Active site studies on Bacillus amyloliquefaciens α-amylase (I)

Summary Modification of liquefying -amylase by diethylpyrocarbonate or its photo-oxidation in the presence of rose bengal caused rapid loss of enzyme activity. The photo-oxidation followed pseudo-first-order kinetics giving maximal value at pH 8.0. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm which was directly proportional to the extent of inactivation. Diethylpyrocarbonate at low concentration at pH 6.0 and 30 ° C completely inactivated a-amylase. Inactivation followed pseudo-first-order kinetics. The reaction order with respect to inactivation by diethylpyrocarbonate was one, thus indicating modification of a single histidine per mole of the enzyme. Diethylpyrocarbonate-modified enzyme showed increased absorbance at 240 nm which was reversed completely upon treatment with NH₂OH at 30 °C for 16 hr. Calculating the histidine residues being modified from the increase in absorbance at 240 nm showed that three residues were ethoxyformylated on treatment with diethylpyrocarbonate, of which only one was found at the active site. Substrate and competitive inhibitor protects the enzyme against both, photo-oxidation, and modification by diethylpyrocarbonate, confirming that histidine plays an essential role at the -amylase active site.     

The Yeast INO80 Complex Operates as a Tunable DNA Length-Sensitive Switch to Regulate Nucleosome Sliding

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Reproductive development and nuclear DNA content in angiosperms

Correlates of nuclear DNA content in angiosperms have been noted previously for a range of features, cellular to geographic. A new hypothesis, the correlation between nuclear DNA content and reproductive developmental features (after Cavalier-Smith, Journal of Cell Science 34, 247–268, 1978) is posed and tested here. Of three features tested (megasporogenesis, microsporogenesis, and endosperm development), megasporogenesis alone was shown to be correlated with nuclear DNA amount. The hypothesis was examined in 107 families of angiosperms using nonparametric statistics, and in 53 families of monocotyledons and outgroups using a phylogenetic test of association. A correlation was found between large genomes and successive microsporogenesis for all angiosperms, but not for monocots and dicots analyzed separately, thus underlining the importance of taking into account phylogenetic relationships in such studies. A correlation between cellular endosperm and large genomes in dicotyledons needs to be confirmed in a phylogenetic context. A tendency for deviations from monosporic megasporogenesis to occur in taxa that have a nuclear DNA content of over 9.0 pg/C was demonstrated using both phylogenetic and nonphylogenetic tests. It is hypothesized that cytoskeleton dynamics are affected in reproductive cells, enabling decoupling between nuclear and cytoplasmic cell cycles and leading to variation in reproductive development.     

In vitro screening procedure for osmotic tolerance in Prunus

Significant growth differences (p<0.01) were observed for two micropropagated Prunus accessions after 14 days in culture when 685 mM mannitol was included in Quoirin and Lepoivre nutrient medium. While there was an 11% growth increase in fresh weight during the 28-day culture period for accession K537-067, explants from New Jersey Plumcot No. 3 increased fresh weight an average of 123%. Similar tests were conducted to determine the repeatability of this short term in vitro screening procedure. Explants of Mananna 2624 were subjected to two levels of mannitol, 275 mM and 550 mM, included in the Quoirin and Lepoivre nutrient medium. Three successive 28-day tests were conducted. Explants were examined at both 14 and 28 days after the onset of the experiment for net growth changes. Addition of mannitol to the nutrient medium at concentrations of 275 mM and 550 mM decreased explant fresh weights of Marianna 2624 to 36% and 28% of controls, respectively, at 28 days past initial culture. Initial fresh weight and fresh weight changes at day 14 were significantly different (p 0.05) between tests. No significant differences existed between tests with regard to weight changes at 28 days past initial culture. This information may aid Prunus breeders in the choice of procedures for inducing drought stress and screening large numbers of plant accessions.Abbreviations K5 K537-067 - M2624 Marianna 2624 - NJPC3 New Jersey Plumcot No. 3 - Q & L Quoirin and Lepoivre     

An expeditious synthesis of blood-group antigens,ABO histo-blood group type II antigens and xenoantigen oligosaccharides with amino type spacer−arms

Blood group oligosaccharides are one of the most clinically important antigen families and they may also act as secondary ligands for bacterial toxins from Escherichia coli and Vibrio cholerae. Herein we report the synthesis of spacered (sp = CH₂CH₂CH₂NH₂) glycosides of A antigen {α-D-GalNAc-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-}, B antigen{α-D-Gal-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-}, LewisX{α-D-Gal-(l→4)-[α-L-Fuc-(l→3)]-β-D-GlcNAc-}, A type-II {α-D-GalNAc-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-(1→4)-β-D-GlcNAc-}, B type-II {α-D-Gal-(l→3)-[α-L-Fuc-(l→2)]-β-D-Gal-(1→4)-β-D-GlcNAc-}, H type-II{α-L-Fuc-(l→2)-β-D-Gal-(1→4)-β-D-GlcNAc-}, xenoantigen {α-D-Gal-(l→3)-β-D-Gal-(1→4)-[α-L-Fuc-(l→2)]-β-D-GlcNAc-} and Linear B Type II {α-D-Gal-(l→3)-β-D-Gal-(1→4)-β-D-GlcNAc-} useful for a range of biochemical investigations. This linker was chosen so as to facilitate the future conjugation of the antigens to proteins or other molecules. We also measured the affinities of some synthesized oligosaccharides against El Tor CTB strain from V. cholera.     

Integration of Stem Cell to Chondrocyte-Derived Cartilage Matrix in Healthy and Osteoarthritic States in the Presence of Hydroxyapatite Nanoparticles

We investigated the effectiveness of integrating tissue engineered cartilage derived from human bone marrow derived stem cells (HBMSCs) to healthy as well as osteoarthritic cartilage mimics using hydroxyapatite (HA) nanoparticles immersed within a hydrogel substrate. Healthy and diseased engineered cartilage from human chondrocytes (cultured in agar gels) were integrated with human bone marrow stem cell (HBMSC)-derived cartilaginous engineered matrix with and without HA, and evaluated after 28 days of growth. HBMSCs were seeded within photopolymerizable poly (ethylene glycol) diacrylate (PEGDA) hydrogels. In addition, we also conducted a preliminary in vivo evaluation of cartilage repair in rabbit knee chondral defects treated with subchondral bone microfracture and cell-free PEGDA with and without HA. Under in vitro conditions, the interfacial shear strength between tissue engineered cartilage derived from HBMSCs and osteoarthritic chondrocytes was significantly higher (p < 0.05) when HA nanoparticles were incorporated within the HBMSC culture system. Histological evidence confirmed a distinct spatial transition zone, rich in calcium phosphate deposits. Assessment of explanted rabbit knees by histology demonstrated that cellularity within the repair tissues that had filled the defects were of significantly higher number (p < 0.05) when HA was used. HA nanoparticles play an important role in treating chondral defects when osteoarthritis is a co-morbidity. We speculate that the calcified layer formation at the interface in the osteoarthritic environment in the presence of HA is likely to have attributed to higher interfacial strength found in vitro. From an in vivo standpoint, the presence of HA promoted cellularity in the tissues that subsequently filled the chondral defects. This higher presence of cells can be considered important in the context of accelerating long-term cartilage remodeling. We conclude that HA nanoparticles play an important role in engineered to native cartilage integration and cellular processes.     

Cytotoxicity of zinc chloride in mice in vivo

Intraperitoneal administration of zinc chloride (ZnCl2) to Swiss albino mice in vivo induced a significant (p less than or equal to 0.05) increase in the frequencies of chromosomal aberrations of the bone-marrow cells at all concentrations used following acute (7.5, 10, 15 mg/kg body weight) and chronic (2.0, 3.0 mg/kg body wt) treatment. The degree of clastogenicity was directly proportional to the concentrations (p less than or equal to 0.05, trend test) and indirectly to the period of treatment (p less than or equal to 0.05, ANOVA test). It induced a dose-dependent, statistically significant increase (Mann-Whitney U statistics, Student's t-test) in sperm-head abnormalities. The data designate ZnCl2 as a potent clastogen and as a toxic chemical at the concentrations used.     

Role of metal ions in goat carboxypeptidase A-catalysed hydrolysis of acyl peptides

The carboxypeptidase A purified from goat pancreas has been found to have a molecular weight of 34,600 +/- 300. The enzyme is a zinc-protein and the molar ratio of zinc to enzyme protein is 1:1. Removal of zinc yields an inactive apocarboxypeptidase A. The loss of activity of the native enzyme and restoration of the activity of the apoenzyme run parallel with the zinc content of the protein, thus showing the essentiality of zinc for the enzymatic activity. The exact role of zinc in the enzyme catalysed hydrolysis of the acylpeptides has been investigated after preparing metallo proteins by substituting the zinc of carboxypeptidase A with Co2+, Mn2+, Ni2+, Fe2+, Cd2+, Hg2+, and Cu2+ and determining the kinetic parameters of such metalloproteins. These studies indicate that the metal ion is involved in both binding the substrate and polarising the peptide bond.     

Effect of altered sterol levels on the transport of amino acids and membrane structure ofMicrosporum gypseum

Ergosterol and cholesterol supplementation resulted in a significant increase (1·5-fold) in the sterol content while phospholipid remained unaffected inMicrosporum gypseum. The levels of phosphatidylethanolamine and phosphatidylcholine increased in ergosterol supplemented cells. However, a decrease in phosphatidylcholine and an increase in phosphatidylethanolamine was observed in cholesterol grown cells. The ratio of unsaturated to saturated fatty acids decreased on ergosterol/cholesterol supplementation. The uptake of amino acids (lysine, glycine and aspartic acid) decreased in sterol supplemented cells. Studies with fluorescent probe l-anilinonaphthalene-8-sulfonate showed structural changes in membrane organisation as evident by increased number of binding sites in such cells.