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RT-PCR Analysis (rt-pcr + analysis)
Kinds of RT-PCR Analysis Selected AbstractsA single nucleotide polymorphism at the splice donor site of the human MYH base excision repair gene results in reduced translation efficiency of its transcriptsGENES TO CELLS, Issue 5 2002Satoru Yamaguchi Background: Adenine paired with 8-hydroxyguanine, a major oxidatively damaged DNA lesion, is excised by mutY homologue (MYH) base excision repair protein in human cells. Since genetic polymorphisms of DNA repair genes associated with the activities and the expression levels of their products may modulate cancer susceptibility of individuals, we investigated the effect of a single nucleotide polymorphism (SNP) in the MYH gene on the difference in the expression levels of its products. Results: An aberrant size of the , type nuclear form transcript was detected in a lung cancer cell line, VMRC-LCD, by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The transcript contained the intron 1 sequence, and it was due to alternative splicing resulting from IVS1+5G/C SNP. The presence of the upstream open reading frame (ORF) on the 5,-side of the native ORF in the , type transcript from the IVS1+5C allele could reduce the translation efficiency of the transcript into the nuclear form protein. Thus, expression vectors bearing the 5,-untranslated region sequence of either the IVS1+5G or 5C allele were constructed. In vitro translation analysis, as well as Western blot and quantitative RT-PCR analyses of the H1299 lung cancer cell line transfected with these vectors, revealed that the translation efficiency of the IVS1+5C transcript into MYH protein was much lower (, 30%) than that of the IVS1+5G transcript. Conclusions: The SNP at the splice donor site of the MYH gene resulted in reduced translation efficiency of its transcripts. This is the fourth case of single nucleotide variations that cause alterations in translation initiation sites and translation efficiencies in human cells. [source] Fusion gene-mediated truncation of RUNX1 as a potential mechanism underlying disease progression in the 8p11 myeloproliferative syndromeGENES, CHROMOSOMES AND CANCER, Issue 7 2007Helena Ågerstam The 8p11 myeloproliferative syndrome (EMS) is a chronic myeloproliferative disorder molecularly characterized by fusion of various 5, partner genes to the 3, part of the fibroblast growth factor receptor 1 (FGFR1) gene at 8p, resulting in constitutive activation of the tyrosine kinase activity contained within FGFR1. EMS is associated with a high risk of transformation to acute myeloid leukemia (AML), but the mechanisms underlying the disease progression are unknown. In the present study, we have investigated a case of EMS harboring a t(8;22)(p11;q11)/BCR-FGFR1 rearrangement as well as a t(9;21)(q34;q22) at the time of AML transformation. FISH and RT-PCR analyses revealed that the t(9;21) leads to a fusion gene consisting of the 5, part of RUNX1 (exons 1,4) fused to repetitive sequences of a gene with unknown function on chromosome 9, adding 70 amino acids to RUNX1 exon 4. The t(9;21) hence results in a truncation of RUNX1. No point mutations were found in the other RUNX1 allele. The most likely functional outcome of the rearrangement was haploinsufficiency of RUNX1, which thus may be one mechanism by which EMS transforms to AML. © 2007 Wiley-Liss, Inc. [source] Diverse roles of 2-arachidonoylglycerol in invasion of prostate carcinoma cells: Location, hydrolysis and 12-lipoxygenase metabolismINTERNATIONAL JOURNAL OF CANCER, Issue 5 2007Michael P. Endsley Abstract Endogenous 2-arachidonoylglycerol (2-AG) is antiinvasive in androgen-independent prostate carcinoma (PC-3) cells. Invasion of PC-3 cells is also inhibited by exogenously added noladin ether, a non-hydrolyzable analog of 2-AG. In contrast, exogenous 2-AG has the opposite effect. Cell invasion significantly increased with high concentrations of exogenous 2-AG. In PC-3 cells, arachidonic acid (AA) and 12-hydroxyeicosatetraenoic acid (12-HETE) concentrations increased along with exogenously added 2-AG, and 12-HETE concentrations increased with exogenously added AA. Invasion of PC-3 cells also increased with exogenously added AA and 12(S)-HETE but not 12(R)-HETE. The exogenous 2-AG-induced invasion of PC-3 cells was inhibited by 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP, an inhibitor of 2-AG hydrolysis) and baicalein (a 12-LO inhibitor). Western blot and RT-PCR analyses indicated expression of 12-HETE producing lipoxygenases (LOs), platelet-type 12-LO (P-12-LO) and leukocyte-type 12-LO (L-12-LO), in PC-3 cells. These results suggest that exogenous 2-AG induced, rather inhibited, cell invasion because of its rapid hydrolysis to free AA, and further metabolism by 12-LO of AA to 12(S)-HETE, a promoter of PC cell invasion. The results also suggest that PC-3 cells and human prostate stromal (WPMY-1) cells released free AA, 2-AG, and 12-HETE. In the microenvironment of the PC cells, this may contribute to the cell invasion. The 2-AG hydrolysis and concentration of 2-AG in microenvironment are critical for PC cell's fate. Therefore, inhibitors of 2-AG hydrolysis could potentially serve as therapeutic agents for the treatment of prostate cancer. © 2007 Wiley-Liss, Inc. [source] An In Vivo Model to Study Osteogenic Gene Regulation: Targeting an Avian Retroviral Receptor (TVA) to Bone With the Bone Sialoprotein (BSP) Promoter,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2005Ling Li Abstract To study bone development in vivo, a transgenic mouse model was established in which an avian retroviral receptor (TVA) gene driven by the BSP promoter was selectively expressed in skeletal tissues. The model was validated by showing suppressed BSP expression and delayed bone and tooth formation after infection with a virus expressing a mutated Cbfa1/Runx2 gene. Introduction: Tissue-specific expression of the avian retroviral (TVA) receptor can be used to efficiently target ectopic expression of genes in vivo. To determine the use of this approach for studies of osteogenic differentiation and bone formation at specific developmental stages, transgenic mice expressing the TVA receptor under the control of a 5-kb bone sialoprotein (BSP) promoter were generated. The mice were first analyzed for tissue-specific expression of the TVA gene and then, after infection with a viral construct, for the effects of a dominant-negative form of the Cbfa1/Runx2 transcription factor on bone formation. Materials and Methods: We first generated transgenic mice (BSP/TVA) in which the TVA gene was expressed under the control of a 4.9-kb mouse BSP promoter. The tissue-specific expression of the TVA gene was analyzed by RT-PCR, in situ hybridization, and immunohistochemistry and compared with the expression of the endogenous BSP gene. A 396-bp fragment of mutated Cbfa1/Runx2 (Cbfa1mu) encoding the DNA-binding domain was cloned into a RCASBP (A) viral vector, which was used to infect neonatal BSP/TVA mice. Results and Conclusion: Expression of the TVA receptor mRNA and protein in the transgenic mice was consistent with the expression of endogenous BSP. Four days after systemic infection with the Cbfa1mu-RCASBP (A) vector, RT-PCR analyses revealed that the expression of BSP mRNA in tibia and mandibles was virtually abolished, whereas a 30% reduction was seen in calvarial bone. After 9 days, BSP expression in the tibia and mandible was reduced by 45% in comparison with control animals infected with an empty RCASBP vector, whereas BSP expression in the membranous bone of calvariae was decreased ,15%. However, after 4 and 8 weeks, there was almost no change in BSP expression in any of the bone tissues. In comparison, a reduction in osteopontin expression was only observed 9 days after viral transfection in the three bones. Histomorphological examination revealed that bone formation and tooth development were delayed in some of the mice infected with mutated Cbfa1. These studies show that BSP/TVA transgenic mice can be used to target genes to sites of osteogenesis, providing a unique system for studying molecular events associated with bone formation in vivo. [source] Selective Blockade of Voltage-Gated Potassium Channels Reduces Inflammatory Bone Resorption in Experimental Periodontal Disease,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2004Paloma Valverde Abstract The effects of the potassium channel (Kv1.3) blocker kaliotoxin on T-cell-mediated periodontal bone resorption were examined in rats. Systemic administration of kaliotoxin abrogated the bone resorption in conjunction with decreased RANKL mRNA expression by T-cells in gingival tissue. This study suggests a plausible therapeutic approach for inflammatory bone resorption by targeting Kv1.3. Introduction: Kv1.3 is a critical potassium channel to counterbalance calcium influx at T-cell receptor activation. It is not known if Kv1.3 also regulates RANKL expression by antigen-activated T-cells, and consequently affects in vivo bone resorption mediated by activated T-cells. Materials and Methods:Actinobacillus actinomycetemcomitans 29-kDa outer membrane protein-specific Th1-clone cells were used to evaluate the expression of Kv1.3 (using reverse transcriptase-polymerase chain reaction [RT-PCR] and Western blot analyses) and the effects of the potassium channel blocker kaliotoxin (0,100 nM) on T-cell activation parameters ([3H]thymidine incorporation assays and ELISA) and expression of RANKL and osteoprotegerin (OPG; flow cytometry, Western blot, and RT-PCR analyses). A rat periodontal disease model based on the adoptive transfer of activated 29-kDa outer membrane protein-specific Th1 clone cells was used to analyze the effects of kaliotoxin in T-cell-mediated alveolar bone resorption and RANKL and OPG mRNA expression by gingival T-cells. Stimulated 29-kDa outer membrane protein-specific Th1 clone cells were transferred intravenously on day 0 to all animals used in the study (n = 7 animals per group). Ten micrograms of kaliotoxin were injected subcutaneously twice per day on days 0, 1, 2, and 3, after adoptive transfer of the T-cells. The control group of rats was injected with saline as placebo on the same days as injections for the kaliotoxin-treated group. The MOCP-5 osteoclast precursor cell line was used in co-culture studies with fixed 29-kDa outer membrane protein-specific Th1-clone cells to measure T-cell-derived RANKL-mediated effects on osteoclastogenesis and resorption pit formation assays in vitro. Statistical significance was evaluated by Student's t -test. Results: Kaliotoxin decreased T-cell activation parameters of 29-kDa outer membrane protein-specific Th1 clone cells in vitro and in vivo. Most importantly, kaliotoxin administration resulted in an 84% decrease of the bone resorption induced in the saline-treated control group. T-cells recovered from the gingival tissue of kaliotoxin-treated rats displayed lower ratios of RANKL and OPG mRNA expression than those recovered from the control group. The ratio of RANKL and osteoprotegerin protein expression and induction of RANKL-dependent osteoclastogenesis by the activated T-cells were also markedly decreased after kaliotoxin treatments in vitro. Conclusion: The use of kaliotoxin or other means to block Kv1.3 may constitute a potential intervention therapy to prevent alveolar bone loss in periodontal disease. [source] FAP-1-mediated activation of NF-,B induces resistance of head and neck cancer to fas-induced apoptosisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2007Eva Wieckowski Abstract Molecular mechanisms responsible for tumor resistance to apoptosis often involve the Fas/FasL pathway. While squamous cell carcinomas of the head and neck (SCCHN) express both Fas and FasL, their resistance to self-induced apoptosis or apoptosis mediated by Fas agonistic antibody (CH-11Ab) was independent of the level of Fas surface expression or the presence of soluble Fas in supernatants of primary or metastatic SCCHN cell lines. By in vitro immunoselection, using PCI-15A cell line treated with successive cycles of CH-11 Ab, Fas-resistant sublines with the parental genotype were selected. Such sublines failed to cleave caspase-8 upon Fas engagement and were resistant to CH-11 Ab, although they remained sensitive to VP-16 or staurosporin. In the presence of cycloheximide, the selected SCCHN sublines become susceptible to CH-11 Ab, and showed cleavage of caspase-8, suggesting that apoptosis resistance was mediated by an inhibitory protein(s) acting upstream of caspase-8. Overexpression of Fas-associated phosphatase 1 (FAP-1), but not cellular FLICE-inhibitory protein (cFLIP) in SCCHN sublines was documented by Western blots and RT-PCR analyses. The FAP-1+ selected sublines also downregulated cell surface Fas. A high phosphorylation level of I,B,, NF,B activation and upregulation of Bcl-2 expression were observed in the FAP-1+ sublines. Treatment with the phosphatase inhibitor, orthovanadate, or silencing of FAP-1 with siRNA abolished their resistance to apoptosis, suggesting that FAP-1 phosphatase activity could be responsible for NF-,B activation and resistance of SCCHN cells to Fas-mediated apoptosis. J. Cell. Biochem. 100: 16,28, 2007. © 2006 Wiley-Liss, Inc. [source] Interleukin-1, induces MMP-9 expression via p42/p44 MAPK, p38 MAPK, JNK, and nuclear factor-,B signaling pathways in human tracheal smooth muscle cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Kao-Chih Liang Matrix metalloproteinases (MMPs) are responsible for degradation of extracellular matrix and play important roles in cell migration, proliferation, and tissue remodeling related to airway inflammation. Interleukin-1, (IL-1,) has been shown to induce MMP-9 production in many cell types and contribute to airway inflammatory responses. However, the mechanisms underlying MMP-9 expression induced by IL-1, in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here, we investigated the roles of p42/p44 MAPK, p38 MAPK, JNK, and NF-,B pathways for IL-1,-induced MMP-9 production in HTSMCs. IL-1, induced production of MMP-9 protein and mRNA in a time- and concentration-dependent manner determined by zymographic, Western blotting, and RT-PCR analyses, which was attenuated by inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), JNK (SP600125), and NF-,B (helenalin), and transfection with dominant negative mutants of MEK1/2, p38 and JNK, respectively. IL-1,-stimulated phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK was attenuated by pretreatment with U0126, SB202190, SP600125, or transfection with these dominant negative mutants of MEK, ERK, p38 and JNK, respectively. Furthermore, IL-1,-stimulated translocation of NF-,B into the nucleus and degradation of I,B-, was blocked by helenalin. Finally, the reporter gene assay revealed that MAPKs and NF-,B are required for IL-1,-induced MMP-9 luciferase activity in HTSMCs. MMP-9 promoter activity was enhanced by IL-1, in HTSMCs transfected with MMP-9-Luc, which was inhibited by helenalin, U0126, SB202190, and SP600125. Taken together, the transcription factor NF-,B, p42/p44 MAPK, p38 MAPK, and JNK that are involved in MMP-9 expression in HTSMCs exposed to IL-1, have now been identified. J. Cell. Physiol. 211: 759,770, 2007. © 2007 Wiley-Liss, Inc. [source] Transactivation of Src, PDGF receptor, and Akt is involved in IL-1,-induced ICAM-1 expression in A549 cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Chih-Chung Lin In previous study, interleukin-1, (IL-1,) has been shown to induce ICAM-1 expression through MAPKs and NF-,B in A549 cells. In addition to these pathways, transactivation of non-receptor tyrosine kinase (Src), PDGF receptors (PDGFRs), and phosphatidylinositol 3-kinase (PI3K)/Akt has been implicated in the expression of inflammatory genes. Here, we further investigated whether these different mechanisms participating in IL-1,-induced ICAM-1 expression in A549 cells. We initially observed that IL-1,-induced ICAM-1 promoter activity was attenuated by the inhibitors of Src (PP1), PDGFR (AG1296), PI3-K (LY294002 and wortmannin), and Akt (SH-5), revealed by reporter gene assay, Western blotting, and RT-PCR analyses. The involvement of Src and PI3-K/Akt in IL-1,-induced ICAM-1 expression was significantly attenuated by transfection of A549 cells with dominant negative plasmids of Src, p85 and Akt, respectively. Src, PDGFR, and PI3K/Akt mediated the effects of IL-1, because pretreatment with PP1, AG1296, and wortmannin also abrogated IL-1,-stimulated Src, PDGFR, and Akt phosphorylation, respectively. Moreover, pretreatment with p300 inhibitor (curcumin) also blocked ICAM-1 expression. We further confirmed that p300 was associated with ICAM-1 promoter which was dynamically linked to histone H4 acetylation stimulated by IL-1,, determined by chromatin immunoprecipitation assay. Association of p300 and histone-H4 to ICAM-1 promoter was inhibited by LY294002. Up-regulation of ICAM-1 enhanced the adhesion of neutrophils onto A549 cell monolayer exposed to IL-1,, which was inhibited by PP1, AG1296, LY294002, wortmannin, and helenalin. These results suggested that Akt phosphorylation mediated through transactivation of Src/PDGFR promotes the transcriptional p300 activity and eventually leads to ICAM-1 expression induced by IL-1,. J. Cell. Physiol. 211: 771,780, 2007. © 2007 Wiley-Liss, Inc. [source] Adenylyl cyclase-5 activity in the nucleus accumbens regulates anxiety-related behaviorJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Kyoung-Shim Kim Abstract Type 5 adenylyl cyclase (AC5) is highly concentrated in the dorsal striatum and nucleus accumbens (NAc), two brain areas which have been implicated in motor function, reward, and emotion. Here we demonstrate that mice lacking AC5 (AC5,/,) display strong reductions in anxiety-like behavior in several paradigms. This anxiolytic behavior in AC5,/, mice was reduced by the D1 receptor antagonist SCH23390 and enhanced by the D1 dopamine receptor agonist, dihydrexidine (DHX). DHX-stimulated c- fos induction in AC5,/, mice was blunted in the dorso-lateral striatum, but it was overactivated in the dorso-medial striatum and NAc. The siRNA-mediated inhibition of AC5 levels within the NAc was sufficient to produce an anxiolytic-like response. Microarray and RT-PCR analyses revealed an up-regulation of prodynorphin and down-regulation of cholecystokinin (CCK) in the NAc of AC5,/, mice. Administration of nor-binaltorphimine (a kappa opioid receptor antagonist) or CCK-8s (a CCK receptor agonist) reversed the anxiolytic-like behavior exhibited by AC5,/, mutants. Taken together, these results suggest an essential role of AC5 in the NAc for maintaining normal levels of anxiety. [source] Glutamate Receptor Subunit ,2 Is Highly Expressed in a Novel Population of Glial-Like Cells in Rat Pineal Glands in CultureJOURNAL OF NEUROCHEMISTRY, Issue 3 2000Shouki Yatsushiro Abstract: The mammalian pineal gland uses L-glutamate as an intercellular chemical transmitter to regulate negatively melatonin synthesis. To receive glutamate signals, pinealocytes express at least three kinds of glutamate receptors: metabotropic receptor types 3 and 5 and an ionotropic receptor, GluR1. In this study, we examined whether or not the fourth class of ionotropic receptor, ,, which is known for its nondefinitive molecular function and its unique expression pattern in brain, is expressed in pineal gland. RT-PCR analyses with specific probes indicated the expression of mRNA of ,2 but not that of ,1 in pineal gland and cultured pineal cells. Western blotting analysis with polyclonal antibodies specific to the carboxyl-terminal region of the ,2 receptor recognized a single 110-kDa polypeptide of cerebellar membranes and specifically immunostained Purkinje cells. The ,2 antibodies recognized a 110-kDa polypeptide of pineal membranes and specifically immunostained huge glial-like cells with the occasional presence of several long, branching processes in a pineal cell culture. ,2 is not uniformly distributed throughout the cells and is relatively abundant at the periphery of the cell bodies and long processes, where the terminals of synaptophysin-positive processes of pinealocytes, a site for glutamate secretion, are frequently present. The ,2-positive cells constitute a very minor population among total pineal cells (,0.03%). Double immunolabeling with ,2 antibodies and antibodies against marker proteins for pineal interstitial cells clearly distinguishes ,2-positive pineal cells and other known interstitial cells, including glial fibrillary acidic protein- or vimentin-positive glial-like cells. These results indicated that the ,2 glutamate receptor is expressed in a novel subpopulation of pineal glial-like cells in culture and suggest the presence of a glutamate-mediated intercellular signal transduction mechanism between pinealocytes and ,2-expressing cells. The pineal cells may provide a good experimental system for studies on the function of glutamate receptor ,2. [source] Transplantation of mesenchymal stem cells in a canine disc degeneration modelJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2008Akihiko Hiyama Abstract Transplantation of mesenchymal stem cells (MSCs) is effective in decelerating disc degeneration in small animals; much remains unknown about this new therapy in larger animals or humans. Fas-ligand (FasL), which is only found in tissues with isolated immune privilege, is expressed in IVDs, particularly in the nucleus pulposus (NP). Maintaining the FasL level is important for IVD function. This study evaluated whether MSC transplantation has an effect on the suppression of disc degeneration and preservation of immune privilege in a canine model of disc degeneration. Mature beagles were separated into a normal control group (NC), a MSC group, and the disc degeneration (nucleotomy-only) group. In the MSC group, 4 weeks after nucleotomy, MSCs were transplanted into the degeneration-induced discs. The animals were followed for 12 weeks after the initial operation. Subsequently, radiological, histological, biochemical, immunohistochemical, and RT-PCR analyses were performed. MSC transplantation effectively led to the regeneration of degenerated discs. FACS and RT-PCR analyses of MSCs before transplantation demonstrated that the MSCs expressed FasL at the genetic level, not at the protein level. GFP-positive MSCs detected in the NP region 8 weeks after transplantation expressed FasL protein. The results of this study suggest that MSC transplantation may contribute to the maintenance of IVD immune privilege by the differentiation of transplanted MSCs into cells expressing FasL. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:589,600, 2008 [source] Expression of zinc finger protein 105 in the testis and its role in male fertilityMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2010Huaxin Zhou Using an in silico approach, we identified a putative zinc finger domain-containing transcription factor (zinc finger protein 105, ZFP105) enriched in the adult mouse testis. RT-PCR analyses showed that Zfp105 was indeed highly expressed in adult mouse testis and that its expression was regulated during postnatal development. To further characterize Zfp105 expression, we generated a Zfp105:,-galactosidase (LacZ) knock-in reporter mouse line (Zfp105LacZ/+) in which a Zfp105:LacZ fusion gene was expressed. Whole-mount LacZ analyses of adult Zfp105LacZ/+ tissues showed robust LacZ staining in the testis, very weak staining in the ovary, and no staining in the spleen, liver, kidney, heart, lung, thymus, adrenal gland, uterus, or oviduct. Sectional LacZ staining showed that ZFP105 was highly expressed in pachytene spermatocytes. ZNF35, the human ortholog of Zfp105, was also highly expressed in human testis. Immunofluorescence analysis showed that ZNF35 was located primarily in the cytoplasm of male germ cells. More importantly, reduced male fertility was observed in adult Zfp105LacZ/LacZ mice. Histological studies showed the presence of undifferentiated spermatogenic cells in the lumen of seminiferous tubules at stage VII and in the epididymal lumen of adult Zfp105LacZ/LacZ mice. Taken together, our results suggest that ZFP105 is a male germ-cell factor and plays a role in male reproduction. Mol. Reprod. Dev. 77: 511,520, 2010. © 2010 Wiley-Liss, Inc. [source] cDNA nucleotide sequence coding for stearoyl-CoA desaturase and its expression in the zebrafish (Danio rerio) embryo,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2003S.L. Hsieh Abstract A cDNA sequence of stearoyl-CoA desaturase (SCD) was determined from zebrafish (Danio rerio) and compared to the corresponding genes in several teleosts. Zebrafish SCD cDNA has a size of 1,061 bp, encodes a polypeptide of 325 amino acids, and shares 88, 85, 84, and 83% similarities with tilapia (Oreochromis mossambicus), grass carp (Ctenopharyngodon idella), common carp (Cyprinus carpio), and milkfish (Chanos chanos), respectively. This 1,061 bp sequence specifies a protein that, in common with other fatty acid desaturases, contains three histidine boxes, believed to be involved in catalysis. These observations suggested that SCD genes are highly conserved. In addition, an oligonucleotide probe complementary to zebrafish SCD mRNA was hybridized to mRNA of approximately 396 bases with Northern blot analysis. The Northern blot and RT-PCR analyses showed that the SCD mRNA was expressed predominantly in the liver, intestine, gill, and muscle, while a lower level was found in the brain. Furthermore, we utilized whole-mount in situ hybridization and real-time quantitative RT-PCR to identify expression of the zebrafish SCD gene at five different stages of development. This revealed that very high levels of transcripts were found in zebrafish at all stages during embryogenesis and early development. Mol. Reprod. Dev. 66: 325,333, 2003. © 2003 Wiley-Liss, Inc. [source] Abscisic acid and late embryogenesis abundant protein profile changes in winter wheat under progressive drought stressPLANT BIOLOGY, Issue 5 2010I. I. Vaseva Abstract Three varieties (cv. Pobeda, Katya and Sadovo) of winter wheat (Triticum aestivum), differing in their agronomic characteristics, were analysed during progressive soil water stress and recovery at early vegetation stages. Changes in abscisic acid content, SDS,PAGE and immunoblot profiles of proteins that remained soluble upon heating were monitored. Initially higher ABA content in control Pobeda and Katya corresponded to earlier expression of the studied late embryogenesis abundant (LEA) proteins. A combination of higher ABA content, early immunodetection of dehydrins, and a significant increase of WZY2 transcript levels were observed in drought-stressed leaves of the tolerant variety Katya. One-step RT-PCR analyses of some acidic dehydrin genes (WCOR410b, TADHN) documented their relatively constant high expression levels in leaves under drought stress during early vegetative development. Neutral WZY2 dehydrin, TaLEA2 and TaLEA3 transcripts accumulated gradually with increasing water deficit. Delayed expression of TaLEA2 and TaLEA3 genes was found in the least drought-tolerant wheat, Sadovo. The expression profile of WZY2 revealed two distinct and separate bands, suggesting alternative splicing, which altered as water stress increased. [source] Proteome analysis of human monocytic THP-1 cells primed with oxidized low-density lipoproteinsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2006Jeong Han Kang Abstract Native low-density lipoprotein (LDL) and oxidized LDL (oxLDL) possess a wide variety of biological properties, and play a central role in atherogenesis. In this study, we used a proteomic analysis of human monocyte THP-1 cells induced with oxLDL or with LDL, to identify proteins potentially involved in atherosclerotic processes. Of the 2500,proteins detected, 93,were differentially expressed as a result of priming with LDL or oxLDL. The proteins were unambiguously identified by comparing the masses of their tryptic peptides with those of all known proteins using MALDI,TOF,MS and the NCBI database. The largest differences in expression were observed for vimentin (94-fold increase), meningioma-expressed antigen,6 (48-fold increase), serine/threonine protein phosphatase,2A (40-fold increase), and beta-1,3-galactosyltransferase (15-fold increase). In contrast, the abundance of an unnamed protein product and phosphogluconate dehydrogenase decreased 30-fold and 25-fold, respectively. The expression of some selected proteins was confirmed by Western blot and RT-PCR analyses. The proteins identified in this study are attractive candidates for further biomarker research. This description of the altered protein profiles induced by oxLDL in human monocytes will support functional studies of the macrophage-derived foam cells involved in the pathogenesis of atherosclerosis. [source] Expression pattern of calcitonin gene-related peptide in the superior colliculus during postnatal development: Demonstration of its intrinsic nature and possible rolesTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 5 2006Inmaculada Gerrikagoitia Abstract Calcitonin gene-related peptide (CGRP) is a widespread neuropeptide with multiple central and peripheral targets. In an analysis on the expression of this peptide throughout the rat brain during postnatal development, we observed a discrepancy between results obtained by immunohistochemistry and by in situ hybridization. In the superior colliculus (SC), only the immunohistochemical signal could be detected (Terrado et al. [1997] Neuroscience 80:951,970). Here we focus our attention on this structure because the temporal pattern of CGRP immunoreactivity observed in the SC suggested the participation of this peptide in the postnatal maturation of the SC. In the present study, we describe in detail the postnatal development of collicular CGRP-immunoreactive structures and their spatiotemporal relationship with cholinergic modules and definitively demonstrate the local expression of CGRP in the SC. CGRP-immunopositive axons and neurons were distributed within the most ventral part of superficial strata and in the intermediate strata of the SC, showing a peak in staining intensity and density at the end of the first postnatal week. At P14, CGRPergic terminal fibers are arranged in small, clearly defined patches in a complementary manner with respect to the cholinergic modules, which start forming at this stage. By using Western blot and RT-PCR analyses, and by means of injections of antisense oligonucleotides, both the presence of CGRP peptide in the SC and the local expression of ,-CGRP transcripts in collicular neurons were demonstrated. A possible role of CGRP is discussed in the context of postnatal modular compartmentalization of collicular afferents. J. Comp. Neurol. 494:721,737, 2006. © 2005 Wiley-Liss, Inc. [source] ORIGINAL ARTICLE: Identification of Toll-Like Receptors in the Rat (Rattus norvegicus): Messenger RNA Expression in the Male Reproductive Tract Under Conditions of Androgen VariationAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 4 2009Barnali Biswas Problem, Although the majority of Toll-like receptors (TLRs) are reported in many species, some of them are not yet described in the rat. Further, factors that govern Tlr expression in the male reproductive tract have received little attention. We attempt to identify and characterize Tlrs in the rat and determine the expression profile under conditions that affect male reproductive tract gene expression. Method of study, Rat Tlr5, Tlr10, and Tlr11 transcript sequences were submitted to GenBank and in silico characterization carried out using bioinformatics tools. RT-PCR analyses using gene specific primers for rat Tlr1,13 were carried out with RNA isolated from reproductive tract tissues of various experimental groups. Results,Tlr5, Tlr10, and Tlr11 identified in this study share features that are characteristic of the known TLRs. Abundant Tlr expression was observed in the male reproductive tract of adult and developing rats. Further, Tlr expression was also observed in the epididymides of androgen ablated rats. Conclusion,Tlr5, Tlr10, and Tlr11 are ubiquitously expressed in the rat. Tlrs seem to be expressed during male reproductive tract development and under conditions of androgen ablation, suggesting the preparedness of the male reproductive tract to detect an infection under all conditions of androgen status. [source] Dominant Negative p63 Isoform Expression in Head and Neck Squamous Cell Carcinoma,THE LARYNGOSCOPE, Issue 12 2004Joseph C. Sniezek MD Abstract Objectives/Hypothesis: p63, a member of the p53 family of genes, is vital for normal epithelial development and may play a critical role in epithelial tumor formation. Although p63 has been identified in various head and neck malignancies, a detailed analysis of which of the six isoforms of the p63 gene is present in normal mucosa and head and neck malignancies has not yet been performed. The study analyzed p63 isoform expression on the RNA and protein level in normal, diseased, and malignant mucosa of the head and neck to examine the differential expression of p63 isoforms in head and neck tumors versus adjacent nonmalignant tissue and to identify the predominant p63 isoform expressed in head and neck squamous cell carcinoma (HNSCC). Study Design: Three experiments were performed. In experiment 1, p63 expression was analyzed by immunohistochemical analysis in 36 HNSCC specimens and matched normal tissue control specimens harvested from the same patient. Western blot analysis was also performed on matched specimens to confirm the identity of the p63 isoforms that were found. In experiment 2, reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed on matched normal and tumor specimens to analyze and quantitatively compare p63 isoform expression at the RNA level. In experiment 3, p63 expression was evaluated by immunohistochemical analysis in oral lichen planus, a benign mucosal lesion marked by hyperdifferentiation and apoptosis. Methods: Immunohistochemical analysis, RT-PCR, and Western blot analysis of p63 were performed on HNSCC specimens and matched normal tissue control specimens. p63 expression in oral lichen planus specimens was also examined by immunohistochemical analysis. Results: In experiment 1, analysis of 36 HNSCC specimens from various head and neck subsites showed p63 expression in all tumors and matched normal tissue specimens (36 of 36). Western blot analyses indicated that dominant negative (,N) isoform p63, (,Np63,) is the major isoform expressed at the protein level in tumors and adjacent normal tissue. In experiment 2, RT-PCR analyses of 10 matched specimens confirmed that, although all three ,Np63 isoforms (,Np63,, ,Np63,, and ,Np63,) are expressed in normal and malignant mucosa of the head and neck, ,Np63, is the predominant transcript expressed. In experiment 3, immunohistochemical analysis of p63 in the pro-apoptotic condition of lichen planus indicated that p63 is underexpressed as compared with normal mucosal specimens. Conclusion: Although all three ,Np63 isoforms are present in HNSCC, ,Np63, protein is the predominant isoform expressed in these malignancies. ,Np63, is also overexpressed in tumors compared with matched normal tissue specimens and is underexpressed in the pro-apoptotic condition of lichen planus. These findings suggest that ,Np63, plays an anti-differentiation and anti-apoptotic role in the mucosal epithelium of the head and neck, possibly playing a pivotal role in the formation of HNSCC. Currently, ,Np63, is an attractive target for mechanistic study aimed at therapeutic intervention. [source] Loss of Nkx3.1 expression in the transgenic adenocarcinoma of mouse prostate modelTHE PROSTATE, Issue 16 2007Carlise R. Bethel Abstract BACKGROUND The transgenic adenocarcinoma of mouse prostate (TRAMP) model has been extensively characterized at the histological and molecular levels, and has been shown to mimic significant features of human prostate cancer. However, the status of Nkx3.1 expression in the TRAMP model has not been elucidated. METHODS Immunohistochemical analyses were performed using dorsal, lateral, and ventral prostate (VP) lobes from ages 6 to 30 weeks. Quantitative RT-PCR analyses were performed to determine relative mRNA expression. RESULTS Heterogeneous loss of Nkx3.1 was observed in hyperplastic lesions of the ventral, dorsal, and lateral lobes. At 6 weeks of age, the ventral lobe displayed profound loss of Nkx3.1. Diminished Nkx3.1 protein was observed in well- to moderately-differentiated cancer lesions of all lobes. Poorly differentiated (PD) tumors stained negatively for Nkx3.1. Quantitative RT-PCR analyses revealed the presence of Nkx3.1 mRNA in each lobe at all ages, albeit reduced to variable levels. CONCLUSIONS These data suggest that disease progression in the TRAMP model may be driven by loss of function of Nkx3.1, in addition to p53 and Rb. Lobe-specific disease progression in the TRAMP model correlates with the reduction of Nkx3.1 protein. Regulation of Nkx3.1 expression during tumorigenesis appears to occur by post-transcriptional and post-translational mechanisms. Prostate 67: 1740,1750, 2007. © 2007 Wiley-Liss, Inc. [source] Nucleotide polymorphisms and the 5,-UTR transcriptional analysis of the bovine growth hormone secretagogue receptor 1a (GHSR1a) geneANIMAL SCIENCE JOURNAL, Issue 5 2010Masanori KOMATSU ABSTRACT Growth hormone secretagogue receptor 1a (GHSR1a) mediates the different actions of its endogenous ligand, ghrelin. Ghrelin-GHSR is involved in many important functions that include growth hormone secretion and food intake. We evaluated the haplotype variety and characterized the microsatellite ((TG)n, 5,-UTR) and nucleotide polymorphisms of the bovine GHSR1a gene. The nucleotide sequencing of this gene (,6 kb) revealed 47 single nucleotide polymorphisms (SNPs), four indels and the microsatellite ((GTTT)n, Intron 1). The 19 haplotypes were constructed from all nucleotide viability patterns and were divided into three major groups. Four SNPs (L24V, nt456(G>A), D191N and nt667(C>T)) and DelR242 in Exon 1 and a haplotype block of approximately 2.2 kb (nt667(C>T) , nt2884 (A>G)) were found in Bos taurus breeds. Breed differences in allele frequencies of the two microsatellites, nt-7(C>A), L24V, and DelR242 loci were found (P < 0.005). A DelR242 was found in the Japanese Shorthorn (frequency: , 0.44), Japanese Brown, five European cattle breeds, the Philippine native cattle, but none detected in the Japanese Black or the Mishima island cattle. Additionally, 5,-rapid amplification of cDNA ends and RT-PCR analyses revealed that there were two different kinds of transcripts: spliced, without a microsatellite within 5,-UTR (GHSR1a); and non-spliced, with the microsatellite (GHSR1b). [source] Ergosterol peroxide from an edible mushroom suppresses inflammatory responses in RAW264.7 macrophages and growth of HT29 colon adenocarcinoma cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2007M Kobori Background and purpose: 5,,8,-Epidioxy-22E -ergosta-6, 22-dien-3,-ol (ergosterol peroxide) is a major antitumour sterol produced by edible or medicinal mushrooms. However, its molecular mechanism of action has yet to be determined. Here, we examine the anticancer and anti-inflammatory effects of ergosterol peroxide. Experimental approach: After treating RAW264.7 macrophages with LPS and purified ergosterol peroxide or ergosterol, we determined LPS-induced inflammatory cytokines, nuclear DNA binding activity of transcription factors and phosphorylation of MAP kinases (MAPKs). HT29 colorectal adenocarcinoma cells were treated with ergosterol peroxide for 5 days. To investigate the antitumour properties of ergosterol peroxide, we performed DNA microarray and RT-PCR analyses and determined the reactive oxygen species (ROS) in HT29 cells. Key results: Ergosterol peroxide suppressed LPS-induced TNF-, secretion and IL-1,/, expression in RAW264.7 cells. Ergosterol peroxide and ergosterol suppressed LPS-induced DNA binding activity of NF-,B and C/EBP,, and inhibited the phosphorylation of p38, JNK and ERK MAPKs. Ergosterol peroxide down-regulated the expression of low-density lipoprotein receptor (LDLR) regulated by C/EBP, and HMG-CoA reductase (HMGCR) in RAW264.7 cells. In addition, ergosterol peroxide showed cytostatic effects on HT29 cells and increased intracellular ROS. Furthermore, ergosterol peroxide induced the expression of oxidative stress-inducible genes, and the cyclin-dependent kinase inhibitor CDKN1A, and suppressed STAT1 and interferon-inducible genes. Conclusion and Implication: Our results suggest that ergosterol peroxide and ergosterol suppress LPS-induced inflammatory responses through inhibition of NF-,B and C/EBP, transcriptional activity, and phosphorylation of MAPKs. Moreover, ergosterol peroxide appears to suppress cell growth and STAT1 mediated inflammatory responses by altering the redox state in HT29 cells. British Journal of Pharmacology (2007) 150, 209,219. doi:10.1038/sj.bjp.0706972 [source] Molecular identification and localization of cellular titin, a novel titin isoform in the fibroblast stress fiberCYTOSKELETON, Issue 6 2007Peter J. Cavnar Abstract We previously discovered a large titin-like protein,c-titin,in chicken epithelial brush border and human blood platelet extracts that binds ,-actinin and organizes arrays of myosin II bipolar filaments in vitro. RT-PCR analysis of total RNA from human megakaryoblastic (CHRF-288-11) and mouse fibroblast (3T3) nonmuscle cells reveal sequences identical to known titin gene exon sequences that encode parts of the Z-line, I-band, PEVK domain, A-band, and M-line regions of striated muscle titins. In the nonmuscle cells, these sequences are differentially spliced in patterns not reported for any striated muscle titin isoform. Rabbit polyclonal antibodies raised against expressed protein fragments encoded by the Z-repeat and kinase domain regions react with the c-titin band in Western blot analysis of platelet extracts and immunoprecipitate c-titin in whole platelet extracts. Immunofluorescent localization demonstrates that the majority of the c-titin colocalizes with ,-actinin and actin in 3T3 and Indian Muntjac deer skin fibroblast stress fibers. Our results suggest that differential expression of titin gene exons in nonmuscle cells yields multiple novel isoforms of the protein c-titin that are associated with the actin stress fiber structures. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Characterization and expression of ATP P2X4 receptor from embryonic chick skeletal muscleDRUG DEVELOPMENT RESEARCH, Issue 1 2001Xuenong Bo Abstract Previous pharmacological experiments have indicated the existence of ATP P2X receptors in chick embryonic skeletal muscles. In this study we cloned a P2X4 -like cDNA encoding a protein of 385 amino acids, which shares 75% and 76% identity with rat and human P2X4 receptors, respectively. Functional studies of this cP2X4 receptor expressed in Xenopus oocytes showed that ATP induced a fast inward current, which was partially desensitized upon prolonged application of ATP. The ATP-induced currents were concentration-dependent, with an EC50 of 9.5 ,M. Adenosine 5,- O -(thio)triphosphate and 2-methylthioATP very weak agonists. ,,,-methyleneATP was almost inactive. In contrast to their potentiating effects on recombinant rat P2X4 receptors, both suramin and pyridoxalphosphate-6-azophenyl-2,,4,-disulfonic acid partially blocked ATP-induced currents. TrinitrophenylATP was able to block ATP-induced response completely, with an IC50 of 4.7 ,M. Northern blot and RT-PCR analysis showed that cP2X4 mRNAs were mainly expressed in skeletal muscle, brain, and gizzard of day 10 chick embryos. Lower levels of expression were also detected in liver, heart, and retina. Whole-mount in situ hybridization showed that cP2X4 mRNAs were expressed in the brain, spinal cord, notochord, gizzard, and skeletal muscle. The physiological functions of cP2X4 receptors in embryonic skeletal muscle remain unclear at present. Drug Dev. Res. 53:22,28, 2001. © 2001 Wiley-Liss, Inc. [source] The role of Fas ligand as an effector molecule in corneal graft rejectionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2005Patrick Abstract Previous studies have shown that the expression of Fas ligand (FasL; CD95L) by donor corneas is critical to their survival when placed on allogeneic recipients. Since there have been reports that the cornea expresses Fas, we tested the idea that FasL on lymphoid cells could be an effector molecule during rejection episodes. When FasL defective BALB/c- gld mice were engrafted with allogeneic corneas, significantly more of these corneas were accepted than by normal BALB/c mice. However, this was not due to impaired FasL-mediated effector function in these mice as the allogeneic corneas did not express detectable Fas by Western blot or RT-PCR analysis. Furthermore, donor corneas without Fas were given no survival advantage, but were rejected similar to wild-type donor allogeneic corneas. Examination of the T cell compartment in gld mice revealed that these cells express higher levels of Fas and are more susceptible to Fas-mediated death than wild-type cells. These results indicate that FasL is not an effector molecule in corneal graft rejection and that gld mice show reduced graft rejection due to greater susceptibility of their T cells to Fas-mediated apoptosis. [source] BimEL as a possible molecular link between proteasome dysfunction and cell death induced by mutant huntingtinEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2010Rebecca Leon Abstract Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the N-terminus of the huntingtin protein. It is characterized by a selective loss of medium spiny neurons in the striatum. It has been suggested that impaired proteasome function and endoplasmic reticulum (ER) stress play important roles in mutant huntingtin (mHtt)-induced cell death. However, the molecular link involved is poorly understood. In the present study, we identified the essential role of the extra long form of Bim (Bcl-2 interacting mediator of cell death), BimEL, in mHtt-induced cell death. BimEL protein expression level was significantly increased in cell lines expressing the N-terminus of mHtt and in a mouse model of HD. Although quantitative RT-PCR analysis indicated that BimEL mRNA was increased in cells expressing mHtt, we provided evidence showing that, at the post-translational level, phosphorylation of BimEL played a more important role in regulating BimEL expression. Up-regulation of BimEL facilitated the translocation of Bax to the mitochondrial membrane, which further led to cytochrome c release and cell death. On the other hand, knocking down BimEL expression prevented mHtt-induced cell death. Taken together, these findings suggest that BimEL is a key element in regulating mHtt-induced cell death. A model depicting the role of BimEL in linking mHtt-induced ER stress and proteasome dysfunction to cell death is proposed. [source] Hypocretin (orexin) in the rat pineal gland: a central transmitter with effects on noradrenaline-induced release of melatoninEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2001Jens D. Mikkelsen Abstract Hypocretin-1 (HCRT-1) and hypocretin 2 (HCRT-2), also known as orexin-A and orexin-B, are two neuropeptides derived from the same precursor. Hypocretinergic neurons have been found exclusively in the hypothalamic dorsolateral area. These neurons are implicated in sleep and feeding through activation of specific G-protein-coupled orexin-1 and orexin-2 receptor (OR-R1 and OR-R2). The purpose of this study was to determine the existence of the HCRT peptides in the central input of the rat pineal gland. Further, OR-R1 and OR-R2 expression was determined in the pineal gland and the effect of HCRT-2 on melatonin synthesis and secretion was analysed in dissociated rat pinealocytes. A large contingent of HCRT-positive nerve fibres and terminals were observed in the epithalamus, many of which entered into the pineal parenchyma. A significant number of nerve fibres endowed with positive boutons were identified in the pineal stalk, though the number of positive fibres decreased along the extension of the stalk. So far, no positive fibres have been found in the superficial pineal gland. RT-PCR analysis revealed the expression of OR-R2 mRNA, whereas OR-R1-receptor mRNA was not detected. When tested alone, HCRT-2 had no effect on secretion of melatonin from cultured rat pinealocytes. However, HCRT-2 partially inhibited (by a maximum of 30%) the ,-adrenergic-induced melatonin secretion. The same effect was seen on activation of N-acetyltransferase activity. The distribution and the large number of HCRT-positive fibres together with the effect on noradrenaline-mediated melatonin release through specific receptors suggests that these peptides may be significant central transmitters in pineal function, probably mediating homeostatic signals to the pineal gland. [source] Capillary supply and gene expression of angiogenesis-related factors in murine skeletal muscle following denervationEXPERIMENTAL PHYSIOLOGY, Issue 3 2005A. Wagatsuma Capillary supply of skeletal muscle decreases during denervation. To gain insight into the regulation of this process, we investigated capillary supply and gene expression of angiogenesis-related factors in mouse gastrocnemius muscle following denervation for 4 months. Frozen transverse sections were stained for alkaline phosphatase to detect endogenous enzyme in the capillary endothelium. The mRNA for angiogenesis-related factors, including hypoxia inducible factor-1, (HIF-1,), vascular endothelial growth factor (VEGF), kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1), fms-like tyrosine kinase (Flt-1), angiopoietin-1 and tyrosine kinase with Ig and epidermal growth factor(EGF) homology domain 2 (Tie-2), was analysed using a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The fibre cross-sectional area after denervation was about 20% of the control value, and the capillary to fibre ratio was significantly lower in denervated than in control muscles. The number of capillaries around each fibre also decreased to about 40% of the control value. These observations suggest that muscle capillarity decreases in response to chronic denervation. RT-PCR analysis showed that the expression of VEGF mRNA was lower in denervated than in control muscles, while the expression of HIF-1, mRNA remained unchanged. The expression levels of the KDR/Flk-1 and Flt-1 genes were decreased in the denervated muscle. The expression levels of angiopoietin-1 but not Tie-2 genes were decreased in the denervated muscle. These findings indicate that reduction in the expression of mRNAs in the VEGF/KDR/Flk-1 and Flt-1 as well as angiopoietin-1/Tie-2 signal pathways might be one of the reasons for the capillary regression during chronic denervation. [source] NtKTI1, a Kunitz trypsin inhibitor with antifungal activity from Nicotiana tabacum, plays an important role in tobacco's defense responseFEBS JOURNAL, Issue 19 2010Hao Huang A cDNA library from tobacco inoculated with Rhizoctonia solani was constructed, and several cDNA fragments were identified by differential hybridization screening. One cDNA clone that was dramatically repressed, NtKTI1, was confirmed as a member of the Kunitz plant proteinase inhibitor family. RT-PCR analysis revealed that NtKTI1 was constitutively expressed throughout the whole plant and preferentially expressed in the roots and stems. Furthermore, RT-PCR analysis showed that NtKTI1 expression was repressed after R. solani inoculation, mechanical wounding and salicylic acid treatment, but was unaffected by methyl jasmonate, abscisic acid and NaCl treatment. In vitro assays showed that NtKTI1 exerted prominent antifungal activity towards R. solani and moderate antifungal activity against Rhizopus nigricans and Phytophthora parasitica var. nicotianae. Bioassays of transgenic tobacco demonstrated that overexpression of NtKTI1 enhanced significantly the resistance of tobacco against R. solani, and the antisense lines exhibited higher susceptibility than control lines towards the phytopathogen. Taken together, these studies suggest that NtKTI1 may be a functional Kunitz trypsin inhibitor with antifungal activity against several important phytopathogens in the tobacco defense response. [source] Down-regulation of reduced folate carrier may result in folate malabsorption across intestinal brush border membrane during experimental alcoholismFEBS JOURNAL, Issue 24 2007Abid Hamid Folate plays a critical role in maintaining normal metabolic, energy, differentiation and growth status of all mammalian cells. The intestinal folate uptake is tightly and diversely regulated, and disturbances in folate homeostasis are observed in alcoholism, attributable, in part, to intestinal malabsorption of folate. The aim of this study was to delineate the regulatory mechanisms of folate transport in intestinal absorptive epithelia in order to obtain insights into folate malabsorption in a rat model of alcoholism. The rats were fed 1 g·kg,1 body weight of ethanol daily for 3 months. A reduced uptake of [3H]folic acid in intestinal brush border membrane was observed over the course of ethanol administration for 3 months. Folate transport exhibited saturable kinetics and the decreased intestinal brush border membrane folate transport in chronic alcoholism was associated with an increased Km value and a low Vmax value. Importantly, the lower intestinal [3H]folic acid uptake in ethanol-fed rats was observed in all cell fractions corresponding to villus tip, mid-villus and crypt base. RT-PCR analysis for reduced folate carrier, the major folate transporter, revealed that reduced folate carrier mRNA levels were decreased in jejunal tissue derived from ethanol-fed rats. Parallel changes were observed in reduced folate carrier protein levels in brush border membrane along the entire crypt,villus axis. In addition, immunohistochemical staining for reduced folate carrier protein showed that, in alcoholic conditions, deranged reduced folate carrier localization was observed along the entire crypt,villus axis, with a more prominent effect in differentiating crypt base stem cells. These changes in functional activity of the membrane transport system were not caused by a general loss of intestinal architecture, and hence can be attributed to the specific effect of ethanol ingestion on the folate transport system. The low folate uptake activity observed in ethanol-fed rats was found to be associated with decreased serum and red blood cell folate levels, which might explain the observed jejunal genomic hypomethylation. These findings offer possible mechanistic insights into folate malabsorption during alcoholism. [source] Two splicing isoforms of the Y-box protein ctYB-1 appear on the same mRNA moleculeFEBS JOURNAL, Issue 1 2007Dmitry Nashchekin Y-box proteins constitute an evolutionarily conserved family of DNA- and RNA-binding proteins involved in the regulation of transcription and translation. In the dipteran Chironomus tentans, a homologue to the vertebrate Y-box protein YB-1 was recently characterized and designated ctYB-1. It is transferred from nucleus to cytoplasm bound to mRNA and is likely to affect translation. It appears in two size variants, p40 and p50. We further analysed the two size variants and their interaction with mRNA. Southern blot analysis, in situ hybridization and RT-PCR analysis suggested that there is just one YB-1 gene, and that the two size variants represent splicing isoforms. In a C. tentans epithelial cell line, only p40 is present, whereas both variants appear together in eight tissues from fourth-instar larvae, although in somewhat different proportions. Furthermore, the appearance of the two isoforms was studied in relation to a specific 35,40 kb mRNA transcript in the salivary glands, the Balbiani ring mRNA. Because of their exceptional size, Balbiani ring messenger ribonucleoprotein particles in nucleoplasm and Balbiani ring polysomes in cytoplasm could be identified and selectively studied. We were able to establish that both isoforms are associated with both nuclear and cytoplasmic Balbiani ring mRNA. In addition, a p50-specific antibody coimmunoprecipitated p40 from Balbiani ring polysomes, suggesting that the two splicing isoforms are located along the same Balbiani ring mRNA molecule. The functional significance of the two isoforms is being discussed. [source] | |||||||||||||||||||||||||||||||||||||