Gonadotropin-releasing hormone action upon luteinizing hormone bioactivity in pituitary gland: role of sulfation

The regulation of rat luteinizing hormone (rLH) bioactivity was studied in an in vitro system using isolated pituitaries from male rats. Stored and released rLH was evaluated in terms of mass (I-LH), bioactivity (B-LH), mobility in nonequilibrium pH gradient electrophoresis, and mannose and sulfate incorporation either in the presence or absence of gonadotropin-releasing hormone (GnRH). GnRH increased the biological potency of stored and released rLH. The pituitary content revealed seven I-LH species (pH 7.2, 7.8, 8.5, 9.0, 9.1, 9.3, and 9.7) and five B-LH species (pH 8.5, 9.0, 9.2, 9.4, and 9.7). The major I-LH and B-LH peaks were at pH 9.0 and 9.2, respectively. I-LH peaks at pH 7.2 and 7.8 are devoid of bioactivity; at these pH values, free rLH subunits are detectable. GnRH increases the amount of both I-LH and B-LH material secreted into the medium, and the major component migrates at pH 8.5 and is probably the alpha beta dimer. [3H]Mannose and [35S]sulfate can be incorporated into stored and released rLH (pH 7.2, 7.8, 9.0, 9.1, and 9.3 and 7.2, 7.8, 8.5, and 9.0, respectively). GnRH decreases [2-3H]mannose incorporation into secreted rLH. [35S]Sulfate was incorporated into I-LH released spontaneously into the medium; the form at pH 7.2 has no biological activity and is probably the free alpha subunit. GnRH decreases the [35S]sulfate-labeled rLH content of the pituitary concomitantly with a 500% increase in [35S]sulfate-labeled released rLH, suggesting that, soon after [35S]sulfate is incorporated, sulfated rLH is released. Sulfatase action on released rLH reveals that sulfation may be related to release of rLH but that sulfate residues are not involved in the expression of rLH bioactivity. In conclusion, GnRH stimulates carbohydrate incorporation and processing of the oligosaccharide residues giving the highest biological potent rLH molecule and also increases sulfation; this step is closely related to the step limiting the appearance of LH in the medium in the absence of GnRH.     

Distribution of extracutaneous melanin pigment in Sparus auratus, Mugil cephalus, and Dicertranchus labrax (Pisces, Teleostei)

The morphological and biochemical characteristics of pigment accumulations found in the kidney, liver, spleen, and mesentery of three different species of teleost fishes have been studied. There are significant differences in number, distribution, and morphology of pigment accumulations in different organs of the three species. Biochemical studies have shown the existence of tyrosinase activity in the mesentery of Mugil cephalus and in the kidney and mesentery of Sparus auratus. No tyrosinase activity was found in any internal organs of Dicertranchus labrax. That activity was assayed using three methods: tyrosine hidroxylation, dopa oxidation, and melanin formation. The morphological and biochemical observations are in agreement. In those organs in which we have demonstrated melanin synthetic activity, the pigment cells are morphologically and like melanophores, while in the organs that show no melanin synthetic activity, the pigment cells resemble macrophages.     

Stimulation by calcium and carbamoylcholine of the ouabain-sensitive uptake of 86Rb+ in isolated rat pancreatic acinar cells

The uptake of 86Rb+ was assayed in isolated rat pancreatic acinar cells to determine the effect of calcium and carbamoylcholine on the ouabain-sensitive and ouabain-insensitive components. The presence of calcium in the medium bathing the cells during the preincubation and the main incubation periods was needed to preserve in optimum conditions the uptake of 86Rb+, the stimulation by carbamoylcholine and the sensitivity to ouabain. In the presence of calcium, the ouabain-sensitive component of 86Rb+ uptake was higher than the ouabain-insensitive. The ouabain-sensitive component was 3-times lower in cells incubated in a medium lacking calcium and containing 1 mM EGTA, as compared to cells incubated in the presence of calcium. Carbamoylcholine, at 5 X 10(-4) M, stimulated the uptake of 86Rb+ and this effect depended on the presence of calcium in the bathing medium. Maximal stimulation by carbamoylcholine was reached at 0.2 mM calcium. The nett stimulation by carbamoylcholine was inhibited up to 85% by 1 mM ouabain. As judged by digitonin-disruption of plasma membrane, the above-indicated effects were limited to a cytoplasmic pool of 86Rb+ and a leaky plasma membrane could be ruled out. The results suggest that in rat pancreatic acinar cells, carbamoylcholine stimulated the ouabain-sensitive uptake of 86Rb+ and required the presence of calcium in the bathing medium.     

The phylogeny of the hominoid primates: a statistical analysis of the DNA-DNA hybridization data

Sibley and Ahlquist compared the single-copy nuclear DNA sequences of the hominoid primates using DNA-DNA hybridization. From this data set they estimated a phylogeny that clusters man and chimpanzees using a distance Wagner procedure. However, no assessment of statistical confidence in this estimated phylogeny was made, despite the fact that their data set contains internal inconsistencies concerning the correct branching order. This paper presents a modification of Pielou's Q- statistic that allows one to make nonparametric tests of phylogenetic relationship from distance data. The results of this analysis indicate that the estimated phylogeny of Sibley and Ahlquist is without statistical significance owing to the internal inconsistencies of the data set. A survey and additional analyses of other types of molecular data indicate that the phylogeny that clusters chimpanzees and gorillas and has the human lineage splitting off earlier is statistically consistent with all the molecular data (including the DNA-DNA hybridization data), whereas the phylogeny estimated by Sibley and Ahlquist can be rejected at the 5% level using the data on restriction- endonuclease sites in the mitochondrial genome.      

A new spectrophotometric assay for dopachrome tautomerase

The existence of a new enzyme involved in mammalian melanogenesis has been recently reported. The names dopachrome oxidoreductase and dopachrome tautomerase have been proposed for the enzyme. So far, this enzyme has been assayed at 475 nm on the basis of its ability to catalyze dopachrome decoloration. This method presents two major problems, derived from the instability of the substrate (dopachrome): (1) dopachrome must be prepared immediately before use, and (2) the rate of dopachrome decoloration in the absence of the enzyme is not negligible, and, furthermore, is enhanced by non-enzymatic agents. In order to overcome these problems, we present a new procedure that combines: (1) a quantitative, fast and easy way to prepare dopachrome from L-dopa by sodium periodate oxidation; (2) a spectrophotometric method in the UV region, at 308 nm, based on following the absorbance increase due to the enzyme-specific tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid as opposed to the absorbance decrease due to the spontaneous decarboxylative transformation of dopachrome into 5,6-dihydroxyindole. The advantages of these methods as compared to the previously used procedures are discussed.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Use of F1 progeny of HolsteinxZebu cross cattle as oocyte donors for in vitro embryo production and gene microinjection

This study was designed to determine the possibility of using F1 crossbreed cattle (Holstein x Zebu) as donors of oocytes for in vitro fertilization (IVF) and for pronuclear gene microinjection into in vitro-produced embryos. In the first part of the experiment oocytes from Bos taurus (Holstein), Bos indicus (Zebu) and F1 crossbred Bos taurus x Bos indicus (Holstein x Zebu) genotypes were inseminated with Bos taurus (Holstein) semen and were allocated for in vitro embryo production using conventional IVF procedures. No differences were observed on the in vitro maturation (IVM) rates between breeds (Holstein x Holstein:85%, Zebu x Holstein:84% and Zebu x Holstein x Holstein:88%). Holstein cows yielded the highest number of cumulus oocyte complexes (6.8 per ovary) for in vitro maturation, differing (P<0.05) from Zebu x Holstein and Zebu x Holstein x Holstein F1 by 5.1 and 5.8, respectively. However, the Holstein breed also yielded the lowest percentage of cleavage (45.1 vs 71.9% for Zebu x Holstein and 65.1% for Zebu x Holstein x Holstein). Of the 3 genotypes, the hybrid F1 breed was the most efficient source of oocytes for the production of embryos capable of reaching morulae and blastocyst stages (76 250 ; P< 0.001). In the second part of the study, 599 oocytes from the F1 breed were fertilized in vitro, 1 group of 150 oocytes was used for the determination of the optimal pronuclear visualization period. The highest number of oocytes with 2 pronuclei was observed between 24 to 28 h after IVF (27 to 42%). The remaining 399 oocytes were microinjected with a gene construct bearing the bacterial lacZ gene as the reporter for gene expression. Survival of embryos to microinjection was 73.8%, and 45.5% of them (50 110 ) cleaved in culture. Of the microinjected embryos, 1 out of 50 showed beta-galactosidase activity. These findings indicate that a tropical crossbreed of cattle (Zebu x Holstein x Holstein) can be used as a source of oocytes for IVF programs and gene microinjection studies.     

Induction of programmed cell death (apoptosis) in mature lymphocytes

The apoptosis of human peripheral blood lymphocytes was analyzed by the breakdown of DNA into oligonucleosome-sized fragments. The mature lymphocytes were rendered sensitive to apoptosis by either the omission of fetal bovine serum in the culture medium or the addition of polymyxin B. In the first case it was counteracted by phorbol myristate acetate. The possible involvement of protein kinase C in cell survival is pointed out.     

The action of luteinizing hormone on the testis

Luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) receptors are coupled to intracellular effector systems, most notably adenylate cyclase, through guanyl nucleotide-binding proteins or G-proteins. The molecular mechanism involved in the dynamic coupling of the LH/hCG receptor however, are not known. It has been postulated that receptor aggregation at the molecular level plays a critical role in this process. There have been attempts to understand the receptor association and dissociation phenomena at the molecular level. One of them involves the participation of the major histocompatibility complex (MHC) class I antigen in the mechanism of receptor activation and/or expression. One molecular basis for these mechanisms consists of a physical interaction between MHC proteins and receptors to form "compound receptors" able to transfer a hormonal signal to the cell. Using a photo-reactive probe we demonstrated that the LH/hCG receptors and the class I antigens are closely associated in the membrane. Thus, it is possible to form covalent complexes of hCG and class I antigens through the binding of the hormone to specific receptors. These findings imply that LH/hCG receptors and the MHC class I antigens may interact at the level of the plasma membrane in the mechanism of LH action. We also performed experiments using a single cell and limiting stimulation to a patch of membrane. The results stimulating the cell in a localized area suggested that even if all components are entirely free to float there is a constraint in the localization of the receptor, G-protein, and/or the effector, supporting the constraint dissociation model. Within a limited area subunits could dissociate, but they would not be free to diffuse throughout the membrane. Moreover the concept of compartmentalization that has been utilized to explain some inconsistencies in second-messenger action now can be proved by experimental design.