[Lipid spectrum and function of kidneys before and after liver transplantation].

BACKGROUND: In patients after liver transplantation cardiovascular complications is the third main reason of death afer allograf failure and infections. The most important factors in the development of cardiovascular diseases are dyslipidemia and impaired renal function. The aim of the study was to investigate the lipid spectrum and renal function in liver recipients in real clinical practice and the correspondence of their correction to current clinical recommendations for the diagnosis and treatment of dyslipidemia and chronic kidney disease (CKD). METHODS: A retrospective analysis of lipid spectrum and renal function in patients who underwent OLT in Research Institute - Regional Clinical Hospital №1, Krasnodar was performed. The level of creatinine, GFR and lipid spectrum was studied before and 36 months after liver transplantation. The GFR was calculated using the formula CKD­EPI (Chronic Kidney Disease Epidemiology Collaboration). Statistical analysis of the study results was made using the program Statistica 10. RESULTS: Liver recipients have a significantly higher total cholesterol by 31.0% (p<0.01) in comparison with the baseline before surgery. Total cholesterol was increased in 13.7% (p<0.01), triglycerides in 12.3% (p<0.01) before transplantation. Tree years after transplantation, the increasion in cholesterol was registered in 42.6% (p<0.01) and triglycerides in 37.9% (p <0.01), respectively. 3 years after transplantation reduction of GFR was observed in comparison with the baseline by 22.6% (p=0.00006). Verification of chronic kidney disease and statin administration in patients were carried out in some cases. The levels of total cholesterol and triglycerides had a reliable inverse correlation with GFR (r = ­0.42; p<0.01 and r = ­0.36; p<0.05). CONCLUSIONS: In the long­term postoperative period there was an impaired lipid metabolism and decreased level of GFR. Dyslipidemia was closely related to the progression of renal dysfunction in liver recipients, an inverse correlation was established between the glomerular filtration rate and the increasion in cholesterol and triglyceride levels. It is necessary to increase the attention of physicians with regard to timely correction of lipid metabolism disorders and detection of initial manifestations of renal dysfunction.

[RESULTS OF DIAGNOSTICS AND TREATMENT OF ADRENOCORTICAL CANCER].

The results of examination and treatment of 96 patients with adrenocortical cancer (ACC) were analyzed. Local forms of ACC (I and II stages (T1-2N0M0) were found in 19 patients, locally advanced forms (III stage (T1-4NIM0; T3-4N0M0) - in 62 cases and metastatic forms of ACC (IV stage (TxNxM1) - in 15 patients. The diagnostic approach to ACC was optimized. It allowed identifying ACC on early stages of oncological process and staging of oncological process preoperatively in order to justify a rational treatment option. Surgical interventions were performed on 85 patients. The authors used an open access in 75 patients and endovideosurgical - in 10. The most common way of surgery was to remove an affected adrenal gland with fat of upper paranephrium and regional for adrenal lymph nodes (n=56). The adrenalectomy and nephrectomy were fulfilled on 23 patients. A removal of the right adrenal with tumor and thrombus of the interior vena cava was carried out in 2 patients. Some patients (n=4) underwent the explorative interventions. Combined treatment was applied in 28 patients with ACC of III stage. This gave a possibility to increase their life-span from 17,5±8,4 to 36,3±6 months. The overall 3-year survival rate for patients with ACC was 41,2% and 5-year survival observed in 18,7%. An application of modified treatment-and-diagnostics algorithm allowed increasing detection of patients with local and locally advanced forms of ACC in 2,5 times. Therefore, the application of rational treatment options have reduced the number of intraoperative complications from 38,8% to 10,2% and postoperative complication rates- from 61,1% to 20,4%, the lethality :rate - from 7,1% to 0% in early postoperative period. These measures have increased the life-span and life quality in 2 times.

Tumor necrosis by controlled ebullism.

In the early days of manned space flight, experiments were done in which dogs and chimpanzees were exposed to near vacuum in anticipation of possible manned space flight accidents. These specimens experienced what was termed "ebullism". This syndrome involved boiling of body fluids resulting in extreme dehydration and circulatory failure. Whereas malignant tumors are typically warmer than normal tissue, it should be possible to destroy them while sparing normal tissue through this phenomenon by subjecting patients to low pressure slightly greater than that which would produce systemic ebullism.

Platelet-derived growth factor activates production of reactive oxygen species by NAD(P)H oxidase in smooth muscle cells through Gi1,2.

Recent findings indicate that platelet-derived growth factor (PDGF) plays a role in the generation of reactive oxygen species (ROS) as second messengers in smooth muscle cells (SMC). To identify the source and signal transduction pathway of ROS formation in SMC, we investigated PDGF-induced ROS formation. Stimulation of SMC with PDGF resulted in a rapid increase of ROS production. Using an inactivating antibody, we identified the increase to be dependent on p22phox, a NAD(P)H-oxidase subunit. ROS release was completely inhibited by the Gi protein inhibitor PTX as well as an antibody against Galphai1,2, however, not by antibodies against Galphai3/0, Gas, and Gbeta1beta2. The effect of PDGF on ROS production in SMC membranes could likewise be mimicked by the use of a recombinant Galphai2 subunit but not by Galphai3, Galphai0, Gas, and Gbetagamma subunits. Immunoaffinity chromatography demonstrated coupling of Galphai1,2 to the PDGF a-receptor, which, after preincubation of the SMC membranes with PDGF, was increased in the absence of GTPgammaS but decreased in the presence of GTPgammaS and prevented by PTX treatment. These data define a novel G protein-dependent mechanism by which PDGF signaling is transduced through direct coupling of the Gai1,2 subunit of the trimeric G proteins to the PDGF tyrosine kinase receptor.

Phosphoinositide 3-kinase isoforms selectively couple receptors to vascular L-type Ca(2+) channels.

Heterodimeric class I phosphoinositide 3-kinase (PI3K) has been shown to be involved in the stimulation of voltage-gated Ca(2+) channels by various mediators. In this study, we bring evidences that vascular L-type Ca(2+) channels can be modulated by both tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks. Purified recombinant PI3Ks increased the peak Ca(2+) channel current density when applied intracellularly. Furthermore, PI3Kalpha-, beta-, and delta-mediated stimulations of Ca(2+) channel currents were increased by preactivation by a phosphotyrosyl peptide, whereas PI3Kgamma- and beta-mediated effects were increased by Gbetagamma. In freshly isolated and cultured vascular myocytes, angiotensin II and Gbetagamma stimulated L-type Ca(2+) channel current. In contrast, platelet-derived growth factor (PDGF)-BB and the phosphotyrosyl peptide did not stimulate Ca(2+) channel current in freshly isolated cells despite the presence of endogenous PDGF receptors and PI3Kalpha and PI3Kgamma. Interestingly, when endogenous PI3Kbeta expression arose in cultured myocytes, both PDGF and phosphotyrosyl peptide stimulated Ca(2+) channels through PI3Kbeta, as revealed by the inhibitory effect of an anti-PI3Kbeta antibody. These results suggest that endogenous PI3Kbeta but not PI3Kalpha is specifically involved in PDGF receptor-induced stimulation of Ca(2+) channels and that different isoforms of PI3K regulate physiological increases of Ca(2+) influx in vascular myocytes stimulated by vasoconstrictor or growth factor.

Phosphoinositide 3-kinase gamma mediates angiotensin II-induced stimulation of L-type calcium channels in vascular myocytes.

Previous results have shown that in rat portal vein myocytes the betagamma dimer of the G(13) protein transduces the angiotensin II-induced stimulation of calcium channels and increase in intracellular Ca(2+) concentration through activation of phosphoinositide 3-kinase (PI3K). In the present work we determined which class I PI3K isoforms were involved in this regulation. Western blot analysis indicated that rat portal vein myocytes expressed only PI3Kalpha and PI3Kgamma and no other class I PI3K isoforms. In the intracellular presence of an anti-p110gamma antibody infused by the patch clamp pipette, both angiotensin II- and Gbetagamma-mediated stimulation of Ca(2+) channel current were inhibited, whereas intracellular application of an anti-p110alpha antibody had no effect. The anti-PI3Kgamma antibody also inhibited the angiotensin II- and Gbetagamma-induced production of phosphatidylinositol 3,4,5-trisphosphate. In Indo-1 loaded cells, the angiotensin II-induced increase in [Ca(2+)](i) was inhibited by intracellular application of the anti-PI3Kgamma antibody, whereas the anti-PI3Kalpha antibody had no effect. The specificity of the anti-PI3Kgamma antibody used in functional experiments was ascertained by showing that this antibody did not recognize recombinant PI3Kalpha in Western blot experiments. Moreover, anti-PI3Kgamma antibody inhibited the stimulatory effect of intracellularly infused recombinant PI3Kgamma on Ca(2+) channel current without altering the effect of recombinant PI3Kalpha. Our results show that, although both PI3Kgamma and PI3Kalpha are expressed in vascular myocytes, the angiotensin II-induced stimulation of vascular L-type calcium channel and increase of [Ca(2+)](i) involves only the PI3Kgamma isoform.

Gbeta 5gamma 2 is a highly selective activator of phospholipid-dependent enzymes.

In this study, Gbeta specificity in the regulation of Gbetagamma-sensitive phosphoinositide 3-kinases (PI3Ks) and phospholipase Cbeta (PLCbeta) isozymes was examined. Recombinant mammalian Gbeta(1-3)gamma(2) complexes purified from Sf9 membranes stimulated PI3Kgamma lipid kinase activity with similar potency (10-30 nm) and efficacy, whereas transducin Gbetagamma was less potent. Functionally active Gbeta(5)gamma(2) dimers were purified from Sf9 cell membranes following coexpression of Gbeta(5) and Ggamma(2-His). This preparation as well as Gbeta(1)gamma(2-His) supported pertussis toxin-mediated ADP-ribosylation of Galpha(i1). Gbeta(1)gamma(2-His) stimulated PI3Kgamma lipid and protein kinase activities at nanomolar concentrations, whereas Gbeta(5)gamma(2-His) had no effect. Accordingly, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), significantly stimulated the lipid kinase activity of PI3Kbeta in the presence or absence of tyrosine-phosphorylated peptides derived from the p85-binding domain of the platelet derived-growth factor receptor. Conversely, both preparations were able to stimulate PLCbeta(2) and PLCbeta(1). However, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), activated PLCbeta(3). Experimental evidence suggests that the mechanism of Gbeta(5)-dependent effector selectivity may differ between PI3K and PLCbeta. In conclusion, these data indicate that Gbeta subunits are able to discriminate among effectors independently of Galpha due to selective protein-protein interaction.

Roles of non-catalytic subunits in gbetagamma-induced activation of class I phosphoinositide 3-kinase isoforms beta and gamma.

By using purified preparations we show that nanomolar concentrations of Gbetagamma significantly stimulated lipid kinase activity of phosphatidylinositol 3-kinase (PI3K) beta and PI3Kgamma in the presence as well as in the absence of non-catalytic subunits such as p85alpha or p101. Concomitantly, Gbetagamma stimulated autophosphorylation of the catalytic subunit of PI3Kgamma (EC(50), 30 nM; stoichiometry >/=0.6 mol of P(i)/mol of p110gamma), which also occurred in the absence of p101. Surprisingly, we found that p101 affected the lipid substrate preference of PI3Kgamma in its Gbetagamma-stimulated state. With phosphatidylinositol as substrate, p110gamma but not p101/p110gamma was significantly stimulated by Gbetagamma to form PI-3-phosphate (EC(50), 20 nM). The opposite situation was found when PI-4,5-bisphosphate served as substrate. Gbetagamma efficiently and potently (EC(50), 5 nM) activated the p101/p110gamma heterodimer but negligibly stimulated the p110gamma monomer to form PI-3,4,5-trisphosphate. However, this weak stimulatory effect on p110gamma was overcome by excess concentrations of Gbetagamma (EC(50), 100 nM). This finding is in accordance with the in vivo situation, where activated PI3K catalyzes the formation of PI-3,4,5-trisphosphate but not PI-3-phosphate. We conclude that p101 is responsible for PI-4, 5-bisphosphate substrate selectivity of PI3Kgamma by sensitizing p110gamma toward Gbetagamma in the presence of PI-4,5-P(2).

Role of the adenovirus 72-kDa DNA binding protein in the rapid decay of early viral mRNA.

Previous experiments have shown that the early adenovirus E1A and E1B mRNAs decay with a half-life of 20 min in a lytic infection dependent on the action of the viral 72-kDa DNA binding protein. In contrast, the same E1A and E1B mRNAs are stable when synthesized in 293 cells, an adenovirus-transformed cell line that is devoid of the 72-kDa protein. If 293 cells are infected with the E1A deletion mutant dl312, the endogenous E1A RNA disappears after 4 hr of infection, a time coincident with the appearance of the 72-kDa protein. The induction of decay is specific since there is no decrease in the level of actin or certain other cellular mRNAs. Thus, the stability of the early RNAs is variable and correlates with the presence of the 72-kDa protein. An interaction of the 72-kDa DNA binding protein with RNA inside the cell has been demonstrated by in vivo crosslinking of protein to RNA. However, the protein is found in association with actin mRNA as well as E1A mRNA. Thus, although the 72-kDa protein appears to be required for the rapid decay of viral mRNA it apparently does not impart specificity to the process.

Effect of adenovirus on metabolism of specific host mRNAs: transport control and specific translational discrimination.

We have studied the adenovirus-induced inhibition of host cell protein synthesis and the effect of infection on the overall metabolism of host cell mRNA during the late phase of adenovirus infection by following the fate of a number of cellular mRNAs complementary to specific cloned DNA segments. At a time in infection when the rate of total cellular protein synthesis is drastically (greater than 90%) reduced, transcription of specific cellular genes is undiminished. However, the transport of newly synthesized cellular mRNA to the cytoplasm is greatly decreased. This decreased appearance of new mRNA in the cytoplasm cannot account for the observed cessation of cell specific protein synthesis, however, since the concentration of several preexisting cellular mRNAs, including the mRNA for actin, remains unchanged throughout the course of infection. The preexisting mRNA is intact, capped, and functional as judged by its ability to direct protein synthesis in vitro in a cap-dependent fashion. The interruption in host translation appears to operate at the level of initiation directly, since we find that fewer ribosomes are associated with a given cellular mRNA after infection than before infection. Furthermore, the in vivo inhibition of cellular protein synthesis does not appear to be the result of competition with viral mRNA, since conditions which prevent the efficient initiation of translation of viral mRNA (infection with a viral mutant) do not result in the recovery of cell translation. Thus, it appears that a late adenovirus gene product directly mediates a shutoff of host protein synthesis.

The stability of early adenovirus mRNA is controlled by the viral 72 kd DNA-binding protein.

H5ts125, a temperature-sensitive mutant of adenovirus type 5, is restricted to the early phase of infection when grown at the nonpermissive temperature. One phenotype of the virus is the overproduction of early viral mRNA at the nonpermissive temperature relative to levels found in wild-type-infected cells, although normal levels are found at the permissive temperature. We have analyzed this phenomenon for the production of RNA from two specific early viral transcription units, E1A and E1B. Transcription rates from both of these regions were found to be the same in ts125-infected cells as in wild-type-infected cells at the nonpermissive temperature. However, when the cytoplasmic stabilities of the E1A and E1B mRNAs were measured in wild-type- and ts125-infected cells, it was found that at the nonpermissive temperature, the RNAs were 3 to 5 times more stable in a ts125 infection than in a wild-type infection. Since the mutation in ts125 maps to the gene for the 72 kd DNA-binding protein, these results imply that a functional 72 kd protein is required for the rapid turnover of early viral mRNA in wild-type-infected cells, indicating that the abundance of early viral mRNA is controlled by the 72 kd DNA-binding protein.

Modification of bovine pancreatic ribonuclease A with the site-specific reagent 4-arsono-2-nitrofluorobenzene. Spectrophotometric titration of arsononitrophenyl ribonuclease A derivatives.

The 4-arsono-2-nitrophenyl chromophore can serve as a versatile spectrophotometric probe of the surface structure of proteins. Values of pK1' and pK2' for the arsonic acid ionizations are near 3 and 8, respectively, and the presence of nearby positive and negative charges produces substantial alterations in the spectral response of the probe. Changes in the extinction at the wavelength of maximum difference are 30-50% of the extinction coefficients, epsilonmax, for each ionization of the arsonic acid moiety. The titration of 41-(4-arsono-2-nitrophenyl)ribonuclease A indicates that the arsonate dianion binds near the active-site histidine residues. With protonation of a carboxylate side chain in the acidic region, presumably aspartic acid-121, the active site is disrupted. The 41-(4-arsono-2-nitrophenyl) group interacts to a greater degree with the histidine-119 side chain than it does with the histidine-12 residue. Interactions of uridine or 3'-cytidylic acid with the ligand-binding region of 41-(4-arsono-2-nitrophenyl) ribonuclease A modify the spectrophotometric response extensively. 3'-Cytidylic acid binds 41-(4-arsono-2-nitrophenyl) ribonuclease A with an affinity 300 times less than that for native ribonuclease A and 17 times lower than that for 41-(2,4-dinitrophenyl) ribonuclease A. The arsononitrophenyl chromophore is responsive to changes in the active site of ribonuclease A induced by such perturbants as ligand binding, chemical modification, and both acid and thermal denaturation.

Early capping of transcripts from the adenovirus major late transcription unit.

The various events which result in modification of a primary RNA transcipt may have a role in choosing transcripts which are to be processed into mRNA. If there is a stepwise and interdependent nature to each of the modifications then it is important to place the various steps in temporal order. We show here that the formation of a capped 5' terminus seems to be a very early event for adenovirus type 2 (Ad-2) nuclear RNA that is initiated at the major late Ad-2 promoter. There is an equally rapid entry of 3H-adenosine into the cap and the first dozen or so adenylate residues in the RNA chain. Taken together with evidence on general mRNA metabolism in Chinese hamster cells, it seems unlikely that capping has any differential role in successful RNA processing rather is an automatic event for all RNA polymerase II products.

The genome-linked protein of picornaviruses. VI. The 5'-terminal protein of poliovirus type 2RNA is covalently linked to a nonanucleotide identical to that of poliovirus type 1 RNA.

Digestion of poliovirus type 2[32P]-RNA with enzymes and analysis of the products by column chromatography and paper electrophoresis provides evidence that the RNA is covalently linked to a small, basic protein, VPg, via a 5'-terminal pU residue. Digestion of the RNA with proteinase K followed by labeling of the peptidyl-RNA with the Bolton and Hunter reagent [iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester] yields 5'-labeled material suitable for rapid sequencing. The 5'-terminal sequence of poliovirion type 2 RNA was determined to be VPg-pUUAAAACAG... which is identical to the sequence at the 5'-terminus of the poliovirus type 1 genome.

Distribution of water between extracellular and intracellular compartments of incised wounds of rabbits.

The distribution of water between the extracellular and intracellular compartments in incised wounds of skin, muscle and stomach has been studied in healthy rabbits and the progress of healing monitored by the determination of tensile strength for 120 days. It has been shown that, after wounding, there is an immediate expansion of the extracellular space. The increase is most marked during the first 24 hours and is maximal by this time in wounds of the skin and the muscle. During the first 30 days of healing, all incised tissues contain similar amounts of extracellular water which constitute approximately one-half of the total tissue mass, irrespective of the size of the extracellular space prior to wounding. There is an inverse relationship between the increase in the amount of extracellular water and the size of the extracellular space prior to wounding. All incised tissues maintain an elevated, and similar, concentration of extracellular water for more than 120 days. The period of maximal gain in tensile strength corresponds to the period of maximal expansion of the extracellular space which signifies a particularly active phase in the wound healing process. The intracellular space in wounds of muscle and stomach is reduced for more than 120 days, but no significant changes are seen in the intracellular water of wounds of skin. It has been concluded that expansion of the extracellular space is essential for wound healing, and it can, therefore, serve as a sensitive indicator of tissue injury.

Prognostic significance of portal pressure in patients with bleeding esophageal varices.

Portal pressure was determined in 76 patients during acute bleeding varices, and the degree of portal hypertension was related to the cessation or noncessation of hemorrhage with nonoperative management. A direct relationship between the severity of hemorrhage and the degree of portal hypertension was established. It is suggested that portal pressure be determined as soon as feasible when bleeding varices are suspected and that other causes of hemorrhage be carefully excluded. The obtained data are of great prognostic significance and might be helpful in planning the course of therapy in these individuals.