Detection of calcitonin gene expression in human infant and monkey carotid body chief cells by in situ hybridization

Calcitonin mRNA was detected in human and monkey carotid bodies by in situ hybridization histochemistry, using a ³⁵S-labeled oligonucleotide probe for human calcitonin. In both human and monkey carotid body, moderate to high hybridization signal for calcitonin mRNA was observed in all cases. The hybridization signal in the formalin-fixed, paraffin-embedded samples was comparable to that obtained from frozen paraformaldehyde-fixed tissue. Our observations extend the finding of calcitonin-like immunoreactivity in the carotid body chief cells and indicate that calcitonin is produced in the carotid body, probably in the chief cells.     

Effect of eicosapentaenoic acid on the different endothelin system components in endothelin-1-induced hypertrophied cardiomyocytes

The cardiovascular benefit of fish oil, including eicosapentaenoic acid (EPA), in humans and experimental animals has been reported. The role of endothelin-1 (ET-1) in cardiac hypertrophy is well known. Endothelin-1 stimulates prepro-ET-1 mRNA expression in cardiomyocytes, and the autocrine/paracrine system of ET-1 is important for cardiomyocyte hypertrophy. Although many studies link EPA to cardiac protection, the effect of EPA on cardiac hypertrophy has yet to be clarified. Recently, we demonstrated that ET-1-induced cardiomyocytic change could be prevented by pretreatment with EPA. The present study investigated the changes of different components of the ET system at the mRNA level in ET-1-administered cardiomyocytes, and examined the effect of EPA pretreatment. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats, cultured in Dulbecco's modified Eagle's medium and Ham F12 supplemented with 0.1% fatty acid-free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into 3 groups: control group, ET-1-treated (0.1 nM) group, and ET-1-treated group pretreated with EPA (10 microM). Twenty-four hours after treatment, the gene expressions of different components of the endothelin system in three experimental groups were evaluated by real-time polymerase chain reaction. Prepro-ET-1 mRNA expression was 53% upregulated in ET-1-induced hypertrophied cardiomyocytes and suppressed in the EPA-pretreated group. Endothelin-converting enzyme-1 (ECE-1) was also increased in ET-1-administered cardiomyocytes by 42% compared with the control group and was reversed in the EPA-pretreated group. The two receptors of ET system, ET(A) and ET(B), tended to be increased in the ET-1-treated group, but no statistical significance was seen among study groups. Endothelin-1 increased prepro-ET-1 and ECE-1 mRNA expression in hypertrophied-neonatal cardiomyocytes, and this was reversed with EPA pretreatment. Thus, EPA may play a crucial role in the regression of ET-1-induced cardiomyocyte hypertrophy, partly through the suppression of ET-1 and ECE-1 expression.     

Abnormal gene expression profiles in human ovaries from polycystic ovary syndrome patients

Polycystic ovary syndrome (PCOS) represents the most common cause of anovulatory infertility and affects 5-10% of women of reproductive age. The etiology of PCOS is still unknown. The current study is the first to describe consistent differences in gene expression profiles in human ovaries comparing PCOS patients vs. healthy normoovulatory individuals. The microarray analysis of PCOS vs. normal ovaries identifies dysregulated expression of genes encoding components of several biological pathways or systems such as Wnt signaling, extracellular matrix components, and immunological factors. Resulting data may provide novel clues for ovarian dysfunction in PCOS. Intriguingly, the gene expression profiles of ovaries from (long-term) androgen-treated female-to-male transsexuals (TSX) show considerable overlap with PCOS. This observation provides supportive evidence that androgens play a key role in the pathogenesis of PCOS. Presented data may contribute to a better understanding of dysregulated pathways in PCOS, which might ultimately reveal novel leads for therapeutic intervention.     

The use ofin situ hybridization histochemistry for the study of neuropeptide gene expression in the human brain

1. The application of in situ hybridization histochemistry to the study of neuropeptide gene expression in human brain postmortem tissues is reviewed. We focus on neuropeptides preferentially expressed in hypothalamus and basal ganglia. 32P-labeled oligonucleotides were used as hybridization probes. 2. Autoradiography combined with computerized image analysis was used to visualize and quantify the hybridization signal. 3. Several criteria were considered in order to ascertain the specificity of the signal, including Northern analysis, use of heterologous probes, competition assays, and thermal stability of the hybrids. 4. In control human striatum high levels of hybridization signal were observed for somatostatin, neuropeptide Y, and preproenkephalin A mRNAs. In contrast, no detectable signal was observed with the cholecystokinin, arginine-vasopressin, and oxytocin probes in this area. In the hypothalamus high levels of oxytocin and arginine-vasopressin mRNAs were visualized in several nuclei. Preproenkephalin A and somatostatin mRNAs were also observed in this region, while cholecystokinin mRNA was not detected. 5. No significant correlations were found between the density of the hybridization signal and parameters such as postmortem delay, age, and gender in the population studied. 6. Finally, alterations of mRNA levels for some of these peptides were found in Parkinson's disease and Huntington's chorea striatal tissues. 7. These results show that in situ hybridization histochemistry can be used to examine at the microscopic level neuropeptide gene expression in postmortem materials.     

Differences in human alpha-, beta- and delta-globin gene expression in monkey kidney cells

We have compared the function of the human alpha-, beta- and delta-globin genes using various plasmid expression vectors derived from pBR322. Amplification of recombinants occurred after their introduction, by calcium-phosphate-mediated DNA transfer, into monkey kidney cells that constitutively produce T antigen (COS cells). The human alpha-globin gene promoter functioned independently, but the beta-globin gene promoter was nearly totally dependent on the enhancing activity of the 72 bp direct repeats from the SV40 genome. Furthermore, when the human alpha- and beta-globin genes were linked in the same vector, the alpha promoter was active but the beta promoter was not. Function of the delta-globin gene promoter also depended on the enhancer element. In vectors containing the 72 bp repeats and the beta- or delta-globin gene, the activity of the beta-globin gene was approximately 50 times greater than that of the delta-globin gene, approximating the ratio of beta and delta mRNA observed in normal human bone marrow cells.     

Nitric oxide in cyclosporine A-induced hypertension: endothelin receptors gene expression.

Cyclosporine A (CsA) is an immunosuppressive agent, which also causes hypertension. The effect of CsA on vascular responses was determined in spontaneously hypertensive rats and isolated rat aortic rings. Male rats weighing 250-300 g were given either CsA (25 mg/kg/day) in olive oil or vehicle by i.p. injection for 7 days. CsA administration produced a 27% increase (P < 0.001) in mean arterial pressure (MAP) which reached a plateau after 3 days. Conversely, the level of nitrate/nitrite, metabolites of nitric oxide (NO), decreased by 44% (P < 0.001) in the urine. In the presence of endothelin (ET) 10(-9) M, thoracic aortic rings from rats treated with olive oil, L-Arginine (L-Arg) or L-Arg+CsA showed a 100% increase (P < 0.001) in tension compared to the aortic rings from rats treated with CsA alone; aortic rings from rats treated with CsA alone did not respond to ET. The effects of CsA were reversed in both in vivo and in vitro by pretreatment with L-Arg (10 mg/kg/day ip), the precursor of NO. There were no changes in MAP and tension in rats treated with L-Arg alone. Possible explanation for lack of response to ET of aortic rings from CsA treated rats may be that CsA affected ET signalling pathway; ET receptors mRNA (messenger ribonucleic acid) gene expression was inhibited in aortic rings of rats treated with CsA. In summary, CsA inhibits endothelial NO formation, with resulting increases in MAP, and this inhibition can be overcome by parenteral administration of L-Arg.     

Presence of Fas-Fas ligand system and bcl-2 gene products in cells and fluids from gonadotropin-stimulated human ovaries

Apoptosis, or programmed cell death, is an important mechanism for the regulation of embryonic development and tissue homeostasis. It is coordinated by a number of molecules including the Fas-Fas ligand (FasL) system and bcl-2. The purpose of this study was to characterize the expression of these molecules in human oocytes and cumulus cells from gonadotropin-stimulated human ovaries and to determine whether the presence of soluble Fas (sFas), soluble FasL, or interferon-gamma in follicular fluid (FF) correlated with apoptosis in cumulus cells, oocyte maturation, and embryo quality. Levels of sFas were significantly higher in FF containing immature oocytes compared with those containing atretic oocytes (P < 0.05; FF containing mature oocytes had highly variable levels of sFas. Levels of sFas in FF did not correlate with either fertilization, embryo quality resulting from fertilized oocytes, or apoptosis rate in cumulus cells. Fas was expressed in both unfertilized oocytes and cumulus cells, whereas FasL expression was not usually detected in these cell types. Messenger RNA for bcl-2 was detectable in both freshly isolated oocytes and cumulus cells but was not demonstrable following 24 h of culture that coincided with a significant increase of apoptosis in cumulus cells. Our results indicate that soluble forms of the Fas-FasL system are present in FF from gonadotropin-stimulated human ovaries and suggest that this system may play a role in preventing oocyte atresia during folliculogenesis but is probably not important for apoptotic events in cumulus cells and oocytes after fertilization failure. Apoptosis in this case may be facilitated by the downregulation of bcl-2. Further studies on the expression of these molecules in follicles containing atretic oocytes and immature oocytes are needed to confirm this new hypothesis.     

The expression of the apolipoprotein A-1 gene in the early stages of human embryogenesis studied by hybridization in situ]

The apolipoprotein A-1 (apo A-1) gene expression at certain stages of human embryo development was studied using an in situ hybridization procedure. By the fifth week of development, the presence of apo A-1 mRNAs was detected in cells of different tissue types such as CNS rudiment, somite myotomes, ear vesicle, nasal placodes, lens, apical areas of the maxillary, mandibular and hyoid arcs, mesenchyme of limb buds, small intestine, mesonephros, genital ridge, and several types of blood cells. At later stages (weeks 8-9) the distribution of these mRNAs showed considerable changes. The high apo A-1 mRNA level was characteristic of liver cells, adrenals, kidney, neural tube and ganglia. These data suggest that changes in tissue-specific apo A-1 gene expression during human embryogenesis may be associated with the development of blood circulatory system and CNS.