Tolerance of 5,10-methenyltetrahydrofolate to ultraviolet radiation

Aeration of aqueous solutions of 5,10-methenyltetrahydrofolic acid (MTHF) during exposure to ultraviolet irradiation (lambda 300-390 nm, 240 W/m2, 30 min) slowed down photolysis in comparison with deaerated solutions. The rate of photolysis in the presence of oxygen depended on buffer composition. It did not exceed 6% of the starting amount of MTHF. Photolysis of MTHF included opening of the imidazoline ring, dehydrogenation of the tetrahydropterin portion, and elimination of the p-aminobenzoylglutamate moiety. 6,7-Dimethyltetrahydropterin was used as a model compound to show that protonation of the reduced pterin heterocycle increased its tolerance to oxidation, and UV irradiation did not accelerate this process. The stabilizing effect of protonation of the pterin portion and the presence of the positively charged imidazoline moiety are assumed to hamper MTHF oxidation and photolysis. It is assumed that these factors favored the choice of MTHF molecules as photosensors in light-sensitive proteins in the course of evolution.     

The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier?

Sepsis-induced vascular leakage is a major underlying cause of the respiratory dysfunction seen in severe sepsis. Here, we studied the role of MLC phosphorylation in LPS-induced endothelial hyperpermeability and assessed how the changes in phospho-MLC distribution affect LPS-induced barrier dysfunction. We demonstrated that the changes in human lung microvascular endothelial permeability are preceded by the increase in intracellular calcium level, and increase in MYPT and MLC phosphorylation. Using the siRNA approach, we showed that both LPS-induced barrier dysfunction and MLC phosphorylation are attenuated by the depletion of the smooth muscle isoform of MLC kinase (MLCK) and Rho kinase 2 (ROCK2). Surprisingly, pharmacological inhibition of both ROCK1 and 2 with Y-27632 exacerbated LPS-induced drop in transendothelial resistance, although significantly decreasing MLC phosphorylation level. We next studied the involvement of protein kinase A (PKA)-dependent pathways in LPS-induced barrier dysfunction. We showed that LPS decreased the level of PKA-dependent phosphorylation in endothelial cells; and the pretreatment with forskolin or PKA activator bnz-cAMP counteracted this effect. Forskolin and bnz-cAMP also attenuated LPS-induced increase in MLC phosphorylation level. As we have shown earlier (Bogatcheva et al., 2009), forskolin and bnz-cAMP provide protection from LPS-induced barrier dysfunction. We compared the effects of bnz-cAMP and Y-27632 on phospho-MLC distribution and observed that while bnz-cAMP increased the association of the phospho-MLC signal with the cortical structures, Y-27632 decreased this association. These data indicate that an overall decrease in MLC phosphorylation could be either beneficial or detrimental to endothelial barrier, depending on the intracellular locale of major phospho-MLC changes.     

A Biochip for Genotyping Polymorphisms Associated with Eye,Hair, Skin Color,AB0 Blood Group,Sex, Y Chromosome Core Haplogroup,and Its Application to Study the Slavic Population

Molecular Biology - This paper presents a method for genotyping a panel of 60 single nucleotide polymorphisms (SNPs) using single-stage PCR followed by hybridization on a hydrogel biochip. The pool...     

Characteristics of the filterable forms of Mycobacterium tuberculosis and their significance in pathology

The results of the present investigation indicate that antituberculosis therapy for a period of 6 months leads to qualitative changes in M. tuberculosis population. This is manifested by the appearance of the filterable forms of M. tuberculosis in pathological material. At the same time these forms retain the initial pathogenicity of M. tuberculosis and induce not only tuberculous, but also nonspecific inflammation. Among the population of these filterable forms organisms carrying the genetic information of the species and capable of replication processes have been detected.     

Regulation of the activity of rabbit skeletal muscle phosphorylase kinase by glycogen

The effects of glycogen on the non-activated and activated forms of phosphorylase kinase were studied. It was found that in the presence of glycogen the activity of non-activated kinase at pH 6.8 and 8.2 and that of the activated (in the course of phosphorylation) form are enhanced. The degree of activation depends on glycogen concentration. At saturating concentrations, this enzyme activity increases 2-3-fold; the enzyme affinity for the protein substrate, phosphorylase b, also shows an increase. The polysaccharide has no effect on the activity of phosphorylase kinase stimulated by limited proteolysis. In the presence of glycogen, the rate of autocatalytic phosphorylation of the enzyme is increased. Glycogen stabilizes the enzyme activity upon dilution. The experimental results suggest that the polysaccharide directly affects the phosphorylase kinase molecule. The maximal binding was shown to occur at the enzyme/polysaccharide ratio of 1:10 (w/w) in the presence of Ca2+ and Mg2+.     

The role of phosphorylase kinase subunits in interaction with glycogen

The interaction of rabbit skeletal muscle phosphorylase kinase with CNBr-activated glycogen results in the formation of a covalent complex. The non-bound kinase was removed by chromatography on DEAE-cellulose and phenyl-Sepharose. The amount of the bound protein increased with an increase in the number of activated groups in the glycogen molecule; the enzyme activity was thereby decreased. The kinase covalently and non-covalently bound to glycogen exhibited a higher affinity for the protein substrate (phosphorylase b) as well as for Mg2+ and Ca2+ than did the kinase in the absence of glycogen. Electrophoresis performed under denaturating conditions showed that the gamma-subunit of phosphorylase kinase is responsible for the enzyme binding to CNBr-glycogen. The effect of cross-linking reagents (glutaric aldehyde, 1.5-difluoro-2.4-dinitrobenzene) on the binding of phosphorylase kinase subunits was studied. Glycogen afforded protection of the gamma-subunit from the cross-linking to other enzyme subunits. An analysis of the subunit composition of phosphorylase kinase covalently bound to CNBr-glycogen and of the enzyme treated with cross-linking reagents in the presence of glycogen-revealed that the gamma-subunit is involved in the specific binding of phosphorylase kinase to glycogen.