[Detection of nonsense and frame shift mutations in human BRCA1 gene using new plasmid vector pPhoA-frame].

The technique for the detection of frame shift and nonsense mutations in BRCA1 gene was suggested. The technique presumes the construction of recombinant plasmids where the tested DNA fragment placed in-frame with alkaline phosphatase gene of Escherichia coli (phoA). A plasmid pPhoA-frame was constructed for such analysis, the plasmid contains DNA fragment coding for alkaline phosphatase of E. coli. Synthetic DNA fragment with BglII, StuI, ApaI and SacII sites was inserted into the DNA fragment coding for alkaline phosphatase of E. coli between Ala218 and Gly219 codons to facilitate the cloning of BRCA1 gene fragments. Occurrence of the frame shift or nonsense mutation in the tested DNA fragment can be detected after transformation of E. coli by the recombinant plasmid containing the tested fragment. E. coli colonies with the newly constructed recombinant plasmids are plated out on the indicator agar. In the case of frame shift or nonsense mutation the colonies are not colored, DNA fragments without such mutations result in the formation of the blue colonies.

[Guest peptide insertions into the surface loops of alkaline phosphatase].

The paper presents the description of the experiments in line with the rational concept for the "safe" insertion of guest polypeptides into the alkaline phosphatase with the minimal influence of the inserts on the enzymatic activity of the protein. Several approaches are described in the paper for the surface loop length estimation and two loops were used as the sites for guest peptides introduction by gene engineering technique. The experiments clearly demonstrate that insertions of several peptides after Ala218 of alkaline phosphatase (the site was selected by loop length analysis) do not block the activity of the enzyme. According the experimental data, the selection of the loops for the guest peptides insertion can be defined by the mobility of backbone dihedral angles during molecular dynamics simulation of alkaline phosphatase. The paper demonstrates the possibility to use in practice the estimation of loop length based on the mobility of backbone dihedral angles during molecular dynamics simulation. Indeed, it looks that the proteins with new features can be constructed by the introduction of new polypeptides into the enzymatically active proteins.

[Possibility of using fusion proteins for detection of nonsense mutations and frame-shift mutations in BRCA1 gene]. / Izuchenie vozmozhnosti ispol'zovaniia slitnykh belkov dlia detektsii nonsens-mutatsii i mutatsii sdviga ramki v gene BRCA1.

The aim of this study was to evaluate the possibility of detecting nonsense and frame-shift mutations in exon 11 of brca1 gene by constructing fusion open reading frame (ORF) "exon 11 ORF-alpha-peptide of beta-galactosidase". The ability/inability of this newly constructed ORF to cause alpha-complementation in E. coli delta M15gal cells transformed by the plasmid with the ORF may reflect the absence/presence of nonsense and frame-shift mutations in the studied fragment. A single ORF fragment of exon 11 of brca1 gene--LacZ' gene was designed in pGEN7Zf plasmid, the plasmid was shown to cause Lac+ phenotype in E. coli delta M15gal. Four frame-shift deletion mutations were introduced into exon 11 sequence in the plasmid. Surprisingly, the frame-shift deletion mutations did not influence the ability of plasmids to induce Lac+ phenotype in E. coli delta M15gal in 3 cases and only one deletion mutation resulted in inability of the plasmid to form Lac+ phenotype in E. coli delta M15gal. We suppose that the phenomenon can be explained by the alpha-peptide translation reinitiation from inframe ATG codons situated within the exon 11 sequence. Seven inframe ATG sequences were found in exon 11, at least two in-frame ATG-containing fragments were demonstrated to cause reinitiation. On the other hand, the only deletion mutation resulted in inability of the plasmid to form Lac+ phenotype in E. coli delta M15gal did not leave LacZ' in-frame ATG in econ 11 sequence. We conclude that it is possible to detect frame-shift mutations by in-frame cloning with the LacZ' reporter gene, but this possibility is strongly impeded by the reinitiation of alpha-peptide translation from the in-frame ATG codons within the studied sequence.

[Role of xenobiotic biotransformation enzymes in susceptibility to bronchial asthma and in formation of its clinical phenotypic features]. / Rol' fermentov biotransfomatsii ksenobiotikov v predraspolozhennosti k bronkhial'noi astme i formirovanii osobennostei ee klinicheskogo fenotipa.

The paper shows the impact of some mutations of CYP1A, GSTM1, GSTT1, and NAT2 genes and different combinations of alleles of these genes on susceptibility to bronchial asthma and its clinical features. Passive smoking modulates these relationships.

[Polymorphism of CYP1A1 and CYP2D6 genes in tundra nentses and European populations of western Siberia]. / Izuchenie polimorfizma genov CYP1A1 i CYP2D6 v populiatsiiakh tundrovykh nentsev i evropeoidov zapadnoi Sibiri.

Data on the first examination of the CYP1A1 and CYP2D6 genes' polymorphism in the populations of Tundra Nentsis (Yamalo-Nenetskii Autonomous District) and migrant population of Western Siberia (Novosibirsk oblast and Altaiskii krai) are presented. The frequency of the 2D6*4 mutant allele in Tundra Nentsis, characterized by a two-component Caucasoid and Mongoloid origin, was shown to be intermediate in Caucasoid and Mongoloid populations. The frequencies of the 2D6*4 and 1A1Val* mutant alleles across migrant inhabitants of Western Siberia (Caucasoid populations) were similar to that reported for the Caucasoid populations overall. Distribution of the CYP1A1 genotypes (Ile/Ile, Ile/Val*, and Val*/Val*) in Tundra Nentsis was similar to that found in Mongoloid groups. However, the frequency of the 1A1Val* allele in Tundra Nentsis was 1.5 times higher than that in the Southern Mongoloid populations (Chinese, Koreans, and Japanese).

Genetic polymorphism of CYP1A1 and CYP2D6 in the Tundra Nentsi population of Siberia.

The purpose of this study was to establish the frequencies of CYP1A1 and CYP2D6 polymorphic genotypes in the Tundra Nentsi population, which is a small indigenous northern people living in Siberia and belonging to the Northern Mongoloid race. The frequencies of Ile/Ile, Ile/Val, and Val/Val genotypes in the Tundra Nentsi population, as determined by means of the allele-specific PCR, were 50.8%, 39.2%, and 10%, respectively. Thus, the Val allele frequency in Tundra Nentsi appeared to be as high (29.5%) as in the Japanese population (25%) reported elsewhere. Those frequencies in the reference group of Siberian Caucasians were in good agreement with the data reported elsewhere for other Caucasians, although the Val allele frequency observed in Siberia inhabitants (5.7%) was somewhat higher than those frequencies obtained for other Caucasian populations. By means of PCR followed by specific-site digestion with MvaI endonuclease, we analysed the frequencies of CYP2D6B allele in the Tundra Nentsi population. The frequencies of 2D6wt/2D6wt and 2D6wt/B in the group of 120 Nentsi were 84.2% and 15.8%, respectively, with no subject possessing the 2D6B/2D6B genotype. The group of Siberian Caucasians represented those frequencies as 67.7%, 27.1%, and 5.2%, respectively. In total, the frequency of CYP2D6B allele in the Tundra Nentsi population was half that in Caucasians (8.3% vs. 19%). Taken together, our data indicate that the frequencies of CYP2D6B and Val allele of CYP1A1 in Tundra Nentsi population are different from those obtained for Caucasians. We also found similarities in the CYP1A1 mutation frequencies in the Tundra Nentsi and Japanese populations.

[Genes and enzymes of the xenobiotic-metabolizing system in cancer pathology]. / Geny i fermenty sistemy metabolizma ksenobiotikov v onkopatologii.

The paper presents the results of study on polymorfisms of xenobiotic biotransformation enzymes (CYP1A1, glutathione S-transferase MI and N-acetyltransferase 2) and p53 tumor suppressor protein in patients with lung, stomach and intestine cancer. The frequency of CYP1A1-Val allele in all studied cancer groups was 3 to 5 times higher than in healthy control group. The carriers of homozygous glutathione S-transferase M1 gene deletion and slow acetylator phenotype were also of higher lung cancer risk. The substantial increase in slow acetylator phenotype frequency was shown also in the group of intestine cancer patients. The p53 Arg/Pro polymorphism study revealed the elevated frequency of Arg allele in lung and stomach cancer groups. The risk of lung cancer for the carriers of susceptible alleles depended on the age and smoking status of the patients. The results testify to a high possibility of studied polymorphic genes to be the markers of susceptibility to oncopathologies.

[Benzonal--a phenobarbital-type of inducer of the mono-oxygenase system]. / Benzonal--induktor monooksigenaznoi sistemy fenobarbitalovogo tipa.

It has been found that the Soviet anticonvulsant drug Benzonal is an inducer of the liver cytochrome P-450 of the phenobarbital type. The drug causes formation of the cytochrome P-450 form which is immunologically identical to the phenobarbital inducible form of the hemoprotein with identical molecular mass determined with the SDS-gel electrophoresis method in PAAG. The microsomes, obtained from the rats treated with Benzonal display increased rates of metabolism of 7-pentoxiresorufin and 16 beta-hydroxylation of androstenedione which are specific substrates for the cytochrome P-450b.

[Comparison of the inducing effect of triphenyldioxan, bis-(dichloropyridyloxy)benzene and phenobarbital on the liver monooxygenase]. / Sravnenie indutsiruiushchego deistviia trifenildioksana, bis-(dikhlorpiridiloksi)benzola i fenobarbitala na monooksigenazu pecheni.

The induction by triphenyldioxane (TPD) of cytochrome P-450 in rat liver microsomes was studied. It was demonstrated that TPD injection in a single dose (10 mg/kg of body mass) is associated with a marked induction of cytochromes P-450 b/e (cytochrome PB-forms) in rat liver microsomes and a significant increase in the benzphetamine-N-demethylase activity typical of cytochrome P-450b. In other words, TPD is a potent inducer of PB-type, the inducing effect being attained by an injection of a single dose of TPD which is by one order of magnitude less than that of phenobarbital. It can be assumed that this compound shows a high affinity for the hypothetical receptor responsible for cytochrome P-450b synthesis. It was shown also that TPD does not induce the monooxygenase system of mouse liver, whereas 1,4-bis[2-(dichloropyridyloxy)]benzene (DPB) is a potent inducer of PB-type in mice, being fairly ineffective in rats. Hence, the species-specific effect of TPD and DPB appears to be opposite.

The phenobarbital-type induction of rat liver microsomal monooxygenases by perfluorodecalin.

Induction of perfluorodecalin (PFD) of the liver microsomal system of metabolism of xenobiotics has been studied and compared with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that PFD increases the content of cytochrome P-450, NADPH-cytochrome c reductase activity. Like PB, PFD induces the activities of benzphetamine-N-demethylase, aldrine-epoxidase, 16 beta-androstendion-hydroxylase. Using specific antibodies against cytochromes P-450b and P-450c (which are the main isoenzymes of cytochrome P-450 in the PB- and MC-microsomes respectively), an immunological identity of the cytochrome P-450 isoforms during PFD and PB induction has been found. According to the rocket immunoelectrophoresis the content of cytochrome P-450 in PFD-microsomes, which is immunologically indistinguishable from P-450b, was approximately 70% of the total cytochrome P-450. Two forms of cytochrome P-450 were isolated from the liver microsomes of PFD-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from the PB-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromotographic behavior on DEAE-Sephacel column, molecular weight determined by sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gel, immunoreactivity, peptide mapping, catalytic activity. The data presented demonstrate that PFD induced in rat liver microsomes the cytochrome P-450 forms whose immunological properties and substrate specificity correspond to those of the PB-type cytochrome P-450. These findings suggest that PFD and PB, which differ in their chemical structure, induce in the rat liver microsomes identical forms of cytochrome P-450.

[Comparative characteristics of isolated forms of cytochrome P-450 induced by phenobarbital and perfluorodecalin]. / Sravnitel'naia kharakteristika izolirovannykh form tsitokhroma P-450, indutsiruemykh fenobarbitalom i perftordekalinom.

Two forms of cytochrome P-450 were isolated from liver microsomes of perfluorodecalin-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from phenobarbital-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromatographic behaviour on 1.8-diaminooctyl-Sepharose 4B and DEAE-Sephacel columns, molecular mass determined by SDS polyacrylamide gel electrophoresis, spectral properties, immunoreactivity, peptide mapping, catalytic activity. These findings suggest that in rat liver microsomes perfluorodecalin and phenobarbital which differ in their chemical structure induce identical forms of cytochrome P-450.

[Comparison of cytochrome P-450 forms induced by phenobarbital and 1,4-bis[3,5-dichloropyridyloxy)]benzene in the mouse and rat liver]. / Sravnenie form tsitokhroma P-450, indutsiruemykh fenobarbitalom i 1,4-bis[2-(3,5-dikhloropiridiloksi)]benzolom v pecheni myshei i krys.

A form of cytochrome P-450 (P-450PB) with a molecular weight of 53.5-54.0 kD possessing a high benzphetamine-N-demethylase activity (100-120 nmol formaldehyde/min/nmol cytochrome) was isolated from liver microsomes of phenobarbital-induced C57Bl/6 mice. This cytochrome P-450 form is immunologically identical to its rat liver counterpart-P-450b (Mr = 52 kD) which is also characterized by a high rate of benzphetamine-N-demethylation. It was shown that 1.4-bis[2-(3.5-dichloropyridyloxy])benzene (TCPOBOP) induces in mouse liver the synthesis of the monoxygenase form whose substrate specificity and immunologic properties are identical to those of cytochromes P-450PB and P-450b. The immunochemically quantitated content of this form makes up to 20% of the total P-450 pool in liver microsomes of phenobarbital- or TCPOBOP-induced mice. Immunochemical analysis of microsomes with the use of antibodies to cytochromes P-450PB and P-450b revealed the presence on the electrophoregrams of phenobarbital-induced rat liver microsomes of two immunologically identical forms of cytochrome P-450, i.e., P-450b and P-450e (the latter had a low ability to benzphetamine N-demethylation). Liver microsomes of phenobarbital- or TCPOBP-induced mice gave only one precipitation band corresponding to cytochrome P-450PB.

[Directed modification of the structure of cytochrome P-450 substrates as a method of generating new inducers of the microsomal monooxygenase system]. / Napravlennaia modifikatsiia struktury substratov tsitokhroma P-450 kak sposob polucheniia novykh induktorov mikrosomnoi monooksigenaznoi sistemy.

Using the previously obtained data on the substrate-type induction of monooxygenase by xenobiotics of phenobarbital type, the method of conversion of typical substrates for cytochrome P-450 into inducers of biosynthesis of this enzymatic system by blocking in the substrate molecule of the position subjected to oxidative conversion in the enzyme active center was tested. The introduction of the methyl group in the omega-1 position of amobarbital, of Cl- into positions 2 and 4 of biphenyl and the substitution of methyl groups for the isopropyl groups in the 4-N(CH3)2 position of aminopyrine provides for marked induction of these derivatives of cytochrome P-450 and some monooxygenase activities.

[Effect of acrylates on the level and isozyme composition of cytochrome P-450 in rat liver microsomes]. / Vliianie akrilatov na soderzhanie i izoformnyi sostav tsitokhroma P-450 mikrosom pecheni krys.

Content of cytochrome P-450 was decreased in rat liver microsomes, pretreated with phenobarbital, after incubation with acrylamide, methyl methacrylate and butyl methacrylate in presence of NADPH in vitro. The phenomenon was not observed in the analogous experiments but carried out without NADPH, thus suggesting the microsomal metabolism of the acrylates. At the same time, after intraperitoneal administration of acrylamide 0.4 mmole/kg, methyl methacrylate 6.0 mmole/kg and butyl methacrylate 9.0 mmole/kg within 3 days into intact and protected with phenobarbital rats alterations in total content of cytochrome P-450 were not observed. All the acrylates studied exhibited the slight inducing effect on cytochrome P-450b but did not affect the cytochrome P-450c. Methyl- and butyl methacrylates decreased distinctly the content of cytochrome P-450 isoenzyme with molecular mass of 48,000 daltons.

[Determination of the monospecificity of antibodies to different forms of cytochrome p-450 using rocket immunoelectrophoresis]. / Vyiavlenie monospetsifichnosti antitel protiv razlichnykh form tsitokhroma p-450 metodom raketnogo immunoélektroforeza.

Antibodies against different forms of cytochrome P-450 were analyzed, using rocket immunoelectrophoresis technique. The technique was found capable of revealing antibody monospecificity. The antibodies against a certain form of cytochrome P-450 may be regarded as monospecific when they form a single precipitation line during the analysis of microsomal preparations which contain a mixture of different forms of cytochrome P-450.

[Induction of the phenobarbital form of cytochrome P-450 by chemically unrelated compounds]. / Induktsiia fenobarbitalovoi formy tsitokhroma P-450 khimicheski nerodstvennymi soedineniiami.

The induction of the phenobarbital form of cytochrome P-450 by xenobiotics (phenobarbital, PB, hexachlorobenzene, HCB; hexachlorocyclohexane. HCCH, and aroclor 1016, Ar) was studied. It was demonstrated that administration of these compounds to animals is accompanied by an increase in the total cytochrome P-450, NADPH-cytochrome P-450 reductase, benzphetamine-N-demethylase and aldrin-epoxidase activities. Using monospecific antibodies against the cytochrome P-450 form isolated from PB-induced microsomes (PB-cytochrome P-450), a double immunodiffusion test revealed immunological identity of cytochrome P-450 forms induced by phenobarbital and other xenobiotics. The content of this form determined by rocket immunoelectrophoresis increased markedly and made up to 20-40% of the total cytochrome P-450 content. Antibodies against PB-cytochrome P-450 inhibited by 50-70% the benzphetamine-N-demethylase and aldrin-epoxidase activities, whereas the antibodies to methylcholanthrene-induced cytochrome P-450 were fairly ineffective. It was concluded that the chemically unrelated compounds induce in liver microsomes a cytochrome P-450 form, whose immunological properties and substrate specificity are close to the PB-form of cytochrome P-450.

Immunochemical evidences that hexachlorobenzene induces two forms of cytochrome P-450 in the rat liver microsomes.

Induction by hexachlorobenzene (HCB) of the liver microsomal system of metabolism of xenobiotics has been studied in comparison with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that HCB increases the content of cytochrome P-450 in the microsomes. Like PB, HCB induces the activities of aminopyrine- and benzphetamine-N-demethylases. At the same time HCB increases also the activities of benzpyrenehydroxylase and 7-ethoxyresorufin-O-deethylase, which are characteristic of the MC-induction. However, sodium dodecyl sulphate (SDS)-electrophoresis on polyacrylamide gel has revealed that HCB, similar to PB, induces protein with Mr = 52 000 (cytochrome P-450), but not the protein with Mr = 56 000, which is the main isoenzyme of cytochrome P-450 in MC-microsomes (P-448). Using specific antibodies to isolated cytochromes P-450 and P-448 (anti-P-450 and anti-P-448) it has been found by rocket immunoelectrophoresis that in HCB-treated microsomes 20% of the total cytochrome P-450 consist of PB-form and about 10% comprise cytochrome P-488. It has also been found that anti-P-448 totally inhibit 7-ethoxyresorufin-O-deethylase activity of HCB-microsomes while anti-P-450 was inactive. The data presented give direct proof that HCB exemplifies an individual chemical compound which is able to initiate the synthesis of both PB-form and MC-form of the cytochrome P-450.

[Isolation and characterization of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats]. / Poluchenie i kharaktteristika mnozhestvennykh form tsitokhroma P-450 iz mikrosom pecheni krys linii wistar, indutsirovannykh 3-metilkholantrenom.

Chromatography on 1.8-diaminooctyl-Sepharose and DEAE-Sephacel resulted in 4 fractions of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats. All the four fractions differed in terms of their absorption maxima in the CO-reduced state, Mr and catalytic activity. Only one cytochrome fraction (cytochrome P-450 C) possessed a high activity upon benz(a)pyrene hydroxylation. All cytochrome P-450 forms were characterized by a low rate of aminopyrine N-demethylation. Antibodies against cytochrome P-450 C (P-448) (anti-P-448) were raised. Cytochromes of fractions A, B1 and B2 in the Ouchterlony reaction of double immunodiffusion did not give precipitation bands with anti-P-448. Neither of the four cytochrome P-450 forms interacted with the antibodies raised against cytochrome P-450 isolated from liver microsomes of rats induced with phenobarbital. The procedure developed is applicable to the isolation of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced rats. Using rocket immunoelectrophoresis, cytochrome P-450 C possessing a high (as compared to benz(a)pyrene metabolism) activity (18 nmol/min/nmol cytochrome) and a high (60-70%) content in 3-methylcholanthrene-induced rat liver microsomes was shown to give a relatively high yield.

[Hexachlorobenzene induction of 2 forms of cytochrome P-450 in the rat liver microsome]. / Induktsiia geksakhlorbenzolom dvukh form tsitokhrom P-450 v mikrosomakh pecheni krys.

Induction of the monooxygenase system of xenobiotic metabolism by means of hexachlorobenzene (HCB) was studied in rat liver microsomes as compared with induction using phenobarbital (PB) and 3-methylcholanthrene (3-MC). HCB was shown to increase the content of cytochrome P-450 as well as aminopyrine- and benzphetamine-N-demethylase activities in liver microsomes. At the same time, HCB increased the benzpyrene hydroxylase activity as it was the case in induction with 3-MC. Electrophoresis in SDS-polyacrylamide gel showed that HCB, as phenobarbital does, induced appearance of the protein with a molecular mass 52,000 (cytochrome P-450) but not of the protein of molecular mass 56,000, which is the main isozyme of cytochrome P-450 in 3-MC microsomes (P-448). Except of cytochrome P-450, cytochrome, immunologically similar to cytochrome-P-448 from 3-MC microsomes, constituted 10% of the total content of cytochrome in HCB microsomes as shown by rocket immunoelectrophoresis using monospecific antibodies towards individual cytochromes P-448 and P-450 (anti-P-448 and anti-P-450). Anti-P-450 and especially anti-P-448 inhibited benzpyrene hydroxylase in HCB microsomes. HCB appears to be an inductor of a "mixed" type.

[Immunological study of the identity of cytochrome P-448 in the mouse and rat liver after induction with 3-methylcholanthrene]. / Immunokhimicheskoe issledovanie identichnosti tsitokhromov P-448 pecheni myshei i krys pri induktsii 3-metilkholantrenom.

As shown by means of Ouchterlony double immunodiffusion technique after induction with 3-methyl cholanthrene the antibodies towards cytochrome P-448 from rat liver tissue developed a clear-cut precipitation line with homologous antigen and interacted also with cytochrome P-448 from mice liver tissue although the precipitation was less distinct. Antibodies towards mice liver cytochrome P-448 reacted also with the rat liver cytochrome P-448 but less distinctly as compared with the mice cytochrome. At the same time, these antibodies inhibited similarly the benz(a)-pyrene-hydroxylase reaction, catalyzed by both mice and rat cytochromes P-448. Partial immunological identity appears to occur in the cytochromes studied. Role of total antigenic determinants in enzymatic reactions catalyzed by cytochrome P-448 is discussed.