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Predominant Expression (predominant + expression)
Selected AbstractsPredominant Expression of Mutant EGFR (EGFRvIII) is Rare in Primary GlioblastomasBRAIN PATHOLOGY, Issue 2 2004Wojciech Biernat EGFR amplification is a frequent genetic alteration in primary (de novo) glioblastomas, and is often associated with structural alterations. Most common is variant III (EGFRvIII), which results from a non-random 801 bp in-frame deletion of exons 2 to 7 of the EGFR gene. We assessed amplification and overexpression of EGFRvIII and wild-type EGFR in 30 glioblastoma biopsies. Immunohistochemically, EGFR overexpression was observed in 20 (67%) of 30 glioblastomas. Eight (27%) cases also showed immunoreactivity to an EGFRvIII antibody. In 6 of these cases, the pattern of EGFR and EGFRvIII overexpression was compared in serial sections: In 4 cases, areas with immunoreactivity to EGFRvIII largely coincided with wild-type EGFR expression. In the other 2 cases, the areas immunoreactive to EGFRvIII were significantly less extensive than EGFR-positive areas. To assess whether EGFRvIII is predominantly amplified in tumors with concurrent wild-type EGFR amplification, we carried out real-time quantitative PCR using 2 sets of primers located in exon 2 and intron 15 of the EGFR gene. A>5-fold ratio of relative copy numbers between intron 15 (present both in wild-type EGFR and EGFRvIII) and exon 2 (present only in wild-type EGFR, but missing in EGFRvIII) suggested predominant amplification of EGFRvIII in only 3 (10%) of 30 glioblastomas. The observation that intratumoral wild-type EGFR overexpression is often more extensive and that predominant amplification of EGFRvIII is a rare event would limit the effectiveness of therapeutic approaches based on selective targeting of EGFRvIII. [source] Genetically Engineered Phage Fibers and Coatings for Antibacterial ApplicationsADVANCED FUNCTIONAL MATERIALS, Issue 2 2010Joan Y. Mao Abstract Multifunctionality can be imparted to protein-based fibers and coatings via either synthetic or biological approaches. Here, potent antimicrobial functionality of genetically engineered, phage-based fibers and fiber coatings, processed at room temperature, is demonstrated. Facile genetic engineering of the M13 virus (bacteriophage) genome leverages the well-known antibacterial properties of silver ions to kill bacteria. Predominant expression of negatively charged glutamic acid (E3) peptides on the pVIII major coat proteins of M13 bacteriophage enables solution-based, electrostatic binding of silver ions and subsequent reduction to metallic silver along the virus length. Antibacterial fibers of micrometer-scale diameters are constructed from such an E3-modified phage via wet-spinning and glutaraldehyde-crosslinking of the E3-modified viruses. Silverization of the free-standing fibers is confirmed via energy dispersive spectroscopy and inductively coupled plasma atomic emission spectroscopy, showing ,0.61,µg cm,1 of silver on E3,Ag fibers. This degree of silverization is threefold greater than that attainable for the unmodified M13,Ag fibers. Conferred bactericidal functionality is determined via live,dead staining and a modified disk-diffusion (Kirby,Bauer) measure of zone of inhibition (ZoI) against Staphylococcus epidermidis and Escherichia coli bacterial strains. Live,dead staining and ZoI distance measurements indicate increased bactericidal activity in the genetically engineered, silverized phage fibers. Coating of Kevlar fibers with silverized E3 phage exhibits antibacterial effects as well, with relatively smaller ZoIs attributable to the lower degree of silver loading attainable in these coatings. Such antimicrobial functionality is amenable to rapid incorporation within fiber-based textiles to reduce risks of infection, biofilm formation, or odor-based detection, with the potential to exploit the additional electronic and thermal conductivity of fully silverized phage fibers and coatings. [source] Myosin16b: The COOH-tail region directs localization to the nucleus and overexpression delays S-phase progressionCYTOSKELETON, Issue 1 2007Richard S. Cameron Abstract Rat Myo16a and Myo16b comprise the founding members of class XVI myosin and are characterized by an N-terminal ankyrin repeat domain thought to mediate an association with protein phosphatase 1 catalytic subunits 1, and 1,. Myo16b is the principal isoform and reveals predominant expression in developing neural tissue. Here, we use COS-7 cells as a model system to develop an understanding of Myo16b function. We find that Myo16b displays predominant localization in the nucleus of cells transitioning through interphase, but is not associated with processes of mitosis. Using a panel of EGFP-Myo16b-expression plasmids in transient transfection studies, we identified the COOH-terminal residues 1616,1912 as necessary and solely sufficient to target Myo16b to the nucleus. We show that the Myo16b-tail region directs localization to a nuclear compartment containing profilin and polymerized actin, which appears to form a three-dimensional meshwork through the depth of the nucleus. Further, we demonstrate that this compartment localizes within euchromatic regions of the genome and contains proliferating cell nuclear antigen (PCNA) and cyclin A, both markers of S-phase of the cell cycle. Cells transiently expressing Myo16b or Myo16b-tail region show limited incorporation of BrdU, delayed progression through S-phase of the cell cycle, and curtailed cellular proliferation. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Over-expression of CCL3,,MIP-1, in a blastoid mantle cell lymphoma with hypercalcemiaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2010Norimichi Hattori Abstract We analyzed a case with the blastoid variant of mantle cell lymphoma (MCL-BV), a rare subtype of B-cell lymphoma, presenting with marked hypercalcemia at diagnosis. Enzyme-linked immunosorbent assay (ELISA) showed elevated serum levels of interleukin-6 (IL-6), tumor necrosis factor-, (TNF-,), macrophage inflammatory protein-1, (MIP-1,), and type I collagen telopeptide, but not parathyroid hormone, calcitriol or parathyroid hormone-related peptide at diagnosis, suggesting local osteoclastic hypercalcemia in this case. By reverse transcription polymerase chain reaction (RT-PCR) analysis, we found predominant expression of mRNA for MIP-1, in addition to those for receptor-activator of nuclear-factor kappa B ligand (RANKL), TNF-,, and IL-6 in lymphoma cells obtained from the patient. Furthermore, recombinant MIP-1, significantly stimulated 3H-thymidine uptake by isolated MCL cells in vitro. Treatment with intravenous fluids, bisphosphonate, and methylprednisolone followed by combination chemotherapy promptly corrects the hypercalcemia and successfully induced complete remission, which was accompanied by a decrease of these cytokines in the serum, including MIP-1,. In the present case, MIP-1,, an osteoclast-activating factor produced by mantle lymphoma cells, may contribute to the development of hypercalcemia. It likely acts through RANKL expression in tumor cells and/or stroma cells, as indicated in multiple myeloma (MM) and adult T-cell leukemia/lymphoma (ATLL). Furthermore, MIP-1, is also involved in the development of an aggressive phenotype on MCL by stimulating proliferation of these lymphoma cells. In summary, the present study demonstrated that MIP-1, is an important factor in the development of both hypercalcemia and an aggressive phenotype in some types of B-cell lymphoma. [source] Evolution and human tissue expression of the Cres/Testatin subgroup genes, a reproductive tissue specific subgroup of the type 2 cystatinsEVOLUTION AND DEVELOPMENT, Issue 3 2010Jessica Frygelius SUMMARY The cystatin family comprises a group of generally broadly expressed protease inhibitors. The Cres/Testatin subgroup (CTES) genes within the type 2 cystatins differs from the classical type 2 cystatins in having a strikingly reproductive tissue-specific expression, and putative functions in reproduction have therefore been discussed. We have performed evolutionary studies of the CTES genes based on gene searches in genomes from 11 species. Ancestors of the cystatin family can be traced back to plants. We have localized the evolutionary origin of the CTES genes to the split of marsupial and placental mammals. A model for the evolution of these genes illustrates that they constitute a dynamic group of genes, which has undergone several gene expansions and we find indications of a high degree of positive selection, in striking contrast to what is seen for the classical cystatin C. We show with phylogenetic relations that the CTES genes are clustered into three original groups, a testatin, a Cres, and a CstL1 group. We have further characterized the expression patterns of all human members of the subfamily. Of a total of nine identified human genes, four express putative functional transcripts with a predominant expression in the male reproductive system. Our results are compatible with a function of this gene family in reproduction. [source] Conservation and variation in Ubx expression among cheliceratesEVOLUTION AND DEVELOPMENT, Issue 6 2001Aleksandar Popadi SUMMARY Chelicerates are an ancient arthropod group with a distinct body plan composed of an anterior (prosoma) and a posterior portion (opisthosoma). The expression of the Hox gene Ultrabithorax (Ubx) has been examined in a single representative of the chelicerates, the spider Cupiennius salei. In spiders, Ubx expression starts in the second opisthosomal segment (O2). Because the first opisthosomal segment (O1) in spiders is greatly reduced relative to other chelicerates, we hypothesized that the observed Ubx expression pattern might be secondarily modified. Shifts in the anterior boundary of the expression of Ubx have been correlated with functional shifts in morphology within malacostracan crustaceans. Thus, the boundary of Ubx expression between chelicerates with different morphologies in their anterior opisthosoma could also be variable. To test this prediction, we examined the expression patterns of Ubx and abdominal-A (collectively referred to as UbdA) in two basal chelicerate lineages, scorpions and xiphosurans (horseshoe crabs), which exhibit variation in the morphology of their anterior opisthosoma. In the scorpion Paruroctonus mesaensis, the anterior border of early expression of UbdA is in a few cells in the medial, posterior region of the O2 segment, with a predominant expression in O3 and posterior. Expression later spreads to encompass the whole O2 segment and a ventral, posterior portion of the O1 segment. In the xiphosuran Limulus polyphemus, early expression of UbdA has an anterior boundary in the segment. Later in development, the anterior boundary moves forward one segment to the chilarial (O1) segment. Thus, the earliest expression boundary of UbdA lies within the second opisthosomal segment in all the chelicerates examined. These results suggest that rather than being derived, the spider UbdA expression in O2 likely reflects the ancestral expression boundary. Changes in the morphology of the first opisthosomal segment are either not associated with changes in UbdA expression or correlate with late developmental changes in UbdA expression. [source] CD44 variant isoform v10 is expressed on tumor-infiltrating lymphocytes and mediates hyaluronan-independent heterotypic cell,cell adhesion to melanoma cellsEXPERIMENTAL DERMATOLOGY, Issue 2 2003T. K. Weimann Abstract: CD44 is a family of cell-surface receptors on human lymphocytes that act as co-stimulatory molecules leading to the induction of effector functions in T cells. We have analyzed primary cutaneous malignant melanomas with clinical and histologic signs of tumor regression using immunohistochemistry and observed the predominant expression of the CD44 variant isoform v10 on CD3 CD4/CD8 co-expressing tumor-infiltrating lymphocytes (TIL). We further analyzed the role of CD44v10 in adhesion of lymphocytes to human melanoma cells. In contrast to CD44, lymphatic cells, CD44v10+ lymphatic cells strongly bound to cultured human melanoma cells and to frozen tissue samples of melanomas. Antibody blocking studies revealed a hyaluronan-, integrin-, and selectin-independent pathway of adhesion. Furthermore, CD44v10+ lymphatic cells exhibited significantly higher invasiveness in three-dimensional collagen matrices as compared with CD44H+ and CD44-negative lymphocytes. These results indicate that expression of CD44v10 on TIL may mediate adhesion to melanoma cells and result in gain of novel invasive properties. [source] Genome-wide identification, classification, evolutionary expansion and expression analyses of homeobox genes in riceFEBS JOURNAL, Issue 11 2008Mukesh Jain Homeobox genes play a critical role in regulating various aspects of plant growth and development. In the present study, we identified a total of 107 homeobox genes in the rice genome and grouped them into ten distinct subfamilies based upon their domain composition and phylogenetic analysis. A significantly large number of homeobox genes are located in the duplicated segments of the rice genome, which suggests that the expansion of homeobox gene family, in large part, might have occurred due to segmental duplications in rice. Furthermore, microarray analysis was performed to elucidate the expression profiles of these genes in different tissues and during various stages of vegetative and reproductive development. Several genes with predominant expression during various stages of panicle and seed development were identified. At least 37 homeobox genes were found to be differentially expressed significantly (more than two-fold; P < 0.05) under various abiotic stress conditions. The results of the study suggest a critical role of homeobox genes in reproductive development and abiotic stress signaling in rice, and will facilitate the selection of candidate genes of agronomic importance for functional validation. [source] The expression of glutathione reductase in the male reproductive system of rats supports the enzymatic basis of glutathione function in spermatogenesisFEBS JOURNAL, Issue 5 2002Tomoko Kaneko Glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) using an NADPH as the electron source. The function of GR in the male genital tract of the rat was examined by measuring its enzymatic activity and examining the gene expression and localization of the protein. Levels of GR activity, the protein, and the corresponding mRNA were the highest in epididymis among testes, vas deferens, seminal vesicle, and prostate gland. The localization of GR, as evidenced by immunohistochemical techniques, reveals that it exists at high levels in the epithelia of the genital tract. In testis, GR is mainly localized in Sertoli cells. The enzymatic activity and protein expression of GR in primary cultured testicular cells confirmed its predominant expression in Sertoli cells. Intracellular GSH levels, expressed as mol per mg protein, was higher in spermatogenic cells than in Sertoli cells. As a result of these findings, the effects of buthionine sulfoximine (BSO), an inhibitor for GSH synthesis, and 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU), an inhibitor for GR, on cultured testicular cells were examined. Sertoli cells were prone to die as the result of BCNU, but not BSO treatment, although intracellular levels of GSH declined more severely with BSO treatment. Spermatogenic cells were less sensitive to these agents than Sertoli cells, which indicates that the contribution of these enzymes is less significant in spermatogenic cells. The results herein suggest that the GR system in Sertoli cells is involved in the supplementation of GSH to spermatogenic cells in which high levels of cysteine are required for protamine synthesis. In turn, the genital tract, the epithelia of which are rich in GR, functions in an antioxidative manner to protect sulfhydryl groups and unsaturated fatty acids in spermatozoa from oxidation during the maturation process and storage. [source] Ca2+ entry through TRPC1 channels contributes to intracellular Ca2+ dynamics and consequent glutamate release from rat astrocytesGLIA, Issue 8 2008Erik B. Malarkey Abstract Astrocytes can respond to a variety of stimuli by elevating their cytoplasmic Ca2+ concentration and can in turn release glutamate to signal adjacent neurons. The majority of this Ca2+ is derived from internal stores while a portion also comes from outside of the cell. Astrocytes use Ca2+ entry through store-operated Ca2+ channels to refill their internal stores. Therefore, we investigated what role this store-operated Ca2+ entry plays in astrocytic Ca2+ responses and subsequent glutamate release. Astrocytes express canonical transient receptor potential (TRPC) channels that have been implicated in mediating store-operated Ca2+ entry. Here, we show that astrocytes in culture and freshly isolated astrocytes from visual cortex express TRPC1, TRPC4, and TRPC5. Indirect immunocytochemistry reveals that these proteins are present throughout the cell; the predominant expression of functionally tested TRPC1, however, is on the plasma membrane. Labeling in freshly isolated astrocytes reveals changes in TRPC expression throughout development. Using an antibody against TRPC1 we were able to block the function of TRPC1 channels and determine their involvement in mechanically and agonist-evoked Ca2+ entry in cultured astrocytes. Blocking TRPC1 was also found to reduce mechanically induced Ca2+ -dependent glutamate release. These data indicate that Ca2+ entry through TRPC1 channels contributes to Ca2+ signaling in astrocytes and the consequent glutamate release from these cells. © 2008 Wiley-Liss, Inc. [source] Identification of the isoforms of Ca2+/calmodulin-dependent protein kinase II and expression of brain-derived neurotrophic factor mRNAs in the substantia nigraJOURNAL OF NEUROCHEMISTRY, Issue 1 2006Akifumi Kamata Abstract Ca2+/calmodulin-dependent protein kinase (CaMK)II is highly expressed in the CNS and mediates activity-dependent neuronal plasticity. Four CaMKII isoforms, ,, ,, , and ,, have a large number of splicing variants. Here we identified isoforms of CaMKII in the rat substantia nigra (SN). Northern blot and RT,PCR analyses revealed that the , and , isoform mRNAs with several splicing variants were predominantly expressed in SN. Immunoblot analysis indicated that the major isoforms were ,A, ,C, ,1 and ,3. An immunohistochemical study also confirmed the preferential localization of , and , isoforms in SN dopaminergic neurons. In dopaminergic neurons, immunoreactivity against anti-CaMKII,1,4 antibody was detected in both nucleus and cytoplasm, in contrast to the predominant expression of , isoforms in the cytoplasm. Furthermore, we showed expression of brain-derived neurotrophic factor (BDNF) mRNAs with exons II and IV in SN. Taken together with our previous observations, the results suggest that the CaMKII,3 isoform is involved in the expression of BDNF in the SN. [source] Characterization of two novel proteins, NgRH1 and NgRH2, structurally and biochemically homologous to the Nogo-66 receptorJOURNAL OF NEUROCHEMISTRY, Issue 3 2003V. Pignot Abstract Nogo-66 receptor (NgR) has recently been identified as the neuronal receptor of the myelin-associated proteins Nogo-A, oligodendrocyte protein (OMgp) and myelin-associated glycoprotein (MAG), and mediates inhibition of axonal regeneration both in vitro and in vivo. Through database searches, we have identified two novel proteins (NgRH1 and NgRH2) that turned out to be homologous in their primary structures, biochemical properties and expression patterns to NgR. Like NgR, the homologues contain eight leucine-rich repeats (LRR) flanked by a leucine-rich repeat C-terminus (LRRCT) and a leucine-rich repeat N-terminus (LRRNT), and also have a C-terminal GPI signal sequence. Northern blot analysis showed predominant expression of NgRH1 and NgRH2 mRNA in the brain. In situ hybridization and immunohistochemistry on rat brain slices revealed neuronal expression of the genes. NgRH1 and NgRH2 were detected on the cell surface of recombinant cell lines as N-glycosylated GPI anchored proteins and, consistent with other GPI anchored proteins, were localized within the lipid rafts of cellular membranes. In addition, an N-terminal proteolytic fragment of NgR comprising the majority of the ectodomain was found to be constitutively secreted from cells. Our data indicate that NgR, NgRH1 and NgRH2 constitute a novel receptor protein family, which may play related roles within the CNS. [source] Expression and molecular diversity of Tcf7l2 in the developing murine cerebellum and brainJOURNAL OF NEUROSCIENCE RESEARCH, Issue 7 2009Tommy A. Nazwar Abstract The Wingless family of secreted proteins impinges on multiple aspects of vertebrate nervous system development, from early global patterning and cell fate decision to synaptogenesis. Here, we mapped the developmental expression of the Tcf7l2, which is key to the canonical Wingless signaling cascade, in the developing cerebellum. The exclusive and transient expression of Tcf7l2 in ventricular and Olig2-defined precursor cells within the cerebellar anlage, and its predominant expression in postmitotic neurons in the midbrain/inferior colliculus allowed us to ask whether cell type,specific differences are also reflected in splice isoform variability. We also included in this analysis intestinal epithelia, where Tcf7l2 function has been intensively studied. Our data reveal extensive variability of Tcf7l2 splicing in the central nervous system. Additional variability in brain-expressed Tcf7l2 is generated by a length polymorphism of expressed mRNAs in a stretch of normally nine adenines found at the beginning of exon 18, reminiscent of variability observed at the same site in cancers with microsatellite instability. A consensus emerging from our data is that the expression of isoforms comprising or lacking the C-clamp motif, which has been linked by in vitro studies to the regulation of cell growth, is indeed tightly correlated with the proliferative status in vivo. © 2009 Wiley-Liss, Inc. [source] Functional Role of ,3-Adrenoreceptors in the BladderLUTS, Issue 2009Masahide HIGAKI The detrusor muscle contains ,-adrenoceptors (,-AR) and three subtypes, such as ,1-AR, ,2-AR, and ,3-AR, which have been identified in most species. There is a predominant expression of ,3-AR messenger RNA in human bladder tissue when compared with the ,1-AR and ,2-AR subtypes. Moreover, the presence of ,1, ,2, and ,3-AR in the human urothelium has been identified. It has also been demonstrated in animals that relaxation mediated through ,s-AR is achieved solely by cAMP-dependent mechanisms in non-contracted detrusor muscles, whereas in KCl precontracted detrusor muscles, cAMP-dependent and -independent mechanisms by way of calcium-activated K +(BK Ca) channels may be involved in ,-adrenergic relaxation. In addition, a recent phase II proof-of-concept study using a novel selective ,3-adrenoceptor agonist (YM178) has shown clinical efficacy in the treatment of overactive bladder (OAB) symptoms, suggesting that ,3-AR should be used as a therapeutic target for the treatment of OAB disorders. [source] A shared promoter region suggests a common ancestor for the human VCX/Y, SPANX, and CSAG gene families and the murine CYPT familyMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2008Martin A. Hansen Abstract Many testis-specific genes from the sex chromosomes are subject to rapid evolution, which can make it difficult to identify murine genes in the human genome. The murine CYPT gene family includes 15 members, but orthologs were undetectable in the human genome. However, using refined homology search, sequences corresponding to the shared promoter region of the CYPT family were identified at 39 loci. Most loci were located immediately upstream of genes belonging to the VCX/Y, SPANX, or CSAG gene families. Sequence comparison of the loci revealed a conserved CYPT promoter-like (CPL) element featuring TATA and CCAAT boxes. The expression of members of the three families harboring the CPL resembled the murine expression of the CYPT family, with weak expression in late pachytene spermatocytes and predominant expression in spermatids, but some genes were also weakly expressed in somatic cells and in other germ cell types. The genomic regions harboring the gene families were rich in direct and inverted segmental duplications (SD), which may facilitate gene conversion and rapid evolution. The conserved CPL and the common expression profiles suggest that the human VCX/Y, SPANX, and CSAG2 gene families together with the murine SPANX gene and the CYPT family may share a common ancestor. Finally, we present evidence that VCX/Y and SPANX may be paralogs with a similar protein structure consisting of C terminal acidic repeats of variable lengths. Mol. Reprod. Dev. 75: 219,229, 2008. © 2007 Wiley-Liss, Inc. [source] ,3 -Adrenoceptors in urinary bladder,,NEUROUROLOGY AND URODYNAMICS, Issue 6 2007Osamu Yamaguchi Abstract The ,-adrenoceptor (AR) is currently classified into ,1, ,2, and ,3 subtypes. A third subtype, ,3 -AR, was first identified in adipose tissue, but has also been identified in smooth muscle tissue, particularly in the gastrointestinal tract and urinary bladder smooth muscle. There is a predominant expression of ,3 -AR messenger RNA (mRNA) in human bladder, with 97% of total ,-AR mRNA being represented by the ,3 -AR subtype and only 1.5 and 1.4% by the ,1 -AR and , 2 -AR subtypes, respectively. Moreover, the presence of ,1 -, ,2 -, and ,3 -AR mRNAs in the urothelium of human bladder has been identified. The distribution of ,-AR subtypes mediating detrusor muscle relaxation is species dependent, the predominant subtype being the ,3 -AR in humans. Recent studies have suggested that cAMP-dependent routes are not exclusive mechanisms triggering the ,-AR-mediated relaxation of smooth muscle. It has been demonstrated in rats detrusor muscle that cAMP plays a greater role in ,-adrenergic relaxation against basal tone than against KCl-induced tone and that conversely calcium-activated K+ channels (BKca channels) play a greater role under the latter circumstances. In rat models, ,3 -AR agonists increase bladder capacity without influencing bladder contraction and have only weak cardiovascular side effects. Although this evidence points toward the clinical utility of ,3 -AR agonists as therapy for overactive bladder (OAB), pharmacological differences exist between rat and human ,3 -ARs. Development of compounds with high selectivity for the human ,3 -AR, identified by screening techniques using cell lines transfected with the human ,1 -, ,2 -, and ,3 -AR genes, may mitigate against such problems. The association between the tryptophan 64 arginine polymorphism in the ,3 -AR gene and idiopathic OAB is discussed. Neurourol. Urodynam. 26:752,756, 2007. © 2007 Wiley-Liss, Inc. [source] Expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic cartilagePATHOLOGY INTERNATIONAL, Issue 11 2007Satoko Naito A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)1, 4, 5, 8, 9 and 15, members of the ADAMTS gene family, have the ability to degrade a major cartilage proteoglycan, aggrecan, at the specific sites, and thus are called ,aggrecanases'. The expression of these ADAMTS species was examined in human osteoarthritic articular cartilage on reverse transcription,polymerase chain reaction. The results demonstrated the predominant expression of ADAMTS4 in osteoarthritic cartilage, while ADAMTS5 was constitutively expressed in osteoarthritic and normal cartilage. ADAMTS9 was expressed mainly in normal cartilage, whereas no or negligible expression of ADAMTS1, 8 and 15 was observed in either osteoarthritic or normal cartilage. In situ hybridization for ADAMTS4 indicated that chondrocytes in osteoarthritic cartilage expressed the mRNA. Two monoclonal antibodies to ADAMTS4 were developed, and immunolocalized ADAMTS4 to chondrocytes in the proteoglycan-depleted zones of osteoarthritic cartilage, showing a direct correlation with the Mankin scores. Immunoblotting indicated a major protein band of 58 kDa in the chondrocyte culture media and osteoarthritic cartilage tissue homogenates. These data demonstrate that among the six ADAMTS species, ADAMTS4 is mainly expressed in an active form in osteoarthritic cartilage, and suggest that ADAMTS4 may play an important role in the degradation of aggrecan in human osteoarthritic cartilage. [source] Expression of Messenger Ribonucleic Acid Encoding for Phosphodiesterase Isoenzymes in Human Female Genital TissuesTHE JOURNAL OF SEXUAL MEDICINE, Issue 6 2007Stefan Uckert PhD ABSTRACT Objectives., The use of inhibitors of phosphodiesterase 5 (PDE5) has been suggested to treat symptoms of female sexual dysfunction (FSD). Nonetheless, there has been a relatively low success rate of PDE5 inhibitors in FSD in comparison with male erectile dysfunction. The elevated expression of PDE5 in the human penile erectile tissue is considered the reason for the high clinical efficacy of PDE5 inhibitors in the pharmacotherapy of male erectile dysfunction. Aim., To evaluate by means of molecular biology the expression of messenger ribonucleic acid expression (mRNA) encoding for cyclic AMP and cyclic GMP PDE isoenzymes in female genital tissues. Main Outcome Measures., The amount of mRNA transcripts specifically encoding for cyclic AMP- and/or cyclic GMP-degrading PDE isoenzymes was determined. Methods., Human clitoral, labial, and vaginal tissue was obtained from four female cadavers (age at death: 18,42 years). The expression of mRNA specifically encoding for PDE1A, 1B, 1C, 2A, 4A, 5A, 10A, and 11A was elucidated by means of real-time polymerase chain reaction (PCR) analysis (TaqMan). Human penile erectile tissue (corpus cavernosum [HCC]) was used as a reference tissue. Results., mRNA encoding for all PDE isoforms mentioned above is expressed in the female genital tissues. Different magnitudes of mRNA expression were observed: a predominant expression of mRNA encoding for PDE1A but only insignificant amounts of PDE1B, 1C, 4A, 10, and 11A mRNA were registered. With PDE1A being the only exception, the mRNA expression was always higher in the HCC than in the female genital tissues. Especially, the expression of mRNA encoding for PDE5 was several-fold higher in the HCC. Conclusion., On the mRNA level, various PDE isoforms are expressed in the clitoris, labia, and vagina. It remains to be established as to whether the low expression of PDE5 in female genital tissue might be a negative predictor for the success of PDE5 inhibitors in the treatment of FSD. Uckert S, Ellinghaus P, Albrecht K, Jonas U, and Oelke M. Expression of messenger ribonucleic acid encoding for phosphodiesterase isoenzymes in human female genital tissues. J Sex Med 2007;4:1604,1609. [source] An Immunomodulatory Role for Follistatin-Like 1 in Heart Allograft TransplantationAMERICAN JOURNAL OF TRANSPLANTATION, Issue 11 2008J. B. Le Luduec Donor-specific tolerance to heart allografts in the rat can be achieved by donor-specific blood transfusions (DST) before transplantation. We have previously reported that this tolerance is associated with strong leukocyte infiltration, and that host CD8+ T cells and TGF, are required. In order to identify new molecules involved in the induction phase of tolerance, we compared tolerated and rejected heart allografts (suppressive subtractive hybridization) 5 days after transplantation. We identified overexpression of Follistatin-like 1 (FSTL1) transcript in tolerated allografts compared to rejected allografts or syngeneic grafts. We show that FSTL1 is overexpressed during both the induction and maintenance phase of tolerance, and appears to be specific to the tolerance model induced by DST. Analysis of graft-infiltrating cells revealed predominant expression of FSTL1 in CD8+ T cells from tolerated grafts, and depletion of these cells prior to transplantation abrogated FSTL1 expression and heart allograft survival. Moreover, overexpression of FSTL1 by adenovirus gene transfer in vivo significantly prolonged allograft survival in association with inhibition of the proinflammatory cytokines, IL6, IL17 A and IFN,. Taken together, these results suggest that FSTL1 could be an active component of the mechanisms mediating heart allograft tolerance. [source] Evaluation of T-Cell Receptor Repertoires in Patients with Long-Term Renal Allograft SurvivalAMERICAN JOURNAL OF TRANSPLANTATION, Issue 4 2005Cristiam M. Alvarez The mechanisms underlying long-term acceptance of kidney allografts in humans under minimal or no maintenance immunosuppression are poorly understood. We analyzed the T-cell receptor (TCR) repertoires in circulating T cells of patients with long-term (,9 years) renal allograft survival with (LTS-IS) and without immunosuppression (LTS-NoIS). T cells of LTS patients exhibited strongly altered TCR Vß usage, including an increased frequency of oligoclonality and a decreased frequency of polyclonality. All 3 LTS-NoIS and 12 of 16 LTS-IS patients demonstrated oligoclonality in at least three or more TCR Vß families, and the frequency of oligoclonality in these patients was significantly higher as compared to patients with well-functioning grafts at 3 years (p < 0.005 both), an uncomplicated course during the first year (p < 0.0001, both), acute rejection (p < 0.0001, both), chronic allograft nephropathy at 7 (p < 0.0001, both) or 13 years (p < 0.0001, both), dialysis patients (p < 0.0001, both) or healthy controls (p < 0.0001, both). In contrast to LTS patients, all other studied patient groups exhibited a polyclonal TCR repertoire. Our data indicate that TCR alteration is a common feature of long-term allograft outcome, which might be explained by clonal deletion, exhaustion of alloreactive T cells or predominant expression of particular T-cell subpopulations, such as regulatory T cells. [source] The role of TASK1 in aldosterone production and its expression in normal adrenal and aldosterone-producing adenomasCLINICAL ENDOCRINOLOGY, Issue 1 2010Edson F. Nogueira Summary Objectives, Aldosterone production in the adrenal glomerulosa is mainly regulated by angiotensin II and K+. Adrenal glomerulosa cells are uniquely sensitive to extracellular K+. Genetic deletion of subunits of K+ -selective leak-channels (KCNK), TASK1 and/or TASK3, in mice generates animals with hyperaldosteronism and histological changes in the adrenal cortex. Herein, we studied the expression of TASK1 in human adrenocortical cells, as well as its role in aldosterone production in H295R cells. Design, TASK1 expression was investigated by comparative microarray analysis of aldosterone-producing adenomas (APA) and normal adrenals (NAs). The effects of TASK1 knockdown by siRNA transfection were investigated in H295R cells. Fluo-4 fluorescent measurements of intracellular Ca2 + and pharmacological inhibition of Ca2 + -dependent calmodulin kinases (CaMK) were performed to better define the effects of TASK1 on Ca2 + signalling pathways. Results, Microarray analysis of APA and NA showed similar expression of TASK1 between these two groups. However, in APA, NA and H295R cells the expression of TASK1 was predominant when compared with other KCNK family members. Knockdown of TASK1 (with siRNA) induced the expression of steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2), and also stimulated pregnenolone and aldosterone production. Cells transfected with siTASK1 had increased intracellular Ca2 + , leading to activation of CaMK and increased expression of CYP11B2. Conclusions, Our study reveals the predominant expression of TASK1 over other KCNK family genes in the human adrenal cortex. Herein, we also described the role of TASK1 in the regulation of human aldosterone production through regulation of intracellular Ca2 + and CaMK signalling pathways. [source] | |||||||||||||