Expression of Mash1 in basal cells of rat circumvallate taste buds is dependent upon gustatory innervation.
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Mash1, a mammalian homologue of the Drosophila achaete-scute proneural gene complex, plays an essential role in differentiation of subsets of peripheral neurons. In this study, using RT-PCR and in situ RT-PCR, we investigated if Mash1 gene expression occurs in rat taste buds. Further, we examined dynamics of Mash1 expression in the process of degeneration and regeneration in denervated rat taste buds. In rat tongue epithelium, Mash1 gene expression is confined to circumvallate, foliate, and fungiform papilla epithelia that include taste buds. In taste buds, Mash1-expressing cells are round cells in the basal compartment. In contrast, the mature taste bud cells do not express the Mash1 gene. Denervation and regeneration experiments show that the expression of Mash1 requires gustatory innervation. We conclude that Mash1 is expressed in cells of the taste bud lineage, and that the expression of Mash1 in rat taste buds is dependent upon gustatory innervation. (+info)
The TATA-box binding protein of Entamoeba histolytica: cloning of the gene and location of the protein by immunofluorescence and confocal microscopy.
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A 309 bp DNA fragment from Entamoeba histolytica was amplified by PCR using primers derived from the Acanthamoeba castellanii consensus TATA-box binding protein amino acid sequence. The amplified fragment was used to isolate cDNA and genomic DNA clones containing an ORF encoding the complete E. histolytica TATA-box binding protein (Ehtbp, 702 bp, 234 aa, molecular mass 26 kDa). The EhTBP functional domain showed 55% sequence identity to that of Homo sapiens, 54% to A. castellanii and 37% to Plasmodium falciparum TBPs. In Southern blot experiments we detected a single Ehtbp band, which was transcribed as a 1.3 kb mRNA containing a 420 nt 5' untranslated region. However, the probe hybridized with the 0.8 and 1.5 Mb chromosomes, suggesting that this sequence is diploid. In situ PCR assays showed two signals in 95% of trophozoites, one located in the nucleus and another in EhkO, the novel DNA-containing organelle recently reported. The recombinant E. histolytica TATA-box binding protein was expressed in Escherichia coli. Antibodies against it recognized two proteins of 26 and 29 kDa in E. histolytica nuclear extracts. Confocal microscopy immunofluorescence analysis located the protein in both the nucleus and EhkO. (+info)
Latent Varicella-zoster virus in human dorsal root ganglia.
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To understand further the molecular events underlying the process of Varicella-zoster virus (VZV) latency in human ganglionic tissues, in situ hybridisation (ISH) for VZV RNA and DNA, and PCR in situ amplification for VZV DNA were used in human dorsal root ganglia from 12 individuals (3 normal and 9 who had died with AIDS). The results showed that (a) two separate regions of the VZV genome, represented by genes 4 and 40, were detected in neurons in two normal and three AIDS ganglia, (b) evidence of transcription of VZV genes 4, 21, 29, and 63 was found in normal and AIDS cases, and (c) VZV DNA and RNA for the same gene (gene 29) was detected in neurons in serial tissue sections in three cases. Thus more than one region of the VZV genome is present in neurons during VZV ganglionic latency, and the presence of both a VZV gene and its corresponding RNA transcript can be shown to occur in the same localised region of DRG tissue. (+info)
Expression of FAS adjacent to fibrotic foci in the failing human heart is not associated with increased apoptosis.
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Fibrosis in the heart may result from loss of myocytes, which are replaced by collagens. Apoptosis is now known to contribute to myocyte loss in the failing human heart. The mechanisms underlying the induction of cardiomyocyte apoptosis, and thus the expansion of fibrotic foci in the failing heart, are poorly understood. We hypothesized that viable heart cells adjacent to fibrotic foci might become "predisposed" to apoptosis by expression of the receptor FAS (APO1, CD95). We therefore studied the spatial relationship of FAS expression and fibrosis in patients with heart failure. Left ventricular biopsies were obtained from seven patients undergoing coronary artery bypass grafting. All patients had reduced ejection fraction but varied in New York Heart Association class score at the time of surgery. Heart cell apoptosis, fibrosis, and FAS expression were studied by propidium iodide and in situ end labeling (ISEL) of DNA, Picrosirius red staining, and immunohistochemistry. All patient samples exhibited, albeit to varying degrees, apoptosis detected by ISEL, chromatin condensation, and nuclear fragmentation. In all samples, fibrosis (collagen) was evident both perivascular and in isolated regions of scarring. Regardless of the extent of fibrosis or detectable apoptosis, FAS expression was observed in regions immediately adjacent to the fibrosis, but not in regions distal to fibrosis, nor in fibrotic areas devoid of nuclei. Expression of FAS was found adjacent to both perivascular and diffuse fibrosis, and ISEL-positive nuclei were found within cells reacting positively with anti-FAS antibodies. However, ISEL-positive nuclei were no more abundant in FAS-positive regions (67.6 +/- 5.8% of total nuclei) than in FAS-negative areas (69.5 +/- 9.8%). We conclude that expression of FAS occurs in remaining heart cells adjacent to fibrosis of either perivascular or presumed reparative origin. Although this phenomenon could contribute to the expansion of fibrotic foci, FAS-induced apoptosis in the failing heart may not be more prevalent than apoptosis initiated by other signaling mechanisms. (+info)
Localization of mariner DNA transposons in the human genome by PRINS.
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Homologous recombination occurring among misaligned repeated sequences is a significant source of the molecular rearrangements resulting in human genetic disease. Studies of the Charcot-Marie-Tooth disease locus on chromosome 17 have implicated the involvement of an ancient DNA transposon of the mariner family (Hsmar2) in the initiation of double-strand break events leading to homologous recombination. In this study, the genomic locations of 109 Hsmar2 elements were determined by primed in situ labeling (PRINS) using primers designed to match the right and left inverted terminal repeats (ITRs) of the transposon. Although the resolution of the PRINS technique is approximately 400 chromosomal Giemsa bands, the data presented here provide the first large-scale mapping study of these elements, which may be involved in initiation of homologous recombination events in the human genome. (+info)
NF-kappa B modulates TNF-alpha production by alveolar macrophages in asymptomatic HIV-seropositive individuals.
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Local TNF-alpha production in different organs may affect HIV replication and pathogenesis. Alveolar macrophages (AMs) obtained by bronchoalveolar lavage from asymptomatic HIV-seropositive and HIV-seronegative individuals did not spontaneously release TNF-alpha, but LPS stimulation of these cells significantly increased TNF-alpha production. We tested whether NF-kappa B affects TNF-alpha production by AMs using N-tosyl-l -phenylalanine chloromethylketone (TPCK) or N-benzoyl-l -tyrosine ethyl ester (BTEE), which inhibit the degradation of I kappa B, or tricyclodecan-9-yl-xanthogenate-potassium (D609), which inhibits phospholipase C. Alveolar macrophages were exposed to LPS alone and with the chemical protease inhibitors TPCK, BTEE, and D609. NF-kappa B DNA binding induced by LPS treatment of AMs was inhibited by TPCK, BTEE, and D609. These agents also inhibited TNF-alpha mRNA and TNF-alpha protein production. After 24 h, the levels of TNF-alpha mRNA reached equilibrium, as assessed by RT-PCR. The levels of NF-kappa B mRNA remained constant under all conditions. The levels of I kappa B-alpha mRNA were similar after 30, 60, and 180 min, but the I kappa B-beta mRNA concentration was initially low and increased over time under all conditions. I kappa B-alpha and I kappa B-beta protein production was not affected by the chemical protease inhibitors. Our data show that TNF-alpha production by LPS-stimulated AMs from asymptomatic HIV-seropositive and -seronegative individuals is regulated via the phospholipase C pathway and by NF-kappa B DNA binding activity without obvious changes in I kappa B-alpha or I kappa B-beta protein concentrations. (+info)
Detection and characterization of micronuclei in a murine liver epithelial cell line, by application of the in vitro cytokinesis block MN assay and PRINS.
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The cytokinesis block micronucleus assay was applied to murine cell line C6, derived from fetal liver, after an optimal protocol had been designed. Micronucleus frequencies were assayed after exposure to three concentrations of colcemid or diepoxybutane. Two-colour primed in situ DNA synthesis (PRINS) was applied to simultaneously label telomeric and centromeric (minor satellite DNA) sequences. Both chemicals induced a highly significant increase in MN and the effect was dose dependent. Diepoxybutane did not appear to significantly increase the frequency of centromere-positive micronuclei. Colcemid, as expected, induced high frequencies of centromere-positive micronuclei at all concentrations tested; in addition a significant increase in centromere-negative micronuclei was observed at 10(-5) M. Many centromere-positive micronuclei carried three or four telomeres, thus indicating that a duplicated (non-disjoined) chromosome with two chromatids was contained in the micronucleus. This observation leads to the conclusion that micronuclei deriving from missegregation could be due to errors occurring before the onset of anaphase. The results obtained on C6 cells are in good agreement with those obtained on other cell systems, indicating that this cell line can be considered for in vitro aneuploidy evaluation. (+info)
Quantitative detection of apoptotic thymocytes in low-dose X-irradiated mice by an anti-single-stranded DNA antibody.
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The quantitative detection of apoptotic cells in low frequency in the thymus of mice irradiated with X-rays using an anti-single-stranded DNA antibody was explored. The antibody against single-stranded DNA (anti-ssDNA) was obtained with rabbits hyperimmunized with complexes of alkaline-denatured calf thymus DNA. AKR female mice were irradiated with 10 to 100 cGy or 4 Gy X-rays; thereafter, thymus sections were prepared at various times after irradiation. The detection and counting of apoptotic cells in the section were performed after histochemical staining using an anti-ssDNA antibody. The results demonstrate that, although sensitive and quantitative detection of apoptotic cells in irradiated thymus using the anti-ssDNA antibody is possible, the sensitivity is lower compared to that of in situ end-labeling methods, such as TUNEL or ISEL. The antibodies could also be used for rat thymus and spleen. In addition, an increase in positively stained cells by both methods was detected as early as 6 min after the irradiation of mice. (+info)