N-Terminal arginylation of proteins in explants of injured sciatic nerves and embryonic brains of rats

Posttranslational modification of proteins by arginine and lysine has been demonstrated in crude extracts of vertebrate nerves and brain but not in intact cells. In the present experiments we have exploited the fact that Arg is added posttranslationally only at the N-terminus of target proteins, to demonstrate these reactions in intact cells of sciatic nerves and embryonic brains of rats. Sciatic nerves were crushed in anaesthesized rats and 2 hrs later segments of nerve, including the site of the crush, were removed and incubated in media containing [³H]Arg. Incorporation of [³H]Arg into total proteins was analyzed by acid precipitation and the presence of label at the N-terminus was determined by a modification of the Edman degradation procedure. Approximately 25% of protein bound [³H]Arg was released from the N-terminus by the Edman reaction indicating that it was added posttranslationally rather than through protein synthesis. N-terminal labeling was not detectable in nerves not crushed prior to explant and incubation. Slices of embryonic day 20 visual cortex, when incubated under similar conditions as injured sciatic nerves, also showed approximately 25% of the protein incorporated [³H]Arg at the N-terminus, while arginylation was not detectable in adult rat brain slices. Since Lys is not added posttranslationally to the N-terminus, we have attempted to observe lysylation of proteins in intact cells by using cycloheximide (Cx) to block protein synthesis without interfering with protein modification. The posttranslational incorporation of Arg/Lys into proteins was found to be insensitive to up to 2.0 mM Cx in tissue extracts (in vitro). However, in intact cells, doses as low as 10 uM Cx completely inhibited the incorporation of [³H]Arg/Lys into proteins. One uM Cx allowed for some incorporation of [³H]Arg/Lys into protein and approximately 40% of the Cx insensitive Arg was incorporated into the N-terminal. These results show that in vivo but not in vitro, Cx can block protein modification, suggesting that either in intact cells protein modification requires protein synthesis, or that Cx has effects other than as an inhibitor of protein synthesis on cells in culture, effects that it does not have on the partially purified components of the reaction.     

First fossil evidence of samaras of Ventilago Gaertn. (Rhamnaceae) from India and its implications

The Ventilago Gaertn. (Rhamnaceae) is widely distributed in pantropical areas of Africa, Asia, and Australia. However, fossil records of this taxon are sparse, which limits understanding of the evolution and biogeographic history of the genus. In the present study, we report and describe two new fossil species of Ventilago, V. siwalika sp. nov. from the Miocene sediments of Himachal Pradesh, western Himalaya, and V. pliocenica sp. nov. from the Pliocene sediments of Jharkhand, eastern India based on single-winged samaras. Ventilago pliocenica is characterized by a prominent midvein, obtuse to sub-round apex with mucronate tip, longitudinal secondary veins extending the full length of the fruit, and reticulate nature of higher-order veins, the presence of equatorial rim, the hypanthium, and short pedicel. On the other hand, V. siwalika is characterized by a prominent midvein, obtuse to sub-round apex with mucronate tip, longitudinal secondary veins extending the full length of the fruit, and reticulate nature of higher-order veins. Our discovery represents the first unambiguous fossil record of single-winged samara of Ventilago from India and provides valuable insights into the evolution of this genus. In this paper, we also review its biogeographic history and add new information to understand its hypothetical migration route. Present and earlier records of Ventilago also suggest that this genus was a common forest element during Neogene (Miocene time) in Asia.     

Protective immunity and granulomatous inflammation is mediated in vivo by T cells reactive to epitopes common to avirulent and listeriolysin-negative mutants of Listeria monocytogenes.

The ability of several listeriolysin O-negative mutants of the EGD and NCTC 7973 strains of Listeria monocytogenes to activate specific T cell responses in vitro and in vivo was determined. T cell lines from different inbred mouse strains and derived T cell clones elicited by L. monocytogenes, strain EGD, which are able to adoptively transfer protection and granuloma formation were examined. Specificity testing revealed no differences between listeriolysin-positive and -negative strains to induce proliferation of the T cell lines and clones. Similar results were obtained when we examined CD4+ T cell-mediated granuloma formation in the livers of mice previously immunized with viable bacteria of the virulent strain. Granulomatous inflammation could be elicited by iv application of heat-killed bacteria of listeriolysin-positive and of -negative bacteria. Protective immunity to listerial infections and granulomatous inflammation therefore appears to be mediated by T cells recognizing epitopes on listerial antigens that are shared by both pathogenic and nonpathogenic Listeria strains.     

A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.

The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes is the sole listerial factor needed for recruitment of host actin filaments by intracellularly motile bacteria. Here we report that following Listeria infection the host vasodilator-stimulated phosphoprotein (VASP), a microfilament- and focal adhesion-associated substrate of both the cAMP- and cGMP-dependent protein kinases, accumulates on the surface of intracytoplasmic bacteria prior to the detection of F-actin 'clouds'. VASP remains associated with the surface of highly motile bacteria, where it is polarly located, juxtaposed between one extremity of the bacterial surface and the front of the actin comet tail. Since actin filament polymerization occurs only at the very front of the tail, VASP exhibits properties of a host protein required to promote actin polymerization. Purified VASP binds directly to the ActA polypeptide in vitro. A ligand-overlay blot using purified radiolabelled VASP enabled us to identify the ActA homologue of the related intracellular motile pathogen, Listeria ivanovii, as a protein with a molecular mass of approximately 150 kDa. VASP also associates with actin filaments recruited by another intracellularly motile bacterial pathogen, Shigella flexneri. Hence, by the simple expedient of expressing surface-bound attractor molecules, bacterial pathogens effectively harness cytoskeletal components to achieve intracellular movement.     

Correlation of DNA fragment sizes within loci in the presence of non-detectable alleles

At present most forensic databases of DNA profiling of individuals consist of DNA fragment sizes measured from Southern blot restriction fragment length polymorphism (RFLP) analysis. Statistical studies of these databases have revealed that, when fragment sizes are measured from RFLP analysis, some of the single-band patterns of individuals may actually be due to heterozygosity of alleles in which fragment size resulting from one allele remains undetected. In this work, we evaluate the effect of such allelic non-detectability on correlation of fragment sizes within individuals at a locus, and its impact on the inference of independence of fragment sizes within loci. We show that when non-detectable alleles are present in a population at a locus, positive correlations of fragment sizes are expected, which increase with the proportion of non-detectable alleles at the locus. Therefore, a non-zero positive correlation is not a proof of allelic dependence within individuals. Applications of this theory to the current forensic RFLP databases within the US show that there is virtually no evidence of significant allelic dependence within any of the loci. Therefore, the assumption that DNA fragment sizes within loci are independent is valid, and hence, the population genetic principles of computing DNA profile frequencies by multiplying binned frequencies of fragment sizes are most likely to be appropriate for forensic applications of DNA typing data.Editor's commentsThe presence of non-detectable alleles for VNTR loci has plagued the use of these highly-discriminating systems in human identification. The authors take explicit account of these alleles, and are able to show independence of the frequencies of detectable alleles. They raise the troubling issue of how to account for occasional significant results when multiple tests are performed. By invoking Bonferroni corrections, they regard all tests, even those performed on different loci, as addressing the same hypothesis—the absence of dependence at any VNTR locus.     

Collagenase of a new streptomycete: Its induction and purification

Rifampin and chloramphenicol inhibited the synthesis of collagenase of Streptomyces sp. A₈, suggesting de novo synthesis. The collagenase was induced by insoluble collagen, its macromolecular fragments, gelatin, peptone, hide powder and yeast extract. Growth as well as collagenase synthesis were dependent on substrate availability. Purification of collagenase by DEAE-cellulose chromatography resulted in approximately 25-fold increase in activity (268.6 μmol glycine equivalents min^?1 mg^?1 protein) relative to the activity of the culture filtrate (10.5 μmol glycine equivalents min^?1 mg^?1 protein).     

Evidence for supercoiled hepatitis B virus DNA in chimpanzee liver and serum Dane particles: possible implications in persistent HBV infection

In chimpanzee hepatitis B virus (HBV) carriers, the mechanism of viral persistence has been examined by analyzing viral DNA molecules in liver and serum. Chimpanzee liver DNA contained two extrachromosomal HBV DNA molecules migrating on hybridization blots at 4.0 kb and 2.3 kb. There was no evidence for integration of HBV DNA into the host genome. The extrachromosomal molecules were distinct from Dane particle DNA and were converted to linear 3.25 kb full-length double-stranded HBV DNA on digestion with Eco RI. Nucleases S1 and Bal 31 converted "2.3 kb" HBV DNA to 3.25 kb via an intermediate of "4.0 kb" apparent length. The HBV DNA molecule that migrated at 2.3 kb represents a supercoiled form I of the HBV genome, and the molecule that migrated at 4.0 kb represents a full-length "nicked," relaxed circular form II. Evidence for supercoiled HBV DNA in serum Dane particles was obtained by production of form II molecules upon digestion with nuclease S1 or Bal 31. It is proposed that most Dane particles represent interfering noninfectious virus containing partially double-stranded DNA circles and that particles containing supercoiled HBV DNA may represent infectious hepatitis B virus.     

Statistical Studies on Protein Polymorphism in Natural Populations. III. Distribution of Allele Frequencies and the Number of Alleles per Locus

With the aim of understanding the mechanism of maintenance of protein polymorphism, we have studied the properties of allele frequency distribution and the number of alleles per locus, using gene-frequency data from a wide range of organisms (mammals, birds, reptiles, amphibians, Drosophila and non-Drosophila invertebrates) in which 20 or more loci with at least 100 genes were sampled. The observed distribution of allele frequencies was U-shaped in all of the 138 populations (mostly species or subspecies) examined and generally agreed with the theoretical distribution expected under the mutation-drift hypothesis, though there was a significant excess of rare alleles (gene frequency, 0 ~ 0.05) in about a quarter of the populations. The agreement between the mutation-drift theory and observed data was quite satisfactory for the numbers of polymorphic (gene frequency, 0.05 ~ 0.95) and monomorphic (0.95 ~ 1.0) alleles.—The observed pattern of allele-frequency distribution was incompatible with the prediction from the overdominance hypothesis. The observed correlations of the numbers of rare alleles, polymorphic alleles and monomorphic alleles with heterozygosity were of the order of magnitude that was expected under the mutation-drift hypothesis. Our results did not support the view that intracistronic recombination is an important source of genetic variation. The total number of alleles per locus was positively correlated with molecular weight in most of the species examined, and the magnitude of the correlation was consistent with the theoretical prediction from mutation-drift hypothesis. The correlation between molecular weight and the number of alleles was generally higher than the correlation between molecular weight and heterozygosity, as expected.     

Oestrous and ovarian activity in a female jaguar (Panthera onca).

The mean +/- s.e.m. duration of oestrus was 12.0 +/- 1.0 days (n = 7) and the oestrous cycle length was 47.2 +/- 5.4 days (n = 6) for a captive jaguar. Ovulation did not occur spontaneously, but was induced by treatment with hCG or LH-RH.