Alternative splicing generates multiple mRNA forms of the acetylcholine receptor gamma-subunit in rat muscle.
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The fetal type acetylcholine receptor, composed of the alphabeta gammadelta subunits, has shown a highly variable channel kinetics during postnatal development. We examine the hypothesis whether such a variability could result from multiple channel forms, differing in the N-terminus of the gamma-subunit. RT-PCR revealed, in addition to the full-length mRNA, three new forms lacking exon 4. One of them in addition lacks 19 nucleotides from exon 5, predicting a complete subunit, with a 43 residues shorter N-terminus. A third one lacking the complete exon 5 predicts a subunit without transmembrane segments. These forms, generated by alternative splicing, may account for the kinetic variability of the acetylcholine receptor channel. (+info)
Canine preprorelaxin: nucleic acid sequence and localization within the canine placenta.
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Employing uteroplacental tissue at Day 35 of gestation, we determined the nucleic acid sequence of canine preprorelaxin using reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Canine preprorelaxin cDNA consisted of 534 base pairs encoding a protein of 177 amino acids with a signal peptide of 25 amino acids (aa), a B domain of 35 aa, a C domain of 93 aa, and an A domain of 24 aa. The putative receptor binding region in the N'-terminal part of the canine relaxin B domain GRDYVR contained two substitutions from the classical motif (E-->D and L-->Y). Canine preprorelaxin shared highest homology with porcine and equine preprorelaxin. Northern analysis revealed a 1-kilobase transcript present in total RNA of canine uteroplacental tissue but not of kidney tissue. Uteroplacental tissue from two bitches each at Days 30 and 35 of gestation were studied by in situ hybridization to localize relaxin mRNA. Immunohistochemistry for relaxin, cytokeratin, vimentin, and von Willebrand factor was performed on uteroplacental tissue at Day 30 of gestation. The basal cell layer at the core of the chorionic villi was devoid of relaxin mRNA and immunoreactive relaxin or vimentin but was immunopositive for cytokeratin and identified as cytotrophoblast cells. The cell layer surrounding the chorionic villi displayed specific hybridization signals for relaxin mRNA and immunoreactivity for relaxin and cytokeratin but not for vimentin, and was identified as syncytiotrophoblast. Those areas of the chorioallantoic tissue with most intense relaxin immunoreactivity were highly vascularized as demonstrated by immunoreactive von Willebrand factor expressed on vascular endothelium. The uterine glands and nonplacental uterine areas of the canine zonary girdle placenta were devoid of relaxin mRNA and relaxin. We conclude that the syncytiotrophoblast is the source of relaxin in the canine placenta. (+info)
Glutathione-independent prostaglandin D2 synthase in ram and stallion epididymal fluids: origin and regulation.
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Microsequencing after two-dimensional electrophoresis revealed a major protein, glutathione-independent prostaglandin D2 synthase (PGDS) in the anterior epididymal region fluid of the ram and stallion. In this epididymal region, PGDS was a polymorphic compound with a molecular mass around 30 kDa and a range of pI from 4 to 7. PGDS represented 15% and 8% of the total luminal proteins present in this region in the ram and stallion, respectively. The secretion of the protein as judged by in vitro biosynthesis, and the presence of its mRNA as studied by Northern blot analysis, were limited to the proximal caput epididymidis. Using a specific polyclonal antibody raised against a synthetic peptide, PGDS was found throughout the epididymis, decreasing in concentration toward the cauda region. PGDS was also detected in the testicular fluid and seminal plasma by Western blotting. Castration and efferent duct ligation in the ram led to a decrease in PGDS mRNA and secretion. PGDS mRNA was not detected in the stallion 1 mo after castration, and it was restored by testosterone supplementation. This study showed that PGDS is present in the environment of spermatozoa throughout the male genital tract. Its function in the maturation and/or protection of spermatozoa is unknown. (+info)
Vitamin D regulates human ectocervical epithelial cell proliferation and insulin-like growth factor-binding protein-3 level.
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The differentiation status of the cervical epithelial cell has an important influence on responsiveness to estrogens and progestins. Several agents, including glucocorticoids and retinoids, are known to influence cervical cell differentiation. However, the effects of vitamin D have not been examined. Vitamin D is known to regulate cell proliferation and gene expression in a variety of epithelial cells. In the present study we investigated the ability of 1alpha25-dihydroxyvitamin D3 (D3) to regulate cell proliferation and expression of insulin-like growth factor-binding protein-3 (IGFBP-3) in human ectocervical epithelial cells. ECE16-1, a non-tumorigenic cervical cell line, was growth inhibited by D3 with maximal inhibition at 1000 nM. IGFBP-3 levels increased in parallel with the growth inhibition. IGFBP-3 levels were half-maximally increased at approximately 10-100 nM and maximally increased (10- to 30-fold) at 1000 nM D3. These studies show that vitamin D regulates cervical epithelial cell gene regulation and cell proliferation and that IGFBP-3 may be an in vivo marker of vitamin D action in the cervix. (+info)
Localization of sodium bicarbonate cotransporter (NBC) protein and messenger ribonucleic acid in rat epididymis.
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An acidic environment is important for sperm maturation in the epididymis and also helps to maintain mature sperm in an immotile state during storage in this organ. Both an Na+/H+ exchanger and an H+ATPase have been implicated in this process. The H+ATPase is concentrated in specialized apical (and/or narrow) and clear cells of the epididymis, while the Na+/H+ exchanger has not yet been localized in situ. As in other proton-secreting epithelia, bicarbonate transport occurs in the epididymis, where it is implicated in luminal acidification. In this study we used an antibody raised against a fusion protein (maltose-binding protein: MBP-NBC-5) from the C-terminus of the recently cloned rat kidney Na+/HCO3- cotransporter (NBC) to localize this protein in the epididymis and vas deferens of the rat. The distribution of the respective mRNA was mapped by in situ hybridization. NBC message was strongly expressed in the initial segment and the intermediate zone of the epididymis, and the NBC-5 antibody gave a strong basolateral staining in both principal cells and apical/narrow cells in this region. Western blotting revealed a single band at about 160 kDa in the epididymis. The intensity of staining as well as mRNA levels decreased in the cauda epididymidis and in the vas deferens, where only weak staining was seen. Basolateral NBC may function in parallel with apical proton secretion to regulate luminal acidification and/or bicarbonate reabsorption in the excurrent duct system. (+info)
Hormonal regulation of messenger ribonucleic acid expression for steroidogenic factor-1, steroidogenic acute regulatory protein, and cytochrome P450 side-chain cleavage in bovine luteal cells.
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To examine hormonal regulation of genes pertinent to luteal steroidogenesis, bovine theca and granulosa cells derived from preovulatory follicles were cultured with various combinations of forskolin and insulin. On Day 8 of culture, progesterone production was measured, and mRNA levels of steroidogenic factor-1 (SF-1), cytochrome P450 side-chain cleavage enzyme (P450scc), and steroidogenic acute regulatory protein (StAR) were determined by means of semiquantitative reverse transcription-polymerase chain reaction. Notably, the combination of forskolin plus insulin stimulated progesterone production in luteinized theca cells. This was probably a result of a synergistic interaction between forskolin and insulin, observed on both StAR and P450scc mRNA levels. However, in luteinized granulosa cells (LGC), forskolin and insulin each independently were able to up-regulate the levels of P450scc and StAR mRNA levels, respectively. Moreover, insulin alone was sufficient to maintain the high steady-state levels of StAR mRNA in LGC. Both insulin and insulin-like growth factor I enhanced StAR gene expression in LGC. SF-1 was constitutively expressed in bovine luteal cells; its amounts did not vary between the two luteal cell types or with hormonal treatments. In summary, this study demonstrates a distinct, cell-type specific regulation of StAR and P450scc mRNA in the two bovine luteal cell types. (+info)
Effects of spinal cord injury on spermatogenesis and the expression of messenger ribonucleic acid for Sertoli cell proteins in rat Sertoli cell-enriched testes.
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The study was an examination of the effects of spinal cord injury (SCI) on spermatogenesis and Sertoli cell functions in adult rats with Sertoli cell-enriched (SCE) testes. The effects of SCI on the seminiferous epithelium were characterized by abnormalities in the remaining spermatogenic cells during the first month after SCI. Three days after SCI, serum testosterone levels were 80% lower, while serum FSH and LH levels were 25% and 50% higher, respectively, than those of sham control SCE rats. At this time, the levels of mRNA for androgen receptor (AR), FSH receptor (FSH-R), and androgen-binding protein (ABP) were normal whereas those for transferrin (Trf) had decreased by 40%. Thereafter, serum testosterone levels increased, but they remained lower than those of the sham control rats 28 days after SCI; and serum FSH and LH levels returned to normal. The levels of mRNA for AR, ABP, and Trf exhibited a biphasic increase 7 days after SCI and remained elevated 28 days after SCI. FSH-R mRNA levels were also elevated 90 days after SCI. Unexpectedly, active spermatogenesis, including qualitatively complete spermatogenesis, persisted in > 40% of the tubules 90 days after SCI. These results suggest that the stem cells and/or undifferentiated spermatogonia in SCE testes are less susceptible to the deleterious effects of SCI than the normal testes and that they were able to proliferate and differentiate after SCI. The presence of elevated levels of mRNA for Sertoli cell FSH-R and AR, as well as of that for the Sertoli cell proteins, in the SCE testes during the chronic stage of SCI suggests a modification of Sertoli cell physiology. Such changes in Sertoli cell functions may provide a beneficial environment for the proliferation of the stem cells and differentiation of postmeiotic cells, thus resulting in the persistence of spermatogenesis in these testes. (+info)
Down-regulation of oxytocin-induced cyclooxygenase-2 and prostaglandin F synthase expression by interferon-tau in bovine endometrial cells.
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Oxytocin (OT) is responsible for the episodic release of luteolytic prostaglandin (PG) F2alpha from the uterus in ruminants. The attenuation of OT-stimulated uterine PGF2alpha secretion by interferon-tau (IFN-tau) is essential for prevention of luteolysis during pregnancy in cows. To better understand the mechanisms involved, the effect of recombinant bovine IFN-tau (rbIFN-tau) on OT-induced PG production and cyclooxygenase-2 (COX-2) and PGF synthase (PGFS) expression in cultured endometrial epithelial cells was investigated. Cells were obtained from cows at Days 1-3 of the estrous cycle and cultured to confluence in RPMI medium supplemented with 5% steroid-free fetal calf serum. The cells were then incubated in the presence or absence of either 100 ng/ml OT or OT+100 ng/ml rbIFN-tau for 3, 6, 12, and 24 h. OT significantly increased PGF2alpha and PGE2 secretion at all time points (p < 0.01), while rbIFN-tau inhibited the OT-induced PG production and reduced OT receptor binding in a time-dependent manner. OT increased the steady-state level of COX-2 mRNA, measured by Northern blot, which was maximal at 3 h (9-fold increase) and then decreased with time (p < 0.01). OT also caused an increase in COX-2 protein, which peaked at 12 h (11-fold increase), as measured by Western blot. Addition of rbIFN-tau suppressed the induction of COX-2 mRNA (89%, p < 0.01) and COX-2 protein (50%, p < 0.01) by OT. OT also increased PGFS mRNA, and this stimulation was attenuated by rbIFN-tau (p < 0.01). To ensure that the decrease in COX-2 was not solely due to down-regulation of the OT receptor, cells were stimulated with a phorbol ester (phorbol 12-myristate 13-acetate; PMA) in the presence and absence of rbIFN-tau. The results showed that rbIFN-tau also decreased PMA-stimulated PG production and COX-2 protein. It can be concluded that rbIFN-tau inhibition of OT-stimulated PG production is due to down-regulation of OT receptor, COX-2, and PGFS. (+info)