CcpA Regulates Arginine Biosynthesis in Staphylococcus aureus through Repression of Proline Catabolism

Staphylococcus aureus is a leading cause of community-associated and nosocomial infections. Imperative to the success of S. aureus is the ability to adapt and utilize nutrients that are readily available. Genomic sequencing suggests that S. aureus has the genes required for synthesis of all twenty amino acids. However, in vitro experimentation demonstrates that staphylococci have multiple amino acid auxotrophies, including arginine. Although S. aureus possesses the highly conserved anabolic pathway that synthesizes arginine via glutamate, we demonstrate here that inactivation of ccpA facilitates the synthesis of arginine via the urea cycle utilizing proline as a substrate. Mutations within putA, rocD, arcB1, argG and argH abolished the ability of S. aureus JE2 ccpA::tetL to grow in the absence of arginine, whereas an interruption in argJBCF, arcB2, or proC had no effect. Furthermore, nuclear magnetic resonance demonstrated that JE2 ccpA::ermB produced ¹³C₅ labeled arginine when grown with ¹³C₅ proline. Taken together, these data support the conclusion that S. aureus synthesizes arginine from proline during growth on secondary carbon sources. Furthermore, although highly conserved in all sequenced S. aureus genomes, the arginine anabolic pathway (ArgJBCDFGH) is not functional under in vitro growth conditions. Finally, a mutation in argH attenuated virulence in a mouse kidney abscess model in comparison to wild type JE2 demonstrating the importance of arginine biosynthesis in vivo via the urea cycle. However, mutations in argB, argF, and putA did not attenuate virulence suggesting both the glutamate and proline pathways are active and they, or their pathway intermediates, can complement each other in vivo.     

3 beta,13-dihydroxylated C20 gibberellins from inflorescences of Rumex acetosa L

Using full scan GC-MS a wide range of gibberellins (GAs) was identified in the young inflorescences of the dioecious species Rumex acetosa L., consistent with the ubiquitous early 13-hydroxylation pathway in both male and female plants. In addition, R. acetosa is the first species in which all three 3beta,13-dihydroxylated C(20)-GAs-GA(18), GA(38) and GA(23)-have been identified in the same organism, suggesting an early 3beta,13-dihydroxylation biosynthesis pathway in this species. Authentic GA(18), GA(38) and GA(23) were synthesized and their effects and that of GA(1), a GA common to both pathways, on the time to inflorescence emergence was investigated. GA(1) accelerated the emergence of inflorescences in both male and female plants. In addition some evidence for biological activity per se of the C(20)-GA(38) was obtained.     

A bifunctional diglycosyltransferase forms the Fucalpha1,2Galbeta1,3-disaccharide on Skp1 in the cytoplasm of dictyostelium

Skp1 is a subunit of the Skp1 cullin-1 F-box protein (SCF) family of E3 ubiquitin ligases and of other regulatory complexes in the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by a pentasaccharide with the type I blood group H antigen (Fucalpha1,2Galbeta1,3GlcNAc-) at its core. Addition of the Fuc is catalyzed by FT85, a 768-amino acid protein whose fucosyltransferase activity maps to the C-terminal half of the protein. A strain whose FT85 gene is interrupted by a genetic insertion produces a truncated, GlcNAc-terminated glycan on Skp1, suggesting that FT85 may also have beta-galactosyltransferase activity. In support of this model, highly purified native and recombinant FT85 are each able to galactosylate Skp1 from FT85 mutant cells. Site-directed mutagenesis of predicted key amino acids in the N-terminal region of FT85 abolishes Skp1 beta-galactosyltransferase activity with minimal effects on the fucosyltransferase. In addition, a recombinant form of the N-terminal region exhibits beta-galactosyltransferase but not fucosyltransferase activity. Kinetic analysis of FT85 suggests that its two glycosyltransferase activities normally modify Skp1 processively but can have partial function individually. In conclusion, FT85 is a bifunctional diglycosyltransferase that appears to be designed to efficiently extend the Skp1 glycan in vivo.     

Biological activity of cytokinins derived from Ortho- and Meta-Hydroxybenzyladenine

To determine the structure-activity relationships of natural aromatic cytokinins, the activity of 6-benzylaminopurine (BAP) and its hydroxylated derivatives was compared in three bioassays based on stimulation of tobacco callus growth, retention of chlorophyll in excised wheat leaves, and dark induction of betacyanin synthesis in Amaranthus cotyledons. The aromatic cytokinins 6-(2-hydroxybenzylamino)purine (ortho-topolin) and 6-(3-hydroxybenzylamino)purine (meta-topolin), their 9-ribosides and 9-glucosides, were synthesized by the condensation of 6-chloropurine and its 9-glycosides with the appropriate hydroxybenzylamine. The activity of free bases, 9-ribosides and 9-glucosides was compared with that of BAP, trans-zeatin and their 9-glycosides. Hydroxylation of the benzyl ring in the meta position increased the activity of BAP and its riboside in tobacco callus and chlorophyll retention bioassays, whereas ortho-hydroxylation decreased the activity. In contrast, in the Amaranthus bioassay meta-hydroxylation of BAP substantially decreased its activity. Ribosylation at position 9 had no significant effect on the activity of zeatin, BAP and both topolins. The activity of 9-glucosides of all cytokinins tested was near zero. The biological activity of meta-topolin and its riboside is comparable to that of the most active isoprenoid cytokinin, zeatin, in tobacco callus growth and senescence bioassays. The results establish the existence of a family of endogenous aromatic cytokinins centered around the highly active compound, meta-topolin. We also report here an improved chlorophyll retention bioassay based on incubation of 2.5 cm long detached wheat leaf segments in microtiter plate wells containing 150 µl of cytokinin solution. The consumption of cytokinin to be tested is 0.1 µmol per assay only. The amount as small as 1.5 pmol of substance can be estimated using this biotest.     

Properties of the chloride-ATPase fromLimonium salt glands: Activation by,and binding to,specific sugars

Summary Attempts to separate membrane fractions enriched in Cl^–-ATPase activity fromLimonium leaf microsomes were hampered because, it seemed, the microsomal membranes were aggregated in clumps. We found hemagglutination activity, specific for N-acetylgalactosamine and to a lesser extent galactose, in the soluble phase of the homogenate, and we were able to prevent membrane aggregation by adding galactose to the microsomes. We discovered that the Cl^–-ATPase activity of the microsomes was increased by galactose and to an even greater extent by N-acetylgalactosamine. We report that the Cl^–-ATPase binds to galactosamine-sepharose, from which it can be eluted in 0.1m galactose, i.e., the enzyme is associated with a saccharide-binding site similar to that of the hemagglutinins. This procedure results in a 100-fold enrichment of the Cl^–-ATPase activity and represents a new way of purifying a membrane-bound enzyme from a heterogeneous membrane preparation in one step. Enzyme isolated by affinity chromatography of Triton-solubilized membranes was essentially free of other ATPase and accounted for a substantial proportion (sometimes all) of the Cl^–-ATPase of the microsomes. This purified preparation of the enzyme shows N-acetylgalactosamine-specific hemagglutination activity. However, we can show that the Cl^–-ATPase and the hemagglutinins are different entities. Thus, material isolated in the same way from salt-free plants showed hemagglutination but not Cl^–-ATPase activity. Also, the hemagglutinins, but not the Cl^–-ATPase, will bind to galactosaminesepharose in the absence of ATP.This is the first report of enzyme activity associated with a carbohydrate receptorspecific protein. Possible roles for saccharide-binding in the control, assembly, and orientation of the chloride-pump are discussed.     

Concentration Gradients of Free and Total γ-Aminobutyric Acid and Homocarnosine in Human CSF: Comparison of Suboccipital and Lumbar Sampling

Abstract: Concentrations of free and total γ-aminobutyric acid (GABA) and homocarnosine were determined in sequential aliquots of the first 30 ml of CSF obtained by lumbar puncture in five patients. Rostrocaudal gradients were calculated and compared to gradients estimated by determining concentrations of these substances in CSF obtained by simultaneous suboccipital and lumbar punctures in four more patients. In the lumbar fractions study, rostrocaudal mean gradients of 0.36, 36, and 21 pmol/ml for free GABA, total GABA, and homocarnosine, respectively, were calculated. In the suboccipital/lumbar study, gradients of 0.33, 30, and 24 pmol/ml for free GABA, total GABA, and homocarnosine, respectively, were estimated. These results indicate that valid comparison of CSF concentrations of these substances is restricted to similar fractions and suggest that in CSF the substances originate largely from brain rather than from peripheral sources.     

Induction of multifunctional human immunodeficiency virus type 1 (HIV-1)-specific T cells capable of proliferation in healthy subjects by using a prime-boost regimen of DNA- and modified vaccinia virus Ankara-vectored vaccines expressing HIV-1 Gag coupled to CD8+ T-cell epitopes

A double-blind randomized phase I trial was conducted in human immunodeficiency virus type 1 (HIV-1)-negative subjects receiving vaccines vectored by plasmid DNA and modified vaccinia virus Ankara (MVA) expressing HIV-1 p24/p17 gag linked to a string of CD8(+) T-cell epitopes. The trial had two groups. One group received either two doses of MVA.HIVA (2x MVA.HIVA) (n=8) or two doses of placebo (2x placebo) (n=4). The second group received 2x pTHr.HIVA followed by one dose of MVA.HIVA (n=8) or 3x placebo (n=4). In the pTHr.HIVA-MVA.HIVA group, HIV-1-specific T-cell responses peaked 1 week after MVA.HIVA vaccination in both ex vivo gamma interferon (IFN-gamma) ELISPOT (group mean, 210 spot-forming cells/10(6) cells) and proliferation (group mean stimulation index, 37), with assays detecting positive responses in four out of eight and five out of eight subjects, respectively. No HIV-1-specific T-cell responses were detected in either assay in the 2x MVA.HIVA group or subjects receiving placebo. Using a highly sensitive and reproducible cultured IFN-gamma ELISPOT assay, positive responses mainly mediated by CD4(+) T cells were detected in eight out of eight vaccinees in the pTHr.HIVA-MVA.HIVA group and four out of eight vaccinees in the 2x MVA.HIVA group. Importantly, no false-positive responses were detected in the eight subjects receiving placebo. Of the 12 responders, 11 developed responses to previously identified immunodominant CD4(+) T-cell epitopes, with 6 volunteers having responses to more than one epitope. Five out of 12 responders also developed CD8(+) T-cell responses to the epitope string. Induced T cells produced a variety of anti-viral cytokines, including tumor necrosis factor alpha and macrophage inflammatory protein 1 beta. These data demonstrate that prime-boost vaccination with recombinant DNA and MVA vectors can induce multifunctional HIV-1-specific T cells in the majority of vaccinees.     

Buzonamine, a new alkaloid from the defensive secretion of the millipede, Buzonium crassipes

GC-MS analysis of the defensive secretion from the millipede Buzonium crassipes showed three volatile components, beta-pinene (35%), limonene (6%) and a new alkaloid, buzonamine (59%). Buzonamine had an HRMS molecular ion at m/z=221.1785 (calculated for C(14)H(23)NO, 221.1781), 14 carbons in the 13C-NMR and 23 hydrogens by SFORD, DEPT and APT experiments. All distinct 1H nuclei were assigned to 13C resonances with 2D 1H-13C COSY data, and the final structure was determined by 1H-1H COSY, 1H-13C HMBC and nOe experiments. Buzonamine contains four rings including an epoxy group and a tertiary nitrogen. beta-Pinene, limonene or buzonamine, kept the mound nesting ant, Formica obscuripes, from eating mealworms (Tenebrio molitor) treated with 1mg of the chemical. A 150mg millipede expels 4mg of secretion.     

The Guanine-Nucleotide Exchange Factor SGEF Plays a Crucial Role in the Formation of Atherosclerosis

The passage of leukocytes across the endothelium and into arterial walls is a critical step in the development of atherosclerosis. Previously, we showed in vitro that the RhoG guanine nucleotide exchange factor SGEF (Arhgef26) contributes to the formation of ICAM-1-induced endothelial docking structures that facilitate leukocyte transendothelial migration. To further explore the in vivo role of this protein during inflammation, we generated SGEF-deficient mice. When crossed with ApoE null mice and fed a Western diet, mice lacking SGEF showed a significant decrease in the formation of atherosclerosis in multiple aortic areas. A fluorescent biosensor revealed local activation of RhoG around bead-clustered ICAM-1 in mouse aortic endothelial cells. Notably, this activation was decreased in cells from SGEF-deficient aortas compared to wild type. In addition, scanning electron microscopy of intimal surfaces of SGEF^−/− mouse aortas revealed reduced docking structures around beads that were coated with ICAM-1 antibody. Similarly, under conditions of flow, these beads adhered less stably to the luminal surface of carotid arteries from SGEF ^−/− mice. Taken together, these results show for the first time that a Rho-GEF, namely SGEF, contributes to the formation of atherosclerosis by promoting endothelial docking structures and thereby retention of leukocytes at athero-prone sites of inflammation experiencing high shear flow. SGEF may therefore provide a novel therapeutic target for inhibiting the development of atherosclerosis.