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Expressed
Terms modified by Expressed Selected AbstractsInhibition of the Activity of Excitatory Amino Acid Transporter 4 Expressed in Xenopus Oocytes After Chronic Exposure to EthanolALCOHOLISM, Issue 7 2008Seung-Yeon Yoo Background:, The extracellular glutamate concentration is tightly controlled by excitatory amino acid transporters (EAATs). EAAT4 is the predominant EAAT in the cerebellar Purkinje cells. Purkinje cells play a critical role in motor coordination and may be an important target for ethanol to cause motor impairments. We designed this study to determine the effects of chronic ethanol exposure on the activity of EAAT4 and evaluate the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in these effects. Methods:, EAAT4 was expressed in Xenopus oocytes following injection of EAAT4 mRNA. Oocytes were incubated with ethanol-containing solution for 24 to 96 hours. Membrane currents induced by l -aspartate were recorded using 2-electrode voltage clamps. Responses were quantified by integration of the current trace and reported in microCoulombs (,C). Results:, Ethanol dose- and time-dependently reduced EAAT4 activity. EAAT4 activity after a 96-hour exposure was significantly decreased compared to the control values at all concentrations tested (10 to 100 mM). Ethanol (50 mM) significantly decreased the Vmax (2.2 ± 0.2 ,C for control vs. 1.6 ± 0.2 ,C for ethanol, n = 18, p < 0.05) of EAAT4 for l -aspartate. Preincubation of ethanol-treated (50 mM for 96 hours) oocytes with phorbol-12-myrisate-13-acetate (100 nM for 10 minutes) abolished the ethanol-induced decrease in EAAT4 activity. While staurosporine (2 ,M for 1 hour) or chelerythrine (100 ,M for 1 hour) significantly decreased EAAT4 activity, no difference was observed in EAAT4 activity among the staurosporine, ethanol, or ethanol plus staurosporine groups. Similarly, EAAT4 activity did not differ among the chelerythrine, ethanol, or ethanol plus chelerythrine groups. Pretreatment of the oocytes with wortmannin (1 ,M for 1 hour) also significantly decreased EAAT4 activity. However, no difference was observed in the wortmannin, ethanol, or ethanol plus wortmannin groups. Conclusions:, The results of this study suggest that chronic ethanol exposure decreases EAAT4 activity and that PKC and PI3K may be involved in these effects. These effects of ethanol on EAAT4 may cause an increase in peri-Purkinje cellular glutamate concentration, and may be involved in cerebellar dysfunction and motor impairment after chronic ethanol ingestion. [source] Is there a SSRI dose response in treating major depression?DEPRESSION AND ANXIETY, Issue 1 2003The case for re-analysis of current data, for enhancing future study design Abstract It has been widely stated that the available research data has not demonstrated a SSRI dose response for major depression. We re-evaluated the methods used to analyze the SSRI data by clarifying two key alternative definitions of dose response and their implications for enhancing analysis of currently available data as well as future study design. We differentiated "potential" dose response, which focuses exclusively on response excluding tolerability effects and asks whether differences in dose can result in significant differences in response, from "expressed" dose response, which incorporates all tolerability effects currently associated with dose (including those caused by study protocol or treatment practice) and asks whether differences in dose do result in significant differences in response. To analyze potential dose response for all studies, one should use a "dose-tolerant" sample, i.e., an ITT sample from which dropouts due to adverse events have been removed. To analyze an expressed dose response, an ITT sample is the optimum sample if the study conforms to several design specifications. In the absence of conformance to these specifications, an ITT sample may be an approximation of the appropriate sample. Given design limitations of currently available studies, a dose-tolerant sample may provide a more informative approximation of an optimal sample to be used in evaluating the expressed dose response that could be expected in the best clinical practice. Future studies of dose-response relations could be enhanced by taking into account the principles noted above, and currently available data should be reanalyzed based on these principles. This re-analysis is performed in a companion article [Baker et al. 2003, Depress Anxiety 17:1-9]. Depression and Anxiety 17:10,18, 2003. © 2003 Wiley-Liss, Inc. [source] Morphometry and sexual dimorphism of the coastal spotted dolphin, Stenella attenuata graffmani, from Bahía de Banderas, MexicoACTA ZOOLOGICA, Issue 4 2004Laura Sanvicente-Añorve Abstract External measurements and size differences between the sexes were examined in the coastal spotted dolphin, Stenella attenuata graffmani, in Bahía de Banderas, on the Mexican Pacific coast. The dolphins were collected by local fishermen and 29 external characteristics were measured by members of the Marine Mammals Laboratory, University of Mexico. The length of each characteristic with respect to total length was analysed through adjustment of the data to a power equation. A stepwise discriminant analysis was applied to the absolute values and to those expressed as proportions to analyse the differences between the sexes. Results indicate that growth in these dolphins is generally negatively allometric, and most of the characteristics measured were, in both absolute and proportional terms, greater in male dolphins than in female dolphins. As found in many species of odontocetes, the discriminant analysis showed that the main differences between the sexes for this coastal subspecies include the relative positions of the umbilicus, the genital aperture and the anus. The morphometric data provided by this study, corresponding to 29 specimens of S. a. graffmani collected in a restricted locality of the Mexican Pacific coast, are particularly interesting to studies documenting latitudinal morphological differences in the coastal spotted dolphin. [source] Competitive binding comparison of endocrine-disrupting compounds to recombinant androgen receptor from fathead minnow, rainbow trout, and humanENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2007Vickie S. Wilson Abstract Typically, in vitro hazard assessments for the identification of endocrine-disrupting compounds (EDCs), including those outlined in the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Tier 1 Screening protocols, utilize mammalian receptors. Evidence, however, exists that fish sex steroid hormone receptors differ from mammalian receptors both structurally and in their binding affinities for some steroids and environmental chemicals. Most of the binding studies to date have been conducted using cytosolic preparations from various tissues. In the present study, we compare competitive binding of a set of compounds to full-length recombinant rainbow trout androgen receptor , (rtAR), fathead minnow androgen receptor (fhAR), and human androgen receptor (hAR), each expressed in COS cells. Saturation binding and subsequent Scatchard analysis using [3H]R1881, a high-affinity synthetic androgen, revealed an equilibrium dissociation constant (Kd) of 0.11 nM for the rtAR, 1.8 nM for the fhAR, and 0.84 nM for the hAR. Compounds, including endogenous and synthetic steroids, known mammalian antiandrogens, and environmental compounds, were tested for competitive binding to each of the three receptors. Overall, agreement existed across receptors as to binding versus nonbinding for all compounds tested in this study. Minor differences, however, were found in the relative order of binding of the compounds to the individual receptors. Studies such as these will facilitate the identification of EDCs that may differentially affect specific species and aid in the development and support of future risk assessment protocols. [source] Metallothionein gene expression and protein levels in triploid and diploid oysters Crassostrea gigas after exposure to cadmium and zincENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2006Véronique Marie Abstract Quantitative real-time polymerase chain reaction (PCR) was used to compare for the first time the differential expression of metallothionein (MT) isoform genes, together with biosynthesis of the total MT proteins, in the gills of triploid and diploid juvenile Pacific oyster Crassostrea gigas in response to cadmium (Cd) and zinc (Zn) exposure. Oysters were exposed to Cd (0.133 ,M), Zn (15.3 ,M), and Cd+Zn for 14 d. Results showed similar response capacities to metal exposures in the two populations. No significant difference was revealed in terms of MT gene expression, MT protein synthesis, and Cd accumulation. However, triploid oysters bioaccumulated Zn 30% less efficiently than diploid oysters. Among the three MT isoform genes, CgMT2 appeared to be more expressed than CgMT1, whereas CgMT3 appeared to be anecdotal (106 times lower than CgMT2). CgMT2 and CgMT1 gene expression levels were increased sevenfold in the presence of Cd, whereas Zn appeared to have no effect. A twofold increase in MT protein levels occurred in response to Cd exposure. Discrepancies between mRNA and protein levels suggest that in C. gigas MT are regulated at the transcriptional level, as well as at the translational level. [source] Lep d 2 polymorphisms in wild and cultured Lepidoglyphus destructor mitesFEBS JOURNAL, Issue 4 2003Liselotte Kaiser We have previously cloned, expressed and characterized two variants of the major allergen Lep d 2 from cultured Lepidoglyphus destructor mites. These variants, Lep d 2.0101 and Lep d 2.0201, differ at 13 amino acid positions. In this study we investigated Lep d 2 sequence diversity between wild and cultured mites. PCR, Southern blot and DNA sequence analysis revealed the presence of two different Lep d 2 genes, one with and one without an intron. In addition, two new variants of Lep d 2, Lep d 2.0102 and Lep d 2.0202, were found at different frequencies in wild and cultured mites. When we expressed the Lep d 2 variants and compared their IgE binding properties by ELISA inhibition, we found that Lep d 2.0102 was a more potent inhibitor than Lep d 2.0101, and to a lesser extent Lep d 2.0202 was more potent than Lep d 2.0201. Long-term cultures of peripheral blood mononuclear cells were used to assess the ability of the expressed Lep d 2 variants to induce cytokine release. Although cells from different individuals released different amounts of interferon-, and interleukin-5, no consistent cytokine release pattern could be linked to any specific Lep d 2 variant. In conclusion, we show that both cultured and wild Lepidoglyphus destructor mites contain the same pattern of polymorphism. Furthermore, this Lep d 2 sequence diversity seems not to have any significant impact on the allergens IgE binding or its ability to induce T cell cytokine release. [source] Characterization of a human alternatively spliced truncated reduced folate carrier increasing folate accumulation in parental leukemia cellsFEBS JOURNAL, Issue 3 2000Stavit Drori Human CEM-7A cells established by gradual deprivation of leucovorin from the growth medium, display 100-fold overexpression of methotrexate transport activity. We found that this was associated with 10-fold reduced folate carrier gene amplification and 50-fold overexpression of both the principal 3 kb reduced folate carrier transcript and, surprisingly, a novel truncated 2 kb reduced folate carrier mRNA poorly expressed in parental CEM cells. The molecular basis for the generation of this truncated reduced folate carrier transcript and its potential functional role in folate accumulation were studied. Reduced folate carrier genomic and cDNA sequencing revealed that the truncated transcript had an internal deletion of 987 nucleotides which was a result of an alternative splicing utilizing a cryptic acceptor splice site within exon 6. This deletion consisted of the 3,-most 480 nucleotides of the reduced folate carrier ORF and the following 507 nucleotides of the 3,-UTR. These resulted in a truncated reduced folate carrier protein, which lacks the C-terminal 160 amino acids, but instead contains 58 new C-terminal amino acids obtained from reading through the 3,-UTR. Consequently, a truncated reduced folate carrier protein is generated that lacks the 12th transmembrane domain and contains a new and much shorter C-terminus predicted to reside at the extracellular face. Western analysis with plasma-membrane fraction from CEM-7A cells revealed marked overexpression of both a broadly migrating , 65,90 kDa native reduced folate carrier and a , 40,45 kDa truncated reduced folate carrier, the core molecular masses of which were confirmed by in vitro translation. However, unlike the native reduced folate carrier, the truncated reduced folate carrier protein failed to bind the affinity labels NHS-[3H]MTX and NHS-[3H]folic acid. Stable transfection of the truncated reduced folate carrier cDNA into mouse L1210 leukemia cells: increased folate accumulation, decreased their leucovorin and folic acid growth requirements, and increased their sensitivity to methotrexate. This constitutes the first documentation of an expressed alternatively spliced truncated reduced folate carrier that, when coexpressed along with the native carrier, augments folate accumulation and consequently decreases the cellular folate growth requirement. The possible mechanisms by which the truncated reduced folate carrier may increase folate accumulation and/or metabolism in cells coexpressing the truncated and native reduced folate carrier are discussed. [source] MLL/GRAF fusion in an infant acute monocytic leukemia (AML M5b) with a cytogenetically cryptic ins(5;11)(q31;q23q23)GENES, CHROMOSOMES AND CANCER, Issue 4 2004Ioannis Panagopoulos More than 30 fusions involving the MLL gene at 11q23 have been reported in acute myeloid leukemia (AML). Some of these chimeras are rather common, such as MLL/MLLT3(AF9), but many are quite rare, with some, for example, MLL/GRAF, described only in a single case. The MLL/GRAF fusion, in which the reciprocal hybrid was not expressed, suggesting that the former transcript was the leukemogenic one, was detected in a juvenile myelomonocytic leukemia with a t(5;11)(q31;q23). Here, we report a second case,an infant acute monocytic leukemia (AML M5b),with an MLL/GRAF fusion. By conventional G-banding, the karyotype was normal. However, Southern blot and fluorescence in situ hybridization analyses revealed that MLL was rearranged and that the 5, part of the MLL gene was inserted into 5q in the vicinity of 5q31, which harbors GRAF. Reverse-transcriptase polymerase chain reaction (PCR) showed that exon 9 of MLL was fused in-frame with exon 19 of GRAF. Extralong genomic PCR with subsequent sequence analysis demonstrated that the breakpoints occurred in intron 9 of MLL, nine base pairs (bp) downstream from exon 9, and in intron 18 of GRAF, 117 bp downstream from exon 18. A 6-bp insertion (ACACTC) of unknown origin was present at the junction. The putative MLL/GRAF fusion protein would retain the AT-hook DNA-binding domain, the DNA methyl transferase motif, the transcription repression domain of MLL, and the SH3 domain of GRAF. As expected, the reciprocal GRAF/MLL was neither expressed nor generated at the genomic level as a consequence of the ins(5;11)(q31;q23q23). On the basis of the now-reported two cases with MLL/GRAF, we conclude that this transcript,but not the reciprocal one,characterizes a rare genetic subgroup of infant AML. © 2004 Wiley-Liss, Inc. [source] Epidermal keratinocytes do not activate peripheral T-cells: interleukin-10 as a possible regulatorIMMUNOLOGY, Issue 3 2008Rocío Isabel Domínguez-Castillo Summary The immunogenicity of allogeneic cultured human epidermal keratinocytes (cHEKs) has been studied in several models with contradictory results. We studied human T-cell activation in an in vitro assay by incubating, for 4 and 24 hr, cHEK confluent sheets with human peripheral blood mononuclear cells (PBMC); parallel HEK cultures were incubated with interferon (IFN)-, to induce the expression of major histocompatibility complex (MHC) molecules before their interaction with PBMC. T-cell activation was evaluated by flow cytometry. T cells neither expressed the early and late activation markers CD69 and CD25, respectively, nor proliferated after incubation with the epidermal sheets, despite the IFN-,-induced expression of MHC and adhesion molecules in cHEKs. Interleukin (IL)-10 was detected in the medium from the co-cultured PBMC and HEK sheets, but not from HEK alone. The results suggest that HEKs are unable to stimulate T lymphocytes through secretion of cytokines that might contribute to the immunosuppressive effect in this in vitro model. [source] Role of functional polymorphisms of NRAMP1 gene for the development of Crohn's diseaseINFLAMMATORY BOWEL DISEASES, Issue 10 2008Maria Gazouli PhD Abstract Background: Crohn's disease (CD) is characterized by chronic activation of macrophages. Natural resistance-associated macrophage protein 1 (NRAMP1) gene exerts many pleiotropic effects on macrophage functions. Hence, NRAMP1 may be also involved in the resistance to intracellular pathogens, and this effector of the innate immunity might be involved in CD pathogenesis. Polymorphic alleles at the NRAMP1 locus have been previously associated with susceptibility both to the putative infectious agents and to autoimmune disorders. Based on these indications, in the present study we investigate its candidacy as a genetic determinant for CD in a Greek population in an association-based study, comparing frequencies of 274 CD patients to these of 200 healthy control subjects. Methods: The 5,(GT)n promoter polymorphism and 9 either single nucleotide (SNPs) or insertion/deletion type polymorphisms were genotyped across the NRAMP1 gene. Reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry were performed in order to investigate the NRAMP1 mRNA levels in RNA isolated from biopsies of CD patients as well as protein expression in tissues. Results: Three NRAMP1 polymorphisms [5,(GT)n, D543N, and INT4G/C] were significantly associated with CD. Consistent with previous autoimmune disease studies, allele 3 at the functional 5,(GT)n promoter region repeat polymorphism, was significantly associated with CD when compared to healthy controls (odds ratio 1.50; 95% confidence interval [CI]: 1.16,1.95; P = 0.002). Interestingly, we observed that CD patients homozygous for allele 3 expressed higher NRAMP1 mRNA levels compared to carriers of allele 2. Furthermore, the protein levels of allele 3 carriers in tissues were also elevated compared to those of allele 2 carriers. Based on these data we can speculate that overrepresentation of allele 3 in CD patients could lead to hyperactivation of bowel-wall macrophages that are chronically exposed to lipopolysaccharide and this could subsequently cause the autoimmune-like phenotype characteristic of CD. Conclusions: Collectively, our data indicate that genetic polymorphisms of NRAMP1 might be associated with susceptibility to CD. (Inflamm Bowel Dis 2008) [source] Selectivity of lynx proteins on insect nicotinic acetylcholine receptors in the brown planthopper, Nilaparvata lugensINSECT MOLECULAR BIOLOGY, Issue 3 2010B. Yang Abstract Neuronal nicotinic acetylcholine receptors (nAChRs) are major excitatory neurotransmitter receptors in both vertebrates and invertebrates. Two lynx proteins (Nl-lynx1 and Nl-lynx2) have been identified in the brown planthopper, Nilaparvata lugens, which act as modulators on insect nAChRs. In the present study, two lynx proteins were found to act on the triplet receptor Nl,1/Nl,2/,2 expressed in Xenopus oocytes, increasing agonist-evoked macroscopic currents, but not changing agonist sensitivity and desensitization properties. Nl-lynx1 and Nl-lynx2 increased Imax (maximum responses) of acetylcholine to 4.85-fold and 2.40-fold of that of Nl,1/Nl,2/,2 alone, and they also increased Imax of imidacloprid to 2.57-fold and 1.25-fold. Although, on another triplet nAChRs Nl,3/Nl,8/,2, Nl-lynx2 increased Imax of acetylcholine and imidacloprid to 3.63-fold and 2.16-fold, Nl-lynx1 had no effects on Imax of either acetylcholine or imidacloprid. The results demonstrate the selectivity of lynx proteins for different insect nAChR subtypes. This selectivity was also identified in native N. Lugens. Co-immunoprecipitation was found between Nl,1/Nl,2-containing receptors and both Nl-lynx1 and Nl-lynx2, but was only found between Nl,3/Nl,8-containing receptors and Nl-lynx2. When the previously identified Nl,1Y151S and Nl,3Y151S mutations were included (Nl,1Y151S/Nl,2/,2 and Nl,3Y151S/Nl,8/,2), the increase in Imax of imidacloprid, but not acetylcholine, caused by co-expression of Nl-lynx1 and Nl-lynx2 was more noticeable than that of their wildtype counterparts. Taken together, these data suggest that two modulators, Nl-lynx1 and Nl-lynx2, might serve as an influencing factor in target site insensitivity in N. lugens, such as Y151S mutation. [source] How different are luminal A and basal breast cancers?INTERNATIONAL JOURNAL OF CANCER, Issue 6 2009François Bertucci Abstract Heterogeneity of breast cancer makes its evolution difficult to predict, and its treatment far from being optimal. At least 5 main molecular subtypes exist. Two major subtypes are luminal A and basal subtypes, which have opposite features, notably survival. To characterize these 2 subtypes better, with the hope of better understanding their different biology and clinical outcome, we have profiled a series of 138 tumours (80 luminal A and 58 basal) using Affymetrix whole-genome DNA microarrays. We have identified 5,621 probe sets as differentially expressed between the 2 subtypes in our series. These differences were validated in 6 independent public series (more than 600 tumours) profiled using different DNA microarrays platforms. Analysis of functions and pathways related to these probe sets, and the extent of the observed differences, confirmed that the 2 subtypes represent very distinct entities. Genes associated with proliferation, cell cycle, cell motility, angiogenesis, and NFkB signalling were overexpressed in basal tumours. Genes involved in fatty acid metabolism, TGFB signalling, and oestrogen receptor (ER) signalling were overexpressed in luminal A samples. Half of the genes overexpressed in luminal tumours contained ER-binding sites. The number of differentially expressed genes was as high as the set of genes discriminating 2 cancers of different anatomical origin (breast and colon) or discriminating acute myeloid and lymphoid leukaemia. We provide a comprehensive list of genes/pathways that define potential diagnostic, prognostic and therapeutic targets for these 2 subtypes, which should be treated differently given the profound differences observed at the molecular level. © 2008 Wiley-Liss, Inc. [source] Expression of multiple human endogenous retrovirus surface envelope proteins in ovarian cancerINTERNATIONAL JOURNAL OF CANCER, Issue 1 2007Feng Wang-Johanning Abstract Individual classes of human endogenous retrovirus (HERV) genes and proteins are expressed in cancer, but expression of more than one type of HERV is rare. We report here the expression of multiple HERV genes and proteins in ovarian cell lines and tissues. Expression of HERV-K env mRNA was greater in ovarian epithelial tumors than in normal ovarian tissues (N = 254). The expression of this protein on the surface and in the cytoplasm of ovarian cancer cells was confirmed using anti-HERV-K specific antibody by flow cytometric analysis. The frequency of expression of HERV-K env protein in multitissue microarrays (N = 641) was determined by immunohistochemistry and a significant correlation with tumor histotype was found. A significantly increased expression of HERV-K was observed in tumors with low malignant potential and low grade, relative to expression in normal ovarian tissues. The increase in expression of HERV-K env protein took place in a stepwise fashion in serous papillary adenocarcinoma. Interestingly, we found that other classes of HERV env mRNAs, including ERV3 and HERV-E, are expressed in the same ovarian cancer tissues that expressed HERV-K. Furthermore, anti-HERV antibodies including anti-ERV3 (30%), anti-HERV-E (40%) and anti-HERV-K (55%) were detected in patients with ovarian cancer, but not in normal female controls. HERV env proteins are frequently transcribed and translated in ovarian epithelial tumors, and multiple HERV families are detectable in ovarian cancer. HERV env proteins, and especially those expressed on the cell surface, may serve as novel tumor targets for detection, diagnosis and immunotherapy of ovarian cancer. © 2006 Wiley-Liss, Inc. [source] Responses of large volcanic eruptions in the instrumental and documentary climatic data over Central EuropeINTERNATIONAL JOURNAL OF CLIMATOLOGY, Issue 4 2006Jan Písek Abstract Responses of large volcanic eruptions in selected long temperature series from Austria, the Czech Republic and Germany as well as in three global radiation series in Central Europe are studied. In the example of seven large tropical eruptions (Krakatau 1883; Pelée, Soufriére and Santa María 1902; Agung, 1963; El Chichón, 1982; Mt Pinatubo, 1991) it has been demonstrated that volcanic signal in regional series is not so strongly expressed as in the hemispheric scale owing to different local effects and circulation patterns. This is also valid in the case of two further discussed eruptions of Tambora (1815) and Katmai (1912). The responses of eruptions in areas closer to Central Europe such as Iceland or Italy are more important. In nine analysed cases with VEI = 4,5 with a single exception of the Hekla eruption (1917), cold seasons were observed to follow the eruption. Responses to the Lakagígar eruption (1783) of Iceland with important impacts are also discussed in detail. Moreover, correlation between temperatures (annual and winter half-year series) and NAOI is prevailingly smaller for the period following eruptions than in the period preceding eruptions. The importance of documentary evidence as a valuable source of the information about the impacts of volcanic eruptions is demonstrated. Copyright © 2006 Royal Meteorological Society. [source] Defective enamel ultrastructure in diabetic rodentsINTERNATIONAL JOURNAL OF PAEDIATRIC DENTISTRY, Issue 4 2004M. ATAR Summary. We investigated six different types of diabetic rodents. Four expressed a genetic obesity resulting in diabetes. One developed diabetes induced by a diet-dependent obesity, and one with genetic diabetes received anti-diabetic medication. The tooth samples were examined under a scanning electron microscope and with an energy dispersive microanalysis (EDX). The electron micrographs showed severe, varying degrees of damage within the six different diabetic animal types, such as irregular crystallite deposition and prism perforations in genetically obese animals compared to less-disordered prism structures in diet-dependent obesity. Anti-diabetic medication resulted in normal enamel ultrastructure. The EDX analysis revealed a reduction in the amount of calcium and phosphorus in all regions affected by diabetes. Based on these animal studies, we suggest that both juvenile diabetes type I (in infants) and adult diabetes type II (in pregnant mothers, affecting the developing foetus) may affect the normal development of teeth in humans. [source] The work of health visitors and school nurses with children with psychological and behavioural problemsJOURNAL OF ADVANCED NURSING, Issue 4 2008Philip Wilson Abstract Title., The work of health visitors and school nurses with children with psychological and behavioural problems Aim., This paper is a report of a study to describe the workload of health visitors and school nurses in relation to children and young people with psychological, emotional or behavioural problems, and to identify perceived challenges, obstacles and sources of satisfaction associated with this aspect of their work. Background., There is little published information on the work performed by non-specialist community nurses with children and young people who have psychological, emotional and behavioural problems. Method., We analysed data from a survey conducted in 2002 , 2003 of 1049 Scottish professionals working with children and young people. Data included quantitative responses and free-text describing the cases seen by respondents. Responses from a sub-sample of 71 health visitors and 100 school nurses were analysed using a combination of descriptive statistics and analysis of themes emerging from the text. Findings., Although community-based nurses saw a relatively small number of children with psychological, emotional or behavioural problems each week, dealing with these problems took up a disproportionate amount of time. The commonest types of problem were self-harm, externalizing behaviours and family difficulties. Few respondents had received specific training in child and adolescent mental health but most expressed a wish to receive such training. Conclusion., The work of health visitors and school nurses in caring for children with mental health problems is substantial and important. Development of their public health role should not be at the expense of this important contribution. There is a need for rigorous evaluation of nursing mental health interventions among children and young people. [source] Differential Expression of Estrogen Receptor-Related Receptor , and Estrogen Receptors , and , in Osteoblasts In Vivo and In Vitro,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2002Edith Bonnelye Abstract The orphan nuclear estrogen receptor-related receptor (ERR) , is expressed by osteoblastic cells, is known to transactivate at least one osteoblast-associated gene osteopontin (OPN) and plays a functional role in osteoprogenitor cell proliferation and differentiation. To dissect further the role of ERR-, in bone formation, we compared its expression to that of the estrogen receptor (ER) , and ER-, in rat calvaria (RC) and fetal tibia in vivo and in RC and rat bone marrow (RBM) cells in vitro. We found that ERR-, is highly and widely expressed in most, if not all, cells in RC cell cultures from early proliferation stages through mineralized nodule formation; ER-, was localized similarly but at lower levels and ER-,, although present, was barely detectable. These patterns of expression in vitro correlated with what we observed in vivo in sections of 21-day fetal RC, in which ERR-, appeared to be more highly expressed than either of the ERs. Interestingly, ERR-, also is highly expressed in RBM cells, while ER-, and ER-, mRNA is expressed, but at lower levels. Moreover, we found that ERR-,, ER-,, and ER-, were all expressed in osteoblasts in fetal and adult tibia whereas they were expressed differentially in calvaria in vivo in subsets of osteoblasts, supporting the hypothesis that ERR-, may interact with one or both of the ERs in those osteoblasts in which they are coexpressed and that all three receptors may be required for bone formation but at different times and for different functions. [source] NFAT expression in human osteoclastsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2005Christopher J. Day Abstract Nuclear factor of activated T-cells cytoplasmic (NFATc) is a family of transcription factors originally identified in T-cells. The gene family is currently known to have four members (NFATc1 through NFATc4) which have roles both within and outside the immune system. We show that NFATc1 is the major induced NFAT in human osteoclasts, with expression greatly exceeding that of NFATc2 through NFATc4. In macrophage-like cells in culture, NFATc1 through NFATc4 are expressed at similar low levels. NFATc1 is comprised of five mRNA transcript variants known to encode three different protein isoforms. The mRNA encoding isoform C (mRNA variant 3) was the most expressed with 38 copies per nanogram followed by isoform B (mRNA variant 5) with 17 copies per nanogram of total RNA. Isoform A (mRNA variant 1) and mRNA variants 2 and 4 made up less than 1% of the total NFATc1 expressed. NFATc1 is activated by calcineurin after calcium-calmodulin signalling. The induction of NFATc1 in osteoclasts was not altered in the presence of cyclosporin A, an inhibitor of calcineurin, suggesting that NFATc1 does not participate in autoregulatory activation of its own promoter. The NFATc1 variants expressed by human osteoclasts are not those normally expressed by effector T-cells but are similar to those seen in naïve T-cells. © 2005 Wiley-Liss, Inc. [source] Expression patterns of hair and epithelial keratins and transcription factors HOXC13, LEF1, and ,-catenin in a malignant pilomatricoma: a histological and immunohistochemical studyJOURNAL OF CUTANEOUS PATHOLOGY, Issue 1 2006Bernard Cribier Background:, We have previously shown that benign pilomatricomas not only maintain the sequential expression of the hair matrix and precortex keratins hHa5 and hHa1 of normal hair follicles in their transitional cell compartment, but also preserve the association of hHa5 expression with that of its regulatory homeoprotein HOXC13 in the lower transitional cell compartment. In contrast, hHa1 expression in the upper transitional cell compartment is uncoupled from the nuclear co-expression of the LEF1/,-catenin complex seen in normal hair follicles (Cribier et al., J Invest Dermatol 2004; 122: 1078). Methods:, Formalin-fixed paraffin sections of the tumor were examined using a panel of mono- and polyclonal hair and epithelial keratin antibodies as well as antibodies against HOXC13, LEF1, and ,-catenin. Results:, Morphologically, the malignant pilomatricoma investigated here clearly deviated from the described major tumor type by a large number of differently sized parakeratotic squamoid whorls emerging within the mass of basaloid cells and surrounded by cells remembering transitional cells, but only rarely containing shadow cells and signs of calcification. We show that hHa5/HOXC13 co-expression was maintained in transitional cell areas, in which hHa1 expression was much stronger than in benign pilomatricomas, but again uncoupled from concomitant nuclear LEF1/,-catenin expression. Surprisingly, however, and in clear contrast to benign pilomatricomas, these transitional cells co-expressed the epithelial keratins K5, K14, and K17, with the latter being as strongly expressed as hHa1, both also staining the entire inner mass of the parakeratotic whorls. Conclusions:, Although the malignant pilomatricoma investigated here was distinctive in that it contained a multitude of parakeratinizing whorls and no signs of calcification, it shared both hHa5/HOXC13 co-expression and disrupted hHa1/,-catenin,LEF1 expression in its transitional cell compartment around the whorls with benign pilomatricomas. However, in clear contrast to the latter, transitional cells of the malignant tumor also strongly expressed the epithelial keratins K5, K14, and K17. We speculate that the observed dominance of the epithelial differentiation pathway over the competing conventional shadow cell differentiation pathway may prevent massive calcification of the tumor. [source] Modification of human papillomavirus-like particle vaccine by insertion of the cross-reactive L2-epitopesJOURNAL OF MEDICAL VIROLOGY, Issue 5 2008Kazunari Kondo Abstract Infection with human papillomavirus 16 (HPV16), which is one of the 15 types of HPV causally associated with cervical cancer and accounts for 50% of the cases, can be prevented in a type-specific manner by an HPV16 virus-like particle (VLP) vaccine comprised of particles of the L1 protein alone. We attempted to modify the VLP vaccine by inserting the HPV16 L2-peptides including cross-neutralization epitopes into the L1 polypeptide. The chimeric L1 had, between L1 amino acids (aa) 430 and 433, the L2 sequence of aa 18,38, 56,75, or 96,115 (with the replacements of S at aa 101 and T at aa 112 with L and S, respectively). The three chimeric L1s were each expressed from the recombinant baculovirus in insect Sf9 cells, and the resultant VLPs were characterized. The chimeric VLPs were shown to present the L2-peptides on their surface. By immunizing rabbits with the VLPs, it was shown that they retained capability to induce the antibody neutralizing HPV16 and acquired capability to elicit antibodies cross-neutralizing the infectious HPV18, 31, 52, and 58 pseudovirions. Although the cross-neutralizing titers were lower than the type-specific neutralizing titer, the results suggest that the chimeric VLPs have potential to serve as a vaccine candidate for a broad spectrum of high-risk HPVs. J. Med. Virol. 80:841,846, 2008. © 2008 Wiley-Liss, Inc. [source] KiSS-1 and GPR54 Genes are Co-Expressed in Rat Gonadotrophs and Differentially Regulated In Vivo by Oestradiol and Gonadotrophin-Releasing HormoneJOURNAL OF NEUROENDOCRINOLOGY, Issue 3 2008N. Richard Kisspeptin, the product derived from KiSS-1, and its cognate receptor, GPR54, both exert a role in the neuroendocrine control of reproduction by regulating gonadotrophin-releasing hormone (GnRH) secretion. In the present study, we demonstrate, using dual immunofluorescence with specific antibodies, that the KiSS-1 and GPR54 genes are both expressed in rat gonadotrophs. All luteinising hormone ,-immunoreactive (LH,-ir) cells were stained by the KiSS-1 antibody but some kisspeptin-ir cells were not LH, positive; thus, we cannot exclude the possibility that kisspeptins are expressed in other pituitary cells. All GPR54-ir are co-localised with LH, cells, but only a subset of LH, cells are stained with the GPR54 antibody. Using the real-time reverse transcription-polymerase chain reaction (RT-PCR), we found that the expression of KiSS-1 and GPR54 is differentially regulated by steroids. In the female, KiSS-1 mRNA levels dramatically decreased following ovariectomy (OVX), and this decrease was prevented by administration of 17,-oestradiol (E2), but not by administration of GnRH antagonist or agonist. Administration of E2 in OVX rats receiving either GnRH antagonist or agonist clearly shows that E2 acts directly on the pituitary to positively control KiSS-1 expression. In OVX rats, administration of the selective oestrogen receptor (ER), ligand propylpyrazoletriol, but not the selective ER, ligand diarylpropionitrile, mimics this effect. By contrast, our study shows that GPR54 expression is positively regulated by GnRH and negatively controlled by chronic exposure to E2. In summary, our data document for the first time that, in the female rat pituitary, KiSS-1 expression is up-regulated by oestradiol, similarly to that seen in the anteroventral periventricular nucleus of the hypothalamus. Conversely, GPR54 is up-regulated by GnRH, which exclusively targets gonadotrophs. [source] Exposure of mouse preosteoblasts to pulsed electromagnetic fields reduces the amount of mature, type I collagen in the extracellular matrixJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2006Yoshitada Sakai Abstract We tested the hypothesis that exposure of a mouse preosteoblast cell line to pulsed electromagnetic fields (PEMF) would affect components of the extracellular matrix. We report that exposure of MC3T3-E1 cells to a single PEMF waveform significantly reduced the amount of mature, ,1(I) collagen in the extracellular matrix (ECM) and the conditioned medium, without affecting the amount of total ECM protein. This decrease was not due to changes in the steady-state level of Col1A1 mRNA or to degradation of mature collagen. We then tested the effect of three distinct PEMF waveforms, two orthogonal coil orientations, and two waveform amplitude levels on the amount of ,1(I) collagen in the conditioned medium. A sequence of factorial ANOVAs and stepwise regression modeling revealed that the period (duration) of the individual pulses accounted for a significant proportion of the variance associated with the amount of ,1(I) collagen in the conditioned medium. The total variance accounted for, however, was small (R2,=,0.155, p,<,0.001 and R2,=,0.172, p,<,0.001, in the horizontal and vertical orientations, respectively). The positive and negative regression coefficients for the coil orientations revealed that the influence of pulse period was significantly different for the orthogonal coil orientations (p,<,0.001). The findings imply that the dominant influence of PEMF on the amount of mature, ,1(I) collagen in the ECM is related to variables other than those expressed in the time-amplitude domain. The results provide objective direction toward identifying waveform characteristics that contribute to the observed between-waveform differences with regard to collagen. Advances in this area may lead toward improving waveforms and waveform delivery protocols. © 2005 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res [source] Cerebellar Gene Expression Profiling and eQTL Analysis in Inbred Mouse Strains Selected for Ethanol SensitivityALCOHOLISM, Issue 9 2005Erik J. MacLaren Background: Inbred Long-Sleep (ILS) and Inbred Short-Sleep (ISS) mice exhibit striking differences in a number of alcohol and drug related behaviors. This study examined the expression levels of more than 39,000 transcripts in these strains in the cerebellum, a major target of ethanol's actions in the CNS, to find differentially expressed (DE) candidate genes for these phenotypes. Methods: Genes that were differentially expressed between the strains were identified using oligonucleotide arrays as well as complimentary DNA arrays. Sequence alignment was used to locate DE genes in the mouse genome assembly. In silico expression QTL (eQTL) mapping was used to identify chromosomal regions likely to control the transcription level of DE genes, and the EASE program identified overrepresented functional themes. The genomic region immediately upstream of the cyclase associated protein homolog 1 (Cap1) gene was directly sequenced from PCR products. Results: Nearly 300 genes were identified as differentially expressed between the cerebella of ILS and ISS. These genes and their corresponding eQTLs map to genomic regions linked to several phenotypes that differ between the ILS and ISS strains, including ethanol preference and cocaine-induced locomotor activation on Chromosomes 4 and 7 respectively. Eight genes were cross-platform validated, four of which are more highly expressed in ILS cerebellum. Three SNPs, one of which disrupts a predicted Sp1 binding site, were found in the upstream region of Cap1, a strong candidate for influencing ethanol phenotypes. Conclusions: Many of these DE genes are candidates to influence ethanol and drug regulated phenotypes because they either map to ethanol related QTLs in the genome or are linked to them through eQTL mapping. Genes involved in calcium ion binding and transcriptional regulation are overrepresented and therefore these gene classes may influence ethanol behaviors in mice and humans. [source] Glycoprotein Ib,IX-mediated activation of integrin ,IIb,3: effects of receptor clustering and von Willebrand factor adhesionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2003M. Arya Summary., The interaction between the platelet glycoprotein (GP) Ib,IX complex and von Willebrand factor (VWF) initiates both hemostasis and pathological thrombosis. This interaction is not only the first adhesive event of platelets at sites of vessel injury, but also facilitates fibrinogen binding to ,IIb,3, which subsequently results in platelet aggregation. Since it has been suggested that GP Ib,IX clustering may promote platelet activation, we investigated the effect of such clustering on both VWF,GP Ib,IX and fibrinogen,,IIb,3 bonds using optical tweezers. In our system, fusion of tandem repeats of FK506-binding protein (FKBP) to the cytoplasmic tail of the GP IX subunit of the GP Ib,IX complex allowed subsequent receptor clustering within the plasma membrane by the bivalent, cell-permeant small molecule ligand AP20187. We measured binding forces between polystyrene beads coated with either plasma-derived VWF or the VWF A1 domain and GP Ib,IX(FKBP)2, and those between fibrinogen-coated beads and ,IIb,3 expressed on Chinese hamster ovary cells. The minimal detachment force between GP Ib,IX(FKBP)2 and A1 or plasma-derived VWF doubled after AP20187 was added. The binding force between immobilized fibrinogen and ,IIb,3 was not changed by the clustering agent; however, the strength of single fibrinogen,,IIb,3 bonds increased significantly after ligation of GP Ib,IX(FKBP)2 by A1. These results demonstrate that GP Ib,IX clustering increases the overall strength of its interaction with VWF. Furthermore, signals from GP Ib,IX can activate ,IIb,3, thereby increasing the strength of its interaction with fibrinogen. [source] Expression of the p16INK4a gene and methylation pattern of CpG sites in the promoter region in rat tumor cell linesMOLECULAR CARCINOGENESIS, Issue 1 2004Kanya Honoki Abstract Loss of p16INK4a protein expression has frequently been related to DNA methylation in association with gene silencing. Although the methylation status of exon1, for p16INK4a involvement in various cancers has been extensively analyzed, it has been pointed out that some inconsistencies existed in its relationship to gene silencing of p16INK4a. In this study, we focused on the expression and methylation status in the regions of nt ,478 to ,201, containing a putative TATA box (nt ,401 to ,396), and nt ,233 to 26, both in a recently cloned 5, upstream region of rat p16INK4a. We showed that rat lung adenocarcinoma RLCNR did not express the p16INK4a gene, whereas rat osteosarcoma COS1NR and malignant fibrous histiocytoma MFH1NR both expressed it at levels similar to normal fibroblasts, even though the region of nt ,233 to 26 was hypermethylated in COS1NR rather than RLCNR. In contrast, the CpG islands near the putative TATA box region were consistently methylated in RLCNR, but not in COS1NR and MFH1NR, as well as in normal fibroblasts. Treatment with 5-aza 2,-deoxycytidine induced expression of p16INK4a gene in RLCNR after 48 h, but no changes were observed in COS1NR and MFH1NR. The results indicated that methylation of CpG islands near a TATA box region played a critical role for gene silencing of the rat p16INK4a gene, rather than that of other regions. © 2003 Wiley-Liss, Inc. [source] Fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) genes expressed during infection of cotton (Gossypium hirsutum),MOLECULAR PLANT PATHOLOGY, Issue 2 2006HELEN G. MCFADDEN SUMMARY We sought to identify Fusarium oxysporum f. sp. vasinfectum (Fov) genes that may be associated with pathogenicity. Initially we utilized microarray and Q-PCR technology to identify Fov genes expressed in root and hypocotyl tissues during a compatible infection of cotton. We identified 218 fungal clones representing 174 Fov non-redundant genes as expressed in planta. The majority of the expressed sequences were expressed in infected roots, with only six genes detected in hypocotyl tissue. The Fov genes identified were predominately of unknown function or associated with fungal growth and energy production. We then analysed the expression of the identified fungal genes in infected roots and in saprophytically grown mycelia and identified 11 genes preferentially expressed in plant tissue. A putative oxidoreductase gene (with homology to AtsC) was found to be highly preferentially expressed in planta. In Agrobacterium tumefaciens, AtsC is associated with virulence. Inoculation of a susceptible and a partially resistant cotton cultivar with either a pathogenic or a non-pathogenic isolate of Fov revealed that the expression of the Fov AtsC homologue was associated with pathogenicity and disease symptom formation. [source] A novel group of multi-GAP-domain proteinsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 10 2008Yibing Ruan Abstract The Rho GTPase-activating proteins (RhoGAPs) play an essential role in regulating various cellular processes. Rat tGAP1 is the first reported protein that has multiple GAP domains. It is exclusively expressed in male germ cells. However, tGAP1 does not possess GAP activities in vitro. No tGAP1 homology has been identified in other species. In this study, we searched the genomic databases and identified many genes whose protein products possess 2,4 GAP domains in rat, mouse and dog. These genes all showed sequence similarity to tGAP1. The rat tGAP gene loci all locate on chromosome 2 and are all expressed in testes in RT-PCR analysis. The mouse tGAP gene loci also clustered on chromosome 3 but RT-PCR analysis showed most are pseudogene loci. Multiple sequence alignment showed that many conserved residues of the "arginine finger" motif within the GAP domains of predicted tGAP proteins have mutated, suggesting that tGAP proteins do not possess GAP activity. We also elucidated the evolutionary relations among the rat tGAP genes. Based on the phylogenetic analysis data, we proposed that tGAP genes and Arhgap20 genes have a common ancestor. Mol. Reprod. Dev. 75: 1578,1589 © 2008 Wiley-Liss, Inc. [source] Protease-activated receptor-4 (PAR4): a role as inhibitor of visceral pain and hypersensitivityNEUROGASTROENTEROLOGY & MOTILITY, Issue 11 2009C. Augé Abstract, Protease-activated receptor-4 (PAR4) belongs to the family of receptors activated by the proteolytic cleavage of their extracellular N-terminal domain and the subsequent binding of the newly released N-terminus. While largely expressed in the colon, the role of PAR4 in gut functions has not been defined. We have investigated the effects of PAR4 agonist on colonic sensations and sensory neuron signalling, and its role in visceral pain. We observed that a single administration of the PAR4 agonist peptide (AYPGKF-NH2), but not the control peptide (YAPGKF-NH2) into the colon lumen of mice significantly reduced the visceromotor response to colorectal distension at different pressures of distension. Further, intracolonic administration of the PAR4 agonist, but not the control peptide, was able to significantly inhibit PAR2 agonist- and transcient receptor potential vanilloid-4 (TRPV4) agonist-induced allodynia and hyperalgesia in response to colorectal distension. Protease-activated receptor-4 was detected in sensory neurons projecting from the colon, and isolated from the dorsal root ganglia, where it co-expressed with PAR2 and TRPV4. In total sensory neurons, PAR4 agonist exposure inhibited free intracellular calcium mobilization induced by the pro-nociceptive agonists of PAR2 and TRPV4. Finally, PAR4 -deficient mice experienced increased pain behaviour in response to intracolonic administration of mustard oil, compared with wild-type littermates. These results show that PAR4 agonists modulate colonic nociceptive response, inhibit colonic hypersensitivity and primary afferent responses to pro-nociceptive mediators. Endogenous activation of PAR4 also plays a major role in controlling visceral pain. These results identify PAR4 as a previously unknown modulator of visceral nociception. [source] Bottom-up cell proliferation with cyclin A and p27Kip1 expression in ulcerative colitis-associated dysplasiaPATHOLOGY INTERNATIONAL, Issue 1 2006Tetuo Mikami To analyze the cell kinetics of ulcerative colitis (UC)-associated dysplasia, cyclin A, cyclin D1, cyclin E, cdk2, cdk4, p21Waf1, and p27Kip1 were immunohistochemically examined, in comparison with sporadic tubular adenomas. Immunohistochemical labeling indices for each marker in formalin-fixed paraffin-embedded tissue sections were assessed in a total of 23 low-grade dysplasias, 27 high-grade dysplasias, and 14 invasive adenocarcinomas associated with UC. For comparison, 21 sporadic tubular adenomas with low-grade dysplasia, 33 with high-grade dysplasia, and 21 invasive adenocarcinomas were also examined. In UC-associated dysplasias, cyclin A and p27Kip1 were located in the lower parts of the crypts and p21Waf1 in the upper regions. In tubular adenomas, cyclin A, cdk4, p27Kip1, and p21Waf1 were all expressed in the upper parts of the crypts. The expression levels of cyclin D1, cyclin E, and cdk2 were low. The cell proliferation zone in UC-associated dysplasia is located towards the bases of the crypts with the strong expression of cyclin A and p27Kip1, in contrast to tubular adenomas, which have their cell proliferation zone in the upper parts of neoplastic crypts. It is considered that tumorigenesis with UC-associated dysplasia is of the bottom-up type, related to altered expression of cyclin A and p27Kip1. [source] Biochemical and immunological characterization of the plant-derived candidate human immunodeficiency virus type 1 mucosal vaccine CTB,MPR649,684PLANT BIOTECHNOLOGY JOURNAL, Issue 2 2009Nobuyuki Matoba Summary Plants are potentially the most economical platforms for the large-scale production of recombinant proteins. Thus, plant-based expression of subunit human immunodeficiency virus type 1 (HIV-1) vaccines provides an opportunity for their global use against the acquired immunodeficiency syndrome pandemic. CTB,MPR649,684[CTB, cholera toxin B subunit; MPR, membrane proximal (ectodomain) region of gp41] is an HIV-1 vaccine candidate that has been shown previously to induce antibodies that block a pathway of HIV-1 mucosal transmission. In this article, the molecular characterization of CTB,MPR649,684 expressed in transgenic Nicotiana benthamiana plants is reported. Virtually all of the CTB,MPR649,684 proteins expressed in the selected line were shown to have assembled into pentameric, GM1 ganglioside-binding complexes. Detailed biochemical analyses on the purified protein revealed that it was N- glycosylated, predominantly with high-mannose-type glycans (more than 75%), as predicted from a consensus asparagine,X,serine/threonine (Asn-X-Ser/Thr) N- glycosylation sequon on the CTB domain and an endoplasmic reticulum retention signal attached at the C-terminus of the fusion protein. Despite this modification, the plant-expressed protein retained the nanomolar affinity to GM1 ganglioside and the critical antigenicity of the MPR649,684 moiety. Furthermore, the protein induced mucosal and serum anti-MPR649,684 antibodies in mice after mucosal prime-systemic boost immunization. Our data indicate that plant-based expression can be a viable alternative for the production of this subunit HIV-1 vaccine candidate. [source] | |||||||||||||||||||||||||||||||