Species-specific diversity among simian immunodeficiency viruses from African green monkeys

The prevalence, natural history, and genetic characteristics of simian immunodeficiency virus (SIV) infections in most feral African monkey species are presently unknown, yet this information is essential to elucidate their origin and relationship to other simian and human immunodeficiency viruses. In this study, a combination of classical and molecular approaches were used to identify and characterize SIV isolates from West African green monkeys (Cercopithecus sabaeus) (SIVagm isolates). Four SIVagm viruses from wild-caught West African green monkeys were isolated and analyzed biologically and molecularly. Amplification, cloning, and sequencing of a 279-bp polymerase fragment directly from uncultured peripheral blood mononuclear cells was facilitated by the use of nested polymerase chain reaction. The results indicated that West African green monkeys are naturally infected with SIVs which are closely related to East African SIVagm isolates. However, structural, antigenic, and genetic differences were observed which strongly suggest that the West African green monkey viruses comprise a phylogenetically distinct subgroup of SIVagm. These findings support our previous hypothesis that SIVagm viruses may have evolved and diverged coincident with the evolution and divergence of their African green monkey host. In addition, this study describes a polymerase chain reaction-based approach that allows the identification and molecular analysis of divergent SIV strains directly from primary monkey tissue. This approach, which does not depend on virus isolation methods, should facilitate future studies aimed at elucidating the origins and natural history of SIVs in feral African green monkey populations.     

Cell-cycle-dependent changes in labelling of specific phosphoproteins by the monoclonal antibody MPM-2 in plant cells

The MPM-2 antibody, which recognizes a mitosis-specific phosphorylated epitope, has been used to study cell-cycle-related proteins in partially synchronized cell suspension cultures and root meristem cells. Immunofluorescence revealed that the epitope recognized by MPM-2 is located in the nucleus in interphase cells. In mitotic cells, MPM-2 labels the prophase nucleus, the spindle and some cytoplasmic components. The relative amount of the epitope changes significantly during the cell cycle. Labelling is lowest in G1 and S-phase cells and increases 2–3-fold during G2. Prophase and metaphase show four to five times the labelling of G1 cells. Labelling decreases rapidly after metaphase and is at a very low level by telophase. One- (1-D) and two-dimensional (2-D) immunoblots showed that MPM-2 labels a family of phosphorylated proteins. The labelling shows significant cell cycle dependence. Subfractionation shows at least one of these proteins is a component of the detergent-insoluble cytoskeleton cell fraction. This component is resolved on 2-D immunoblots to two to three spots of slightly different isoelectric point, possibly charge isomers, at a relative molecular mass of approximately 65 kDa. The same spots are labelled by IFA, an antibody against intermediate filament proteins. Another three of the spots at lower relative molecular mass are labelled on 2-D immunoblots of the nuclear matrix fraction.     

Reactive synaptogenesis following degeneration of photoreceptor terminals in the fly's optic lobe: a quantitative electron microscopic study.

Following photo-ablation of receptor cells in the retina of the housefly's compound eye, their synaptic terminals degenerate with a timecourse which we have followed over 8 d. Degeneration deprives the monopolar interneurons in the first optic neuropile, the lamina, of their main synaptic input. Simultaneously it deprives one monopolar interneuron (L2) of one of its synaptic targets, as L2 makes numerous feedback synaptic contacts at which it is pre-synaptic upon receptor terminals. Because the feedback synapses are dyadic, input still remains available to the second element post-synaptic at the dyad, which does not degenerate. This element is T1, a higher-order interneuron from the next most proximal neuropile (the medulla). Some of the original feedback synaptic sites soon disappear as a consequence of the photo-ablation, but their loss is partly offset by the production of new synaptic contacts. The new pre-synaptic ribbons resemble those at the original sites except for being smaller. The sites are, moreover, monadic, with T1 now the sole post-synaptic partner. These results show that interneurons in the fly's lamina retain a dynamic capacity for synaptogenesis throughout much of adult life, normally a few weeks in Musca, and that during this synaptogenesis they re-enact the same cell preferences expressed earlier in development.     

Dictyostelium discoideum gene family contains a long internal amino acid repeat

Two different cDNA clones denoted pTO270-6 and pTO270-11 represent two mRNAs that are developmentally regulated during spore germination in Dictyostelium discoideum. The respective mRNAs are found only during early germination and are not present in other stages of growth or multicellular development. Four different genomic clones that hybridize to sequences that are common to both of the 270 cDNA clones were isolated from Dictyostelium libraries and sequenced. Two are the genes for the two cDNAs, and the other two represent genes that do not seem to be transcribed. All four genomic sequences possess a very unusual internal feature in the deduced protein sequences composed of a monotonous repeat of the tetrapeptide threonine-glutamic acid-threonine-proline. The other portions of the proteins have no homology among themselves. The deduced protein corresponding to the 270-6 gene is very similar to avocado (Persea americana) cellulase. Since cellulose in the spore wall has to be digested during spore germination this suggests that this protein may function as an endo-(1,4)-beta-D-glucanase during germination.     

Binding protein dependent transport of C4-dicarboxylates in Rhodobacter capsulatus

The characteristics of malate transport into aerobically grown cells of the purple photosynthetic bacterium Rhodobacter capsulatus were determined. A single transport system was distinguished kinetically which displayed a K_t value of 2.9 ± 1.2 μM and V_max of 43 ± 6 nmol · min^-1 · mg^-1 protein. Competition experiments indicated that the metabolically related C4-dicarboxylates succinate and fumarate are also transported by this system. Malate uptake was sensitive to osmotic shock and evidence from the binding of radiolabelled malate and succinate to periplasmic protein fractions indicated that transport is mediated by a dicarboxylate binding protein. The activity of the transport system was studied as a function of external and internal pH and it was found that a marked activation of uptake occurred at intracellular pH values greater than 7. The use of a high affinity binding protein dependent system to transport a major carbon and energy source suggests that Rhodobacter capsulatus would be capable of obtaining growth sustaining quantities of C4-dicarboxylates even if these were present at very low concentrations in the environment.     

Comparison of transforming growth factor β and a human tumour-derived suppressor factor

Summary Serum-free supernatants from the human melanoma cell line G361 contain a factor that can potently suppress the generation of tumouricidal lymphokine-activated killer (LAK) cells in response to interleukin-2. To characterise the suppressive factor of tumour origin we performed a number of physicochemical and functional comparisons with another immunosuppressive protein, transforming growth factor (TGF). The bioactivity of tumour-derived suppressor factor (TDSF), assayed by suppression of LAK cell generation, was unaffected by a reducing agent but lost when denatured with a chaotropic agent. In contrast, TGF was inactivated by reduction but not denaturation. TDSF lost bioactivity in conditions of pH less than 4, whereas TGF showed no loss of activity. The TDSF moiety has an estimated pI of 4.3 and a molecular mass of 69–87 kDa. This differs from published values of pI 9.5, and 25 kDa molecular mass for TGF. Anti-TGF antiserum reversed the effects of TGF but did not affect the suppression of LAK cell generation caused by TDSF. These findings provide compelling evidence that the TDSF moiety is not TGF, and may be a novel immunoregulatory cytokine.     

The functional cell surface glycoprotein CD9 is distinguished by being the major fatty acid acylated and a major iodinated cell-surface component of the human platelet

We showed that a 22 kDa protein (which comigrated with the leukocyte differentiation antigen CD9 as determined by immunoblotting with the platelet-activating mAb 50H.19) is a major iodinated component of the platelet surface. The iodinated protein was identified as CD9 by limited proteolysis analysis. The major acylated protein in platelets incubated with [3H]palmitic acid also had a mobility of 22 kDa. The radiolabelled fatty acid in CD9 appears to be ester bonded, as it is removed by treatment with hydroxylamine. Non-enzymatic ligation of the fatty acid is not involved. Since platelets lack protein synthetic capacity, the palmitolation of a surface protein indicates the existence of a plasma-membrane located transacylase which functions independently of protein synthesis. Limited proteolysis analysis of the palmitylated protein obtained by immunoprecipitation with mAb 50H.19 confirmed its identity as CD9. An additional novel minor component of 27 kDa was detected in platelets by immunoprecipitation of 125I-surface-labelled, or [3H]palmitic acid-labelled protein, and by immunoblotting with mAb 50H.19. The analogous cleavage patterns obtained by the limited proteolysis analysis of the 22, 24 and 27 kDa glycoproteins suggest that they may be differently modified variants of a single polypeptide.     

The expression of human glycerol-3-phosphate dehydrogenase in human/rodent somatic-cell hybrids

Our previous studies using rodent/human somatic-cell hybrids suggested that the expression of human mitochondrial glycerol-3-phosphate dehydrogenase (GPDM) is dependent on the presence of human mitochondria. This has now been tested directly by analysis of GPDM activity in a series of nine hybrid-cell lines, four segregating human chromosomes and five losing rodent chromosomes (reverse segregants). The chromosome composition of the hybrids was deduced from analysis of biochemical markers and examination of G- and G11-banded metaphase spreads and the mitochondrial content was determined by Southern blot analysis, using cloned mouse and human mtDNA sequences as probes. We found that the mtDNA species present in these hybrids correlated exactly with the pattern of chromosome segregation such that the conventional hybrids contained rodent mtDNA and the reverse segregants human mtDNA. However, the pattern of GPDM expression was not directly correlated with the species of chromosomes or mitochondria present: all the hybrids showed strong rodem GPDM activity and two from each class of hybrid also showed human GPDM activity but the other hybrids were negative for human GPDM. We conclude that rodent GPDM readily integrates into human mitochondria, that the expression of rodent GPDM is not dependent on the presence of rodent mitochondria, and that GPDM is not coded by mtDNA. Human GPDM either is not capable of being inserted into the rodent mitochondrial membrane or is regulated in some way in the hybrid cells by an unidentified rodent factor.     

Teleocidin and phorbol ester tumor promoters exert similar mitogenic effects on human lymphocytes

The tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) affects a wide variety of cellular functions via its binding to protein kinase C (PKC). The TPA molecule contains a diacylglycerol (DAG)-like structure, which may explain its ability to mimic DAG in PKC activation. Teleocidin (TCD) is a different tumor promoter which can compete with TPA in binding to its cell surface receptors even though structurally unrelated to TPA or DAG. Since TCD may use an additional receptor system and/or be distinguished from TPA in its effect on cells, we compared the effects of TPA and TCD on human peripheral blood lymphocytes (PBL). Both tumor promoters preferentially enhanced cell proliferation of sheep erythrocyte-rosetted lymphocytes, which were enriched for T cells. Additionally, TPA and TCD both induced a high density of cell surface receptors for interleukin 2 (IL2) and transferrin, but not synthesis or production of IL2. However, either of the tumor promoters synergized with T cell mitogens to induce high level IL2 production by PBL. In dose response and kinetic studies, matching concentrations of TPA and TCD induced similar effects in PBL. The results thus demonstrate that TPA and TCD are alike in mitogenic capacity, and suggest that structural similarity between the tumor promoter and DAG, the physiological activator of PKC, is not an essential property for promoting tumors or affecting a wide variety of cellular functions.