Flagellar adenosine triphosphatases from annelid spermatozoa: electrophoretic identification of dyneins

Axonemes of sperm flagella were prepared from the annelid, Tylorrhynchus heterochaetus. Dialysis of the axonemes against 1 mm Tris-HCl buffer (pH 8.3)-0.1 mm EDTA-0.1 mm dithiothreitol (Tris-EDTA solution) caused disintegration of typical 9 + 2 microtubules into each doublet, resulting in extraction of one-third of the protein and almost all ATPase activity. Agarose polyacrylamide gel electrophoresis of the extract showed the presence of three kinds of dyneins actively stained for ATPase (designated as bands I, II, and III) and two non-ATPase proteins (bands IV, V). The polypeptide components of each dynein molecule and intact axoneme were analyzed by subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis to obtain the following results: (1) In the highmolecular-weight region, the intact axonemes yield two major polypeptides with molecular weights of 365,000 and 345,000 (designated as bands A and B, respectively) and three minor polypeptides, 310,000, 290,00, and 270,00 (C1, C2, C3). (2) All three dyneins contain A-band polypeptide as a common polypeptide component. In addition, band I dynein and band II dynein also contain B and C1 polypeptides, and C3 polypeptide, respectively, as high-molecular-weight components. (3) Band III dynein also contains four polypeptides in the lower molecular-weight region, which migrate similarly with those of 21 S dynein from sea urchin sperm flagella or 18 S dynein from Chlamydomonas.     

Stress-induced testicular hyposensitivity to gonadotropin in rats. Role of the pituitary gland

The time course of stress-induced testicular hyposensitivity to gonadotropins was studied in hypophysectomized or naloxone-treated rats exposed to various periods of immobilization. Blood was collected from a chronically indwelling intra-atrial catheter every hour for luteinizing hormone (LH) and testosterone (T) measurement. Eight hours of immobilization completely suppressed T secretion without significant effect on LH. Human chorionic gonadotropin (hCG, 5 IU/rat, i.m.) induced a marked increase in plasma T levels in normal control groups 3 h post-injection while in immobilized rats the response was completely abolished, even after only 30 min of stress. In hypophysectomized rats, as expected, plasma T levels were undetectable, but, contrary to results obtained in normal animals, hCG induced a similar increase of plasma T levels both in control and stressed rats. Immobilization stress failed to inhibit plasma T values in hypophysectomized rats pretreated for 4 days with human menopausal gonadotropin (hMG) + hCG, while it did so in similarly treated normal animals. Naloxone induced a rise of plasma LH and T levels in control rats, but did not antagonize the stress-induced fall of plasma T concentration. In all groups, steroid testicular content mimicked variations of plasma T values. In particular, in stressed animals the lack of accumulation of testicular 17-hydroxyprogesterone probably reflected a normal activity of 17-20 lyase. These results indicate that stress induces very rapidly a state of Leydig cell hyposensitivity to gonadotropins and a blockade of T biosynthesis. The causal relationship between the two effects is presently not clear but these events seem to be due to stress-induced release of an inhibitory factor of pituitary origin other that endorphin.     

Evaluation of factors responsible for the inability of insulin to antagonize lipolysis due to high concentrations of catecholamines

Previous studies using rat adipocytes have shown that the ability of insulin to antagonize lipolysis induced by physiological concentrations of catecholamines is diminished at high concentrations of these hormones. Since such high concentrations of catecholamines cause an accumulation of free fatty acids, a decrease in cellular ATP level and a ‘short lived’ increase in cAMP (that is many fold higher than required to activate lipolysis maximally), we studied which of these modulates the antilipolytic activity of insulin. We found that inhibition of adenylate cyclase by virazole (2 mM), which lowers the initial cyclic AMP burst by about 70%, enables insulin to antagonize lipolysis at high isoproterenol concentrations. In contrast, reduction of cellular ATP level by 40% and 70%, using cyanide ion, or increasing free fatty acids in the medium to a level that suppresses the effects of insulin on glucose metabolism, failed to compromise the antilipolytic activity of the hormone. These data indicate that the inability of insulin to antagonize lipolysis induced by high isoproterenol concentrations is the direct consequence of the initial, larger burst of cyclic AMP.     

Macrophage-mediated cytolysis of erythrocytes in the guinea pig. II. Role of oxidative burst products in macrophage cytotoxicity activated by lectins and phorbol ester derivatives

Guinea pig peritoneal macrophages (GPPM) exhibited enhanced production of O₂^? and H₂O₂, and cytolytic activity toward erythrocytes, in response to reagents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), its methylated derivative 4-O-MeTPA, Con A, wheat germ agglutinin (WGA), and opsonized zymosan. In order to examine the possible role of oxidative burst products such as O₂^? and H₂O₂ in the cytolytic process, we used reagents and enzymes which influence the balance of O₂^? and H₂O₂ outside and inside the GPPM cells. Macrophage-mediated cytolysis (MMC) of erythrocytes in the presence of the activators and modulators was assessed by ⁵¹Cr release assay. MMC activated by TPA and 4-O-MeTPA was inhibited by scavengers of H₂O₂ such as catalase and α-tocopherol, and was augmented by the catalase inhibitor 3-amino-1,2,4-triazole, and by horseradish peroxidase. TPA- and 4-O-MeTPA-activated MMC was only partially inhibited by the O₂^? scavenger cytochrome c and the enzyme superoxide dismutase and unaffected by cytochalasin D (an inhibitor of phagocytosis). MMC activated by the lectins Con A and WGA was unaffected by the scavengers and enzymes used, but markedly inhibited by cytochalasin D. Activation of MMC by TPA, WGA, and phagocytosis of opsonized zymosan, as well as O₂^? and H₂O₂ generation triggered by these reagents, were markedly inhibited by chlorpromazine. The results indicate that GPPM-mediated cytolysis activated by lectins, phorbol ester derivatives, and phagocytosis of opsonized zymosan, is dependent on the generation of oxidative burst products, mainly H₂O₂. TPA- or 4-O-MeTPA-activated MMC is mainly an extracellular event, while lectin-activated MMC may take place within the macrophages.     

Natural enemies ofAcraea terpsicore [Lep.: Nymphalidae] in ghana,with particular reference to the parasiteCharops diversipes [Hym.: Ichneumonidae]

The arthropod parasites and predators ofAcraea terpsicore (L.) were determined in the forest zone of Ghana. An unidentified mite was predatory on the very young larvae. The pentatomidsPlatynopus rostratus Dru. andMacrorhaphis acuta (Dall.) were also predatory on the larvae.Telenomus sp. parasitized the eggs. The tachinidCarcelia normula (Curran) and the ichneumonidCharops diversipes Roman were parasitic on the larvae.C. diversipes was hyperparasitized by the chalcididBrachymeria feae Masi and the eulophidPediobius taylori Kerrich. Laboratory tests showed that parasitism ofA. terpsicore byC. diversipes was significantly highest in the 1st instar, followed by the 2nd, 3rd and 4th. The 5th instar was not parasitized. These results seemed to reflect both host susceptibility and parasite preference. Only a singleC. diversipes larva developed in a host. The developmental period of the parasite egg and larva varied inversely with the age of the host at which it was parasitized. A femaleC. diversipes could oviposit immediately after emergence.     

Genetic recombination in Micromonospora rosaria by protoplast fusion

Auxotrophic strains of Micromonospora rosaria were isolated by N-methyl-N'-nitro-N'-nitrosoguanidine mutagenesis and used in intraspecific recombination by protoplast fusion. High-frequency fusion of protoplasts of M. rosaria strains was induced by polyethylene glycol (molecular weight, 1,000) (PEG 1,000). The optimum concentration of PEG 1,000 for fusion of M. rosaria was 50% (wt/vol). PEG 4,000 was slightly better than PEG 1,000 at concentrations lower than 50% (wt/vol). The recombinant frequency did not increase after treatment with PEG 1,000 (50% [wt/vol]) for longer than 20 min. Under these conditions, fusion with many auxotrophic strains of M. rosaria resulted in a high frequency of formation of true recombinants (sometimes more than 10%). Additionally, when ros (rosamicin nonproducing) strains were crossed by protoplast fusion; about 5% of the resultant prototrophic recombinants were shown to have the ros+ (rosamicin producing) characteristic restored. Rosamicin production by M. rosaria colonies was clearly distinguished by the broth overlay method. The results of fusion experiments between ros and ros+ strains indicated that either the chromosomal mutation or pleiotrophic effect of some auxotrophic markers is involved.     

Microbial Resolution of alpha-Hydroxy Acids by Enantiospecifically Dehydrogenating Bacteria from Soil

Thirty-three bacterial strains were isolated from soil, utilizing optically asymmetric degradation of dl-2-hydroxy-4-methylpentanoic acid (dl-HMPA) as the screening probe. Those strains were distributed in the following group and genera: Coryneform and Bacillus, Pseudomonas, and Streptomyces. Among them, the most potent strains, Bacillus freudenreichii NRS-137KH20B and Brevibacterium albidum NRS-130KH20B, could perform the resolution of more than 30 g of dl-HMPA per liter within 4 to 5 days of fermentation. Optically pure l- and d-HMPA enantiomers were obtained in more than 80% theoretical yield, whereas the transformed enantiomer was almost quantitatively recovered as 2-oxo-4-methyl-pentanoic acid in the culture broth. The enantiospecific dehydrogenation responsible for this resolution reaction had a rather wide substrate specificity on straight or branched aliphatic C(4) to C(16) 2-hydroxy acids, exhibiting the optima at chain lengths of either C(7) or C(5), although the enantiospecificity was not changed by chain length. The process was thus successfully extended to the preparation of optically pure C(5) to C(9) 2-hydroxy acids.     

Monoclonal antibody reactive with the human epidermal-growth-factor receptor recognizes the blood-group-A antigen

The hybridoma antibody TL5, which precipitates the EGF receptor from the human epidermoid carcinoma cell line A431, has been shown to recognize the blood-group-A carbohydrate structure. This conclusion has been reached from studies of (a) the binding of the antibody to glycoproteins and haemagglutination of erythrocytes with known blood-group-antigen activities and (b) the inhibition of binding of the antibody to a radiolabelled blood-group-A-active glycoprotein by structurally defined oligosaccharides.     

Correspondence of homologies in amino acid sequence and tertiary structure of protein molecules

According to the method developed previously (Kubota, Y., Takahashi, S., Nishikawa, K. and Ooi, T. (1981) J. Theor, Biol. 91, 347-361), homology among proteins may be estimated quantitatively. We extended the method to investigate the relationship of an amino acid sequence to its teritary structure and identify homologous segments which have homologous native conformations in proteins. First, we selected proper indices for the computation of correlation coefficients from 32 properties inherent to amino acids, such as hydrophobicity. The arithmetic average of correlation coefficients using six indices gave rise to a good correlation for the CD- and EF-hand regions (Ca2+ binding sites) in carp parvalbumin, but poor ones for other segments. We then applied the method to homologous proteins, the three-dimensional structures of which are known: horse hemoglobin alpha-chain and beta-chain; cytochrome c and c2; serine proteases, chymotrypsinogen and elastase; alpha-lytic protease and protease A from prokaryotic organisms. The results show that the sequence homology estimated by the present method has a good correspondence to the homology in three-dimensional structures and therefore the method is promising for the identification of important sites in sequences which have similar native conformations. For an example of the application of the method, two sequences of human interferon, one from fibroblast and the other from leukocyte, are compared, suggesting functional sites in the molecule.