A role for inhibin in the control of follicle-stimulating hormone secretion in male rats

We examined the relationship of testosterone (T) and porcine follicular fluid (pFF) in the negative feedback control of FSH and LH secretion in adult male rats. Either at the time of castration (acute) or at least 30 days after castration (chronic), we implanted T-filled Silastic capsules, which were 2 mm, 10 mm, or 30 mm long; empty capsules (30 mm) served as controls. Seven days later, we injected either 0.15 ml of pFF or saline (i.v.), decapitated the rats 6 hours later, and collected trunk blood for subsequent serum analysis of FSH, LH, and T by RIA. In the acute groups, T implants suppressed the postcastration rises in plasma FSH and LH levels in a dose-dependent manner, with only the largest implant, 30 mm, able to return them to intact levels. PFF injection significantly suppressed FSH levels in intact and acute rats but had no effect on serum LH. In chronic rats, T therapy for 7 days suppressed plasma LH levels in a dose-dependent relationship, yet did not do so to plasma FSH levels. FSH levels were significantly higher in rats with the 30 mm T implants than in intact rats, but were significantly suppressed as compared to chronic controls. PFF significantly suppressed serum FSH levels in all chronic groups with the chronic controls showing the greatest amount of suppression. We conclude that the role for inhibin in the normal control of FSH secretion is that of a secondary modulator which is superimposed on, yet independent of, the steroid feedback mechanism. At any given moment this modulation is dependent upon the secretory activity of the FSH gonadotrope.     

Glycoprotein biosynthesis in B lymphocytes: induction of protein N-glycosylation, RNA synthesis, and DNA synthesis by phorbol ester plus ionomycin is blocked by protein kinase inhibitors

The combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin produces a dramatic increase in the incorporation of [2-3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and glycoprotein, and the induction of RNA and DNA synthesis in murine splenic B lymphocytes (B cells). The kinetics of the induction processes and the concentrations of PMA and ionomycin required for the optimal response have been defined. While the levels of induction of RNA and DNA synthesis by PMA + ionomycin were similar to the mitogenic response to bacterial lipopolysaccharide, activation by PMA and the calcium ionophore resulted in a threefold higher stimulation in dolichol-linked oligosaccharide biosynthesis and protein N-glycosylation. These results indicate that all signalling mechanisms that trigger RNA and DNA synthesis may not be sufficient to produce maximal induction of the N-glycosylation apparatus. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C inhibitor, prevented the induction of protein N-glycosylation activity (IC50 = 11 microM), as well as RNA (IC50 = 18 microM) and DNA synthesis (IC50 = 12 microM), two common indices of B cell activation. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) also inhibited the induction of oligosaccharide-lipid intermediate, glycoprotein, RNA, and DNA synthesis, but required higher concentrations than H-7 for 50% inhibition. N-(2-Guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a potent inhibitor of cyclic nucleotide-dependent protein kinases, had little effect on the activation of the B cell metabolic processes. The H-7-sensitive reactions involved in the induction of RNA and DNA synthesis occurred within 4 h, but induction of lipid intermediate and glycoprotein biosynthesis remained sensitive to H-7 for 10 h after exposure to PMA and ionomycin. Direct in vitro assays in the presence of 0.6% Brij 58 reveal that a cytosolic, phospholipid-dependent protein kinase activity is translocated to a membrane site(s) after treatment with PMA and ionomycin, and the translocated protein kinase is sensitive to H-7. The relative order of potency of the protein kinase inhibitors on the metabolic processes strongly supports the hypothesis that protein kinase C, acting synergistically with Ca2+ mobilization, plays a key regulatory role in the early stages of B cell activation. The synthesis of oligosaccharide-lipid intermediates and protein N-glycosylation are also shown to be induced in B cells activated by PMA + ionomycin.     

Biogenesis of the endoplasmic reticulum in activated B lymphocytes: temporal relationships between the induction of protein N-glycosylation activity and the biosynthesis of membrane protein and phospholipid.

An earlier report from this laboratory documented a substantial increase in the rates of dolichol-linked oligosaccharide intermediate synthesis and protein N-glycosylation in purified murine splenic B lymphocytes (B cells) activated by treatment with bacterial lipopolysaccharide (LPS). In this study the developmental patterns for the induction of lipid-mediated protein N-glycosylation, membrane protein, and phosphatidylcholine (PC) biosynthesis were compared during the proliferative response of B cells to LPS. By electron microscopy it could be seen that a distinct endoplasmic reticulum (ER) network began to develop by 24-48 h after exposure of the purified B cells to LPS. The rate of synthesis of membrane protein increased markedly during the first 10 h after activation, reaching a maximum at 30-40 h. The induction of protein N-glycosylation was delayed slightly relative to membrane protein synthesis, with glycoprotein synthesis increasing sharply approximately 20 h after activation. When phospholipid synthesis was monitored by measuring [CH3-3H]choline incorporation into PC, the rate of labeling increased slowly during the first 35 h, but more substantially between 35 and 90 h. The incorporation of labeled choline into PC was drastically reduced by 5'-deoxy-5'-isobutylthio-3-deazaadenosine, an inhibitor of CDP-choline synthesis, indicating that the incorporation of radiolabeled choline is primarily a measurement of the rate of de novo synthesis of PC. In vitro assays revealed that while choline kinase activity was virtually unchanged, CDP-choline synthetase activity increased gradually throughout the activation period. Diacylglycerol cholinephosphotransferase activity, an ER-associated enzyme, was present at low levels between 0 and 35 h, but increased fivefold between 35 and 90 h. On the basis of the developmental patterns for the rates of protein N-glycosylation, membrane protein insertion, and PC biosynthesis determined by metabolic labeling experiments, we tentatively conclude that all of the ER-associated membrane proteins involved in these biosynthetic processes are not induced concurrently during the activation of B cells by LPS.     

MEASUREMENTS OF NATURAL SELECTION ON FLORAL TRAITS IN WILD RADISH (RAPHANUS RAPHANISTRUM). I. SELECTION THROUGH LIFETIME FEMALE FITNESS

Although the role of natural selection in the evolution of floral traits has been of great interest to biologists since Darwin, studies of selection on floral traits through differences in lifetime fitness have been rare. We measured selection acting on flower number, flower size, stigma exsertion, and ovule number per flower using field data on lifetime female fitness (seed production) in wild radish, Raphanus raphanistrum. The patterns of selection were reasonably consistent across three field seasons, with strong directional selection for increased flower production in all three years, weaker selection for increased ovule number per flower in two years, and selection for increased flower size in one year. The causes of the selection were investigated using path analysis combined with multiplicative fitness components. Increased flower production increased fruit production directly, and increased numbers of ovules per flower increased the number of seeds per fruit in all three years; pollinator visitation did not influence either of these fitness components. Increased flower size was associated with increases in both the number of fruit and the number of seeds per fruit in one year, with the latter relationship being stronger. Total lifetime seed production was affected more strongly by differences in fruit production than by differences in either the number of seeds per fruit or the proportion of fertilized seeds that were viable, but all three fitness components were positively correlated with total seed production.     

The effect of wild radish floral morphology on pollination efficiency by four taxa of pollinators

The effects of floral morphology on rates of pollen removal and deposition by different pollinators in generalist plant species are not well known. We studied pollination dynamics in wild radish, Raphanus raphanistrum, a plant visited by four groups of pollinators: honey bees, small native bees, butterflies, and syrphyd flies. The effects of anther position and other factors on pollen removal during single visits by all four pollinator taxa were measured. Flowers with high anther exsertion (i.e., anthers placed higher above the opening of the corolla tube) tended to have the highest numbers of pollen grains removed, but this effect was strongest for honey bees and butterflies. For all pollinator taxa, pollen removal increased with the number of pollen grains available on a flower and whowed a positive, decelerating relationship with the duration of the visit. The effects of stigma position and other factors on pollen deposition during single visits by honey bees and butterflies were also studied. The nectar-feeding butterflies had a higher pollination efficiency (percentage of pollen grains removed from anthers that were subsequently deposited on a stigma) than the nectar- and pollen-feeding honey bees. Flowers with intermediate stigma exsertion had the highest numbers of pollen grains deposited on their stigmas by butterflies, but stigma exsertion had no effect on deposition by honey bees. For both butterflies and honey bees, pollen deposition on the recipient flower increased with the amount of pollen removed from the donor flower, and there was a positive, decelerating relationship between deposition and time spent at the flower; these results are analogous to those for pollen removal. The effects of anther and stigma exsertion on pollen removal and denosition did not fit predictions based on patterns of floral correlations, but results for morphology, pollen availability, time spent per visit, and pollinator efficiency are in broad agreement with previous studies, suggesting the possible emergence of some general rules of pollen transfer.     

The potassium A-current,low firing rates and rebound excitation in Hodgkin-Huxley models

It is widely believed, following the work of Connor and Stevens (1971,J. Physiol. Lond. 214, 31–53) that the ability to fire action potentials over a wide frequency range, especially down to very low rates, is due to the transient, potassium A-current (I _A). Using a reduction of the classical Hodgkin-Huxley model, we study the effects ofI _A on steady firing rate, especially in the near-threshold regime for the onset of firing. A minimum firing rate of zero corresponds to a homoclinic bifurcation of periodic solutions at a critical level of stimulating current. It requires that the membrane's steady-state current-voltage relation be N-shaped rather than monotonic. For experimentally based genericI _A parameters, the model does not fire at arbitrarily low rates, although it can for the more atypicalI _A parameters given by Connor and Stevens for the crab axon. When theI _A inactivation rate is slow, we find that the transient potassium current can mediate more complex firing patterns, such as periodic bursting in some parameter regimes. The number of spikes per burst increases asg _A decreases and as inactivation rate decreases. We also study howI _A affects properties of transient voltage responses, such as threshold and firing latency for anodal break excitation. We provide mathematical explanations for several of these dynamic behaviors using bifurcation theory and averaging methods.     

Satellite DNA repeat sequence variation is low in three species of burying beetles in the genus Nicrophorus (Coleoptera: Silphidae)

Three satellite DNA families were identified in three species of burying beetles, Nicrophorus orbicollis, N. marginatus, and N. americanus. Southern hybridization and nucleotide sequence analysis of individual randomly cloned repeats shows that these satellite DNA families are highly abundant in the genome, are composed of unique repeats, and are species-specific. The repeats do not have identifiable core elements or substructures that are similar in all three families, and most interspecific sequence similarity is confined to homopolymeric runs of A and T. Satellite DNA from N. marginatus and N. americanus show single-base-pair indels among repeats, but single-nucleotide substitutions characterize most of the repeat variability. Although the repeat units are of similar lengths (342, 350, and 354 bp) and A + T composition (65%, 71%, and 71%, respectively), the average nucleotide divergence among sequenced repeats is very low (0.18%, 1.22%, and 0.71%, respectively). Transition/transversion ratios from the consensus sequence are 0.20, 0.69, and 0.70, respectively.      

Function,expression, specificity,diversity and incompatibility of actinobacteriophage parABS systems

More than 180 individual phages infecting hosts in the phylum Actinobacteria have been sequenced and grouped into Cluster A because of their similar overall nucleotide sequences and genome architectures. These Cluster A phages are either temperate or derivatives of temperate parents, and most have an integration cassette near the centre of the genome containing an integrase gene and attP. However, about 20% of the phages lack an integration cassette, which is replaced by a 1.4 kbp segment with predicted partitioning functions, including plasmid‐like parA and parB genes. Phage RedRock forms stable lysogens in Mycobacterium smegmatis in which the prophage replicates at 2.4 copies/chromosome and the partitioning system confers prophage maintenance. The parAB genes are expressed upon RedRock infection of M. smegmatis, but are downregulated once lysogeny is established by binding of RedRock ParB to parS‐L, one of two centromere‐like sites flanking the parAB genes. The RedRock parS‐L and parS‐R sites are composed of eight directly repeated copies of an 8 bp motif that is recognized by ParB. The actinobacteriophage parABS cassettes span considerable sequence diversity and specificity, providing a suite of tools for use in mycobacterial genetics.     

The Bacteriohopanepolyol Inventory of Novel Aerobic Methane Oxidising Bacteria Reveals New Biomarker Signatures of Aerobic Methanotrophy in Marine Systems

Aerobic methane oxidation (AMO) is one of the primary biologic pathways regulating the amount of methane (CH₄) released into the environment. AMO acts as a sink of CH₄, converting it into carbon dioxide before it reaches the atmosphere. It is of interest for (paleo)climate and carbon cycling studies to identify lipid biomarkers that can be used to trace AMO events, especially at times when the role of methane in the carbon cycle was more pronounced than today. AMO bacteria are known to synthesise bacteriohopanepolyol (BHP) lipids. Preliminary evidence pointed towards 35-aminobacteriohopane-30,31,32,33,34-pentol (aminopentol) being a characteristic biomarker for Type I methanotrophs. Here, the BHP compositions were examined for species of the recently described novel Type I methanotroph bacterial genera Methylomarinum and Methylomarinovum, as well as for a novel species of a Type I Methylomicrobium. Aminopentol was the most abundant BHP only in Methylomarinovum caldicuralii, while Methylomicrobium did not produce aminopentol at all. In addition to the expected regular aminotriol and aminotetrol BHPs, novel structures tentatively identified as methylcarbamate lipids related to C-35 amino-BHPs (MC-BHPs) were found to be synthesised in significant amounts by some AMO cultures. Subsequently, sediments and authigenic carbonates from methane-influenced marine environments were analysed. Most samples also did not contain significant amounts of aminopentol, indicating that aminopentol is not a useful biomarker for marine aerobic methanotophic bacteria. However, the BHP composition of the marine samples do point toward the novel MC-BHPs components being potential new biomarkers for AMO.     

16, 16 Dimethyl prostaglandin E2 decreases the formation of collagen in fibrotic rat liver slices

The effect of 16, 16 dimethyl prostaglandin E₂ (DMPG) on fibrogenesis was studied in slices from normal and fibrotic rat liver. Rats received a cirrhogenic diet for seven months; supplemented controls received a diet with the deficient nutrients restored. Slices from fibrotic livers incorporated more ¹⁴C-proline and produced more ¹⁴C-hydroxyproline in TCA precipitable proteins than slices from control livers. DMPG (10^−10M) decreased the incorporation of labeled proline and the synthesis of labeled hydroxyproline in slices from fibrotic livers to the same extent, suggesting that DMPG did not affect the hydroxylation of proline per se. The magnitude of the DMPG induced decrease in labeled proline incorporation correlated with the hydroxyproline content in the liver (i.e. with increasing fibrosis there was a greater effect of DMPG; while in control rat liver slices, DMPG had no effect). DMPG did not change the size of the proline pool, its specific activity, or the activity of proline oxidase. We conclude that under these conditions of enhanced fibrogenesis, DMPG decreases the formation of collagen in vitro, possibly by lowering the incorporation of proline into collagen precursors. This may explain, at least in part, the inhibition of fibrogenesis by DMPG in vivo.