An ancient common origin of aboriginal Australians and New Guinea highlanders is supported by alpha-globin haplotype analysis.

The origins of aboriginal Australians and their relationship with New Guineans and neighboring Southeast Asians remains controversial. We have studied the alpha-globin haplotype composition of an aboriginal tribe from central Australia, to address some of the ambiguities of previous studies. Australians have a haplotype repertoire that is shared with New Guinea highlanders, a fact that strongly supports a common origin of these two populations. Further, Australians and New Guinea highlanders have a different set of alpha haplotypes from Southeast Asians and a lower genetic diversity. This, coupled with the presence of many locally specific central Australian haplotypes, suggests that much of the original diversity was lost in a population bottleneck prior to or during the early colonization of Sahul and that subsequent recovery of diversity has been accompanied by the generation of new haplotypes. These conclusions contrast with some previous genetic studies suggesting links between Australians, coastal New Guineans, and present-day Southeast Asians. Much of this discrepancy appears to be due to more recent Southeast Asian admixture on the north coast of Australia.     

Identification of the 5-Hydroxytryptamine2C Receptor as a 60-kDa N-Glycosylated Protein in Choroid Plexus and Hippocampus

Abstract: The rat 5-hydroxytryptamine_2C (5-HT_2C) receptor was identified as N -glycosylated polypeptide of 60-kDa apparent molecular mass using antibodies against its putative third and fourth (C-terminal) cytoplasmic domain. To show that the polypeptides detected on western blots and by immunoprecipitation represent the 5-HT_2C receptor, binding studies of the 5-HT_2C ligand [³H]-mesulergine to immunoprecipitates from extracts of pig choroid plexus were performed. We demonstrate the presence of a signal sequence that was cleaved off during membrane insertion resulting in a 38-kDa polypeptide. During further maturation, the receptor was N -glycosylated at two sites via a 48-kDa intermediate. This intermediate was far more abundant in choroid plexus than in hippocampus and may represent an intracellular receptor reserve. After transfection of 5-HT_2C cDNAs into cultured cells, polypeptides were observed that differed from the ones found in vivo due to abnormal N -glycosylation and possibly other alterations depending on the system used. Thus the 5-HT_2C receptor expressed in cell lines may also differ in function from the receptor in its native tissue.     

Cloning of theHelicobacter pylori recA gene and functional characterization of its product

The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned arecA homolog fromHelicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to theCampylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus σ⁷⁰ promoter sequence was found upstream ofrec A. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream ofrecA. Compared to the wild-type strains, isogenicH. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinantH. pylori RecA protein produced inEscherichia coli strain GC6 (recA ^?) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein inH. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of theH. pylori mutant using the clonedrecA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA inH. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced inE. coli, giving rise to a smaller but inactive protein.     

Glutamine aspartate transaminase modified at cysteine 390 with enriched carbon-13 cyanide.

The supernatant isozyme of pig heart glutamate aspartate transaminase (EC 2.6.1.1.) has been modified “syncatalytically” at cysteine 390 with 90% enriched [¹³C] cyanide. The C-13 NMR spectra have been recorded under conditions of pH changes and addition of substrates and substrate analogs when the probe environment may be affected. Under these conditions the C-13 probe revealed no changes in the region surrounding cysteine 390.     

Purification and characterization of a sialidase fromClostridium chauvoei NC08596

The sialidase secreted byClostridium chauvoei NC08596 was purified to apparent homogeneity by ion-exchange chromatography, gel filtration, hydrophobic interaction-chromatography, FPLC ion-exchange chromatography, and FPLC gel filtration. The enzyme was enriched about 10 200-fold, reaching a final specific activity of 24.4 U mg^–1. It has a relatively high molecular mass of 300 kDa and consists of two subunits each of 150 kDa. The cations Mn²⁺, Mg²⁺, and Ca²⁺ and bovine serum albumin have a positive effect on the sialidase activity, while Hg²⁺, Cu²⁺, and Zn²⁺, chelating agents and salt decrease enzyme activity. The substrate specificity, kinetic data, and pH optimum of the enzyme are similar to those of other bacterial sialidases.Abbreviations FPLC fast protein liquid chromatography - NCTC National Collection of Type Cultures - ATCC American Type Culture Collection - MU-Neu5Ac 4-methylumbelliferyl--d-N-acetylneuraminic acid - buffer A 0.02m piperazine, 0.01m CaCl₂, pH 5.5 - buffer B 0.02m piperazine, 0.01m CaCl₂, 1.0m NaCl, pH 5.5 - buffer C 0.1m sodium acetate, 0.01m CaCl₂, pH 5.5 - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - Neu5Ac N-acetylneuraminic acid - BSM bovine submandibular gland mucin - GD1a IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer - GM1 II³Neu5Ac-GgOse₄Cer - MU-Neu4,5Ac₂ 4-methylumbelliferyl--d-N-acetyl-4-O-acetylneuraminic acid - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - EDTA ethylenediamine tetraacetic acid - EGTA ethylene glycol bis(2-aminoethyl-ethen)-N,N,N,N-tetraacetic acid - BSA bovine serum albumin - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - IEF isoelectric focusing - IEP isoelectric point     

Production, purification and characterization of hemolysins from Listeria ivanovii and Listeria monocytogenes Sv4b

In culture supernatants of both Listeria ivanovii and Listeria monocytogenes Sv4b, for the first time a hemolysin of molecular weight 58 kDa was identified, which had all the characteristics of an SH-activated cytolysin, and which was therefore identified as listeriolysin O (LLO). In the case of L. ivanovii a second major supernatant protein of molecular weight 24 kDa co-purified with LLO. However, the function of this protein has to be determined. In culture supernatants of L. ivanovii a sphingomyelinase and a lecithinase activity could be detected, both enzymatic activities together contributing to the pronounced hemolysis caused by L. ivanovii. The N-terminal amino acid sequences of LLO and the 24 kDa from L. ivanovii are shown.     

Genetic deletion of apolipoprotein A-I increases airway hyperresponsiveness,inflammation, and collagen deposition in the lung

The relationship between high-density lipoprotein and pulmonary function is unclear. To determine mechanistic relationships we investigated the effects of genetic deletion of apolipoprotein A-I (apoA-I) on plasma lipids, paraoxonase (PON1), pro-inflammatory HDL (p-HDL), vasodilatation, airway hyperresponsiveness and pulmonary oxidative stress, and inflammation. ApoA-I null (apoA-I^−/−) mice had reduced total and HDL cholesterol but increased pro-inflammatory HDL compared with C57BL/6J mice. Although PON1 protein was increased in apoA-I^−/− mice, PON1 activity was decreased. ApoA-I deficiency did not alter vasodilatation of facialis arteries, but it did alter relaxation responses of pulmonary arteries. Central airway resistance was unaltered. However, airway resistance mediated by tissue dampening and elastance were increased in apoA-I^−/− mice, a finding also confirmed by positive end-expiratory pressure (PEEP) studies. Inflammatory cells, collagen deposition, 3-nitrotyrosine, and 4-hydroxy-2-nonenal were increased in apoA-I^−/− lungs but not oxidized phospholipids. Colocalization of 4-hydroxy-2-nonenal with transforming growth factor β-1 (TGFβ-1 was increased in apoA-I^−/− lungs. Xanthine oxidase, myeloperoxidase and endothelial nitric oxide synthase were increased in apoA-I^−/− lungs. Dichlorodihydrofluorescein-detectable oxidants were increased in bronchoalveolar lavage fluid (BALF) in apoA-I^−/− mice. In contrast, BALF nitrite+nitrate levels were decreased in apoA-I^−/− mice. These data demonstrate that apoA-I plays important roles in limiting pulmonary inflammation and oxidative stress, which if not prevented, will decrease pulmonary artery vasodilatation and increase airway hyperresponsiveness.     

Two new species of oribatid mites of the genus Truncozetes (Acari,Oribatida, Epactozetidae) from?Ecuador

Two new oribatid mite species of the genus Truncozetes (Oribatida, Epactozetidae), Truncozetes ecuadoriensis sp. n. and Truncozetes monodactylus sp. n., are described from the Ecuadorian soils. The morphology of the gnathosoma and the legs is presented in detail for the first time for the species of Truncozetes. An identification key to all known species of the family Epactozetidae is given.     

MDHM, a macrophage-activating product of Mycoplasma fermentans, stimulates murine macrophages to synthesize nitric oxide and become tumoricidal

Abstract In continuation of previous work on macrophage activation by a Mycoplasma fermentans -derived product, originally named “mycoplasma-derived high mol. wt. material” (MDHM), we have investigated whether MDHM was capable of inducing synthesis of the reactive nitrogen intermediate nitric oxide (NO), thus rendering macrophages cytocidal. Mycoplasmas were first delipidated with acetone, and MDHM activity was then extracted with 50 mM 1-O-octyl-β- d -glucopyranoside to yield a particularly active new preparation of MDHM which we have named MDHM-D (D for detergent). In combination with IFN-γ, MDHM-D activated macrophages to produce reactive nitrogen intermediates and kill P815 mastocytoma cells in co-culture. P815 target cells were chosen because they are TNF-resistant. Macrophages from the LPS-low responder strain C3H/HeJ were used to minimize interference from possible LPS contamination. MDHM-D activity in this system was strictly IFN-γ-dependent. In the presence of 25 U/ml IFN-γ MDHM-D gave a half maximal response at a dilution of 1/100 000, showing a parallel concentration dependency for nitrite production and cytocidal activity.