Identification of a membrane-localized cysteine cluster near the substrate-binding sites of the Streptococcus equisimilis hyaluronan synthase

The membrane-bound hyaluronan synthase (HAS) from Streptococcus equisimilis (seHAS), which is the smallest Class I HAS, has four cysteine residues (positions 226, 262, 281, and 367) that are generally conserved within this family. Although Cys-null seHAS is still active, chemical modification of cysteine residues causes inhibition of wild-type enzyme. Here we studied the effects of N-ethylmaleimide (NEM) treatment on a panel of seHAS Cys-mutants to examine the structural and functional roles of the four cysteine residues in the activity of the enzyme. We found that Cys226, Cys262, and Cys281 are reactive with NEM, but Cys367 is not. Substrate protection studies of wild-type seHAS and a variety of Cys-mutants revealed that binding of UDP-GlcUA, UDP-GlcNAc, or UDP can protect Cys226 and Cys262 from NEM inhibition. Inhibition of the six double Cys-mutants of seHAS by sodium arsenite, which can cross-link vicinyl sulfhydryl groups, also supported the conclusion that Cys262 and Cys281 are close enough to be cross-linked. Similar results indicated that Cys281 and Cys367 are also very close in the active enzyme. We conclude that three of the four Cys residues in seHAS (Cys262, Cys281, and Cys367) are clustered very close together, that these Cys residues and Cys226 are located at the inner surface of the cell membrane, and that Cys226 and Cys262 are located in or near a UDP binding site.     

Differential apoptotic and redox regulatory activities of curcumin and its derivatives

We have synthesized different bioconjugates of curcumin, which were tested for their pro- and antioxidant properties. In the present study five representative derivatives of curcumin, i.e., 4,4'-di-(O-acetyl) curcumin, 4,4'-di-(O-glycinoyl) curcumin, 4,4'-di-(O-glycinoyl-di-N-piperoyl) curcumin, 4,4'-di-(O-piperoyl) curcumin, and 4,4'-(O,O-cystinoyl)-3,3'-dimethoxydiphenyl-1,6-heptadiene-3,5-dione, were used for testing their apoptotic potential on tumor cells. Dipiperoyl and diglycinoyl derivatives showed higher apoptotic activity at lower concentrations, whereas diacetyl curcumin had slightly lower apoptotic activity on tumor cells. On the other hand, diglycinoyl-dipiperoyl and cystinoyl heptadiene derivatives had lost their apoptotic potential significantly. The apoptotic activity of these derivatives correlated very well with the generation of ROS by the tumor cells, whereas GSH levels remained unaltered. Our studies also indicate downregulation of Bcl-2 and participation of caspase-3 in the apoptotic death of tumor cells.     

Immunomodulation by 'Immuplus (AquaImmu)' in giant freshwater prawn, Macrobrachium rosenbergii (De Man)

ImmuPlus, a polyherbal commercial formulation was used to modulate the immune system of commercially important giant freshwater prawn M. rosenbergii. The prawns were fed with basal diet supplemented with ImmuPlus at 1g/kg feed for 4 weeks. Results showed that the phenoloxidase activity (PO), haemagglutination and lysozyme activities were significantly elevated in ImmuPlus-fed prawn up to 3 weeks of feeding and declined after 4 weeks of feeding. The total protein level in ImmuPlus-fed prawn raised up to 2nd week of feeding. Incorporation of ImmuPlus at the rate of 1g/kg feed in the diet of prawn for 3 weeks may be beneficial in raising the immune status of prawn.     

Acetoxy drug: protein transacetylase of buffalo liver-characterization and mass spectrometry of the acetylated protein product

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3-3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K(m) (1667 microM) and V(max) (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3-3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3-3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3-3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3-3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys(51), Lys(82), Lys(123), Lsy(181), Lys(191) and Lys(210)) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions.     

Group B Streptococcus: global incidence and vaccine development

An ongoing public health challenge is to develop vaccines that are effective against infectious diseases that have global relevance. Vaccines against serotypes of group B Streptococcus (GBS) that are prevalent in the United States and Europe are not optimally efficacious against serotypes common to other parts of the world. New technologies and innovative approaches are being used to identify GBS antigens that overcome serotype-specificity and that could form the basis of a globally effective vaccine against this opportunistic pathogen. This Review highlights efforts towards this goal and describes a template that can be followed to develop vaccines against other bacterial pathogens.     

Simulated microgravity‐induced epigenetic changes in human lymphocytes

Real space flight and modeled microgravity conditions result in changes in the expression of genes that control important cellular functions. However, the mechanisms for microgravity‐induced gene expression changes are not clear. The epigenetic changes of DNA methylation and chromatin histones modifications are known to regulate gene expression. The objectives of this study were to investigate whether simulated microgravity alters (a) the DNA methylation and histone acetylation, and (b) the expression of DNMT1, DNMT3a, DNMT3b, and HDAC1 genes that regulate epigenetic events. To achieve these objectives, human T‐lymphocyte cells were grown in a rotary cell culture system (RCCS) that simulates microgravity, and in parallel under normal gravitational conditions as control. The microgravity‐induced DNA methylation changes were detected by methylation sensitive‐random amplified polymorphic DNA (MS‐RAPD) analysis of genomic DNA. The gene expression was measured by Quantitative Real‐time PCR. The expression of DNMT1, DNMT3a, and DNMT3b was found to be increased at 72 h, and decreased at 7 days in microgravity exposed cells. The MS‐RAPD analysis revealed that simulated microgravity exposure results in DNA hypomethylation and mutational changes. Gene expression analysis revealed microgravity exposure time‐dependent decreased expression of HDAC1. Decreased expression of HDAC1 should result in increased level of acetylated histone H3, however a decreased level of acetylated H3 was observed in microgravity condition, indicating thereby that other HDACs may be involved in regulation of H3 deacetylation. The findings of this study suggest that epigenetic events could be one of the mechanistic bases for microgravity‐induced gene expression changes and associated adverse health effects. J. Cell. Biochem. 111: 123–129, 2010. © 2010 Wiley‐Liss, Inc.     

Genetic control of leaf-blade morphogenesis by the <Emphasis Type="Italic">INSECATUS</Emphasis> gene in <Emphasis Type="Italic">Pisum sativum</Emphasis>

To understand the role of INSECATUS (INS) gene in pea, the leaf blades of wild-type, ins mutant and seven other genotypes, constructed by recombining ins with uni-tac, af, tl and mfp gene mutations, were quantitatively compared. The ins was inherited as a recessive mutant allele and expressed its phenotype in proximal leaflets of full size leaf blades. In ins leaflets, the midvein development was arrested in distal domain and a cleft was formed in lamina above this point. There was change in the identity of ins leaflets such that the intercalary interrupted midvein bore a leaf blade. Such adventitious blades in ins, ins tl and ins tl mfp were like the distal segment of respective main leaf blade. The ins phenotype was not seen in ins af and ins af uni-tac genotypes. There was epistasis of uni-tac over ins. The ins, tl and mfp mutations interacted synergistically to produce highly pronounced ins phenotype in the ins tl mfp triple mutant. The role(s) of INS in leaf-blade organogenesis are: positive regulation of vascular patterning in leaflets, repression of UNI activity in leaflet primordia for ectopic growth and in leaf-blade primordium for indeterminate growth of rachis, delimitation of proximal leaflet domain and together with TL and MFP homeostasis for meristematic activity in leaflet primordia. The variant apically bifid shape of the affected ins leaflets demonstrated that the leaflet shape is dependent on the venation pattern.     

Establishment of baseline susceptibility data to various insecticides for Homalodisca coagulata (Homoptera: Cicadellidae) by comparative bioassay techniques

Homalodisca coagulata Say, adults from three locations in California were subjected to insecticide bioassays to establish baseline toxicity. Initially, two bioassay techniques, petri dish and leaf dip, were compared to determine the most useful method to establish baseline susceptibility data under laboratory and greenhouse conditions. Comparative dose-response data were determined by both techniques to endosulfan, dimethoate, cyfluthrin, and acetamiprid. Toxic values were similar to some insecticides with both techniques but not for all insecticides, revealing susceptibility differences among the three populations of H. coagulata. In subsequent tests, the petri dish technique was selected to establish baseline susceptibility data to various contact insecticides. A systemic uptake bioassay was adapted to estimate dose-mortality responses to a systemic insecticide, imidacloprid. A 2-yr comparison of toxicological responses showed all three populations of H. coagulata to be highly susceptible to 10 insecticides, including chlorpyrifos, dimethoate, endosulfan, bifenthrin, cyfluthrin, esfenvalerate, fenpropathrin, acetamiprid, imidacloprid, and thiamethoxam. In general, two pyrethroids, bifenthrin and esfenvalerate, were the most toxic compounds, followed by two neonicotinoids, acetamiprid and imidacloprid. The LC50 values for all insecticides tested were lower than concentrations used as recommended field rates. Baseline data varied for the three geographically distinct H. coagulata populations with the petri dish technique. Adult H. coagulata collected from San Bernardino County were significantly more susceptible to select pyrethroids compared with adults from Riverside or Kern counties. Adults from San Bernardino County also were more sensitive to two neonicotinoids, acetamiprid and imidacloprid. The highest LC50 values were to endosulfan, which nonetheless proved highly toxic to H. coagulata from all three regions. In the majority of the tests, mortality increased over time resulting in increased susceptibility at 48 h compared with 24 h. These results indicate a wide selection of highly effective insecticides that could aid in managing H. coagulata populations in California.