Ridostin induces transcription of a wide spectrum of interferon genes in human cells.

The effects of Ridostin on the transcription of IFN family genes in human fibroblasts and lymphocytes were studied by quantitative real-time PCR. The degree of gene induction by Ridostin was most pronounced in fibroblasts, and was significantly higher than the induction by Kagocel: transcription of IFN-ß, oligoadenylate synthetase, and double-stranded RNA-dependent protein kinase genes increased by about 2000, 100, and 20 times, respectively. In lymphocytes, Ridostin also activated a wide variety of IFN family genes, including genes of IFN-ß, IFN-γ, and IFN-dependent enzymes, but this induction was less pronounced than in the fibroblasts. It was shown that gene response in lymphocyte from a child with cancer is reduced in comparison with that of adult healthy participant. Ridostin, and even more so Reaferon up-regulated activities of ß-actin, glycerophosphate dehydrogenase, and ß2-microglobulin genes, thus making impossible or limiting their use as constitutive stable reference genes (standards) in PCR-assays of IFN and their inductors.

[Mutation scanning of short amplicons: optimization of DNA melting conditions].

High resolution melting analysis (HRMA) using special "saturating" fluorescent dyes is a new and very effective approach to genotyping and mutation scanning. HRMA, which is carried out usually just after PCR without any intermediate manipulations (the "closed tube" format), is simple and high-throughput method excluding sample cross-contaminations. The "closed tube" format makes, however, HRMA dependent on PCR mixes and, as such, limits its capability. The "open tube" format (post-PCR amplicon shortening and optimization of the ionic medium) proposed by us earlier, although somewhat more laborious, significantly increases sensitivity of the method and makes it possible to scan mutations in the short amplicons using conventional SYBR Green I dye and a standard (not adapted specifically for HRMA) real-time PCR instrument. Detection of mutant K-RAS in DNA of clinical specimens (tumor tissues, formalin-fixed paraffin-embedded samples) reveals equal, at least, sensitivity of this method as compared with the HRMA and much higher as compared with Sanger sequencing. The problem of false-negative results in mutation scanning of K-RAS, which is highly important in some forms of cancer, is discussed.

Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases.

The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed.

[Formation of nonculturable Mycobacterium tuberculosis and their regeneration]. / Obrazovanie "nekul'tiviruemykh" kletok Mycobacterium tuberculosis i ikh ozhivlenie.

Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long post-stationary phase. These cells were small (0.6-0.8 micron) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosis culture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.

Formation and resuscitation of "non-culturable" cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary phase.

After growth of Rhodococcus rhodochrous in Sauton's medium, and further incubation for about 60 h in stationary phase, there was a transient (up to 5 log) decrease in the c.f.u. count, whereas the total count remained similar to its initial value. At the point of minimal viability, the most probable number (MPN) count was 10 times greater than the c.f.u. count. This difference was further magnified by 3-4 logs (giving values close to the total count) by incorporating supernatant taken from growing cultures. A small protein similar to Rpf (resuscitation-promoting factor of Micrococcus luteus) appeared to be responsible for some of the activity in the culture supernatant. The formation of "non-culturable" cells of the "Academia" strain of Mycobacterium tuberculosis was similarly observed following growth in Sauton's medium containing Tween 80 in sealed culture vessels, and further incubation for an extended stationary phase. This resulted in the formation, 4-5 months post-inoculation, of a homogeneous population of ostensibly "non-culturable" cells (zero c.f.u.). Remarkably, the MPN count for these cultures was 10(5) organisms ml(-1), and this value was further increased by one log using supernatant from an actively growing culture. Populations of "non-culturable" cells of Mycobacterium tuberculosis were also obtained by the filtration of "clumpy" cultures, which were grown in the absence of Tween 80. These small cells could only be grown in liquid medium (MPN) and their viability was enhanced by the addition of culture supernatant or Rpf. The "non-culturable" cells that accumulated during prolonged stationary phase in both the R. rhodochrous and the Mycobacterium tuberculosis cultures were small ovoid and coccoid forms with an intact permeability barrier, but with undetectable respiratory activity. The authors consider these non-culturable bacteria to be dormant. The observed activity of culture supernatants and Rpf with "non-culturable" bacterial suspensions invites the speculation that one, or more, of the cognate Mycobacterium tuberculosis Rpf-like molecule(s) could be involved in mechanisms of latency and reactivation of tuberculosis in vivo.

[Restriction polymorphism in patients with lipid metabolism disorders and ischemic heart disease]. / Issledovanie restriktsionnogo polimorfizma genov apoB u bol'nykh s narusheniiami lipidnogo obmena i ishemicheskoi bolezn'iu serdtsa.

Using the RELP analysis we studied the frequency of X2 allele of apoB gene in three groups of patients: 1) men at the age of 20-59 with lipid metabolism disorders revealed in population inspection of Oktyabrsky district in Moscow; 2) men with ischaemic heart disease and 3) healthy men. It was established that in individuals suffering from type IIa hyperlipidemia the frequency of X2 allele was significantly higher than in healthy donors from Moscow population. Homozygotes for X2 allele of XbaI RELP had 7-9% higher serum cholesterol levels, than homozygotes for X1 allele. The study suggests the X2 allele of the apoB gene to be associated with the development of high plasma cholesterol level. No significant difference in X2 allele frequencies was found between patients with ischaemic heart disease and healthy donors. There was also no association found between cholesterol and triglyceride levels and the presence of X2 allele in this group of patients.

[Nucleotide sequence of a mutant allele and wild type allele SUP1 and comparison of transcripts of SUP1 and SUP2 genes]. / Nukleotidnaia posledovatel'nost' mutantnoi alleli i alleli dikogo tipa SUP1 i sravnenie transkriptov genov SUP1 i SUP2.

Nucleotide sequences of the yeast recessive suppressor gene SUP1 and one of its mutant alleles (supl-ts36) are compared. One open reading frame is found in the gene able to code 438 amino acid residues. The reading frame is not interrupted by introns. Mutant allele supl-ts36 contains one nucleotide change at position + 101 (T----C) inducing the exchange of leucine on serine in N-terminal part of the polypeptide product. The homology between the structure of SUP1 gene and two groups of proteins is found (1.tRNA-binding or nucleotide-binding proteins; 2. mitochondrial proteins coded by mitochondrial genome). The size of transcript for SUP1 gene is 1600 nucleotides corresponding to the coding region of the gene. For SUP2 gene two stable transcripts are found corresponding to approximately 2500 and approximately 1400 nucleotides. The homology between SUP1 and SUP2 genes is not found. The absence of splicing for both SUP1 and SUP2 genes transcripts is demonstrated in the experiments with RNA2 conditional mutants with impaired splicing.

Absence of structural homology between sup1 and sup2 genes of yeast Saccharomyces cerevisiae and identification of their transcripts.

The results of Southern blotting demonstrate that sup2 is a unique gene in Saccharomyces cerevisiae that does not possess homologous sequences in the yeast genome. The direct hybridization of DNA fragments, containing cloned sup1 and sup2 genes, did not reveal any structural homology between these two genes. By Northern blotting analysis the sizes of the transcripts were determined to be 1.6 kb for sup1 gene and 2.5 and 1.4kb for sup2 gene. Experiments with RNA isolated from yeast mutant with impaired splicing demonstrated that sup1 and sup2 genes do not contain introns.