[The past, present, and future of GosNIIgenetika].
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Academies and Institutes↗
Biomedical subjects
V G Debabov
Publications and source records attributed to V G Debabov.
[A search for association between the polymorphic markers of PON1 and PON2 genes and diabetic nephropathy in patients with type I diabetes mellitus].
Alleles and genotypes of polymorphic markers of paraoxonase 1 and paraoxonase 2 genes (PON1 and PON2) encoding enzymes of the body antioxidative defense were compared in type 1 diabetes mellitus patients with or without diabetic nephropathy. The patients with nonoverlapping ("polar") phenotypes constituted different groups. The first group contained patients with diabetic nephropathy (DN+, n = 62), clinical proteinuria (albuminuria above 300 mg per day), and at least 15-year disease duration. In control group, the patients had no diabetic nephropathy (DN-, n = 68), their albuminuria was below 200 mg per day, and disease duration was at least 20 years. Comparative analysis with exact Fisher's test revealed no significant differences in frequencies of alleles and genotypes of the PON1 gene polymorphic marker Gln192Arg and of PON2 gene polymorphic markers Ala148Gly and Cys311Ser. Our results suggest that the polymorphic markers studied are not associated with diabetic nephropathy among Russian patients in Moscow.
Aryldialkylphosphatase↗
[Associative analysis of the connection between mutation C1167T in the catalase gene and polymorphic marker D6S392 nearby the Mn-dependent superoxide dismutase gene with diabetic microangiopathies].
AIM: To study allele polymorphism of two variable regions [C1167T substitution in the catalase (CAT) gene D6S392 microsatellite near the Mn-dependent superoxide dismutase (SOD2) gene] was studied in insulin-dependent diabetic (IDDM) patients with (n = 36) or without (n = 56) diabetic nephropathy, and with (n = 30) or without (n = 44) diabetic retinopathy. MATERIAL AND METHODS: Both polymorphic regions were amplified using polymerase chain reaction (PCR). PCR products were separated using polyacrylamide (D6S366) or agarose (C1167T) gel electrophoresis. In a case of C1167, PCR-amplified products were digested with BstXI restriction endonuclease before electrophoresis. A significance of the difference between allele distributions in complicated and uncomplicated IDDM patients was estimated using the exact Fisher's test. RESULTS: No significant difference was observed in allele and genotype frequencies in complicated and uncomplicated IDDM subjects. CONCLUSION: C1167 polymorphism in the CAT gene and D6S366 near the SOD2 gene are not associated with the development of diabetic nephropathy and diabetic retinopathy in IDDM.
Adolescent↗
[Design of the human tumor-associated VNTR(Muc1) antigen gene, fused with streptavidin, its expression in Escherichia coli. Study of properties of the hybrid protein].
A gene of human tumor-associated antigen VNTR(MUC1) bound to streptavidin, an expression plasmid, and a highly effective hybrid protein-producing strain were constructed. It was shown that the streptavidin leader peptide ensures an effective secretion of the hybrid protein into the periplasmic space of Escherichia coli cells. The hybrid protein was isolated in a homogeneous state and its immunogenic properties were studied.
Amino Acid Sequence↗
Isolation and primary characterization of an amidase from Rhodococcus rhodochrous.
Amidase (EC 3.5.1.4) was purified to homogeneity from Rhodococcus rhodochrous M8 using isopropanol fractionation and exchange chromatography on Mono Q. The isolated amidase consists of four identical subunits with molecular weight 42+/-2 kD. The activity of the enzyme is maximal at 55-60 degrees C and within the pH range 5-8. The amidase from R. rhodochrous M8 is highly sensitive to such sulfhydryl reagents as Hg2+ and Cu2+. Chelators (EDTA and o-phenanthroline) and serine proteinase inhibitors (PMSF and DIFP) did not inhibit the activity of the enzyme. The enzyme exhibits hydrolytic and acyl transferase activity and does not possess urease activity. Aliphatic amides (acetamide and propionamide) were the best substrates for the amidase from R. rhodochrous M8, whereas bulky aromatic amides were poor substrates of this enzyme. The properties of the isolated enzyme are similar to those found in the corresponding amidase from Arthrobacter sp. J-1 and an amidase with wide substrate specificity from Brevibacterium sp. R312.
Amidohydrolases↗
[Cloning of streptavidin gene from Streptomyces avidinii and its expression in Escherichia coli. Secretion of streptavidin by E. coli cells].
The streptavidin gene from Streptomyces avidinii was cloned, an expression plasmid constructed, and a highly effective strain producer of streptavidin created. It was shown that the leader peptide of streptavidin ensures the effective secretion of this protein into the periplasmic space of Escherichia coli cells. The degradation site of the leader peptide was detected. Upon treatment with the total fraction of proteases secreted by S. avidinii into the culture medium, "core" streptavidin was obtained, which retained the biotin-binding function.
Cloning, Molecular↗
[Polymorphism of gene encoding vascular angiotensin II receptor and microangiopathies in patients with insulin-dependent diabetes mellitus].
Polymorphism A1166C of the AT1R gene encoding angiotensin vascular receptor [replacement of C (cytosine) for A (adenine)) at position 1166] was compared in patients with insulin-dependent diabetes mellitus (IDDM) complicated by diabetic nephropathy (DN) and in noncomplicated patients (n = 27 and n = 41, respectively) and also in patients with IDDM complicated by diabetic retinopathy (DR) and in correspondent noncomplicated individuals (n = 30 and n = 44, respectively). The frequency of AT1R gene alleles and genotypes in patients with IDDM complicated by DN did not differ significantly from that observed in patients with noncomplicated IDDM. In contrast, in patients with IDDM complicated by retinopathy, a significant decrease in the content of A allele (68.3% against 82.6%) and a significant increase in the content of C allele (31.7% against 17.4%) was found as compared with the control group. Thus, in the Moscow population, A1166C polymorphism of the AT1R gene is not associated with diabetic renal complications but indeed associated with diabetic retinal complications. C allele is a risk factor of DR (the relative risk, RR, is equal to 2.17), and A allele is, in contrast, a protective factor against early retinopathy development (RR is equal to 0.49).
Adolescent↗
[A new inhibitor of pyrimidine phosphorylase].
5,5-Bis(hydroxymethyl)-2-oxo-[1-(2-trifluoromethyl)-3,3,3- trifluoropropionamido)-1-trifluoromethyl-2,2,2-trifluoroethyl- 1,3,2-dioxaphosphan (CA-423) is an in vitro inhibitor of the Escherichia coli uridine and thymidine phosphorylases. Unlike widely studied nucleoside analogues, this compound binds to the enzymes irreversibly. Its LD50 in mice was 40 mg/kg. Due to the involvement of pyrimidine phosphorylases in carcinogenesis and the relatively low toxicity of CA-423, it is promising for anticancer therapy.
Animals↗
Isolation and initial characterization of the uridine phosphorylase from Salmonella typhimurium.
The structural udp gene encoding uridine phosphorylase (UPase) was cloned from the Salmonella typhimurium chromosome and overexpressed in E. coli cells. The S. typhimurium UPase was purified to an apparently homogeneous state, and some physicochemical characteristics of the enzyme were studied. The molecular weight of one subunit of UPase is 27.5 kD, and the optimal pH for its activity is 7.2--7.4. The native S. typhimurium UPase consists of six identical subunits, and its molecular weight is about 165 kD. According to these parameters, the S. typhimurium UPase is similar to the E. coli UPase. However, these enzymes differ substantially from one another by the substrate sensitivity and sensitivity to polarity of the medium. The S. typhimurium UPase has much higher phosphorylation activity toward thymidine, deoxyuridine, and 5;-bromide- or 5;-fluoride-containing analogs of nucleosides than that of E. coli UPase.
Cloning, Molecular↗
[Construction and characteristics of a cosmid library of genes of the bacterium Cornyebacterium glutamicum ATSS13032].
A representative genomic library of the Corynebacterium glutamicum ATCC 13032 genes in a cosmid vector Lorist6 was created. The cosmids contain inserts of bacterial DNA obtained by partial digestion with the Sau3A I restrictase. Five hundred and thirty individual primary recombinant clones were transferred into the wells of microtiter plates, where they are now being preserved. The average size of the bacterial DNA inserts determined via a sum of restriction fragment sizes of recombinant molecules is about 38 kb. The capacity of the obtained gene library is 8.4 equivalents of the C. glutamicum genome, i.e., every fragment of the genome is on average represented by eight clones and is presented in at least one clone with the probability > 99%. Clone grids (sets of recombinant clones located on the hybridization membrane in regular and reproducible order) were created. Specificity of the created clone library and its representativeness were confirmed experimentally by hybridization of clone grids with DNA probes corresponding to unique regions of the Corynebacterium genome. A plasmid containing the pheA prephenate dehydratase gene, olygonucleotide corresponding to the lysC gene, and the 21 RNA probe obtained from the insert ends in different cosmids were used as probes. The created set of clones allows the construction of a cosmid contig overlapping the C. glutamicum genome and a physical genetic map on its base.
Corynebacterium↗
Behavior of uridine phosphorylase from Escherichia coli K-12 in hydrated reversed micelles of surfactant in organic solvent.
The catalytic activity of uridine phosphorylase from Escherichia coli K-12 entrapped in hydrated reversed micelles of aerosol OT (AOT) in octane has been studied as a function of the degree of hydration of micelles. It was shown that the catalytic activity reaches maximum values at ratios [H2O]/[AOT] equal to 8.4, 12.8, 16.1, and 18.6. On the basis of sedimentation data the conclusion has been made that the maximums of the catalytic activity of uridine phosphorylase correspond to monomeric, dimeric, trimeric, and tetrameric forms of the enzyme.
Bacterial Proteins↗
Denaturation of uridine phosphorylase from Escherichia coli K-12 by guanidine hydrochloride.
Denaturation of uridine phosphorylase from Escherichia coli K-12 by guanidine hydrochloride results in red shift of the maximum in the protein fluorescence spectrum, dissociation of the hexameric enzyme molecule into monomers, and the loss of the enzymatic activity. The initial rate of the enzyme reactivation after the dilution of the enzyme preincubated with guanidine hydrochloride has the second order with respect to protein. It is assumed that the rate of the reactivation process is limited by the reassociation of monomers possessing low enzymatic activity to dimers followed by the rapid step of hexamer formation.
Enzyme Activation↗
Denaturation of uridine phosphorylase from Escherichia coli K-12 with guanidine hydrochloride: kinetics of inactivation, dissociation, and reactivation of the enzyme.
Denaturation of uridine phosphorylase from Escherichia coli K-12 by guanidine hydrochloride is accompanied by the displacement of the maximum in the protein fluorescence spectrum (lambda max) from 331 to 348 nm. The half-maximal change in the lambda max position is observed at 1.18 M guanidine hydrochloride. For this concentration of denaturant, the sedimentation pattern consists of two boundaries, one of which corresponds to the motion of the hexameric enzyme form (s20,w = 8.2 S) and other represents a monomer (s20,w = 2.6 S). In the presence of 2 M guanidine hydrochloride the enzyme moves as a monomer. The kinetics of inactivation of uridine phosphorylase by guanidine hydrochloride are complex (minima and maxima are observed on the kinetic curves). The initial rate of the enzyme reactivation after dilution of the enzyme preincubated with guanidine hydrochloride is second order with respect to protein. It is assumed that the rate of the reactivation process is limited by the reassociation of low-activity monomers into dimers followed by a rapid hexamer formation. The second-order rate constant for the reassociation of the enzyme is 3.0.10(4) M-1.sec-1 (50 mM borate buffer, pH 7.7, containing 100 mM inorganic phosphate; 20 degrees C). Thiol groups become accessible to titration by 5,5'-dithiobis-(2-nitrobenzoic acid) after treatment of uridine phosphorylase with guanidine hydrochloride. Uridine and uracil inhibit the unfolding of the protein globule by guanidine hydrochloride.
Enzyme Reactivators↗
Use of hydrated reversed micelles of surfactant in organic solvent for stabilization of individual oligomeric forms of uridine phosphorylase from Escherichia coli K-12.
The catalytic activity of uridine phosphorylase from Escherichia coli K-12 entrapped in hydrated reversed micelles of aerosol OT (AOT) in octane has been studied as a function of the degree of hydration of the micelles. It was shown that the catalytic activity of uridine phosphorylase reached maximum values at [H2O/[AOT] ratios equal to 8.4, 12.9, 16.1 and 18.6. Based on the sedimentation data the conclusion has been made that the maxima of the catalytic activity correspond to the monomeric, dimeric, trimeric and tetrameric forms of the enzyme. The measurements of the rate of the enzymatic reaction catalyzed by uridine phosphorylase entrapped in hydrated reversed micelles at various concentrations of AOT indicate that the monomeric enzyme form, in contrast to the trimeric and tetrameric forms, exhibits the membranotropic properties.
Aerosols↗
Modification with tetranitromethane of an essential tyrosine residue in uridine phosphorylase from Escherichia coli.
Treatment with tetranitromethane (TNM) rapidly and irreversibly inactivates uridine phosphorylase (UPase) from E. coli under mildly alkaline conditions. Modification of one of the four tyrosine residues decreases enzyme activity to 10%, while modification of all tyrosines decreases it to 8%. The second-order rate constant for the inactivation is 1250 +/- 50 M-1 min-1 at pH 8.0. Phosphate (0.1 M) does not affect the inactivation rate, while 5 mM uridine, or uridine plus phosphate nearly completely protect the enzyme against inactivation. Free sulfhydryl groups of UPase are not oxidized by TNM. A single modified peptide was isolated from tryptic digest by reverse-phase HPLC. The mass to charge ratio and the sequence determined are consisted with modification of Tyr-169, which corresponds to tryptic peptide 169Tyr-Asp-Thr-Tyr-Ser-Gly-Arg175. Tyrosine nitration leads to a significant decrease in the pKa of the phenolic hydroxy group without significantly affecting enzyme structure. Comparison of the pH dependence of activity and inactivation by diethylpyrocarbonate for the native and modified UPase reveals interaction between the modified tyrosine residue and an essential histidine residue (Drabikowska, A.K. and Wozniak, G (1990) Biochem. J. 270, 319-323). It is suggested that Tyr-169 takes part in the stabilization of the imidazole ring of the essential histidine in UPase.
Amino Acid Sequence↗
Complete inactivation of Escherichia coli uridine phosphorylase by modification of Asp5 with Woodward's reagent K.
Woodward's reagent K (WRK) completely inactivated Escherichia coli uridine phosphorylase by reversible binding in the active site (Ki = 0.07 mM) with subsequent modification of a carboxyl (k2 = 1.2 min-1). Neither substrate alone protected uridine phosphorylase from inactivation. The presence of phosphate did not affect the Ki and k2 values. The addition of uracil or uridine led to a significant increase of both Ki (to 2.5 or 2.1 mM, respectively) and k2 (to 6.1 or 4.8 min-1, respectively) values. Thus, WRK could react in accordance with slow (high affinity) and fast (low affinity) mechanisms. Combined addition of phosphate and uracil completely protected uridine phosphorylase. Tryptic digestion yielded a single modified peptide (Ser4-Asp(WRK)-Val-Phe-His-Leu-Gly-Leu-Thr-Lys13). Treatment of the modified enzyme with hydroxylamine led to removal of the bulky WRK residue and replacement of the Asp5 carboxyl by a hydroxamic group. The enzyme thus obtained recovered about 10% of initial specific activity, whereas its substrate binding ability changed only moderately; the Km values for phosphate and uridine were changed from 5.1 and 0.19 mM (or 7.3 and 0.14 mM according to Leer et al. (Leer, J.C., Hammer-Jespersen, K., and M. Schwartz (1977) Eur. J. Biochem. 75, 217-224)) to 22.6 and 0.12 mM, respectively. The hydroxamic enzyme had higher thermostability than the native enzyme. The results obtained demonstrated the importance of the carboxyl at position 5. The loss of activity after selective group replacement is due to impaired stabilization of the transition state rather than to a decline in substrate affinity or change of the active site structure.
Amino Acid Sequence↗
[Chemical synthesis of a proteinase inhibitor (eglin C) gene without use of T4-DNA-ligase and its expression in E. coli].
A synthetic gene for a proteinase inhibitor (eglin C) that was obtained by direct amplification with oligonucleotides without using DNA ligase and polynucleotide kinase of T4 phage was cloned into expression vectors. A high yield of the polypeptide (110-130 mg/l) was attained in E. coli strains.
Amino Acid Sequence↗