Effects of small heat shock proteins on the thermal denaturation and aggregation of F-actin

Effect of recombinant chicken small heat shock protein with molecular mass 24 kDa (Hsp24) and recombinant human small heat shock protein with molecular mass 27 kDa (Hsp27) on the heat-induced denaturation and aggregation of skeletal F-actin was analyzed by means of differential scanning calorimetry and light scattering. All small heat shock proteins did not affect thermal unfolding of F-actin measured by differential scanning calorimetry, but effectively prevented aggregation of thermally denatured actin. Small heat shock protein formed stable complexes with denatured (but not with intact) F-actin. The size of these highly soluble complexes was smaller than the size of intact F-actin filaments. It is supposed that protective effect of small heat shock proteins on the cytoskeleton is at least partly due to prevention of aggregation of denatured actin.     

Heat shock protein (hsp90) interacts with smooth muscle calponin and affects calponin-binding to actin

Interaction of smooth muscle calponin with 90 kDa heat shock protein (hsp90) was analyzed by means of native gel electrophoresis and affinity chromatography. Under conditions used, calponin and hsp90 form a complex with an apparent dissociation constant in the micromolar range. The major hsp90-binding site is located in the N-terminal (residues 7-144) part of calponin. Addition of calponin to actin-tropomyosin complex results in formation of actin bundles. Hsp90 partially prevents bundle formation without affecting the molar ratio calponin/actin in single actin filaments or actin bundles. At low ionic strength, calponin induces polymerization of G-actin. Hsp90 decreases calponin-induced polymerization of G-actin. It is supposed that hsp90 may be involved in the assembly of actin filaments.     

Sequence analysis of hexon gene from adenovirus KR95 inducing hydropericardium syndrome in chickens

The nucleotide sequence of a part of the HindIII-D fragment (3300 b.p.) of adenovirus KR95 DNA has been determined. Analysis of the nucleotide sequence disclosed a continuous ORF for hexon gene (2814 b.p.) coding the 937 residue protein, part of ORF for the C-terminal region of pVI polypeptide, including 114 residues and the beginning of ORF coding 25 N-terminal residues for viral endoproteinase. Comparison of predicted KR95 hexon sequence and 8 mammalian and avian adenovirus hexon sequences revealed the highest homology between KR95 strain and avian adenoviruses FAV10 and FAV1 (91.1 and 80.1%, respectively). The results were used for creating a test system on the basis of the polymerase chain reaction. The system was used in analysis of fowl samples obtained from 12 poultry farms in Russia. The sequences of hexon gene amplified fragments in the isolated strains and similar fragments of other mammalian and avian adenoviruses have been compared.     

Real-time expression profiling of microRNA precursors in human cancer cell lines

Our previous study described a real-time PCR method to quantify microRNA (miRNA) precursors using SYBR green detection [T. D. Schmittgen, J. Jiang, Q. Liu and L. Yang (2004) Nucleic Acids Res., 32, e43]. The present study adapted the assay to a 384-well format and expanded it to include primers to 222 human miRNA precursors. TaqMan minor groove binder probes were used to discriminate nearly identical members of the let-7 family of miRNA isoforms. The miRNA precursor expression was profiled in 32 human cell lines from lung, breast, colorectal, hematologic, prostate, pancreatic, and head and neck cancers. Some miRNA precursors were expressed at similar levels in many of the cell lines, while others were differentially expressed. Clustering analysis of the miRNA precursor expression data revealed that most of the cell lines clustered into their respective tissues from which each cell line was ostensibly derived. miRNA precursor expression by PCR paralleled the mature miRNA expression by northern blotting for most of the conditions studied. Our study provides PCR primer sequences to all of the known human miRNA precursors as of December 2004 and provides a database of the miRNA precursor expression in many commonly used human cancer cell lines.     

Use of Trinitrophenylation for Quantification of Protease and Peptidase Activities

A sensitive and precise method for quantifying protease and peptidase activities is suggested. N-Terminal amino groups of peptides which are formed during hydrolysis of the substrates react with trinitrobenzenesulfonic acid (TNBS), and the trinitrophenyl (TNP) derivatives are determined spectrophotometrically. Spontaneous hydrolysis of TNBS is considerably diminished on trinitrophenylation at pH 7.4 rather than at pH 9-10 as is usually used. The trinitrophenylation method can be used to determine the initial rate of hydrolysis and the kinetics of reactions catalyzed by proteases and peptidases.     

A source of energy for survival and amplification of heterotrophic microorganisms in the absence of organic substrate. I. Formulation of a hypothesis

The dynamic characteristics of populations of heterotrophic microorganisms Escherichia coli incubated in a medium devoid of organic substrate were studied. It was found that the population dynamics is related to the electromagnetic environment. A mathematical and physical models of cell multiplication in a medium free of organic substrate were constructed. In these models the natural electromagnetic background near the Earth surface serves as a source of free energy.     

Hydrolysis of myelin basic protein by polyclonal catalytic IgGs from the sera of patients with multiple sclerosis

Various catalytic antibodies or abzymes have been detected recently in the sera of patients with several autoimmune pathologies, where their presence is most probably associated with autoimmunization. Recently we have shown that DNase, RNase, and polysaccharide-hydrolyzing activities are associated with IgGs from the sera of patients with multiple sclerosis (MS). Here we present evidence demonstrating that highly purified MS IgGs (but not Igs from the sera of healthy individuals) catalyze specifically hydrolysis of human myelin basic protein (hMBP). In contrast to many known proteases, IgGs do not hydrolyze many other different proteins. Specific inhibitors of acidic and thiol proteases have no remarkable effect on proteolytic activity of IgGs. However, specific inhibitor of serine (PMSF, AEBSF, and benzamidin) and metal-dependent (EDTA) proteases significantly inhibit activity of proteolytic abzymes. Interestingly, the ratio of serine-like and metal-dependent activities of MS IgGs varied very much from patient to patient. The findings speak in favor of the generation by the immune systems of individual MS patients of a variety of polyclonal anti-MBP IgGs with different catalytic properties.     

Structure and properties of small heat shock proteins (sHsp) and their interaction with cytoskeleton proteins

The modern classification of small heat shock proteins (sHsp) is presented and peculiarities of their primary structure and the mechanism of formation of oligomeric complexes are described. Data on phosphorylation of sHsp by different protein kinases are presented and the effect of phosphorylation on oligomeric state and chaperone activity of sHsp is discussed. Intracellular location of sHsp under normal and stress conditions is described and it is emphasized that under certain condition sHsp interact with different elements of cytoskeleton. The literature concerning the effect of sHsp on polymerization of actin in vitro is analyzed. An attempt is made to compare effects of sHsp on polymerization of actin in vitro with the results obtained on living cells under normal conditions and after heat shock or hormone action. The literature concerning possible effects of sHsp on cell motility is also analyzed.