[Engineering of a System for the Production of Mutant Human Alpha-Fetoprotein in the Methylotrophic Yeast Pichia pastoris].

A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

[Neuroprotective effects of lipoic acid and superoxide dismutase in the rat model of cerebral ischemia].

BACKGROUND: the present study was aimed at investigation of neuroprotective effects of alpha-lipoic acid (LA) and superoxide dismutase (SOD) in brain ischemia in rats. METHODS: two models were used to produce brain ischemia: focal ischemia (permanent left middle cerebral artery occlusion) and permanent ligation of both CCA without subsequent reperfusion. Cu/Zn-SOD at a dose 5 mg/kg, i.v. and LA at a dose of 20 mg/kg, i.p. were injected 30 minutes prior or 5 minutes after onset of ischemia. The end-points of the study were histochemically determined: infarction size, ultrastructural changes in the cerebral tissue, and survival rate. RESULTS: LA administration 30 minutes prior to ischemia dramatically reduced infarction size (p < 0.001). Injection of LA 5 minutes after beginning of ischemia did not affect the infarction size. Besides, infarction ion size was unchanged after injection of SOD 30 minutes prior to ischemia. CONCLUSION: the LA treatment regimen used in this study resulted in significant cerebral protection against ischemia. In contrast, SOD did not show any protective effects in focal and forebrain ischemia.

[Protective effect of meloxicam against cerebral ischemia/reperfusion injury in normo- and hypertensive].

Normotensive and hypertensive male Wistar rats were subjected to the 3 h bilateral carotid artery occlusion followed by 72 h of reperfusion. The selective cycloxygenase-2 inhibitor meloxicam was administered intramuscularly after the cerebral reperfusion at a daily dose of 15 mg/kg. Doppler ultrasound was used to evaluate the cerebral blood flow during the reperfusion phase. The level of malondialdehyde (MDA) within the brain tissue homogenate was determined using spectrophotometry. The presence of arterial hypertension caused the altered response of brain tissue to ischemia/reperfusion. The meloxicam treatment significantly decreased the MDA level in normotensive rats subjected to ischemia-reperfusion. The protective effect of meloxicam against cerebral ischemia/reperfusion injury was more pronounced in hypertensive rats as compared to normotensive animals.

Effectiveness of combined treatment with superoxide dismutase and Reamberin during skin ischemia.

Experimental skin ischemia in rats was induced by suturing a skin fold on the back with a silk thread. Combined pretreatment with superoxide dismutase (intraperitoneally) and Reamberin (intravenously) in doses of 0.01 and 6.25 mg/kg (by succinate concentration), respectively, produced a strong protective effect on the skin. The index of cytolysis decreased by 39%. The more pronounced antinecrotic effect of combined treatment with superoxide dismutase and Reamberin compared to the effect of Reamberin alone was related to a sharp increase in the reserve capacity of the antioxidant system. After combined therapy, activity of antioxidant defense enzymes not only increased, but even exceeded the normal level. The increase in activity of endogenous superoxide dismutase under the influence of combined therapy was accompanied by suppression of superoxide anion production.

Antinecrotic and antioxidant effects of superoxide dismutase during skin ischemia.

Antinecrotic activity of SOD was studied in rats with experimental skin ischemia. Treatment with SOD increased activity of endogenous SOD in skin homogenates (by 70 and 26% compared to the ischemic and intact skin, respectively). However, the rate of superoxide anion generation remained unchanged after SOD treatment. Creatine phosphate content and NAD/NADH redox potential increased by 16 and 21%, respectively, on day 3 after SOD administration. The increase in functional activity of the energy supply system and rise in the reserve capacity of the antioxidant protection system contribute to inhibition of lactate dehydrogenase and creatine phosphokinase and decrease in the cytolysis index under the influence of SOD. Our results indicate that SOD produces the antinecrotic effect and holds much promise for the therapy of skin ischemia.

Ointments with superoxide dismutase and interleukin-1beta: effect on reparative processes and impedance of burn wound.

We found that tissue impedance can serve as a reliable criterion of the severity of wound process and efficiency of burn treatment. Ointment with superoxide dismutase effectively promoted wound reparation and recovery of tissue structure after thermal skin burn compared to ointments containing interleukin-1beta or mixture of interleukin-1beta and superoxide dismutase.

Effect of Erysod (erythrocyte superoxide dismutase) on blood concentration of reactive oxygen species in patients with severe burns and burn shock.

The dynamics of blood concentrations of reactive oxygen species and LPO products in patients with thermal injuries of different severity was studied. Monitoring of these parameters by chemiluminescent and spectrophotometric techniques helps to predict the course of burn shock and prevent complications. Erysod (0.004% solution, 33-66 microg/min, daily dose 24-32 mg) added to antishock infusion therapy during the early periods after injury suppressed generation of free radicals (by 20% after 15 min and by 30-40% after 24 h), promoted normalization of their contents, and reduced damage to visceral organs during acute period of thermal injury.

Erysod, erythrocyte superoxide dismutase preparation: effects on LPO processes and morphological changes in the viscera of rats with burns under conditions of delayed antishock infusion therapy.

Biochemical and morphological changes in the kidneys, liver, heart, and lungs were studied in rats with deep burns of 20% body surface. Erysod (0.47 mg/kg/day) added to antishock therapy notably reduced the intensity of LPO processes in tissues both in cases when infusion therapy was started immediately (by 8-20% 12 h and by 5-24% 24 h after the injury) and when this therapy was 6 h delayed (by up about 36% after 12 h). This prevented injuries to visceral organs during the acute period of thermal injury and prevented the development of burn disease.

Inactivation and oxidative modification of Cu,Zn superoxide dismutase by stimulated neutrophils: the appearance of new catalytically active structures.

Bovine superoxide dismutase (SOD) was inactivated during incubation with phorbol myristate acetate-stimulated neutrophils. In addition, stimulated neutrophils were able to disrupt the SOD structure. Inactivation and structural damage were dependent on the action of hypochlorous acid, an oxidant generated by the myeloperoxidase-hydrogen peroxide-chloride system of neutrophils. Incubation of SOD with stimulated neutrophils lead to long-wavelength fluorescence (ex, 350 nm; em, 450 nm) and the appearance of new structural forms with other isoelectric points. These additional forms possess catalytic activity. Generation of catalytically active new forms of SOD demonstrates the inaccessibility of the active centre of SOD to hypochlorite and may be a reason for the successful application of SOD during anti-inflammatory therapy.

[Oxidative modification and inactivation of superoxide dismutase by hypochlorite]. / Okislitel'naia modifikatsiia i inaktivatsiia superoksiddismutazy gipokhloritom.

The inactivating effect of hypochlorite on Cu, Zn-superoxide dismutase (SOD) from bovine erythrocytes has been studied. According to SDS gel electrophoresis and isoelectric focusing data, oxidation is associated with the degradation of the polypeptide chain, formation of aggregates, and appearance of new isoforms. These protein fractions differ from native SOD by the electric charge and molecular mass but possess a catalytic activity. Modified SOD isoforms occur as a result of intramolecular crosslinking of amino groups and aldehydes which is confirmed by the appearance of fluorescence maxima in the longwave region characteristic of such links. It is assumed that the mechanism of SOD inactivation is coupled to the oxidation of amino acids located outside the active center of the enzyme.

[Hydrolysis of microcrystalline cellulose by multienzyme cellulase complexes of various origins]. / Gidroliz mirokristallicheskoi tselliulozy pod deistviem polifermentnykh tselliulaznykh kompleksov razlichnogo proiskhozhdeniia.

The kinetic regularities of glucose and cellobiose formation from microcrystalline cellulose (MCC) under the action of cellulase complexes from eight different sources were studied. By means of successive addition of selected components of the cellulase complexes (endoglucanase and cellobiase) to the reaction system the rate-limiting steps for multienzymatic hydrolysis of MCC were determined. It was shown that in most cases the rate-limiting step of glucose formation (via hydrolysis of the intermediate cellobiose) is the cellobiase action. In a single case only (with a cellulase complex from Aspergillus foetidus enriched with cellobiase) the rate of glucose formation from MCC was limited by the endoglucanase action. In accordance with the kinetic theory developed it was shown that the addition of cellobiase excess to the reaction system resulted in changes of the rate-limiting step over to endoglucanase attack on the non-soluble cellulose for all cellulase complexes under study. Under the given experimental conditions a linear correlation between the steady-state ready of glucose formation from MCC under the action of all cellulase complexes on the on hand, and the endoglucanase activity of these complexes, on the other, was established. It was shown that the action of all cellulase (arbitrarily selected ones) is described by principally the same kinetic regularities, which, in turn, is indicative of identical mechanisms for hydrolysis of the insoluble cellulose under effects of cellulase complexes of various origin.

[Purification and properties of low molecular weight endoglucanase of the cellulase complex from Trichoderma koningii]. / Ochistka i svoistva nizkomolekuliarnoi endogliukanazy tselliulaznogo kompleksa iz Trichoderma koningii.

A homogenous low molecular weight 1,4-beta-glucan glucanohydrolase (endoglucanase) has been isolated from a crude commercial preparation of cellokoningine P10X of T. koningii origin. The molecular weight of the enzyme as determined by polyacrylamide gel electrophoresis is 13 000. The endoglucanase was obtained as a lyophylized preparation free of the cellobiase activity. It was shown that cellobiose or methylcellobioside activate the effect of the low molecular weight endoglucanase (measured by the viscometric technique with respect to CMC hydrolysis) and at the same time almost completely suppress the activity of high molecular weight endoglucanases from the sane source. A detailed kinetic study of the effects showed that the low molecular weight enzyme is activated by a transglycosylation mechanism, where cellobiose acts as an added nucleophile. The activation is 6-fold at saturating concentrations of cellobiose (Ks = 15 mM). It was shown that diverse kinetic behaviour of cellobiose which can act both as activatory and inhibitor for endoglucanases from different sources can be explained, firstly, by different ratios of low to high molecular weight endoglucanases in the cellulase complexes, and, secondly, by their ability to catalyze transglycosylation reactions, which, in turn, results in a transfer of reducing end groups of the reaction products onto cellobiose as an added nucleophile.

[New ultrasonic method for studying the composition and properties of multienzyme systems: enzymes of the cellulase complex]. / Novyi ul'trazvukovoi metod izucheniia sostava i svoistv polifermentnykh sistem: fermenty tselliulaznogo kompleksa.

A new ultrasonic method for determination of the composition and properties of individual components of multienzyme systems without their resolution has been developed. The method is based on a determination of the pH-profiles of the first order rate constants for inactivation of the enzymatic components in a complex by ultrasonic cavitation. The method was used for studying a cellulase complex from Geotrichum candidum. It was shown that the cellulase complex contains at least four cellulolytic enzymes, i. e. endoglucanase, exoglucosidase, cellobiase and aryl-beta-glucosidase, which differ in their pK values for the ionogenic groups controlling the pH-profiles of ultrasonic inactivation and in the inactivation rate constants at the plateau of a pH-rate profile.

[Cellobiose as a regulator of endoglucanase activity of cellulase complexes. Mechanism of the regulation]. / Tsellobioza-peguliator aktivnosti endogliukanaz tselliulaznykh kompleksov. Mekhanizm peguliatsii.

Cellobiose may exert different effects on the activities of various endoglucanases. The endoglucanases of T. reesei and Rapidase are noticeably suppressed by cellobiose at concentrations above 3 mM. On the other hand, a low molecular weight endoglucanase from T. koningii is activated by cellobiose, whereas high molecular weight endoglucanases from the same source are inhibited by cellobiose. A detailed kinetic analysis of the effects showed that the low molecular weight endoglucanase is activated by a transglycosylation mechanism, in which cellobiose acts as an additional nucleophile. At saturating concentrations of cellobiose (Ks = 15 mM) the enzyme activity is increased 6-fold. Such a specific mechanism of activation manifests itself in an acceleration of random cleavage of CM-cellulose by the low molecular weight endoglucanase, which can be recorded by a viscosimetric technique. However, its action does not accelerate the production of soluble reducing sugars.