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Blot Analysis (blot + analysis)
Kinds of Blot Analysis Terms modified by Blot Analysis Selected AbstractsModulation of DNA hypomethylation as a surrogate endpoint biomarker for chemoprevention of colon cancerMOLECULAR CARCINOGENESIS, Issue 2 2004Lianhui Tao Abstract Surrogate end-point biomarkers are being developed as indicators of the efficacy of chemopreventive agents. These biomarkers are molecular and biological end-points that can be modulated by chemopreventive agents in accordance with their efficacy to prevent cancer. DNA hypomethylation is a common alteration found in colon tumors that has the potential of being modulated by chemopreventive agents and thus being useful as a surrogate end-point biomarker. Agents that were either effective or ineffective in preventing colon cancer were evaluated for the ability to modulate DNA hypomethylation in azoxymethane-induced colon tumors in male F344 rats. DNA methylation was determined by Dot Blot Analysis using a mouse monoclonal anti-5-methylcytosine antibody. Colon tumors had a 70% reduction in DNA methylation relative to normal colonic mucosa. DNA methylation in the tumors was increased by 7 days of treatment with agents that have been shown to prevent colon cancer (calcium chloride, ,-diflouromethylornithine [DFMO], piroxicam, and sulindac), whereas agents shown not to prevent colon cancer in rats (low dose aspirin, 2-carboxyphenyl retinamide [2-CPR], quercetin, 9- cis retinoic acid, and rutin) did not increase DNA methylation. The results suggest that the ability to reverse the DNA hypomethylation in colon tumors could be useful as a surrogate end-point biomarker for chemoprevention of colon cancer. © 2004 Wiley-Liss, Inc. [source] Germ-line transformation of pink bollworm (Lepidoptera: Gelechiidae) mediated by the piggyBac transposable elementINSECT MOLECULAR BIOLOGY, Issue 3 2000J. J. Peloquin Abstract The pink bollworm, Pectinophora gossypiella, is a world-wide pest of cultivated cotton. In certain growing regions populations are suppressed by a sterile release strategy. Efforts to improve the sterile insect technique as well as our understanding of lepidopteran biology could benefit greatly from a germ-line transformation system. We report transformation of pink bollworm with a piggyBac transposable element carrying the enhanced green flourescent protein (EGFP) marker gene. This vector,marker system resulted in recovery of transgenics at a rate of approximately 3.5%. Integration of the transforming construct that was typical of piggyBac was demonstrated by Southern analysis and sequence determination of transposon flanks. Expression of the EGFP marker was visualized by fluorescent microscopy and Western Blot analysis. Maintenance of transformed strains indicates that the transgene segregates in a Mendelian fashion and has been stable over fourteen generations to date. [source] Monoclonal antibody detection of naphthalene dioxygenase from Pseudomonas aeruginosa 2NRLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2000M. Civilini A monoclonal antibody, designated mAb,(CT), was generated against a peptide of the ISPNAP,-subunit of the naphthalene dioxygenase (NDO) enzyme of Pseudomonas aeruginosa. Since NDO expression is induced by aromatic hydrocarbons, its detection is important as a tool for environmental biomonitoring. This antibody is highly specific and works well both in an indirect ELISA assay and Western Blot analysis, allowing the detection of Pseudomonas spp. expressing the NDO inducible enzyme. The detection threshold for the ELISA assay developed in this work was 104 colony forming units (cfu) per ml. Thus, this mAb could represent a powerful tool to test for pollutants in soil, groundwater, and other natural environments. [source] Proopiomelanocortin gene expression and ,-endorphin localization in the pituitary, testis, and epididymis of stallionMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2006L. Soverchia Abstract Proopiomelanocortin (POMC) is a precursor protein that contains the sequences of several bioactive peptides including adrenocorticotropin (ACTH), ,-endorphin (,-EP), and melanocyte-stimulating-hormone (MSH). POMC is synthesized in the pituitary gland, brain, and many peripheral tissues. Immunoreactive POMC-derived peptides as well as POMC-like mRNA have been evidenced in several nonpituitary tissues, thus suggesting that POMC is actively synthesized by these tissues. The present study was aimed at evaluating if also in the case of stallion POMC-derived peptide, ,-EP, is produced locally in the testis, thus playing effects in a paracrine/autocrine fashion. To investigate this hypothesis the POMC gene expression was analyzed using 3, RACE-PCR and Northern Blot approaches in the testis and epididimys of stallion; moreover, immunocytochemical localization for ,-EP was also performed through confocal laser microscopy. The immunofluorescence results showed a positive ,-EP reaction not only in cellular nest of pituitary but also in the testis and genital tract of stallion, which function could be related with sperm mobility. Such role seem not to be no dependent on the peptide synthesized locally, because the molecular biology approach demonstrated the presence of POMC transcript in the pituitary only. In fact the Northern Blot analysis showed the presence of a single POMC transcript in the pituitary while no signal was detected in the testis and epididimys. The same results were obtained by applied 3, RACE-PCR analysis. In conclusion, opioid-derived peptide ,-EP is present in the genital tract of stallion, but is not locally produced as in other mammalian, and nonmammalian models; its possible biological function at testicular level could be linked to a long-loop feed-back mechanisms. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Is GRP78/BiP a potential salivary biomarker in patients with rheumatoid arthritis?PROTEOMICS - CLINICAL APPLICATIONS, Issue 3 2010Laura Giusti Abstract Purpose: In the last few years, serum and joint synovial fluid have been extensively analyzed for the proteomic research of rheumatoid arthritis (RA) biomarkers. Nonetheless, to date, there have been no studies investigating salivary biomarkers in this condition. Therefore, aim of this study is to investigate the presence of potential biomarkers of RA in human whole saliva. Experimental design: We combined 2-DE and MS to analyze the whole saliva protein profile of 20 RA patients in comparison with 20 sex- and age-matched healthy subjects. Results: Eight salivary proteins resulted differentially expressed, namely calgranulin A, calgranulin B, apolipoprotein A-1, 6-phosphogluconate dehydrogenase, peroxiredoxin 5, epidermal fatty acid-binding protein, 78,kDa glucose-regulated protein precursor (GRP78/BiP), and 14-3-3 proteins. It is particularly interesting that chaperone GRP78/BiP showed the greatest increase in RA patients. This finding was validated by Western Blot analysis and the over-expression of GRP78/BiP appear to be distinctive of RA and drugs treatment independent. Conclusions and clinical relevance: This study provides a rationale for further studies aimed at evaluating any correlation between GRP78/BiP and different clinical/serological aspects of the disease in order to improve the diagnostic algorithms of RA. [source] p53 is dispensable for the induction of apoptosis after inhibition of protein kinase CK2THE PROSTATE, Issue 2 2010Carolin C. Schneider Abstract BACKGROUND Protein kinase CK2 is a ubiquitously expressed heterotetramer consisting of two catalytic ,/,, and two regulatory , subunits. Expression of CK2 is highly elevated in tumor cells where it protects cells from apoptosis. A variety of different compounds were tested as inhibitors of protein kinase CK2 in order to find new therapy strategies. To analyze the role of p53 in the response to CK2 inhibition we used one of the most specific CK2 inhibitors available, TBB, in different prostate cancer cell lines. METHODS We treated prostate cancer cells with the CK2 inhibitor TBB and determined its effect on CK2 activity by an in vitro phosphorylation assay and its effect on viability by an MTT assay. Furthermore, we analyzed changes in the expression of p53 and PARP cleavage by Western Blot analysis. RESULTS Inhibition of CK2 by TBB led to a decrease in cell viability and apoptosis in two cell lines which express wild-type p53 whereas two other cell lines expressing mutant or no p53 failed to show signs of apoptosis. Moreover, cell lines expressing wild-type p53 showed an increase of the amount of p53 and of its transactivation efficiency. However, down-regulation of p53 by RNAi showed that p53 is not necessary for the induction of apoptosis. CONCLUSIONS Wild-type p53 is not necessary for the induction of apoptosis by TBB in prostate cancer cells. Prostate 70: 126,134, 2010. ©2009 Wiley-Liss, Inc. [source] A co-operative interaction between Neisseria gonorrhoeae and complement receptor 3 mediates infection of primary cervical epithelial cellsCELLULAR MICROBIOLOGY, Issue 9 2002Jennifer L. Edwards Summary Little is known about the pathogenesis of gonococcal infection within the lower female genital tract. We recently described the distribution of complement receptor 3 (CR3) on epithelia of the female genital tract. Our studies further indicate that CR3-mediated endocytosis serves as a primary mechanism by which N. gonorrhoeae elicits membrane ruffling and cellular invasion of primary, human, cervical epithelial cells. We have extended these studies to describe the nature of the gonococcus,CR3 interaction. Western Blot analysis demonstrated production of alternative pathway complement components by ecto- and endocervical cells which allows C3b deposition on gonococci and its rapid conversion to iC3b. Anti-iC3b and -factor I antibodies significantly inhibited adherence and invasion of primary cervical cells, suggesting that iC3b covalently bound to the gonococcus serves as a primary ligand for CR3 adherence. However, gonococcal porin and pili also bound to the I-domain of CR3 in a non-opsonic manner. Binding of porin and pili to CR3 were required for adherence to and invasion of cervical epithelia. Collectively, these data suggest that gonococcal adherence to CR3 occurs in a co-operative manner, which requires gonococcal iC3b-opsonization, porin and pilus. In conjunction, these molecules facilitate targeting to and successful infection of the cervical epithelium. [source] Sexual dimorphism of g-protein subunit Gng13 expression in the cortical region of the developing mouse ovaryDEVELOPMENTAL DYNAMICS, Issue 7 2007Akihiro Fujino Abstract In our search for genes required for the development and function of mouse gonads, we identified Gng13 (guanine nucleotide binding protein 13, gamma), a gene with an embryonic expression pattern highly restricted to the ovary. Based on reverse transcriptase-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization, Gng13 is expressed in both XX and XY gonads at embryonic day (E) 11.5, but becomes up-regulated in the XX gonad by E12.5. Expression is retained after treatment with busulfan, a chemical known to eliminate germ cells, pointing to the soma as a site of Gng13 transcription. In situ hybridization of embryonic ovarian tissue sections further localized the expression to the cortex of the developing XX gonad. Gng13 expression in the adult is also highly restricted. Northern blot analyses and Genomic Institute of the Novartis Research Foundation expression profiling of adult tissues detected very high expression in the cerebrum and cerebellum, in addition to, a weaker signal in the ovary. Gng13 belongs to a well-known family of signal transduction molecules with functions in many aspects of development and organ physiology. Here, we report that, in the developing mouse embryo, expression of Gng13 mRNA is highly restricted to the cortex of the XX gonad during sexual differentiation, suggesting a role for this gene during ovarian development. Developmental Dynamics 236:1991,1996, 2007. © 2007 Wiley-Liss, Inc. [source] Sex differences in the level of Bcl-2 family proteins and caspase-3 activation in the sexually dimorphic nuclei of the preoptic area in postnatal ratsDEVELOPMENTAL NEUROBIOLOGY, Issue 13 2006Shinji Tsukahara Abstract In developing rats, sex differences in the number of apoptotic cells are found in the central division of the medial preoptic nucleus (MPNc), which is a significant component of the sexually dimorphic nucleus of the preoptic area, and in the anteroventral periventricular nucleus (AVPV). Specifically, male rats have more apoptotic cells in the developing AVPV, whereas females have more apoptotic cells in the developing MPNc. To determine the mechanisms for the sex differences in apoptosis in these nuclei, we compared the expression of the Bcl-2 family members and active caspase-3 in postnatal female and male rats. Western blot analyses for the Bcl-2 family proteins were performed using preoptic tissues isolated from the brain on postnatal day (PD) 1 (day of birth) or on PD8. In the AVPV-containing tissues of PD1 rats, there were significant sex differences in the level of Bcl-2 (female > male) and Bax (female < male) proteins, but not of Bcl-xL or Bad proteins. In the MPNc-containing tissues of PD8 rats, there were significant sex differences in the protein levels for Bcl-2 (female < male), Bax (female > male), and Bad (female < male), but not for Bcl-xL. Immunohistochemical analyses showed significant sex differences in the number of active caspase-3-immunoreactive cells in the AVPV on PD1 (female < male) and in the MPNc on PD8 (female > male). We further found that active caspase-3-immunoreactive cells of the AVPV and MPNc were immunoreactive for NeuN, a neuronal marker. These results suggest that there are sex differences in the induction of apoptosis via the mitochondrial pathway during development of the AVPV and MPNc. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Genetic, immunological and biochemical evidence for a Rnf complex in the acetogen Acetobacterium woodiiENVIRONMENTAL MICROBIOLOGY, Issue 6 2009Eva Biegel Summary Acetogenic bacteria grow by the oxidation of various substrates coupled to the reduction of carbon dioxide (acetogenesis) or other electron acceptors but the mechanisms of energy conservation are still enigmatic. Here, we report the presence of a rnf gene cluster rnfCDGEAB in Acetobacterium woodii that is speculated to encode a novel, energy-conserving ferredoxin:NAD+ -oxidoreductase complex composed of at least six different subunits. Transcriptional analysis revealed that the genes constitute an operon. RnfC and RnfG were heterologously produced and antibodies were generated. Western blot analyses demonstrated that these subunits were produced and are associated with the cytoplasmic membrane. The subunits were present in cells respiring with either carbon dioxide or caffeate. A preparation with NADH dehydrogenase activity was obtained from detergent solubilized membranes that contained RnfC and RnfG. [source] Fitness drift of an atrazine-degrading population under atrazine selection pressureENVIRONMENTAL MICROBIOLOGY, Issue 3 2008Marion Devers Summary Pseudomonas sp. ADP harbouring the atrazine catabolic plasmid ADP1 was subcultured in liquid medium containing atrazine as sole source of nitrogen. After approximately 320 generations, a new population evolved which replaced the initial population. This newly evolved population grew faster and degraded atrazine more rapidly than the initial population. Plasmid profiles and Southern blot analyses revealed that the evolved strain, unlike the ancestral strain, presented a tandem duplication of the atzB gene encoding the second enzyme of the atrazine catabolic pathway responsible for the transformation of hydroxyatrazine to N-isopropylammelide. This duplication resulted from a homologous recombination that occurred between two direct repeats of 6.2 kb flanking the atzB gene and constituted by the insertion sequences IS1071, ISPps1 and a pdhL homologous sequence. This study highlights the IS-mediated plasticity of atrazine-degrading potential and demonstrates that insertion sequences not only help to disperse the atrazine-degrading gene but also improve the fitness of the atrazine-degrading population. [source] Dose-dependent Induction of Cytochrome P450 (CYP) 3A4 and Activation of Pregnane X Receptor by TopiramateEPILEPSIA, Issue 12 2003Srikanth C. Nallani Summary:,Purpose: In clinical studies, topiramate (TPM) was shown to cause a dose-dependent increase in the clearance of ethinyl estradiol. We hypothesized that this interaction results from induction of hepatic cytochrome P450 (CYP) 3A4 by TPM. Accordingly, we investigated whether TPM induces CYP3A4 in primary human hepatocytes and activates the human pregnane X receptor (hPXR), a nuclear receptor that serves as a regulator of CYP3A4 transcription. Methods: Human hepatocytes were treated for 72 h with TPM (10, 25, 50, 100, 250, and 500 ,M) and known inducers, phenobarbital (PB; 2 mM), and rifampicin (10 ,M). The rate of testosterone 6,-hydroxylation by hepatocytes served as a marker for CYP3A4 activity. The CYP3A4-specific protein and mRNA levels were determined by using Western and Northern blot analyses, respectively. The hPXR activation was assessed with cell-based reporter gene assay. Results: Compared with controls, TPM (50,500 ,M),treated hepatocytes exhibited a considerable increase in the CYP3A4 activity (1. 6- to 8.2-fold), protein levels (4.6- to 17.3-fold), and mRNA levels (1.9- to 13.3-fold). Comparatively, rifampicin (10 ,M) effected 14.5-, 25.3-, and a 20.3-fold increase in CYP3A4 activity, immunoreactive protein levels, and mRNA levels, respectively. TPM (50,500 ,M) caused 1.3- to 3-fold activation of the hPXR, whereas rifampicin (10 ,M) caused a 6-fold activation. Conclusions: The observed induction of CYP3A4 by TPM, especially at the higher concentrations, provides a potential mechanistic explanation of the reported increase in the ethinyl estradiol clearance by TPM. It also is suggestive of other potential interactions when high-dose TPM therapy is used. [source] Long-term suppression of tyrosinase by terrein via tyrosinase degradation and its decreased expressionEXPERIMENTAL DERMATOLOGY, Issue 6 2009Seo-Hyoung Park Abstract:, Previously, we reported that a fungal metabolite, terrein, decreases melanin synthesis via downregulation of microphthalmia-associated transcription factor (MITF). In the present study, we further investigated the long-term hypopigmenting action of terrein in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Treatment with terrein at a concentration of 50 ,m strongly decreased melanogenesis in a time-dependent manner. Interestingly, the decreased tyrosinase protein levels lasted for at least 7 days, even though the MITF protein levels were restored after 3 days of treatment. In accordance with the results of Western blot analyses, the tyrosinase mRNA levels were found to be continuously decreased for at least 7 days, even though recovery of the MITF mRNA levels began after 3 days of terrein treatment. Therefore, we evaluated tyrosinase downregulation to determine if it is caused by proteasomal degradation. We found that the reduction in tyrosinase levels that was induced by terrein was clearly recovered by MG-132, a proteasome inhibitor. Moreover, ubiquitination of tyrosinase increased following treatment with terrein in the presence of MG-132. Taken together, these results suggest that terrein decreases melanogenesis through ubiquitin-dependent proteasomal degradation as well as via decreased expression of its mRNA. [source] A novel Takeout-like protein expressed in the taste and olfactory organs of the blowfly, Phormia reginaFEBS JOURNAL, Issue 18 2006Kazuyo Fujikawa In insects, the functional molecules responsible for the taste system are still obscure. The gene for a 28.5 kDa protein purified from taste sensilla of the blowfly Phormia regina belongs to a gene family that includes takeout of Drosophila melanogaster. Molecular phylogenetic analysis revealed that the Phormia Takeout-like protein is most similar to the protein encoded by a member of the Drosophila takeout gene family, CG14661, whose expression and function have not been identified yet. Western blot analyses revealed that Phormia Takeout-like protein was exclusively expressed in antennae and labellum of the adult blowfly in both sexes. Immunohistochemical experiments demonstrated that Takeout-like protein was localized around the lamella structure of the auxiliary cells and in the sensillar lymph of the labellar taste sensillum. In antennae, Takeout-like protein was distributed at the base of the olfactory sensilla as well. No significant differences in Takeout-like protein expression were found between the sexes. Our results suggest that Phormia Takeout-like protein is involved in some early events concerned with chemoreception in both the taste and olfactory systems. [source] Parvalbumin deficiency in fast-twitch muscles leads to increased ,slow-twitch type' mitochondria, but does not affect the expression of fiber specific proteinsFEBS JOURNAL, Issue 1 2006Peter Racay Parvalbumin (PV), a small cytosolic protein belonging to the family of EF-hand calcium-binding proteins, is highly expressed in mammalian fast-twitch muscle fibers. By acting as a ,slow-onset' Ca2+ buffer, PV does not affect the rapid contraction phase, but significantly contributes to increase the rate of relaxation, as demonstrated in PV,/, mice. Unexpectedly, PV,/, fast-twitch muscles were considerably more resistant to fatigue than the wild-type fast-twitch muscles. This effect was attributed mainly to the increased fractional volume of mitochondria in PV,/, fast-twitch muscle, extensor digitorum longus, similar to levels observed in the slow-twitch muscle, soleus. Quantitative analysis of selected mitochondrial proteins, mitochondrial DNA-encoded cytochrome oxidase c subunit I and nuclear DNA-encoded cytochrome oxidase c subunit Vb and F1-ATPase subunit , revealed the PV,/,tibialis anterior mitochondria composition to be almost identical to that in wild-type soleus, but not in wild-type fast-twitch muscles. Northern and western blot analyses of the same proteins in different muscle types and in liver are indicative of a complex regulation, probably also at the post-transcriptional level. Besides the function in energy metabolism, mitochondria in both fast- and slow-twitch muscles act as temporary Ca2+ stores and are thus involved in the shaping of Ca2+ transients in these cells. Previously observed altered spatio-temporal aspects of Ca2+ transients in PV,/, muscles are sufficient to up-regulate mitochondria biogenesis through the probable involvement of both calcineurin- and Ca2+/calmodulin-dependent kinase II-dependent pathways. We propose that ,slow-twitch type' mitochondria in PV,/, fast muscles are aimed to functionally replace the slow-onset buffer PV based on similar kinetic properties of Ca2+ removal. [source] Three pheromone-binding proteins in olfactory sensilla of the two silkmoth species Antheraea polyphemus and Antheraea pernyiFEBS JOURNAL, Issue 10 2000Rosario Maida Females of the sibling silkmoth species Antheraea polyphemus and A. pernyi use the same three sex pheromone components in different ratios to attract conspecific males. Accordingly, the sensory hairs on the antennae of males contain three receptor cells sensitive to each of the pheromone components. In agreement with the number of pheromones used, three different pheromone-binding proteins (PBPs) could be identified in pheromone-sensitive hairs of both species by combining biochemical and molecular cloning techniques. MALDI-TOF MS of sensillum lymph droplets from pheromone-sensitive sensilla trichodea of male A. polyphemus revealed the presence of three major peaks with m/z of 15702, 15752 and 15780 and two minor peaks of m/z 15963 and 15983. In Western blots with four antisera raised against different silkmoth odorant-binding proteins, immunoreactivity was found only with an anti-(Apol PBP) serum. Free-flow IEF, ion-exchange chromatography and Western blot analyses revealed at least three anti-(Apol PBP) immunoreactive proteins with pI values between 4.4 and 4.7. N-Terminal sequencing of these three proteins revealed two proteins (Apol PBP1a and Apol PBP1b) identical in the first 49 amino acids to the already known PBP (Apol PBP1) [Raming, K., Krieger, J. & Breer, H. (1989) FEBS Lett.256, 2215,2218] and a new PBP having only 57% identity with this amino-acid region. Screening of antennal cDNA libraries with an oligonucleotide probe corresponding to the N-terminal end of the new A. polyphemus PBP, led to the discovery of full length clones encoding this protein in A. polyphemus (Apol PBP3) and in A. pernyi (Aper PBP3). By screening the antennal cDNA library of A. polyphemus with a digoxigenin-labelled A. pernyi PBP2 cDNA [Krieger, J., Raming, K. & Breer, H. (1991) Biochim. Biophys. Acta1088, 277,284] a homologous PBP (Apol PBP2) was cloned. Binding studies with the two main pheromone components of A. polyphemus and A. pernyi, the (E,Z)-6,11-hexadecadienyl acetate (AC1) and the (E,Z)-6,11-hexadecadienal (ALD), revealed that in A. polyphemus both Apol PBP1a and the new Apol PBP3 bound the 3H-labelled acetate, whereas no binding of the 3H-labelled aldehyde was found. In A. pernyi two PBPs from sensory hair homogenates showed binding affinity for the AC1 (Aper PBP1) and the ALD (Aper PBP2), respectively. [source] Ribonucleases expressed by human pancreatic adenocarcinoma cell linesFEBS JOURNAL, Issue 5 2000Ester Fernández-Salas Human ribonucleases have been considered as a possible tumor marker for pancreatic cancer, and elevated serum levels of ribonuclease activity in patients with pancreatic cancer have been reported by many authors. The reason for this elevation is unknown. In this study, we demonstrate that human pancreatic adenocarcinoma cell lines synthesize and secrete different ribonucleases. We isolated and characterized human pancreatic, or secretory, ribonuclease (RNase 1) from the conditioned media of the human pancreatic adenocarcinoma cell lines Capan-1, MDAPanc-3, IBF-CP3 and Panc-1, and the ampullary adenocarcinoma cell line MDAAmp-7, which represent a wide range of differentiation stages. Only one of these cell lines, Panc-1, produces significant amounts of nonsecretory ribonuclease. We then established a purification procedure for both secretory and nonsecretory ribonucleases, consisting of concentration of the supernatant by tangential filtration, anion-exchange and cation-exchange liquid chromatography and C4 RP-HPLC. Ribonuclease activity fractions were monitored using both the spectrophotometric and negative-staining zymogram techniques. The results of N-terminal sequence analysis, kinetic analysis and endoglycosidase digestion studies indicate that the main ribonuclease secreted by all the cell lines is the secretory-type ribonuclease and that it is composed of several differently N -glycosylated forms. Northern blot analyses confirm that some of the cell lines express secretory ribonuclease mRNA. The mRNA levels produced by Panc-1 and MDAPanc-28 are too low to be detected. Similar levels of expression of nonsecretory ribonuclease are found by Northern blot analysis in all the cell lines except Panc-1, which expresses higher levels. Here, we describe, for the first time, that several human pancreatic cancer cell lines with different degrees of differentiation express and secrete ribonucleases. This fact indicates that one origin of the elevated serum RNase levels in patients with pancreatic cancer are tumor cells. Analysis of the oligosaccharide moiety of the RNase 1 secreted by Capan-1 shows that it is highly glycosylated and its N -glycan chains are significantly different from that of the RNase 1 produced by normal pancreas. These results renew the possibility of using human serum RNase 1 determination as a tumor marker. [source] The use of the green fluorescent protein as a biomarker for sapstain fungiFOREST PATHOLOGY, Issue 3 2002S. LEE To understand wood colonization by sapstain fungi and their potential biocontrol agents, it is necessary to differentiate these organisms directly on their natural substrates. In the present study the feasibility of transforming with the green fluorescent protein (GFP), the sapstain fungus Ophiostoma piceae and a potential biocontrol agent Cartapip®, an Ophiostoma piliferum albino strain was assessed. Transformants of the two fungal species were screened by polymerase chain reaction and Southern blot analyses. The GFP was expressed in spores, synnemata and mycelia of the transformants grown in artificial media or wood. The growth, pigmentation and wood colonization of the transformants were similar to that of the non-transformants, suggesting that the presence of the gfp gene had no negative effect on the biology of the transformants. Using fluorescence and confocal microscopy, the GFP-expressing fungi were easily differentiated from the wild-type strains and other fungal species in wood, even 4 months after inoculation. The results show that the use of the GFP system is feasible to monitor Ophiostoma fungi in wood. Utilisation de la protéine fluorescente verte (GFP) comme marqueur biologique des champignons de bleuissement du bois Pour comprendre la colonisation du bois par les champignons de bleuissement et par les agents de lutte biologique potentiels, il est nécessaire de distinguer ces organismes directement dans leur substrat naturel. Nous avons évalué la possibilité de transformation par la protéine fluorescente verte (GFP) du champignon de bleuissement Ophiostoma piceae et d'une souche albinos de Ophiostoma piliferum, agent de lutte biologique potentiel Cartapip®. Des transformants des deux espèces fongiques ont été triés par analyses PCR et Southern blot. La GFP a été exprimée dans les spores, les synnemas et le mycélium des transformants cultivés sur milieux artificiels et sur bois. Avec les transformants, la croissance, la pigmentation et la colonisation du bois étaient semblables à celles des non transformants, ce qui suggère que la présence du gène gfp n'a pas d'effet négatif sur la biologie des transformants. Par microscopie confocale à fluorescence, les champignons exprimant la GFP ont été facilement distingués des souches de type sauvage et d'autres espèces fongiques dans le bois, même 4 mois après inoculation. Nos résultats montrent que l'utilisation de la GFP est possible pour suivre les Ophiostoma dans le bois. Verwendung des Grünen Fluoreszenzproteins als Biomarker für Bläuepilze Um die Besiedelung von Holz durch Bläuepilze und ihre möglichen Antagonisten zu verstehen, muss man diese Organismen direkt auf ihrem natürlichen Substrat unterscheiden können. Es wurde überprüft, ob sich der Bläuepilze Ophiostoma piceae und der mögliche Antagonist Cartapip®, ein Albinostamm von Ophiostoma piliferum, mit dem Grünen Fluoreszenzprotein (GFP) transformieren lassen. Transformierte Stämme der beiden Pilzarten wurden mit PCR und Southern Blot Analysen untersucht. Das GFP wurde in Sporen, Synnemata und Myzelien der transformierten Stämme exprimiert. Dies war auf künstlichen Medien ebenso wie auf Holz der Fall. Wachstum, Pigmentierung und Holzbesiedelung waren bei den transformierten Stämmen ähnlich wie bei den nichttransformierten; somit dürfte die Präsenz des gfpGens keine negativen Auswirkungen auf die Biologie der transformierten Stämme haben. Mit Hilfe der Fluoreszenz- und Konfokal-Mikroskopie konnten die GFP exprimierenden Pilze leicht von den Wildtyp-Stämmen und anderen Pilzarten auf Holz unterschieden werden. Dies war auch noch vier Monate nach der Inokulation der Fall. Die Ergebnisse zeigen, dass das GFP-System zur Beobachtung von Ophiostoma -Arten im Holz geeignet ist. [source] Critical roles of LGN/GPSM2 phosphorylation by PBK/TOPK in cell division of breast cancer cellsGENES, CHROMOSOMES AND CANCER, Issue 10 2010Chikako Fukukawa To investigate the molecular mechanism of mammary carcinogenesis and identify novel molecular targets for breast cancer therapy, we analyzed genome-wide gene expression profiles of 81 clinical breast cancer samples. Here, we report the critical role of LGN/GPSM2 (Leu-Gly-Asn repeat-enriched protein/G-protein signaling modulator 2) in the growth of breast cancer cells. Semiquantitative RT-PCR and Northern blot analyses confirmed upregulation of LGN/GPSM2 in a large proportion of breast cancers. Immunocytochemical staining identified LGN/GPSM2 at the spindle in cells at metaphase, and at midzone and midbody in cytokinetic cells. Western blot analysis indicated the highest expression and the phosphorylated form of LGN/GPSM2 protein in G2/M phase. Treatment with small-interfering RNAs (siRNAs) targeting LGN/GPSM2 caused incompletion of cell division and resulted in significant growth suppression of breast cancer cells. We found that the 450th threonine (Thr450) of LGN/GPSM2 was phosphorylated by the serine/threonine kinase PBK/TOPK during mitosis. Overexpression of LGN/GPSM2-T450A in which Thr450 was substituted with alanine induced growth suppression and aberrant chromosomal segregation. These findings imply an important role of LGN/GPSM2 in cell division of breast cancer cells and suggest that the PBK/TOPK-LGN/GPSM2 pathway might be a promising molecular target for treatment of breast cancer. © 2010 Wiley-Liss, Inc. [source] Chemokine expression in the white matter spinal cord precursor niche after force-defined spinal cord contusion injuries in adult ratsGLIA, Issue 8 2010Friederike Knerlich-Lukoschus Abstract Inflammatory cascades induced by spinal cord injuries (SCI) are localized in the white matter, a recognized neural stem- and progenitor-cell (NSPC) niche of the adult spinal cord. Chemokines, as integrators of these processes, might also be important determinants of this NSPC niche. CCL3/CCR1, CCL2/CCR2, and SDF-1,/CXCR4 were analyzed in the ventrolateral white matter after force defined thoracic SCI: Immunoreactivity (IR) density levels were measured 2 d, 7 d, 14 d, and 42 d on cervical (C 5), thoracic (T 5), and lumbar (L 5) levels. On day post operation (DPO) 42, chemokine inductions were further evaluated by real-time RT-PCR and Western blot analyses. Cellular phenotypes were confirmed by double labeling with markers for major cell types and NSPCs (nestin, Musashi-1, NG2, 3CB2, BLBP). Mitotic profiles were investigated in parallel by BrdU labeling. After lesion, chemokines were induced in the ventrolateral white matter on IR-, mRNA-, and protein-level. IR was generally more pronounced after severe lesions, with soaring increases of CCL2/CCR2 and continuous elevations of CCL3/CCR1. SDF-1, and CXCR4 IR induction was focused on thoracic levels. Chemokines/-receptors were co-expressed with astroglial, oligodendroglial markers, nestin, 3CB2 and BLBP by cells morphologically resembling radial glia on DPO 7 to DPO 42, and NG2 or Musashi-1 on DPO 2 and 7. In the white matter BrdU positive cells were significantly elevated after lesion compared with sham controls on all investigated time points peaking in the early time course on thoracic level: Here, chemokines were co-expressed by subsets of BrdU-labeled cells. These findings suggest an important role of chemokines/-receptors in the subpial white matter NSPC niche after SCI. © 2010 Wiley-Liss, Inc. [source] Host's innate immune response to fungal and bacterial agents in vitro: up-regulation of interleukin-15 gene expression resulting in enhanced natural killer cell activityIMMUNOLOGY, Issue 2 2003Phay Tran Summary Natural killer (NK) cells play an important role in the first line of defence against viral infections. We have shown earlier that exposure of human peripheral blood mononuclear cells (PBMC) to viruses results in rapid up-regulation of NK cell activity via interleukin-15 (IL-15) induction, and that this mechanism curtails viral infection in vitro. By using Candida albicans, Escherichia coli and Staphylococcus aureus, we now show here that exposure of PBMC to fungi and bacteria also results in an immediate increase of NK cytotoxicity. Reverse transcriptase,polymerase chain reaction and Western blot analyses as well as the use of antibodies against different cytokines revealed that IL-15 induction played a predominant role in this NK activation. These results indicate that IL-15 is also involved in the innate immune response against fungal and bacterial agents. [source] Advanced glycation end products accumulate in the reproductive tract of men with diabetesINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 4 2009C. Mallidis Summary Light microscopic studies comparing sperm parameters show little association between diabetes and male fertility. However, with the introduction of new analytical techniques, evidence is now emerging of previously undetectable effects of diabetes on sperm function. Specifically, a recent study has found a significantly higher sperm nuclear DNA fragmentation in diabetic men. As advanced glycation end products (AGEs) are important instigators of oxidative stress and cell dysfunction in numerous diabetic complications, we hypothesized that these compounds could also be present in the male reproductive tract. The presence and localization of the most prominent AGE, carboxymethyl-lysine (CML), in the human testis, epididymis and sperm was determined by immunohistochemistry. Parallel ELISA and Western blot analyses were performed to ascertain the amount of CML in seminal plasma and sperm from 13 diabetic and nine non-diabetic subjects. CML immunoreactivity was found throughout the seminiferous epithelium, the nuclei of spermatogonia and spermatocytes, in the basal and principle cells cytoplasm and nuclei of the caput epididymis and on most sperm tails, mid pieces and all cytoplasmic droplets. The acrosomal cap, especially the equatorial band, was prominently stained in diabetic samples only. The amount of CML was significantly higher (p = 0.004) in sperm from non-diabetic men. Considering the known detrimental actions of AGEs in other organs, the presence, location and quantity of CML, particularly the increased expression found in diabetic men, suggest that these compounds may play a hitherto unrecognized role in male infertility. [source] JKT-1 is not a human seminoma cell lineINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 4 2007Jeroen de Jong Summary The JKT-1 cell line has been used in multiple independent studies as a representative model of human testicular seminoma. However, no cell line for this specific tumour type has been independently confirmed previously; and therefore, the seminomatous origin of JKT-1 must be proven. The genetic constitution of the JKT-1 cells was determined using flow cytometry and spectral karyotyping, as well as array comparative genomic hybridization and fluorescent in situ hybridization. Marker profiling, predominantly based on differentially expressed proteins during normal germ cell development, was performed by immunohistochemistry and Western blot analyses. Moreover, genome wide affymetrix mRNA expression and profiling of 157 microRNAs was performed, and the status of genomic imprinting was determined. A germ cell origin of the JKT-1 cells was in line with genomic imprinting status and marker profile (including positive staining for several cancer-testis antigens). However, the supposed primary tumour, from which the cell line was derived, being indeed a classical seminoma, was molecularly proven not to be the origin of the cell line. The characteristic chromosomal anomalies of seminoma, e.g. gain of the short arm of chromosome 12, as well as the informative marker profile (positive staining for OCT3/4, NANOG, among others) were absent in the various JKT-1 cell lines investigated, irrespective of where the cells were cultured. All results indicate that the JKT-1 cell line is not representative of human seminoma. Although it can originate from an early germ cell, a non-germ cell derivation cannot be excluded. [source] A polymorphic major histocompatibility complex class II-like locus maps outside of both the chicken B-system and Rfp-Y-systemINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 2 2000H. R. Juul-Madsen Chickens have two major regions encoding major histocompatibility complex (MHC) class I, genes and MHC class IIß genes, the serological and functional B-system and the Rfp-Y-system. Recently, they have been shown to assort in a genetically independent way although still located on the same microchromosome. Moreover, the monomorphic MHC class II, gene maps at a third locus located 5 c m from the nearest class IIß genes, located in the B-system ( Kaufman et al., 1995 ). A pedigree family was studied in three generations in order to assign MHC class IIß restriction fragments observed in Southern blot analyses to either the B-system, the Rfp-Y-system or the B-L, locus. In this study, we demonstrate by classical genetic testing of chickens within this fully pedigreed family the existence of an MHC class II-like polymorphic restriction fragment that segregates independently of the B-system, the Rfp-Y-system and of the B-L, locus. [source] Telomerase activity and telomere length in acute leukemia: correlations with disease progression, subtypes and overall survivalINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 2 2010Y. WANG Summary The progressive shortening of telomeres and the activation of telomerase are considered to be one of the important mechanisms in cellular immortalization and disease progression. Bone marrow samples were collected from 148 patients with acute leukemia (AL). Based on the stage of the disease, patients were divided into the newly diagnosed group, the relapsed group and the complete remission (CR) group. telomerase activity (TA) was examined by PCR-ELISA, and telomere length (TL) was examined by Southern blot analyses. TA and TL were analyzed in relation to AL stage and subtype. Five-year survival was analyzed using Kaplan,Meier survival curve. TA in AL patients was higher than healthy individuals. TA level was the highest in the relapsed group, followed by the newly diagnosed group, and then the CR group. TA had no difference between acute nonlymphocytic leukemia (ANLL) group and acute lymphocytic leukemia (ALL) group. But TA in group of subtype M3 was lower than other subtypes of ANLL. TL in AL group was shorter than the control group. TL was the shortest in the relapsed group, followed by the newly diagnosed group, and finally the CR group. TL exhibited an inverse correlation with TA. The group of patients with high TA had a significantly poorer five-year-survival than that of low TA group. TA is elevated and TL is shortened in AL patients. There is a significant inverse correlation between TL and TA. Patients in late-stage disease had shorter TL and higher TA than those in early stages. The shortened TL and elevated TA correlated with disease progression and relapse, and they may serve as prognostic factors for AL patients with poor outcome. M3 subtype is special with relative lower TA and long-lasting survival than other subtypes. [source] Modulation of angiogenesis is effective in a model of rheumatoid arthritisJOURNAL OF ANATOMY, Issue 5 2002A. O. Afuwape A feature of rheumatoid arthritis (RA) is prominent hyperplasia of the synovium, which results in an increased distance between the invasive pannus and the existing synovial vasculature. Concomitantly the hyperplastic tissue imposes an augmented metabolic demand on the pre-existing vasculature. As a consequence the synovium in RA becomes hypoxic, resulting in an increased rate of formation of new blood vessels, to supply nutrients and oxygen. Targeting the vasculature in RA is a potential therapeutic approach in RA. VEGF, a key vascular permeability and angiogenic factor, is expressed in RA. In this study we utilised adenovirus expressing the secreted form of the extracellular domain of the Flt-1 VEGF receptor (sFlt-1) to inhibit VEGF in the collagen-induced arthritis (CIA) model, to determine whether blocking the effects of vegf might be an effective treatment for RA. AdvsFlt-1, administered intravenously on the first day of arthritis, significantly suppressed CIA. For example, on d 6 of arthritis the mean increase in paw thickness, which reflects oedema, for untreated and null adenovirus-treated animals was 0.23 ± 0.05 mm and 0.38 ± 0.08, respectively, compared to 0.07 ± 0.05 for AdvsFlt-1-treated mice (P < 0.001 vs. Adv0-treated and untreated mice by 2-way anova). Western blot analyses revealed the presence of a 100-kDa band, corresponding to human sFlt-1, in liver extracts from arthritic mice infected with AdvsFlt-1 at 24 h but not 72 h after infection. This band was absent in liver extracts from Adv0-infected mice and all synovial extracts. Measurement of protein levels by ELISA demonstrated the presence of sFlt-1 in liver, synovium and serum, although levels declined by 72 h post infection. These data suggest efficient but transient expression of sFlt-1. Sera from adenovirus infected mice were found to contain antiviral antibodies and additionally, sera from AdvsFlt-1-infected but not Adv0-infected mice recognised human recombinant sFlt-1. These observations demonstrate that adenoviral mediated delivery of human sFlt-1 leads to transient gene expression and suppression of CIA. This effect is reduced later in the course of disease due to the expression of antiadenovirus as well as antisFlt-1 antibodies. Future studies will assess the effect of combination treatment, using AdvsFlt-1 together with anti-TNF(antibody, to prolong the beneficial effects of VEGF blockade. These results suggest that blocking the pro-angiogenic and permeability action of VEGF may be beneficial for treatment of RA. [source] Serological analysis of patients treated with a new surgical hemostat containing bovine proteins and autologous plasma ,JOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 6 2001Patricia A. Nelson Abstract A randomized, controlled clinical study of the management of diffuse bleeding with CoStasis® surgical hemostat, a new hemostat containing bovine thrombin and collagen with the patient's own plasma, included patients undergoing cardiac, hepatic, iliac, and general surgery. Sera from 92 patients treated with CoStasis and 84 control patients were collected preoperatively and at a post surgical follow-up of 8 weeks. Among the control group, 57 patients were treated with Instat® collagen sponge in noncardiac indications. Results showed that antibody responses in the CoStasis clinical study were similar to the reported literature for all antigens screened and were not associated with any adverse reactions. The bovine thrombin preparations in CoStasis and other commercially available thrombins were compared with the use of SDS-PAGE and Western blot analyses. Within this clinical study, CoStasis was shown to be a safe and effective hemostatic product containing bovine thrombin and bovine collagen and no pooled human blood products. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res (Appl Biomater) 58: 710,719, 2001 [source] PTHrP Signaling Targets Cyclin D1 and Induces Osteoblastic Cell Growth Arrest,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2005Nabanita S Datta PhD Abstract PTHrP control of the MC3T3-E1 cell cycle machinery showed that, during differentiation, PTHrP induced G1 growth arrest. Cyclin D1 was a critical mediator as a downstream effector of cAMP, PKC, and MAPK signaling, and the process was PKA-independent. The involvement of JunB has been found critical for PTHrP effects. Introduction: PTH-related protein (PTHrP) has been implicated in the control of bone cell turnover, but the mechanisms underlying its effect on osteoblast proliferation and differentiation have not been clearly defined. The mechanisms by which PTHrP impacts cell cycle proteins and the role of signaling pathways in differentiated osteoblasts were studied. Materials and Methods: To elucidate the role of PTHrP, flow cytometric analyses were performed using MC3T3-E1 and primary mouse calvarial cells. Relative protein abundance (Western blot), physical association of partners (immunoprecipitation), and kinase activities (in vitro kinase assays using either GST-Rb or H1-histone as substrates) of cell cycle-associated proteins in vehicle and PTHrP-treated 7-day differentiated cells were determined. ELISA and/or Northern blot analyses were done to evaluate JunB and cyclin D1 expression. SiRNA-mediated gene silencing experiments were performed to silence JunB protein. Finally, inhibitors of cAMP, protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) were used to determine involvement of different signaling pathways. Results: PTHrP inhibited cyclin D1 protein expression 7-fold in a dose- and time-dependent manner and increased the level of p16 protein in differentiated osteoblasts. Additionally, PTHrP reduced cyclin D1-CDK4/CDK6 and CDK1 kinase activities. Forskolin, a cAMP agonist, mimicked PTHrP action, and the PKC inhibitor, GF109203X, slightly blocked downregulation of cyclin D1, implying involvement of both cAMP and PKC. U0126, a MAPK inhibitor, alone decreased cyclin D1 protein, suggesting that the basal cyclin D1 protein is MAPK dependent. H-89, a PKA inhibitor, did not alter the effect of PTHrP on cyclin D1, suggesting a PKA-independent mechanism. Finally, expression of JunB, an activating protein-1 transcription factor, was significantly upregulated, and silencing JunB (siRNA) partially reversed the cyclin D1 response, implying involvement of JunB in the PTHrP-mediated growth arrest of MC3T3-E1 cells. Conclusion: PTHrP upregulates JunB and reduces cyclin D1 expression while inducing G1 cell cycle arrest in differentiated osteoblasts. Such regulation could be an important determinant of the life span and bone-forming activity of osteoblasts. [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] Thiazide Diuretics Affect Osteocalcin Production in Human Osteoblasts at the Transcription Level Without Affecting Vitamin D3 ReceptorsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2000D. Lajeunesse Abstract Besides their natriuretic and calciuretic effect, thiazide diuretics have been shown to decrease bone loss rate and improve bone mineral density. Clinical evidence suggests a specific role of thiazides on osteoblasts, because it reduces serum osteocalcin (OC), an osteoblast-specific protein, yet the mechanisms implicated are unknown. We therefore investigated the role of hydrochlorothiazide (HCTZ) on OC production by the human osteoblast-like cell line MG-63. HCTZ dose-dependently (1,100 ,M) inhibited 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]- induced OC release by these cells (maximal effect, ,40,50% and p < 0.005 by analysis of variance [ANOVA]) as measured by ELISA. This effect of HCTZ on OC release was caused by a direct effect on OC gene expression because Northern blot analysis revealed that OC messenger RNA (mRNA) levels were reduced in the presence of increasing doses of the diuretic (,47.2 ± 4.0%; p < 0.0001 by paired ANOVA with 100 ,M HCTZ). HCTZ (100 ,M) also stimulated calcium (Ca2+) uptake (8.26 ± 1.78 pmol/mg protein/15 minutes vs. 13.6 ± 0.49 pmol/mg protein/15 minutes; p < 0.05) in MG-63 cells. Reducing extracellular Ca2+ concentration with 0.5 mM EDTA or 0.5 mM ethylene glycol-bis(,-amino ethyl ether)- N,N,N',N' -tetraacetic acid (EGTA) only partly prevented the inhibitory effect of the diuretic on OC secretion (maximal effect, ,22.5 ± 6.9%), suggesting that thiazide-dependent Ca2+ influx is not sufficient to elicit the inhibition of OC secretion. Because OC production is strictly dependent on the presence of 1,25(OH)2D3 in human osteoblasts, we next evaluated the possible role of HCTZ on vitamin D3 receptors (VDR) at the mRNA and protein levels. Both Northern and Western blot analyses showed no effect of HCTZ (1,100 ,M) on VDR levels. The presence of EGTA in the culture media reduced slightly the VDR mRNA levels under basal condition but this was not modified in the presence of increasing levels of HCTZ. The OC gene promoter also is under the control of transcription factors such as Yin Yang 1 (YY1) and cFOS. Western blot analysis revealed no changes in YY1 levels in response to HCTZ either in the presence or in the absence of 0.5 mM EGTA in the culture media. In contrast, HCTZ induced a dose-dependent increase in cFOS levels (p < 0.002 by ANOVA), a situation prevented by incubation with EGTA. These studies indicate that HCTZ inhibits OC mRNA expression independently of an effect on VDR, YY1, or extracellular Ca2+ levels but involves changes in cFOS levels. As OC retards bone formation/mineralization, the inhibition of OC production by HCTZ could explain its preventive role in bone loss rate. (J Bone Miner Res 2000;15:894,901) [source] | |||||||||||||||||||||||||||||||||||||