Peptide synthesis in organic media with the use of subtilisin 72 immobilized on a poly(vinyl alcohol) cryogel

Subtilisin 72 serine protease (EC 3.4.21.14) immobilized on a poly(vinyl alcohol) cryogel was used as a catalyst in the syntheses of N-protected peptide p-nitroanilides of the general formulas Z(or Boc)-Xaa-Phe-pNA (Xaa = Leu or Ala), Z-Ala-Xaa-Yaa-pNA (Xaa = Leu or Ala; Yaa = Leu or Phe), and Z-Ala-Ala-Xaa-Yaa-pNA (Xaa = Leu, Arg, or Gly; Yaa = Phe, Leu, Gly, Asp, or Glu). The syntheses were carried out in DMF-acetonitrile mixtures. A number of protected di-, tri-, and tetrapeptides were prepared in yields up to 99%. The syntheses were found to retain stereoselectivity under the conditions studied. The activation of carboxyl group of the acylating component was shown to have a positive effect upon the coupling rate. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.     

Conformational Properties of the CalliFMRF-Amide Series Neuropeptides

Conformational properties of five neuropeptides belonging to the calliFMRF-amide series with the Xaa-Pro-Yaa-Gln-Asp-Phe-Met-Arg-Phe-NH₂ homologous sequences were studied by the method of theoretical conformational analysis. Three members of these group [(1) (Xaa = Thr, Yaa = Gln), (2) (Xaa = Thr, Yaa = Ser), and (3) (Xaa = Yaa = Ser)] can stimulate the saliva secretion from the separated salivary gland of the Calliphora vomitoria fly, whereas two other calliFMRF-amides [(4) (Xaa = Lys, Yaa = Asn) and (5) (Xaa = Ala, Yaa = Gly)] are inactive in this biological test. Low-energy spatial structures of the studied compounds were determined by a conformational analysis. A comparison of the stable structures of the biologically active and inactive neuropeptides revealed a similarity in their conformational properties and allowed determination of the role of separate residues in the peptide folding. The calculations demonstrated that the C-terminal hexapeptide fragment identical in all the five peptides tends to form -helical structure, whereas the variable N-terminal tripeptide regions of calliFMRF-amides (1)–(5) form more conformationally flexible structures.     

WNT10A variants in relation to nonsyndromic hypodontia in eastern Slovak population

Nonsyndromic hypodontia is a congenital absence of less than six permanent teeth, with a most common subtype maxillary lateral incisor agenesis (MLIA). Mutations in several genes have been described in severe tooth agenesis. The aim of this study was to search for the variants in wingless-type MMTV-integration site family member (WNT10A), paired box 9 (PAX9) and axis inhibitor 2 (AXIN2) genes, and investigate their potential role in the pathogenesis of non-syndromic hypodontia. Clinical examination and panoramic radiograph were performed in the cohort of 60 unrelated Slovak patients of Caucasian origin with nonsyndromic hypodontia including 37 MLIA cases and 48 healthy controls. Genomic DNA was isolated from buccal swabs and Sanger sequencing of WNT10A, PAX9 and AXIN2 was performed. Altogether, we identified 23 single-nucleotide variants, of which five were novel. We have found three rare nonsynonymous variants in WNT10A (p.Gly165Arg; p.Gly213Ser and p.Phe228Ile) in eight (13.33%) of 60 patients. Analysis showed potentially damaged WNT10A variant p.Phe228Ile predominantly occurred only in MLIA patients, and with a dominant form of tooth agenesis (odds ratio \(({\hbox {OR}}_{\mathrm{dom}}) = 9.841\); \(P=0.045\); 95% confidence interval (CI) 0.492–196.701; \({\hbox {OR}}_{\mathrm{rec}} = 0.773\); \(P =1.000\); 95% CI 0.015–39.877). In addition, the WNT10A variant p.Phe228Ile showed a trend associated with familial nonsyndromic hypodontia (\(P =0.024\); OR = 1.20; 95% CI 0.97–1.48). After Bonferroni correction, these effects remained with borderline tendencies. Using a 3D WNT10A protein model, we demonstrated that the variant Phe228Ile changes the protein secondary structure. In PAX9 and AXIN2, common variants were detected. Our findings suggest that the identified WNT10A variant p.Phe228Ile could represent risk for the inherited nonsyndromic hypodontia underlying MLIA. However, further study in different populations is required.     

Phenotypic variability and identification of novel <Emphasis Type="Italic">YARS2</Emphasis> mutations in YARS2 mitochondrial myopathy,lactic acidosis and sideroblastic anaemia

Background

Mutations in the mitochondrial tyrosyl-tRNA synthetase (YARS2) gene have previously been identified as a cause of the tissue specific mitochondrial respiratory chain (RC) disorder, Myopathy, Lactic Acidosis, Sideroblastic Anaemia (MLASA). In this study, a cohort of patients with a mitochondrial RC disorder for who anaemia was a feature, were screened for mutations in YARS2.

Methods

Twelve patients were screened for YARS2 mutations by Sanger sequencing. Clinical data were compared. Functional assays were performed to confirm the pathogenicity of the novel mutations and to investigate tissue specific effects.

Results

PathogenicYARS2 mutations were identified in three of twelve patients screened. Two patients were found to be homozygous for the previously reported p.Phe52Leu mutation, one severely and one mildly affected. These patients had different mtDNA haplogroups which may contribute to the observed phenotypic variability. A mildly affected patient was a compound heterozygote for two novel YARS2 mutations, p.Gly191Asp and p.Arg360X. The p.Gly191Asp mutation resulted in a 38-fold loss in YARS2 catalytic efficiency and the p.Arg360X mutation did not produce a stable protein. The p.Phe52Leu and p.Gly191Asp/p.Arg360X mutations resulted in more severe RC deficiency of complexes I, III and IV in muscle cells compared to fibroblasts, but had relatively normal YARS2 protein levels. The muscle-specific RC deficiency can be related to the increased requirement for RC complexes in muscle. There was also a failure of mtDNA proliferation upon myogenesis in patient cells which may compound the RC defect. Patient muscle had increased levels of PGC1-α and TFAM suggesting mitochondrial biogenesis was activated as a potential compensatory mechanism.

Conclusion

In this study we have identified novel YARS2 mutations and noted marked phenotypic variability among YARS2 MLASA patients, with phenotypes ranging from mild to lethal, and we suggest that the background mtDNA haplotype may be contributing to the phenotypic variability. These findings have implications for diagnosis and prognostication of the MLASA and related phenotypes.

     

The effects of mepiquat chloride on the lateral root initiation of cotton seedlings are associated with auxin and auxin-conjugate homeostasis

Background

Mepiquat chloride (MC) is a plant growth regulator widely used in cotton (Gossypium hirsutum L.) production to suppress excessive vegetative growth, increase root growth and avoid yield losses. To increase root growth, cotton seeds were treated with MC to increase the number of lateral root (LRs) and improve drought resistance. An increased indole-3-acetic acid (IAA) pool appeared to correlate with LR growth, and the principal source of IAA in germinating seeds is IAA conjugates. Here, the role of IAA homeostasis and signaling was investigated in cotton seedlings treated with MC.

Results

In the present research, MC significantly increased endogenous IAA levels in the roots, which promoted lateral root initiation (LRI) by upregulating GhARF7/19 and GhLBD18s and subsequently increasing LR quantity and elongation. The levels of IAA-amide conjugates significantly decreased in MC-treated seedlings compared with untreated control seedlings. Sixteen members of the cotton IAA amidohydrolase (IAH) gene family were identified, of which GhIAR3a, GhIAR3b, GhILR1, GhILL3 and GhILL6 were expressed during cotton seed germination. Compared with those in untreated control seedlings, the expression levels of GhIAR3a, GhIAR3b, GhILR1 and GhILL6 in the MC-treated seedlings were markedly elevated. The GhIAR3a/b and GhILR1 genes were cloned and expressed in Escherichia coli; these recombinant proteins exhibited hydrolytic activity that could cleave IAA-phenyalanine (Phe), IAA-methionine (Met), IAA-glycine (Gly) and IAA-leucine (Leu) in vitro, while only GhIAR3a hydrolyzed IAA-alanine (Ala) efficiently. The content of GhIAR3a, as detected via an established sandwich enzyme-linked immunosorbent assay (ELISA), increased in the MC-treated seedlings compared with the untreated control seedlings. In addition, the Arabidopsis iar3 mutant was less responsive to MC-induced LR growth than was wild type.

Conclusions

These findings suggested that MC application could mediate IAA homeostasis via increased IAA levels from IAA-amide conjugate hydrolysis by accelerating IAH gene expression, which might promote LRI and increase the LR quantity and elongation.

     

The spectrum of mutations in genes associated with resistance to rifampicin,isoniazid, and fluoroquinolones in the clinical strains of <Emphasis Type="Italic">M. tuberculosis</Emphasis> reflects the transmissibility of mutant clones

To study the transmissibility of drug resistant mutant clones, M. tuberculosis samples were isolated from the patients of the clinical department and the polyclinic of the Central TB Research Institute (n = 1455) for 2011–2014. A number of clones were phenotypically resistant to rifampicin (n = 829), isoniazid (n = 968), and fluoroquinolones (n = 220). We have detected 21 resistance-associated variants in eight codons of rpoB, six variants in three codons of katG, three variants in two positions of inhA, four variants in four positions of ahpC, and nine variants in five codons of gyrA, which were represented in the analyzed samples with varied frequencies. Most common mutations were rpoB 531 Ser→Leu (77.93%), katG 315 (Ser→Thr) (94.11%), and gyrA 94 (Asp→Gly) (45.45%). We found that the mutations at position 15 of inhA (C→T) (frequency of 25.72%) are commonly associated with katG 315 (Ser→Thr). This association of two DNA variants may arise due to the double selection by coexposure of M. tuberculosis to isoniazid and ethionamide. The high transmissibility of mutated strains was observed, which may be explained by the minimal influence of the resistance determinants on strain viability. The high transmissibility of resistant variants may also explain the large populational prevalence of drug-resistant TB strains.     

Drug resistance mutations and susceptibility phenotypes of <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> isolates in Russia

Steady growth in the degree of antimicrobial resistance in Neisseria gonorrhoeae calls for the control of the spreading of resistance mutations. Here we present the data describing drug resistance mutations, the results of antimicrobial susceptibility tests, and molecular genotypes of 128 recent N. gonorrhoeae isolates collected across 9 regions of the Russian Federation. The mutations in chromosome genes penA, ponA, rpsJ, gyrA, parC, which determine the susceptibility of N. gonorrhoeae to penicillins, tetracyclines, and fluoroquinolones were detected by multiplex amplification followed by hybridization on a hydrogel microarray. The most frequent mutation was an insertion of an aspartate at position 345 of penA gene (76.6%), whereas mutations Leu421Pro in ponA gene, Val57Met in rpsJ gene, Ser91Phe in gyrA gene, Asp95Gly in gyrA gene, and Ser87Arg in parC gene were detected in 32.8–36.7% of strains. One third of studied N. gonorrhoeae isolates harbored multiple drug resistance mutations in bacterial chromosome, resulting in the bimodal distribution of mutation profiles and related patterns of antimicrobial susceptibility. The spread of multiple resistance could be explained by the vertical transfer of the mutations resulting in the clonality of the N. gonorrhoeae population.     

Genetic Testing of Non-familial Deaf Patients for <Emphasis Type="Italic">CIB2</Emphasis> and <Emphasis Type="Italic">GJB2</Emphasis> Mutations: Phenotype and Genetic Counselling

CIB2 and GJB2 genes variants contribute significantly in familial cases of prelingual recessive hearing loss (HL). This study was aimed to determine the CIB2 and GJB2 variants and associated phenotype in 150 non-familial individuals with HL. After getting informed consent, 150 non-familial deaf patients were enrolled and blood samples were obtained for DNA extraction. Pure tone air conduction audiometry was performed. Coding exons of CIB2 and GJB2 genes were Sanger sequenced. A tetra primer ARMS assay was developed for recurrent CIB2 variant. Four bi-allelic GJB2 variants, c.71G>A p.(Trp24*), c.231G>A p.(Trp77*), c.235delC p.(Leu79Cysfs3*) and c.35delG p.(Gly11Leufs24*), were found in nine hearing impaired individuals. We also found four homozygotes and five carriers of c.380G>A p. (Arg127His) variant of controversial clinical significance. CIB2 sequencing revealed single recurrent variant c.272T>C p. (Phe91Ser) segregating with HL in ten individuals. Among our patients, c.71G>A (p.Trp24*) was the most common variant, accounted for 45% of GJB2 variants. Two known GJB2 variants, c.235delC p. (Leu79Cysfs3*) and c.310del14 p. (Lys105Argfs2*), are reported here for the first time in Pakistani population. Our data further support the benign nature of c.380G>A p. (Arg127His) variant. For CIB2, c.272T>C p. (Phe91Ser) is the second common cause of HL among our sporadic cases. Phenotypically, in our patients, individuals homozygous for GJB2 variants had profound HL, whereas CIB2 homozygotes had severe to profound prelingual HL. Our results suggest that GJB2 and CIB2 are common cause of HL in different Pakistani ethnicities.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

A Novel High-Molecular-Mass Bacteriocin Produced by <Emphasis Type="Italic">Enterococcus faecium</Emphasis>: Biochemical Features and Mode of Action

Discovery of a novel bacteriocin is always an event in sciences, since cultivation of most bacterial species is a general problem in microbiology. This statement is reflected by the fact that number of bacteriocins is smaller for tenfold comparing to known antimicrobial peptides. We cultivated Enterococcus faecium on simplified medium to reduce amount of purification steps. This approach allows to purify the novel heavy weight bacteriocin produced by E. faecium ICIS 7. The novelty of this bacteriocin, named enterocin-7, was confirmed by N-terminal sequencing and by comparing the structural-functional properties with available data. Purified enterocin-7 is characterized by a sequence of amino acid residues having no homology in UniProt/SwissProt/TrEMBL databases: NH2 - Asp - Ala - His - Leu - Ser - Glu - Val - Ala - Glu - Arg - Phe - Glu - Asp - Leu - Gly. Isolated thermostable protein has a molecular mass of 65 kDa, which allows it to be classified into class III in bacteriocin classification schemes. Enterocin-7 displayed a broad spectrum of activity against some Gram-positive and Gram-negative microorganisms. Fluorescent microscopy and spectroscopy showed the permeabilizing mechanism of the action of enterocin-7, which is realized within a few minutes.     

<Emphasis Type="Italic">CDKN2A</Emphasis> and <Emphasis Type="Italic">MC1R</Emphasis> variants found in Cypriot patients diagnosed with cutaneous melanoma

The prevalence of genetic variants associated to cutaneous melanoma (CM) has never been determined within Cypriot melanomas. This study evaluates the frequency of variants in cyclin-dependent kinase inhibitor 2A (CDKN2A) and melanocortin-1 receptor (MC1R) in 32 patients diagnosed with CM. Other characteristics and risk factors were also assessed. CDKN2A p.Ala148Thr was detected in three of 32 patients, while the control group revealed no variations within CDKN2A. MC1R screening in 32 patients revealed the following variations: p.Val60Leu in 11 patients, p.Arg142His in four patients, p.Thr314Thr in one patient, p.Arg160Trp in one patient, p.Val92Met/p.Thr314Thr in one patient and p.Val92Met/p.Arg142His/p.Thr314Thr in one patient. The control group revealed only p.Val60Leu (in 10 of 45 individuals), which is frequently found in general populations. Two unrelated patients carried CDKN2A p.Ala148Thr in combination with MC1R p.Arg142His, suggesting digenic inheritance that may provide evidence of different gene variants acting synergistically to contribute for CM development. This study confirms the presence of CDKN2A and MC1R variants among Cypriot melanomas and supports existing evidence of a role for these variants in susceptibility to melanoma.     

Kinetics and Mechanism of the Peptide Synthesis in Solution

The kinetics of the reaction of Boc-Xaa fluorophenyl esters (where Xaa = Ala, Val, Phe, Ser, Leu, Gly, Met, Pro, or Ile) with leucinamide was studied in order to measure changes in fluorescence emission at 375 nm of the fluorophenyl chromophore accompanying the reaction. It was found that the experimental kinetic data could not be described by a simple scheme of the second order reaction. Measurements of the kinetic parameters of the reaction at various initial concentrations of reagents indicated that the reaction rate can be expressed as: = kC _N ^a C _AE ^b , where k is the reaction rate constant, C _N is the concentration of leucinamide, and C _AE is the concentration of fluorophenyl ester. The a and b reaction orders were close to 1/2 and 3/2 for Xaa = Ala, Val, Phe, Ser, or Leu, 1/2 and 1 for Gly, Met, or Pro, and 1 and 2 for Ile. The experimental equations for the reaction rate can theoretically be derived from a single scheme of chain reactions with various deactivation ways for active intermediates.     

Evaluation of <Emphasis Type="Italic">GPx1</Emphasis> Pro198Leu polymorphism in idiopathic male infertility

Infertility is defined as failure to conceive a child after 1 year of unprotected regular sexual intercourse. Approximately half of all cases of infertility are caused by factors related to the male. In nearly 50% of infertile men it is not possible to determine the cause of infertility and this situation has been defined as unexplained or idiopathic. Oxidative stress plays an important role in the pathophysiology of male infertility. Oxidative stress results from an imbalance in free radicals and antioxidant defense mechanisms of the body. Genetic variations in the antioxidant gene coding for GPx enzyme may lead to decreased or impaired regulation of its enzymatic activity and alter reactive oxygen species (ROS) detoxification. We have investigated the possible association between polymorphism GPx1 Pro198Leu and idiopathic male infertility. One hundred patients with idiopathic male infertility and one hundred fifty healthy volunteers were enrolled. Genomic DNA was extracted from blood samples. Genotyping for the GPx1 Pro198Leu polymorphism was done by PCR–restriction fragment length polymorphism (RFLP) using ApaI. The genotype frequencies were 11% (Leu/Leu), 76% (Pro/Leu) and 13% (Pro/Pro) in the patient group and 8.7% (Leu/Leu), 67.3% (Pro/Leu) and 24% (Pro/Pro) in the control group. The genotype and allele frequencies of GPx1 Pro198Leu did not differ between the patient group and the control group (P = 0.09 and P = 0.1, respectively). In conclusion, there is no correlation between idiopathic male infertility and the GPx1 codon Pro198Leu polymorphism. Further studies are needed to investigate other genetic factors that influence the development of idiopathic male infertility.     

Conformational properties of the CalliFMRF-amide series neuropeptides

Conformational properties of five neuropeptides belonging to the calliFMRF-amide series with the Xaa-Pro-Yaa-Gln-Asp-Phe-Met-Arg-Phe-NH2 homologous sequences were studied by the method of theoretical conformational analysis. Three members of these group (1) (Xaa = Thr, Yaa = Gln), (2) (Xaa = Thr, Yaa = Ser), and (3) (Xaa = Yaa = Ser) can stimulate the saliva secretion from the separated salivary gland of the Calliphora vomitoria fly, whereas two other calliFMRF-amides (4) (Xaa = Lys, Yaa = Asn) and (5) (Xaa = Ala, Yaa = Gly) are inactive in this biological test. Low-energy spatial structures of the studied compounds were determined by a conformational analysis. A comparison of the stable structures of the biologically active and inactive neuropeptides revealed a similarity in their conformational properties and allowed determination of the role of separate residues in the peptide folding. The calculations demonstrated that the C-terminal hexapeptide fragment identical in all the five peptides tends to form alpha-helical structure, whereas the variable N-terminal tripeptide regions of CalliFMRF-amides (1)-(5) form more conformationally flexible structures.     

Batten disease: biochemical and molecular characterization revealing novel <Emphasis Type="Italic">PPT1</Emphasis> and <Emphasis Type="Italic">TPP1</Emphasis> gene mutations in Indian patients

Background

Neuronal ceroid lipofuscinoses type I and type II (NCL1 and NCL2) also known as Batten disease are the commonly observed neurodegenerative lysosomal storage disorder caused by mutations in the PPT1 and TPP1 genes respectively. Till date, nearly 76 mutations in PPT1 and approximately 140 mutations, including large deletion/duplications, in TPP1 genes have been reported in the literature. The present study includes 34 unrelated Indian patients (12 females and 22 males) having epilepsy, visual impairment, cerebral atrophy, and cerebellar atrophy.

Methods

The biochemical investigation involved measuring the palmitoyl protein thioesterase 1 and tripeptidy peptidase l enzyme activity from the leukocytes. Based on the biochemical analysis all patients were screened for variations in either PPT1 gene or TPP1 gene using bidirectional Sanger sequencing. In cases where Sanger sequencing results was uninformative Multiplex Ligation-dependent Probe Amplification technique was employed. The online tools performed the protein homology modeling and orthologous conservation of the novel variants.

Results

Out of 34 patients analyzed, the biochemical assay confirmed 12 patients with NCL1 and 22 patients with NCL2. Molecular analysis of PPT1 gene in NCL1 patients revealed three known mutations (p.Val181Met, p.Asn110Ser, and p.Trp186Ter) and four novel variants (p.Glu178Asnfs*13, p.Pro238Leu, p.Cys45Arg, and p.Val236Gly). In the case of NCL2 patients, the TPP1 gene analysis identified seven known mutations and eight novel variants. Overall these 15 variants comprised seven missense variants (p.Met345Leu, p.Arg339Trp, p.Arg339Gln, p.Arg206Cys, p.Asn286Ser, p.Arg152Ser, p.Tyr459Ser), four frameshift variants (p.Ser62Argfs*19, p.Ser153Profs*19, p.Phe230Serfs*28, p.Ile484Aspfs*7), three nonsense variants (p.Phe516*, p.Arg208*, p.Tyr157*) and one intronic variant (g.2023_2024insT). No large deletion/duplication was identified in three NCL1 patients where Sanger sequencing study was normal.

Conclusion

The given study reports 34 patients with Batten disease. In addition, the study contributes four novel variants to the spectrum of PPT1 gene mutations and eight novel variants to the TPP1 gene mutation data. The novel pathogenic variant p.Pro238Leu occurred most commonly in the NCL1 cohort while the occurrence of a known pathogenic mutation p.Arg206Cys dominated in the NCL2 cohort. This study provides an insight into the molecular pathology of NCL1 and NCL2 disease for Indian origin patients.

     

The <Emphasis Type="Italic">GPX1</Emphasis> Pro<Superscript>198</Superscript>Leu polymorphism in gastric cancer patients with and without <Emphasis Type="Italic">Helicobacter pylori</Emphasis> infection

Helicobacter pylori infection could induce oxidative stress. Oxidative stress is involved in the pathogenesis of gastric diseases. Glutathione peroxidase 1 (GPX1), is part of the enzymatic antioxidant defense, preventing oxidative damage. The aim of the present study was to assess the association of GPX1 Pro¹⁹⁸Leu genotypes with gastric cancer in patients with and without H. pylori infection in a population of Northern Iran. The present case-control study consisted of 50 patients with gastric cancer and 78 cancer-free subjects as controls. Extraction of DNA was performed on bioptic samples and the GPX1 genotypes were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The frequencies of GPX1 Pro/Pro, Pro/Leu and Leu/Leu genotypes in controls were 21.8, 71.8 and 6.4%, respectively. However, in gastric cancer patients, the frequencies of 34, 56 and 10% were observed for Pro/Pro, Pro/Leu and Leu/Leu genotypes, respectively (p?=?0.185). In 38 (76%) patients infected with H. pylori, the frequencies of Pro and Leu alleles were 94.7 and 3.3%, respectively. There was a higher frequency of combined genotype of Pro/Leu?+?Leu/Leu (94.7%) in H. pylori positive patients than that in patients without H. pylori infection (75%, p?=?0.047). The presence of this genotype tended to increase the risk of H. pylori related gastric cancer by 5.88–fold (p?=?0.069) in our population. Our findings indicated the absence of association between the GPX1 Pro¹⁹⁸Leu polymorphism and the risk of gastric cancer in an Iranian population. However, we detected an association between H. pylori related gastric cancer with GPX1 Pro/Leu?+?Leu/Leu genotype.     

Is there a relationship between <Emphasis Type="Italic">PPARD</Emphasis> T294C/<Emphasis Type="Italic">PPARGC1A</Emphasis> Gly482Ser variations and physical endurance performance in the Korean population?

The peroxisome proliferator-activated receptor δ (PPARD) and peroxisome proliferator-activated receptor γ coactivator 1α (PPARGC1A) genes recently have been suggested to have an association with athletic performance and physical endurance. These gene products are reported to be crucial components in training-induced muscle adaptation, since they are related with mRNA and/or protein activity in coordinated response to exercise. To assess the possible contribution of the PPARD T294C/PPARGC1A Gly482Ser polymorphism to differences in physical endurance, we performed a population-based study of 111 Korean athletes and 145 healthy controls based on their genotype distribution of the genes. The two loci were found to be not deviated from Hardy–Weinberg equilibrium. There were no differences in genotype distribution of PPARD T294C and PPARGC1A Gly482Ser between the athletic group and controls (p > 0.05). In contrast, we found a significant association between the PPARGC1A Gly482Ser polymorphism and the 20 m shuttle run activity (a measure of endurance performance) in the athletic group (p = 0.003). The result showed a remarkable increase in the numbers of shuttle run ratio from subjects with the PPARGC1A Gly/Gly genotype (85.29 ± 28.80) than those with the Gly/Ser (58.05 ± 32.76) and Ser/Ser (68.38 ± 30.47) genotypes. Thus, our data imply that the PPARGC1A Gly/Gly genotype may provide a beneficial effect on elite-level endurance status, although functional studies with larger sample sizes are necessary to elucidate these findings.     

Effect of Active Site Pocket Structure Modification of <Emphasis Type="SmallCaps">d</Emphasis>-Stereospecific Amidohydrolase on the Recognition of Stereospecific and Hydrophobic Substrates

d-Stereospecific amidohydrolase (DAH) from Streptomyces sp. 82F2 has potential utility for the synthesis of d/l configuration dipeptides by an aminolysis reaction. Structural comparison of DAH with substrate-bound d-amino acid amidase revealed that three residues located in the active site pocket of DAH (Thr145, Ala267, and Gly271) might be involved in interactions with d-phenylalanine substrate. We substituted Ala267 and Gly271, which are located at the bottom of the hydrophobic pocket of DAH, with Phe and observed changes in the stereoselectivity and specific activity toward the free and acetylated forms of d/l-Phe-methyl esters. In contrast, the mutation of Thr145, which likely supplies negative charge for recognition of the amino group of the substrate, hardly affected the stereoselectivity of the enzyme. A similar effect was observed in an investigation of hydrolysis and aminolysis reactions using the acetylated forms of d/l-Phe-methyl esters and 1,8-diaminooctane as an acyl-donor and acyl-acceptor, respectively. Substrate binding by DAH was disrupted by the mutation of Ala267 to Val or Trp and kinetic analysis showed that the hydrophobicity of the bottom of the active site pocket (Ala267 and Gly271) is important for both stereoselectivity and recognition of hydrophobic substrates.     

Analysis of <Emphasis Type="Italic">GJB6</Emphasis> (Сx30) and <Emphasis Type="Italic">GJB3</Emphasis> (Сx31) genes in deaf patients with monoallelic mutations in <Emphasis Type="Italic">GJB2</Emphasis> (Сx26) gene in the Sakha Republic (Yakutia)

Тhe DNA testing of autosomal recessive deafness type 1A (DFNB1A, MIM 220290) is complicated when deaf patients have only monoallelic (heterozygous) recessive mutations in the GJB2 (Сх26) gene that is uninformative for establishment of diagnosis. Such patients may be “random” heterozygous carriers of GJB2 mutations as well as have the mutant allele in a cis-regulatory region of GJB2 gene, in element genes encoding other connexins: GJB6 (Сх30) or GJB3 (Сх31). Previous studies of genetic causes of hearing loss in patients from Yakutia were directed to search for only mutations in the GJB2 gene, and the DNA diagnostics was uninformative for 9.7% (38/393) of the patients with monoallelic GJB2 mutations. In this work the search for mutations in genes GJB3 and GJB6 and two deletions с.del(GJB6-D13S1830) and с.del(GJB6-D13S1854) to the cis-regulatory region of GJB2 gene was conducted in 35 patients with GJB2 monoallelic mutations and in 104 normal hearing individuals. The genes studied are two synonymous substitution c.489G>A (р.Leu163Leu) (GJB6) and c.357C>T (р.Asn119Asn) (GJB3) have been found, probably do not have clinical significance, and two nonsynonymous substitution c.301G>A (p.Glu101Lys) (GJB6) and с.580G>A (p.Ala194Thr) (GJB3). Additional experimental evidences are needed for confirmation of pathogenic significance of detected nonsynonymous substitutions in development of hearing loss in studied patients. Diagnosis of the DFNB1A was confirmed in only one patient, who was discovered by the deletion с.del(GJB6-D13S1830) (GJB2) in combination with a recessive mutation с.35delG (GJB2). In general, our results indicate low contribution of mutations in genes GJB6 and GJB3 in hearing loss etiology in Yakutia.     

Kinetics and mechanism of peptide synthesis in solution

The kinetics of the reaction of Boc-Xaa fluorophenyl esters (where Xaa = Ala, Val, Phe, Ser, Leu, Gly, Met, Pro, or Ile) with leucinamide was studied measuring changes in the fluorescence emission at 375 nm of the fluorophenyl chromophore accompanying the reaction. It was found that the experimental kinetic data couldn't be described by a simple scheme of the second order reaction. The measurements of the kinetic parameters of the reaction at various initial concentrations of reagents indicated that the reaction rate can be expressed as: v = kCNaCAEb, where k is the reaction rate constant, CN is the concentration of leucinamide, and LeuNH2, CAE is the concentration of fluorophenyl ester. The a and b reaction orders were close to 1/2 and 3/2 for Xaa = Ala, Val, Phe, Ser, or Leu, 1/2 and 1 for Gly, Met, or Pro, and 1 and 2 for Ile. The experimental equations for the reaction rate can theoretically be derived from a single scheme of chain reactions with various deactivation ways for active intermediates. The English version of the paper.