Characterization of fucosyltransferase activity during mouse spermatogenesis: evidence for a cell surface fucosyltransferase

Fucosyltransferase activity was quantified in mouse germ cells at different stages of spermatogenesis. Specifically, fucosyltransferase activities of pachytene spermatocytes, round spermatids, and cauda epididymal sperm were compared. Fucosyltransferase activity of mixed germ cells displayed an apparent Vmax of 17 pmol (mg of protein)-1 min-1 and an apparent Km of approximately 13 microM for GDP-L-[14C]fucose in the presence of saturating amounts of asialofetuin at 33 degrees C. Under these conditions, cellular fucosyltransferase activity was found to increase during spermatogenesis. In agreement with assays of intact cells, examination of subcellular fractions indicated that a large fraction of fucosyltransferase activity was associated with the cell surface. The fraction of fucosyltransferase activity that was associated with the cell surface progressively increased throughout spermatogenesis and epididymal maturation so that nearly all of the fucosyltransferase in epididymal sperm was on the cell surface. Specifically, by comparison of activities in the presence and absence of the detergent NP-40, the fraction of fucosyltransferase activity that was associated with the cell surface in pachytene spermatocytes, round spermatids, and epididymal sperm was 0.36, 0.5, and 0.85, respectively. These results suggest that a cell surface fucosyltransferase may be important during differentiation of spermatogenic cells in the testis as well as during epididymal maturation and fertilization.     

Protein carboxyl methyltransferase activity specific for age-modified aspartyl residues in mouse testes and ovaries: Evidence for translation during spermiogenesis

An antiserum prepared against the purified protein carboxyl methltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age-damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell-deficient ovaries and testes obtained from mutant atrichosislatrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4–5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5-fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.     

Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies

We have identified three germ cell surface carbohydrate antigens that exhibit a common, stage-specific pattern of expression during spermatogenesis in the mouse. IgM-class monoclonal antibodies designated "J1," "C6," and "A5" were absorbed by adult testis, but not by any adult somatic tissue tested. In indirect immunofluorescence assays using collagenase-dissociated prepuberal and adult testicular cells, these antibodies labeled the surfaces of early and late pachytene spermatocytes and round spermatids. Gonocytes from fetal and neonatal testes were not labeled. In paraffin sections of prepuberal and adult testes, sialidase treatment exposed antigens recognized by antibodies C6 and A5 on preleptotene, leptotene, and zygotene spermatocytes located near the perimeter of seminiferous tubules. The determinants recognized by antibodies J1, C6, and A5 were characterized partially using a sugar hapten inhibition assay. The binding of J1 to adult testicular cells was inhibited specifically by N-acetylglucosamine and the binding of both C6 and A5 was inhibited by N-acetyllactosamine. The glycoconjugates recognized by J1, C6, and A5 eluted from gel filtration columns with an apparent molecular weight greater than 1 X 10(6) and were sensitive to endo-beta-galactosidase (keratanase) treatment. The apparent high molecular weight of these glycoconjugates was confirmed by immunolabeling Western blots of testis extracts separated by SDS-polyacrylamide gel electrophoresis. The results suggest that polylactosamine (keratan) glycoconjugates of high molecular weight are associated with the plasma membranes of meiotic and haploid male germ cells. The effects of sialidase on antibody labeling patterns suggest that changes in cell surface sialylation accompany the transition of early meiotic germ cells to pachytene spermatocytes during spermatogenesis.     

Using simulations to evaluate Mantel‐based methods for assessing landscape resistance to gene flow

Mantel‐based tests have been the primary analytical methods for understanding how landscape features influence observed spatial genetic structure. Simulation studies examining Mantel‐based approaches have highlighted major challenges associated with the use of such tests and fueled debate on when the Mantel test is appropriate for landscape genetics studies. We aim to provide some clarity in this debate using spatially explicit, individual‐based, genetic simulations to examine the effects of the following on the performance of Mantel‐based methods: (1) landscape configuration, (2) spatial genetic nonequilibrium, (3) nonlinear relationships between genetic and cost distances, and (4) correlation among cost distances derived from competing resistance models. Under most conditions, Mantel‐based methods performed poorly. Causal modeling identified the true model only 22% of the time. Using relative support and simple Mantel r values boosted performance to approximately 50%. Across all methods, performance increased when landscapes were more fragmented, spatial genetic equilibrium was reached, and the relationship between cost distance and genetic distance was linearized. Performance depended on cost distance correlations among resistance models rather than cell‐wise resistance correlations. Given these results, we suggest that the use of Mantel tests with linearized relationships is appropriate for discriminating among resistance models that have cost distance correlations <0.85 with each other for causal modeling, or <0.95 for relative support or simple Mantel r. Because most alternative parameterizations of resistance for the same landscape variable will result in highly correlated cost distances, the use of Mantel test‐based methods to fine‐tune resistance values will often not be effective.     

Combined allosteric and competitive interaction between extracellular Na(+) and K(+) during ion transport by the alpha(1), alpha(2), and alpha(3) isoforms of the Na, K-ATPase

A combined allosteric and competitive model describes the interaction between extracellular Na(+) and Rb(+) during ion transport mediated by the Na, K-ATPase. The model was developed from experiments based on (86)Rb uptake by whole cells transfected with rat isoforms of the enzyme. In the absence of Na(+), only a single transport site for extracellular Rb(+) exists. After the occupation of the Na(+)-specific allosteric site, the Rb(+) transport pocket opens to allow occupation by an additional Rb(+) and the subsequent transport of the two Rb(+) ions into the cells. Na(+) can also directly compete with Rb(+) for binding to at least one of the transport sites. While the model derived here applies to each of the three rat isoforms of the Na, K-ATPase expressed in HeLa cells, subtle differences exist among the isoforms. The alpha(3)* isoform has an increased intrinsic affinity for Rb(+) and a lower affinity for the allosteric Na(+) site than alpha(1) or alpha(2)*. The stimulation of uptake observed according to the best-fit model is due to the displacement by Rb(+) of inhibitory Na(+) bound to the transport site.     

Effect of selected antimicrobial compounds on the radiosensitization of Salmonella Typhi in ground beef

Aims:  In this study, we extended our previous work to determine the efficiency of antimicrobial compounds in increase of relative radiosensitivity of Salmonella Typhi in medium fat ground beef (23% fat) by testing 41 different essential oils (EOs), oleoresins and food sauces.
Methods and Results:  Ground beef samples inoculated with Salmonella Typhi (10⁶ CFU g⁻¹ ) were treated with each antimicrobial compound at a concentration of 0·5% (w/w). Then, the samples (25 g each) were packaged under air and irradiated in a ⁶⁰Co irradiator at doses from 0 to 1·75 kGy. Radiosensitivity was evaluated by calculating relative radiation sensitivity, defined as the ratio of radiation D ₁₀ value in the absence/presence of antimicrobial compound.
Conclusions:  Depending on the compound tested, the addition of antimicrobial compound decreased the D ₁₀ value of Salmonella Typhi, resulting in an increase of the radiation sensitivity up to more than four times. Among these antimicrobial compounds, Chinese cinnamon EO, clove EO and trans -cinnamaldehyde were most effective to increase the radiosensitivity of Salmonella Typhi in ground beef.
Significance and Impact of the Study:  These observations demonstrate that some active compounds can function as radiosensitizers of Salmonella Typhi.     

Testosterone may increase selective attention to threat in young male macaques

Animal studies indicate that sex hormones have widespread effects on the brain, cognition and emotion, but findings in humans are inconsistent. Well-controlled studies in nonhuman primates are crucial to resolve these discrepancies. In this study, we examined the effects of testosterone (T) on emotion in male rhesus monkeys. Six young adult males were tested on two emotional tasks during three hormonal conditions in a crossover design: when intact at baseline and when pharmacologically hypogonadal with add-back of T or placebo. The emotional tasks were the Approach–Avoidance task, which tested behavioral responses to three categories of objects (familiar, novel, and negative) and a Social Playback task which tested behavioral responses to scenes of unfamiliar conspecifics engaged in three types of social activities (neutral, positive, or negative). Following a 4-week baseline period, monkeys were treated with Depot Lupron, 200 μg/kg before being randomly assigned to one of two treatment groups: Depot Lupron + Testosterone Enanthate (TE, 20 mg/kg) or Depot Lupron + oil vehicle. In each treatment group, monkeys received one injection of Lupron and one injection of TE or one injection of Lupron and one injection of oil at the onset of a 4-week testing period, before crossing over to the alternate treatment for an additional 4 weeks of testing. TE treatment had no effect on behavioral measures in the Approach–Avoidance task. For the Social Playback task, however, TE significantly increased watching time of video clips which depicted fights between unfamiliar conspecifics. The enhancing effect of T on watching time for negative social scenes is consistent with human data suggesting that T decreases aversion or facilitates approach to threatening social stimuli. Further studies are needed to understand the mechanisms by which T may mediate responsiveness to social threat in male primates.