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Polymerase Chain Reaction Analysis (polymerase + chain_reaction_analysis)
Kinds of Polymerase Chain Reaction Analysis Selected AbstractsCytogenetic, FISH, and molecular studies in a case of B-cell chronic lymphocytic leukemia with karyotypic evolutionEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5-6 2002Christian Chena Abstract:, We report the clinical, cytogenetic, fluorescence in situ hybridization (FISH) and molecular findings in a 54-yr-old male patient diagnosed with B-cell chronic lymphocytic leukemia (B-CLL), who showed progression to a diffuse large B-cell lymphoma (Richter's syndrome). Genetic studies were performed at diagnosis and during the Richter's transformation (RT). A clonal karyotype with two dicentric chromosomes, psu dic(12,21)(q24;q10) and dic(17,18)(p11.2;p11.2), was found. Both rearrangements were confirmed by FISH. Molecular cytogenetics analysis using p53 probe showed monoallelic loss of this tumor suppressor gene in 43.8% and 77.3% of cells for the first and the second studies, respectively). In both studies, deletions of D13S319 (18% and 12% of cells) and D13S25 loci (13% and 12% of cells) at 13q14 were found. Polymerase chain reaction analysis showed the MBR/JH rearrangement of the bcl-2 gene. FISH studies using LSI bcl-2/IgH probe allowed quantifying the clonal cell population with this rearrangement (4% and 6.6% of cells at diagnosis and RT, respectively). To our knowledge, this is the first case with a psu dic(12,21) described in B-CLL. The low percentage of cells with the 13q14 deletion and bcl-2/IgH rearrangement suggests that they were secondary events that resulted from clonal evolution. Our patient had a short survival (9 months) and a clear lack of response to several therapeutic agents, confirming the association of p53 gene deletion and karyotypic evolution with disease progression. [source] Fatal infectious mononucleosis with evidence suggestive of the development of B cell lymphomaPATHOLOGY INTERNATIONAL, Issue 9 2003Kenzo Hiroshima A 4-year-old girl presented to a local hospital in August 1999 with fever and cervical lymphadenopathy. A diagnosis of Epstein,Barr virus (EBV) infection was made and the patient was treated with corticosteroids. One month later she developed dyspnea secondary to tonsilar swelling, and underwent tonsillectomy and adenoidectomy. Her dyspnea increased, however, and by mid September she required mechanical ventilation. Six weeks later, she was transferred to Chiba Children's Hospital (Chiba, Japan). Despite vigorous treatment, she died within four weeks of admission. At autopsy, microscopic examination revealed numerous histiocytes with frequent hemophagocytosis in her lungs, liver, spleen, thymus, and lymph nodes. The tentative diagnosis was EBV-associated hemophagocytic syndrome (EBVAHS). A proliferation of atypical lymphocytes was observed in the lymph nodes, the majority of which stained positive with CD79a antibody. A whitish nodule, 8 mm in diameter, was noted in her right ovary. It consisted of a proliferation of pleomorphic lymphoid cells expressing CD79a antigen. In situ hybridization detected EBV RNA within CD79a antigen-positive cells in the lungs, spleen, thymus, bone marrow, lymph nodes, and the right ovary. Polymerase chain reaction analysis of DNA from the ovarian nodule demonstrated a monoclonal rearrangement of the immunoglobulin heavy chain gene indicating that it consisted of a clone of B lymphocytes. We suggest that EBVAHS develops into polyclonal and monoclonal lymphoproliferative disorder in a short period, and that EBVAHS is a preneoplastic condition that may result in B cell lymphoma. [source] Radiomutants of chrysanthemum (Dendranthema grandiflora Tzvelev) of the Lady group: RAPD analysis of the genetic diversityPLANT BREEDING, Issue 3 2004J. Lema-Rumińska Abstract Random amplified polymorphic DNA (RAPD) markers were used to study the molecular characterization of 10 new radiomutants of chrysanthemum. The original cultivar ,Richmond' differed in genetic distance from its Lady group mutants. The analysis of genetic similarity indices revealed low diversity within the radiomutants. The dendrogram obtained after cluster analysis separated the new cultivars as a group that differed from the original cultivar ,Richmond'. The Lady group cultivars, derived from one original cultivar by radiomutation, could be distinguished from each other by using RAPD markers of only a single primer or sets of two or three primers. Polymerase chain reaction analysis proved the efficiency of the RAPD method for DNA fingerprinting of the original cultivar ,Richmond' and its new radiomutants. [source] Characterization of the quantitative trait locus for haloperidol-induced catalepsy on distal mouse chromosome 1GENES, BRAIN AND BEHAVIOR, Issue 2 2008J. R. Hofstetter We report here the confirmation of the quantitative trait locus for haloperidol-induced catalepsy on distal chromosome (Chr) 1. We determined that this quantitative trait locus was captured in the B6.D2- Mtv7a/Ty congenic mouse strain, whose introgressed genomic interval extends from approximately 169.1 to 191.3 Mb. We then constructed a group of overlapping interval-specific congenic strains to further break up the interval and remapped the locus between 177.5 and 183.4 Mb. We next queried single nucleotide polymorphism (SNP) data sets and identified three genes with nonsynonymous coding SNPs in the quantitative trait locus. We also queried two brain gene expression data sets and found five known genes in this 5.9-Mb interval that are differentially expressed in both whole brain and striatum. Three of the candidate quantitative trait genes were differentially expressed using quantitative real-time polymerase chain reaction analyses. Overall, the current study illustrates how multiple approaches, including congenic fine mapping, SNP analysis and microarray gene expression screens, can be integrated both to reduce the quantitative trait locus interval significantly and to detect promising candidate quantitative trait genes. [source] Human papillomavirus-negative ileostomal chronic papillomatous dermatitisJOURNAL OF CUTANEOUS PATHOLOGY, Issue 4 2003Christy M. Williams Background:, Papillomatous stoma-related skin lesions may result from irritant reactions or infection with epidermodysplasia verruciformis human papillomavirus (HPV) types. Methods:, ,We report upon a papillomatous lesion at the ileostoma of a 63-year-old male with familial adenomatous polyposis and colorectal adenocarcinoma. We thoroughly tested the lesion for HPV using immunohistochemistry, transmission electron microscopy, and polymerase chain reaction analyses. Results:, ,The lesion was a fleshy, multilobulated, and verrucous plaque, with hyperkeratosis, hypergranulosis, acanthosis and marked papillomatosis. The clinical and light microscopic features were suggestive of a condyloma. However, no HPV was detected. Conclusions:, We suggest that the lesion most likely represents chronic papillomatous dermatitis, a reaction to mechanical and/or chemical irritation usually associated with urostomies and only rarely observed with ileostomies. This case highlights the clinical, diagnostic and therapeutic aspects of an unusual cutaneous morbidity associated with ileostomies. [source] Altered gene expression in the brain and liver of female fathead minnows Pimephales promelas Rafinesque exposed to fadrozoleJOURNAL OF FISH BIOLOGY, Issue 9 2008D. L. Villeneuve The fathead minnow Pimephales promelas is a small fish species widely used for ecotoxicology research and regulatory testing in North America. This study used a 2000 gene oligonucleotide microarray to evaluate the effects of the aromatase inhibitor, fadrozole, on gene expression in the liver and brain tissue of exposed females. Reproductive measures, plasma vitellogenin and gene expression data for the brain isoform of aromatase (cytP19B), vitellogenin precursors and transferrin provided evidence supporting the efficacy of the fadrozole exposure. Unsupervised analysis of the microarray results identified 20 genes in brain and 41 in liver as significantly up-regulated and seven genes in brain and around 45 in liver as significantly down-regulated. Differentially expressed genes were associated with a broad spectrum of biological functions, many with no obvious relationship to aromatase inhibition. However, in brain, fadrozole exposure elicited significant up-regulation of several genes involved in the cholesterol synthesis, suggesting it as a potentially affected pathway. Gene ontology-based analysis of expression changes in liver suggested overall down-regulation of protein biosynthesis. While real-time polymerase chain reaction analyses supported some of the microarray responses, others could not be verified. Overall, results of this study provide a foundation for developing novel hypotheses regarding the system-wide effects of fadrozole, and other chemical stressors with similar modes of action, on fish biology. [source] Cervical sympathectomy causes alveolar bone loss in an experimental rat modelJOURNAL OF PERIODONTAL RESEARCH, Issue 6 2009Y. Kim Background and Objective:, Periodontal disease, a pathological destructive inflammatory condition, is characterized by alveolar bone loss. Recent studies have suggested a correlation between the sympathetic nervous system and bone remodeling. To confirm the importance of the sympathetic nervous system in bone resorption, we investigated the effects of superior cervical ganglionectomy and oral challenge with Porphyromonas gingivalis on alveolar bone loss in rats. Material and Methods:, Rats were divided into three groups: group A underwent a sham operation as the control group; group B underwent superior cervical ganglionectomy; and group C underwent a sham operation and oral challenge with P. gingivalis. Horizontal alveolar bone loss was evaluated by measuring the distance between the cemento-enamel junction and the alveolar bone crest. Cytokine gene expression in the gingival tissues was assessed using reverse transcription,polymerase chain reaction analyses. The furcation areas of the mandibular molars were examined histologically. Results:, Both superior cervical ganglionectomy and oral challenge with P. gingivalis resulted in accelerated alveolar bone loss. Gingival tissues in the superior cervical ganglionectomy group showed increased expression of the cytokines interleukin-1alfa, tumor necrosis factor-alfa and interleukin-6. The density of neuropeptide Y-immunoreactive fibers was decreased following superior cervical ganglionectomy. Osteoclasts were observed in the superior cervical ganglionectomy and P. gingivalis- challenged groups. Conclusion:, Both superior cervical ganglionectomy and oral challenge with P. gingivalis induced alveolar bone loss. These results provide new information on the occurrence of alveolar bone loss, in that both oral challenge with P. gingivalis and superior cervical ganglionectomy are important accelerating factors for alveolar bone loss. Thus, we suggest that the sympathetic nervous system is linked with the prevention of alveolar bone loss. [source] The knockdown of endogenous replication factor C4 decreases the growth and enhances the chemosensitivity of hepatocellular carcinoma cellsLIVER INTERNATIONAL, Issue 1 2009Masaaki Arai Abstract Aims: To identify differentially expressed genes and thereby detect potential molecular targets for future therapies directed against hepatocellular carcinoma (HCC). Methods: To isolate differentially expressed genes between HCC and adjacent non-cancerous liver tissues, cDNA microarray and quantitative reverse transcriptase polymerase chain reaction analyses were performed. Gene knockdown experiments in HepG2 cells were also performed using small interfering RNAs (siRNAs). Proteins were detected by immunostaining, and cell proliferation was analysed using the MTT/WST-8 assay. Apoptosis and cell cycle analyses were performed using flow cytometry. Results: After an intensive screening for differentially expressed genes in HCC tissues, we isolated 23 upregulated genes in these lesions. Among these, we focused on the replication factor C4 (RFC4) gene. The expression of endogenous RFC4 proteins in HepG2 cells was found to be significantly reduced by RFC4 -specific siRNA. This inhibition of RFC4 expression correlated with a decrease in cellular proliferation, increased levels of apoptosis and a sensitizing of the cells to the DNA-damaging chemotherapeutic agents, doxorubicin and camptothecin. Conclusion: The replication factor C4 gene may be a novel target for developing cancer therapeutics, which can enhance the antitumour activity of chemotherapeutic agents that induce DNA damage. [source] How many symbionts are provided by mothers, acquired by offspring, and needed for successful vertical transmission in an obligate insect,bacterium mutualism?MOLECULAR ECOLOGY, Issue 24 2007TAKAHIRO HOSOKAWA Abstract Vertical symbiont transmission is among the most pivotal processes for maintenance of symbiotic associations. However, it is poorly understood whether and how the levels of resource allocation and investment upon vertical transmission are regulated. The stinkbug Megacopta punctatissima is obligatorily associated with the gut symbiotic bacterium ,Candidatus Ishikawaella capsulata', whose transmission is mediated by a unique mechanism called ,symbiont capsule'. We investigated the population dynamics of the symbiont during vertical transmission in the host,symbiont mutualism. The stinkbug mothers produced one capsule for around 3.6 eggs irrespective of clutch size, suggesting a strict maternal control over symbiont supply for the offspring. However, experimental manipulation of egg/capsule ratios revealed that one capsule is sufficient for symbiont transmission to six nymphs. Quantitative polymerase chain reaction analyses demonstrated that a capsule contains 1.2 × 108 symbionts, a newborn nymph possesses 2 × 107 symbionts from a capsule, and thus one capsule certainly contains a sufficient amount of symbiont cells for six nymphs. These results indicated that the stinkbug mothers produce 1.7 times more symbiont capsules than needed. The newborn nymphs consistently harboured around 2 × 107 symbionts, also suggesting a nymphal control over symbiont transmission. The threshold symbiont titre minimally needed for successful vertical transmission was estimated to be 1.9 × 106 symbionts, which is only 1/10 of the actual symbiont titre detected in a newborn nymph. These results illuminate several ecological factors that may be relevant to parental and offspring controls over symbiotic resource allocation through host insect generations. [source] Detection of serotype k Streptococcus mutans in Thai subjectsMOLECULAR ORAL MICROBIOLOGY, Issue 5 2009J. Lapirattanakul Introduction:,Streptococcus mutans, known to be a pathogen of dental caries as well as bacteremia and infective endocarditis, is classified into four serotypes, c, e, f and k, based on the structures of serotype-specific polysaccharides. Serotype k was recently designated using blood isolates from Japanese subjects and such strains are considered to be virulent in the bloodstream. The purpose of the present study was to analyse the serotype distribution of strains isolated from Thai subjects and determine whether serotype k strains were present. Methods:, A total of 250 S. mutans strains were isolated from 50 Thai subjects, and serotypes of all strains were determined. Then, molecular and biological analyses were carried out for serotype k strains. Results:, Immunodiffusion and polymerase chain reaction analyses showed that serotype c was the most prevalent (70%), followed by serotypes e (22.8%), f (4.4%) and k (2.8%), which indicated that serotype k S. mutans strains occurred in Thai individuals at a similar rate to that previously reported for Japanese and Finnish populations. Molecular analyses of the seven serotype k strains showed extremely low expression of rgpE, which is related to glucose side-chain formation in serotype-specific rhamnose-glucose polymers, similar to previous reports for those other populations. In addition, analysis of the biological properties of the seven serotype k strains demonstrated low levels of sucrose-dependent adhesion, cellular hydrophobicity, dextran-binding activity and phagocytosis susceptibility by human polymorphonuclear leukocytes, which are characteristics similar to those of serotype k strains previously isolated in Japan. Conclusion:, Our results indicate the possibility of a worldwide prevalence of serotype k strains with properties in common with those of previously reported strains. [source] Inhibition of lymphangiogenesis and lymphatic drainage via vascular endothelial growth factor receptor 3 blockade increases the severity of inflammation in a mouse model of chronic inflammatory arthritisARTHRITIS & RHEUMATISM, Issue 9 2009Ruolin Guo Objective This study was undertaken to investigate the effect of lymphatic inhibition on joint and draining lymph node (LN) pathology during the course of arthritis progression in mice. Methods Tumor necrosis factor (TNF),transgenic mice were used as a model of chronic inflammatory arthritis. Mice were subjected to contrast-enhanced magnetic resonance imaging to obtain ankle and knee joint synovial volumes and draining popliteal LN volumes before and after 8 weeks of treatment with vascular endothelial growth factor receptor 3 (VEGFR-3) neutralizing antibody, VEGFR-2 neutralizing antibody, or isotype IgG. Animals were subjected to near-infrared lymphatic imaging to determine the effect of VEGFR-3 neutralization on lymph transport from paws to draining popliteal LNs. Histologic, immunohistochemical, and reverse transcriptase,polymerase chain reaction analyses were used to examine lymphatic vessel formation and the morphology of joints and popliteal LNs. Results Compared with IgG treatment, VEGFR-3 neutralizing antibody treatment significantly decreased the size of popliteal LNs, the number of lymphatic vessels in joints and popliteal LNs, lymphatic drainage from paws to popliteal LNs, and the number of VEGF-C,expressing CD11b+ myeloid cells in popliteal LNs. However, it increased the synovial volume and area of inflammation in ankle and knee joints. VEGFR-2 neutralizing antibody, in contrast, inhibited both lymphangiogenesis and joint inflammation. Conclusion These findings indicate that lymphangiogenesis and lymphatic drainage are reciprocally related to the severity of joint lesions during the development of chronic arthritis. Lymphatic drainage plays a beneficial role in controlling the progression of chronic inflammation. [source] BAFF synthesis by rheumatoid synoviocytes is positively controlled by ,5,1 integrin stimulation and is negatively regulated by tumor necrosis factor , and toll-like receptor ligandsARTHRITIS & RHEUMATISM, Issue 10 2007Ghada Alsaleh Objective It was recently demonstrated that synoviocytes (FLS) from rheumatoid arthritis (RA) patients express BAFF transcripts that are up-regulated by tumor necrosis factor , (TNF,) and interferon-, (IFN,). Thus, BAFF increases in RA target cells might be related to activation of the receptors of innate immunity. The purpose of this study was to determine whether ligands of Toll-like receptor 2 (TLR-2), TLR-4, TLR-9, and ,5,1 integrin are able to induce BAFF synthesis by RA FLS. Methods Quantitative reverse transcription,polymerase chain reaction analyses and enzyme-linked immunosorbent assays were performed to evaluate BAFF messenger RNA induction and BAFF release from FLS after stimulation by ligands for TLR-2, TLR-4, TLR-9, ,5,1 integrin (bacterial lipopeptide [BLP] palmitoyl-3-cysteine-serine-lysine-4, lipopolysaccharide [LPS], CpG, and protein I/II, respectively), TNF,, and IFN,. Results In contrast to IFN,, neither TNF,, LPS, BLP, nor CpG induced the de novo synthesis and release of BAFF by FLS. Priming of cells with IFN, did not have a synergistic effect on BAFF synthesis by FLS stimulated with bacterial products known as pathogen-associated molecular patterns. Moreover, we found that IFN,-induced BAFF synthesis is inhibited by simultaneous stimulation with either TLR ligands or TNF,. We also showed that interplay between TLRs, TNF receptors, and IFN, signaling induces the expression of suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 and reduces IFN,-dependent STAT-1 phosphorylation, which might explain this inhibition. In contrast, we demonstrated that stimulation of ,5,1 integrin can induce BAFF synthesis and release per se and that stimulation of this pathway has no inhibitory effect on IFN,-induced BAFF synthesis. Conclusion Our findings indicate that BAFF secretion by resident cells in target organs of autoimmunity is tightly regulated by innate immunity, with positive and negative controls, depending on the receptors and the pathways triggered. [source] Maternal environment affects endogenous virus induction in the offspring of type 1 diabetes model non-obese diabetic miceCONGENITAL ANOMALIES, Issue 3 2005Yukiko Kagohashi ABSTRACT Type 1 diabetes results from the destruction of pancreatic b-cells (insulitis). It is a multifactorial disease involving genetic and environmental factors, including the maternal environment. Viruses have also been implicated in the pathogenesis of human type 1 diabetes as well as in its model non-obese diabetic (NOD) mice during the perinatal period, as endogenous viruses and/or as infectious agents vertically transmitted from mothers. However, the role of virus as genetic or environmental factor and its interaction with other maternal factors remain unclear. In a series of experiments, we transplanted preimplantation-stage NOD embryos into the uterus of recipient Institute of Cancer Research (ICR) mice, which are without diabetic genetic predisposition, and NOD mice, which did not exhibit overt diabetes during the experiment, and designated offspring as NOD/ICR and NOD/NOD, respectively. We previously observed that NOD/ICR offspring developed insulitis significantly earlier than NOD/NOD offspring. To assess the role of viruses in the development of insulitis, we examined the appearance of viral particles and expression of retroviruses between NOD/ICR and NOD/NOD. NOD/ICR showed earlier expression of env region of the xenotropic type C retrovirus by polymerase chain reaction analysis than NOD/NOD, while the retrovirus-like particles were observed in the islet b-cells similarly in both groups by electron microscopy. Serum corticosterone level, which is suggested to enhance retroviral induction, was significantly higher in the ICR than in the NOD surrogate mothers. These findings suggest that the observed virus is endogenous and that maternal environmental factors, including hormone levels, affect the induction of endogenous viruses and cause the earlier onset of insulitis. [source] Epigenetic regulation of the imprinted U2af1-rs1 gene during retinoic acid-induced differentiation of embryonic stem cellsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2006Noelia Andollo Epigenetic modifications such as DNA methylation and changes in chromatin structure are changes in the chemical composition or structure of DNA that work by regulating gene expression. Their mechanisms of action have been generally studied in imprinted genes. The present work analyzes the involvement of these mechanisms in the expression of the U2af1-rs1 imprinted gene during the differentiation process of embryonic stem (ES) cells induced by retinoic acid. By DNA digestion with methylation-dependent or independent restriction enzymes and consecutive Southern blot, we have found that methylation of the U2af1-rs1 gene increases in differentiated ES cells and in embryoid bodies. However, northern blot and real-time reverse transcription,polymerase chain reaction analysis showed a higher expression of the U2af1-rs1 gene in differentiated ES cells and in embryoid bodies than in undifferentiated ones. On the other hand, the sensitivity to DNase-I assay demonstrated an open chromatin conformation for differentiated cells with regard to undifferentiated ES cells. Our results suggest that the expression of the U2af1-rs1 gene would be regulated by changes in chromatin structure rather than by DNA methylation during the RA-induced process of differentiation of ES cells. [source] Identification of asymmetrically localized transcripts along the animal,vegetal axis of the Xenopus eggDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 8 2005Kensuke Kataoka In many organisms, proper embryo development depends on the asymmetrical distribution of mRNA in the cytoplasm of the egg. Here we report comprehensive screening of RNA localized in the animal or vegetal hemisphere of the Xenopus egg. Macroarrays including over 40 000 independent embryonic cDNA clones, representing at least 17 000 unigenes, were differentially hybridized with labeled probes synthesized from the mRNA of animal or vegetal blastomeres. After two rounds of screening, we identified 33 clones of transcripts that may be preferentially distributed in the vegetal region of the early stage embryo, but transcripts localized in the animal region were not found. To assess the array results, we performed northern blot and quantitative real-time reverse transcription,polymerase chain reaction analysis. As a result, 21 transcripts of the 33 were confirmed to be localized in the vegetal region of the early stage embryo. Whole-mount in situ hybridization analysis revealed that 11 transcripts, including 7 previously reported genes, were localized in the vegetal hemisphere of the egg. These 11 transcripts were categorized into three groups according to their expression patterns in the egg. The first group, which contained four transcripts, showed uniform expression in the vegetal hemisphere, similar to VegT. The second group, which contained three transcripts, showed gradual expression from the vegetal pole to the equator, similar to Vg1. The last group, which contained three transcripts, was expressed at the germ plasm, similar to Xdazl. One transcript, Xwnt11, showed both the second and the third expression patterns. [source] Microarray analysis of retinoid-dependent gene activity during rat embryogenesis: Increased collagen fibril production in a model of retinoid insufficiencyDEVELOPMENTAL DYNAMICS, Issue 4 2004George R. Flentke Abstract Retinoic acid (RA) is an essential mediator of embryogenesis. Some, but not all, of its targets have been identified. We previously developed a rat model of gestational retinoid deficiency (RAD; Power et al. [1999] Dev. Dyn. 216:469,480) and generated embryos with developmental impairments that closely resemble genetic and dietary models of retinoid insufficiency. Here, we used microarray analysis and expression profiling to identify 88 transcripts whose abundance was altered under conditions of retinoid insufficiency, as compared with normal embryos. Among these, the induction by RAD of genes involved in collagen I synthesis (COL1A1, IA2 and VA2, prolyl-4-hydroxylase-,1) and protein galactosylation (galactokinase, ABO galactosyltransferase, UDP-galactose transporter-related protein) was especially noteworthy because extracellular matrix regulates many developmental events. We also identified several genes involved with stress responses (cathepsin H, UBC2E, IGFBP3, smoothelin). Real-time polymerase chain reaction analysis of selected candidates revealed excellent agreement with the array findings. Further validation came from the demonstration that these genes were similarly dysregulated in two genetic models of retinoid insufficiency, the retinol binding protein null-mutant embryo and the Raldh2 null-mutant embryo. In situ hybridization of RAD embryos found increased collagen IA1 and IGFBP3 mRNA within the connective mesenchyme and vasculature, respectively, and a failure to repress the growth factor midkine within the RAD neural tube. Many of the identified genes were not known previously to respond to retinoid status and will provide new insights to retinoid roles and to the consequences of retinoid insufficiency. Developmental Dynamics 229:886,898, 2004. © 2004 Wiley-Liss, Inc. [source] Microbiological investigation of methane- and hydrocarbon-discharging mud volcanoes in the Carpathian Mountains, RomaniaENVIRONMENTAL MICROBIOLOGY, Issue 4 2006Karine Alain Summary Paclele Mici is a terrestrial mud volcano field located in the Carpathian Mountains (Romania), where thermal alteration of sedimentary organic compounds leads to methane, higher hydrocarbons and other petroleum compounds that are continuously released into the environment. The hydrocarbons represent potential substrates for microorganisms. We studied lipid biomarkers, stable isotope ratios, the effect of substrate (methane, other organic compounds) addition and 16S rRNA genes to gain insights into the hitherto unknown microbial community at this site. Quantitative real-time polymerase chain reaction analysis demonstrated that bacteria were much more abundant than archaea. Phylogenetic analyses of 16S rDNA clone sequences indicated the presence of bacterial and archaeal lineages generally associated with the methane cycle (methanogens, aerobic and anaerobic methanotrophs), the sulfur cycle (sulfate reducers), and groups linked to the anaerobic degradation of alkanes or aromatic hydrocarbons. The presence of sulfate reducers, methanogens and methanotrophs in this habitat was also confirmed by concurrent surveys of lipid biomarkers and their isotopic signatures. Incubation experiments with several common and complex substrates revealed the potential of the indigenous microbial community for sulfate reduction, methanogenesis and aerobic methanotrophy. Additionally, consistently to the detection of methane-oxidizing archaea (ANME) and 13C-depleted archaeal lipids, a weak but significant activity of anaerobic methane oxidation was measured by radiotracer techniques and in vitro. This survey is the first to report the presence and activity of ANME in a terrestrial environment. [source] An evaluation of the etiology of reduced CYP1A1 messenger RNA expression in the Atlantic tomcod from the Hudson River, New York, USA, using reverse transcriptase polymerase chain reaction analysisENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 5 2001Nirmal K. Roy Abstract Adult Atlantic tomcod, Microgadus tomcod, from the Hudson River, New York State, USA, exhibit reduced inducibility of hepatic cytochrome P4501A1 (CYP1A1) mRNA compared with adult tomcod from the cleaner Miramichi River, New Brunswick, Canada, when treated with coplanar polychlorinated biphenyl (PCB) congeners or 2,3,7,8-tetrachlorodibenzo- p -dioxin. In contrast, little difference in CYP1A1 inducibility is observed between tomcod from these two rivers when treated with polycyclic aromatic hydrocarbons (PAHs). We sought to determine if impaired hepatic CYP1A1 inducibility in Hudson River tomcod results from a multigenerational, genetic adaptation or a single generational, physiological acclimation. Embryos and larvae from controlled experimental crosses of Hudson River and Miramichi River parents were exposed for 24 h to water-borne PCB congener 77 (10 ppm), benzo[a]pyrene (BaP; 10 ppm), or dimethysulfoxide, and CYP1A1 expression was assessed in individual larva using competitive reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The CYP1A1 mRNA was significantly induced in larvae from both populations by BaP (47- and 52-fold) and PCB 77 (9- and 22-fold), although levels of expression were higher in offspring of Miramichi matings. Most important, CYP1A1 mRNA was significantly induced by PCB 77 in larvae from Hudson River parents. Concentrations of dioxin, furan, and PCB congeners were measured in livers and eggs of female tomcod from these two locales to quantify the extent of maternal transfer of contaminants. For both rivers, wet-weight contaminant concentrations were significantly higher (4,7 times) in livers than in eggs of the same females, suggesting that a threshold level of contaminants may have to be reached before CYP1A1 transcription is impaired. We conclude that reduced inducibility of hepatic CYP1A1 mRNA in adult tomcod from the Hudson River is most consistent with single-generational acclimation. [source] Human mast cells express androgen receptors but treatment with testosterone exerts no influence on IgE-independent mast cell degranulation elicited by neuromuscular blocking agentsEXPERIMENTAL DERMATOLOGY, Issue 3 2010WenChieh Chen Please cite this paper as: Human mast cells express androgen receptors but treatment with testosterone exerts no influence on IgE-independent mast cell degranulation elicited by neuromuscular blocking agents. Experimental Dermatology 2010; 19: 302,304. Abstract:, Women predominate in the anaphylactic reactions to neuromuscular blocking agents (NMBA). The expression of oestrogen receptors has been demonstrated in mast cells and oestrogen treatment can enhance mast cell degranulation, but the influence of androgens remains largely unclear. Our immunocytochemical study showed the expression of androgen receptor (AR) in mast cells isolated from human foreskin as well as in two human mast cell lines, HMC-1 and LAD2. The amount of AR was most abundant in human skin mast cells as determined by real-time polymerase chain reaction analysis. Treatment of the HMC-1 mast cells with testosterone or 17,-oestradiol, alone or in combination with different NMBA, did not affect mast cell degranulation as measured by the release of ,-hexosaminidase. Our study shows for the first time the expression of AR in human skin mast cells. Further studies using primary human mast cell cultures are needed to understand whether and how sex hormones can influence mast cell activation. [source] Immunomodulatory effects of probiotic bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cellsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2003Karen Manon Lammers Abstract A new therapeutic approach for inflammatory bowel diseases is based on the administration of probiotic bacteria. Prokaryotic DNA contains unmethylated CpG motifs which can activate immune responses, but it is unknown whether bacterial DNA is involved in the beneficial effects obtained by probiotic treatment. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with pure DNA of eight probiotic strains and with total bacterial DNA from human feces collected before and after probiotic ingestion. Cytokine production was analyzed in culture supernatants. Modification of human microflora after probiotic administration was proven by polymerase chain reaction analysis. Here we show that Bifidobacterium genomic DNA induced secretion of the antiinflammatory interleukin-10 by PBMC. Total bacterial DNA from feces collected after probiotic administration modulated the immune response by a decrease of interleukin-1, and an increase of interleukin-10. [source] Major histocompatibility complex class II, fetal skin dendritic cells are potent accessory cells of polyclonal T-cell responsesIMMUNOLOGY, Issue 2 2000A. Elbe-Bürger Summary Whereas dendritic cells (DC) and Langerhans cells (LC) isolated from organs of adult individuals express surface major histocompatibility complex (MHC) class II antigens, DC lines generated from fetal murine skin, while capable of activating naive, allogeneic CD8+ T cells in a MHC class I-restricted fashion, do not exhibit anti-MHC class II surface reactivity and fail to stimulate the proliferation of naive, allogeneic CD4+ T cells. To test whether the CD45+ MHC class I+ CD80+ DC line 80/1 expresses incompetent, or fails to transcribe, MHC class II molecules, we performed biochemical and molecular studies using Western blot and polymerase chain reaction analysis. We found that 80/1 DC express MHC class II molecules neither at the protein nor at the transcriptional level. Ultrastructural examination of these cells revealed the presence of a LC-like morphology with indented nuclei, active cytoplasm, intermediate filaments and dendritic processes. In contrast to adult LC, no LC-specific cytoplasmic organelles (Birbeck granules) were present. Functionally, 80/1 DC in the presence, but not in the absence, of concanavalin A and anti-T-cell receptor monoclonal antibodies stimulated a vigorous proliferative response of naive CD4+ and CD8+ T cells. Furthermore, we found that the anti-CD3-induced stimulation of naive CD4+ and CD8+ T cells was critically dependent on the expression of Fc,R on 80/1 DC and that the requirement for co-stimulation depends on the intensity of T-cell receptor signalling. [source] cDNA of an arylphorin-type storage protein from Pieris rapae with parasitism inducible expression by the endoparasitoid wasp Pteromalus puparumINSECT SCIENCE, Issue 3 2009Jia-Ying Zhu Abstract, This report presents the cDNA cloning of a storage protein, PraAry, from Pieris rapae and investigates its expression regulated by parasitism of an endoparasitoid wasp Pteromalus puparum. The full-length cDNA of PraAry is 2 270 nucleotides and contains a 2 121 nucleotide open reading frame encoding 707 amino acids with calculated molecular weights of approximately 83 kDa. Analysis of the primary protein sequence revealed that it possesses a signal peptide of 16 amino acids at the N-terminus and contains two highly conserved storage protein signature motifs. According to both phylogenetic analysis and the criteria for amino acid composition, PraAry belongs to the subfamily of arylphorin-type storage protein (1.42% methionine and 18.82% aromatic amino acids). Reverse transcription , polymerase chain reaction analysis indicated that the transcriptional level of PraAry mRNA in P. rapae pupae fat body is inducible in response to parasitism by P. puparum. [source] Induction of vascular endothelial growth factor expression in human pulp fibroblasts stimulated with black-pigmented BacteroidesINTERNATIONAL ENDODONTIC JOURNAL, Issue 9 2004L.-C. Yang Abstract Aim, To investigate the effect of black-pigmented Bacteroides on the expression of vascular endothelial growth factor (VEGF) gene in human pulp fibroblasts. Methodology, The supernatants of Porphyromonas endodontalis, Porphyromonas gingivalis and Prevotella intermedia were used to evaluate VEGF gene expression in human pulp fibroblasts. The levels of mRNAs were measured by the quantitative reverse-transcriptase polymerase chain reaction analysis. Results, Black-pigmented Bacteroides induced significantly high levels of VEGF mRNA gene expression in human pulp fibroblasts (P < 0.05). In addition, the expression of VEGF depended on the bacteria tested. Conclusions, Black-pigmented Bacteroides may be involved in developing pulpal disease through the stimulation of VEGF production that would lead to the expansion of the vascular network coincident to progression of the inflammation. [source] NO-sulindac inhibits the hypoxia response of PC-3 prostate cancer cells via the Akt signalling pathwayINTERNATIONAL JOURNAL OF CANCER, Issue 1 2009Grant D. Stewart Abstract Nitric oxide-donating non-steroidal anti-inflammatory drugs are safer than traditional NSAIDs and inhibit the growth of prostate cancer cells with greater potency than NSAIDs. In vivo, prostate cancer deposits are found in a hypoxic environment which induces resistance to chemotherapy. The aim of this study was to assess the effects and mechanism of action of a NO-NSAID called NO-sulindac on the PC-3 prostate cancer cell line under hypoxic conditions. NO-sulindac was found to have pro-apoptotic, cytotoxic, and anti-invasive effect on PC-3 cells under normoxia and hypoxia. NO-sulindac was significantly more cytotoxic than sulindac at all oxygen levels. The sulindac/linker and NO-releasing subunits both contributed to the cytotoxic effects of NO-sulindac. Resistance of PC-3 cells to NO-sulindac was induced as the oxygen concentration declined. Hypoxia-induced chemoresistance was reversed by knocking-down hypoxia-inducible factor-1, (HIF-1,) mRNA using RNAi. Nuclear HIF-1, levels were upregulated at 0.2% oxygen but reduced by treatment with NO-sulindac, as was Akt phosphorylation. NO-sulindac treatment of hypoxic PC-3 cells transfected with a reporter construct, downregulated activation of the hypoxia response element (HRE) promoter. Co-transfection of PC-3 cells with the HRE promoter reporter construct and myr-Akt (constitutively active Akt) plasmids reversed the NO-sulindac induced reduction in HRE activation. Real-time polymerase chain reaction analysis of hypoxic, NO-sulindac treated PC-3 cells showed downregulation of lysyl oxidase and carbonic anhydrase IX mRNA expression. Collectively, these novel findings demonstrate that NO-sulindac directly inhibits the hypoxia response of PC-3 prostate cancer cells by inhibiting HIF-1, translation via the Akt signalling pathway. The ability of NO-sulindac to inhibit tumour adaption to hypoxia has considerable relevance to the future management of prostate cancer with the same cellular properties as PC-3. © 2008 Wiley-Liss, Inc. [source] Automated detection of malaria-associated intraleucocytic haemozoin by Cell-Dyn CD4000 depolarization analysisINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 2 2003C.S. Scott Summary Laboratory tests for malaria are only performed if there is clinical suspicion of the disease, and a missed diagnosis contributes substantially to morbidity and mortality. Malaria parasites produce haemozoin, which is able to depolarize light and this allows the automated detection of malaria during routine complete blood count analysis (CBC) with some Abbott Cell-Dyn instruments. In this study, we evaluated the Cell-Dyn CD4000 with 831 blood samples submitted for malaria investigations. Samples were categorized as malaria negative (n = 417), convalescent malaria (n = 64) or malaria positive (n = 350) by reference to thin/thick film microscopy, ,rapid test' procedures, polymerase chain reaction analysis and clinical history. With regard to CD4000 depolarization analysis, a malaria positive CD4000 pattern was ascribed to samples that showed one or more abnormal depolarizing purple events, which corresponded to monocytes containing ingested malaria pigment (haemozoin). Positive CD4000 patterns were observed in 11 of 417, 50 of 64 and 281 of 350 of malaria negative, convalescent malaria and malaria positive samples respectively. The specificity and positive predictive values for malaria (active and convalescent) were very high (97.4 and 96.8%, respectively), while sensitivity and negative predictive values were 80.0 and 83.0% respectively. Depolarization analysis was particularly effective for Plasmodium falciparum malaria but there was lower detection sensitivity for White compared with Black African patients. CD4000 90° depolarization vs 0° analysis revealed a proportion of samples with small nonleucocyte-associated depolarizing particles. Appearance of such events in the form of a discrete cluster was associated with P. vivax rather than P. falciparum infection. [source] Mechanisms involved in hydroxycamptothecin-induced apoptosis of gastric cancer cellsJOURNAL OF DIGESTIVE DISEASES, Issue 1 2002Shui Ping TU OBJECTIVE: Hydroxycamptothecin (HCPT) is a unique antitumor drug that acts directly on topoisomerase I and inhibits its activity. However, the mechanism of HCPT-induced apoptosis is unclear. In the present study, the mechanism of HCPT-induced apoptosis in gastric cancer cells was investigated by detecting the expression of p53, c - myc, bcl-2, bcl-xl and bcl-xs genes in gastric cancer cells. METHODS: Apoptosis of gastric cancer cells (SGC-7901, MKN-45) was determined by terminal deoxyribonucleotidyl transferase-mediated dUTP, digoxigenin nick-end labeling (TUNEL) and flow cytometry. The mRNA and protein levels of the p53, c-myc, bcl-2, bcl-xl and bcl-xs genes were tested by reverse transcription,polymerase chain reaction analysis and immunocytochemical stain, respectively. RESULTS: Hydroxycamptothecin may induce apoptosis in different differentiated gastric cancer cells. The effect of HCPT-induced apoptosis on gastric cancer cells was dependent on the dose of HCPT used and the time of exposure to HCPT. Both SGC-7901 and MKN-45 cells manifested some morphological features of apoptosis after 12 h exposure to HCPT, including cell shrinkage, nuclear condensation, DNA fragmentation and formation of apoptotic bodies. Some typical subdiploid peaks before the G0/G1 phase were observed. The apoptotic rates induced by 10 ,g/mL HCPT in SGC-7901 and MKN-45 cells were 21.88 and 12.34%, respectively. The mRNA and protein levels of p53 and bcl-2 were downregulated after treatment with HCPT in SGC-7901 cells. However, the c-myc, bcl-xl and bcl-xs protein levels were unchanged in SGC-7901 cells. In MKN-45 cells, the mRNA and protein levels of p53 increased after treatment with HCPT, whereas the protein levels of bcl-2, c-myc, bcl-xl and bcl-xs remained unchanged. CONCLUSIONS: Our results indicate that HCPT-induced apoptosis in gastric cancer cells may be regulated through modulation of the expression of p53 and bcl-2 genes. [source] Role of M2, M3, and M4 muscarinic receptor subtypes in the spinal cholinergic control of nociception revealed using siRNA in ratsJOURNAL OF NEUROCHEMISTRY, Issue 4 2009You-Qing Cai Abstract Muscarinic acetylcholine receptors (mAChRs) are involved in the control of nociception in the spinal cord. The M2, M3, and M4 mAChR subtypes are present in the spinal dorsal horn. However, the role of the individual subtypes in the anti-nociceptive effect produced by mAChR agonists is uncertain. Here, we determined the contribution of M2, M3, and M4 subtypes to spinal muscarinic analgesia by using small-interference RNA (siRNA) targeting specific mAChR subtypes in rats. The neuronal uptake and distribution of a chitosan-siRNA conjugated fluorescent dye in the spinal cord and dorsal root ganglion were confirmed after intrathecal injection. The control and gene-specific siRNA-chitosan complexes were injected intrathecally for three consecutive days. Quantitative reverse-transcription polymerase chain reaction analysis showed that treatment with siRNA targeting M2, M3, or M4 subtype produced a large reduction in the corresponding mRNA levels in the dorsal root ganglion and dorsal spinal cord. Also, the protein levels of the mAChR subtypes in the spinal cord were significantly down-regulated by siRNA treatment, as determined by the immunoprecipitation and receptor-binding assay. Treatment with the M2 -siRNA caused a large reduction in the inhibitory effect of muscarine on the nociceptive withdrawal threshold. Furthermore, M4 knockdown at the spinal level significantly reduced the anti-nociceptive effect of muscarine. However, the anti-nociceptive effect of muscarine was not significantly changed by the M3 -specific siRNA. Our study suggests that chitosan nanoparticles can be used for efficient delivery of siRNA into the neuronal tissues in vivo. Our findings also provide important functional evidence that M2 and M4, but not M3, contribute to nociceptive regulation by mAChRs at the spinal level. [source] Characterization of CD8-positive macrophages infiltrating the central nervous system of rats with chronic autoimmune encephalomyelitisJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2009Keiko Hiraki Abstract CD8+ macrophages appear in the central nervous system (CNS) under various pathological conditions such as trauma and ischemia. Furthermore, macrophages expressing CD8 were found in CNS lesions of chronic, but not acute, experimental autoimmune encephalomyelitis (EAE). To further characterize cells with this phenotype, we examined CD8+ macrophages/monocytes in the CNS and peripheral organs during the course of acute and chronic EAE that had been induced by immunization of rats with myelin basic protein and myelin oligodendrocyte glycoprotein, respectively. Counting CD8+ macrophages in CNS lesions revealed that their numbers increased reaching about 60% of total infiltrating macrophages in chronic EAE, while CD8+ macrophages remained less than 5% throughout the course of acute EAE. Unexpectedly, however, higher abundance of CD8+ monocytes/macrophages in the peripheral blood was found in both acute and chronic EAE. Real-time polymerase chain reaction analysis revealed no significant difference in the levels of chemokines and chemokine receptors of blood CD8+ monocytes between acute and chronic EAE. mRNA expression of perforin, a cytotoxic substance, was up-regulated in CD8+ monocytes compared with that of CD8, monocytes in both acute and chronic EAE. These findings suggest that activated CD8+ macrophages may play a cytotoxic role in chronic EAE lesions and that cells other than CD8+ monocytes/macrophages determined the difference in CNS pathology between acute and chronic EAE. Analysis of CD8+ monocytes/macrophages may provide useful information to permit further dissect the pathomechanisms of multiple sclerosis and to develop effective immunotherapies against autoimmune diseases in the CNS. © 2008 Wiley-Liss, Inc. [source] Histamine-induced Ca2+ entry in human astrocytoma U373 MG cells: Evidence for involvement of store-operated channelsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 15 2008Margarita Barajas Abstract Glial and glia-derived cells express a variety of receptors for neurotransmitters and hormones, the majority of which evoke both Ca2+ release from intracellular stores and Ca2+ entry across the plasma membrane. We investigated the links between histamine H1 receptor activation, Ca2+ release from intracellular stores and Ca2+ influx in human astrocytoma U373 MG cells. Histamine, through a H1 receptor-mediated effect, evoked an increase in cytoplasmic free calcium concentration ([Ca2+]i) that occurred in two phases: an initial, transient, increase owing to Ca2+ mobilization from intracellular pools, and a second, sustained increase dependent on both Ca2+ influx and continuous receptor occupancy. The characteristics of histamine-induced increases in [Ca2+]i were similar to the capacitative entry evoked by emptying of the Ca2+ stores with thapsigargine, and different from that observed when Ca2+ influx was activated with OAG (1-oleoyl-2-acetyl- sn -glycerol), a diacylglycerol (DAG) analog. OAG application or increased endogenous DAG, resulting from DAG kinase inhibition, reduced the histamine-induced response. Furthermore, activation of the DAG target, protein kinase C (PKC), by TPA (12-O-tetradecanoyl 4,-phorbol 13,-acetate) resulted in inhibition of the histamine-induced Ca2+ response, an action prevented by PKC inhibitors. By using reverse transcriptase,polymerase chain reaction analysis, mRNAs for transient receptor potential channels (TRPCs) 1, 4, and 6 as well as for STIM1 (stromal-interacting molecule) and Orai1 were found to be expressed in the U373 MG cells, and confocal microscopy using specific antibodies revealed the presence of the corresponding proteins. Therefore, TRPCs may be candidate proteins forming store-operated channels in the U373 MG cell line. Further, our results confirm the involvement of PKC in the regulation of H1 receptor-induced responses and point out to the existence of a feedback mechanism acting via PKC to limit the increase in [Ca2+]i. © 2008 Wiley-Liss, Inc. [source] Identification of genes related to mechanical stress in human periodontal ligament cells using microarray analysisJOURNAL OF PERIODONTAL RESEARCH, Issue 1 2007R. M. S. De Araujo Background and Objective:, Differential expression of genes in human periodontal ligament (PDL) under mechanical stress, such as orthodontic force, is thought to be involved in the remodeling of PDL cells and periodontal tissues. However, little is known about the genes expressed in PDL cells under mechanical stress. Material and Methods:, We employed microarray analysis to assess, in a comprehensive manner, the gene expression profiles in PDL cells compressed by a static force using an in vitro three-dimensional culture system. Six genes were selected and validated by quantitative real-time polymerase chain reaction analysis, consistent with the microarray data. Results:, The microarray data revealed that 108 of 30,000 genes tested were differentially expressed by mechanical force loading. Among them, 85 genes were up-regulated by mechanical stress, while 23 genes were down-regulated, judging by the thresholds of a two-fold increase/decrease compared with the controls. Thirty-two of the up-regulated and eight of the down-regulated genes, well-characterized in protein function, were involved in numerous biological processes including cell communication, cell signaling, cell cycle, stress response, and calcium release. However, several genes differentially expressed in our microarray data have not been well defined as stress-response molecules. Conclusion:, Our microarray is the first to show the gene profile in PDL cells caused by mechanical stress; however, further studies to clarify the physiological function of these molecules in PDL cells are required. [source] | |||||||||||||||||||||||||||||||