Crystal structure of malonyl CoA-Acyl carrier protein transacylase from Xanthomanous oryzae pv. oryzae and its proposed binding with ACP

Xanthomonas oryzae pv. oryzae (Xoo) is a plant bacterial pathogen that causes bacterial blight (BB) disease, resulting in serious production losses of rice. The crystal structure of malonyl CoA-acyl carrier protein transacylase (XoMCAT), encoded by the gene fabD (Xoo0880) from Xoo, was determined at 2.3 Å resolution in complex with N-cyclohexyl-2-aminoethansulfonic acid. Malonyl CoA-acyl carrier protein transacylase transfers malonyl group from malonyl CoA to acyl carrier protein (ACP). The transacylation step is essential in fatty acid synthesis. Based on the rationale, XoMCAT has been considered as a target for antibacterial agents against BB. Protein-protein interaction between XoMCAT and ACP was also extensively investigated using computational docking, and the proposed model revealed that ACP bound to the cleft between two XoMCAT subdomains.     

Development of quenching and washing protocols for quantitative intracellular metabolite analysis of uninfected and baculovirus-infected insect cells

Metabolomics refer to the global analysis of small molecule metabolites in a biological system, and can be a powerful tool to elucidate and optimize cellular processes, particularly when integrated into a systems biology framework. Determining the endometabolome in cultured animal cells is especially challenging, due to the conflicting demands for rapid quenching of metabolism and retention of membrane integrity, while cells are separated from the complex medium. The challenge is magnified in virus infected cells due to increased membrane fragility. This paper describes an effective methodology for quantitative intracellular metabolite analysis of the baculovirus–insect cell expression system, an important platform for the production of heterologous proteins and baculovirus-based biopesticides. These two applications were represented by Spodoptera frugiperda (Sf9) and Helicoverpa zea (HzAM1) cells infected with recombinant Autographa californica and wild-type Helicoverpa armigera nucleopolyhedroviruses (AcMNPV and HaSNPV), respectively. Specifically, an ice-cold quenching solution comprising 1.1% w/v NaCl and 0.2% w/v Pluronic® F-68 (NaCl + P) was found to be efficacious in preserving cell viability and minimizing cell leakage during quenching and centrifugation-based washing procedures (prior to extraction using cold 50% v/v acetonitrile). Good recoveries of intracellular adenosine triphosphate, total adenosine phosphates and amino acids were obtained after just one wash step, for both uninfected and infected insect cells. The ability to implement wash steps is critical, as insect cell media are metabolites-rich, while infected insect cells are much more fragile than their uninfected counterparts. Hence, a promising methodology has been developed to facilitate endometabolomic analysis of insect cell–baculovirus systems for bioprocess optimization.     

The thrH gene product of Pseudomonas aeruginosa is a dual activity enzyme with a novel phosphoserine:homoserine phosphotransferase activity

The thrH gene product of Pseudomonas aeruginosa has been shown to complement both homoserine kinase (thrB gene product) and phosphoserine phosphatase (serB gene product) activities in vivo. Sequence comparison has revealed that ThrH is related to phosphoserine phosphatases (PSP, EC 3.1.3.3) and belongs to the l-2-haloacid dehalogenase-like protein superfamily. We have solved the crystal structures of ThrH in the apoform and in complex with a bound product phosphate. The structure confirms an overall fold similar to that of PSP. Most of the catalytic residues of PSP are also conserved in ThrH, suggesting that similar catalytic mechanisms are used by both enzymes. Spectrophotometry-based in vitro assays show that ThrH is indeed a phosphoserine phosphatase with a K(m) of 0.207 mm and k(cat) of 13.4 min(-1), comparable with those of other PSPs. More interestingly, using high pressure liquid chromatography-based assays, we have demonstrated that ThrH is able to further transfer the phosphoryl group to homoserine using phosphoserine as the phosphoryl group donor, indicating that ThrH has a novel phosphoserine:homoserine phosphotransferase activity.     

Pyrrole and furan oligoglycosides from the starfish Asterina batheri and their inhibitory effect on the production of pro-inflammatory cytokines in lipopolysaccharide-stimulated bone marrow-derived dendritic cells

Three new pyrrole oligoglycosides, astebatheriosides A–C (1–3), and a new furan oligoglycoside, astebatherioside D (4), were isolated from the starfish Asterina batheri by various chromatographic methods. Their structures were elucidated by spectroscopic and chemical methods. Compounds 2, 3, and 4 moderately inhibited IL-12 p40 production in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells (BMDCs) with IC₅₀ values of 36.4, 31.6, and 22.8 μM, respectively.     

Simulated Microgravity Induces the Proliferative Inhibition and Morphological Changes in Porcine Granulosa Cells

Astronauts are always faced with serious health problems during prolonged spaceflights. Previous studies have shown that weightlessness significantly affects the physiological function of female astronauts, including a change in reproductive hormones and ovarian cells, such as granulosa and theca cells. However, the effects of microgravity on these cells have not been well characterized, especially in granulosa cells. This study aimed to investigate the effects of simulated microgravity (SMG) on the proliferation and morphology of porcine granulosa cells (pGCs). pGC proliferation from the SMG group was inhibited, demonstrated by the reduced O.D. value and cell density in the WST-1 assay and cell number counting. SMG-induced pGCs exhibited an increased ratio of cells in the G0/G1 phase and a decreased ratio of cells in the S and G2/M phase. Western blot analysis indicated a down-regulation of cyclin D1, cyclin-dependent kinase 4 (cdk4), and cyclin-dependent kinase 6 (cdk6), leading to the prevention of the G1-S transition and inducing the arrest phase. pGCs under the SMG condition showed an increase in nuclear area. This caused a reduction in nuclear shape value in pGCs under the SMG condition. SMG-induced pGCs exhibited different morphologies, including fibroblast-like shape, rhomboid shape, and pebble-like shape. These results revealed that SMG inhibited proliferation and induced morphological changes in pGCs.     

Anticoagulant properties of dextranmethylcarboxylate benzylamide sulfate (DMCBSu); a new generation of bioactive functionalized dextran

Dextranmethylcarboxylate benzylamide sulfate (DMCBSu), a functionalized dextran, exhibits anticoagulant properties. Its synthesis involves three steps: a carboxymethylation with monochloroacetic acid in alkaline water-iso-propanol, a benzylamidification of some of the methylcarboxylate groups with benzylamine in the presence of a water soluble carbodiimide and a partial sulfation of the remaining hydroxyl groups with SO3-pyridine in dimethylformamide. This procedure yields reproducibly DMCBSu with degrees of substitution in methylcarboxylate (MC), benzylamide (B) and sulfate (Su) groups, respectively, up to 1.61, 0.35 and 1.5, each obtained in one step. For a degree of substitution of methylcarboxylate ca. 1, the presence of sulfate groups is absolutely necessary to confer anticoagulant activities to the samples. In addition, the anticoagulant ability is higher for derivatives bearing benzylamide groups. The anticoagulant ability of DMCBSu increases with the degree of sulfation, reaching 20% of heparin activity for a degree of substitution of Su groups about 1.3.     

Purification and characterization of a lymph node sulfotransferase responsible for 6-O-sulfation of the galactose residues in 2'-fucosyllactose and other sialyl LewisX-related sugars

A microsomal galactose-6-O-sulfotransferase (Gal-6-O-Stase) from porcine lymph nodes, able to transfer the sulfate group from adenosine 3'-phosphate 5'-phosphosulphate (PAPS) onto 2'-fucosyllactose (2'-FL) and other sialyl LewisX (sLex)-related sugars, has been purified and characterized. The enzyme was purified to about 35,000-fold by a combination of conventional and affinity chromatographic steps. The purified enzyme preparation exhibited two protein bands at around 80-90 and 170 kDa on 7.5% SDS-PAGE under reducing conditions. Both of these protein bands always comigrated in the gel when peak fractions containing Gal-6-O-Stase activity from the 3',5'-ADP-agarose column were subjected to 6% SDS-PAGE under reducing conditions. These protein bands also showed similar binding patterns to WGA (wheat germ agglutinin), Con A (concanvalin A), and EBA (elderberry agglutinin). Similarly, when the enzyme preparation after the hydroxylapatite step was photolabeled with 8-azido-[32P]-PAPS, both 80-90 and 170 kDa protein bands were labeled in a specific manner. These results suggest a possible association of these two protein bands with the enzyme activity. The carbohydrate substrate specificity of this enzyme suggests that it is well suited to catalyze the sulphonation at the C-6 position of the galactose residues of oligosaccharides that are structurally similar to sLex. Furthermore, a survey of several porcine organs revealed that this enzyme was selectively expressed in lymphoid tissues such as lymph nodes (peripheral and mesenteric) and spleen. These findings suggest that this enzyme may be involved in the assembly of 3'-sialyl-6'-sulfo Lewisx, the major capping group of HEV-ligands for L-selectin.     

Characterization of Bacillus probiotics available for human use

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.     

Gene knockdown of gamma-glutamylcysteine synthetase by RNAi in the parasitic protozoa Trypanosoma brucei demonstrates that it is an essential enzyme

The parasitic protozoa Trypanosoma brucei utilizes a novel cofactor (trypanothione, T(SH)2), which is a conjugate of GSH and spermidine, to maintain cellular redox balance. gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the first step in the biosynthesis of GSH. To evaluate the importance of thiol metabolism to the parasite, RNAi methods were used to knock down gene expression of gamma-GCS in procyclic T. brucei cells. Induction of gamma-GCS RNAi with tetracycline led to cell death within 4-6 days post-induction. Cell death was preceded by the depletion of the gamma-GCS protein and RNA and by the loss of the cellular pools of GSH and T(SH)2. The addition of GSH (80 microM) to cell cultures rescued the RNAi cell death phenotype and restored the intracellular thiol pools to wild-type levels. Treatment of cells with buthionine sulfoximine (BSO), an enzyme-activated inhibitor of gamma-GCS, also resulted in cell death. However, the toxicity of the inhibitor was not reversed by GSH, suggesting that BSO has more than one cellular target. BSO depletes intracellular thiols to a similar extent as gamma-GCS RNAi; however, addition of GSH did not restore the pools of GSH and T(SH)2. These data suggest that BSO also acts to inhibit the transport of GSH or its peptide metabolites into the cell. The ability of BSO to inhibit both synthesis and transport of GSH likely makes it a more effective cytotoxic agent than an inhibitor with a single mode of action. Finally the potential for the T(SH)2 biosynthetic enzymes to be regulated in response to reduced thiol levels was studied. The expression levels of ornithine decarboxylase and of S-adenosylmethionine decarboxylase, two essential enzymes in spermidine biosynthesis, remained constant in induced gamma-GCS RNAi cell lines.