Detection of prophospholipase A2 in human spermatozoa

Prophospholipase A2 (proPA2) has been isolated from human spermatozoa after acid extraction and chromatography on hydrophobic WP-Butyl (C4) and ion-exchange (SP 5PW) columns. The addition of benzamidine, a noncompetitive synthetic trypsin inhibitor, to semen samples has kept a portion of the sperm phospholipase A2 (PA2) in its zymogen form and allowed its isolation after acid extraction. When radioactive phosphatidylcholine (PC) or phosphatidylethanolamine (PE) were used as substrates, an identical elution profile of this enzyme was obtained on a C4 column. The proenzyme was separated from active PA2 on the C4 column. Human sperm proPA2 exhibited a less cationic charge than active PA2 on the SP 5PW column. Porcine pancreatic proPA2 had the same chromatographic behavior on high performance liquid chromatography (HPLC) (SP 5PW) as human sperm proPA2. The purification procedure resulted in the isolation of proPA2 which, upon activation by proteolysis, presented the same chromatographic elution profile on HPLC as active PA2 of human spermatozoa and porcine pancreas. Thus, a zymogen form of PA2 exists in human spermatozoa.     

The primary structure of human apolipoprotein A-IV

Human apolipoprotein (apo) A-IV was purified from chylous ascites fluid. Proteolytic peptides produced by trypsin and Staphylococcus aureus V8 proteinase digestions were purified by high-performance liquid chromatography and sequenced. Human apoA-IV contains 376 amino acid residues. The peptide-derived sequence generally matches two previously reported DNA-derived amino acid sequences except for discrepancies in five positions. In order to examine these discrepancies further, one complete apoA-IV cDNA clone and another partial clone were sequenced. Comparison of all the available information indicates that the peptide-derived sequence reported here is accurate. Sequencing errors probably account for some of the discrepancies between the two primary sequences predicted by earlier nucleotide analyses. In certain positions, however, bona fide sequence heterogeneity or cloning artifact cannot be excluded.     

Modulation of phosphoinositide metabolism in rat brain slices by excitatory amino acids,arachidonic acid,and GABA

In rat brain slices the synthesis of [³H]phosphoinositides and the production of [³H]inositol monophosphate (IP₁) induced by norepinephrine (NE) were inhibited by glutamate. Calcium concentrations were varied to test if these inhibitory effects of glutamate were mediated by a calcium-dependent process. Although reducing calcium or addition of the calcium antagonist verpamil reduced the inhibitory effects of glutamate, these results were equivocal because reduced calcium directly decreased agonist-induced [³H]phosphoinositide synthesis. The inhibitory effects of glutamate were mimicked by quisqualate in a dose-dependent manner, but none of a variety of excitatory amino acid receptor antagonists modified the inhibition caused by quisqualate. It is suggested that glutamate activates a quisqualate-sensitive receptor (for which an antagonist is not available) and causes inhibition of phosphoinositide hydrolysis mediated in part by a direct or indirect inhibitory effect of calcium on phosphoinositide synthesis. Modulatory effects of arachidonic acid were examined because glutamate and calcium can activate phospholipase A₂. Arachidonic acid caused a rapid and dose-dependent inhibition of [³H]phosphoinositide synthesis and of NE-stimulated [³H]IP₁ production. A similar inhibition of the response to carbachol also occurred. The inhibition caused by arachidonic acid was unchanged by addition of inhibitors of cyclooxygenase or lipoxygenase. Activation of phospholipase A₂ with melittin caused inhibitory effects similar to those of arachidonic acid. Inhibitors of phospholipase A₂ were found to impair phosphoinositide metabolism, likely due to their lack of specificity for phospholipase A₂. Further studies were carried out in slices that were prelabelled with [³H]inositol in an attempt to separate modulatory effects on [³H]phosphoinositide synthesis and agonist-stimulated [³H]IP₁ production. Several excitatory amino acid agonists inhibited NE-stimulated [³H]IP₁ production. This inhibitory inter-action could be due to impaired synthesis of [³H]phosphoinositides because, even though the slices were prelabeled, addition of unlabelled inositol reduced NE-stimulated [³H]IP₁ production, indicating that continuous regeneration of [³H]phosphoinositides is required. In contrast to the inhibitory effects of the excitatory amino acids, gamma-aminobutyric acid (GABA) enhanced the response to NE in cortical and hippocampal slices. GABA also enhanced the response to carbachol in hippocampal and striatal slices and to ibotenic acid in hippocampal slices. Baclofen potentiated the response to NE similarly to the effect of GABA and baclofen partially blocked the inhibitory effect of arachidonic acid but did not alter that of quisqualate.Abbreviations AMPA -amino-3-hydroxy-5-methyl-4-isoxazolepropionic - acid AP₄ dl-2-amino-4-phosphonobutyric acid - BPB bromphenacyl bromide - BSA bovine serum albumin - CNQX 6-cyano-7-nitroquinoxaline-2,3-dione - DFMO -difluoromethylornithine - DIDS diisothiocyanotostilbene-2,2-disulfonic acid - EGTA ethyleneglycol-bis-N - N, N N-tetraacetic acid - GABA -aminobutyric acid - GDEE glutamate diethyl ether - -GG -glutamylglycine - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - NDGA nordihydroguaiaretic acid - NE norepinephrine - NMDA N-methyl-d-aspartate     

Infectivity inhibition of HIV prototype strains by antibodies directed against a peptide sequence of mycoplasma

Antibodies prepared against a peptide corresponding to the site of cyto-adherence of Mycoplasma genitalium adhesine inhibit or reduce the infectivity of the HIV-1BRU and HIV-2ROD strains of Human Immunodeficiency Virus in lymphoid cells. These results strengthen the hypothesis that some mycoplasmas may play an important part in HIV replication and pathogenicity.     

Interaction of alpha-N-Acetyl-beta-endorphin and calmodulin

Acetylation at the -amino terminal is a common post-translational modification of many peptides and proteins. In the case of the potent opiate peptide -endorphin, -N-acetylation is a known physiological modification that abolishes opiate activity. Since there are no known receptors for -N-acetyl--endorphin, we have studied the association of this peptide with calmodulin, a calcium-dependent protein that binds a variety of peptides, phenothiazines, and enzymes, as a model system for studying acetylated endorphin-protein interactions. Association of the acetylated peptide with calmodulin was demonstrated by cross-linking with bis(sulfosuccinimidyl)suberate; like -endorphin, adducts containing 1 mol and 2 mol of acetylated peptide per mole calmodulin were formed. Some of the bound peptides are evidently in relatively close proximity to each other since, in the presence of amidated (i.e., lysine-blocked) calmodulin, cross-linking yielded peptide dimers. The acetylated peptide exhibited no appreciable helicity in aqueous solution, but in trifluoroethanol (TFE) considerable helicity was formed. Also, a mixture of acetylated peptide and calmodulin was characterized by a circular dichroic spectrum indicative of induced helicity. Empirical prediction rules, applied earlier to -endorphin, suggest that residues 14–24 exhibit -helix potential. This segment has the potential of forming an amphipathic helix; this structural unit is believed to be important in calmodulin binding. The acetylated peptide was capable of inhibiting the calmodulin-mediated stimulation of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity with an effective dose for 50% inhibition of about 3 µM; this inhibitory effect was demonstrated using both an enzyme-enriched preparation as well as highly purified enzyme. Thus, acetylation at the -amino terminal of -endorphin, although abolishing opiate activity, does not interfere with the binding to calmodulin. Indeed, -endorphin and the -N-acetylated peptide behave very similarly with respect to calmodulin association.Portions of this work are in partial fulfillment of the requirements for the Ph.D. degree from Vanderbilt University.     

Biosynthesis of Territrems by Aspergillus terreus

Different radioactive precursors were added to 8-day potato-dextrose liquid cultures of Aspergillus terreus 23-1. Territrems were isolated from chloroform extracts of the cultures at day 14 and purified by thin-layer chromatography and high-pressure liquid chromatography. The territrem B obtained was treated with alkaline hydrogen peroxide, and 3, 4, 5-trimethoxy benzoic acid was isolated from an ethyl acetate extract of the reaction mixture and purified by thin-layer chromatography and high-pressure liquid chromatography. By comparison of the specific radioactivities of territrem B and its cleaved aromatic product (disintegrations per minute per micromole of compound), it was demonstrated that the radioactivity of territrem B was located mainly on its aromatic moiety when [U-C]shikimate, l-[methyl-C]methionine, and l-[methyl-H]methionine were precursors; however, the radioactivity of territrem B was located mainly on its nonaromatic moiety when [2-C]mevalonate was the precursor. Mevinolin, a specific inhibitor of beta-hydroxyl beta-methyl glutaryl coenzyme A reductase, was shown to inhibit production of territrems by A. terreus 23-1. When [U-C]acetate was used as a precursor, mevinolin inhibited the incorporation of radioactive carbon into territrem but mevinolin did not inhibit incorporation of radioactive carbon from [2-C]mevalonate into territrem.     

Studies on the growth of the fetal sheep. Effects of surgical reduction in placental size, or experimental manipulation of uterine blood flow on plasma sulphation promoting activity and on the concentration of insulin-like growth factors I and II

The effect of long- and short-term manipulations of uterine blood flow on fetal plasma levels of IGF-I and -II have been studied in sheep at days 125-139 of pregnancy and compared with those in near term rats and guinea pig. The primary objective is to show that both long- and short-term reduction of uterine blood flow is associated with increase in the fetal plasma concentration of IGF-II while that of IGF-I falls. In the pregnant sheep long-term depression of utero-placental blood flow was caused by surgical reduction in placental mass (carunclectomy) prior to conception. This reduced fetal weight to 2.42 +/- 0.49 kg (SD) compared with 3.41 +/- 0.46 in controls; the respective values for uterine blood flow being 1694 +/- 558 and 913 +/- 324 ml/min respectively. This was associated with a fall in fetal plasma IGF-I concentration from 22.6 +/- 3.4 ng/ml to 14.9 +/- 1.31 ng/ml and a rise in IGF-II from 1952 +/- 284 ng/ml to 3360 +/- 914 ng/ml respectively. Similar changes in the plasma concentrations of IGF peptides were observed in fetal rats and guinea pigs in response to uterine artery ligation. Short-term reduction (60 min) of the uterine blood flow was caused either by compression of the common uterine artery to depress flow from 1491 +/- 375 to 648 +/- 216 ml/min or through intraarterial infusion of adrenaline at 35 ug/min to lower flow from 1628 +/- 339 to 1195 +/- 128 ml/min. Such falls in uterine blood flow had no significant effect on fetal plasma IGF-I levels but increased IGF-II levels by 30 to 60%.     

Characterization of base-pair opening in deoxynucleotide duplexes using catalyzed exchange of the imino proton

Using nuclear magnetic resonance line broadening, longitudinal relaxation and magnetization transfer from water, we have measured the imino proton exchange times in the duplex form of the 10-mer d-CGCGATCGCG and in seven other deoxy-duplexes, as a function of the concentration of exchange catalysts, principally ammonia. All exchange times are catalyst dependent. Base-pair lifetimes are obtained by extrapolation to infinite concentration of ammonia. Lifetimes of internal base-pairs are in the range of milliseconds at 35 degrees C and ten times more at 0 degrees C. Lifetimes of neighboring pairs are different, hence base-pairs open one at a time. Lifetimes of d(G.C) are about three times longer than those of d(A.T). The nature of neighbors usually has little effect, but lifetime anomalies that may be related to sequence and/or structure have been observed. In contrast, there is no anomaly in the A.T base-pair lifetimes of d-CGCGA[TA]5TCGCG, a model duplex of poly[d(A-T)].poly[d(A-T)]. The d(A.T) lifetimes are comparable to those of r(A.U) that we reported previously. End effects on base-pair lifetimes are limited to two base-pairs. The low efficiency of exchange catalysts is ascribed to the small dissociation constant of the deoxy base-pairs, and helps to explain why exchange catalysis had been overlooked in the past. This resulted in a hundredfold overestimation of base-pair lifetimes. Cytosine amino proteins have been studied in the duplex of d-CGm5CGCG. Exchange from the closed base-pair is indicated. Hence, the use of an amino exchange rate to evaluate the base-pair dissociation constant would result in erroneous, overestimated values. Catalyzed imino proton exchange is at this time the safest and most powerful, if not the only probe of base-pair kinetics. We propose that the single base-pair opening event characterized here may be the only mode of base-pair disruption, at temperatures well below the melting transition.     

Tubulin isotypes and their interaction with microtubule associated proteins

Summary To assay the functional significance of the multiple but closely related - and -tubulin polypeptides (termed isotypes) that are expressed in mammalian cells, we have generated a number of sera that uniquely discriminate among these isotypes. These sera have been used to demonstrate that there is no subcellular sorting of either - or -tubulin isotypes among microtubules of diverse function, either in cells growing in culture or in tissues consisting of cell types that contain specialized kinds of microtubule. In spite of this failure to segregate between functionally distinct kinds of microtubule, the fact that isotype-specific amino acid sequences have been strictly conserved over extensive periods of evolutionary time argues persuasively for a functional role for the different tubulin gene products. One possibility is that they are required for specific interactions with microtubule associated proteins (MAPs), and that tubulin isotypes have coevolved with different cell type-specific MAPs with which they must interact. We have tested this hypothesis by examining the distribution of -tubulin isotypes in mammalian cerebellum in relationship to the known patterns of expression of a number of MAPs, and find that these patterns correlate in the case of M 2 and MAP 3, and M 6 and MAP 1 a. These data, plus emerging data based on a structural analysis of tau, MAP 1 b and MAP 2 obtained via sequence determination of cloned cDNAs, are discussed in terms of the possible functional significance of tubulin isotype/MAP interactionsin vivo.