The study of nucleoside triphosphate-binding center of DNA-dependent RNA-polymerase of phage T7 using GTP analogs

The NTP binding site of bacteriophage T7 DNA-dependent RNA polymerase was studied using GTP analogs. For four analogs the irreversible inhibition was demonstrated. The kinetic parameters for competitive (Ki) and irreversible (KI and k3) inhibition were determined. One of the analogs, 5'[2-hydroxy(4-iodoacetamido)benzoyl]guanosine, was shown to inactivate the enzyme rapidly due to the modification of SH-groups. Some suggestions on the structure of the RNA polymerase active site have been made.     

Modulation of glutamate-induced uncompetitive blocker binding to the NMDA receptor by temperature and by glycine

The effect of temperature on the binding of [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) to the ion channel of the N-methyl-D-aspartate (NMDA) receptors was studied in washed rat brain-cortex membranes. Raising the temperature from 5 to 33 degrees C resulted in a significant increase in the association rates of [3H]TCP binding measured in the presence of 1 microM glutamate and 1 microM glycine, but was less effective in the absence of the added agonists. No such effects of temperature on the dissociation rates of [3H]TCP-receptor complexes were observed. In the absence of agonists, neither the association nor the dissociation binding components varied with temperature, suggesting a diffusion-controlled limitation of access of the ligand to its site within the nonactivated NMDA receptor. No evidence was found for a temperature-dependent change in the density of [3H]TCP binding sites or for heterogeneity of [3H]TCP binding sites associated with the NMDA receptor, even though when approaching equilibrium the binding kinetics in the presence of glutamate and glycine deviated from an ordinary bimolecular reaction scheme. The data were fitted instead to a two-exponent binding function, comprising the sum of a fast and a slow binding component. Their corresponding time constants exhibited an increase with temperature, and the increase of each one was correlated significantly with the corresponding decrease in the equilibrium binding constant; however, there was no temperature-related change in the relative proportions of the two components, with the fast binding component (alpha) accounting for 50-70% of the site population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of pyruvate decarboxylase by 3-hydroxypyruvate.

Pyruvate decarboxylase from Zymomonas mobilis is inhibited by 3-hydroxypyruvate, which can also act as a poor substrate. While catalysing the decarboxylation of this alternative substrate, the enzyme undergoes a progressive but partial inactivation over several hours. The extent of inactivation depends upon the pH and upon the concentration of 3-hydroxypyruvate. After partial inactivation and removal of unchanged 3-hydroxypyruvate, enzymic activity recovers slowly. We suggest that inactivation results from accumulation of enzyme-bound glycollaldehyde, which is relatively stable, possibly because it is dehydrated to form an acetyl group.     

Effects of hyperthermia and photodynamic therapy on BALB/c mice bearing subcutaneous tumors

The therapeutic effects of photodynamic therapy and hyperthermia on mice bearing subcutaneous tumors were investigated. Ehrlich ascites tumor cells (1 x 10(7)) were implanted subcutaneously into the femoral area of BALB/c mice. A total of 134 tumor-bearing mice were treated with photodynamic therapy, i.e., administration of laser irradiation (514.5 nm, 112.5 mW/cm2 for 11.12 min with a total energy 75 J/cm2) after injection (i.p.) of hematoporphyrin derivative (HPD, 7.5 and 10.0 mg/kg body weight) and/or hyperthermia (by electric heating needles to 44 and 45 degrees C for 30 min) once a day for three successive days. The results revealed that the therapeutic effects of the combination of photodynamic therapy and hyperthermia were improved when compared with photodynamic therapy or hyperthermia alone. A combination of photodynamic therapy (10.0 mg HPD/kg body weight and 75 J/cm2 of total laser irradiation energy) and hyperthermia (44 degrees C for 30 min) had the best therapeutic effect, indicating that the mortality rate within 120 days (MR120) was 12.5% and the mean survival time (MST120) was 113.8 days.     

Calcium mediates the interconversion between two states of the liver inositol 1,4,5-trisphosphate receptor

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) regulates intracellular Ca2+ by mobilizing Ca2+ from a non-mitochondrial store. We have investigated the effects of Ca2+ on the binding of [32P]Ins (1,4,5)P3 to permeabilized rat hepatocytes and a liver plasma membrane-enriched fraction. Increasing the free Ca2+ concentration in the medium from 0.1 nM to 0.7 microM increased the capacity of a high affinity binding component (KD = 2-3 nM) in permeabilized cells by a factor of 10. If the membrane fraction was preincubated at 37 degrees C before binding was measured at 4 degrees C, all of the Ins(1,4,5)P3 receptors were transformed to a low affinity state (KD = 65 +/- 12 nM, Bmax = 3.1 +/- 0.1 fmol/mg, n = 4). When 0.7 microM of Ca2+ was added, the receptors were totally transformed to a high affinity state (KD = 2.8 +/- 0.4 nM, Bmax = 2.7 +/- 0.4 fmol/mg, n = 4). The EC50 of the Ca2(+)-induced interconversion of the Ins(1,4,5)P3 receptor was 140 nM. This Ca2(+)-induced transformation of the Ins(1,4,5)P3 receptor from a low affinity to a high affinity state was associated with an inhibition of the Ins(1,4,5)P3-induced Ca2+ release in permeabilized hepatocytes. These data suggest that the Ins(1,4,5)P3-dependent hormones, by increasing the intracellular Ca2+ concentration, induce a reversible transformation of the receptor from its low affinity state, coupled to the Ca2+ release, to a desensitized high affinity state. Transformation of the receptor may play a role in the oscillatory release of Ca2+ observed in single isolated hepatocytes.     

Chemo- and karyotaxonomic studies on some rhizomatous species of the genusSymphytum (Boraginaceae)

InS. tuberosum subspp.tuberosum andnodosum, S. grandiflorum andS. ibericum the presence of the pyrrolizidine alkaloids lycopsamine, echimidine and symphytine could be demonstrated. The taxonS. tuberosum contains an unknown compound that seems to be specific for this taxon. This compound is not the pyrrolizidine alkaloid anadoline which has previously been reported for this species. It is possibly represented by a peak on GC/MS with a molecular ion peak at m/z 623 (as TMS derivative) and can be used as a chemotaxonomic marker for the speciesS. tuberosum. The pyrrolizidine alkaloid pattern of the two subspecies ofS. tuberosum reinforces the close relationship. Fresh material ofS. tuberosum contained the triterpene isobauerenol, but in herbarium material isobauerenol was lacking. InS. grandiflorum, neither fresh nor dried material contains isobauerenol. In herbarium material ofS. ibericum also no isobauerenol could be found. More extensive chemotaxonomical research is necessary to support the view thatS. abchasicum is more closely related toS. ibericum than toS. grandiflorum.     

Purification and partial characterization of an iron regulated outer membrane protein of B. fragilis under non-denaturing conditions

The CHAPS-PAGE gelsystem we applied gave a good separation of the proteins of Bacteroides fragilis under non-denaturing conditions. We succeeded with preparative CHAPS-PAGE in purifying an iron regulated outer membrane protein (a 44 kDa polypeptide on SDS-PAGE) of B. fragilis. This integral membrane protein proved to be a lipopolysaccharide binding protein with an isoelectric point of approximately pH 5.5. This method of purifying membrane proteins could be an important step in research into the function of membrane proteins.     

The indices of antipneumococcal immunity in children with chronic pneumonia

In 76 children with chronic pneumonia the levels of serum antibodies to pneumococcal capsular polysaccharide antigens (serotypes 1, 3, 6B, 8, 9N, 15F, 23F), O-polysaccharide and pneumococcal protein somatic antigens were determined by ELISA techniques. The study showed that in sick children the content of antipneumococcal antibodies in the blood increased with age. No correlation between the content of total immunoglobulins and that of antipneumococcal antibodies in the blood of the patients was established, but a sharp decrease in the concentration of antibodies was registered in a child with hypoglobulinemia. No increase in the level of antibodies to pneumococcal antigens was observed in cases of the exacerbation of Pneumococcus-induced inflammatory process in the lungs.