Differential proteomic analysis of a polymicrobial biofilm

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia exist in a polymicrobial biofilm associated with chronic periodontitis. The aim of this study was to culture these three species as a polymicrobial biofilm and to determine proteins important for bacterial interactions. In a flow cell all three species attached and grew as a biofilm; however, after 90 h of culture P. gingivalis and T. denticola were closely associated and dominated the polymicrobial biofilm. For comparison, planktonic cultures of P. gingivalis and T. denticola were grown separately in continuous culture. Whole cell lysates were subjected to SDS-PAGE, followed by in-gel proteolytic H(2)(16)O/H(2)(18)O labeling. From two replicates, 135 and 174 P. gingivalis proteins and 134 and 194 T. denticola proteins were quantified by LC-MALDI TOF/TOF MS. The results suggest a change of strategy in iron acquisition by P. gingivalis due to large increases in the abundance of HusA and HusB in the polymicrobial biofilm while HmuY and other iron/haem transport systems decreased. Significant changes in the abundance of peptidases and enzymes involved in glutamate and glycine catabolism suggest syntrophy. These data indicate an intimate association between P. gingivalis and T. denticola in a biofilm that may play a role in disease pathogenesis.     

Correlation between protein stability and crystal properties of designed ROP variants

Six variants of the ROP protein, designed with the aim to analyze by X-ray crystallography loop formation and core packing interactions in 4-α-helical bundles- have been purified and a search of their crystallization conditions has been carried out. Five mutants yield crystals that are suitable for medium to high resolutionX-ray diffraction studies. For all mutants crystal size- sensitivity to X-irradiation and diffraction limit are correlated to their stability as determined by differential scanning calorimetry- in a manner which is not yet understood in detail. © Wiley-Liss, Inc.     

Biochemical and pharmacological characterization of human α/β-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12)

In the central nervous system, three enzymes belonging to the serine hydrolase family are thought to regulate the life time of the endocannabinoid 2-arachidonoylglycerol (C20:4) (2-AG). From these, monoacylglycerol lipase (MAGL) is well characterized and, on a quantitative basis, is the main 2-AG hydrolase. The postgenomic proteins α/β-hydrolase domain containing (ABHD)6 and ABHD12 remain poorly characterized. By applying a sensitive fluorescent glycerol assay, we delineate the substrate preferences of human ABHD6 and ABHD12 in comparison with MAGL. We show that the three hydrolases are genuine MAG lipases; medium-chain saturated MAGs were the best substrates for hABHD6 and hMAGL, whereas hABHD12 preferred the 1 (3)- and 2-isomers of arachidonoylglycerol. Site-directed mutagenesis of the amino acid residues forming the postulated catalytic triad (ABHD6: S148-D278-H306, ABHD12: S246-D333-H372) abolished enzymatic activity as well as labeling with the active site serine-directed fluorophosphonate probe TAMRA-FP. However, the role of D278 and H306 as residues of the catalytic core of ABHD6 could not be verified because none of the mutants showed detectable expression. Inhibitor profiling revealed striking potency differences between hABHD6 and hABHD12, a finding that, when combined with the substrate profiling data, should facilitate further efforts toward the design of potent and selective inhibitors, especially those targeting hABHD12, which currently lacks such inhibitors.     

Structure of the hepatitis A virion: peptide mapping of the capsid region.

Milligram amounts of highly purified hepatitis A virus (HAV) were obtained from persistently infected cell cultures. The HAV polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for detection by an enzyme-linked immunotransfer blot procedure. The HAV nucleotide-derived amino acid sequence was subjected to computer analysis to identify potential immunogenic regions within the HAV capsid polypeptides. Synthetic peptides corresponding to selected regions of each of the larger putative capsid polypeptides were coupled to keyhole limpet hemocyanin and used to immunize rabbits. Four of six anti-HAV peptide sera were strongly reactive. Antipeptide serum generated against amino acids (a.a.) 75 through 82 reacted with the 27,000-molecular-weight (MW) polypeptide; serum against a.a. 279 through 285 reacted with the 29,000-MW HAV polypeptide; and sera against a.a. 591 through 602 and 606 through 618 reacted with the 33,000-MW HAV polypeptide. These reactions enabled the identification of the gene order of the larger HAV P1 region gene products. Our data indicate the following molecular weights: HAV VP2 or 1B, 27,000; HAV VP3 or 1C, 29,000; and HAV VP1 or 1D, 33,000.     

Effect of host plant on cornucopia of mango fruit flies (Diptera: Tephritidae) and their triumphant management in context of climate change

A study was performed to assess the preference of fourteen mango cultivars for fruit flies and their management by bagging. So the choice of Tephritid flies to mango cultivars during fruiting phase is crucial. Fourteen different cultivars of mango viz., ‘Dusehri’, ‘Malda’, ‘Langra’ early cultivars, ‘Chaunsa’, ‘Fajri Klan’, ‘Sensation’ medium whereas ‘Sanglakhi’, ‘Retaul-12’, ‘Mehmood Khan’, ‘Tukhmi’, ‘Kala Chaunsa’, ‘Chitta Chaunsa’, ‘Dai Wala’ and ‘Sobey De Ting’ late cultivars were assessed for their suitability for fruit flies. The results indicate that the population density of fruit flies was higher on late cultivars like ‘Sanglakhi’ (20.61 percent), ‘Mehmood Khan’ (20.22 percent) and ‘Reutal-12’ (19.92 percent) were proved to be highly susceptible to fruit flies. Among these the cultivar ‘Reutal-12’ was selected being commercial and future cultivar for the management of fruit flies through bagging. The results reported that the attack of tephritid fruit flies and other insect pests were zero in bagged fruits as compared with control. It was further recorded that the bagged fruits has maximum average fruit weight i.e. 203.50 and 197.83 g per fruit was noted in those treatments where butter paper bag and brown paper bag was wrapped with better coloration as compared with un-bagged fruit with 159.5 g per fruit. Similarly, on an average fruit length were more i.e. 90.17, 91.33 mm in bagged fruit and 85.33 in un-bagged fruits. Furthermore, bagged fruits have zero incidence of disease with reduced fruit crack, fruit sunburn, mechanical damage, bird damage, fruit blemished and agrochemical residues on the fruit. So, it is concluded that the special attention should be given on ‘Reutal-12’ for the management of fruit flies when devising an IPM program for the control of fruit flies. Further, bagging has proved to be the good agricultural practices for the production of quality mango.     

Synthesis and chemical behaviour of D-glucosyl esters of glutamic acid having the side-chain carboxyl group involved in the glycosidic linkage.

Simultaneous and stepwise deprotection of the fully benzylated D-glucosyl esters of 1-benzyl N-benzyloxycarbonyl- and N-tert-butyloxycarbonyl-L-glutamic acid (1 and 5, respectively) have been examined. Catalytic hydrogenation of 1 led to intramolecular aminolysis to give pyroglutamic acid and D-glucose, but similar treatment in the presence of trifluoroacetic acid afforded both anomers of 1-O-(L-gamma-glutamyl)-D-glucopyranose, which were characterized as trifluoroacetates (2alpha and 2beta) and converted into 2,3,4,6-tetra-O-acetyl-1-O-[1-methyl N-(acetyl)-L-glutam-5-oyl]-D-glucopyranose (4) which was also prepared by a definitive method. Hydrogenolysis of 5 gave both anomers of 1-O-[N-(tert-butyloxycarbonyl)-L-gamma-glutamyl]-D-glucopyranose (6), which, upon treatment with trifluoroacetic acid at - 10 degrees, afforded 2alpha and 2beta, respectively. The structure of 6beta was established by its conversion into 2,3,4,6-tetra-O-acetyl-1-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-beta-D-glucopyranose (7beta), whereas similar treatment of 6alpha gave a mixture of 1,3,4,6-tetra-O-acetyl-2-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-alpha-D-glucopyranose (9) and 7alpha. A 1 leads to 2 acyl migration occurred during esterification of the aglycon carboxyl group of 6alpha with diazomethane to give 2-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-alpha-D-glucopyranose (8).     

Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli

S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial proteins in E. coli.