Relatedness structure detected by microsatellite analysis and attempted pedigree reconstruction in an endangered marsupial, the northern hairy-nosed wombat Lasiorhinus krefftii

The northern hairy-nosed (NHN) wombat is perhaps Australia's most endangered mammal. Being fossorial and nocturnal as well as rare, NHN wombats are difficult to observe in the wild. Hence little is known of their social biology, such as their mating and dispersal systems. A hypothesis has been advanced that adult females of the species disperse post-breeding, leaving their young to inhabit the natal burrow. Female-biased dispersal is expected to result in higher relatedness amongst males in a burrow cluster than amongst females in a burrow cluster. The usefulness of a panel of microsatellite markers in estimating the relatedness structure, and in reconstructing pedigrees for, the sole known population of NHN wombats was assessed. Microsatellite genotypes at eight or nine loci were obtained from 58 of the 85 known individuals, and used to estimate pairwise individual relatedness using Queller & Goodnight's (1989) RELATEDNESS 4.2. Our analysis gave the unexpected result that both males and females were significantly more closely related to their same-sex burrow cluster mates than random, while opposite-sex animals sharing burrows were only slightly (nonsignificantly) more related than random. This raises the possibility of dispersal patterns which lead to association of same-sex relatives. The observed relatedness structure is not expected to make likely a high incidence of inbred matings, as close relatives of the opposite sex are not significantly associated in space. Parentage analysis was attempted using genetic exclusion and LOD likelihood ratios, but proved difficult because of low genetic variation, incomplete sampling of potential parents, and paucity of ecological data such as known mother/offspring pairs and ages of individuals.     

Visualization of detyrosination along single microtubules reveals novel mechanisms of assembly during cytoskeletal duplication in trypanosomes

We have been able to use immunogold labeling with monoclonal antibodies specific for tyrosinated alpha-tubulin to define new microtubule assembly within the T. brucei pellicular cytoskeleton. Using this approach, we have been able to visualize and define the detyrosination gradient along single microtubules in vivo. New microtubules are seen to invade the cytoskeletal array early in the cell cycle between old microtubules. In post-mitotic cells, a unique form of microtubule assembly occurs, with very short microtubules being intercalated in the array. We propose that these are nucleated by lateral interaction with the MAPs on existing adjacent microtubules. This construction pattern suggests a templated morphogenesis of microtubule arrays with semi-conservative distribution to the daughter cells.     

The treatment of cancer patients with human lymphoblastoid interferon

Summary Highly purified human lymphoblastoid interferon (HLBI) derived from virus-stimulated Namalwa cells was administered by 6-h IV infusion or IM injection to 40 patients with a variety of disseminated malignancies refractory to standard therapy. Each patient received doses escalating from 0.1 to 50×10⁶ U for up to 5 weeks. Extensive monitoring for clinical effect, toxicity, and pharmacokinetics has revealed higher peak serum interferon levels and somewhat more pronounced systemic toxicity for the IV than for the IM route of administration. Objective evidence of tumor regression was observed in two patients receiving HLBI IV.     

Identification of the C-terminally alpha-amidated amino acid in peptides by high-performance liquid chromatography

A sensitive method for the rapid identification of the C-terminally amidated amino acid in peptides is described. Peptides containing the alpha-amide group at the C-terminus were cleaved with endopeptidases. The fragments released (oligopeptides, amino acids and the C-terminally amidated residue) are coupled to phenylisothiocyanate. The phenylthiocarbamoyl derivative of the amino acid alpha-amide is selectively extracted from the mixture by alkaline butyl acetate and identified by a high-performance liquid chromatography system that enables rapid and complete separation of the derivatives of 17 amino acid amides at a detection limit of 20-50 pmol. The C-terminal alpha-amides of neurokinin-A (Met-NH2), mammalian secretin (Val-NH2), pancreatic polypeptide (Tyr-NH2) and peptide HI (Ile-NH2) are unequivocally determined at a level of 0.5-2 nmol per peptide. This method was used to characterize a crude peptide fraction prepared from porcine brain. Cholecystokinin-58 was identified in this fraction by detection of phenylthiocarbamoyl-phenylalaninamide. The method is suitable for the identification of the C-terminal alpha-amidated residue of purified peptides, but can also be used as a screening strategy to isolate from complex biological extracts novel peptides containing an alpha-amidated amino acid at the C-terminus.     

Delineation of a Particulate Thyrotropin-Releasing Hormone-Degrading Enzyme in Rat Brain by the Use of Specific Inhibitors of Prolyl Endopeptidase and Pyroglutamyl Peptide Hydrolase

The degradation of thyrotropin-releasing hormone in rat brain homogenates was studied in the presence of N-benzyloxycarbonyl-prolyl-prolinal and pyroglutamyl diazomethyl ketone, specific and potent active-site-directed inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase, respectively. Substantial TRH degradation was observed, suggesting the presence of another thyrotropin-releasing hormone-degrading enzyme(s). Reports of a thyrotropin-releasing hormone-degrading enzyme with narrow specificity that cleaves the pGlu-His bond of this tripeptide led us to develop a coupled assay using pGlu-His-Pro-2NA as the substrate to measure this activity. Cleavage of the pGlu-His bond of this substrate under conditions in which pyroglutamyl peptide hydrolase is not expressed occurred in the particulate fraction of a rat brain homogenate. This particulate pyroglutamyl-peptide cleaving enzyme was not inhibited by pyroglutamyl diazomethyl ketone but was inhibited by metal chelators such as EDTA and o-phenanthroline. The particulate pyroglutamyl-peptide cleaving enzyme was found predominantly in the brain. Activity in brain regions varied widely with highest levels present in cortex and hippocampus and very low levels in pituitary. The data suggest that degradation of thyrotropin-releasing hormone by the particulate fraction of a brain homogenate is catalyzed mainly by an enzyme that cleaves the pGlu-His bond of thyrotropin-releasing hormone but is distinct from pyroglutamyl peptide hydrolase.     

A multicatalytical protease complex from pituitary that forms enkephalin and enkephalin containing peptides

The pituitary contains a high molecular weight (M.W.～700,000) neutral endopeptidase complex that behaves in solution as a single protein, but on polyacrylamide gel electrophoresis under dissociating conditions shows the presence of five components with molecular weights of 24,000 to 28,000. The complex exhibits trypsinlike, chymotrypsinlike and peptidylglutamyl-peptide bond hydrolysing activities. Experiments indicate that each of these activities is associated with a separate component of the complex. The chymotrypsinlike and trypsinlike activities efficiently generate Leu-enkephalin, Leu-enkephalin-Arg⁶ and Leu-enkephalin-Arg⁶-Phe⁷ from a single synthetic precursor. Selective inhibition of the chymotrypsinlike activity enhances the trypsinlike activity and alters the relative proportion of opioid peptides formed.     

The association between testosterone, sexual arousal, and selective attention for erotic stimuli in men

Twenty-six, eugonadal men between the ages of 18 and 27 participated in this investigation of the relationship between sexual arousal, testosterone (T) levels, and the processing of sexual information. At each of the two test sessions, subjects gave a blood sample, listened to an erotic or neutral priming audiotape, and completed a dichotic listening task designed to assess selective attention for sexual stimuli. Subjective levels of sexual arousal to the audiotape and sexual attitudes and sexual experience were assessed by self-report measures. Contrary to our hypothesis, there was no relationship between levels of free T and the strength of the selective attention bias for sexual stimuli. However, men who were more distracted by the sexual material in the task reported higher levels of sexual arousal to erotic imagery than men who were less distracted by the sexual material in the task (P less than 0.01). Moreover, men who were more sexually aroused by the erotic audiotape made significantly less shadowing errors in the erotic prime condition then they did during the neutral prime condition (P less than 0.05). There was a negative association between T and shadowing errors in the erotic prime condition (P less than 0.05). These results suggest that lower thresholds for sexual arousal are associated with a greater bias to attend to sexual information and that T may have effects on cognitive-motivational aspects of sexual behavior by enhancing attention to relevant stimuli.     

The characterization of somatomedin A, isolated by microcomputer-controlled chromatography, reveals an apparent identity to insulin-like growth factor 1

The polypeptide termed somatomedin A (SMA) was isolated from outdated human plasma by a new purification procedure, not using acid ethanol extraction. Fractions containing SMA were monitored by a placenta radioreceptorassay and a radioimmunoassay for SMA. The purification method utilized a microcomputer-controlled chromatography system, yielding both SMA (identified as insulin-like growth factor 1 (IGF-1) or a deamidated derivative) and insulin-like growth factor 2 (IGF-2). The first step of CM-Affigel blue adsorbed at neutral pH the majority of somatomedins detectable by the radioreceptorassay for SMA. Exclusion chromatography on Sephadex G-50 in 0.1 M acetic acid separated this active material from albumin and NaCl. Separation between SMA and IGF-2 was achieved on two different cation-exchange columns, but not in the final high-performance liquid chromatography step. The isoelectric points, determined by chromatofocusing, were 8.0 for SMA and 6.2 for IGF-2. The amino acid compositions of the two isolated peptides were indistinguishable from the known compositions of IGF-1 and IGF-2. Sequence analysis up to position 39 of the peptide with a pI of 6.2 also proved identity with IGF-2 for all positions examined. The peptide with a pI of 8.0, corresponding to SMA, was degraded directly as well as after CNBr cleavage. The results show that it is identical to IGF-1, with the possible exception of acid/amide assignment, which could correspond to a deamidation. If occurring in the native preparation before analysis, it could explain the chromatographic properties and isoelectric point of SMA versus IGF-1 isolated by other techniques.