Formate dehydrogenase in a reversed micelle system: regulation of catalytic activity and oligomeric composition of the enzyme

Formate dehydrogenases from the methylotrophic bacteria Pseudomonas sp. 101 and Mycobacterium vaccae N10 were studied in a system of Aerosol OT reversed micelles in octane. Three peaks of the catalytic activity were found on the plot of activity versus surfactant hydration degree (the size of the micellar inner cavity) which corresponded to functions of the enzyme in various oligomeric forms: monomeric, dimeric, and octameric. Kinetic data were confirmed by results of sedimentation analysis. The enzyme was chemically modified by a bifunctional reagent (dimethyl suberimidate) to obtain a catalytically active non-dissociating dimeric molecule of formate dehydrogenase. In the case of the covalently-linked non-dissociating dimeric enzyme, the peak which corresponded to the monomeric form of the enzyme was found to disappear from the catalytic activity curve.     

Medico-genetic study of residents of Marii El republic: burden of hereditary diseases in four regions of the republic

A medical genetic study of Orshanskii, Morkinskii, Sovetskii, and Semurskii raions (districts) of the Marii El Republic was performed. The total number of subjects examined was 115,743. Meadow Maris and Russians accounted for the most part of the populations of the districts studied. A total of 147 families with presumably autosomal dominant (AD) pathology and 150 families with presumably autosomal recessive (AR) or X-linked pathology (270 and 169 affected persons, respectively) were revealed. Segregation analysis demonstrated a good agreement between the observed and expected segregation frequencies for both AR and AD diseases, as well as a considerable number of sporadic cases of presumably AD diseases. The incidence of hereditary diseases was estimated separately for different population groups. Significant differences in this incidence were revealed between the urban and rural, as well as between the Russian and Mari populations; the average incidence was 2.33 affected subjects per 1000 people. The incidence of AR diseases was significantly higher in Maris than in Russians (1.34 x 10(-3) and 0.82 x 10(-3), respectively). The populations studied exhibited a significant, high correlation between the incidence of AR diseases and the levels of random and local inbreeding. The incidence of X-linked recessive diseases was approximately the same as in Russian populations studied earlier. Its average value was 0.5 per 1000 men; the incidence in the Mari and Russian populations did not differ significantly. The higher AD incidence in the total population studied and the higher AR incidence in the Meadow Mari population compared to the populations studied earlier are discussed.     

Generation of tryptic maps of alpha- and beta-globin chains by capillary electrophoresis in isoelectric buffers

A novel method for generating peptide maps, following tryptic digests of proteins, is reported here: capillary zone electrophoresis in the presence of isoelectric buffers as the sole buffering species. A typical buffer composition comprises 50 mM aspartic acid (pH = pI = 2.77), 0.5% hydroxyethyl cellulose (added as a dynamic coating agent for preventing peptide adsorption to weakly ionized silanols), 5% trifluoroethanol and 1% zwitterionic detergent (CHAPS). With this buffer composition, a high-voltage gradient can be applied (typically 600 V/cm in 75 microns I.D. and 900 V/cm in 50 microns I.D. capillaries), thus drastically reducing the analysis times. The method is applied to the generation of peptide maps of alpha- and beta-globin chains from human adult hemoglobin. In the case of beta-peptides, at an operative pH of 2.77, which represents a cross-over point in the titration curve of peptides T2 and T9, the two analytes merge into a single peak. However it is shown that it is possible to change the pH of the zwitterionic buffer by adjusting its concentration in solution. In 30 mM Asp (pH 3.0) or 20 mM Asp (pH 3.1) resolution of these two peptides is fully restored. Isoelectric, amphoteric buffers thus seem to represent a novel, powerful buffer system able to offer high resolution and high selectivity.     

The oxidant and antioxidant systems in patients with stable stenocardia and with a history of small- and large-focus myocardial infarcts

A study was made as well as a comparison of values of the cholesterol (Chs) lipid fractions concentrations, and of those of the lipid peroxidation processes activity (LPP) and the gluthathione antioxidant system (AOS) status in male subjects aged from 45 to 59 years, healthy individuals and patients with stable angina pectoris and myocardial infarction (micro- and macrofocal) during cicatrizing stage (health resort rehabilitation stage). In patients with various forms of IHD total cholesterol is raised as are atherogenic lipoproteins, and atherogenicity index, intensification being noted of LPO along with a considerable decrease in functional potencies of AOS. Similar pattern of changes of LPO and AOS in patients with stable angina pectoris and myocardial infarction (MI) during cicatrization stage certainly warrants correction of these systems not only in acute phase of MI but at the stage of rehabilitation as well.     

Characterization of recombinant heparin cofactor II expressed in insect cells

Recombinant human heparin cofactor II (rHCII) was expressed as a fully active protein in the High-Five insect cell line. A maximal protein concentration of 6 micrograms/10(6) cells was achieved 2 days postinfection. Approximately 40 micrograms of partially purified rHCII was routinely recovered from 50 ml of media after sequential heparin and Q-Sepharose affinity adsorption. rHCII had a slightly lower apparent molecular weight than blood plasma HCII (pHCII) due to differences in N-glycosylation. Like pHCII, rHCII formed a stable bimolecular complex with thrombin when assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The thrombin and chymotrypsin inhibitory properties of rHCII and pHCII were quite similar. In the absence of glycosaminoglycan, the thrombin inhibition rate (k2 x 10(-4) M-1 min-1) was 2.29 +/- 0.36 for rHCII and 3.38 +/- 0.34 for pHCII. Chymotrypsin inhibition rates (k2 x 10(-5) M-1 min-1) were 6.2 +/- 2.0 for rHCII and 8.0 +/- 2.6 for pHCII. In the presence of glycosaminoglycans, the maximal thrombin inhibition rate (k2 x 10(-3) M-1 min-1) for rHCII was 10.4 +/- 2.5 at 100 micrograms/ml heparin and 16.0 +/- 4.3 at 1000 micrograms/ml dermatan sulfate compared to 9.0 +/- 0.7 at 200 micrograms/ml heparin and 18.5 +/- 5.3 at 1000 micrograms/ml dermatan sulfate for pHCII. HCII inhibition of thrombin was blocked by a synthetic sulfated hirudin peptide in both the presence and the absence of glycosaminoglycan. The present report describes for the first time the expression and characterization of HCII in a baculovirus system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rHCII for future structure-function studies.     

The interaction of homogeneous motor conditioned reflexes during conditioned-reflex switchover: the transfer of learning and interference

The dynamics of elaboration of the conditioned switchover (CSw) of homogeneous motor conditioned reflexes (CR) with different lateralization of the motor skill was studied in rabbits. Learning of CSw was shown to pass three basic stages characterized by different degree of consolidation of the CRs, by interaction between the two CRs, and by manifestation of CSw. Stages I and II reflected the process of afferent and efferent generalization of CRs and CSw, stage III was associated with specialization of CRs and CSw. Both positive and negative transfer of motor learning as well as interference between the two motor skills took place during CSw elaboration. The highest rate of CSw acquisition was observed in rabbits displaying positive motor transfer.     

Clinical pharmacokinetic and pharmacodynamic studies with the nonclassical antifolate thymidylate synthase inhibitor 3, 4-dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)-quinazolone dihydrochloride (AG337) given by 24-hour continuous intravenous infusion

3,4-Dihydro-2-amino-6-methyl-4-oxo-5-(4-pyridylthio)-quinazolon e dihydrochloride (AG337) is a nonclassical inhibitor of thymidylate synthase (TS) designed to avoid potential resistance mechanisms that can limit the activity of classical antifolate antimetabolites. A clinical pharmacokinetic and pharmacodynamic study of AG337 given as a 24-h i.v. infusion was performed. Thirteen patients received 27 courses over the dose range 75-1350 mg/m2. Plasma AG337 concentrations were achieved which, in preclinical models, were associated with antitumor effects. AG337 clearance was saturable, and the pharmacokinetics of the drug at doses above 300 mg/m2 was best described by a one-compartment model with saturable elimination (median Km = 6.5 microgram/ml; range, 4.1-13 microgram/ml; median Vmax = 2.0 microgram/ml/h/m2; range, 0.96-5.6 microgram/ml/h/m2). Following the end of the infusion, AG337 was cleared rapidly (t1/2, 53-193 min), and levels were less than 0.2 microgram/ml in all patients by 48 h. Plasma protein binding was 96-98%, and the urinary excretion of AG337 as unchanged drug did not exceed 30% of the dose administered. Measurements of plasma deoxyuridine (dUrd) concentrations showed that doses of 600 mg/m2 and above of AG337 produced a consistent elevation in plasma dUrd levels (60-290%), suggesting that TS inhibition was being achieved in patients. However, in all cases dUrd concentrations had returned to pretreatment levels 24 h after the end of the infusion, suggesting that TS inhibition was not maintained. Local toxicity, probably due to the infusate pH, was the only significant adverse effect observed. These studies have shown that cytotoxic AG337 plasma concentrations can be readily achieved without acute toxicity and that these concentrations are associated with elevations in plasma dUrd levels. The lack of prolonged dUrd elevations indicates that extended administration should be explored using central line or p.o. administration to avoid local toxicity.     

Method of assessing the functional state of the human body

The article demonstrates an algorithm to evaluate functional state of operators exposed to ecologic hazards. The evaluation of the functional state includes analysis of the heart rhythm registered for 2 minutes. The functional state is assessed through an Indicator of Regulatory Systems Activity (IRSA), obtained by summation of all the values in modulus. IRSA values define 5 functional states: norm; functional strain moderate, marked and extreme; asthenia of regulatory systems. The algorithm suggested differs from the known ones in some features as follows. The diagnosis of heterotopic arrhythmia serves for more precise code demonstrating the heart automatism; formed on the basis of variation coefficient, the code demonstrating stability of the regulation proves the status of stable or disordered regulation; the code demonstrating activities of subcortical neural centers is formed on the basis of measured relative capacity of primary and secondary respiratory waves.