[Mutation analysis of functional role of amino acid residues in domain IV of elongation factor G].

Seven variants of elongation factor G (EF-G) from Thermus thermophilus with mutations Glu494Ile, Gly495Asp, Lys496Ile, His509Leu, Lys564Ile and Tyr568Lys located in the beta-sheet of its domain IV and mutation Gly553Asp in a loop between domain III and IV were constructed using polymerase chain reaction. Functional tests demonstrated that only mutation Lys496Ile, located in the vicinity of the loop 501-504, inhibits translocation effectiveness, in the presence of the mutated EF-G. The functional analysis of all mutations constructed up to now in domain IV reveals that only those located in loops 501-504 and 573-578 markedly decrease the translocation activity of EF-G. These loops are located at the tip of domain IV and close to the decoding center of the 30S ribosomal subunit upon EF-G interaction with the ribosome. The functional role of EF-G and its domain IV in ribosomal translocation is discussed.

Antioxidants attenuate early up regulation of retinal vascular endothelial growth factor in streptozotocin-diabetic rats.

AIMS/HYPOTHESIS: A strong positive correlation has been found between lipid peroxidation product and vascular endothelial growth factor concentrations in the vitreous of patients with proliferative diabetic retinopathy. To establish a causal relation between diabetes-associated enhanced oxidative stress and vascular endothelial growth factor production, we evaluated two antioxidants, DL-alpha-lipoic acid and taurine, on retinal vascular endothelial growth factor protein and mRNA expression and on parameters of oxidative stress in streptozotocin-diabetic rats. METHODS: Our experiments were on control rats and streptozotocin-diabetic rats with a 6-week duration of diabetes, treated with or without DL-alpha-lipoic acid (100 mg x kg(-1) x d(-1), i.p.) or taurine (1% in the diet) starting from induction of diabetes. Vascular endothelial growth factor protein in retinal homogenates was assessed by sandwich ELISA with an affinity-purified polyclonal antibody and vascular endothelial growth factor mRNA by ribonuclease protection assay. Retinal lipid peroxidation products i.e. malondialdehyde plus 4-hydroxyalkenals were quantified with N-methyl-2-phenylindole. Retinal reduced and oxidized glutathione, ascorbate, dehydroascorbate, and sorbitol pathway intermediates were measured spectrofluorometrically, and taurine by reverse-phase HPLC. RESULTS: Vascular endothelial growth factor protein concentration (means +/- SD) was increased in diabetic rats compared with control rats (33+/-7 vs 19+/-5 pg/mg total protein, p < 0.01) This increase was attenuated by taurine (26+/-8, p < 0.05) and prevented by DL-alpha-lipoic acid (21+/-4, p < 0.01). Vascular endothelial growth factor mRNA abundance was reduced by 1.4-fold in diabetic rats compared with control rats and this decrease was attenuated but not completely prevented by both antioxidants. Malondialdehyde plus 4-hydroxyalkenal concentration was increased in diabetic rats compared with control rats, and both antioxidants arrested accumulation of lipid peroxidation products. Taurine, reduced glutathione, oxidized glutathione, ascorbate, dehydroascorbate and sorbitol pathway intermediate concentrations as well as oxidized glutathione/reduced glutathione and dehydroascorbate/ascorbate ratios were similar in control and diabetic rats treated with or without taurine. CONCLUSION/INTERPRETATION: Oxidative stress is directly involved in up regulation of vascular endothelial growth factor protein in the retina during early diabetes.

Pulmonary tissue factor mRNA expression during murine traumatic shock: effect of P-selectin blockade.

Tissue factor (TF) is the primary cellular initiator of the coagulation protease cascade and serves as a cell surface receptor and a specific cofactor for plasma factors VII/VIIa. Because there is evidence that TF is regulated by a P-selectin dependent gene, we examined TF mRNA expression in the lungs during murine traumatic shock in the presence and absence of recombinant soluble P-selectin glycoprotein ligand-1 (rsPSGL.Ig) by using ribonuclease protection assays. Moreover, we studied the level of TF mRNA expression in mice with their P-selectin gene deleted (P-selectin -/-). Our data show that TF mRNA was significantly increased (+143%; P < 0.001) in the lungs 2 h after trauma compared with control rats subjected to sham trauma, which exhibited reduced TF mRNA expression (-34%; P < 0.001) after systemic administration of rsPSGL.Ig. The expression of TF mRNA was also significantly decreased (-29%; P < 0.05) in the lungs of P-selectin -/- mice compared with wild-type control C57B16 mice. The present results provide evidence for a P-selectin-dependent mechanism that enhances TF gene expression in traumatic shock. The major support for this mechanism is that either blockade of P-selectin by rsPSGL.Ig or deletion of the P-selectin gene leads to significant decreases in TF mRNA expression in the lung. These results are consistent with the concept that TF interacting with P-selectin may play a significant role in the pathophysiology of trauma.

Tissue factor expression in vital organs during murine traumatic shock: role of transcription factors AP-1 and NF-kappaB.

BACKGROUND: Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation that has been shown to have a role in the pathophysiology of sepsis and reperfusion injury. The purpose of this study was to investigate TF expression in vital organs and to determine possible regulatory mechanisms of TF expression in the lung during traumatic shock in rats. METHODS: Noble-Collip drum trauma was induced in anesthetized Sprague-Dawley rats. Anesthetized rats without trauma served as controls. TF activity was measured in plasma and lung tissue. TF messenger RNA (mRNA) was measured in the lung, liver, and small intestine using ribonuclease protection assays. Electromobility shift assays were used to quantify binding of nuclear extracts from lung to TF-specific consensus domains for transcription factors NF-kappaB and AP-1. RESULTS: TF activity in plasma increased up to 14-fold and +232% in the lung (P < 0.001 for plasma and lung) 2 h after trauma. TF mRNA level was significantly increased in the lungs (P < 0.01), small intestine (P < 0.01), and liver (P < 0.05) 1 h after trauma compared to sham-operated control rats. TF mRNA expression continued to increase in the lungs and the liver (both, P < 0.001) 2 h after trauma TF sequence-specific complex binding to AP-1 and NF-kappaB domains was enhanced in the lungs of trauma rats (+395%, P < 0.001 and +168%, P < 0.001, respectively). CONCLUSIONS: These results suggest that TF may play an important role in the pathophysiology of severe trauma and that regulatory elements AP-1 and NF-kappaB may be involved in the regulation of TF mRNA expression in traumatic shock.

Endothelin-1, endothelin receptors and ecNOS gene transcription in vital organs during traumatic shock in rats.

Endothelin-1 (ET-1) is a vasoconstrictor peptide that may play an important role in the pathophysiology of severe trauma. We examined ET-1 gene expression in vital organs (i.e., heart, lungs, kidneys, liver and small intestine) during murine traumatic shock using ribonuclease protection assays. Our data show that ET-1 mRNA was significantly increased in the lungs two hours after trauma when compared with control anesthetized rats. There was also a significant increase in ET-1 transcripts occurring in the kidneys, heart and liver. During these experimental conditions, we also observed statistically significant increased endothelin type B (ET(B)) receptor mRNA expression in the lung, heart, liver, kidney and small intestine. Expression of endothelial constitutive nitric oxide synthase (ecNOS) gene, which is functionally coupled to ET(B) receptor, also was increased in vital organs during traumatic shock. Endothelin type A (ET(A)) receptor gene expression was slightly decreased in the lung, liver and small intestine. These results suggest that ET-1 and ET(B) mRNA expression are mainly increased in the lung and other vital organs and may play a functional role in the pathophysiology of murine traumatic shock.

Essential role of P-selectin in the initiation of the inflammatory response induced by hemorrhage and reinfusion.

Resuscitation from hemorrhage induces profound pathophysiologic alterations and activates inflammatory cascades able to initiate neutrophil accumulation in a variety of tissues. This process is accompanied by acute organ damage (e.g., lungs and liver). We have previously demonstrated that significant leukocyte-endothelium interactions occur very early in other forms of ischemia/reperfusion (i.e., splanchnic ischemia/reperfusion and traumatic shock) which are largely mediated by increased expression of the adhesion molecule, P-selectin, on the vascular endothelium. Here we postulated that increased endothelial expression of P-selectin in the microvasculature would play an essential role in initiating the inflammatory signaling of hemorrhagic shock. Using intravital microscopy, we found that hemorrhagic shock significantly increased the number of rolling and adherent leukocytes in the mouse splanchnic microcirculation. In contrast, mice genetically deficient in P-selectin, or wild-type mice given either an anti-P-selectin monoclonal antibody or a recombinant soluble P-selectin glycoprotein ligand (PSGL)-1 immunoglobulin, exhibited markedly attenuated leukocyte-endothelium interaction after hemorrhagic shock. Thus, activation of P-selectin protein on the microvascular endothelium is essential for the initial upregulation of the inflammatory response occurring in hemorrhagic shock. Moreover, endogenous levels of PSGL-1 mRNA were significantly increased in the lung, liver, and small intestine of wild-type mice subjected to hemorrhagic shock. Since PSGL-1 promotes adhesive interactions largely through P-selectin expressed on the vascular endothelium, this result further supports the crucial role played by P-selectin in the recruitment of leukocytes during hemorrhagic shock.

Effect of recombinant soluble P-selectin glycoprotein ligand-1 on leukocyte-endothelium interaction in vivo. Role in rat traumatic shock.

-Traumatic shock induces profound pathophysiological alterations and initiates inflammatory reactions in many tissues, thus resulting in acute multiple organ damage (eg, intestine, pancreas, and liver). In the rat, Noble-Collip drum trauma increases P-selectin expression on the vascular endothelium as a result of loss of endothelium-derived NO. Here we postulated that blockade of the earliest steps in leukocyte adhesion (ie, leukocyte rolling) via administration of a recombinant soluble form of P-selectin glycoprotein ligand-1 (PSGL-1; the recombinant soluble form is rsPSGL.Ig) would attenuate selectin-mediated events observed in the rat during traumatic shock. Using intravital microscopy of the rat mesenteric microvasculature, we found that intravenous infusion of rsPSGL.Ig significantly inhibited leukocyte-endothelium interaction (ie, leukocyte rolling, adherence, and transmigration) induced by traumatic shock as well as by activation of the microvascular endothelium with 50 micromol/L NG-nitro-L-arginine methyl ester. Immunohistochemical detection of P-selectin on the mesenteric venular endothelial surface demonstrated that rsPSGL.Ig functionally neutralizes effects of P-selectin on the endothelial cell surface rather than attenuating P-selectin expression. Systemic administration of rsPSGL.Ig to traumatized rats prolonged survival time and survival rate, significantly attenuating ileal myeloperoxidase activity and significantly preserving mesenteric endothelial function. Furthermore, PSGL-1 mRNA levels were significantly increased in the blood of traumatized rats and were reduced after systemic administration of rsPSGL.Ig. Thus, soluble recombinant forms of PSGL-1 are able to ameliorate acute shock states by suppressing selectin-mediated leukocyte-endothelium interaction at both the functional and molecular levels.

P-selectin is up-regulated in vital organs during murine traumatic shock.

Murine traumatic shock is associated with increased adherence of neutrophils to the vascular endothelium resulting in neutrophil infiltration and tissue damage. We examined the effects of trauma on the expression of the adhesion molecule P-selectin in several vital organs (i.e., heart, lungs, liver, kidneys, and small intestine) 2 h after induction of Noble-Collip drum trauma in anesthetized rats. Total RNA was extracted from these organs and P-selectin mRNA was quantified by RNase protection assays. P-selectin mRNA was significantly increased over control nontraumatized, anesthetized rats in all vital organs (P<0.05 or less), with the largest increase occurring in the lung (P<0.01). Immunohistochemical analysis showed increased expression of P-selectin protein in postcapillary venules of all vital organs after trauma. To further investigate the possible mechanisms of increased P-selectin mRNA transcription promoter activity during trauma, we quantified binding of proteins from nuclear extracts to the kappaB site (-218GGGGGTGACCC[-207]) of the P-selectin gene by electrophoretic mobility shift assay. We confirmed the results of NF-kappaB binding by demonstrating increases in p50 and p52, as well as decreases in IkappaB in cytoplasmic and nuclear extracts from the lungs of trauma rats by Western blotting. Increased activity of the transcription factor, nuclear factor kappaB (NF-kappaB), occurred in all vital organs of the trauma rats compared to sham-operated controls. Our findings suggest that severe trauma results in up-regulation of P-selectin at the transcriptional level, which is partly controlled by an NF-kappaB-responsive element in the region of the P-selectin promoter. This increased activation of NF-kappB binding may contribute to the widespread increases in P-selectin expression observed in several vital organs 2 h after trauma, which in turn may play a key role in the pathogenesis of traumatic shock.

Regulation of P-selectin expression in human endothelial cells by nitric oxide.

P-selectin translocation to the surface of endothelial cells is increased after exposure to the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), resulting in increased endothelial adhesiveness. L-NAME (3 mM) was added to human cultured iliac vein endothelial cells for 1, 2, 4, and 6 h, and P-selectin mRNA expression was quantified by a ribonuclease protection assay. In parallel experiments, the NO donor, SPM-5185 (10 microM), was added to human iliac venous endothelial cells, and P-selectin mRNA expression quantified. P-selectin protein synthesis was quantified by Western blot analysis. L-NAME caused increased expression of P-selection RNA at 2-4 h, whereas D-NAME, the stereoisomer lacking NO synthase-inhibitory activity, had no effect. The stimulatory effect of L-NAME was reversed by addition of 3 mM L-arginine. SPM-5185 decreased P-selectin mRNA over the same time period (P < 0.02). The increased P-selectin mRNA expression induced by L-NAME was paralleled by an increase in P-selectin protein synthesis. The effects of SPM-5185 and L-arginine were also paralleled by decreases in P-selectin protein synthesis and in decreased adherence of human neutrophils to human iliac venous endothelial cells. The peak effect of inhibition of NO synthesis or addition of exogenous NO occurred at 2-4 h. These results suggest a regulatory effect of NO on endothelial P-selectin expression that modulates early leukocyte-endothelial cell interactions to preserve vascular homeostasis.

[The intensity of protein biosynthesis in the periodontium]. / Intensivnost' biosinteza belka v parodonte.

The intensity of protein biosynthesis in clinically healthy gingival tissues is the maximal at young age (20 to 30) and reduced in older subjects (aged 40 to 50). In generalized periodontitis the intensity of protein biosynthesis is reliably increased, more markedly in exacerbations and less so in a chronic course. An acute course is characterized by an approximately similar increases in the activity of protein synthesis in disease of varying severity. In an acute course the intensity of biosynthesis was the minimal in cases with the slight and the maximal in patients with grave generalized periodontitis. The content of sulfhydryl groups in the gingiva in generalized periodontitis is increased; it does not depend on the disease severity in an acute course of the disease, whereas in a chronic course this value is the maximal in cases with slight and the minimal in grave generalized periodontitis.

[The intensity of protein biosynthesis in the periodontium]. / Intensivnost' biosinteza belka v parodonte.

Protein biosynthesis intensity in clinically healthy gingival tissues is the highest at the age 20-30 and reduces by 40-50. In generalized periodontitis this parameter glows, particularly so in exacerbations. An exacerbation is characterized by similar rises of protein synthesis activity in various degrees of the disease. In chronic periodontitis biosynthesis intensity is the minimal in slight and maximal in severe generalized periodontitis. Increased content of sulfhydryl groups in the gingiva in generalized periodontitis does not depend on the disease severity in an exacerbation; in a chronic course their level is the maximal in slight and minimal in severe generalized periodontitis.

[Plasma corticosterone level in experimental mild cranio-cerebral injury in rabbits]. / Soderzhanie kortikosterona v plazme krovi krolikov v dinamike éksperimental'noi legkoi cherepno-mozgovoi travmy.

The content of corticosterone in the rabbits blood plasma in dynamics of experimental brain concussion was studied. We found two peaks of elevating the content of corticosterone during two weeks (15 minutes-2 hours, 7 days). Level of corticosterone more than twice exceeded the control values after 3 months of concussion. We can conclude that the brain concussion distinct from the stress action was more deep and in some cases with irreversible changes in neuro-humoral status.

[Mysteries of mitochondrial protein synthesis]. / Zagadki mitokhondrial'nogo belkovogo sinteza.

Mitochondria possess an endogenous system of translation, in which all constituent components are unique. An electrophoretic analysis of mitochondrial translation products revealed that the content of polypeptides in mitochondria is two times as high as that of mitochondrial DNA genes. The electrophoretically determined molecular mass of proteins synthesized in mitochondria is much less than that calculated from gene sequencing data. The average amino acid composition of the proteins synthesized in mitochondria differs significantly from that encoded by the nucleotide sequence of corresponding mitochondrial genes. These enigmas of mitochondrial protein synthesis await further solution.

[The time-dependent stimulating action of hydrocortisone on mitochondrial RNA biosynthesis in the rat liver]. / Zavisimoe ot vremeni stimuliruiushchee deistvie gidrokortizona na biosintez mitokhonrial'nykh RNK v pecheni krys.

Mitochondrial RNA biosynthesis was studied at different time after hydrocortisone administration. Hydrocortisone induced liver mitochondrial RNA biosynthesis in vivo and in isolated organellae 15 min., 1.5 and 4 h after hormone injection. As shown by means of molecular hybridization of RNA-DNA, an increase in liver mitochondrial RNA biosynthesis induced by hydrocortisone administration was due to an increase in the expression of all mitochondrial genes simultaneously rather than to selective enhancement of individual gene transcription. Hydrocortisone changed the biosynthesis and transport of cytoplasmic RNA in the liver of intact rats. The incorporation of 3H-UTP into RNA by isolated liver mitochondria of control and hydrocortisone-treated rats was inhibited by ethidium bromide. RNA biosynthesis in isolated rat liver mitochondria was unchanged in 15-300 min. of 0.9% NaCl administration.

[Characteristics of the action of dexamethasone and hydrocortisone on the biosynthesis of mitochondrial and cytoplasmic RNA in the rat liver]. / Osobennosti deistviia deksametazona i gidrokortizona na biosintez mitokhondrial'nykh i tsitoplazmaticheskikh RNK v pecheni krys.

In experiments on intact rats it was established that dexamethasone and hydrocortisone induce biosynthesis of mitochondrial RNA in a similar way, the, action being of the phase character. Three maxima of RNA biosynthesis induction in the liver mitochondria were found. The first increase of RNA biosynthesis intensity in organelles was noted 15 minutes after administration of glucocorticoids, it was short-term and less pronounced than the subsequent ones. The maximal increase of mitochondrial RNA synthesis was observed at the 4th hour of the action of glucocorticoids (the 3rd phase). The increase of mitochondrial RNA synthesis intensity was associated with the decrease of the specific radioactivity of cytoplasmic RNA.

[Metabolism of 3H-corticosterone in the liver cells of intact and adrenalectomized rats]. / Metabolizm 3H-korikosterona v kletkakh pecheni intaktnykh i adrenaléktomirovannykh krys.

Subcellular distribution of 3H-corticosterone and its metabolites in liver subcellular fractions of intact and adrenalectomized rats was studied. Corticosterone metabolites were found in the cytosol, microsomes, nuclei and mitochondria of rat liver cells in intact and adrenalectomized rats. The content of metabolites was different in all subcellular fractions. In adrenalectomized rats it was enhanced in the liver microsomes, nuclei and mitochondria which might be accounted for by a raised retention of metabolites by the endoplasmic reticulum membranes, mitochondria and nuclei.

[Characteristics of the effect of hydrocortisone on biosynthesis of mitochondrial proteins in the liver of thyroidectomized rats]. / Osobennosti deistviia gidrokortizona na biosintez mitokhondrial'nykh belkov v pecheni tireoidéktomirovannykh krys.

Biosynthesis of different groups of mitochondrial proteins was studied in liver cells of control and thyroidectomized rats after hydrocortisone administration. The hormone augmented protein biosynthesis in liver mitochondria within 15 min but the effect was less distinct in thyroidectomized rats. After hydrocortisone administration into intact rats the rate of liver mitochondrial proteins biosynthesis, coded by mitochondrial genes, was considerably higher as compared with those effects of nuclear genes, while biosynthesis of mitochondrial proteins was similarly increased in thyroidectomized rats. Hydrocortisone stimulated protein biosynthesis in matrix, inner and outer mitochondrial membranes of intact and thyroidectomized rats, whereas the biosynthesis was altered less distinctly in outer membranes. These data suggest that the effect of hydrocortisone on biosynthesis of proteins in mitochondria depended on content of thyroid hormones.

Effect of hydrocortisone on biosynthesis of mitochondrial and cytoplasmatic RNA in liver of adrenalectomized rats.

The effect of hydrocortisone on the biosynthesis of mitochondrial and cytoplasmic RNA in the liver of adrenalectomized rats was studied. A biphasic effect of hydrocortisone on the induction of RNA biosynthesis was observed: a short-term increase at 10th and 90th minute and more prolonged increase with a maximum at 210 min. At the same the intramitochondrial pool of labelled precursors of RNA did not change. According to hybridization data the expression of mitochondrial URF 5 and 6 genes linked to the effect of glucocorticoids was selectively increased already at the initial stage of hormone effect, but the resulting remarkable increase of RNA biosynthesis did not further result in changes of the spectrum of mitochondrial RNA. Specific activity of cytoplasmic RNA was decreasing at 10 min and increasing at 1, 2 and 4 h after the administration of hydrocortisone. The size of cytoplasmic acid soluble pool of labelled RNA precursors was increased only at 4 h after the administration of the hormone. It seems that a decrease of specific activity of cytoplasmic RNA during the initial period of hormone action may result from the changes of RNA transport from the nuclei to the cytoplasm.

[Molecular mechanisms of the action of insulin]. / Molekuliarnye mekhanizmy deistviia insulina.

Molecular mechanisms of insulin action on metabolism such as interaction with membrane receptor, autophosphorylation of receptor, internalization of insulin-receptor complexes into cells, changes in the processes of transcription, RNA release from nuclei, translation and post-translation modification of proteins and key enzymes are considered. The role of mediators in realization of biological effects of insulin is analyzed. It is emphasized that insulin action on the gene expression is an important link of its influence on metabolism.