Internal GTP stimulates the speract receptor mediated voltage changes in sea urchin spermatozoa membrane vesicles

A voltage-sensitive Na+/H+ exchanger in the flagellar membrane is responsible for regulating the intracellular pH of the sea urchin spermatozoa. A previous study has shown that the egg peptide speract can modulate this Na+/H+ exchanger through its hyperpolarizing effect on the membrane potential. The effect of GTP on this speract receptor mediated process is investigated in this study. Plasma membrane vesicles with an outwardly directed K+ gradient were prepared from the isolated flagella by osmotic lysis. Vesicular membrane potential was monitored by a cationic probe, diS-C3-(5), and an anionic probe, diS-BA-C2-(3). Results show that the presence of internal GTP greatly stimulated the speract induced membrane hyperpolarization in this vesicle system. The analog GTP gamma S was not only active but could, by itself, induce partial hyperpolarization which was further enhanced by speract addition. Internal GDP was partially active in supporting the speract effect, whereas GDP beta S, cGMP, GMP, and ATP were all inactive. The ionic selectivity of the speract effect was investigated by increasing the external concentration of various cations. K+ and Rb+ abolished the hyperpolarization while Cs+ had no effect. These results indicate that internal GTP is involved in the coupling between the speract receptor and the membrane hyperpolarization, which is most likely due to the activation of K+ selective channels.     

Purification and characterization of an N alpha-acetyltransferase from Saccharomyces cerevisiae

N alpha-Acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of proteins and peptides, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 4,600-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, hydroxylapatite, DE52 cellulose, and Affi-Gel blue. The Mr of the native enzyme was estimated to be 180,000 +/- 10,000 by gel filtration chromatography, and the Mr of each subunit was estimated to be 95,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 9.0, and its pI is 4.3 as determined by chromatofocusing on Mono-P. The enzyme catalyzed the transfer of an acetyl group to various synthetic peptides, including human adrenocorticotropic hormone (ACTH) (1-24) and its [Phe2] analogue, yeast alcohol dehydrogenase I (1-24), yeast alcohol dehydrogenase II (1-24), and human superoxide dismutase (1-24). These peptides contain either Ser or Ala as NH2-terminal residues which together with Met are the most commonly acetylated NH2-terminal residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). Yeast enolase, containing a free NH2-terminal Ala residue, is known not to be N alpha-acetylated in vivo (Chin, C. C. Q., Brewer, J. M., and Wold, F. (1981) J. Biol. Chem. 256, 1377-1384), and enolase (1-24), a synthetic peptide mimicking the protein's NH2 terminus, was not acetylated in vitro by yeast acetyltransferase. The enzyme did not catalyze the N alpha-acetylation of other synthetic peptides including ACTH(11-24), ACTH(7-38), ACTH(18-39), human beta-endorphin, yeast superoxide dismutase (1-24). Each of these peptides has an NH2-terminal residue which is rarely acetylated in proteins (Lys, Phe, Arg, Tyr, Val, respectively). Among a series of divalent cations, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate and N-bromosuccinimide.     

X-Linked Female-Sterile Loci in DROSOPHILA MELANOGASTER

We have examined the number of X-linked loci specifically required only during oogenesis. Complementation analyses among female-sterile (fs) mutations obtained in two mutagenesis screens--GANS' and MOHLER's--indicate that any fs locus represented by two or more mutant alleles in GANS' collection are usually present in MOHLER's collection. However, when a locus is represented by a single allele in one collection, it is generally not present in the other collection. We propose that this discrepancy is due to the fact that most "fs loci" represented by less than two mutant alleles are, in fact, vital (zygotic lethal) genes, and that the fs alleles are hypomorphic mutations of such genes. In support of this hypothesis we have identified lethal alleles at 12 of these "fs loci." The present analysis has possibly identified all maternal-effect lethal loci detectable by mutations on the X chromosome and has allowed us to reevaluate the number of "ovary-specific fs" loci in the Drosophila genome. Finally, germline clone analysis of a large number of fs mutations was performed in order to estimate the relative contribution of germline and somatic cell derivatives to oogenesis and to embryonic development. All the maternal-effect lethal loci tested are germline-dependent.     

Improved Listeria monocytogenes selective agar.

By increasing the LiCl concentration to 5 g/liter and adding 20 mg of moxalactam per liter to modified McBride agar base, it was possible to inhibit the growth of many bacteria which interfered with the recovery of Listeria monocytogenes from beef.     

Demonstration of the GC-rich common arm in yeast ribosomal 5.8S RNA via 500-MHz proton nuclear magnetic resonance and Overhauser enhancements

In this paper we report the first 1H NMR study of the base-paired secondary structure of yeast 5.8S RNA. On the basis of a combination of homonuclear Overhauser enhancements and temperature dependence of the proton 500-MHz NMR spectrum, we are able to identify and assign eight of the nine base pairs in the most thermally stable helical arm: G116.C137-C117.G136-C118.G135- C119.G134-C120.G133-U121.G132- U122.A131-G123.C130. This arm contains an unusually temperature-stable (to 71 degrees C) segment of four consecutive G.C base pairs. This work constitutes the most direct evidence to date for the existence and base-pair sequence of the GC-rich helix, which is common to most currently popular secondary structural models for eukaryotic 5.8S ribosomal RNA.     

Endocrine differences in rams after genetic selection for testis size

Testis diameter and body weight were recorded from 6 to 76 weeks of age in ram lambs from two established lines selected for high (H) and low (L) testis size. While testis growth was greater in the H line up to 14 weeks of age (P less than 0.001), body weight was significantly lower, with the L line rams being 10 kg heavier by 76 weeks. There were no differences in plasma LH up to 20 weeks of age, but FSH concentrations were significantly lower at 14 and 20 weeks in the H line. Testosterone concentrations were not significantly higher in the H line from 6 to 20 weeks. In lambs castrated at birth, significantly higher FSH values were recorded from 6 to 20 weeks of age in the H line (P less than 0.001) whereas there was no difference in LH concentration at 6 and 10 weeks of age between the lines. At 14 and 20 weeks, however, the concentrations of LH were greater in the H than L line lambs (P less than 0.05). After hemicastration at 6 weeks of age, the rate of growth of the remaining testis in the L line lambs was significantly faster than in entire lambs of that line from 10 to 20 weeks (P less than 0.05 at 10 weeks to P less than 0.001 at 20 weeks). There was no difference in the rate of testis growth between the the entire and hemicastrated lambs from the H line from 6 to 12 weeks of age. It can be concluded that there is an underlying genetic difference in pituitary gland and/or hypothalamic activity in ram lambs from the two selected lines.     

Ultrastructure of proteinaceous bladder plugs in male rats

Proteinaceous plugs in the bladder (bladder plugs) were found in male rats with an incidence of 14.1 to 17.8% in ages ranging from 10 weeks to 2 years. No evidence of urinary obstruction was found due to the plugs, but they appeared to irritate the bladder epithelium mechanically causing denudation. Consequently, exfoliated epithelial cells were incorporated into the plugs. Early in development, the plugs consisted of loosely organized eosinophilic masses with fine eosinophilic granules and fenestrated filaments in which eosinophilic globules were suspended. The components of plugs were similar to that contained in seminal vesicles. Subsequently, the plugs became more compact in structure with formation of densely interwoven amphophilic trabeculae containing exfoliated cells and spermatozoa. The periphery of the plugs was surrounded by exfoliated cells, cellular debris, eosinophilic granular materials and spermatozoa. Under electron microscopy, the eosinophilic granules surrounding the plugs were dense aggregates of electron-dense globules and vesicles derived from disintegrated bladder epithelium. The amphophilic trabeculae had a dense compact granular structure consisting of densely aggregated protein globules with a filamentous network. The intertrabecular proteinaceous material had a spongy like structure consisting of sparsely scattered protein globules with fine fenestrated filaments. Proteinaceous plugs having exfoliated cells and spermatozoa were found also in the male accessory sex glands. The plugs in the urinary bladder or male sex accessory glands appeared to be developed from back-flow of semen following ejaculatory disturbance.     

Activating and 'anxiogenic' effects of corticotropin releasing factor are not inhibited by blockade of the pituitary-adrenal system with dexamethasone

Central administration of corticotropin releasing factor (CRF) in rats produces pituitary-adrenal activation and a variety of "anxiogenic-like" effects. The present study was designed to explore the contribution of the peripheral pituitary-adrenocortical axis in mediating these CRF responses. Intraventricularly administered CRF produced suppression of responding in the conflict test and a marked locomotor activation. Neither behavioral effect was altered by the prior administration of dexamethasone in a dose that blocked pituitary-adrenal activation to CRF. These results support the hypothesis that behavioral effects of CRF are mediated by its action at central sites and not via an action on the pituitary-adrenocortical system.     

Is the large-male mating advantege in anurans an epiphenomenon?

Summary This study examines the idea that variation in forelimb length among male anurans influences reproductive success, and does so independently of body size. Analyses of covariance and multivariate analyses of morphological data for five species of explosives breeders are used to test the prediction that for species in which male-male displacements contribute to variation in male reproductive success, amplectant males have longer forelimbs than do non-amplectant males at any body length. The findings for four of five species are in agreement with expectation. The results lend support to the suggestion that for explosive-breeding anurans, the large-male mating advantage may not be a consequence of large body size per se, but rather is due to the fact that large males have longer forelimbs with which to grasp females securely during amplexus and thus resist displacements by competing males.