Structural and functional organization of the gene encoding the human thyrotropin-releasing hormone receptor.

The thyrotropin-releasing hormone (TRH) receptor (TRHR) is widely distributed throughout the central and peripheral nervous systems. In addition to its role in controlling the synthesis and secretion of thyroid-stimulating hormone and prolactin from the anterior pituitary, TRH is believed to act as a neurotransmitter as well as a neuromodulator. We have isolated genomic lambda and P1-derived artificial chromosome clones encoding the human TRHR. The gene was found to be 35 kb with three exons and two introns. A 541-bp intron 1 (-629 to -89 relative to the translation start site) is conserved between human and mouse. A large intron 2 of 31 kb disrupts the open reading frame (starting in position +790) in the sequence encoding the supposed junction between the third intracellular loop and the putative sixth transmembrane domain. A similar intron was found in chimpanzee and sheep but not in rat and mouse. Promoter analysis of upstream regions demonstrated cell type-specific reporter activation, and sequencing of 2.5 kb of the promoter revealed putative cis-acting regulatory elements for several transcription factors that may contribute to the regulation of the TRHR gene expression. Functional analysis of potential response elements for the anterior pituitary-specific transcription factor Pit-1 revealed cell type-specific binding that was competed out with a Pit-1 response element from the GH gene promoter.

Testing migration patterns and estimating founding population size in Polynesia by using human mtDNA sequences.

The hypervariable 1 region of human mtDNA shows markedly reduced variability in Polynesians, and this variability decreases from western to eastern Polynesia. Fifty-four sequences from New Zealand Maori show that the mitochondrial variability with just four haplotypes is the lowest of any sizeable human group studied and that the frequency of haplotypes is markedly skewed. The Maori sequences, combined with 268 published sequences from the Pacific, are consistent with a series of founder effects from small populations settling new island groups. The distributions of haplotypes were used to estimate the number of females in founding population of New Zealand Maori. The three-step simulation used a randomly selected founding population from eastern Polynesia, an expansionary phase in New Zealand, and finally the random selection of 54 haplotypes. The results are consistent with a founding population that includes approximately 70 women (between 50 and 100), and sensitivity analysis shows that this conclusion is robust to small changes in haplotype frequencies. This size is too large for models postulating a very small founding population of "castaways," but it is consistent with a general understanding of Maori oral history as well as the results of recent canoe voyages recreating early trans-oceanic voyages.

Advanced glycation end products and their receptors co-localise in rat organs susceptible to diabetic microvascular injury.

Advanced glycation end products (AGEs) are believed to play an important role in the development of diabetic complications. AGEs are increased in experimental diabetes and treatment with the inhibitor of advanced glycation end products, aminoguanidine, has been shown to attenuate the level of these products in tissues undergoing complications. Recently, an AGE-binding protein has been isolated from bovine lung endothelial cells and termed the receptor for advanced glycated end products (RAGE). The present study sought to determine the distribution of AGE and RAGE in tissues susceptible to the long-term complications of diabetes including the kidney, eye, nerve, arteries as well as in a tissue resistant to such complications, the lung. Using polyclonal antisera both AGE and RAGE were found to co-localize in the renal glomerulus. AGE staining was clearly increased with age and was further increased by diabetes. Aminoguanidine treatment reduced AGE accumulation in the kidney. Co-localisation of AGE and RAGE was demonstrated in the inner plexiform layer and the inner limiting membrane of the retina and in nerve bundles from mesenteric arteries. In the aorta, both AGE and RAGE were found in the intima, media and adventitia. Medial staining was increased in diabetes and was reduced by aminoguanidine treatment. A similar pattern was observed for RAGE in the aorta. In the lung, RAGE was found widely distributed throughout the lung whereas the distribution of AGE staining was more limited, primarily localising to macrophages. The co-localisation of AGEs and RAGE in sites of diabetic microvascular injury suggests that this ligand-receptor interaction may represent an important mechanism in the genesis of diabetic complications.

Effect of cortisol on percentage of non-sex-hormone-bound steroid: implications for distribution of steroids on binding proteins in serum.

We previously observed that in men concentrations of serum testosterone (T) not bound to sex-hormone-binding globulin (n-SHBGT) decreased as concentrations of cortisol increased in early morning. This led us to investigate in vitro the influence of several steroids on protein-bound T. Steroids were added to late-evening sera containing low concentrations of cortisol. Changes were measured in percent T or estradiol not bound to SHBG (%n-SHBGT or %n-SHBGE). Results were compared with computer simulations of a mass action model describing current understanding of steroid binding to serum proteins. In vitro measurements confirmed changes observed in vivo. Cortisol at 600 nmol/L reduced %n-SHBGT to 61% +/- 5% of basal, but this was reversed with cortisol at 2000 nmol/L. Progesterone reduced %n-SHGBT less, and dexamethasone had no effect. Free T rose with added cortisol. Increasing estradiol to 900 nmol/L caused an increase in %n-SHBGT. The %n-SHBGE rose with added cortisol (121% +/- 5% of basal with cortisol at 1000 nmol/L). Simulation predicted all behaviors except the marked initial decrease in %n-SHBGT as cortisol concentrations increased and the absolute values of %n-SHBGT and %n-SHBGE. A possible explanation for the former is that T is displaced from corticosteroid-binding globulin (CBG) by added cortisol, more T is bound to CBG than expected, and T displaced from CBG associates with SHBG rather than albumin. Alternatively, current understanding about steroid binding to serum proteins has other major deficiencies.

Tumor necrosis factor-alpha is expressed by glomerular visceral epithelial cells in human membranous nephropathy.

The role of tumor necrosis factor alpha (TNF-alpha) was examined in biopsy-proven glomerulonephritis by immunohistochemistry, in situ hybridization, immunogold electron microscopy, immunoassay in serum and urine, and urinary immunoblot. Striking glomerular capillary wall and visceral glomerular epithelial cell TNF-alpha protein staining was observed in all cases of membranous nephropathy and membranous lupus nephropathy. Staining was less frequently observed in crescentic glomerulonephritis and in isolated cases of other histological subtypes of glomerulonephritis, usually in association with glomerular macrophages. By immunogold electron microscopy TNF-alpha was localized in membranous nephropathy within the visceral glomerular epithelial cells, and also in the glomerular basement membrane, especially in relation to immune deposits. In situ hybridization localized TNF-alpha mRNA exclusively to glomerular epithelial cells in all biopsies with membranous morphology but not in other histological subtypes. Concentrations of TNF-alpha were significantly increased compared with normal controls in the urine of patients with membranous nephropathy and with crescentic glomerulonephritis. The expression of TNF-alpha by glomerular epithelial cells exclusively and universally in biopsies showing a membranous morphology strongly suggests this cytokine has a role in the pathogenesis of membranous nephropathy.

Angiotensin II and acetylcholine differentially activate mobilization of inositol phosphates in Xenopus laevis ovarian follicles.

Angiotensin II (AII) evokes a Ca(2+)-dependent Cl- current in Xenopus laevis ovarian follicles that appears to involve a pertussis-toxin-sensitive G protein mediating phosphoinositide hydrolysis and Ca2+ mobilization from intracellular stores. Follicle responses to AII closely resemble the two-component response stimulated by acetylcholine (ACh) in this tissue. Intraoocyte injections of phytic acid, heparin, and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], acting as inhibitors of Ins(1,4,5)P3-induced Ca(2+)-release, resulted in loss of responsiveness to AII and ACh. As previously reported for ACh [Moriarty et al. (1988) Proc Natl Acad Sci USA 85: 8865-8869], pertussis toxin and microinjected GTP[gammaS] were found to inhibit follicle responses to AII, implying the involvement of a G protein. However, ACh and AII responses differ strikingly in the way they mobilize inositol phosphates and in densitization characteristics. We have previously been unable to find significant increases in inositol phosphates after 60 min stimulation (with Li+) by AII, although ACh potently activated increases in these [McIntosh and McIntosh (1990) Arch Biochem Biophys 283: 135-140]. In the present paper, AII was found to activate rapid increases in inositol bis- and trisphosphates after 1 min stimulation without Li+. ACh and AII also exerted different actions on follicle adenylate-cyclase-dependent responses. We conclude that at least two separate inositol-phosphate-linked receptor mechanisms may exist in ovarian follicles, resulting from involvement of one or more pertussis-toxin-sensitive G protein(s).

Bovine inhibin immediately inhibits the electrophysiological response to chorionic gonadotrophin in ovarian follicles of Xenopus laevis.

Inhibin was originally isolated from ovarian tissue using, as a bioassay, its ability to diminish synthesis and secretion of gonadotrophins in pituitary cells after several days. Inhibin is known also to modify responses to gonadotrophins in the ovary after stimulation times of hours or days. Here we show that action for less than 4 min of purified bovine inhibin on ovarian follicles from Xenopus laevis causes a specific, dose-responsive (greater than 10 U/ml) inhibition of the membrane hyperpolarization produced by stimulation with chorionic gonadotrophin. The CG-induced change in follicle membrane potential has previously been shown to be caused by potassium efflux produced by rises in intracellular cAMP. Inhibin did not affect similar hyperpolarization stimulated by 10 microM adenosine. The apparent Ca(2+)-dependent Cl- currents which are often associated with CG action, were not inhibited by inhibin. At concentrations up to 2000 U/ml, inhibin did not significantly alter the timing of CG-induced germinal vesicle breakdown in the oocytes, which may suggest functional action on other developmental processes.