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Immunohistochemistry Experiments (immunohistochemistry + experiment)
Selected AbstractsVery high conservation between Cyp6a2 from Drosophila melanogaster and its ortholog Cyp6a26 from D. simulansINSECT SCIENCE, Issue 1 2007SOPHIE TARÈS Abstract Although Drosophila simulans is closely related to D. melanogaster, very few cytochrome P450 genes have been studied in this species until now. As Cyp6a2 from D. melanogaster is a major gene implicated in the detoxification of xenobiotic molecules, we decided to look for its ortholog in D. simulans. The isolated gene, Cyp6a26, presents structural characteristics very similar to those of Cyp6a2: an identical size of 1 590-bp comprising two exons separated by a 69-bp intron and a nucleotide sequence homology of 95%. Many putative transcriptionally important motifs were identified in the upstream DNAs of the two genes but only 16 elements are in common positions. Treatment of flies with phenobarbital leads to an increased production of Cyp6a26 mRNAs. The expression of Cyp6a26 mRNAs varies following developmental stages in the same manner as Cyp6a2. Immunohistochemistry experiments of phenobarbital-treated adult drosophila show that the spatial expression pattern of the two proteins is also conserved between the two species. All these data argue in favor of the conservation of the function of these homologous genes between the two Drosophila species. [source] A genome-wide association study identifies an osteoarthritis susceptibility locus on chromosome 7q22,ARTHRITIS & RHEUMATISM, Issue 2 2010Hanneke J. M. Kerkhof Objective To identify novel genes involved in osteoarthritis (OA), by means of a genome-wide association study. Methods We tested 500,510 single-nucleotide polymorphisms (SNPs) in 1,341 Dutch Caucasian OA cases and 3,496 Dutch Caucasian controls. SNPs associated with at least 2 OA phenotypes were analyzed in 14,938 OA cases and ,39,000 controls. Meta-analyses were performed using the program Comprehensive Meta-analysis, with P values <1 × 10,7 considered genome-wide significant. Results The C allele of rs3815148 on chromosome 7q22 (minor allele frequency 23%; intron 12 of the COG5 gene) was associated with a 1.14-fold increased risk (95% confidence interval 1.09,1.19) of knee and/or hand OA (P = 8 × 10,8) and also with a 30% increased risk of knee OA progression (95% confidence interval 1.03,1.64) (P = 0.03). This SNP is in almost complete linkage disequilibrium with rs3757713 (68 kb upstream of GPR22), which is associated with GPR22 expression levels in lymphoblast cell lines (P = 4 × 10,12). Immunohistochemistry experiments revealed that G protein,coupled receptor protein 22 (GPR22) was absent in normal mouse articular cartilage or synovium. However, GPR22-positive chondrocytes were found in the upper layers of the articular cartilage of mouse knee joints that were challenged with in vivo papain treatment or methylated bovine serum albumin treatment. GPR22-positive chondrocyte-like cells were also found in osteophytes in instability-induced OA. Conclusion Our findings identify a novel common variant on chromosome 7q22 that influences susceptibility to prevalence and progression of OA. Since the GPR22 gene encodes a G protein,coupled receptor, this is potentially an interesting therapeutic target. [source] Prothrombin kringle-2 induces death of mesencephalic dopaminergic neurons in vivo and in vitro via microglial activationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 7 2010Sang Ryong Kim Abstract We have shown that prothrombin kringle-2 (pKr-2), a domain of human prothrombin distinct from thrombin could activate cultured rat brain microglia in vitro. However, little is known whether pKr-2-induced microglial activation could cause neurotoxicity on dopaminergic (DA) neurons in vivo. To address this question, pKr-2 was injected into the rat substantia nigra (SN). Tyrosine hydroxylase (TH) immunohistochemistry experiments demonstrate significant loss of DA neurons seven days after injection of pKr-2. In parallel, pKr-2-activated microglia were detected in the SN with OX-42 and OX-6 immunohistochemistry. Reverse transcription PCR and double-label immunohistochemistry revealed that activated microglia in vivo exhibit early and transient expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and several proinflammatory cytokines. The pKr-2-induced loss of SN DA neurons was partially inhibited by the NOS inhibitor NG -nitro-L-arginine methyl ester hydrochloride, and the COX-2 inhibitor DuP-697. Extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were activated in the SN as early as 1 hr after pKr-2 injection, and localized within microglia. Inhibition of these kinases led to attenuation of mRNA expression of iNOS, COX-2 and several proinflammatory cytokines, and rescue of DA neurons in the SN. Intriguingly, following treatment with pKr-2 in vitro, neurotoxicity was detected exclusively in co-cultures of mesencephalic neurons and microglia, but not microglia-free neuron-enriched mesencephalic cultures, indicating that microglia are required for pKr-2 neurotoxicity. Our results strongly suggest that microglia activated by endogenous compound(s), such as pKr-2, are implicated in the DA neuronal cell death in the SN. © 2009 Wiley-Liss, Inc. [source] A novel Phe171Cys mutation in integrin ,IIb causes Glanzmann thrombasthenia by abrogating ,IIb,3 complex formationJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2004N. Rosenberg Summary.,Background: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation induced by most agonists. The disease is caused by mutations in either ,IIb[glycoprotein (GP) IIb] or ,3 (GPIIIa) genes that lead to a lack or dysfunction of the integrin ,IIb,3 which serves as a fibrinogen receptor. Patients Mucocutaneous bleeding manifestations and platelet dysfunction consistent with GT were observed in three members of a Cypriot family: a 3-year-old proband, her father and her paternal uncle. Objective: To determine the molecular basis of GT in this family and to characterize possible biochemical and structural defects. Results: Analysis of the patients' platelets by fluorescence-activated cell sorting demonstrated trace amounts of ,3, no ,IIb and no ,IIb,3 on the membrane. Sequence analysis revealed a novel T607G transversion in exon 5 of the ,IIb gene predicting a Phe171Cys alteration that created a PstI recognition site. All three patients were homozygous for the mutation, the mother and paternal grandparents of the proband were heterozygous, whereas 110 healthy subjects lacked this transversion. Chinese hamster ovary cells cotransfected with cDNAs of mutated ,IIb and wild-type ,3 failed to express ,IIb,3 as shown by immunoprecipitation and immunohistochemistry experiments. Structural analysis of the ,IIb,3 model, which was based on the crystal structure of ,v,3, indicated that Phe171 plays an essential role in the interface between the ,-propeller domain of ,IIb and the ,A domain of ,3. Conclusions: A novel Phe171Cys mutation in the ,IIb gene of patients with GT is associated with abrogation of ,IIb,3 complex formation. [source] Identification, molecular cloning, and cellular distribution of the rat homolog of minichromosome maintenance protein 7 (MCM7) in the rat testis,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 7 2006Emmanuelle Com Abstract As part of a program to decipher the rat testicular proteome, we studied spermatogonia and identified numerous proteins including the human homolog of the Minichromosome Maintenance Protein 7 (MCM7). MCM7 has been implicated in DNA replication in various species, but had not been detected in the testis. Here we describe the cellular distribution of MCM7 transcripts and protein, and their testicular ontogenetic expression. The full-length coding region of the rat MCM7 was also characterized. Northern blot analyses showed that MCM7 transcripts are more abundant in the testis than other organs and confirmed the presence of the 2.4 kb MCM7 transcript at all ages studied. Interestingly, two additional transcripts of 3.2 and 1.6 kb were found from 26 days post partum onwards, when spermatocytes and spermatids accumulate within the tubules. This was confirmed in isolated cell types: the three MCM7 transcripts were observed in meiotic and post-meiotic germ cells. The 3.2 kb isoform has an extended 5, untranslated region (UTR) and the 1.6 kb transcript is the result of alternative splicing of five exons. Western blot and immunohistochemistry experiments evidenced abundant MCM7 in proliferating gonocytes and Sertoli cells in the fetal testis. In the adult testis, an intense signal was observed in spermatogonia and primary spermatocytes. We conclude that the Mcm7 is one example of genes that are differently transcribed and translated in somatic and spermatogenetic cells in mammals. Further work is required to determine the roles of MCM7 in spermatogonia and germ lineage. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] Altered expression of TRPV1 and sensitivity to capsaicin in pulmonary myelinated afferents following chronic airway inflammation in the ratTHE JOURNAL OF PHYSIOLOGY, Issue 23 2008Guangfan Zhang Vagal pulmonary myelinated afferents are normally not activated by capsaicin, a selective agonist of transient receptor potential vanilloid type 1 (TRPV1) receptors. This study was carried out to investigate whether the expression of TRPV1 in these afferents is altered when chronic airway inflammation is induced by ovalbumin (Ova) sensitization. Two groups of Brown,Norway rats (sensitized and control) were exposed to aerosolized Ova and vehicle, respectively, 3 days per week for 3 weeks. After the C-fibre conduction in both vagus nerves was blocked, right-atrial injection of capsaicin elicited augmented breaths in sensitized rats breathing spontaneously, but not in control rats, indicating a stimulation of rapidly adapting receptors (RARs) by capsaicin. Single-unit fibre activities of RARs and slow adapting receptors (SARs), identified by their firing behaviour and adaptation indexes in response to lung inflation, were recorded in anaesthetized, vagotomized and artificially ventilated rats. Capsaicin injection evoked either negligible or no response in both RARs and SARs of control rats. However, in striking contrast, the same dose of capsaicin evoked an immediate stimulatory effect on these myelinated afferents in sensitized rats. Furthermore, the immunohistochemistry experiments showed that there was a significant increase in the proportion of TRPV1-expressing pulmonary neurones in nodose ganglia of sensitized rats; this increase in TRPV1 expression was found mainly in neurofilament-positive (myelinated) neurones. In conclusion, allergen-induced airway inflammation clearly elevated capsaicin sensitivity in myelinated pulmonary afferents, which probably resulted from an increased expression of TRPV1 in these sensory nerves. 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