Host-driven diversification of gall-inducing Acacia thrips and the aridification of Australia

Background  

Insects that feed on plants contribute greatly to the generation of biodiversity. Hypotheses explaining rate increases in phytophagous insect diversification and mechanisms driving speciation in such specialists remain vexing despite considerable attention. The proliferation of plant-feeding insects and their hosts are expected to broadly parallel one another where climate change over geological timescales imposes consequences for the diversification of flora and fauna via habitat modification. This work uses a phylogenetic approach to investigate the premise that the aridification of Australia, and subsequent expansion and modification of arid-adapted host flora, has implications for the diversification of insects that specialise on them.     

Isolation and characterization of a benzoylformate decarboxylase and a NAD/NADP-dependent benzaldehyde dehydrogenase involved in d-phenylglycine metabolism in Pseudomonas stutzeri ST-201

Following induction with d-phenylglycine both d-phenylglycine aminotransferase activity and benzoylformate decarboxylase activity were observed in cultures of Pseudomonas stutzeri ST-201. Induction with benzoylformate, on the other hand, induced only benzoylformate decarboxylase activity. Purification of the benzoylformate decarboxylase, followed by N-terminal sequencing, enabled the design of probes for hybridization with P. stutzeri ST-201 genomic DNA libraries. Sequencing of two overlapping genomic DNA restriction fragments revealed two open reading frames which were denoted dpgB and dpgC. Sequence alignments suggested that the genes encoded a thiamin-diphosphate-dependent decarboxylase and an aldehyde dehydrogenase, respectively. Both genes were isolated and expressed in Escherichia coli. The dpgB gene product was confirmed as a benzoylformate decarboxylase while the dpgC gene product was characterized as a NAD⁺/NADP⁺-dependent benzaldehyde dehydrogenase. In keeping with their high sequence identities (both greater than 85%) the kinetic properties of the two enzymes were similar to those of the homologous enzymes in the mandelate pathway of Pseudomonas putida ATCC 12633. However, Pseudomonas stutzeri ST-201 was unable to grow on either isomer of mandelate, and sequencing indicated that the dpgB gene did not form part of an operon. Thus it appears that the two enzymes form part of a d-phenylglycine, rather than mandelate, degrading pathway.     

The actin cytoskeleton regulates exocytosis of all neutrophil granule subsets

A comprehensive analysis of the role of the actin cytoskeleton in exocytosis of the four different neutrophil granule subsets had not been performed previously. Immunoblot analysis showed that, compared with plasma membrane, there was less actin associated with secretory vesicles (SV, 75%), gelatinase granules (GG, 40%), specific granules (SG, 10%), and azurophil granules (AG, 5%). Exocytosis of SV, SG, and AG was measured as increased plasma membrane expression of CD35, CD66b, and CD63, respectively, with flow cytometry, and GG exocytosis was measured as gelatinase release with an ELISA. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated exocytosis of SV, GG, and SG with an ED₅₀ of 15, 31, and 28 nM, respectively, with maximal response at 10^–7 M FMLP by 5 min, while no exocytosis of AG was detected. Disruption of the actin cytoskeleton by latrunculin A and cytochalasin D induced a decrease in FMLP-stimulated CD35 expression after an initial increase. Both drugs enhanced the rate and extent of FMLP-stimulated GG, SG, and AG exocytosis, while the EC₅₀ for FMLP was not altered. We conclude that the actin cytoskeleton controls access of neutrophil granules to the plasma membrane, thereby limiting the rate and extent of exocytosis of all granule subsets. Differential association of actin with the four granule subsets was not associated with graded exocytosis. human; cell activation     

Phylogenetic clustering increases with succession for lianas in a Chinese tropical montane rain forest

Previous research found that phylogenetic clustering increased with disturbance for tropical trees, suggesting that community assembly is mainly influenced by abiotic factors during early succession. Lianas are an important additional component of tropical forests, but their phylogenetic community structure has never been investigated. Unlike tropical trees, liana abundance is often high in disturbed forests and diversity can peak in old secondary forest. Therefore, phylogenetic structure along a disturbance gradient might also differ from tropical tree communities. Here we determined phylogenetic community structure of lianas along a disturbance gradient in a tropical montane forest in China, using the net relatedness index (NRI) from 100 equivalent phylogenies with varying branch length that were constructed using DNA‐barcode sequences. Three additional phylogenetic indices were also considered for comparison. When NRI was used as index phylogenetic clustering of liana communities decreased with decreasing tree basal area, suggesting that liana competitive interactions dominate during early succession, which is in contrast to the pattern reported for trees. Liana communities in mature forests, on the other hand, were phylogenetic clustered, which could be caused by dispersal limitation and/or environmental filtering. The three additional phylogenetic indices identified different, sometimes contradicting predictors of phylogenetic community structure, indicating that caution is needed when generalizing interpretations of studies based on a single phylogenetic community structure index. Our study provides a more nuanced picture of non‐random assembly along disturbance gradients by focusing on a non‐tree forest component.     

Studies on the conformational properties of CP-10(42-55), the hinge region of CP-10, using circular dichroism and RP-HPLC.

The conformational properties of CP-10(42-55), a peptide corresponding to the hinge region of CP-10, were investigated using circular dichroism spectroscopy and reverse-phase high-performance liquid chromatography (RP-HPLC). The circular dichroism studies indicated that CP-10(42-55) formed considerable secondary structure in the presence of hydrophobic solution environments including 50% acetonitrile, 50% trifluoroethanol and 200 mM sodium dodecyl sulfate, which comprised a mixture of alpha-helix and beta-sheet. The effect of temperature on the conformation of CP-10(42-55) was investigated between 5 and 40 degrees C, with very small changes in the spectra being observed. RP-HPLC was then used to investigate the effect of temperature on the conformation of CP-10(42-55) in the presence of a hydrophobic surface. Using a C18-adsorbent, CP-10(42-55) exhibited a conformational transition at 25 degrees C, which was associated with an increase in the chromatographic contact area and the binding affinity of the peptide for the stationary phase. In addition, near-planar bandbroadening behaviour indicated that conformational species interconverted with rapid rate constants compared with the chromatographic time scale. These results indicated that the conformational change at 25 degrees C in the RP-HPLC system most likely corresponds to the unfolding of an alpha-helical and/or beta-sheet structure to an extended coil structure. Therefore, the strong chemotactic properties of this peptide may be attributed to its ability to form considerable secondary structure in the presence of a hydrophobic environment.     

Identification of 14-3-3zeta as a protein kinase B/Akt substrate

Protein kinase B/Akt (PKB/Akt) is a member of the ACG kinase family, which also includes protein kinase C, that phosphorylates a number of 14-3-3-binding proteins. 14-3-3 protein regulation of protein kinase C activity is modulated by 14-3-3 phosphorylation. We examined the hypothesis that PKB/Akt interacts with and phosphorylates 14-3-3zeta, leading to modulation of dimerization. By glutathione S-transferase pull-down, Akt precipitated recombinant 14-3-3zeta and endogenous 14-3-3zeta from HEK293 cell lysates. Recombinant active PKB/Akt phosphorylated recombinant 14-3-3zeta in an in vitro kinase assay. Transfection of active PKB/Akt into HEK293 cells resulted in phosphorylation of 14-3-3zeta. Based on a motif search of 14-3-3zeta, a potential PKB/Akt phosphorylation site, Ser-58, was mutated to alanine. PKB/Akt was unable to phosphorylate this mutant protein. Incubation of 14-3-3zeta with recombinant active PKB/Akt resulted in phosphorylation of 45% of the protein, as determined by a pI shift on two-dimensional electrophoresis, but 14-3-3zeta dimerization was not altered. These data indicate that PKB/Akt phosphorylates Ser-58 on 14-3-3zeta both in vitro and in intact cells. The functional relevance of this phosphorylation remains to be determined.     

IFN-gamma enhances expression of formyl peptide receptors and guanine nucleotide-binding proteins by HL-60 granulocytes.

Exposure to IFN-gamma increases the respiratory burst of polymorphonuclear leukocytes stimulated by the chemoattractant FMLP. However, the mechanism by which IFN-gamma alters the response to FMLP is unclear. We addressed the hypothesis that IFN-gamma enhances the response to FMLP by regulating the expression of elements of the formyl peptide receptor transmembrane-signaling pathway. HL-60 granulocytes were used as a model of FMLP transmembrane signaling. Formyl peptide receptor number and affinity were studied in isolated plasma membranes prepared from control HL-60 cells (CM) and cells exposed to IFN-gamma 100 U/ml for 24 h (IFN-M). Formyl peptide receptors were significantly increased on IFN-M compared with CM (1473 +/- 300 vs 3209 +/- 924). FMLP stimulates increased guanine nucleotide-binding protein (G protein) activation in IFN-M as evidenced by enhanced guanosine 5'-[gamma-thio]triphosphate binding and GTPase activity. Gi sub-unit content was increased in IFN-M as measured by pertussis toxin-catalyzed ADP-ribosylation and immunoblotting with antibodies against alpha i2 and alpha i3 G protein subunits. Guanosine 5'-[gamma-thio]triphosphate equilibrium binding demonstrated an increased number of G proteins coupled to formyl peptide receptors on IFN-M. We conclude that IFN-gamma increases expression of both formyl peptide receptors and G proteins coupled to these receptors, thereby enhancing FMLP-stimulated transmembrane signaling. Regulation of transmembrane signaling element expression may be a significant mechanism by which IFN-gamma regulates cellular functions.     

Time-dependent inactivation of human phenylethanolamine N-methyltransferase by 7-isothiocyanatotetrahydroisoquinoline

Inhibitors of phenylethanolamine N-methyltransferase [PNMT, the enzyme that catalyzes the final step in the biosynthesis of epinephrine (Epi)] may be of use in determining the role of Epi in the central nervous system. Here we describe the synthesis and characterization of 7-SCN tetrahydroisoquinoline as an affinity label for human PNMT.     

Alteration in immune complex glomerulonephritis by arachidonic acid

We employed a model of immune complex glomerulonephritis produced in mice by the daily injection of apoferritin to study the effect of treatment with arachidonic acid (AA). Apoferritin injections produced demonstrable glomerular damage by light microscopy associated with deposition of immunoglobulin along peripheral capillary loops. Treatment with AA 100μg daily resulted in significantly less glomerular damage and a shift in the location of immune complex deposition to the mesanguim. The amount of anti-apoferritin antibody was determined by hemagglutination and found to be significantly decreased in mice treated with AA. Separate studies employing this dose of AA revealed that the number of IgM antibody producing cells to SRBC was not altered by AA.