Effects of Vinpocetine on mitochondrial function and neuroprotection in primary cortical neurons.

Vinpocetine (ethyl apovincaminate), a synthetic derivative of the Vinca minor alkaloid vincamine, is widely used for the treatment of cerebrovascular-related diseases. One of the proposed mechanisms underlying its action is to protect against the cytotoxic effects of glutamate overexposure. Glutamate excitotoxicity leads to the disregulation of mitochondrial function and neuronal metabolism. As Vinpocetine has a binding affinity to the peripheral-type benzodiazepine receptor (PBR) involved in the mitochondrial transition pore complex, we investigated whether neuroprotection can be at least partially due to Vinpocetine's effects on PBRs. Neuroprotective effects of PK11195 and Ro5-4864, two drugs with selective and high affinity to PBR, were compared to Vinpocetine in glutamate excitotoxicity assays on primary cortical neuronal cultures. Vinpocetine exerted a neuroprotective action in a 1-50microM concentration range while PK11195 and Ro5-4864 were only slightly neuroprotective, especially in high (>25microM) concentrations. Combined pretreatment of neuronal cultures with Vinpocetine and PK11195 or Ro5-4864 showed increased neuroprotection in a dose-dependent manner, indicating that the different drugs may have different targets. To test this hypothesis, mitochondrial membrane potential (MMP) of cultured neurons was measured by flow cytometry. 25microM Vinpocetine reduced the decrease of mitochondrial inner membrane potential induced by glutamate exposure, but Ro5-4864 in itself was found to be more potent to block glutamate-evoked changes in MMP. Combination of Ro5-4864 and Vinpocetine treatment was found to be even more effective. In summary, the present results indicate that the neuroprotective action of vinpocetine in culture can not be explained by its effect on neuronal PBRs alone and that additional drug targets are involved.

Adenovirus type 2 endoprotease: isoforms and redox effects.

The cysteine protease encoded by adenovirus type 2 contains eight cysteines, some of which are involved in catalysis and enzyme activation. Here we investigated the effects of oxidation, mercapto-ethanol, dithiothreitol, diamide and protein disulphide isomerase on wild-type and mutant enzymes. Three isoforms of the enzyme were detected in infected cells and a fourth in preparations of purified recombinant enzyme. The latter isoform was absent in preparations of enzyme mutated at any of the three conserved cysteines, C-104, C-122 and C-126. Enzyme activity could be stimulated by agents other than the authentic activating peptide (pVIc), such as cysteamine, though less efficiently. Diamide at low concentrations stimulated the activity of the ts1 enzyme, but inhibited both ts1 and wild-type enzyme at higher concentrations. Protein disulphide isomerase failed to restore enzyme activity to the oxidized isoform. The present studies in combination with previous results using mutants appeared to rule out amino acids C-67, C-122, C-126 and C-127, leaving the two remaining semi-conserved C-17 and C-40 and the conserved C-104 as potential candidates for binding peptide pVIc.

Tissue concentrations and correlations of prostaglandins in healthy and inflamed human esophageal and jejunal mucosa.

The PGE2, PGF2 alpha, PGI2, and TXB2 content in biopsies of healthy esophageal mucosa and inflamed mucosa and from subjects with chronic esophagitis was measured and statistically analyzed. No significant differences were found between the tissue concentrations of prostaglandins in the inflamed and the healthy mucosa, except for elevated PGI2 content in the inflamed esophageal mucosa in comparison to healthy mucosa. The prostaglandin content of jejunal mucosa was unchanged in jejunitis and in atrophy compared to findings in healthy subjects. Regression analysis revealed a significant negative correlation between the PGF2 alpha and PGI2 content in both inflamed esophageal and inflamed jejunal mucosa. In healthy mucosa, no correlation was found between the tissue concentrations of these two prostaglandins, either in the esophagus or in the jejunum. These results suggest the redistribution of cyclic endoperoxide metabolism under certain pathological conditions.

Identification of active-site residues of the adenovirus endopeptidase.

Multiple sequence alignment of the 12 adenovirus endopeptidases known to date identified a number of conserved residues which might be important for enzyme activity. Eleven mutants were created in the cloned gene by site-directed mutagenesis to identify the active site of this thiol endopeptidase. Analysis of the proteolytic activity in a crude system using viral precursor proteins, as well as in a purified system with activated proteinases using a new chromophoric octapeptide substrate, yielded results consistent with Cys-104 and His-54 being two members of the active site. This result was confirmed by the carboxymethylation of the reactive Cys-104 and its prevention by the active-thiol-specific agent E64. Although Cys-122 and Cys-126 were also reactive cysteines, mutation of these residues did not affect enzyme activity. Replacement of the active-site Cys-104 by serine converted the enzyme into a serine-like proteinase, sensitive to serine proteinase inhibitors. The absence of homology to other proteinases, particularly at the active-site cysteine, coupled with the requirement for activation by a substrate cleavage fragment, indicates that the adenovirus endoproteinase may represent a new subclass of cysteine proteinases.

Isolation and properties of adenovirus type 2 proteinase.

We have cloned and expressed the human adenovirus type 2 proteinase gene in Escherichia coli. The expressed proteinase was isolated by a four-step chromatographic procedure. Purity and identity of the recombinant protein was established by two-dimensional gel electrophoresis, N-terminal sequencing, and specific antisera. The pure enzyme did not contain disulfide bridges, and it consisted of one subunit with a pI of 10.2. It did not show any sign of autocleavage. Labeled iodoacetate bound the pure enzyme while labeled diisopropyl fluorophosphate did not. The protease readily cleaved the viral pVII protein, ovalbumin, fibrin, and actin but had no effect on synthetic penta-, octa-, or nonapeptides carrying the consensus sequence for cleavage. The inhibitory profile of the isolated proteinase and the affinity labeling clearly indicate that the human adenovirus type 2 proteinase is a cysteine rather than a serine proteinase as previously believed. The most likely candidate for an active site residue is one of the two conserved cysteines, Cys-104 or Cys-122.

Gene regulation during anaerobiosis in soya roots.

The proteins of soybean roots undergoing anaerobiosis can be grouped into three classes. Class 1 proteins are induced severalfold and at least 28 of these were identified by in vivo labeling. These proteins include the enzymes alcohol dehydrogenase (ADH), fructose aldolase, pyruvate decarboxylase, phosphoglucomutase, and lactate dehydrogenase. Class 2 proteins include such enzymes as glucose phosphate isomerase, sucrase, and malate dehydrogenase; their specific activity remains constant in aerobiosis or anaerobiosis. The third class of proteins includes those enzymes such as peroxidase whose activity decreases more than 90% after just 1 day in anaerobiosis. Immunoblotting coupled with two-dimensional chromatography of in vitro translated plant extracts demonstrated that ADH level during anaerobiosis is controlled by its mRNA concentration. Little or no mRNA for ADH was detected in aerobically grown roots. This suggests that the increased level of ADH activity is due to de novo synthesis of the mRNA rather than activation of a sequestered mRNA or superactivation of the protein.

Soybean L-(+)-lactate dehydrogenases: purification, characterization, and resolution of subunit structure.

Lactate dehydrogenase (LDH) (EC 1.1.1.27) from soybean (Glycine max) was purified 2360-fold to homogeneity using ion-exchange, hydroxyapatite, affinity, and hydrophobic chromatographies. The molecular weight of the holoenzyme is 150,000 +/- 5000. Two-dimensional (isoelectrofocusing-sodium dodecyl sulfate) gel electrophoresis reveals two polypeptides subunits of 5.9 and 6.5 pI and of 36,000 +/- 1000 and 37,000 +/- 1000 Mr, respectively. Nondissociating electrophoresis and isoelectric focusing of LDH resolved five tetrameric isoenzymes with pI's between 6.0 and 6.5. The data suggest that these LDH isoenzymes are derived from random association of the products of two different, but most probably related, genes. Kinetic measurements revealed substrate inhibition at high concentrations of lactic acid and biphasic kinetics with NAD.

Mucosal prostaglandin levels of the gastric stump.

The endogenous mucosal PG levels (PGE2, PGF2 alpha, PGI2, TXB2) of the gastric stump were investigated from biopsy materials, after partial gastrectomy made for ulcer disease. The mucosa of the stomach remnant were found to contain mainly PGE2 and PGI2. The PG contents of the mucosa of gastric stump were not influenced by the type of resection. Mucosal PG concentrations on the greater curvature were not dependent on the patients' age, the indication of the gastrectomy, the duration of postoperative interval, or the sex of the patient. There was no relation between the secretion capacity of the resected stomach and the mucosal PG contents of the greater curvature. After Billroth I and II gastrectomy procedures equivalently fair correlations have been established between the mucosal levels of PGE2 and PGF2 alpha, PGE2 and TXB2, PGF2 alpha and TXB2, PGI2 and TXB2 on the greater curvature, respectively. After both types of gastrectomy the mean PGI2 mucosal concentration of the greater curvature was significantly lower than those of the gastroenteroanastomosis and lesser curvature below the cardia, which in turn did not differ from each other. Biliary reflux does not cause characteristic alterations of the mucosal PG levels on the greater curvature. No definite relation between the histological findings of the mucosa and the PG concentrations was observed, which suggests a secondary role of the endogenous PGs in the pathogenesis of light microscopic mucosal alterations of the resected stomach.

Relation of renal thromboxane A2 production to urine flow, electrolyte excretion and plasma renin activity in control state and drug induced hypotension.

The functional importance of renal TxB2 generation in the maintenance of elevated arterial blood pressure in essential hypertension was followed in 22 patients, using the method of sustained blood pressure decrease by i.v. sodium nitroprusside infusion. Linear correlation between urinary excretion of TxB2, urine flow, and sodium excretion could be established in both control and hypotensive periods. Presumably, changes in urinary excretion of TxB2 reflect a secondary intrarenal counterregulatory response.

Release of prostacyclin (PGI2) from trophoblast in tissue culture: the effect of glucose concentration.

It is known that short term cell culture system offers a reliable and reproducible means for measuring placental PGI2 production in vitro, in which factors controlling its production and metabolism can be studied. The aim of the present investigation was to study the effect of glucose on generation of PGI2 by trophoblast obtained from early pregnancy in short term cell culture. Trophoblast was cultured using the method of Jogee et al and the concentration of 6-oxo-PGF1 alpha in culture supernatans was measured by a specific direct radioimmunoassay (New England Nuclear, USA). There was a significant decrease in 6-oxo-PGF1 alpha production by trophoblast cells when incubating with increased glucose concentrations (300 and 600 mg/dl) compared to controls (without glucose). These data show for the first time that high concentrations of glucose inhibit PGI2 production by cultured trophoblast cells obtained from early pregnancy. The implication of these findings for the mechanism of development of congenital anomalies in diabetic pregnant women is discussed.

Platelet sensitivity to adenosine diphosphate and to prostacyclin in diabetic pregnancies.

Platelet sensitivity to adenosine diphosphate (ADP) and to prostacyclin (PGI2) was studied in normal and diabetic pregnant women. The threshold concentrations of ADP inducing the second phase of aggregation were used to determine the platelet sensitivity to PGI2. The sensitivity of platelets to ADP increased in both groups in the second trimester, thereafter it decreased both in normal and diabetic pregnancies. In contrast, sensitivity to PGI2 increased in the last trimester of pregnancy. No difference could be observed between diabetic and normal groups. The similarity of the results between the two groups could be explained by the normoglycaemic state of well-controlled diabetic pregnant women.

Effect of acetylsalicylic acid on prostacyclin production in trophoblast.

In attempt to elucidate whether acetylsalicylic acid (ASA) has an in vivo effect on prostacyclin (PGI2)-like activity released from trophoblast we have evaluated PGI2-like activity in pregnant women scheduled for pregnancy termination after ASA ingestion. Following subjects were studied: Group I: 7 healthy pregnant women who were treated with 1.5 g ASA for two days; Group II: 18 control pregnant women who received placebo for two days. Trophoblast specimens were obtained by legal abortions; PGI2-like activity in trophoblast was measured by the method of Moncada. In normal pregnant women (8-10 weeks gestation) treated with ASA the mean PGI2-like activity of trophoblast significantly decreased compared to the controls. These data indicate that treatment with ASA of early pregnant women might have a harmful effect on trophoblast and the problem should be further explored before allowing the administration of cyclooxygenase inhibiting drugs during early pregnancy.

Mitochondrial substrate oxidation-dependent protection against lipid peroxidation.

The effect of mitochondrial substrate oxidation on the NADPH-dependent lipid peroxidation of intact mitochondria, microsomes and of homogenate from rat liver was studied. It was found that addition of either succinate or beta-OH-butyrate decreased the rate of malondialdehyde production of mitochondria. The effect of succinate was found to be marked (80-90% inhibition). Addition of succinate strongly inhibited the lipid peroxidation of a reconstituted system containing both mitochondria and microsomes. Increasing the amount of mitochondria in this system resulted in enhancement of the inhibition. The same succinate effect on malondialdehyde formation in liver homogenate was also observed. These findings suggest that mitochondria supplied with respiratory substrates may play a role in the protection against the lipid peroxidation in the liver cell.