[Possible use of the growing liver biological set for hepatic recovery after toxic damage (an experimental study)]. / Vozmozhnosti biologicheskoi kombinatsii, poluchennoi iz rastushchei pecheni, dlia ee vosstanovleniia pri toksicheskom povrezhdenii (éksperimental'noe issledovanie).

The lack of acceptable pharmacological approaches for restoration of the injured liver is associated with complex of mechanisms involved in hepatic regeneration and with difficulty of the target selection. The aim of this research was to study the hepatoprotective function of the extract from both the growing and regenerating liver containing a natural set of factors crucial for the hepatic restoration. Extracts from both regenerating liver of rats after 70% hepatic resection and the growing liver of neonatal pigs were obtained using own original technique. The set of resultant extracts was named as the hepatic regeneration set (HRS). HRS fractionation was carried out using the Toyopearl HW-50S sorbent. The efficiency of HRS and its fractions was estimated using a model of the mouse liver thioacetamide injury and monitoring hepatic enzyme activity in blood serum. The activities of AST and ALT in intact animals were 50 U/l and 80 U/l, respectively; after thioacetamide administration they increased to 2059±212 U/l and 4280±440 E/l, respectively (p<0.05). Treatment of injured animals with HRS from the rat regenerating liver resulted in a significant decrease of transaminase activities to 924±148 U/l (AST; p<0.05) and 1633±308 U/l (ALT; p<0.05). A similar effect was observed after treatment with HRS from the neonatal pig liver: the AST decreased to 937±138 U/l (p<0.05), while ALT activity decreased to 1710±237 U/l (p<0.05). HRs fractionation resulted in identification two active fractions characterized by much higher (8-29) hepatotropic effect that that of the whole extract. These fractions contained peptide/protein components with the range of molecular mass of 3-60 kDa (fraction 1) and 3-25 kDa (fraction 2a). Fraction 1 also contained some polynucleotides in fraction 1. Subsequent studies of these fractions exceeding the hepatotropic effect of original HRS is clearly needed to identify their individual components by immunochromatography methods, ELISA, MRM mass spectrometry and quantitative PCR.

Tankyrase Activity in Organs and Tissues of Mice.

Tankyrase, one of the NAD+ ADP-ribosyltransferases, is a target for drugs developed for their anticancer and other pharmacological activities. We designed an assay for estimation of the inhibition or activation of the enzyme in preclinical studies. In mice, the highest specific activity of tankyrase was observed in thymus, spleen, pancreas, and bone marrow. In murine liver, tankyrase is active in ontogenesis and during reparative regeneration; however, the basal activity is hardly detectable in normal liver and most of other organs of adult animals. We suggest that tankyrase is a part of the tissue growth and repair machinery, while its age-dependent inhibition, when an organism stops growing, turns on phenoptosis.

[Relationship of tumor necrosis factor alpha expression with activation of sphingomyelinase and lipid peroxidation after removal of cholestatic factor].

The goal of this work was to study the expression of tumor necrosis factor alpha (TNFalpha), sphingomyelin cycle activation, and lipid peroxidation (LPO) processes after the removal of a cholestatic factor in the liver subjected to different durations of cholestasis. Restored bile flow after a 9-day hepatic cholestasis normalized sphingomyelinase (SMase) activity and levels of TNFalpha and LPO products. The removal of a cholestatic factor after a 12-day cholestasis did not normalize the studied parameters: SMase activity and the levels of TNFalpha and LPO products remained much higher compared to control. A significant positive correlation between TNFalpha expression, SMase activity, and LPO rate has been revealed. The obtained data indicate that hepatocyte apoptosis after bile outflow restoration in late cholestasis can be due to the activation of the sphingomyelin cycle, LPO, and TNFalpha expression. The synergistic interaction can sharply increase the proapoptotic capacity of each of these factors since TNFalpha activates SMase and LPO, SMase activity depends on the LPO rate, while ceramide, an SMase-produced secondary messenger of apoptosis, can induce oxidative stress.

[Changes in sphingomyelinase activity, tumor necrosis factor alpha level, and lipid peroxidation rate in the course of development of cholestatic liver injury].

Changes in sphingomyelinase activity, tumor necrosis factor alpha expression, and lipid peroxidation rate in the course of development of cholestatic liver injury have been studied. The same type phase shifts in the parameters analyzed were observed, which included a marked decrease at the early stages of cholestasis (days 3-6) and a pronounced increase at the later stages (days 12-16), i.e., under the conditions of developed pathology. There is a significant positive linear correlation between tumor necrosis factor alpha expression, sphingomyelinase activity, and lipid peroxidation rate during cholestatic injury. The changes detected may reflect balance between the effects of the two major bile components--bilirubin, which is accumulated in the liver at the early stages of cholestasis, and bile acids, whose influence dominates at the later stages of pathologic process. Our results indicate that tumor necrosis factor alpha overexpression, the sphingomyelin cycle activation, and lipid peroxidation intensification may cause apoptosis of hepatocytes at the late stages of cholestasis.

Expression of prolactin receptors in human liver during cholestasis of different etiology and secondary liver cancer.

Indirect immunoperoxidase assay and computer analysis of photographic images revealed more intensive expression of prolactin receptors in hepatocytes of women compared to men. The intensity of expression was maximum in secondary liver cancer, high in obstructive jaundice of different etiology, and less pronounced in cholelithiasis. The expression of prolactin receptors in cholangiocytes was higher than in hepatocytes and was maximum during obstructive jaundice of different etiology. Cells of secondary tumors were characterized by low expression, while distant hepatocytes most intensively expressed prolactin receptors.

Role of tumor necrosis factor alpha and sphingomyelin cycle activation in the induction of apoptosis by ischemia/reperfusion of the liver.

The signal transduction pathways triggering apoptotic mechanisms after ischemia/reperfusion may involve TNF-alpha secretion, ceramide generation, and initiation of lipid peroxidation. In the present study involvement of the TNF-alpha, sphingomyelin cycle, and lipid peroxidation in the initiation of apoptosis induced in liver cells by ischemia and reperfusion was investigated. Wistar rats were subjected to total liver ischemia (for 15, 30 min, and 1 h) followed by subsequent reperfusion. Ischemia caused sharp decrease of neutral sphingomyelinase activity. Activity of acidic sphingomyelinase initially decreased (during 15-30 min ischemia) but then increased (after 1 h of ischemic injury). Reperfusion of the ischemic lobe of the liver caused increase in neutral sphingomyelinase activity and decrease in acidic sphingomyelinase activity. A small amount of TNF-alpha detected by immunoblotting analysis was accumulated in the ischemic area of liver rapidly and the content of this cytokine dramatically increased after the reperfusion. TNF-alpha is known to induce free radical production. We found that the accumulation of TNF and increase of sphingomyelinase activity during the development of ischemic/reperfusion injury coincided with increase in content of lipid peroxidation products (conjugated dienes) and DNA degradation detected by gel electrophoresis. Recently it was shown that superoxide radicals are used as signaling molecules within the sphingomyelin pathway. This suggests the existence of cross-talk between the oxidation system and the sphingomyelin cycle in cells, which may have important implications for the initial phase and subsequent development of post-ischemic injury.

Role of endogenous TNF-alpha and sphingosine in induced DNA synthesis in regenerating rat liver after partial hepatectomy.

Cytokine-stimulated metabolism of sphingomyelin results in the accumulation of ceramide and sphingosine which play a part in the regulation of cell proliferation, differentiation, and reception, as well as in oncogenesis. Formation of TNF-alpha (a member of the cytokine family), accumulation of sphingosine, and DNA synthesis (measured by immunoblotting, HPLC, and [3H]thymidine incorporation, respectively) were studied in rat liver after partial hepatectomy. The content of TNF-alpha was found to increase during 12 h following hepatectomy. The maximum of sphingomyelinase activity and accumulation of sphingosine precede the maximum of DNA synthesis. Sphingosine is known to inhibit protein kinase C. On the other hand, it stimulates the metabolism of phosphatidylinositol, thus causing accumulation of diacylglycerol and inositol-1,4,5-triphosphate, which in turn activate protein kinase C. Hence, the release of TNF-alpha in regenerating liver may modulate DNA synthesis through the accumulation of sphingosine which is involved in regulation of protein kinase C activity and of phosphatidylinositol turnover.

[Changes in the activity of neutral and acidic isoforms of sphingomyelinase in hepatoma-22, regenerating and ischemic liver]. / Izmenenie aktivnosti neitral'noi i kisloi izoform sfingomielinazy v gepatome-22, v regeneriruiushchei i ishemizirovannoi pecheni.

Activity of neutral and acidic sphingomyelinases (N- and A-SMases) were studied in regenerating liver after partial hepatectomy (during 48 hrs after operation), in ischemic liver during 15, 30 min and 1 and 2 hrs ischemia and during following reperfusion (from 5 min up to 2 hrs), in hepatoma- 22 after 15 days of transplantation and in liver of tumor bearing animals. It was shown that activity of N-SMase is increased in hepatoma-22 and in regenerating liver and it is decreased in ischemic liver. Following reperfusion of ischemic liver area activity of enzyme was found to have returned to baseline in dependence on time of ischemia and reperfusion. Activity of A-SMase is decreased in tumor, is not changed in regenerating liver and increased after long time of ischemia. It was supposed that N-SMase is involved in cell proliferation, but A-SMase is connected with cell damage.

Results of pancreatic blood shunting into the systemic blood flow in insulin-dependent diabetics.

A new surgical method of treating patients with unstable insulin-dependent diabetes (IDD) has been developed--that of surgically shunting pancreatic blood into the systemic blood flow with the purpose of creating a more optimal interaction of subcutaneously administered insulin and pancreas-secreted glucagon. The long term results of the operation depend on the patency of a splenorenal anastomosis. This has been studied by following up 137 patients over periods from half a year to three years. Anastomotic patency was determined by renal and splenic venography and celiacy arteriography, which revealed a patent anastomosis in 114 patients, and an obliterated one in 23. Patients with patent anastomoses showed a lowering of glycosylated hemoglobin (HbA1c) from 13.3 +/- 0.3% to 9.3 +/- 0.6%, p < 0.05, a decrease of the injected insulin dose from 0.97 +/- 0.04 to 0.72 +/- 0.03 U/kg, p < 0.05, disappearance or considerable abatement of pain in the lower extremities, and of hypoglycemia. Improvement of clinical status was accompanied by an increase of glucagon in the systemic blood stream from 60.8 +/- 10.1 to 91.5 +/- 9.4 pg/ml, p < 0.05, a rise of tissue oxygen pressure, pO2, from 49.2 +/- 2.4 to 58.1 +/- 1.9 mm Hg, p < 0.05. In patients with oblivious anastomoses postoperative HbA1c levels did not change from preoperative values: 12.9 +/- 0.4% and 12.8 +/- 0.7%, p < 0.05, respectively; the insulin dose remained the same--0.91 +/- 0.07 U/kg and 0.85 +/- 0.07 U/kg, p < 0.05, no rise of the systemic blood glucagon content was noted, and former complaints continued. The suggested method is not an alternative for insulin therapy, but considerably enhances its potential.

[Urinary enzymes in insulin-dependent diabetes mellitus]. / K voprosu o fermenturii pri insulinzavisimom sakharnom diabete.

In patients suffering from insulin-dependent diabetes mellitus (IDDM) with or without preclinical and clinical signs of diabetic nephropathy, the degree of epithelial cell lesions in the renal tubules was assessed from the urinary activities of enzymes at various sites, such as lysosomal (N-acetyl-beta-D-glucosaminidase (NAG) and beta-galactosidase (beta-GA)), brush edge membranous (alanine aminopeptidase (AAP), and cytosolic (alpha-glucosidase (alpha-GL)). Patients from Groups 1 and 2 had no preclinical and clinical signs of nephropathy. In Group 1 patients, the magnitude of enzymuria was not different from that in normalcy. However, Group 2 patients exhibited significant increases in urinary NAG and beta-GA activities as compared to Group 1 patients and healthy individuals. In Group 3 patients with microproteinuria from 0.05 to 0.5 mg protein per ml urine, displayed a further enhancement of NAG and beta-GA activities as compared to Group 2 patients and significantly higher activity than did Groups 1 and 2 patients and healthy individuals. In Group 4 patients with macroproteinuria of > 0.5 mg/ml), greater increases in the activities of NAG, beta-GA, and AAP were not found, however, there was a significant increase in alpha-G1 activity. The findings suggest the varying degrees of epithelial cell damage in the renal tubules in patients of different groups and the possibility of early detection of lesion in the proximal portion of nephronic tubules in IDDM patients as assessed from urinary enzyme levels.

[Evaluation of proteinuria by ponceau S dye in patients with insulin-dependent diabetes mellitus]. / Otsenka proteinurii s ispol'zovaniem krasitelia ponso S u bol'nykh insulinzavisimym sakharnym diabetom.

Protein content was measured using ponceau S stain in "protein-free" urine (as shown by clinical analysis with sulfosalicylic acid) of 34 patients with insulin-dependent diabetes mellitus. In 15 patients (group 1) protein levels did not differ from those in healthy subjects. In 19 patients (group 2) protein levels surpassed the normal value by 3.6 times, on an average (p < 0.001). Measurements of urinary albumin revealed microalbuminuria in group 2 and normo-albuminuria in group 1. The data indicate that protein measured with the use of ponceau S stain in group 2 patients is not a false-positive result, but reflects the presence of proteins of plasma origin in the urine. Hence, measurements of urinary protein with the use of ponceau S stain may be used for identification of early stages of diabetic nephropathy.

[Two forms of aldose reductase in erythrocytes from patients with insulin-dependent diabetes mellitus]. / Dve formy al'dozoreduktazy v éritrotsitakh bol'nykh insulinzavisimym sakharnym diabetom .

Erythrocytes of patients with insulin-dependent diabetes mellitus contained two forms of aldose reductase a and b (EC No. 1.1.1.21) (the key enzyme in the sorbitol pathway of glucose utilization) as compared with those of healthy donors in whom the form b of the enzyme was only found. The aldose reductase a exhibited a higher maximal rate (Vmax = 16.7 +/- 3.2 IU/D280) and a lower substrate affinity (Km = 6.5 = 19 mM) than the enzyme b, Vmax = 43.8 +/- 0.6 IU/D280, Km = 3.0 = 4.0 mM, respectively. More severe development of the disease was observed in the patients whose erythrocytes contained only aldose reductase a (HbA1c = 14.56 +/- 0.69%) as compared with those in whom the enzyme a and b were found (HbA1c = 11.5 +/- 0.4%). Kinetic parameters of the enzyme showed that aldose reductase a may be active in hyperglycemia of diabetes mellitus, thus contributing to intensification of the sorbitol pathway in these patients.

[Determination of glycosylated hemoglobin by affinity chromatography on boron phenylagarose]. / Opredelenie glikozilirovannogo gemoglobina metodom affinnoi khromatografii na borfenilagaroze.

When Hb AIc was estimated by affinity chromatography where boron phenyl-agarose was used as a sorbent, the highest rate of Hb AIc binding with the sorbent was detected at pH 8.5-9.0 while the maximal elution of the protein occurred at 0.3-0.5 M concentration of sorbitol used as an eluent. Content of Hb AIc, estimated in 56 patients with insulin-dependent diabetes mellitus, constituted 12.3 +/- 0.4%, while in 20 healthy volunteers it was equal to 5.4 +/- 0.2%, which are consistent with the literature data obtained by other methods. At the same time, the data of Hb AIc estimation by means of affinity chromatography correlated exactly with the results of ion exchange chromatography (r = 0.98), thus corroborating the validity of the procedure used. Only the stable ketoamine fraction of Hb AIc was found to interact with boron phenyl-agarose.

[The status of various indicators of the protease inhibitor system in blood and synovial fluid in rheumatoid arthritis]. / Sostoianie nekotorykh pokazatelei proteazno-ingibitornoi sistemy krovi i sinovial'noi zhidkosti pri revmatoidnom artrite.

Elastase-like activity, alpha 1-inhibitor of proteinases and acid stable antitryptic activity were studied in blood of 26 patients and in synovial fluid of 11 patients with rheumatoid arthritis during acute stage of the disease and after treatment with salase pyridasine and orthophene. The higher values of the elastase-like activity were detected in synovial fluid as compared with that of blood. In blood of these patients, hyperproduction of alpha 1-inhibitor of proteinases and acid stable inhibitors was observed, while deficiency of these substances occurred in synovial fluid. Distinct decrease in the patterns of the blood protease-inhibitory system studied simultaneously with clear positive clinical effect were observed after treatment with salase pyridasine together with orthophene; estimation of these patterns may be used in the evaluation of the therapy.

Components of plasma and tissue kallikrein-kinin system in the urine of patients with latent, nephrotic and hypertonic forms of glomerulonephritis.

Activities of main components of KKS were estimated in the urine of patients with latent, nephrotic and hypertonic forms of chronic glomerulonephritis (ChGN) and compared to those parameters in urine of healthy persons. The data obtained allow to make a conclusion concerning the pathogenetic and the compensatory role of plasma KKS in the nephrotic form of ChGN.

[Activity of elastase-like proteinases and their inhibitors in indigent nearly healthy residents in various biogeochemical conditions of the Chuvash ASSR]. / Aktivnost' élastazopodobnykh proteinaz i ikh ingibitorov u korennykh prakticheski zdorovykh zhitelei v raznykh biogeokhimicheskikh usloviiakh Chuvashskoi ASSR.

A proteinase-inhibitory balance of blood (elastase-like activity, alpha 1-proteinase inhibitor and alpha 2-macroglobulin activities) was studied in practically healthy inhabitants of the Chuvash ASSR two subregions--Sura river basin and Cubninocivil region, which are distinctly dissimilar in all the biogeochemical parameters involving macro- and microtrace compositions. The higher activity of elastase-like proteinases and decreased content of alpha 1-proteinase inhibitor were detected in practically healthy inhabitants of the river Sura basin, where high incidence of myocardial infarction was found, as compared with those of the Cubninocivil people. The similar alterations in the proteinase-inhibitory balance were observed in blood of experimental animals maintained on a diet containing fresh water from these subregions. The data obtained suggest that there exists causative relationship between biogeochemical parameters and development of imbalance in the proteinase-inhibitor system in practically healthy inhabitants of the river Sura basin. This imbalance is considered as a pathogenetic factor responsible for development of atherosclerosis.

[A substrate inhibitor analysis of the urinary excretion of tissue and plasma kallikreins in patients with chronic glomerulonephritis]. / Substratno-ingibitornyi analiz mochevoi ékskretsii tkanevogo i plazmennogo kallikreinov u bol'nykh khronicheskim glomerulonefritom.

The usage of substrate inhibitor analysis made it possible to estimate the levels of excretion of plasma proteinases, including plasma kallikrein in the urinary DValLeuArgpNA (S-2266)- and DProPheArgpNA (S-2302)-amidase activity in patients with latent and nephrotic types of chronic glomerulonephritis (CGN). The soya bean trypsin inhibitor, an inhibitor of plasma kallikrein and other plasma proteinases, such as that of the blood coagulative factors XIa and XIIa, and the high selective plasma kallikrein inhibitor DPhePheArgCH2Cl were used as those differentiating kallikreins of tissue and plasma origin. The S-2266 and S-2302-amidase activity of the urine from healthy subjects was shown to be determined by only tissue (renal) kallikrein. The urine from the patients with a latent CGN type displayed the activity of plasma proteinases, but plasma kallikrein made no significant contribution to the urine amidase activity in these patients. With a nephrotic CGN type, great quantities of trypsin-like proteinases were secreted from the plasma through the glomerular filter into the urine, the proportion of plasma kallikrein in the urinary S-2266 and S-2302-amidase activities being approximately 27%. The compensatory and pathogenetic role of plasma kallikrein is discussed if there is lower excretion of tissue (renal) kallikrein in CGN with the nephrotic syndrome.

[Components of kallikrein-kinin system in the urine of patients with glomerulonephritis]. / Komponenty kallikrein-kininovoi sistemy v moche bol'nykh glomerulonefritom.

Activities of main components of the kallikrein-kinin system: tissue (renal) and blood plasma kallikreins, kininases I and II, bradykinin-activating activity as well as free kinins, were estimated in urine of patients with latent and nephrotic forms of glomerulonephritis as compared with those parameters in urine of healthy persons. Content of tissue kallikrein was decreased in urine of patients with nephrotic form of glomerulonephritis as distinct from the disease latent form and healthy persons. At the same time, urine of these patients with nephrotic form contained elevated amount of blood plasma kallikrein and other proteinases of blood plasma origin. Bradykinin-inactivating activity as well as activities of kininase I and II were increased in urine of patients with the disease latent form 2-, 7- and 2.5-fold, respectively, and in urine of patients with nephrotic form--40-, 45- and 40-fold, respectively as compared with normal state. Daily excretion of free kinins with urine of healthy persons constituted 6.6 +/- 0.9 micrograms-eqv of bradykinin (BK), in patients with latent and nephrotic forms of glomerulonephritis--1.7 +/- 0.3 and 2.8 +/- microgram-eqv BK, respectively. Three kinins were found in all the examined persons when urine was collected in acid medium: BK, lysyl-BK and Met-Lys-BK; content of BK was minimal in urine of healthy persons and maximal--in urine of patients with nephrotic form of the disease. Clinical importance of the patterns studied and their role in correction of treatment course of patients with nephrotic form of glomerulonephritis are discussed.

[Resistance of heart valve bioprosthesis to the proteolytic effect of collagenase and elastase]. / Izuchenie ustoichivosti bioprotezov klapanov serdtsa k proteoliticheskomu vozdeistviiu kollagenazy i élastazy.

Proteolytic destruction of pig aortal valves was studied at various steps of their preparation to implantation (native tissue, the tissue treated with terrylytine as well as with terrylytine and glutaric aldehyde) using pig pancreatic elastase and bacterial collagenase. The rate of the tissue destruction was estimated by means of monitoring an increase in content of protein and amino nitrogen in the hydrolysates. The tissue treated with terrylytine and glutaricaldehyde was 10-40-fold more resistant to proteolysis as compared with native heart valve tissue. Inadequate stabilizing effect of glutaric aldehyde on elastin, as compared with that on collagen, was found, when proteolysis of native and modified with glutaric aldehyde elastin from bovine cervical ligament and of calf skin collagen was studied using elastase and collagenase, respectively.