The effect of zinc and/or vitamin E supplementation on biochemical parameters of selenium-overdosed rats.

The normotensive (Wistar) and spontaneously hypertensive (SHR) rats were examined to assess the response of the organism to selenium (Se) overdose. Moreover, the effect of zinc (Zn) and vitamin E, i.e. dietary components interacting in many biochemical processes with Se, on the Se uptake was evaluated. The control group was fed an untreated diet, and the diets of two other groups were overdosed with Se in the form of sodium selenite (9 mg/kg) and supplemented with Zn (13 mg/kg). Two experimental groups were fed a diet supplemented with Zn (13 mg/kg) and Se at an adequate level (0.009 mg/kg); a half of the animals was supplemented with vitamin E. The results showed significant differences in the Se contents between the rat strains in case of Se-overdosed groups, where in the liver and kidney tissue Se contents of SHR rats exceeded 3- and 7-fold the normotensive ones. The Se uptake was altered by the vitamin E; no effect of Zn was observed. Activities of antioxidant enzymes were determined in the animal tissues indicating different patterns according to rat strain, tissue analysed, and administered Se dose. Thus, Se overdose, for instance, via an incorrectly prepared dietary supplement, can result in serious imbalances of the biochemical status of the animals.

Autocrine effects of visfatin on hepatocyte sensitivity to insulin action.

Visfatin was originally described as an adipokine with insulin mimetic effects. Recently, it was found that visfatin is identical with the Nampt (nicotinamide phosphoribosyltransferase) gene that codes for an intra- and extracellular NAD biosynthetic enzyme and is predominantly expressed outside the adipose tissue. In the current study, we found strong protein and mRNA expression of visfatin in rat heart, liver, kidney, and muscle, while the expression of visfatin in visceral fat was significantly lower and undetectable in subcutaneous fat. The insulin-mimetic effects of visfatin (extracellular form of Nampt or eNampt) are controversial and even less is known about autocrine effects of visfatin (intracellular form of Nampt or iNampt). Since liver plays a major role in glucose metabolism, we studied visfatin effects on insulin-stimulated cellular glucose uptake in Fao rat hepatocytes using RNA interference (RNAi). RNAi-mediated downregulation of visfatin expression in Fao cells was associated with significantly reduced NAD biosynthesis (0.3+/-0.01 vs. 0.5+/-0.01 mmol/h/g, P<0.05) and with significantly decreased incremental glucose uptake after stimulation with insulin when compared to controls with normal expression of visfatin (0.6+/-0.2 vs. 2.2+/-0.5 nnmol/g/2 h, P=0.02). These results provide evidence that visfatin exhibits important autocrine effects on sensitivity of liver cells to insulin action possibly through its effects on NAD biosynthesis.

CD36 regulates fatty acid composition and sensitivity to insulin in 3T3-L1 adipocytes.

In the current study, we tested a hypothesis that CD36 fatty acid (FA) transporter might affect insulin sensitivity by indirect effects on FA composition of adipose tissue. We examined the effects of CD36 downregulation by RNA interference in 3T3-L1 adipocytes on FA transport and composition and on sensitivity to insulin action. Transfected 3T3-L1 adipocytes, without detectable CD36 protein, showed reduced neutral lipid levels and significant differences in FA composition when levels of essential FA and their metabolites were lower or could not be detected including gamma linolenic (C18:3 n6), eicosadienic (C20:2 n6), dihomo-gamma linolenic (C20:3 n6), eicosapentaenoic (EPA) (C20:5 n3), docosapentaenoic (DPA) (C22:5 n3), and docosahexaenoic (DHA) (C22:6 n3) FA. Transfected 3T3-L1 adipocytes exhibited a significantly higher n6/n3 FA ratio, reduced 5-desaturase and higher 9-desaturase activities. These lipid profiles were associated with a significantly reduced insulin-stimulated glucose uptake (4.02+/-0.1 vs. 8.42+/-0.26 pmol.10(-3) cells, P=0.001). These findings provide evidence that CD36 regulates FA composition thereby affecting sensitivity to insulin action in 3T3-L1 adipocytes.

Effect of protein kinase inhibitors on protein phosphorylation and germination of aerial spores from Streptomyces coelicolor.

In vitro phosphorylation reaction using extracts prepared from cells in the exponential phase of growth and aerial spores of Streptomyces coelicolor displayed the presence of multiply phosphorylated proteins. Effect of protein kinase inhibitors (PKIs) (geldanamycin, wortmannin, apigenin, genistein, roscovitine, methyl 2,5-dihydroxycinnamate, rapamycin, staurosporine) was determined on protein phosphorylation and on germination of spores. The in vitro experiments showed differences in phosphoprotein pattern due to the presence of PKIs. Cultivation of aerial spores with PKIs led to a significant delay in germ tube emergence and filament formation. However, none of the tested PKIs completely blocked the germination process. These results indicate that protein kinases of spores form complex networks sharing common modulating site that plays an important role in proper timing of early developmental events.

tmRNA abundance in Streptomyces aureofaciens, S. griseus and S. collinus under stress-inducing conditions.

tmRNA and protein SmpB are the main components required for rescue of stalled ribosomes incapable of properly elongating or terminating the polypeptide chain. We examined the tmRNA level and protein synthesis in Streptomyces aureofaciens, S. griseus and S. collinus synthesizing tetracycline, streptomycin and kirromycin, respectively, during various stress conditions. Downshift in temperature caused a decrease in protein synthesis but the level of tmRNA increased. Shift up in temperature induced decay of tmRNA in all strains and in S. collinus led to stimulation and in S. aureofaciens and S. griseus to inhibition of protein synthesis. At high NaCl concentrations protein synthesis was inhibited and tmRNA decayed. Shift in pH from 7.0 to 5.0 had no pronounced effect on the tmRNA level while upshift to pH 9.0 in S. collinus and S. aureofaciens caused inhibition of protein synthesis and decay of tmRNA in S. collinus. In contrast, protein synthesis and tmRNA level increased in S. griseus at the alkaline pH. Our data show that tmRNA abundance is important for survival of streptomycetes under certain unfavorable conditions.

Activity of translation system and abundance of tmRNA during development of Streptomyces aureofaciens producing tetracycline.

Transition from exponential phase of growth to stationary phase in Streptomyces aureofaciens is characterized by a decrease in the rate of translation and induction of tetracycline (Ttc) biosynthesis. In exponential phase, no significant changes were found in the activity of ribosomes at binding of ternary complex Phe-tRNA.EF-Tu.GTP to the A-site on ribosomes. Overexpression of Ttc in stationary phase is accompanied by a decrease in the binding of the ternary complex Phe-tRNA.EF-Tu.GTP to the A-site of ribosome and a formation of an aggregate with Ttc by part of the ribosomes. Antibiotics that cause ribosome to stall or pause could increase the requirement for tmRNA in the process called trans-translation. We found differences in the level of tmRNA during the development of S. aureofaciens. Subinhibitory concentrations of Ttc, streptomycin and chloramphenicol induced an increase in the tmRNA level in cells from the exponential phase of growth. In vitro trans-translation system of S. aureofaciens was sensitive to Ttc at a concentration of > 15 micromol/L; the trans-translation system can thus be considered to contribute to resistance against Ttc produced only at sublethal concentrations. These experiments suggest that the main role of the rising tmRNA level at the beginning of the Ttc production is connected with ribosome rescue.

Structure and functional properties of prokaryotic small noncoding RNAs.

Most biochemical, computational and genetic approaches to gene finding assume the Central Dogma and look for genes that make mRNA and have ORFs. These approaches essentially do not work for one class of genes--the noncoding RNA. In all living organisms RNA is involved in a number of essential cell processes. Functional analysis of genome sequences has largely ignored RNA genes and their structures. Different RNA species including rRNA, tRNA, mRNA and sRNA (small RNA) are important structural, transfer, informational, and regulatory molecules containing complex folded conformations that participate in recognition and catalytic processes. Noncoding RNAs play an number of important structural, catalytic and regulatory roles in the cell. The size of the sRNA genes ranges from 70 to 500 nucleotides. Several transcripts of these genes are processed by RNAases and their final products are smaller. The encoding genes are localized between two ORFs and do not overlap with ORFs on the complementary DNA strand. As aptamers, some sRNA bind small molecular components (metal ions, peptides and nucleotides). This review summarizes recent data on the functions of prokaryotic sRNAs and approaches to their identification.

Changes in ribosome function induced by protein kinase associated with ribosomes of Streptomyces collinus producing kirromycin.

Protein kinase associated with ribosomes of streptomycetes phosphorylates 11 ribosomal proteins. Phosphorylation activity of protein kinase reaches its maximum at the end of exponential phase of growth. When (32)P-labeled cells from the end of exponential phase of growth were transferred to a fresh medium, after 2 h of cultivation ribosomal proteins lost more than 90% of (32)P and rate of polypeptide synthesis increases twice. Protein kinase cross-reacting with antibody raised against protein kinase C was partially purified from 1 M NH(4)Cl wash of ribosomes and used to phosphorylation of ribosomes. Phosphorylation of 50S subunits (L2, L3, L7, L16, L21, L23, and L27) had no effect on the integrity of subunits but affects association with 30 to 70S monosomes. In vitro system derived from ribosomal subunits was used to examine the activity of phosphorylated 50S at poly(U) translation. Replacement unphosphorylated 50S with 50S possessed of phosphorylated r-proteins leads to the reduction of polypeptide synthesis of about 52%. The binding of N-Ac[(14)C]Phe-tRNA to A-site of phosphorylated ribosomes is not affected but the rate of peptidyl transferase is more than twice lower than that in unphosphorylated ribosomes. These results provide evidence that phosphorylation of ribosomal proteins is involved in mechanisms regulating the translational system of Streptomyces collinus.

Characterization of the rplB gene from Streptomyces collinus and its protein product by mass spectrometry.

Ribosomal protein L2 is the largest protein components of 50S subunits. The protein is implicated in peptidyl transferase activity and binds to functionally important domains of 23S rRNA. The rplB gene, which codes for ribosomal protein L2 was cloned from Streptomyces collinus. The gene rplB was isolated from BamHI fragment (3.0 kb) of chromosomal DNA possessing two partial and four complete ORF's in the order from 5' to 3': rplC, rplD, rplW, rplB, rpsS, and rplV. The gene organization corresponds to the S10 operon. Gene rplB (834 bp) encodes a polypeptide chain of 278 amino acids. The molecular mass calculated from genomic structure is 30.5 kDa and pI 11.87. Protein L2 is rich in positively charged amino acids (Arg 36, Lys 20, and His 11). N-terminal domain possesses topology similar to the oligonucleotide/oligosaccharide binding OB folds. The availability of genome sequence makes it possible to identify L2 protein by mass spectrometry, moreover it facilitates the characterization of its potential posttranslational modifications. To confirm the protein sequence derived from the rplB gene the tryptic peptides of L2 were analyzed by mass spectrometric techniques. The obtained data matched exactly with the results of DNA sequencing.

Isolation and characterization of dcw cluster from Streptomyces collinus producing kirromycin.

A 4.5-kb BamHI fragment of chromosomal DNA of Streptomyces collinus containing gene ftsZ was cloned and sequenced. Upstream of ftsZ are localized genes ftsQ, murG, and ftsW, and downstream is yfiH. Gene ftsA is not adjacent to ftsZ or other genes of the cloned fragment. Protein FtsZ was isolated and characterized with respect to its binding to GTP and GTPase activity. The binding of GTP to FtsZ was Ca(2+) or Mg(2+) dependent with an optimum at 10 mM. The rate of GTP hydrolysis by FtsZ was stimulated by KCl. The presence of Ca(2+) (3-5 mM) resulted in a significant increase of GTPase activity. Higher concentrations of Ca(2+) than 5 mM had an inhibitory effect on GTPase activity. These results indicate that divalent ions (Ca(2+) or Mg(2+)) can be involved in regulation of GTP binding and hydrolysis of FtsZ. The maximum level of FtsZ was detected in aerial mycelium when spiral loops and sporulation septa were formed. FtsZ is degraded after finishing sporulation septa.

Protein kinase associated with ribosomes of streptomycetes.

Protein kinases can be classified into two main superfamilies on the basis of their sequence similarity and substrate specificity. The protein His kinase superfamily which autophosphorylate a His residue, and superfamily Ser/Thr and Tyr protein kinases, which phosphorylate Ser, Thr or Tyr residues. During the last years genes encoding Ser/Thr protein kinases have been identified in several microorganisms. Phosphorylation of proteins on Ser/Thr residues can be involved in many functions of prokaryotic cells including cell differentiation, signal transduction and protein biosynthesis. Phosphorylation of prokaryotic protein-synthesizing systems showed that the phosphorylation of initiation and elongation factors is subject to alteration during cell differentiation or bacteriophage infection. Protein kinase associated with ribosomes of streptomycetes phosphorylate the elongation factor Tu and 11 ribosomal proteins even in bacteriophage-uninfected cells. After phosphorylation of ribosomal proteins, ribosomes lose about 30% of their activity at the translation of poly(U).

Expression of the Csp protein family upon cold shock and production of tetracycline in Streptomyces aureofaciens.

A shift down in temperature causes in Streptomyces aureofaciens a transient repression of polypeptide synthesis. During the acclimation phase 32 proteins were synthesized. The addition of tetracycline (200 microg/ml) to cells from exponential phase of growth leads to induction of 27 novel proteins and 17 upregulated proteins migrated in 2-D gel as proteins expressed upon cold shock. Immunoblot analysis using antibodies raised against CspB, CspC, and CspD of Bacillus subtilis revealed five cross-reactive proteins of the Csp family. Proteins CspB and CspD are predominantly induced at low temperature or by the presence of tetracycline. Expression of Csp proteins during the acclimation phase is regulated on the transcription level. Proteins of the Csp family have been shown to be associated with ribosomes and can be removed by 1 M NH(4)Cl. As expression of Csp proteins differs during development or temperature shift down, these proteins can be considered as trans-acting factors to form contacts with the coding region of specific mRNAs.

The role of GTP-binding proteins in mechanochemical movements of microorganisms and their potential to form filamentous structures.

Prokaryotic cells contain proteins which form extended chains or multimers that oscillate between monomers and oligomers of varying length. Hydrolysis of nucleoside triphosphates combined with site-specific disposition of substrates and products to monomers and multimers is the driving force of dynamic instability of these molecules. Polymeric structures are connected in some manner to a variety of signaling systems that adhere to the polymeric matrix, including the GTP-binding protein(s), protein kinases and phosphatases, and other proteins or systems that communicate between the cytoplasmic membrane and the cytosol. Flexible organization allowing regulated dynamic movement is one of the key elements in all living cells. In eukaryotic cells actin and tubulin are the two main components of dynamically controlled spatial system. These proteins are noteworthy for their ability to polymerize, reversibly, into filaments or microtubules in association with hydrolysis of ATP or GTP, respectively. As such, they regulate most of the mechanics of cell movement including cell division, cell differentiation, phagocytosis and other dynamic phenomena. Recent evidence revealed that microbial cells create functional domains at specific sites of the cells and can form cytoplasmic tubules and fibers.

Characterization of ftsZ gene and its protein product from Streptomyces collinus producing kirromycin.

The FtsZ protein is required for septation and conversion of aerial mycelium into chains of streptomycete spores. We have cloned and sequenced the ftsZ gene from Streptomyces collinus. The ftsZ of S. collinus is not in juxtaposition with ftsA and IpxC as in Escherichia coli or Bacillus subtilis. The gene encodes a polypeptide of 402 amino acid residues with a molecular mass of 41.3 kDa. N-terminus shares a high level of sequence similarity with FtsZ of S. coelicolor and S. griseus, respectively. C-terminal part is variable both in length and sequence. The purified protein binds GTP. Using polyclonal antisera against FtsZ, we have found that the protein is expressed at the beginning of germination of spores and is present in vegetative cells and aerial mycelium, but not in spores.

Pattern of phosphoproteins during cell differentiation in Streptomyces collinus.

Transition from vegetative cells to aerial mycelium and spores of Streptomyces collinus is accompanied by changes in the pattern of proteins phosphorylated. Preparation from spores exhibits lower phosphorylation activity than those of vegetative cells and aerial mycelium. Phosphorylation of proteins from aerial mycelium was markedly stimulated by the presence of Mn2+. Our data indicate that phosphorylation of proteins on Ser/Thr residues is involved in transition of vegetative cells to aerial mycelium.

Protein kinase associated with ribosomes phosphorylates ribosomal proteins of Streptomyces collinus.

Protein kinase activity associated with ribosomes of a kirromycin-producing strain of Streptomyces collinus was detected. The enzyme utilizes [gamma-32P]ATP to phosphorylate proteins, yielding acid-stable phosphoamino acids. Two-dimensional electrophoresis of proteins from a crude ribosomal fraction revealed 17 phosphoproteins. Eleven of the phosphoproteins exhibited electrophoretic mobility identical to that of S. collinus ribosomal proteins S3, S4, S12, S13, S14, S18, L2, L7, L16, L17, and L23. Protein L2 was identified by microsequencing of internal peptide fragments. Immunodetection with monoclonal antibodies indicated that the ribosomal proteins are phosphorylated on serine and threonine residues. Phosphorylation of ribosomal proteins led to the reduction of activity of ribosomes in the translation of poly(U). These results provide the first evidence of phosphorylation of ribosomal proteins in bacteriophage-uninfected cells of eubacteria.

Sequencing of the tuf1 gene and the phosphorylation pattern of EF-Tu1 during development and differentiation in Streptomyces collinus producing kirromycin.

We have cloned and sequenced the tuf1 gene from a kirromycin-producing strain of Streptomyces collinus. The gene encodes a polypeptide of 396 amino acid residues with a molecular weight of 43,849. The protein shows 97% identity with EF-Tu1 of S. coelicolor and is sensitive to kirromycin. EF-Tu-dependent translation of poly(U) was reduced to 50% in the presence of 0.25 microM kirromycin. Using high resolution two-dimensional electrophoresis and specific immunodetection with monoclonal antibodies we found that the EF-Tu1 is phosphorylated on threonine and that serine is the second phosphate-accepting amino acid. EF-Tu1 phosphorylated on threonine and serine residues was detected among the S150 supernatant proteins of vegetative cells, aerial mycelium and spores. The level of phosphorylated EF-Tu1 varied during the growth and differentiation.

Translation initiation factors of a tetracycline-producing strain of Streptomyces aureofaciens.

Protein synthesis initiation factors from a tetracycline-producing strain of Streptomyces aureofaciens were purified and characterized. Two forms of IF3 (M(r) = 24,000 and 22,500) were found. By Western blot analysis, only one form of protein IF2 cross-reactive with anti-IF2 of Escherichia coli was revealed. The molecular mass of purified IF2 was 69,000 as determined by SDS-polyacrylamide gel electrophoresis. In spite of differences in molecular mass between the IF2 forms of E. coli and the factor from S. aureofaciens, the latter could substitute IF2 of E. coli in the stimulation of codon-specific binding of initiator tRNA. In contrast to the reported absence of IF1 in some Gram-positive microorganisms, we found "protein IF1" (M(r) = 9,000) in S. aureofaciens that increased the IF2-dependent binding of initiator tRNA to ribosomes.

GroEL-like protein complex of thermophilic bacterium Thermus aquaticus.

GroEL-like particles of Thermus aquaticus are homo-oligomeric complexes of two stacked seven member rings, sedimenting in the gradient at 20S. The apparent molecular mass of the native particles is 820,000 (+/- 30,000). The protein complex is composed with one polypeptide of M(r) 59,000. Immunoblotting results and N-terminal amino acid analysis indicate that the complex is significantly related to the chaperonins. No proteolytic activity was identified in the purified GroEL-like particles. In the presence of Mg2+ and K+ the complex exhibits temperature dependent ATPase activity. Under optimum temperature (75 degrees C) GroEL is stable and hydrolyze ATP with a specific activity of 0.47 mumol min-1 mg-1.

Ribulose-1,5-bisphosphate carboxylase of thermophilic hydrogen-oxidizing microorganism Bacillus schlegelii.

Ribulose-1,5-bisphosphate carboxylase was isolated from thermophilic hydrogen-oxidizing Bacillus schlegelii. Molecular mass of the native enzyme is 560,000 and optimal reaction temperature is 70 degrees C. Km value for ribulose 1,5-bisphosphate is 0.27 mM. The carboxylase activity of the enzyme is dependent on Mg2+ with the optimum at 10 mM. The enzyme is an oligomer of L8S8 type with Mr of large subunits and small subunits of 56,000 and 14,000, respectively. Negatively stained enzyme has regular polygonal shape in top view, 12 nm in diameter, with central electron dense patch.