Bacteriophages that infect the cellulolytic ruminal bacterium Ruminococcus albus AR67.

AIM: To isolate bacterial viruses that infect the ruminal cellulolytic bacterium Ruminococcus albus. METHODS: Four phages infecting R. albus AR67 were isolated under anaerobic conditions using the soft-agar overlay technique. The phages were characterized on morphology, solvent stability, nucleic acid type and digestion characteristics. Two phages, phiRa02 and phiRa04 comprised icosahedral virions with linear double-stranded DNA and appeared to belong to the family Podoviridae [corrected] The other two phages are most likely filamentous phages with circular single-stranded DNA of the family Inoviridae. SIGNIFICANCE OF THE STUDY: Viruses of the family Inoviridae [corrected] have not previously been isolated from rumen bacteria. The phages isolated in this study are the first phages shown to infect the cellulolytic bacteria of the rumen. This suggests that the cellulolytic populations of the rumen are subject to lytic events that may impact on the ability of these bacteria to degrade plant fibre and on the nutrition of the animal.

Zfx mutation results in small animal size and reduced germ cell number in male and female mice.

The zinc-finger proteins ZFX and ZFY, encoded by genes on the mammalian X and Y chromosomes, have been speculated to function in sex differentiation, spermatogenesis, and Turner syndrome. We derived Zfx mutant mice by targeted mutagenesis. Mutant mice (both males and females) were smaller, less viable, and had fewer germ cells than wild-type mice, features also found in human females with an XO karyotype (Turner syndrome). Mutant XY animals were fully masculinized, with testes and male genitalia, and were fertile, but sperm counts were reduced by one half. Homozygous mutant XX animals were fully feminized, with ovaries and female genitalia, but showed a shortage of oocytes resulting in diminished fertility and shortened reproductive lifespan, as in premature ovarian failure in humans. The number of primordial germ cells was reduced in both XX and XY mutant animals at embryonic day 11.5, prior to gonadal sex differentiation. Zfx mutant animals exhibited a growth deficit evident at embryonic day 12.5, which persisted throughout postnatal life and was not complemented by the Zfy genes. These phenotypes provide the first direct evidence for a role of Zfx in growth and reproductive development.

The multiple murine 3 beta-hydroxysteroid dehydrogenase isoforms: structure, function, and tissue- and developmentally specific expression.

The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) is essential for the biosynthesis of all active steroid hormones. To date five distinct isoforms have been identified in the mouse. The different isoforms are indicated by roman numerals (I-V) in the chronological order in which they have been isolated. The different isoforms are expressed in a tissue- and developmentally specific manner and fall into two functionally distinct groups. 3 beta-HSD I, II, and III function as NAD(+)-dependent dehydrogenaselisomerases, and IV and V function as NADPH-dependent 3-keto steroid reductases. These latter two isoforms, therefore, are not involved in the biosynthesis of steroid hormones, but most likely in the inactivation of steroid hormones. In the adult mouse 3 beta-HSD I is expressed in the classical steroidogenic tissues, the adrenal glands and the gonads. 3 beta-HSD II and III are expressed in the liver and kidney, with III being the major isoform expressed in the adult liver. 3 beta-HSD IV is expressed almost exclusively in the kidney of both sexes, and expression of 3 beta-HSD V is observed only in the male liver starting late in puberty. In the fetal liver of both sexes, 3 beta-HSD I is the major or only isoform expressed at 13.5 days postconception and remains the major isoform until the day of birth, after which 3 beta-HSD III becomes the major isoform. Expression of 3 beta-HSD I in the liver decreases after birth and ceases by day 20 postnatally. Thus the liver expresses four distinct isoforms of 3 beta-HSD, I, II, III, and V, at different times during development. The mouse 3 beta-HSD genes, Hsd3b, have been mapped to a small region on mouse chromosome 3. Analysis of two yeast artificial chromosome (YAC) libraries identified one clone that contains the entire Hsd3b locus within a 1400-kb insert. Hybridization by Southern blot analysis of restriction-enzyme-digested YAC DNA using an 18-base oligonucleotide that hybridizes without mismatch to all known Hsd3b sequences indicates that there are a total of seven Hsd3b genes or pseudogenes in the mouse genome. Further analysis of mouse genomic DNA by pulse field gel electrophoresis suggests that all of the Hsd3b gene family is found within a 400-kb fragment.

Systemically administered gonadotrophin-releasing hormone enhances copulatory behaviour in castrated, testosterone-treated hyperprolactinaemic male rats.

Hyperprolactinaemic male rats exhibit deficits in copulatory behaviour which can be reversed by a single injection of GnRH. We tested whether systemically administered GnRH can stimulate copulatory behaviour independently of LH-mediated increases in plasma testosterone levels. Gonadectomized, pituitary-grafted adult male Fischer 344 rats bearing implants of 5, 10 or 20 mm capsules of testosterone were administered a single injection of 500 ng GnRH or saline s.c., 30 min prior to copulation tests. Pituitary-grafted castrates displayed copulatory deficits, relative to sham-operated castrates with identical levels of testosterone replacement. Administration of 500 ng GnRH to pituitary-grafted castrates bearing 10 mm testosterone implants significantly increased the proportion of rats that mounted, intromitted and ejaculated during a 30 min test. This treatment also reduced significantly the latency of intromission and ejaculation, and increased significantly the frequency of intromission. The copulatory behaviour of the sexually unresponsive, pituitary-grafted castrates bearing 5 mm testosterone implants, or of the more sexually responsive castrates bearing 20 mm testosterone implants, was not altered significantly by GnRH injections. These results support the hypothesis that copulatory deficits in moderately hyperprolactinaemic rats are due in part to reduced hypothalamic GnRH release, and suggest that GnRH can stimulate sexual behaviour in these animals via mechanisms that are independent of luteinizing hormone-induced testosterone release. However, a threshold level of testosterone (achieved with 10 mm implants) appears to be required for GnRH to elicit this effect.

Isolation and characterization of several members of the murine Hsd3b gene family.

The enzyme 3 beta-hydroxysteroid dehydrogenase (3 betaHSD) is essential for all steroid hormone biosynthesis. Six distinct 3 betaHSD cDNAs in the mouse (3 betaHSD I-VI) have been isolated previously. This study reports the isolation of genes or partial genes encoding the 3 betaHSDI, II, III, and IV isoforms. Characterization of the genes revealed that they consist of four exons, the same structure that has been observed for characterized human 3 betaHSD genes. Primer extension and nuclease S1 analysis identified the start sites of transcription of Hsd3b -1 and -4. The proximal promoter regions of Hsd3b-1 and -4 were sequenced and putative cis-acting sequences were determined. Previously, we reported that the then known 3 betaHSD genes (3 betaHSD I-IV) were located in a small region of mouse chromosome 3. To analyze this locus further, six yeast artificial chromosome clones containing the 3 betaHSD sequence were identified. One clone appears to contain the complete 3 betaHSD locus within its 1,400-kbp insert. Further analysis of this YAC, along with analysis of mouse genomic DNA by pulsed-field gel electrophoresis, suggests all members of the 3 betaHSD gene family may be contained on a 400-kbp fragment.

The murine 3 beta-hydroxysteroid dehydrogenase multigene family: structure, function and tissue-specific expression.

The classical form of the enzyme 5-ene-3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD), expressed in adrenal glands and gonads, catalyzes the conversion of 5-ene-3 beta-hydroxysteroids to 4-ene-3-ketosteroids, an essential step in the biosynthesis of all active steroid hormones. To date, four distinct mouse 3 beta HSD cDNAs have been isolated and characterized. These cDNAs are expressed in a tissue-specific manner and encode proteins of two functional classes. Mouse 3 beta HSD I and III function as 3 beta-hydroxysteroid dehydrogenases and 5-en-->4-en isomerases using NAD+ as a cofactor. The enzymatic function of 3 beta HSD II has not been completely characterized. Mouse 3 beta HSD IV functions only as a 3-ketosteroid reductase using NADPH as a cofactor. The predicted amino acid sequences of the four isoforms exhibit a high degree of identity. Forms II and III are 85 and 83% homologous to form I. Form IV is most distant from the other three with 77 and 73% sequence identity to I and III, respectively. 3 beta HSD I is expressed in the gonads and adrenal glands of the adult mouse. 3 beta HSD II and III are expressed in the kidney and liver with the expression of form II greater in kidney and form III greater in liver. Form IV is expressed exclusively in the kidney. Although the amino acid composition of forms I, III and IV predicts proteins of the same molecular weight, the proteins have different mobilities on SDS-polyacrylamide gel electrophoresis. This characteristic allows for differential identification of the expressed proteins. The four structural genes encoding the different isoforms are closely linked within a segment of mouse chromosome 3 that is conserved on human chromosome 1.

A novel mouse kidney 3 beta-hydroxysteroid dehydrogenase complementary DNA encodes a 3-ketosteroid reductase instead of a 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) is essential for the biosynthesis of all steroid hormones. The enzyme catalyzes the conversion of delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids. We report the isolation of a novel mouse 3 beta HSD cDNA, 3 beta HSD IV, and describe the tissue-specific expression of its mRNA, the enzyme characteristics of the 3 beta HSD IV protein, and the structural and functional relationships it has to other 3 beta HSD isoforms previously characterized in the mouse and rat. The predicted amino acid sequence of mouse 3 beta HSD IV is 77% and 73% identical to that of mouse 3 beta HSD I and III, respectively. Comparison of the nucleotide and amino acid sequences of the four isoforms characterized to date show that 3 beta HSD IV is more distantly related to I, II, and III than these three forms are to each other. 3 beta HSD IV mRNA is only expressed in mouse kidney. In situ hybridization of mouse kidney indicates that expression is found only in the cortex and appears to be associated with the proximal tubules. Transiently expressed 3 beta HSD IV protein could not convert the delta 5-3 beta-hydroxysteroids, pregnenolone or dehydroepiandrosterone, to their respective delta 4-3-ketosteroids, progesterone or androstenedione, nor did it have the capacity to convert 5 alpha-androstane-3 beta, 17 beta-diol to dihydrotestosterone, characteristic enzymatic activities of expressed mouse 3 beta HSD I and III. 3 beta HSD IV could only catalyze the conversion of dihydrotestosterone to 5 alpha-androstanediol in the presence of the cofactor NADPH. Thus, 3 beta HSD IV is a 3-ketosteroid reductase rather than a 3 beta-hydroxysteroid dehydrogenase/isomerase despite its homology to the other members of the 3 beta HSD family. Mouse 3 beta HSD IV is functionally and structurally most closely related to rat 3 beta HSD III, an isoform expressed predominantly in male rat liver.

Enzyme characteristics of two distinct forms of mouse 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase complementary deoxyribonucleic acids expressed in COS-1 cells.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-Isomerase (3 beta HSD) catalyzes the conversion of delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids, an essential step in the biosynthesis of all biologically active steroid hormones. We previously reported the isolation of three distinct mouse cDNAs for 3 beta HSD (3 beta HSD I, II, and III) and tissue-specific expression of their mRNAs. 3 beta HSD I is expressed only in gonads and adrenal glands, and 3 beta HSD II and III are expressed in both liver and kidneys. In the current study, we present data which demonstrate that transiently expressed 3 beta HSD I and 3 beta HSD III proteins can catalyze the conversion of the delta 5-steroids, pregnenolone and dehydroepiandrosterone, to their respective delta 4-3-ketosteroids, progesterone and androstenedione. They also can dehydrogenate the 3 beta-hydroxy group of the 5 alpha-reduced steroid 5 alpha-androstanediol to yield dihydrotestosterone in the presence of the cofactor NAD+. The Km values of the expressed 3 beta HSD I (for each of these substrates) were all below 0.2 microM. Km values of 3 beta HSD III were greater for all substrates, with the greatest increase observed for pregnenolone, which was over 10-fold greater. Both forms of expressed protein can catalyze the reduction of dihydrotestosterone to 5 alpha-androstanediol in the presence of the cofactor NADH, but with considerably higher Km values (5.5 microM for form I and 6.8 microM for form III). The observed maximum velocity of form I was much higher for all substrates examined. RNase protection and immunoblot analysis of expressed 3 beta HSD I and III indicate that the difference in maximum velocity reflect differences in the steady state levels of mRNA and amounts of protein. In addition, the expressed 3 beta HSD III protein analyzed by Western blot has a lower mobility than the 3 beta HSD I protein, both similar in mol wt to the 3 beta HSD proteins detected in mouse liver and adrenal glands, respectively. These data demonstrate that an isoform of 3 beta HSD expressed in liver and kidney has the capacity to convert delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids. The data suggest that a homologous human 3 beta HSD isoform could play an important role in cases of genetic deficiency of the gonadal and adrenal isoform.

The genes encoding gonadal and nongonadal forms of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase are closely linked on mouse chromosome 3.

The biosynthesis of steroid hormones in the gonads and adrenal glands requires the activities of the enzyme 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD) which catalyzes the NAD(+)-dependent dehydrogenation and subsequent delta 5-->delta 4 isomerization of delta 5-3 beta-hydroxysteroids to delta 4-3-ketosteroids. The mouse expresses four isoforms of 3 beta HSD. 3 beta HSD I is expressed in gonads and adrenal glands and appears to be the major steroidogenic form, 3 beta HSDs II and III are expressed in both liver and kidneys, and 3 beta HSD IV has been detected only in kidneys. To determine the genetic relationship between the 3 beta HSD isoforms, we have mapped the chromosomal locations of the four genes by linkage analysis using gene-specific probes derived from the 3' untranslated regions of 3 beta HSD cDNA clones. The four 3 beta HSD structural genes (Hsd3b) are closely linked within a segment of chromosome 3 that is conserved on human chromosome 1. The order of markers on Chr 3 surrounding Hsd3b is: centromere-Gba-(4.4 +/- 2.2)-Hsd3b-(3.3 +/- 1.9)-Tshb-(6.7 +/- 2.7)-Amy-1.

Multiple forms of mouse 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase and differential expression in gonads, adrenal glands, liver, and kidneys of both sexes.

Observations of patients deficient in the steroidogenic enzyme 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase (3 beta HSD) have suggested the presence of distinct 3 beta HSD structural gene(s) that are expressed at peripheral sites, possibly the liver. We now report the isolation of cDNA clones representing three forms of 3 beta HSD from mouse Leydig cell and liver libraries. The three forms share significant identify but differ from each other by 5-10% within their coding regions. RNA that hybridizes to radiolabeled 3 beta HSD probes is present in the gonads, adrenal glands, liver, and kidneys of both sexes. Ribonuclease protection analysis using antisense probes derived from each of the three forms demonstrates that one form, 3 beta HSD I, is restricted to steroidogenic tissues. Two other forms, 3 beta HSD II and III, are expressed in liver and kidney but are not detected in steroidogenic tissues. A polyclonal antibody raised against the human placental form of 3 beta HSD recognizes a 42-kDa protein in gonadal and adrenal tissue and a 45-kDa protein in liver. The antibody recognizes a 42-kDa protein in kidney only weakly. 3 beta HSD enzyme activity is present in testicular, adrenal, hepatic, and renal tissue, with adrenal tissue possessing the highest specific activity. When expressed as total 3 beta HSD activity for whole organ mass, activity is greatest in the liver. The results demonstrate that the mouse liver is a significant site of 3 beta HSD activity and demonstrate the existence of multiple 3 beta HSD structural genes in the mouse.

Dexamethasone-induced catabolism and insulin resistance in L6 myoblasts are reversed by the removal of serum.

1. One hundred nanomolar dexamethasone reduced protein synthesis by 16% and also decreased the accretion of protein and RNA in L6 myoblasts when foetal calf serum was present; these effects were reversed when serum was omitted from the medium. 2. Insulin (100 microU/ml) increased protein synthesis, protein accretion and RNA accretion both in the presence and the absence of serum. 3. Dexamethasone inhibited the effects of 100 microU insulin/ml in the presence of serum and induced insulin resistance; in the presence of 25 or 100 nM dexamethasone insulin was ineffective at concentrations below 250 microU and 1 mU/ml respectively.

The cyclo-oxygenase inhibitors indomethacin and ibuprofen inhibit the insulin-induced stimulation of ribosomal RNA synthesis in L6 myoblasts.

Insulin stimulated total RNA accretion and the incorporation of [3H]uridine into RNA in L6 skeletal-muscle myoblasts. Incorporation of uridine into the rRNA was measured after either separation of 18 S and 28 S rRNA species by agarose-gel electrophoresis or separation of dissociated 40 S and 60 S ribosomal subunits on sucrose density gradients. Both methods showed a stimulation by insulin of uridine incorporation into the RNA of the two subunits. Two non-steroidal anti-inflammatory drugs, indomethacin and ibuprofen, which inhibit the metabolism of arachidonic acid by the cyclo-oxygenase pathway, inhibited the insulin-induced accretion of total cellular RNA and the incorporation of uridine into the RNA of both ribosomal subunits. The effect of insulin was observed both by using a tracer dose of [3H]uridine (5 microM) and in the presence of a high concentration (1 mM) of uridine to minimize possible changes in intracellular precursor pools. Neither insulin nor indomethacin was found to affect the incorporation of uridine into the total intracellular nucleotide pool, or the conversion of uridine into UTP. The ability of inhibitors of arachidonic acid metabolism to prevent insulin-induced increases in RNA metabolism suggests that a prostaglandin or other eicosanoid is involved in the signal mechanism whereby insulin stimulates RNA synthesis.

Indomethacin inhibits the insulin-induced increases in RNA and protein synthesis in L6 skeletal muscle myoblasts.

Rates of accretion of RNA and protein and rates of protein synthesis were measured in sub-confluent cultures of L6 myoblasts. Insulin (100 microU/ml) stimulated protein synthesis by 15% within 30 min and by 40% at two and six hours. By six hours insulin also increased the accretion of RNA (+15%). The cyclo-oxygenase inhibitor indomethacin did not reduce the basal rate of RNA or protein accretion in L6 cells but reduced the rate of protein synthesis by 16%. When added together with insulin, indomethacin inhibited the hormonally-stimulated rate of protein synthesis and also significantly reduced the accretion of RNA. Indomethacin still reduced the effects of insulin on protein synthesis when added to the cells two hours after the hormone. Synthesis of RNA measured by the incorporation of [3H]-uridine was also stimulated by insulin but was inhibited by indomethacin only when the drug was present throughout the incubation. Inhibition of protein synthesis by cyclo-oxygenase inhibitors may be the result of both a direct action on translational efficiency and an effect on RNA synthesis.

Hyperprolactinemia disrupts neuroendocrine responses of male rats to female conspecifics.

An increase in plasma luteinizing hormone levels which occurs in the male rat in response to the presence of a receptive female was associated with increased norepinephrine metabolism in several regions of the hypothalamus and reduced norepinephrine metabolism in preoptic area and olfactory bulbs. Hyperprolactinemia induced by grafting three anterior pituitary glands beneath the kidney capsule blocked female-induced changes in norepinephrine metabolism and attenuated the increase in luteinizing hormone release. These results suggest that endocrine and behavioral responses of male rats to the presence of a female are mediated by changes in catecholamine metabolism in several brain regions. The ability of hyperprolactinemia to induce deficits in male sexual behavior may be due to the blockade of these CNS responses to exteroceptive stimuli originating from the female.

The effect of luteinizing hormone releasing hormone on the copulatory behavior of hyperprolactinemic male rats.

The present study was undertaken to test the hypothesis that the deficits in copulatory behavior observed in hyperprolactinemic male rats may be related to a reduction in hypothalamic release of luteinizing hormone releasing hormone (LHRH). Adult male Fischer 344 rats were made hyperprolactinemic by ectopic pituitary grafts or were sham operated and 30 min prior to being tested for copulatory performance received a single subcutaneous injection of 500 ng LHRH, 100 ng LHRH, or saline. On different occasions, testosterone (T) levels were measured in plasma collected 30 min following identical treatments. Plasma prolactin (PRL) levels were determined in samples collected 30 min after injection of 500 ng LHRH. Pituitary grafting produced the expected, significant increase in plasma PRL levels and significant deficits in copulatory behavior. Treatment of hyperprolactinemic subjects with 500 ng LHRH significantly reduced both the time to first intromission and the time to ejaculation to times comparable with those of sham-operated subjects. The 100-ng dose produced a significant reduction in mount frequency. Plasma T levels were significantly elevated following either dose of LHRH. These results demonstrate that exogenous LHRH can restore normal copulatory performance in hyperprolactinemic male rats and support the hypothesis that a reduction in hypothalamic LHRH release is responsible for the behavioral deficits observed in those animals.

The effect of insulin and intermittent mechanical stretching on rates of protein synthesis and degradation in isolated rabbit muscle.

Tyrosine balance and protein synthesis were studied during the same incubation in isolated rabbit forelimb muscles. From these measurements, protein degradation was calculated. Isolated muscles were usually in a state of negative amino acid balance, principally as a result of the 75% decrease in protein synthesis. Muscles from rabbits starved for 18 h had lower rates of both protein synthesis and degradation compared with muscles from normally fed rabbits. Intermittent mechanical stretching and the addition of insulin at 100 microunits/ml increased rates of both protein synthesis and degradation. Increases in the rate of protein synthesis were proportionately greater in the muscles from starved animals. In muscles from both fed and starved donors, increases in protein-synthesis rates owing to intermittent stretching and insulin were proportionately greater than the increases in degradation rates. For example, insulin increased the rate of protein synthesis in the muscles from starved donors by 111% and the rate of degradation by 31%. Insulin also increased the rate of protein synthesis when added at a higher concentration (100 munits/ml); at this concentration, however, the rate of protein degradation was not increased. The suppressive effect of insulin on high rates of protein degradation in other skeletal-muscle preparations may reflect a non-physiological action of the hormone.