Study of human cord blood lymphocytes by immobilized metal ion affinity partitioning

The potential of immobilized metal ion affinity partitioning (IMAP) using dextran-PEG+PEG-IDA-M(II) systems to separate mononuclear cells from cord blood has been evaluated. The distribution of B cells, T cells, monocytes and hematopoietic stem cells between PEG and dextran phases was determined by flow cytometry with fluorochrome-labelled specific antibodies. Comparing these values with the post-Ficoll repartition resulted in the determination of enrichment factors, for each subpopulation, in the different phases. We were able to distinguish the partition pattern of B cells, T cells, monocytes and stem cells in different IMAP systems. Their partition was affected by the nature and the concentration of the metal used, but no specificity in distribution for the subpopulations was found.     

Lipoprotein profile during perchlorate toxicity

Perchlorate administration to rats for 45 days alters the lipoprotein profile in plasma. The levels of cholesterol, phospholipids and triglycerides in HDL, LDL and VLDL fractions are significantly increased in perchlorate-treated rats. Post-heparin lipolytic activity of plasma of sodium perchlorate-treated rats is decreased. The risk factor, i.e. the total cholesterol/HDL cholesterol, increases in the experimental animals, indicating that the treatment of rats with perchlorate may develop the susceptibility of the animals to cardiac heart disease.     

Structural study of porcine pancreatic elastase complexed with 7-amino-3-(2-bromoethoxy)-4-chloroisocoumarin as a nonreactivatable doubly covalent enzyme-inhibitor complex

The complex of porcine pancreatic elastase (PPE) with 7-amino-3-(2-bromoethoxy)-4-chloroisocoumarin, a potent mechanism-based inhibitor, was crystallized and the crystal structure determined at 1.9-A resolution with a final R factor of 17.1%. The unbiased difference Fourier electron density map showed continuous density from O gamma of Ser 195 to the benzoyl carbonyl carbon atom and from N epsilon 2 of His 57 to the carbon atom at the 4-position of the isocoumarin ring in the inhibitor. This suggested unambiguously that the inhibitor was doubly covalently bound to the enzyme. It represents the first structural evidence for irreversible binding of an isocoumarin inhibitor to PPE through both Ser 195 and His 57 in the active site. The PPE-inhibitor complex is only partially activated in solution by hydroxylamine and confirms the existence of the doubly covalently bound complex along with the acyl enzyme. The benzoyl carbonyl oxygen atom of the inhibitor is not situated in the oxyanion hole formed by the amide (greater than NH) groups of Gly 193 and Ser 195. The complex is stabilized by the hydrogen-bonding interactions in the active site (from the N epsilon 2 of Gln 192 to the bromine atom in the inhibitor and the amino group at the 7-position of the isocoumarin ring to the carbonyl oxygen of Thr 41) and by van der Waals interactions. The inhibition rates of several 7-substituted 4-chloro-3-(bromoalkoxy)isocoumarins toward PPE were measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein adsorption on histidyl-aminohexyl-Sepharose 4B: II. Application to the negative one-step affinity purification of human β2-microglobulin and Immunoglobulin G

The adsorption of two human proteins, β2-microglobulin and Immunoglobulin G, from uremic patient’s blood ultrafiltrate and plasma, respectively, was investigated on the histidyl-aminohexyl-Sepharose 4B adsorbent. Both target proteins could be adsorbed on the gel through a low affinity for immobilized histidine ligand. However, a fine adjustment of the operating conditions (ionic strength, buffer, pH) prevented their adsorption and thus allowed their “negative affinity” purification (purity estimated by silver nitrate SDS–PAGE) by the removal of the contaminating proteins. This simple and efficient method provides purification under gentle chromatographic conditions and a further characterization of both molecules.     

Characterization of dehydromonacolin-MV2 from Monascus purpureus mutant

AIM: Characterization of dehydromonacolin-MV2, a bioactive metabolite isolated from Monascus purpureus mutant (CFR 410-11). METHODS AND RESULTS: Chloroform extract of rice, fermented with a hyperpigment-producing mutant of M. purpureus (CFR 410-11) was found to contain metabolites that inhibited the growth of Bacillus, Pseudomonas and Streptococcus in agar gel diffusion assays. The extract inhibited lipid peroxidation and scavenged 2,2-diphenyl-1-pycrylhydrazyl and hydroxyl radicals. The active compound purified by silica gel column chromatography was characterized by NMR. The carbon, proton and 2D HSQCT assignments identified dehydromonacolin-MV2 as the bioactive metabolite. CONCLUSIONS: Dehydromonacolin-MV2 apparently originated in the mutant by hydroxylation and oxidation of monacolin-J, an intermediate of monacolin biosynthetic pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of the production of dehydromonacolin-MV2 by M. purpureus mutant (CFR 410-11) is new to literature. Bioactive properties of the compound suggested its pharmaceutical applications.     

Separable Crossover-Promoting and Crossover-Constraining Aspects of Zip1 Activity during Budding Yeast Meiosis

Accurate chromosome segregation during meiosis relies on the presence of crossover events distributed among all chromosomes. MutSγ and MutLγ homologs (Msh4/5 and Mlh1/3) facilitate the formation of a prominent group of meiotic crossovers that mature within the context of an elaborate chromosomal structure called the synaptonemal complex (SC). SC proteins are required for intermediate steps in the formation of MutSγ-MutLγ crossovers, but whether the assembled SC structure per se is required for MutSγ-MutLγ-dependent crossover recombination events is unknown. Here we describe an interspecies complementation experiment that reveals that the mature SC is dispensable for the formation of Mlh3-dependent crossovers in budding yeast. Zip1 forms a major structural component of the budding yeast SC, and is also required for MutSγ and MutLγ-dependent crossover formation. Kluyveromyces lactis ZIP1 expressed in place of Saccharomyces cerevisiae ZIP1 in S. cerevisiae cells fails to support SC assembly (synapsis) but promotes wild-type crossover levels in those nuclei that progress to form spores. While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery. Moreover, K. lactis Zip1-mediated crossovers rely on S. cerevisiae synapsis initiation proteins Zip3, Zip4, Spo16, as well as the Mlh3 protein, as do the crossovers mediated by S. cerevisiae Zip1. Surprisingly, however, K. lactis Zip1-mediated crossovers are largely Msh4/Msh5 (MutSγ)-independent. This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast. Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates.     

An efficient two step purification and molecular characterization of beta-galactosidases from Aspergillus oryzae

Beta-galactosidases (beta-D-galactoside-galactohydrolases (EC 3.2.1.23), lactases) are important industrial enzymes used for the hydrolysis of lactose from milk and milk whey. These enzymes are produced by different organisms and purified by multi-step procedures. The multi-step purification schemes are cost and time ineffective which can also lead to poor yield, denaturation and loss of enzymatic activity. In our study, extracellular beta-galactosidase from mutant strain Aspergillus oryzaeH26-10-7 was purified by a two step procedure, Metal-ion Affinity Chromatography (IMAC) followed by size-exclusion separation. Purified enzyme was characterized by sodium dodecyl Sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and zymographic analysis. This fungal beta-galactosidase was characterized as a protein corresponding to 113 kDa. Enzyme from mutant strain was found to have five times higher catalytic activity on the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside (ONPG) compared to the wild type enzyme. Moreover, the mutant enzyme was more thermo resistant compared to the wild type. This highly important technological characteristic can be exploited in food industry. Moreover, based on the IMAC patterns of wild type and mutant enzymes, similarities in their His topography were supposed.     

UV protectants for the biopesticide based on Bacillus sphaericus Neide and their role in protecting the binary toxins from UV radiation

The UV protectant properties of 26 natural and synthetic compounds were investigated for a biopesticide based on an indigenously isolated strain (ISPC-8) of Bacillus sphaericus Neide. In initial screening, spores of ISPC-8 with 0.1% (w/w for solid and v/w for liquid materials) concentration of different compounds were exposed to UV-B radiation (4.9 × 10⁵ J/m²) for 6 h and their spore viability and larvicidal activity were studied. The larvicidal activity was evaluated against third-instar larvae of Culex quinquefasciatus Say. There was a complete loss of spore viability (1.4% viable spores) and partial reduction in larvicidal activity (57.7% of original activity) after exposure of spores to UV-B for 6 h. However, spore viability as well as larvicidal activity protected significantly when spores were mixed with different compounds before exposing them to UV-B. Among the different compounds tested benzaldehyde, congo red, para-aminobenzoic acid (PABA) and cinnamaldehyde were found to be promising in protecting the spores from UV-B radiation. The presence of binary toxins (41.9 kDa and 51.4 kDa) in protected and unprotected samples were examined by SDS–PAGE. The binary toxin bands disappeared in unprotected spores after 24 h of exposure to UV-B, whereas toxin bands were distinctly visible when spores with benzaldehyde and cinnamaldehyde were exposed to UV-B for 96 h and 120 h, respectively. Congo red and PABA were found to be most effective in protecting binary toxins even after 168 h of exposure to UV-B. Incorporation of these promising UV protectant compounds in biopesticides would help in protecting the spores from the adverse effects of UV radiation and prolong the persistence of biopesticides under field conditions.