Purification and concentration of alkaline phosphatase by selective permeabilization of Escherichia coli using reverse micellar solutions

Recovery of alkaline phosphatase (AP) from the periplasm of Escherichia coli using reverse micellar solutions (RMSs) of sodium dioctyl sulfosuccinate (AOT) in aliphatic hydrocarbons has been attempted. A variety of surface-active agents, solvents, and reverse micellar conditions were screened, and an excellent recovery of the enzyme in a concentrated form, with a high purification factor, was obtained in a single-step process. The permeabilization process strongly depended on the water content of the RMS as well as on the amount of water coating the microbial cell surface. The product was almost free from nucleic acids. In addition, because of the low affinity of AOT and the organic solvent for the aqueous phase, contamination by the permeabilizing agents would also be negligible.     

Polyamine-assisted rapid and clean cleavage of oligonucleotides from cis-diol bearing universal support

Polyamine-assisted deprotection conditions have been developed for the rapid and clean cleavage of oligonucleotide chains from a cis-diol group bearing universal polymer support, making it compatible with modern oligonucleotide synthesis via all types of phosphoramidite synthons, including base labile protecting group bearing synthons as well. The synthesized oligonucleotides were found to be comparable with the corresponding standard oligomers with respect to their retention time on HPLC, mass on MALDI-TOF and biological activity in PCR amplification.     

Molecular pathology of dityrosine cross-links in proteins: Structural and functional analysis of four proteins

The dityrosine bond (DT) is an oxidative covalent cross-link between two tyrosines. DT cross-linking is increasingly identified as a marker of oxidative stress, aging and disease, and has been detected in diverse pathologies. While DT cross- linked proteins have been documented, the consequences of the DT link on the structure and function of the so modified proteins are yet to be understood. With this in view, we have studied the properties of intermolecular DT-dimers of four proteins of diverse functions, namely the enzyme ribonuclease A, the signal protein calmodulin, and the eye lens proteins alpha- and gamma B-crystallins. We find that DT is formed through radical reactions and type I photosensitization (including OH, O₂ ^– and OONO^–), but not by ¹O₂ and NO₂ ^– (which modify his, trp and met more readily). Tyr residues on the surface of the protein make DT bonds (intra- and intermolecular) most readily and preferentially. The conformation of each of these DT-dimers, monitored by spectroscopy, is seen not to be significantly altered in comparison to that of the parent monomer, but the structural stability of the DT cross-linked molecule is lower than that of the parent native monomer. The DT-dimer is denatured at a lower temperature, and at lower concentrations of urea or guanidinium chloride. The effect of DT-cross-linking on the biological activities of these proteins was next studied. The enzymatic activity of the DT-dimer of ribonuclease A is not lost but lowered. DT-dimerization of lens alpha-crystallin did not significantly affect the chaperone-like ability; it inhibits the self-aggregation and precipitation of target proteins just as well as the parent, unmodified alpha-crystallin does. DT-dimerization of gamma B-crystallin is however seen to lead to more ready aggregation and precipitation, a point of interest in cataract. In the case of calmodulin, we could generate both intermolecular and intramolecular DT cross-linking, and study both the DT-dimer and DT-monomer. The DT-dimer binds smooth muscle light chain kinase and also Ca²⁺, but less efficiently and over a broad concentration range than the native monomer. The intramolecular DT-monomer is weaker in all these respects, presumably since it is structurally more constrained. These results suggest that DT cross-linking of globular proteins weakens their structural stability and compromises (though does not abolish) their biological activity, both of which are pathologically relevant. The intramolecular DT cross-link would appear to lead to more severe structural and functional consequences.     

Molecular pathology of dityrosine cross-links in proteins: structural and functional analysis of four proteins

The dityrosine bond (DT) is an oxidative covalent cross-link between two tyrosines. DT cross-linking is increasingly identified as a marker of oxidative stress, aging and disease, and has been detected in diverse pathologies. While DT cross- linked proteins have been documented, the consequences of the DT link on the structure and function of the so modified proteins are yet to be understood. With this in view, we have studied the properties of intermolecular DT-dimers of four proteins of diverse functions, namely the enzyme ribonuclease A, the signal protein calmodulin, and the eye lens proteins alpha- and gamma B-crystallins. We find that DT is formed through radical reactions and type I photosensitization (including .OH, O2- and OONO-), but not by 1O2 and NO, (which modify his, trp and met more readily). Tyr residues on the surface of the protein make DT bonds (intra- and intermolecular) most readily and preferentially. The conformation of each of these DT-dimers, monitored by spectroscopy, is seen not to be significantly altered in comparison to that of the parent monomer, but the structural stability of the DT cross-linked molecule is lower than that of the parent native monomer. The DT-dimer is denatured at a lower temperature, and at lower concentrations of urea or guanidinium chloride. The effect of DT-cross-linking on the biological activities of these proteins was next studied. The enzymatic activity of the DT-dimer of ribonuclease A is not lost but lowered. DT-dimerization of lens alpha-crystallin did not significantly affect the chaperone-like ability; it inhibits the self-aggregation and precipitation of target proteins just as well as the parent, unmodified alpha-crystallin does. DT-dimerization of gamma B-crystallin is however seen to lead to more ready aggregation and precipitation, a point of interest in cataract. In the case of calmodulin, we could generate both intermolecular and intramolecular DT cross-linking, and study both the DT-dimer and DT-monomer. The DT-dimer binds smooth muscle light chain kinase and also Ca2+, but less efficiently and over a broad concentration range than the native monomer. The intramolecular DT-monomer is weaker in all these respects, presumably since it is structurally more constrained. These results suggest that DT cross-linking of globular proteins weakens their structural stability and compromises (though does not abolish) their biological activity, both of which are pathologically relevant. The intramolecular DT cross-link would appear to lead to more severe structural and functional consequences.     

Effect of amino acids and vitamins on laccase production by the bird's nest fungus Cyathus bulleri

Various amino acids, their analogues and vitamins have shown stimulatory as well as inhibitory effects on laccase production by Cyathus bulleri. DL-methionine, DL-tryptophan, glycine and DL-valine stimulated laccase production, while L-cysteine monohydrochloride completely inhibited the enzyme production. Among vitamins tested biotin, riboflavin and pyridoxine hydrochloride were found to induce laccase production.     

A CD45 polymorphism associated with abnormal splicing is absent in African populations

The CD45 antigen is essential for normal antigen receptor-mediated signalling in lymphocytes, and different patterns of splicing of CD45 are associated with distinct functions in lymphocytes. Abnormal CD45 splicing has been recognized in humans, caused by a C77G transversion in the gene encoding CD45 (PTPRC). Recently the C77G polymorphism has been associated with multiple sclerosis and increased susceptibility to HIV-1 infection. These studies suggest that the regulation of CD45 splicing may be critical for the proper function of the immune system. Because of these data we examined the frequency of the C77G allele in African and Asian populations from countries with high or low prevalence of HIV infection. Here we report that the variant CD45 C77G allele is absent in African populations. We further show that populations living in the Pamir mountains of Central Asia have a very high prevalence of the C77G variant.     

Impact of lead pollution on the status of other trace metals in blood and alterations in hepatic functions

Lead pollution and its impact on the status of four other trace elements—Fe, Zn, Br, and Rb—have been studied in the whole blood samples of different population groups employing energy dispersive X-ray fluorescence technique. These population groups included normal, automobile workers and lead battery manufacturers. The maximum increase in the concentration of trace elements in the blood samples of automobile workers and battery manufacturers was observed for Pb, when compared with normal Pb-B levels. The effect of lead pollution had significantly reduced Zn levels in automobile workers. Fe-B levels in automobile workers had been found to be reduced significantly as compared to control, whereas in battery workers the reduction was not significant. The concentration of Br was greatly enhanced in the blood samples of automobile workers, whereas Rb-B levels were significantly higher in both the automobile and battery workers. Oral administration of lead acetate (100 mg/kg body wt) to experimental rats significantly decreased the activities of hepatic transaminases after 3 and 4 mo of treatment, whereas the activity of hepatic alkaline phosphatase decreased significantly after 4 mo of treatment. It is concluded from this study that higher Pb-B levels greatly influence the levels of other trace elements in human blood samples and also the activities of hepatic transaminases as well as alkaline phosphatase in experimental rats.