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Biomedical subjects

V V Kravchenko

Publications and source records attributed to V V Kravchenko.

At least 19 recordsLinked to original sources
PubMed1995

Platelet-activating factor induces NF-kappa B activation through a G protein-coupled pathway.

The capability of platelet-activating factor (PAF) to induce transcription factor activation was examined. In stably transfected Chinese hamster ovary cells expressing the PAF receptor (CHO-PAFR), PAF stimulation resulted in the nuclear expression of a DNA binding activity with specificity to the kappa B sequence. The p50 and p65 proteins, constituents of the prototypic nuclear factor kappa B (NF-kappa B), were identified as components of the DNA protein complexes by antipeptide antibodies in gel supershift as well as UV cross-linking experiments. PAF induced an initial decrease and subsequent increase of cytoplasmic I kappa B alpha levels, accompanied by up-regulation of the I kappa B alpha messenger RNA, a feature of NF-kappa B activation. PAF-induced kappa B binding activity was detected within 15 min after agonist stimulation, peaked at 30-40 min, and remained detectable by 2.5 h. SR 27417, a PAF receptor antagonist, blocked PAF-induced kappa B binding activity but not that induced by tumor necrosis factor-alpha (TNF alpha). Cholera toxin treatment markedly reduced PAF-induced kappa B binding activity, whereas pertussis toxin had no significant inhibitory effect. Neither of the two toxins affected the kappa B binding activity induced by TNF alpha in the same cells. In addition to the CHO-PAFR cells, PAF stimulated kappa B binding activity in the murine P388D1 macrophage and the human ASK.0 B cell lines that express endogenous PAF receptors. These results imply a potential role of PAF in the regulation of gene expression through a G protein-coupled transcription factor activation pathway.

Animals↗
PubMed1995

Platelet-activating factor stimulates transcription of the heparin-binding epidermal growth factor-like growth factor in monocytes. Correlation with an increased kappa B binding activity.

Human peripheral blood monocytes responded to stimulation of platelet-activating factor (PAF) with up-regulation of the transcript for heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen for vascular smooth muscle cells. This function of PAF was observed at nanomolar concentrations of the ligand, starting at 30 min after stimulation. The PAF-induced up-regulation of HB-EGF mRNA was accompanied by an increase in kappa B binding activity. These functions of PAF appeared to be mediated through the cell surface PAF receptors, as two PAF receptor antagonists, WEB 2086 and L-659,989, blocked both the up-regulation of HB-EGF mRNA and kappa B binding activity induced by PAF. The antagonists, however, had no effect on phorbol ester-induced up-regulation of HB-EGF mRNA and kappa B binding activity. Pretreatment of monocytes with pertussis toxin inhibited these functions of PAF, whereas cholera toxin had no inhibitory effect. Pyrrolidine dithiocarbamate, an inhibitor for NF-kappa B activation, markedly reduced PAF-stimulated kappa B binding activity as well as up-regulation of HB-EGF mRNA. These results suggest a potential role of PAF in HB-EGF expression and provide evidence that this stimulation may occur through increased kappa B binding activity.

Bacterial Toxins↗
PubMed1995

Murine CD14 gene expression in vivo: extramyeloid synthesis and regulation by lipopolysaccharide.

A murine model system was used to study the distribution and regulation of CD14 gene expression in vivo. Western blot analysis failed to detect CD14 in plasma from untreated CB6 (BALB/c x C57Bl6) mice, but showed markedly increased levels of CD14 in plasma from mice treated with lipopolysaccharide (LPS). Plasma levels of CD14 increased in a time- and dose-dependent manner, reaching a maximum between 8 and 16 h. Northern blot analysis of total RNA extracted from mouse tissues revealed low, but significant, levels of CD14 mRNA in many tissues of untreated animals with the highest levels in uterus, adipose tissue, and lung. After intraperitoneal injection of LPS, induction of CD14 gene expression was detected in all organs examined with the extent of induction varying between organs. Induction of CD14 mRNA was both time and dose dependent. Maximum induction in the heart and lung was observed 2-4 h after injection of LPS, while liver and kidney showed maximal induction between 8 and 16 h. In situ hybridization showed that CD14 mRNA was expressed in myeloid cells in many tissues, and that expression in these cells was upregulated by LPS. Unexpectedly, CD14 mRNA was also detected in other cells within tissues, including epithelial cells, and expression in these cell types also was upregulated by LPS. Immunochemical analysis revealed that CD14 antigen colocalized to the cytoplasm of cells expressing CD14 mRNA. These studies demonstrate that CD14 gene expression is not restricted to myeloid cells, and that the level of expression of CD14 is influenced by exposure to LPS.

Animals↗
PubMed1994

CD14 is a pattern recognition receptor.

Septic shock caused by a diverse group of bacterial pathogens is a serious human disease. Recognition of bacterial envelope constituents is one mechanism used by mammalian cells to initiate responses leading to bacterial killing or, unfortunately, responses that also cause fatal septic shock. Here we show that CD14 plays a key role in initiating cell activation by a group of bacterial envelope components from Gram-negative and Gram-positive microorganisms, as well as mycobacteria. We propose that CD14 is a receptor used by mammalian cells to recognize and signal responses to a diverse array of bacterial constituents. This finding defines the molecular basis for innate microbial immunity; implicit in these findings are new possibilities for therapeutics.

Animals↗
PubMed1990

[Model polycistron operon for studying conjugated translation in E. coli cells. The role of a stream of ribosomes].

The role of "stream" of ribosomes upon translation of polycistronic mRNAs has been studied using an artificial polycistron. It has been found that the levels of activation of cistron Ci + 1 out of two adjacent cistrons (Ci and Ci + 1) depends, in addition to earlier described effects of mutual arrangement of initiation and termination signals, also on efficiency of translation of the foregoing cistron Ci. The results obtained lead to the conclusion that in polycistronic systems the levels of translation of cistron Ci + 1 can be regulated by "stream" of ribosomes resulted from translation of the proximal cistron Ci.

Base Sequence↗
PubMed1989

Expression vector with two-step control by the cI-pR-Q-p'R-qut-t'R module of coliphage lambda.

A plasmid expression vector (pCEQ3), using temperature-regulated transcription from the p'R promoter of bacteriophage lambda, has been constructed. The vector is derived from pBR327 in which the EcoRI-ClaI fragment of plasmid DNA is replaced with a 2.2-kb DNA module cI857-pR-Q-p'R-qut-t'R, consisting of two regions of the lambda genome. The first region contains the repressor gene cI857 and promoter pR; the second one contains gene Q and the late promoter p'R. When the repressor protein, product of the cI857 gene, becomes temperature-inactivated, it allows the promoter pR to initiate the transcription of the Q gene. The product of the Q gene, in turn, acts as a positive regulator of transcription from promoter p'R. The promoter activity of pR is fully repressed at a low temperature (30 degrees C) and transcription from p'R is terminated in the absence of Q gene product, but the shift of temperature up to 37 degrees C is sufficient to make the transcription from the p'R promoter highly active. Foreign genes can be inserted into the single EcoRI site downstream from the p'R promoter. The resultant constructions express extremely high levels of the cloned gene product in Escherichia coli.

Bacteriophage lambda↗
PubMed1988

[Expression in Escherichia coli of DNA coding for human tumor necrosis factor].

The variants of expression in Escherichia coli of artificial DNA coding for human tumor necrosis factor, an important immune modulator with selective cytotoxic action on a number of transformed cell lines have been described. The DNA was placed under control of either phage M13 promoter of gene for main coat protein or tandem of pair of E. coli tryptophane promoters. It has been shown that E. coli cells harbouring plasmids described with artificial TNF gene provide good level of protein biosynthesis. The protein has been purified by anion exchange chromatography near to homogeneity and used for preparation of monoclonals. As result three hybridomas effectively produced high affinity monoclonal anti-TNF antibodies have been obtained and characterized.

Amino Acid Sequence↗
PubMed1988

[Location of the initiating codon AUG in relation to the 5'-end of mRNA mediates the effectiveness of translation in E. coli cells].

A semisynthetic gene for beta-galactosidase (lacZ) and a synthetic DNA fragment containing the "ideal" promoter sequence were used for construction of an artificial operon including translation initiation codon ATG and no SD sequence. Cloning this artificial operon into pBR322 vector resulted in a number of pV plasmids; ATG positions were varied by insertions of synthetic oligonucleotides between lacZ coding sequence and starting point of transcription. It was found that efficiency of beta-galactosidase synthesis in E. coli cells harbouring pV plasmids strongly depended on the relative position of AUG and mRNA 5'-end. High level of the synthesis was provided by translation of mRNA with AUG codon in 5'-terminal position. Amounts of synthesized beta-galactosidase diminished with increase of the distance (2, 4, and 5 nucleotides) between 5'-end of lacZ mRNA and AUG codon.

Base Sequence↗
PubMed1988

[Effectiveness of distal gene translation in polycistrons depends upon the arrangement of regulatory signals on a template].

The role of the translational terminator and initiator signals arrangement for two adjacent genes in polycistronic mRNA has been studied. Semisynthetic beta-galactosidase gene (lacZ) of E. coli and fragment of phage M13 DNA (with promoter PVIII, gene IX, and part of gene VIII) were used for constructing of the IX-VIII-lacZ artificial polycistronic operon. Cloning of the constructs into pBR322 vector resulted in a number of pLZ381N plasmids differing by the mutual arrangement of gene VIII translation terminator codon and SD site and initiator codon (SD-ATG-region) of lacZ gene. The mutual arrangement of gene VIII terminator codon and SDlacZ-ATG region has been altered by means of deletions and insertions that have not affected lacZ translation initiation signals. The beta-galactosidase (beta-Gal) synthesis in E. coli harbouring different types of pLZ381N plasmids has been found to depend on type of cistron coupling (gene VIII and lacZ). The overlapping of terminator and initiator codons (ATGA) for genes VIII and lacZ (type I of polycistrons) provide approximately equal translational level for both cistrons. On the other side, levels of beta-Gal synthesis in case of polycistrons type II (gene VIII stop-codon position at the beginning of SDlacZ or 10 nucleotides upstream) were 20-30 times as high as for type I. Differences in beta-Gal levels have also been found for variants of VIII-lacZ coupling in types IV and III polycistrons (the SDlacZ-ATG region in 27-50 nucleotides downstream from the proximal cistron VIII stop-codon, which, in turn, is 41 nucleotides upstream this terminator). These data cannot be explained on the basis of possible secondary structure including the SDlacZ-ATG region and other parts of polycistronic mRNA. In all these cases similarly stable stem-loop structures have been found. Therefore, the arrangement of the translation termination and initiation signals for two adjacent genes in essential for distal gene translation efficiency. One can imagine that ribosome or its 30S subpartical, stalling on the proximal gene terminator codon, affects the distal gene translation initiation.

Coliphages↗
PubMed1987

[Construction and properties of the expression vector based on the temperature-regulated P'R promoter in phage lambda DNA].

Plasmid expression vector using the temperature-regulated promoter P'R of bacteriophage lambda is described. The vector carries a combination of two regions of lambda cI857indgenome, that contain: 1) gene cI and promoter PR, and 2) gene Q and promoter P'R. Transcription or gene Q is initiated at promoter PR, which is controlled by cI857 repressor. The Q gene product acts as a positive regulator of RNA synthesis from P'R. At 37 degrees C, sufficient amounts of protein Q are synthesised to initiate the expression of the cloned gene from P'R. Inactivation of Q gene (by elimination of a single NcoI site) results in the loss of P'R expression activity in the vector. E. coli beta-galactosidase gene (lacZ) and human leukocyte interferon alpha 2 gene (ifn alpha 2) were cloned into a single EcoRI cleavage site under the control of P'R. These constructs express high levels of beta-galactosidase and interferon alpha 2 in E. coli at 37 degrees C.

Bacteriophage lambda↗
PubMed1987

[Formation and properties of artificial polycistrons containing truncated genes for E. coli tryptophan operon and phage M13 envelope protein].

Using gene fragments encoding the leader peptide of E. coli tryptophane operon (as duplicated fragment HhaI-140) or M13 phage coat protein (as TaqI-381 or HaeIII-1623 fragments) and basing on pDS1 family of plasmids, expression vectors have been constructed which contained transcription promoters Ptrp, PVIII, and Pv + PVIII, respectively. An artificial gene for human leukocyte interferon alpha 2 (ifn-alpha 2) has been cloned into these plasmids, so that its transcription was a part of polycistronic mRNA and preceding translation was terminated upstream to the ribosome binding site and starting codon of the interferon gene. E. coli cells harbouring these recombinant plasmids provided high level of the interferon biosynthesis. The effect of the mRNA length on the amount of protein synthesised under control of the M13 coat protein transcription-translation signals has been found.

Base Sequence↗
PubMed1987

[Duplication of a synthetic gene for human leukocyte interferon and its expression in polycistron mRNA with coupled translation system].

Using a chemically synthesised adapter, the coding part of an artificial gene for human leukocyte alpha 2 interferon (ifn-alpha 2) has been duplicated. The adapter contained a termination signal of the first gene (TAA) within the Shine-Dalgarno sequence of the second gene (TAAGGA), distance between the terminating codon and starting codon of the second gene being 11 nucleotides. In another case this distance was 69 nucleotides, with the same SD sequence. The expression of the tandems as a part of polycistrons has been studied under control of promoters Plac, (Ptrp)2 of E. coli, and PVIII of M13 phage. It was found that tandems of ifn-alpha 2 genes in polycistronic structures trp L-ifn-ifn and IX-VIII-ifn-ifn under control of promoters (Ptrp)2 and PVIII, respectively, provided high level of the interferon biosynthesis, thus differing from the tandem under Plac promoter control, which had only ifn-ifn translation coupling.

Escherichia coli↗