production traits in the centromeric part of BTA14. These results are in contrast to Grisart et al. [DGAT1 K232A genotype did erase the effect of any other polymorphism at the DGAT1 locus. However, Grisart et al. did not consider the DGAT1 promoter VNTR. In our study, the K232A polymorphism did not significantly affect milk protein yield in a model that also included the promoter VNTR genotype. Compared to previous results obtained in an independent data set [DGAT1 promoter VNTR showed an identical pattern and were very consistent in magnitude with allele 5 being superior for milk fat and milk protein content. In an independent cattle breed, Sanders et al. [DGAT1 K232A mutation and the DGAT1 promoter VNTR for milk fat yield and content also supporting effects of those loci on milk production traits. Recently, Fürbass et al. [DGAT1 promoter VNTR motif. This observation and the confirmed trait association of DGAT1 promoter VNTR alleles presented in this study provide further evidence that the alleles at the DGAT1 promoter VNTR indeed may exert a direct effect on milk production traits. However, in spite of those data, it cannot be formally excluded that another, yet undetected gene outside the DGAT1 locus may be the molecular background for the additional QTL alleles in this genomic region.In our study, the findings ,9 demonst et al. , who dess et al.  detecteds et al.  proved SDGAT1 locus focused on additive gene substitution effects for the QTL affecting milk production traits on BTA14  as well as in models combining the DGAT1 K232A and promoter VNTR genotype effects. Analogous to the non-additive effects of the alleles at the DGAT1 K232A locus, we obtained indication on dominance effects for the alleles 4 and 5 at the DGAT1 promoter VNTR locus. Interestingly, for the 232K allele and the promoter VNTR allele 5, which exhibit a similar pattern of effects on milk production traits, the pattern of dominance was also similar predominantly affecting milk fat and milk protein content. In contrast, recessive effects for the DGAT1 promoter VNTR allele 4 were identified primarily on yield traits. Our data represent the first report on dominance effects on milk production traits for alleles at the DGAT1 locus.Previous publications on the effect of the 14 (e.g. ) as wellDGAT1 effects. Because Fisher and Spelman [et al. [DGAT1 232K/232K included in their study prevented a powerful analysis of DGAT1 K232A effects with respect to dominance. The dominant physiological effects of the DGAT1 232K variant on milk fat synthesis may be caused by the high efficiency of the 232K DGAT1 protein for triacylglycerol synthesis [max of the 232K DGAT1 molecules might result in the supply of substrates becoming the limiting factor for milk fat synthesis, thus preventing a further substantial difference in milk fat synthesis between individuals carrying one or two copies of the DGAT1 232K allele.Some studies ,6,10,18  Spelman ,6. Howevynthesis . The higDGAT1 allele effects have an impact on allele substitutions effects evaluated by regression analysis on the number of DGAT1 K232A or promoter VNTR alleles. They can alter during selection processes in dependency of the magnitude of dominance and allele frequencies. However, allele effects obtained by regression in two independent populations (this study and [Dominant tudy and ) showed DGAT1 alleles demonstrates that although indirect phenotypes like breeding values or daughter yield deviations may have an increased statistical power to detect QTL, the analyses of additional designs with direct phenotypes may be necessary for a precise estimation of the allele effects at causal mutations.The discovery of dominant/recessive effects of DGAT1 promoter VNTR – K232A haplotypes regarding effects on milk yield, milk fat content and milk protein yield. The results for the differences between paternally and maternally inherited haplotypes are in line with results looking at alleles at the DGAT1 K232A locus only. These differences of effects between paternally and maternally inherited DGAT1 alleles/haplotypes have to be evaluated carefully to exclude artificial effects, e.g. due to the applied model including fixed effects of the sires, but not of the dams. This might result in a reduced variance of the paternally inherited alleles/haplotypes, because within the model the paternal DGAT1 haplotype effect might already be included in the sire effect. However, for the DGAT1 3 – 232K haplotype the associated effect on milk yield was larger when paternally inherited than when maternally inherited, while for 1 – 232A the contrast was opposite. This result indicates that it is quite unlikely that a systematic bias due to reduced variance of paternally inherited alleles is the only source for the observed differences. We interpret these differences of haplotype contrasts for paternally and maternally inherited DGAT1 K232A – promoter VNTR haplotypes as parent-of-origin effects. If the DGAT1 locus indeed were a target for parent-of-origin effects, differences between paternally and maternally inherited alleles/haplotypes are expected for the DGAT1 promoter VNTR – K232 haplotype as well as for the DGAT1 K232A mutation alone as we found in our study.Our study revealed a difference between the paternally and maternally inherited DGAT1 gene is not located in a chromosomal region homologous to known imprinted genome areas in mice or human [2 resource population [DGAT1 studies.It has to be noted that the or human . On the or human ,21. Howepulation , which wDGAT1 locus in cattle and also provides further support for a functional role of the DGAT1 promoter VNTR additional to the DGAT1 K232A alleles on milk synthesis. The precise mechanisms behind the dominance and parent-of-origin effects require further targeted functional studies to be fully understood. Additionally, the DGAT1 allele effects deviating from the commonly applied hypothesis of additive QTL effects highlight the need towards efficient ways to include non-additive effects into QTL mapping models [DGAT1 locus, which seems to be responsible for a substantial proportion of the genetic variance of milk production traits [DGAT1 loci in cattle will be a useful model to study the biological background of dominance and parent-of-origin effects in mammals.Our study indicates dominance and parent-of-origin effects at the g models . The DGADGAT1 K232A and promoter VNTR polymorphisms. Nine of the sires were proven sires with high genetic merit for milk production traits with their daughters born after the sires finished their progeny test, whereas ten sires were young sires with their daughters originating from the progeny test itself. The cows themselves were unselected, except for their paternal ancestor.The data set of the study included a total of 1035 German Holstein cows sampled on ordinary dairy farms located in nine different regions of Germany. The cows descended from 19 different sires . The sires were pre-selected to represent a large variety of genotypes at the DGAT1 gene the non-conservative polymorphism K232A in exon 8 giving rise to a lysine => alanine amino acid substitution as well as the non-coding VNTR in the promoter were included. The DGAT1 K232A mutation was genotyped according to [DGAT1UP 5'-GCACCATCCTCTTCCTCAAG-3'; DGAT1DN 5'-GGAAGCGCTTTCGGATG-3') amplified a 411 bp fragment that was digested by the restriction enzyme CfrI . The resulting fragments  were separated on a 2% agarose gel. A PCR fragment containing the VNTR polymorphism at position 1465 in the promoter of the bovine DGAT1 gene (Genbank no. AJ318490) was amplified by the following primers: DGAT1proUP 5'-TCAGGATCCAGAGGTACCAG-3', DGAT1proDN 5'-GGGGTCCAAGGTTGATACAG-3'. The PCR reaction was carried out in a 10 μl volume under the following conditions: 50 ng genomic DNA, 5 pmol of DGAT1proUP, 10 pmol of DGAT1proDN, 1.5 mM MgCl2, 0.2 mM of each dNTP, and 0.2 U Taq Polymerase . A touchdown protocol was performed starting at 70°C with 2°C steps to the final annealing temperature of 60°C. One microliter of the reaction volume was run on an automated fragment analysis system  under denaturing conditions. Allele nomenclature for the DGAT1 promoter VNTR was identical to allele classification in [Within the DGAT1 promoter VNTR – K232A haplotypes of sires were derived from the genotypes of the respective group of daughters. Consecutively, the paternally inherited haplotype of each daughter was determined by assuming no recombination between DGAT1 promoter VNTR and K232A polymorphism due to the close genomic co-localization of both loci . The maternally inherited haplotype of a cow was deduced from subtracting the paternally inherited haplotype from the daughters' genotype. For daughters sharing their sires' genotype at the DGAT1 K232A as well as the promoter VNTR locus, it was not possible to assign paternally or maternally inherited haplotypes, respectively.DGAT1 alleles. Records were only included, if the cow had a minimum of eight milk recordings within the first lactation. Yield deviations for milk fat percentage and milk protein percentage were calculated according to [For the 1035 cows included in the analysis, first lactation yield deviations (YD)  for milkDGAT1 K232A and promoter VNTR genotypes to ensure a high variety of alleles and haplotypes in the analysis. Instead, the maternally inherited alleles and haplotypes of the cows were used to receive an overview of the DGAT1 allele distributions in the population.The allele and haplotype frequencies for the German Holstein population cannot be estimated from the genotypes of the cows, because the sires of the cows included in the study had been selected with respect to DGAT1 promoter VNTR allele 5 displayed the most pronounced effect on milk production traits compared to the other VNTR alleles, particularly on milk fat percentage [DGAT1 promoter VNTR effects estimated in that study with the effects within the German Holstein cow data set of the present study, the allele substitution effect of DGAT1 promoter VNTR allele 5 compared to all other alleles was calculated using a regression analysis with general linear model procedures  applying the following model:In a previous study, daughter yield deviations (DYDs) of German Holstein bulls were analyzed, and the rcentage . To enabyijk is the YD of daughter j within sire i carrying the DGAT1 K232A genotype k, μ is the overall mean, si is the fixed effect of sire i, K232Ak is the fixed effect of the DGAT1 K232A genotype k; zij is the number of alleles 5 at DGAT1 promoter VNTR  of daughter j within sire i, b is the regression coefficient representing the allele substitution effect of VNTR allele 5 compared to all other alleles, and eijk is the random residual effect. The respective analysis was performed including all individuals as well as considering a subset restricted only to the homozygous DGAT1 232A/232A cows.where DGAT1 232A/232A individuals only to evaluate DGAT1 promoter VNTR effects [DGAT1 K232A and promoter VNTR effects jointly by including all DGAT1 K232A genotypes. General linear model procedures  were used to estimate the effects of both DGAT1 locus genotypes on milk yield, milk fat yield, milk protein yield, milk fat percentage, and milk protein percentage with the following model and a type III sum of squares test:While the previous study design consisted of  effects , the preijkl is the YD of daughter j within sire i carrying the DGAT1 genotypes k at K232A and l at the promoter VNTR, respectively, μ is the overall mean, si is the fixed effect of sire i, K232Ak is the fixed effect of the DGAT1 K232A genotype k, VNTRl is the fixed effect of the DGAT1 promoter VNTR genotype l, and eijkl is the random residual effect. Only DGAT1 promoter VNTR genotypes represented by more than ten daughters in the data set were included in the analysis. For comparison to the outcomes of previous studies, a submodel of eq. 2 without the fixed effect of the DGAT1 promoter VNTR genotype was also used.where y232K allele and for the DGAT1 VNTR alleles 4 and 5 defining three genotype classes with zero, one or two copies of the respective allele in each case.For both, the model and the submodel, the magnitude of the dominance was calculated by contrasting the estimated coefficients for heterozygotes to the average of the coefficient of homozygous genotypes. This was done for the DGAT1 promoter VNTR – K232A haplotypes on milk production traits were estimated separately applying the following model:Finally, the effects of the maternally and paternally inherited ijkl is the YD of daughter j of sire i with paternally inherited DGAT1 promoter VNTR – K232A haplotype k and maternally inherited DGAT1 promoter VNTR – K232A haplotype l, μ is the overall mean, si is the fixed effect of sire i, DGAT1_patk * DGAT1_matl is the combined fixed effect of the paternally inherited DGAT1 promoter VNTR – K232A haplotype k and the maternally inherited DGAT1 promoter VNTR – K232A haplotype l, and eijkl is the random residual effect. To test for a parent-of-origin effect, a set of linear hypotheses of the estimated effect coefficients were formulated that compare the estimates of the group that received a specific haplotype from the father to the estimates for the group that received it from the mother . Each linear hypothesis was tested separately (for each haplotype) and finally the whole set of hypotheses was tested simultaneously. This last test is interpreted as an overall test for the parent-of-origin effect. In this analysis, only observations with haplotypes occurring in both parent sexes were included.where yThe author(s) declares that there are no competing interests.CK conceived, designed and coordinated the study, carried out the molecular genetic studies, performed a part of the statistical analyses and drafted the manuscript. RW participated in the molecular analysis and helped drafting the manuscript. CE performed the statistical analyses and GT participated in the statistical analysis and design of the study and helped drafting the manuscript. All authors read and approved the final manuscript.DGAT1 genotype effects on milk production traits. Least square mean values (S.E.) for milk production traits for DGAT1 genotypes in a German Holstein cow data set from a combined model with DGAT1 K232A and VNTR genotype effects and from a reduced model including DGAT1 K232A genotype effects only.Click here for file


The finding of adenomatous polyps of the gallbladder is a rare occurrence and an unusual clinicalproblem.		Among 2,145 patients who underwent cholecystectomy for gallbladder disease only 9 (0.4 per cent)presented with adenomatous polyps. There were 6 women and 3 men, aged 17 to 70 years. Preoperativeultrasonographic diagnosis was made in only 1 of 7 patients with gallstones, in contrast polypoid lesionswithin a gallbladder without stones were easily confirmed by both ultrasonography and oral cholecystographyin the remaining 2 patients. All polyps were 1.0 cm or less in size and without histologic evidenceof malignant change. The clinical significance of this rare condition is discussed, with particularreference to a possible role in development of gallbladder carcinoma. Surgical treatment should beadvocated regardless of clinical manifestation when the polyp exceeds 1.0 cm in diameter or rapidgrowth of the lesion is seen on ultrasonographic follow-up examinations.


The effect of preheating EMT6 cells in vitro on their response to cytotoxic agents of either 43 degrees C or 37 degrees C has been investigated. Preheating for 3 h at 40 degrees C produced measurable protection  to subsequent treatment for 1 h at 43 degrees C. This preheat treatment was further found to reduce cell killing by BLM and BCNU (drug tolerance) present during 1 h at 43 degrees C. In contrast, no such heat-induced drug tolerance was seen with ADR. An additional effect with ADR was the apparent elimination of heat-induced thermal tolerance at toxic drug doses. However, preheating under these conditions had no effect on the subsequent cytotoxicity of any of these drugs at 37 degrees C. Also, preheating for 1 h at 43 degrees C was found to sensitize cells to BLM and BCNU toxicity at 37 degrees C but to protect against ADR toxicity. The results are discussed in relation to known mechanisms of cell killing by heat and of thermal tolerance.


Biological response modifiers such as interleukin 2 (IL2) may be most effective in the setting of minimal residual disease. In a phase I-II clinical trial, IL2 was administered to 10 patients in remission of acute myeloid leukaemia and three with multiple myeloma 1-4 weeks after treatment with ablative chemotherapy or chemotherapy and autologous bone marrow transplantation. The aim was to assess the capacity of these patients to tolerate IL2 after intensive therapy and to determine whether regenerating lymphocytes were capable of responding to IL2 with the generation of anti-leukaemic effector cells. Toxicity was severe in two patients treated with escalating doses of IL2 and 19 subsequent infusions administered to 11 patients on a fixed dose schedule for periods of 3-5 days were well tolerated. Major toxicity was confined to hypotension (two courses) which responded rapidly to treatment cessation. No patients required intensive care unit support. IL2 infusions produced no significant adverse effects on marrow regeneration; while there were transient falls in platelet counts there were no episodes of clinical bleeding and neutrophil counts increased from a mean of 1.1 pre-infusion to 2.5 x 10(9)l-1 during the infusion (P = 0.004). A significant biochemical abnormality was hypokalaemia which responded rapidly to correction. Cells with activity against leukaemic progenitor cells appeared in peripheral blood within 48 h of beginning treatment. We conclude that IL2 may be used in minimal residual haematological malignancy, and by producing anti-neoplastic effector cells has the potential, as yet unproven, to prolong disease-free survival of patients entering remission.


Meiosis is a specialized form of cell division involving one round of chromosome replication followed by two rounds of segregation, thereby producing daughter cells with half the genomic equivalent of the progenitor. In most organisms, double-strand breaks (DSBs) are introduced into the genome following premeiotic S-phase. These breaks are repaired almost exclusively from the homologous chromosome via repair pathways that yield either a crossover or non-crossover recombination product It should be no surprise then that most eukaryotes possess a sophisticated mechanism to control meiotic recombination. Consider the situation in an individual mouse meiocyte. More than 200 DSBs are made; however, only a subset of these precursors are repaired as crossovers, while the rest are repaired as non-crossovers  [4]. ThuAlmost a century after the first observation of crossover control PLoS Genetics identify a role for the Pachytene Checkpoint gene, PCH2, in crossover control in Saccharomyces cerevisiaePCH2, which encodes a putative AAA+-ATPase, was initially identified in yeast as a checkpoint factor due to suppression of a zip1Δ arrest in a pch2Δ mutant. This and other observations led to the hypothesis that Pch2 helps monitor chromosome synapsis during meiotic prophase PCH2 is widely conserved in organisms that construct a synaptonemal complex and exhibit crossover interference, but is absent from organisms such as Schizosaccharomyces pombe that do not exhibit these features pch2 mutants at the HIS4LEU2 recombination hotspot Two articles in this issue of PLoS Genetics, the Alani and Börner groups pch2Δ strains. Importantly, these analyses demonstrated that the crossovers formed in pch2Δ mutants show reduced interference.In studies published in this issue of pch2Δ strains carrying various hypomorphic alleles of the topoisomerase-like protein, Spo11. These hypomorphic alleles decrease the number of DSBs pch2 mutation has little or no effect on spore viability on its own, introducing a spo11 mutation that reduces DSB activity by ∼20% significantly reduced viability despite approximately wild-type crossover frequencies spo11 hypomorphs that reduce DSB activity up to 80%. Although this is an indirect method of measuring crossover homeostasis, these findings provide compelling evidence that Pch2 has a role in multiple aspects of crossover control during yeast meiosis.Recent studies have indicated that decreased crossover interference is associated with a concomitant decrease in crossover homeostasis pch2Δ mutants, Hop1/Red1 and Zip1 exhibit a more uniform axial localization pattern than is observed in wild type. Joshi et al. now demonstrate that chromosome domains that are enriched for Hop1 and Red1 tend to colocalize with future sites of crossover formation, leading to the hypothesis that Pch2 functions to stabilize alternating domains enriched for either Hop1/Red1 or Zip1. Such domains are proposed to be modules that mediate crossover designation and interference. Interestingly, when PCH2 is deleted, not only is axial organization of Hop1/Red1 and Zip1 compromised, but appearance of both crossover and non-crossover products is delayed to similar extents pch2 mutant phenotype are consequences of the same molecular defect, nor is it yet clear precisely how Pch2 protein functions in wild-type cells. Nonetheless, the current findings provide new support for the idea that higher order chromosome structure plays a key role in crossover control So what role could Pch2 play in this process? Pch2 is required for differential organization of chromosome structural proteins Hop1 and Red1 relative to the synaptonemal complex central element protein Zip1


BRCA1 and BRCA2 to breast cancer incidence in outbred populations have been based on studies that are either small or have selected for cases diagnosed at an early age. Only one of these has reported an estimate of the breast cancer risk associated with a mutation in these genes, and there is no published ovarian cancer risk estimate derived from a population-based case series. We screened a population-based series of breast cancer cases diagnosed before the age of 55 for mutations in BRCA1 and BRCA2. Pedigree information from the mutation carriers was used to estimate penetrance and the proportion of familial risk of breast cancer due to BRCA1 and BRCA2. We identified eight (0.7%)BRCA1 and 16 (1.3%)BRCA2 mutation carriers in 1220 breast cancer cases . Mutation prevalence was substantially higher in cases diagnosed before 35 years-of-age and with increasing number of relatives affected with breast or ovarian cancer. However, most mutation carriers were diagnosed in the older age groups, and a minority reported a first-degree relative with breast cancer. Breast cancer penetrance by age 80 was estimated to be 48% (95% CI 7–82%) for BRCA1 mutation carriers and 74% (7–94%) for BRCA2 mutation carriers. Ovarian cancer penetrance for BRCA1 and BRCA2 combined was 22% (6–65%) by age 80. 17% of the familial risk of breast cancer was attributable to BRCA1 and BRCA2. At birth, the estimated prevalence of BRCA1 mutation carriers was 0.07% or 0.09% depending on the penetrance function used for the calculation. For BRCA2 the birth prevalence estimates were 0.14% and 0.22%. Mutations in the genes BRCA1 and BRCA2 are rare in the population and account for a small fraction of all breast cancer in the UK. They account for less than one fifth of the familial risk of breast cancer. Eligibility criteria for BRCA1 and BRCA2 mutation testing based on family history and age of onset will identify only a small proportion of mutation carriers. © 2000 CancerResearch CampaignEstimates of the contribution of


Two patients with relapsed Wilms' tumour and renal failure requiring dialysis were given carboplatin and etoposide by pharmacokinetically guided dosing. The target area under the drug plasma concentration vs time curve (AUC) was 6 mg ml-1 min for carboplatin and 18 and 21 mg ml-1 min for etoposide. On course 1 measured AUCs of carboplatin and etoposide were 6 and 20 mg ml-1 min for patient 1 and 6 and 21 mg ml-1 min for patient 2 respectively. Peritoneal dialysis did not remove carboplatin or etoposide from the plasma, however carboplatin but not etoposide was cleared by haemodialysis. Therapy with carboplatin and etoposide is possible in children and adults with renal failure who require dialysis, but in this situation pharmacokinetic monitoring is essential.


Bmp4 is conditionally deleted within the inner ear, as well as chicken models in which Bmp signaling is knocked down specifically in the cristae, we show that Bmp4 is essential for the formation of all three cristae and their associated canals. Our results indicate that Bmp4 does not mediate the formation of sensory hair and supporting cells within the cristae by directly regulating genes required for prosensory development in the inner ear such as Serrate1 (Jagged1 in mouse), Fgf10, and Sox2. Instead, Bmp4 most likely mediates crista formation by regulating Lmo4 and Msx1 in the sensory region and Gata3, p75Ngfr, and Lmo4 in the non-sensory region of the crista, the septum cruciatum. In the canals, Bmp2 and Dlx5 are regulated by Bmp4, either directly or indirectly. Mechanisms involved in the formation of sensory organs of the vertebrate inner ear are thought to be analogous to those regulating sensory bristle formation in Drosophila. Our results suggest that, in comparison to sensory bristles, crista formation within the inner ear requires an additional step of sensory and non-sensory fate specification.Angular head movements in vertebrates are detected by the three semicircular canals of the inner ear and their associated sensory tissues, the cristae. Bone morphogenetic protein 4 (Bmp4), a member of the Transforming growth factor family (TGF-β), is conservatively expressed in the developing cristae in several species, including zebrafish, frog, chicken, and mouse. Using mouse models in which  Bmp4 in mice affects the formation of not only the sensory regions but also their associated canals. These results demonstrate for the first time that a single gene, Bmp4, is required for the formation of the entire sensory apparatus for detecting angular head movements.Disruption of the sense of balance is highly debilitating, causing vertigo and nausea. Maintenance of proper balance requires sensory inputs from many body parts, including the inner ears and the eyes. Within the inner ear, the vestibular apparatus plays a key role in the sense of balance and is responsible for detecting head orientation and movements. The portion of the vestibular apparatus that detects angular head movements consists of three fluid-filled, semicircular canals oriented at right angles to each other. At one end of each canal is an enlargement that houses the sensory tissue, crista ampullaris, consisting of sensory hair cells and supporting cells. Bone morphogenetic protein 4 (Bmp4), a secreted signaling molecule, is expressed in these sensory regions during development. However, the lack of  The ability to detect angular head movements in vertebrates lies within the vestibular apparatus of the inner ear All the sensory patches within the vertebrate inner ear including the presumptive cristae are thought to arise from a common prosensory  region at the otic placode and otocyst stages . This prDlx and Hmx, have also been implicated Wnt, Dlx, or Hmx gene functions all result in an early disorganization or absence of Bmp4 expression within the presumptive cristae Multiple factors are thought to regulate the formation of the vestibular apparatus Bmp4 in the presumptive cristae is conserved among several vertebrate species including the zebrafish, frog, chicken, and mouse . All animal procedures were approved and conducted according to the NIH Animal Use and Care Committee guidelines.The sequence . Bmp4Tm1at birth . TherefoNoggin cDNA Noggin is driven by the immediate early Cytomegalovirus promoter (Clontech). Chicken Smad6 cDNA in the pCab-IRES-GFP vector Smad6 is driven under the chicken β-actin promoter and the immediate early enhancer of Cytomegalovirus pSmad6, pNoggin and their respective control vectors at a concentration of 4 to 6 mg/ml were injected into the lumen of chicken otocysts at E3.5. Plasmids were electroporated into the anterior region of the otocyst using a positive and negative electrode flanking the anterior and posterior poles of the otocyst, respectively. Two 50 milli-second pulses at 10 volts were applied using a CUY21 electroporator.Chicken embryos were staged according to Hamburger and Hamilton Paint-fill analyses and in situ hybridizations were performed as described http://mathforum.org/library/drmath/view/57605.html). A total of 70 to 190 cells were counted per treatment.Anti-hair cell specific antigen (HCA) antibodies (gift of Guy Richardson) were used at 1:5000 dilution, and staining was performed as previously described Bmp4 null embryos, we used three different mouse lines. The first, cre/+Foxg1, was made by inserting cre into the endogenous Foxg1 gene which is expressed in tissues such as the embryonic otocysts, eyes, and foregut cre allele has been demonstrated by crossing cre/+Foxg1 mice with the Rosa26R reporter line Tm1Bmp4 is a null allele of Bmp4loxPBmp4 conditional allele was generated as described  embryos that are severely delayed in development, (2) embryos with eye malformations that are either normal or slightly smaller in their body size, and (3) embryos that are morphologically indistinguishable from loxP/+Bmp4 littermates  using an RNA probe (B4-del) generated against exons 3 and 4 of Bmp4. Half of the embryos analyzed at 9.5 dpc displayed abnormal Bmp4 expression patterns (n = 4/8). By 10.5 dpc, a higher percentage of cre/+Foxg1; loxP/Tm1Bmp4 embryos show no or reduced Bmp4 expression in tissues such as the eyes and otocysts where Foxg1 is normally transcribed .We evaluated the tissue specificity of Bmp4 is transcribed in an anterior streak of tissue and a posterior focus . By this age, Bmp4 is also expressed in the non-sensory region of the growing cochlear duct Bmp4 expression in the cochlear duct appears normal in all of the cre/+Foxg1; loxP/Tm1Bmp4 specimens examined (data not shown).In a normal otocyst, or focus . The antor focus , arrow aanterior , arrow. cre/+Foxg1; loxP/Tm1Bmp4 inner ears at 13.5 dpc was examined by paint filling the membranous labyrinth. Consistent with the variable Bmp4 expression patterns, the paint-filled cre/+Foxg1; loxP/Tm1Bmp4 specimens also show a range of inner ear phenotypes (loxP/+Bmp4 (n = 3/14) embryos, or display only a lateral canal truncation . Therefore, this milder phenotype observed in cre/+Foxg1; loxP/Tm1Bmp4 embryos is probably due to insufficiency of Bmp4 caused by the presence of both of the Tm1 and the un-recombined floxed Bmp4 allele rather than an incomplete penetrance of the cre activity. Cochlear ducts of cre/+Foxg1; loxP/Tm1Bmp4 embryos show some variability in length . Taken together, inner ear-specific deletion of Bmp4 using two independent cre lines indicates that Bmp4 is required for the formation of the three cristae and semicircular canals, and possibly the utricle and saccule.We also conditionally deleted uncation ; n = 5. uncation ; n = 5. Tm1/loxPBmp4 embryos generated by breeding TgPax2cre; +/Tm1Bmp4 with loxP/loxPBmp4 mice also display lateral canal truncation as well (n = 4/7), suggesting that a combination of Tm1 and loxP alleles can generate hypomorphs depending on the genetic background. Notably, our Tm1/+Bmp4 mice in Black Swiss background do not circle but a small percentage of Bmp4 heterozygous mice in C57BL/6 background do Some of the cre/+Foxg1; loxP/Tm1Bmp4 are already apparent at 11.5 dpc , other crista markers are also present.Analysis of paint-filled ears of younger embryos indicates that the vestibular defects in 11.5 dpc . To bettBmp4 expression in the inner ear also resulted in the absence of semicircular canal formation. The overall size of the vertical canal pouch is usually smaller than normal, particularly in the posterior region  encoding Smad6 or Noggin translationally coupled to GFP were electroporated into the developing anterior crista region in ovo at E3.5, a time when these crista-associated genes are co-expressed in the presumptive crista. Smad6 is an intracellular inhibitor that competes with Smad4 for binding to phosphorylated Smad1/Smad5/Smad8 proteins, thus preventing their subsequent translocation to the nucleus and activation of Bmp target genes Smad6 expression has been used successfully to address the roles of Bmps in neural induction and placode formation Bmp4-positive anterior crista region , Fgf10 (n = 0/6), Lmo4 (n = 0/9) and Ser1 (n = 0/5) are not affected , but quite consistently seen in response to pNoggin . Since Noggin is a secreted molecule, down-regulation of Gata3 and Msx1 in the mesenchyme near the site of electroporation is also observed  or pNoggin (non-cell autonomous) treatments Bmp4 will require further investigation  syndrome DrosophilaGata3 may have a more global effect on cell fate specification in vertebrate crista beyond formation of the cruciatum (see below). Furthermore, the conserved Gata3 expression in the mesenchyme surrounding the presumptive cristae between chicken and mouse Gata3 expression in the mesenchyme by pNoggin, but not by pSmad6, may contribute to the more severe phenotype caused by pNoggin.Drosophila embryogenesis, pannier (homolog of Gata1) is induced by dpp  in the dorsal embryo dpp becomes dependent on pannierGata3 expression appears to begin before that of Bmp4. However, the relatively normal Bmp4 expression in Gata3 null inner ears does not support Gata3 functioning upstream of Bmp4Gata3 in the cristae is dependent on Bmp4.In other systems, Bmp and Gata pathways are thought to interact. For example, during Gata3, considered to be a non-sensory gene, is expressed in the prospective lateral crista of the mouse . Therefore, both Fgfs and Bmp4 could be involved in mediating canal formation.We have proposed that the sensory cristae may induce the formation of their associated semicircular canals Bmp2 in canal formation, Dlx5 is also a key player in canal formation, and its activity is directly or indirectly regulated by Bmp4 as well. Even though the canal phenotypes in Dlx5-/- mutants are milder than those in Bmp4 conditional mutants, the phenotypes in Dlx5-/-; Dlx6-/- double mutants appear to be more severe Bmp4, and in turn, their activities are maintained by Bmp4. It is also interesting that the expression of Dlx5 in the canal pouch is more susceptible than Hmx3 to the lack of Bmp4. Even though both Dlx and Hmx pathways are required for canal formation, regulation of these two pathways appears distinct. This notion is also supported by studies of Gbx2-/- and Wnt1-/-; Wnt3a-/- mutant embryos, in which Dlx5 expression is down-regulated but Hmx3 expression is relatively normal In addition to the postulated role of Dlx, Hmx, Tbx1, Eya1 and Six1 regulate its activity, directly or indirectly Click here for additional data file.Table S1Summary of phenotypes.(0.04 MB DOC)Click here for additional data file.


The complex is selectively antimitochondrial and inhibits the growth of a number ofyeast strains in non-fermentable media at concentrations as low as 2.5 μΜ and induces themitochondrial mutation petite The effect is largely reversed by the presence of aspirin. The complexis shown to be stable in the cell culture media and in the presence of glutathione, but readily reactswith disulfides of oxidized glutathione and serum albumin. Surprisingly, neither [Au(eppe)2]Cl nor[Au(eppe)2]Cl  (dppe = Ph2PCH2CH2PPh2) showed any mitochondrial selectivity in the samescreening protocol.The silver(I) complex [Ag(eppe)


Stem cell differentiation is accompanied by a gradual cellular morphogenesis and transcriptional changes. Identification of morphological regulators that control cell behavior during differentiation could shed light on how cell morphogenesis is coupled to transcriptional changes during development. By analyzing cellular behavior during differentiation of mouse embryonic stem cells (ESCs), we uncover a role of Borg5 (binder of Rho guanosine 5′-triphosphatase 5) in regulating trophectoderm (TE) cell morphogenesis. We report that differentiation of ESCs toward TE is accompanied by enhanced actin protrusion and cell motility that require upregulation of Borg5. Borg5 interacts with both Cdc42 and atypical protein kinase C (aPKC) and functions downstream of Cdc42 to enhance TE cell motility. Borg5 is required for the sorting of differentiating TE to the outside of ESCs in vitro. In developing embryos, Borg5 protein localizes to cell–cell contacts and the cytoplasm after compaction. It exhibits higher levels of expression in outer cells than in inner cells in morula and blastocysts. Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation. Since Cdx2 and Borg5 facilitate each other's expression as ESCs differentiate toward TE, we propose that cell morphogenesis is coupled with transcriptional changes to regulate TE differentiation. Our studies also demonstrate the utility of ESCs in identifying morphological regulators important for development. Lineage segregation during development and stem cell differentiation depends on transcriptional regulation and is accompanied by cell morphological changes. Much is known about how transcriptional programs regulate cell differentiation and how general cell morphological regulators control cell motility, polarity, and adhesion during development. By contrast, little is known about how lineage specific transcriptional regulation is coupled to correspondingly unique cell morphological changes during the building of tissues and organs. Understanding this coupling at the cellular level is important because it would help to define the steps that stem and progenitor cells take to differentiate.Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of preimplantation mammalian blastocysts , which cESCs can be induced to differentiate into different cell lineages in vitro and they have been used extensively to study transcriptional changes during differentiation . Here, wAll ESCs were cultured in the absence of feeder cells  on gelatin-coated dishes. To image ESC differentiation in a LiveCell™ Chamber , time-lapse recording at multiple positions per well were acquired on an inverted microscope  controlled by IPLab4.0 every 5 minutes for 2 days. Closed-loop feedback from a Z Encoder Probe  was used to control focus drifts. For additional detailed materials and methods, please refer to the Supporting Information.Oct3/4, Sox2, or Nanog , which has also been referred to as Cdc42EP1 (Cdc42 effector protein 1), encodes a protein that binds to RhoGTPases. Since RhoGTPases bind to various effector proteins to regulate cell morphogenesis, Borg5 is likely a morphological regulator that could influence TE specific cell morphogenesis and transcription during differentiation.To identify genes that are upregulated early during TE differentiation, we carried out microarray analyses 12 hours after Tc addition and found that 13 genes were upregulated by at least twofold , 17. AmoBorg5 and found that Borg5 mRNA is strongly upregulated after reduction of Oct3/4 in both ZHBTc4 . Thus, Oct3/4 is likely to indirectly suppress Borg5 expression in ESCs.Using quantitative real-time polymerase chain reaction (qRT-PCR), we characterized the expression of h ZHBTc4  and E14 h ZHBTc4 , but noth ZHBTc4 . Therefo in ESCs , 19 and Borg5 protein is expressed at low levels in ESCs, but becomes upregulated shortly after Oct3/4 reduction in both ZHBTc4  and E14 Borg5 cDNA lacking the 5′-untranslated region  or Borg5 cDNA carrying silent mutations (Borg5-ins) that render it insensitive to RNAi (We reasoned that as a RhoGTPase binding protein  Borg5 mi to RNAi  rescued Since Borg5 contains the Cdc42/Rac interactive binding (CRIB) motif, we next asked whether it functions downstream of Cdc42 to regulate cell motility during TE differentiation. We first confirmed that Borg5 interacts with the guanosine triphosphate-bound form of Cdc42 (Cdc42Q61L)  20–22]..20–22]. Borg5 is upregulated during TE differentiation as early as Cdx2, we asked whether it could influence Cdx2 expression. We created ZHBTc4 ESC lines that stably express either S4 short-hairpin RNA (shRNA) or control shRNA (Supporting Information). Both cell lines expressed Oct3/4 and were responsive to Tc, but ZHBTc4-S4 cells failed to upregulate Borg5 as expected  plays a role in TE differentiation upstream of Cdx2 , 23. UsiTE cells are polarized epithelia that occupy the outside position in the blastocyst to enclose the ICM. We designed a cell-sorting assay to test whether Borg5 could be involved in this process. E14 ESCs were mixed with either ZHBTc4 ESCs expressing control (ZHBTc4-Con) or S4 shRNA (ZHBTc4-S4) and Tc was added to initiate TE differentiation from the ZHBTc4 ESCs (Supporting Information). ZHBTc4-Con and ZHBTc4-S4 cells also express GFP, which serves as a marker for identifying these cells. After 42–48 hours of Tc addition, cells were fixed and stained with antibodies to GFP and Oct3/4.In the absence of Tc, ZHBTc4-Con or ZHBTc4-S4 ESCs  intermingled with the E14 ESCs in colonies . HoweverThe above study led us to test whether Borg5 regulates blastocyst formation during preimplantation development. Immunofluorescence staining using affinity purified Borg5 antibodies generated in chicken revealed that Borg5 is localized along the cell–cell borders and in the cytoplasm in the post compaction embryos . PreabsoBorg5 and Cdx2 mRNAs were upregulated during eight-cell to morula stage  differentiate toward different lineages, it is possible to uncover cell morphology regulators that influence transcriptional regulation and vice versa. By analyzing early ESCs differentiation live, we have shown that unique cell morphogenesis indeed accompanies specific lineage differentiation from ESCs. By focusing on the differentiation of the trophectoderm lineage from ESCs, we have identified Borg5 as a morphological regulator that undergoes a similar strong up-regulation to Cdx2, a key transcription factor for trophectoderm differentiation. Our studies strongly suggest that Borg5 and Cdx2 reciprocally regulate each other to couple epithelial morphogenesis to transcriptional changes during development. Further study how Borg5 and Cdx2 regulate each other should help to decipher the steps of cell differentiation and tissue morphogenesis.