Follicle-stimulating hormone and human chorionic gonadotropin induced changes in granulosa cell glycosyl-phosphatidylinositol concentration

In the present investigation, a hCG sensitive glycosyl-phosphatidylinositol (GPI) was isolated from cultured rat granulosa cells obtained from the ovaries of diethylstilbestrol (DES) implanted immature rats. The inositol-phosphoglycan (IPG) moiety of the GPI-lipid contains galactose, glucosamine, and myoinositol as demonstrated by metabolic labelling of granulosa cells for different time periods (5–96 h) with [³H]galactose, [³H]glucosamine, or [³H]myoinositol and treatment of the purified [³H]GPI with phosphatidylinositol-specific phospholipase C. Labelling equilibrium of the GPI-lipid was achieved after 24 h ([³H]galactose and [³H]myoinositol) or 72 h ([³H]glucosamine) incubation, whereas incorporation of other labelled carbohydrates tested ([³H]galactosamine, [³H]mannose, and [³H]sorbitol) was negligible throughout the time period studied. The glucosamine C-1 appears to be linked through a glycosidic bond to the myoinositol molecule of the IPG moiety as revealed by the generation of phosphatidylinositol (PtdIns) after nitrous acid deamination of dual labelled ([³H]glucosamine/[¹⁴C]palmitate or [³H]glucosamine/[¹⁴C]myristate) glycosyl-phosphatidylinositol. To investigate the fatty acid composition of the diacylglycerol (DAG) backbone of the GPI, granulosa cells were also labelled (5–72 hr) with [¹⁴C]linoleate, [³H]myristate, [³H]-oleate, [³H]palmitate, or [³H]stearate and the radioactivity associated with the purified glycosyl-phosphatidylinositol determined. Incorporation of [³H]palmitate and [³H]myristate into the GPI-lipid peaked after 8 h and 24 h of labelling, respectively, and both fatty acids were partially released after PLA₂ treatment of the dual labelled ([³H]glucosamine/[¹⁴C]palmitate or [³H]glucosamine/[¹⁴C]myristate) GPI. In parallel experiments no significant incorporation of labelled stearate, oleate, or linoleic acid into the DAG backbone of the glycosylphosphatidylinositol could be detected. Granulosa cells were also labelled with [³H]glucosamine in the presence of FSH (30 ng/ml), cholera toxin (1 μg/ml), or the membrane permeable cAMP analog (but)₂ cAMP (1 mM). Time related increases in GPI-labelling were apparent after 48 h and reached a maximum level (3-, 5-, and 7-fold for FSH, CT, and (but)₂ cAMP, respectively) after 72 h in culture. In another set of experiments, granulosa cells were labelled for 72 h with [³H]glucosamine in the presence of (but)₂cAMP (1 mM), TPA (10^?7 M), or combination thereof. The effect of treatment with the membrane permeable cAMP analog on GPI labelling was prevented in the presence of TPA, whereas no differences in [³H]GPI content could be observed in untreated granulosa cells or cells cultured in the presence of the protein kinase C-activating phorbol ester alone. In cells differentiated with FSH (30 ng/ml for 3 days) to induce LH receptors, treatment with hCG (100 ng/ml) induced a rapid (60 sec) and transient (5 min) decrease in the GPI content, whereas no efect of the hormone on undifferentiated granulosa cells could be observed. The rapid effect elicited by hCG on GPI content and turnover may be an early transduction mechanism involved in the biological effects of LH/hCG in differentiated granulosa cells. © 1993 Wiley-Liss, Inc.     

Impact of Holoshestes heterodon Eigenmann (Pisces: Characidae) on the plankton community of a subtropical reservoir: the importance of predation by Chaoborus larvae

We analyzed the effects of planktivorous Holeshestes heterodon Eigenmann (Characidae) predation on the plankton community of a small subtropical reservoir, using four enclosures (volume about 17.5 m³), open to the sediment, established in the littoral zone. Two enclosures were stocked with fish (mean TL 5.7 cm), at a density of about 4–5 fish m^–3 (approx. 8 g m^–3), whereas two remained fishless. The experiment lasted a little longer than one month. In the fish enclosures, most Crustacea and Chaoborus larvae remained scarce, probably as a result of visually selective fish predation. In both fishless enclosures, Chaoborus larvae became abundant. However, in only one of these did large individuals become relatively numerous; this discrepancy in the demographic structure of the Chaoborus populations between the two fishless enclosures is unexplained. Only in the fishless enclosure without appreciable numbers of large Chaoborus did densities of Crustacea increase greatly. It is suggested that in the enclosure containing large Chaoborus individuals, crustacean populations were prevented from developing due to predation pressure, while the small Chaoborus larvae of the other enclosure could not readily consume these prey. Rotifers were low in abundance in the absence of fish, probably as a consequence of Chaoborus predation. Phytoplankton density increased in all four enclosures, due probably to the lack of water flow. Only in the fishless enclosure with high densities of crustaceans did phytoplankton abundance decrease markedly at the end of the experiment, perhaps because of grazing losses.     

Survey of bovine mycotic mastitis in dairy herds in the State of São Paulo,Brazil

The purpose of this study was to isolate fungi from the quarter milk of cow udders from several dairy herds and to identify the different genera and species involved in mastitis. A total of 2078 milk samples from normal, clinical and subclinical mastitis quarters from 22 dairy herds of 16 districts in the State of São Paulo, Brazil was utilized in this survey. Two hundred and fifty one (12.07%) fungi were isolated from the samples. Two hundred and eight of these (82.86%) were yeasts and 30 (11.95%) were moulds. The fungi were isolated in pure culture (24.77%) or in cultures mixed with bacteria (72.22%). The yeasts isolated were:Cryptococcus spp. (71 strains),Rhodotorula spp. (40),Candida spp. (68),Trichosporon cutaneum (21),Aureobasidium pullulans (7), andPichia ohmeri (1). Moulds classified in following genera were also isolated:Aspergillus (3),Penicillium (3),Alternaria (3),Phoma (3),Epicoccum (2), andGeotrichum (16).     

Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325° to 333deg;

In the framework of the European project aimed at the sequencing of the Bacillus subtilis genome the DNA region located between gerB (314°) and sacXV (333°) was assigned to the Institut Pasteur. In this paper we describe the cloning and sequencing of a segment of 97 kb of contiguous DNA. Ninety-two open reading frames were predicted to encode putative proteins among which only forty-two were found to display significant similarities to known proteins present in databanks, e.g. amino acid permeases, proteins involved in cell wall or antibiotic biosynthesis, various regulatory proteins, proteins of several dehydrogenase families and enzymes II of the phosphotransferase system involved in sugar transport. Additional experiments led to the identification of the products of new B. subtilis genes, e.g. galactokinase and an operon involved in thiamine biosynthesis.     

Assessment of Morpho-Physiological and Biochemical Responses of Perennial Ryegrass to Gamma-Aminobutyric Acid (GABA) Application Under Salinity Stress Using Multivariate Analyses Techniques

Journal of Plant Growth Regulation - Salinity stress is one of the most important global problems that afflicts and limits the growth and development of turfgrass in arid and semi-arid areas....     

A STAT factor mediates the sexually dimorphic regulation of hepatic cytochrome P450 3A10/lithocholic acid 6 beta-hydroxylase gene expression by growth hormone.

Adult male rodents have a pulsatile profile of growth hormone (GH) release, whereas female rodents have a relatively steady-state pattern with uniform, albeit lower levels of GH. The expression of a number of sexually differentiated hepatic proteins is primarily determined by these plasma GH profiles and only secondarily regulated by gonadal hormones. An important subset of these sexually dimorphic proteins is cytochrome P450s. CYP3A10/6 beta-hydroxylase is a cytochrome P450 that catalyzes the 6 beta-hydroxylation of lithocholic acid. CYP3A10/6 beta-hydroxylase is expressed only in male hamsters; however, mimicking the male GH secretion pattern in females induces expression of the gene to male levels. Using chimeric CYP3A10/6 beta-hydroxylase promoter/luciferase reporter genes transfected into hamster primary hepatocytes, we have shown a GH-mediated induction of promoter activity. A combination of 5'-deletion constructs, heterologous promoter constructs, and specific mutagenesis was used to localize the DNA element involved in the GH-mediated regulation of CYP3A10/6 beta-hydroxylase promoter activity, which resembles a STAT binding site. Footprint and gel shift analyses confirmed that the expression of the protein binding to this site is regulated by GH and that the DNA-protein complex can be partially supershifted by anti-STAT-5 antibodies. This protein is 50% more abundant in male than in female hamster livers, is absent in hypophysectomized female livers, and is restored when hypophysectomized females are injected with GH in a manner that masculinizes female hamsters in terms of CYP3A10/6 beta-hydroxylase expression. The system characterized and described here is ideally suited for dissecting the molecular details governing the sexually dimorphic expression of liver-specific genes.     

A new survey of Brazilian marine algae for agglutinins

Aqueous protein extracts from 30 Brazilian marine algae were examined for haemagglutinating activity using native and enzyme-treated rabbit, chicken, sheep and human erythrocytes. Most extracts agglutinated at least one of the blood cells used. Sheep and rabbit erythrocytes were more suitable for detection of the agglutinating activity. The minimum protein concentration necessary to produce positive agglutination was usually lower with enzyme-treated erythrocytes than native ones. The five algal protein extracts showing the greatest haemagglutination titre were tested for sugar-binding specificity. Only the activity present in the green alga Cauler pacupressoides was inhibited by simple sugars and not by the glycoproteins tested. The activity of the other four extracts was inhibited by at least one of the glycoproteins utilised. This revised version was published online in August 2006 with corrections to the Cover Date.     

The membrane-bound high-molecular-mass cytochromes c from Desulfovibrio gigas and Desulfovibrio vulgaris Hildenborough; EPR and Mössbauer studies

The high-molecular-mass cytochromes c (Hmcs) from the sulfate-reducing bacteria Desulfovibrio gigas and Desulfovibrio vulgaris (Hildenborough) were found to be strongly bound to the cytoplasmic membrane. After detergent solubilization they were shown to be water soluble and to be similar to those previously isolated from the soluble fractions in terms of N-terminal sequence, molecular mass, UV-visible and EPR spectroscopies. In D. gigas, higher amounts of Hmc can be obtained from the membranes than from the soluble fraction. This enabled further characterization of both cytochromes. The apparent heme reduction potentials of both Hmcs, determined at pH 7.5 through visible and EPR redox titrations, span a large range of redox potentials, approximately between 0 and –280?mV, and can be roughly divided into three groups: four to five hemes have E ⁰s of –30?mV to –100?mV, three to four hemes have E ⁰s around –170?mV, and seven to eight hemes have a lower E ⁰ of –250 to –280?mV. Several of these redox potentials are strongly pH dependent. Mössbauer studies of oxidized and reduced D. vulgaris Hmc show that this protein contains two high-spin hemes in both oxidation states. The rate of reduction of both Hmcs with the periplasmic hydrogenases from the corresponding organisms is extremely slow.     

Hemagglutination properties of Enterococcus

In total, 86 enterococcal strains including representatives of most of the described species were tested for the ability to agglutinate human, sheep, and rabbit erythrocytes. Five strains did not react with any of the erythrocytes tested, and 81 (94.2%) strains agglutinated only rabbit erythrocytes. The hemagglutination titers ranged from 2 to 64. Loss of the hemagglutination activity was observed when rabbit erythrocytes were treated with trypsin or neuraminidase. Trypsin treatment of the bacterial suspensions also caused loss of the agglutination ability. On the other hand, heat treatment of bacterial suspensions increased the efficiency of the interactions, and higher titers were obtained. Assays for inhibition of hemagglutination were performed with -d-fucose, -d-galactose, -d-galactose, d-glucose, N-acetyl-galactosamine, N-acetyl-glucosamine, N-acetylneuraminic acid, N-acetylneuraminic acid-lactose, and fetuin. Only fetuin was able to inhibit the hemagglutination reactions. The results showed that hemagglutination properties are common to the different enterococcal species tested. They also suggest that enterococci possess hemagglutinins of proteic and non-proteic nature that are involved in the attachment to sialic acid-containing receptors on the surface of rabbit erythrocytes.