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Monocytes play a key role in the development of metabolic syndrome, and especially obesity. Given the complex features of their development from progenitor cells, whose regulation is mediated by their interactions with bone marrow adipocytes, the importance of a detailed study of the heterogeneous composition of monocytes at the molecular and systemic levels becomes clear. Research argues for monocytes as indicators of changes in the body's metabolism and the possibility of developing therapeutic strategies to combat obesity and components of metabolic syndrome based on manipulations of the monocyte compound of the immune response. An in-depth study of the heterogeneity of bone-marrow-derived monocytes and adipocytes could provide answers to many questions about the pathogenesis of obesity and reveal their therapeutic potential.

Obese individuals are at high risk for developing type 2 diabetes mellitus, cardiovascular diseases, and nonalcoholic fatty liver disease. The aim of this review was to analyze the scientific literature and databases to reveal the fundamental role of neuregulin 4 (NRG4) and its receptors in the development of obesity-associated metabolic disorders. This review demonstrates that NRG4 and its receptors are promising therapeutic targets for the treatment of socially significant obesity-associated pathologies. The review contains nine chapters. Information on the structure of ERBB4 and NRG4 splice isoforms and subsequent activation of downstream targets is presented. The tissue-specific features of the NRG4 and ERBB4 genes and protein production are also highlighted. The role of NRG4 and ERBB3/4 in the pathophysiological mechanisms of the development of metabolic disorders in obesity is discussed in detail. The final chapter of the review is devoted to the miRNA-dependent regulation of NRG4 and ERBB4. Recent studies have shown that several miRNAs regulate ERBB4 expression, but no information was found on the interaction of NRG4 with miRNAs. We now demonstrate the putative relationships between NRG4 and let-7a-5p, let-7c-5p, miR-423-5p, miR-93-5p, miR-23a-3p, and miR-15b-5p for the first time. In addition, we found SNP mutations affecting the interaction of NRG4 and ERBB4 with miRNA in these genes as well as in miRNAs. In summary, this review provides a detailed and comprehensive overview of the role of NRG4 in obesity-associated metabolic disorders. The review summarizes all current studies on this topic and opens perspectives for future research., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s). Published by IMR Press.)

Objective: The aim: This article aims to show the role of local budgets in financing health care in communities, and to initiate a discussion on the possibility of expanding powers of local government bodies in the sphere of health care., Patients and Methods: Materials and methods: This study is based on the normative acts of the Republic of Poland and Ukraine, conclusions, reports of the Regional Chambers of Audit (Regionalnych Izb Obrachunkowych) in the Republic of Poland, OECD Indicators and budget gminas. Dialectical, comparative, analytical methods and the method of system analysis were used., Conclusion: Conclusions: Health care is among own tasks of gminas. Special legislation in the sphere of health care clarifies and narrows the tasks of gminas to monitoring and assessing public health, as well as to development, implementation and evaluation of special policy programs in the sphere of health care, according to identified needs of community residents and projects aimed at informing people about factors harmful to health and about their consequences. These programs can be financed from the budgets of gminas. This approach reduces and limits potential of local governments in the health care sector. Only in the event of an emergency gminas have the right to assign additional tasks to a medical entity and to finance these tasks from their own budget. In order to improve the quality and clarity of legislation own tasks of gminas in the sphere of health care (point 5, Part 1 Art. 7 of the Law of the Republic of Poland dated 8 March, 1990 "On municipal Government") should be specified taking into account point 1 Art. 2 of the Law of the Republic of Poland dated 11 September, 2015 "On Health Care".

For a partial differential field K , we show that the triviality of the first differential Galois cohomology of every linear differential algebraic group over K is equivalent to K being algebraically, Picard-Vessiot, and linearly differentially closed. This cohomological triviality condition is also known to be equivalent to the uniqueness up to an isomorphism of a Picard-Vessiot extension of a linear differential equation with parameters., (© 2019 Taylor & Francis Group, LLC.)

Angiogenesis is a common feature observed in advanced atherosclerotic lesions. We hypothesized that oxidized phospholipids (OxPLs), which accumulate in atherosclerotic vessels can stimulate angiogenesis. We found that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) stimulated the formation of sprouts from endothelial cell spheroids and promoted growth of capillaries into Matrigel plugs in mice. OxPLs stimulated expression of vascular endothelial growth factor (VEGF) in vivo and in several normal and tumor cell types in vitro. In addition, OxPAPC upregulated cyclooxygenase (COX)-2 and interleukin (IL)-8. COX-2 inhibitors, as well as blocking antibodies to IL-8 suppressed activation of sprouting by OxPAPC. We conclude that OxPAPC stimulates angiogenesis via autocrine mechanisms involving VEGF, IL-8, and COX-2-generated prostanoids. Our data suggest that accumulation of OxPLs may contribute to increased growth of blood capillaries in advanced lesions, thus leading to progression and destabilization of atherosclerotic plaques.

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.

One of the key mediators of the hypoxic response in animal cells is the hypoxia-inducible transcription factor-1 (HIF-1) complex, in which the alpha-subunit is highly susceptible to oxygen-dependent degradation. The hypoxic response is manifested in many pathophysiological processes such as tumor growth and metastasis. During hypoxia, cells shift to a primarily glycolytic metabolic mode for their energetic needs. This is also manifested in the HIF-1-dependent up-regulation of many glycolytic genes. Paradoxically, tumor cells growing under conditions of normal oxygen tension also show elevated glycolytic rates that correlate with the increased expression of glycolytic enzymes and glucose transporters (the Warburg effect). A key regulator of glycolytic flux is the relatively recently discovered fructose-2,6-bisphosphate (F-2,6-P2), an allosteric activator of 6-phosphofructo-1-kinase (PFK-1). Steady state levels of F-2,6-P2 are maintained by the bifunctional enzyme PFK-2/F2,6-Bpase, which has both kinase and phosphatase activities. Herein, we show that one isozyme, PFKFB3, is highly induced by hypoxia and the hypoxia mimics cobalt and desferrioxamine. This induction could be replicated by the use of an inhibitor of the prolyl hydroxylase enzymes responsible for the von Hippel Lindau (VHL)-dependent destabilization and tagging of HIF-1 alpha. The absolute dependence of the PFKFB3 gene on HIF-1 was confirmed by its overexpression in VHL-deficient cells and by the lack of hypoxic induction in mouse embryonic fibroblasts conditionally nullizygous for HIF-1 alpha.

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.

Hypoxia induces the stabilization and transcriptional activation of the hypoxia-inducible factor 1alpha (HIF-1alpha) protein, the regulatory member of the HIF-1 complex. The molecular mechanisms that are responsible for oxygen sensing and the downstream pathways utilized by the hypoxic signal are still poorly understood. One hypothesis for oxygen sensing has postulated that reactive oxygen species generated at mitochondrial complex III are the initiators of the hypoxic signal. Here we find that mitochondrial DNA-less (rho(o)) cells have a normal response to hypoxia, measured at the level of HIF-1alpha protein stabilization, nuclear translocation, and its transcriptional activation activity. Furthermore, overexpression of catalase, either in the mitochondria or in the cytosol, fails to modify the hypoxia response indicating that hydrogen peroxide is not a signaling molecule in the hypoxic signaling cascade that culminates with HIF-1 activation.

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.

Vascular endothelial growth factor (VEGF) mediates angiogenic activity in a variety of estrogen target tissues. To determine whether estrogen has a direct transcriptional effect on VEGF gene expression, we developed a model system by transiently transfecting human VEGF promoter-luciferase reporter constructs into primary human endometrial cells and into Ishikawa cells, derived from a well-differentiated human endometrial adenocarcinoma. In primary endometrial epithelial cells, treatment with 17beta-estradiol (E(2)) resulted in a 3.8-fold increase in luciferase activity, whereas a 3. 2-fold induction was demonstrated for stromal cells. Our Ishikawa cells had less than 100 functional estrogen receptors (ER)/cell and were therefore cotransfected with expression vectors encoding either the alpha- or the beta-form of the human ER. In cells cotransfected with ERalpha, E(2) induced 3.2-fold induction in VEGF-promoter luciferase activity. A 2.3-fold increase was observed in cells cotransfected with ERbeta. Through specific deletions, the E(2) response was restricted to a single 385-bp PvuII-SstI fragment in the 5' flanking DNA. Cotransfection of this upstream region with a DNA binding domain ER mutant, or site-directed mutagenesis of a variant ERE within this fragment, resulted in the loss of the E(2) response. Electromobility shift assays demonstrated that this same ERE sequence specifically binds estradiol-ER complexes. These studies demonstrate that E(2)-regulated VEGF gene transcription requires a variant ERE located 1.5 kb upstream from the transcriptional start site. Site-directed mutagenesis of this ERE abrogated E(2)-induced VEGF gene expression.

Endothelin-1 (Et-1) is a vasoconstrictor peptide that plays an important role in the pathophysiology of hypertension, myocardial ischemia, and other diseases. We examined the mechanism of regulation the Et-1 mRNA expression in human microvascular endothelial cells (HMEC-1) in response to hypoxia and cobalt. To determine whether the 5'-flanking region of Et-1 gene mediate transcriptional responses to cellular hypoxia, we constructed reporter plasmids in which Et-1 5'-flanking sequences of Et-1 gene were fused to luciferase coding sequences. Constructs, which contain native Et-1 sequence 5'-AACGTGCA-3', located between -118 and -125 in the opposite orientation as the transcriptional unit, mediate transcriptional response to hypoxia and cobalt. This responsiveness was inhibited by genistein, a tyrosine kinase selective inhibitor. Both hypoxia and cobalt induced binding of HIF-1 (hypoxia inducible-1 factor) to this Et-1 hypoxia responsive element in gel shift assays. Mutation in this sequence eliminated both the hypoxia-induced HIF-1 binding and luciferase expression. Using the supershift assay we have shown that this hypoxia responsive element binds HIF-1alpha and HIF-1beta proteins. Interestingly, genistein only slightly affected HIF-1 binding. These results indicate that the Et-1 gene contains HIF-1 binding hypoxia responsive elements which mediate transcriptional responses to hypoxia and cobalt in microvascular endothelial cells. Genistein appears to inhibit this response by affecting the transcriptional activity of the HIF-1 complex, without significantly affecting its DNA-binding properties.

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.

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.

-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.

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.

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.

Significant elevations in endothelin (ET)-1 levels accompany many diseases, but the underlying regulatory mechanisms are unclear. To investigate the in vivo regulation of human preproendothelin-1 (PPET-1), we examined the activity of the PPET-1 promoter in transgenic mice exposed to hypoxia. Mice expressing one of three PPET-1 promoter-luciferase (PPET-1/LUC) reporter transgenes (approximately 2.5 kb, 138 bp, or none of the 5'-flanking sequences of the PPET-1 gene) were generated. LUC expression was reduced in mice with a truncated 138-bp PPET-1 promoter. Exposure of mice bearing the 2.5-kb PPET-1/LUC transgene to hypoxia (10% O2 for 24 h) increased LUC expression sixfold in pulmonary tissue but only twofold in other tissues. In situ hybridization revealed the strongest transgene expression in the pulmonary vasculature and bronchiolar epithelium. These data are consistent with the hypothesis that hypoxic induction of the PPET-1 gene leads to increased pulmonary production of ET-1 in diseases associated with low O2 tension.

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 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.

Background: Vascular endothelial growth factor (VEGF) is a specific endothelial cell mitogen with potent angiogenic properties. In tumors, VEGF has been localized to the most necrotic and ischemic areas of the tissues, suggesting that local hypoxia is a potent inducer of VEGF production. Initial experiments in vitro confirmed the stimulatory effect of hypoxia on VEGF expression. The extent of this response and the mechanisms involved in oxygen sensing are poorly characterized., Experimental Design: Confluent monolayers of malignant cell lines or primary cultures of fibroblast or endothelial cells were exposed to hypoxia or incubated with either cobalt chloride, a stimulator of erythropoietin gene expression, or sodium azide, an inhibitor of oxydative phosphorylation. VEGF expression was analyzed by Northern blot or RNase protection assays. The expression VEGF in vivo was studied in animals subjected to hypobaric hypoxia or functional anemia., Results: Hypoxia greatly stimulated VEGF expression in tumor cell lines and primary fibroblast cultures. Endothelial cells, that expressed very low constitutive levels of VEGF, were resistant to hypoxic stimulation. RNase protection analysis showed that hypoxia primarily stimulated the induction of smaller and medium VEGF isoforms, i.e., the same ones expressed under normal conditions. The stimulatory effect of hypoxia on VEGF could be reproduced in vitro by cobalt chloride but not with sodium azide. In vivo, both hypoxia and anemia were found to be potent inducers of VEGF expression in several organs including heart, brain, liver, kidney, and muscle. As in vitro, cobalt was also found to be a potent stimulator of VEGF in vivo., Conclusions: Hypoxia is a potent inducer of VEGF expression in malignant as well as normal cultured cells. It is also a stimulator of VEGF expression in vivo. The VEGF gene appears to respond to hypoxia like the erythropoietin gene, and the mechanism of oxygen sensing probably is mediated by a heme-containing protein.

Vascular endothelial growth factor (VEGF) expression is highly stimulated by hypoxia, both in vitro and in vivo. Recent findings suggest that the VEGF gene utilizes an oxygen sensing mechanism similar to the one used by the erythropoietin gene. The genomic sequences that control the VEGF response to hypoxia are, however, largely unknown. In utilizing transient transfection assays in HeLa cells we determined that hypoxia/cobalt responsive enhancer elements are present at the 5' and 3' flanking regions of the human VEGF gene. The 3' enhancer is contained in a 160 bp fragment located about 60 bp downstream of the polyadenylation site. It contains a sequence stretch of about 12 bp which are highly homologous to sequences in the erythropoietin hypoxia-responsive enhancer. The 5' flanking enhancer is contained in a 100 bp fragment located about 800 bp upstream of the start site. This fragment does not contain significant homologies with the erythropoietin enhancer. Thus, it appears that the response to hypoxia of the VEGF gene is controlled by two regulatory elements; one which may be related to the erythropoietin enhancer and a second, which appears to be a completely unrelated sequence.

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.

To assess the possible benefits of combined hypolipidemic therapy (acipimox+marine fish oil) on lipid and lipoprotein metabolism, male Wistar rats were fed for 14 days a high sucrose diet (70 cal% sucrose) alone or a high sucrose diet supplemented with acipimox (0.2 g/100 g diet) and/or fish oil (1 ml orally daily; 30 wt% of n-3 PUFA). Feeding a high sucrose diet increased (control: 61 +/- 6 vs HS: 110 +/- 8 nmol.min-1.mg-1, p < 0.001) the activity of acetyl CoA carboxylase in the liver, this was normalized by fish oil but not acipimox alone (HS+FO: 68 +/- 4; HS+ACI: 95 +/- 4; HS+ACI+FO: 71 +/- 2 nmol.min-1.mg-1). Increased triglyceride concentration in serum and muscle tissue (m. soleus and heart) of high sucrose-fed animals was suppressed equally by fish oil, acipimox, and/or both. The cholesterol-lowering effect of fish oil was also present in the liver (p < 0.005). The cholesterol-lowering action of acipimox was accompanied by the accumulation of cholesterol in the liver (p < 0.005), whereas the combination of acipimox+fish oil did not change the liver cholesterol content. After fish oil the LDL binding capacity of liver plasma membranes was increased 1.6-fold (p < 0.001). LDL receptor activity was significantly decreased in HS+ACI group (p < 0.05), but remained unchanged in HS+FO+ACI-fed animals. In summary, (a) the hypotriglyceridemic effect of fish oil in high sucrose-induced HTG is due to its inhibitory effects at the level of fatty acid synthesis; (b) decreased triglyceride production and output from the liver prevent triglyceride accumulation in muscle tissue; (c) the cholesterol-lowering action of acipimox but not fish oil was accompanied by an accumulation of cholesterol in the liver; (d) the latter phenomenon may be due to the opposite effects of both drugs on cholesterol catabolism via hepatic LDL receptors.

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.

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.

A study was made of the metabolism of 3H-cortisol in the kidney, spleen and thymus cytosol and microsomes of intact and adrenalectomized rats. Cortisone and tetrahydrocortisone were found to be the main cortisol metabolites in the kidney cytosol and microsomes in these two groups of animals. In intact rats, the tetrahydrocortisone level in the kidney cytosol was hizher than that in the microsome fraction. After adrenalectomy opposite changes were noted: the tetrahydrocortisone level was increased in the microsome fraction and significantly lowered in the cytosol which might be accounted for by a decreased rate of cortisol metabolism and a raised retention of tetrahydrocortisone by the endoplasmic reticulum membranes. Cortisone and tetrahydrocortisone turned out to be the main metabolites of cortisol the rat spleen and thymus cytosol but their content was much lower than that in the kidneys.

We have identified and mapped nine discrete poly(A)-containing RNAs (RNA-2 to RNA-10) transcribed from rat liver mitochondrial DNA. The 5'-terminal fragments have been sequenced in five of these. The transcriptional map of rat mitochondrial DNA is homologous to those for mitochondrial DNAs of man and mice. The 5'-terminal nucleotide sequences of poly(A)-containing RNAs from rat liver mitochondria are similar in structure to those of HeLa cells studied by other authors; none of them have leader sequences.

In vivo labelled liver mitochondrial RNA of normal and alloxan-diabetic rats are hybridized with mitochondrial DNA with similar efficiency. The liver mitochondrial RNAs have equal ability to competition with N-specific complementary RNA for hybridization on mitochondrial DNA and its fragments cloned in the composition of bacterial plasmids and characterized by a definite set of genes. Differences between concurrent ability of the liver mitochondrial RNAs of normal and alloxan-diabetic animals were not observed. There were no electrophoretic distinctions in mitochondrial RNAs in both groups of animals during RNA electrophoresis in agaroso-acrylamide gel, as well. The results obtained indicate a close set of RNA molecules in the liver mitochondria of normal and diabetic rats. Thus, a previously observed RNA biosynthesis decrease in the liver mitochondria in diabetes is caused not by a selected inhibition of definite gene expression but by a gradual fall of transcription of all the mitochondrial genome parts.

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.

During electrophoresis in polyacrylamide gels containing 7M urea the major discrete components of preparations of rat liver mitochondrial poly(A)+ and poly(A)- RNA species have similar mobilities. Poly(A)- RNA components hybridize to the 16S rRNA gene of mtDNA. Analysis of 5'-terminal sequences of these components revealed their identity to the 5'-terminal sequence of 16S rRNA. These results show that poly(A)- RNA components are fragmentation products of 16S rRNA. Fragmentation occurs nonrandomly from the 3'-end of the original rRNA molecules and lead to formation of products with electrophoretic mobilities similar to those of poly(A)+ RNA components.

Mitochondrial poly(A)-containing RNAs from the liver of normal and adrenalectomized rats were analyzed electrophoretically in agarose gel with urea. It was revealed that after the glucocorticoid hormone level descended, the content of a single high-molecular poly(A)-containing RNA in liver mitochondria was significantly lowered. This RNA is mapped on mitochondrial DNA as RNA 5 by the blotting-hybridization method (according to the classification of Amalric et al., 1971).

In liver mitochondria from alloxan diabetic rats the biosynthesis of RNA (as measured by 14C-orotic acid incorporation and RNA polymerase activity) and protein (as measured by 14C-leucine incorporation) were decreased. In contrast, insulin (20 U kg-1) injected to intact or diabetic rats increased both these measures. However, no change of mitochondrial DNA biosynthesis (as measured by incorporation of 3H-thymidine) was found. It was concluded that in liver cells insulin plays an important role in regulation of biosynthesis not only of nuclear (cytoplasmic) but also of mitochondrial RNA and proteins.

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.

Mitochondrial gene expression was studied in hypothyroid state and after triiodothyronine administration to thyroidectomized rats. Liver mitochondrial RNA biosynthesis decreased after thyroidectomy and increased after triiodothyronine administration. A mitochondrial genome region whose expression was selectively decreased in hypothyroid state and increased by triiodothyronine in liver cells of thyroidectomized rats, was identified.

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.

The incorporation of 14C-orotic acid into mitochondrial RNA from the liver of alloxan diabetic (AD) rats was increased after a single or repeated administration of hydrocortisone (HC; 5 mg kg-1). while the increase in normal animals was much less. The activity of RNA-polymerase in isolated mitochondria was also considerably increased in AD rats after HC administration, but this was not related to any changes of mitochondrial membrane permeability for precursors. Electrophoretic analysis of total and poly(A)-containing RNA did not show any substantial changes in liver mitochondrial RNA of AD rats treated with HC. No changes in the transcription of liver mitochondrial DNA from intact, AD or AD plus HC treated rats were found after the hybridization of mitochondrial RNA with a rat mitochondrial DNA or DNA from recombinant plasmids, containing inserts of restriction fragments of mitochondrial DNA. In addition, an increase in the rate of protein synthesis was observed after a repeated administration of HC to intact animals, while that in AD rats was decreased and started to increase only after a single administration of HC. In contrast, after a repeated administration of HC the rate of protein synthesis was decreased. It was concluded that the mechanism of RNA and protein biosynthesis in liver mitochondria of AD rats is much more sensitive to the action of glucocorticoids as compared to intact animals and that the biosynthesis of all populations of mitochondrial RNA in the liver of AD rats is increased after HC treatment in a similar degree, the quality of mitochondrial gene expression being not changed.

Sodium ribonucleate (per os application) increased the biosynthesis of mitochondrial, nuclear and cytoplasmic RNA in the rat liver and transportation of nuclear RNA into the cytoplasm. Administration of ribonucleate changed the ultrastructure of hepatocytes, increased nuclear and nucleolar diameter, the number of rough endoplasmic elements, reticulum and glycogen particles and decreased the size of the mitochondria. This suggests that sodium ribonucleate can stimulate the biosynthesis of RNA and change the ultrastructure of hepatocytes also with application per os.

Biosynthesis of RNA was studied in mitochondria of heart muscle, liver, kidney and spleen tissues of normal rats and of the animals with experimental alloxane diabetes. In the diabetes biosynthesis of RNA was distinctly decreased in mitochondria of all the tissues studied, especially in the mitochondria of heart muscle. As shown by means of molecular hybridization of RNA-DNA, using total DNA from mitochondria and DNA of recombinant plasmides containing restrictional fragments of rat mitochondrial DNA, the decrease in mitochondrial RNA biosynthesis under conditions of the diabetes was not due to selective inhibition of individual genes transcription but it was determined by a decrease in expression of all the mitochondrial genes simultaneously.

Experiments were made on albino rats with alloxan diabetes. Half of the animals received sodium ribonucleate (20 mg/100 g) per os daily for two weeks. The intensity of RNA biosynthesis was judged from 32P incorporation. It was established that insulin deficiency abolished the biosynthesis of mitochondrial, cytoplasmic and nuclear RNA in the liver and kidneys, of mitochondrial and cytoplasmic RNA in the heart muscle, and of total RNA in the aorta tissue. Oral administration of sodium ribonucleate gave rise to the increased biosynthesis of RNA in these organs. A conclusion is drawn that oral application of the drug may be used in combined treatment of diabetes mellitus in general, and of that complicated by angiopathy, in particular.