A single Gly114Arg mutation stabilizes the hexameric subunit assembly and changes the substrate specificity of halo-archaeal nucleoside diphosphate kinase

Nucleoside diphosphate kinase from extremely halophilic archaeon (HsNDK) requires above 2M NaCl concentration for in vitro refolding. Here an attempt was made to isolate mutations that allow HsNDK to refold in low salt media. Such a screening resulted in isolation of an HsNDK mutant, Gly114Arg, which efficiently refolded in the presence of 1M NaCl. This mutant, unlike the wild type enzyme, was expressed in Escherichia coli as an active form. The residue 114 is in close proximity to Glu155 of the neighboring subunit in the three dimensional hexameric structure of the HsNDK. It is thus possible that the attractive electrostatic interactions occur between Arg114 and Glu155 in the mutant HsNDK, stabilizing the hexameric subunit assembly.     

Directed evolution of Bacillus licheniformis lipase for improvement of thermostability

Lipases catalyze the hydrolysis of carboxylic acid esters and owing to their vast substrate specificity, they have many industrial applications. Due to the demand of thermostable lipases in industrial applications, we have enhanced the thermostability of lipase from Bacillus licheniformis RSP-09. The thermostable mutant lipases of Bacillus licheniformis RSP-09 were isolated following two rounds of directed evolution using error-prone PCR. The best mutant lipases obtained after first and second round of error-prone PCR were purified and characterized. The mutant lipases showed increased thermostability and retained catalytic function. The best mutant lipase (eP-231-51) showed 13.5-fold increase in percentage thermal stability (% remaining activity after incubation of purified enzyme at 60 °C for 1 h) than wild-type lipase. Also, this mutant lipase (ep-231-51) showed 30% improved catalytic efficiency compared with the wild-type which is due to significant decrease in K_m and marginal increase in k_cat. In addition, the thermostable mutant lipases have shown resistance to hydrophobic organic solvents. The role of mutations in the best mutant lipases of second round i.e. eP-231-51 (Asp72Gly, Asp61Gly, Tyr129His, and Thr101Pro) and eP-231-137 (Leu49Arg, Thr101Pro, Asp72Gly), that led to thermostability have been postulated after the comparison of molecular models of wild-type and mutated enzymes.     

Influence of DNA-repair gene variants on the micronucleus frequency in thyroid cancer patients

The role of different DNA-repair genes (OGG1, XRCC1, XRCC2 and XRCC3) on both the spontaneous and the induced frequency of micronuclei (MN) has been studied in the lymphocytes of a group of 114 patients with differentiated thyroid cancer (DTC). Induction of MN was achieved by treatment of the lymphocytes with 0.5 Gy of gamma-radiation.The selected genes are involved in base-excision repair (BER) (OGG1, Ser326Cys; XRCC1, Arg280His and Arg399Gln), and in homologous recombination repair (HRR) (XRCC2, Arg188His and XRCC3, IVS5-14G). Genotyping was carried out by use of the iPLEX (Sequenom) technique.Results indicate that only the OGG1-Ser326Cys polymorphism was able to modulate the MN frequency. This effect was only observed in the spontaneous MN frequency (P = 0.016), but not in the MN frequency induced after irradiation. In addition, a strong correlation was observed between spontaneous and induced MN frequency, which would suggest an underlying genetic background.     

Thr-114 is an important functional residue of fibroblast growth factor 10 identified by structure-based mutational analysis

Fibroblast growth factor 10 (FGF10) plays important roles in vertebrate limb development, lung branching morphogenesis, and epidermis regeneration. The receptor (FGFR2b) binding specificity is an essential element in regulating the diverse functions of FGF10. Analyzing the FGF10:FGFR2b complex we found that Thr-114 in β4 of FGF10 could form specific interactions with D3 of FGFR2b. To investigate the role of Thr-114 played on functions of FGF10, two mutants of FGF10 were constructed, named TA (Thr-114 → Ala) and TR (Thr-114 → Arg), respectively. The biological activity assays showed that the receptor-binding affinity, the stimulating growth effect on rat tracheal epithelium (RTE) cells, and the inducing ability in receptor phosphorylation of both mutants were decreased, which were consistent with the interaction analysis of the TA:FGFR2b and TR:FGFR2b complexes. These results suggested that Thr-114 is a crucial functional residue for FGF10, and mutating Thr-114 to Ala or Arg would lead to great decrease in receptor-binding affinity and biological activity of FGF10.     

Haplotype analysis of p53 polymorphisms: Arg72Pro,Ins16bp and G13964C in Tunisian patients with familial or sporadic breast cancer

Background: The p53 polymorphisms have been extensively studied as putative breast cancer susceptibility variants. The present study was undertaken to investigate the association of p53 Arg72Pro, Ins16bp and G13964C polymorphisms and their haplotypes with breast cancer risk in Tunisian women. Methods: Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on 159 patients and 132 controls. Results: The G13964C intronic variant was significantly associated with familial breast cancer risk (p = 0.0018) while the genotypic distribution was similar for p53 Arg72Pro and Ins16bp in patients and controls. Moreover, the (NoIns-C), (Arg-C) and (NoIns-Arg-C) haplotypes were significantly associated with familial breast cancer risk (p = 0.0021, p = 0.0096 and p = 0.0084, respectively) while there was a trend of association between the (Ins-Arg) and (Ins-Arg-G) haplotypes and the risk of sporadic breast cancer. Only the G/C genotype as well as the (NoIns-C) haplotype remained significant after correction for multiple testing. Conclusion: Our data revealed an association between the G/C genotype and the (NoIns-C) haplotype and the risk of familial breast cancer in Tunisian women. However, these observations need to be confirmed due to the limited statistical power of our study and the small number of cases.     

Association of TP53 codon 72 and CDH1 genetic polymorphisms with colorectal cancer risk in Bangladeshi population

Till now no pharmacogenetic study of TP53 codon 72 (Arg72Pro) and CDH1 rs16260 (-160C<A) genes has been reported on Bangladeshi population relating those with colorectal cancer. So the aim of the study is to determine whether there is an elevated risk of colorectal cancer development with TP53 codon 72 and CDH1 rs16260 genetic polymorphism in Bangladeshi population for the first time. To investigate the association of these two SNPs, we conducted a case-control study with 288 colorectal cancer patients and 295 healthy volunteers by using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. We found an increased risk of association between Arg/Pro heterozygosity (adjusted OR = 2.58, 95% CI = 1.77–3.77, p < 0.05) and Pro/Pro mutant homozygosity (adjusted OR = 2.92, 95% CI = 1.78–4.78, p < 0.05) along with the combined genotype (Arg/Pro + Pro/Pro) (adjusted OR = 2.70, 95% CI = 1.90–3.82, p < 0.05) and colorectal cancer predisposition. In case of CDH1 rs16260 polymorphism, C/A heterozygous and A/A mutant homozygous are significantly (p < 0.05) found to be associated with colorectal cancer risk with adjusted OR of 1.94 and 2.63, respectively. The combined genotype of C/A and A/A was also found to be strongly associated with colorectal cancer risk compared to C/C genotype (adjusted OR = 2.02, 95% CI = 1.42–2.87, p < 0.05). In conclusion, heterozygosity and mutant homozygosity as well as the combination of both TP53 Arg72Pro and CDH1 rs16260 polymorphisms are responsible to increase the risk of colorectal cancer development in Bangladeshi population.     

Engineering the activity of thermophilic xylose isomerase by site-directed mutation at subunit interfaces

Some of the conserved residues at subunit interfaces of thermophilic xylose isomerases (XIs) were selected by means of both multiple sequences alignment and subunit interactions analysis of XIs, and then were mutated for improving the activity of Thermus thermophilus xylose isomerase (TtXI). By site-directed mutagenesis, single (D375G, K355A, V144A) and double (D375G/V385A) mutations were introduced into TtXI containing a N91D mutation site, namely, TtXI-N91D. It was shown that the specific activities of mutants D375G, K355A and V144A were remarkably increased over a temperature range of 40–90 °C at pH 7.0. The activities of mutants D375G/V385A, D375G, V144A and K355A were 1.14-, 1.62-, 2.49- and 3.02-fold greater than that of TtXI-N91D at 75 °C, respectively. Over the pH range of 5.0–9.0, the activities of mutants D375G, K355A and V144A were greater than that of TtXI-N91D at 60 °C. The thermostability of all mutants, except K355A, was lower than that of TtXI-N91D. The results suggest that the activity of TtXI could be engineered by site-directed mutagenesis on the conserved residues at subunit interfaces. This method could be employed for improving the activity of other thermophilic XIs.     

Allelic variants of glutathione S-transferase P1-1 differentially mediate the peroxidase function of peroxiredoxin VI and alter membrane lipid peroxidation

The dual-functioning antioxidant enzyme peroxiredoxin VI (Prdx6) detoxifies lipid peroxides particularly in biological membranes, and its peroxidase function is activated by glutathione S-transferase Pi (GSTP). The GSTP gene is polymorphic in humans, with the wild-type GSTP1-1 A (Ile105, Ala114) and three variants: GSTP1-1B (Ile105Val, Ala114), GSTP1-1C (Ile105Val, Ala114Val), and GSTP1-1D (Ile105, Ala114Val). The focus of this study was to determine the influence of these polymorphisms on Prdx6 peroxidase function. Using extracellular generation of OH radicals and fluorescence (DPPP dye) detection, we found a fast (∼300 s) onset of lipid peroxidation in membranes of MCF-7 cells transfected with a catalytically inactive Y7F mutant of GSTP1-1 and either GSTP1-1B or GSTP1-1D. However, this effect was not detected in cells expressing either GSTP1-1A or GSTP1-1C. Imaging of DPPP-labeled MCF-7 cells showed fluorescence localized in the plasma membrane, but intensity was substantially diminished in the GSTP1-1 A- and GSTP1-1C-expressing cells. Moreover, in the Y7F mutant of GSTP1-1 A-, GSTP1-1B-, and GST1-1D-expressing cells OH generation resulted (after 36 h) in plasma membrane-permeability-related cell death, whereas GSTP1-1A- and GSTP1-1C-expressing cells had significantly better survival. We used FRET analyses to measure in vitro binding of purified GSTP1-1 allelic variant proteins to purified recombinant Prdx6. The affinities for Prdx6 binding to GSH-loaded GSTP1-1's either mirrored their observed peroxidase activities (using phospholipid hydroperoxide as a substrate), GSTP1-1A>GSTP1-1C (K_D=51.0 vs 57.0 nM), or corresponded to inactivation, GSTP1-1B (GSTP1-1D) (K_D=101.0 (94.0) nM). In silico modeling of the GSTP1-1–Prdx6 heterodimer revealed that the sites of GSTP1-1 polymorphism (Ile105 and Ala114) are in close proximity to the binding interface. Thus, there is a hierarchy of effectiveness for polymorphic variants of GSTP1-1 to regulate Prdx6 peroxidase function, a feature that may influence human population susceptibilities to oxidant stress.     

Characterization and site-directed mutagenesis of a novel class II 5-enopyruvylshikimate-3-phosphate (EPSP) synthase from the deep-sea bacterium Alcanivorax sp. L27

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroA_A.sp. A phylogenetic analysis revealed that AroA_A.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The K_M for PEP and IC₅₀ [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroA_A.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC₅₀ [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroA_A.sp. The specific activity of the wild-type AroA_A.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity.     

LaaA,a novel high-active alkalophilic alpha-amylase from deep-sea bacterium Luteimonas abyssi XH031T

Alpha-amylase is a kind of broadly used industrial enzymes, most of which have been exploited from terrestrial organism. Comparatively, alpha-amylase from marine environment was largely undeveloped. In this study, a novel alkalophilic alpha-amylase with high activity, Luteimonas abyssi alpha-amylase (LaaA), was cloned from deep-sea bacterium L. abyssi XH031^T and expressed in Escherichia coli BL21. The gene has a length of 1428 bp and encodes 475 amino acids with a 35-residue signal peptide. The specific activity of LaaA reached 8881 U/mg at the optimum pH 9.0, which is obvious higher than other reported alpha-amylase. This enzyme can remain active at pH levels ranging from 6.0 to 11.0 and temperatures below 45 °C, retaining high activity even at low temperatures (almost 38% residual activity at 10 °C). In addition, 1 mM Na⁺, K⁺, and Mn²⁺ enhanced the activity of LaaA. To investigate the function of potential active sites, R227G, D229K, E256Q/H, H327V and D328V mutants were generated, and the results suggested that Arg227, Asp229, Glu256 and Asp328 were total conserved and essential for the activity of alpha-amylase LaaA. This study shows that the alpha-amylase LaaA is an alkali-tolerant and high-active amylase with strong potential for use in detergent industry.     

Enhanced catalysis of l-asparaginase from Bacillus licheniformis by a rational redesign

l-Asparaginase (3.5.1.1) being antineoplastic in nature are used in the treatment of acute lymphoblastic leukemia (ALL). However glutaminase activity is the cause of various side effects when used as a drug against acute lymphoblastic leukemia (ALL). Therefore, there is a need of a novel l-asparaginase (L-ASNase) with low or no glutaminase activity. Such a property has been observed with L-ASNase from B. licheniformis (BliA). The enzyme being glutaminase free in nature paved the way for its improvement to achieve properties similar to or near to the commercially available L-ASNases. Rational enzyme engineering approach resulted in four mutants: G238N, E232A, D103V and Q112H. Among these the mutant enzyme, D103V, had a specific activity of 597.7 IU/mg, which is higher than native (rBliA) (407.65 IU/mg). Moreover, when the optimum temperature and in vitro half life were studied and compared with native BliA, D103V mutant BliA was better, showing tolerance to higher temperatures and a 3 fold higher half life. Kinetic studies revealed that the mutant D103V L-ASNase has increased substrate affinity, with K_m value of 0.42 mM and V_max of 2778.9 μmol min⁻¹.     

A model for the 77 K excited state dynamics in Chlamydomonas reinhardtii in state 1 and state 2

The regulatory mechanism of state transitions was studied in Chlamydomonas reinhardtii (C.r.) wild type (WT) as well as mutant strains deficient in the photosystem I (PSI) or the photosystem II (PSII) core. Time-resolved fluorescence measurements were obtained on instantly frozen cells incubated beforehand in the dark in aerobic or anaerobic conditions which leads to state 1 (S1) or state 2 (S2). WT data contains information on the light-harvesting complex (LHC) connected to PSI and PSII. The mutants' data contain information on either LHCII-LHCI-PSI or LHCII-PSII, plus information on LHC antennas devoid of a PS core. In a simultaneous analysis of the data from all strains under S1 or S2 conditions a unified model for the excited state dynamics at 77 K was created. This yielded the completely resolved LHCII-LHCI-PSI and LHCII-PSII dynamics and quantified the state transitions. In WT cells the fraction of light absorbed by LHCII connected to PSII decreases from 45% in S1 to 29% in S2, while it increases from 0% to 16% for LHCII connected to PSI. Thus (16/45 =) 36% of all LHCII is involved in the state transition. In the mutant strains deficient in the PSI core, the red most species peaking at 716 nm disappears completely, indicating that this far red Chl pigment is located in the PSI core. In the mutant strain deficient in the PSII core, red shifted species with maxima at 684 and 686 nm appear in the LHCII antenna. LHCII-684 is quenched and decays with a rate of (310 ps)^? 1.     

Structure and function of sheep hemoglobin Chios: A novel allele at the HBBB locus with two Lys → Arg substitutions at positions β66(E10) and β144(HC1)

A novel hemoglobin variant was observed in pure sheep (Ovis aries) breeds of the island of Chios (Greece), Egypt and Hungary. This silent variant was identified by gel electrophoresis and RP-HPLC of dissociated globin chains. Two Arg for Lys substitutions were detected, by means of MALDI TOF electrospray mass spectrometric analysis for the intact globins, at positions β66(E10) and β144(HC1) of a globin chain having the sequence of the β^B chain. Sequencing of the β-globin gene confirmed the variant gene as being an allele of the HBBB locus having the AAG → AGG and the AAA → AGA mutations at codons 66 and 144, respectively, both corresponding to the Lys → Arg substitution. The intrinsic oxygen affinity of the variant Hb (logP₅₀ = 0.79 at pH 7.0) was found to be intermediate between that of the sheep Hb B (logP₅₀ = 0.92) and that of Cypriot mouflon (O. a. ophion) Hb (logP₅₀ = 0.53), the latter having only the Lys → Arg change at β144, whereas nearly no differences were observed in the presence of the Cl⁻ physiological effector. Result supports the indication that Arg at β144 enhances the role of the ligand in decreasing oxygen affinity, this effect being partially counteracted when Arg is at β66. Data also shows that the Lys → Arg change at β66 is responsible for 1.49 fold reduction in the intrinsic oxygen affinity. This hitherto undescribed variant increases to seven the number of alleles at the sheep HBBB locus. Following the nomenclature used for human Hb variants, the new allele was termed as the Hb Chios or [β^B66(E10) Lys → Arg, 144(HC1)Lys → Arg], whereas the proposed genetic nomenclature of the locus is HBBK.     

Evaluation of red seaweed Gracilaria arcuata as dietary ingredient in African catfish,Clarias gariepinus

The aim of this study was to evaluate the use of dried marine seaweed, Gracilaria arcuata for the first time as dietary ingredient in partial substitution of fishmeal on the growth performance, feed utilization and body composition of African catfish, Clarias gariepinus. Four experimental diets were formulated: D1 as a control group; D2; D3 and D4 which included 10%, 20% and 30% G. arcuata meal respectively. One hundred and eighty African catfish weighing 9.62 ± 0.42 g, (mean ± SE) was divided into four groups corresponding to the different feeding regimes. The final body weight of the fishes showed significant differences (P < 0.05) between the control (D1); D2 and other treated groups D3 and D4, with weights of 66.98, 59.60, 47.34 and 30.73 g recorded for D1, D2, D3 and D4, respectively. Significant differences (P < 0.05) were also evident in weight gain, specific growth rate, and feed utilization between treatment and control groups. However, no significant differences (P > 0.05) were observed between the control group and fishes fed D2 for all previous parameters. Protein productive value, protein efficiency ratio, daily dry feed intake and total feed intake were also significantly lower in fish fed with a diet containing G. arcuata than in the control group and D2 which contains 10% of G. arcuata. Overall, the results of the experiment revealed that African catfish fed a diet with G. arcuata included in 20% and 30% levels showed poorer growth and feed utilization than the control group and D2. However, the study recommended that C. gariepinus can accept this ingredient up to 10% in their diets. More defined experiments therefore seem to be necessary in order to determine the maximum level of this marine seaweed in diets with amino acid supplementation for African catfish.     

Association of p53 and MDM2 polymorphisms with risk of human papillomavirus (HPV)-related esophageal squamous cell carcinoma (ESCC)

PurposeThough polymorphisms of the tumor suppressor gene p53 have been extensively investigated in numerous tumors, particularly tumors associated with human papillomavirus (HPV) infection. However, the results remain controversial. Our previous study showed that HPV serostatus is not an independent risk factor for esophageal squamous cell carcinoma (ESCC) in nonsmokers and nondrinkers. Given the roles of p53 and HPV E6 as well as MDM2 oncoproteins in p53 degradation, we validated the association of p53 and MDM2 polymorphisms with ESCC risk stratified by HPV16 sero-status.MethodsSingle nucleotide polymorphisms of p53 Arg72Pro (rs1042522) and MDM2 (rs937283) in 307 ESCC patients and 311 healthy controls were genotyped. The presence or absence of HPV16 in serum was measured by enzyme-linked immunosorbent assay. Multivariable logistic regression analysis was used to evaluate the possible associations of p53 and MDM2 polymorphisms with ESCC risk stratified by HPV16 sero-status.ResultsPatients carrying p53 Arg/Arg or Arg/Pro had a higher risk of esophageal SCC (P < 0.001, Odds ratio [OR] 4.98, 95% confidential interval [CI] 3.46–7.17), however, not found in MDM2 rs937283. The risk of esophageal SCC increased significantly among patients carrying p53 Arg/Arg, or Arg/Pro and HPV16-seropositivity (P < 0.001, OR 9.33, 95% CI 5.44–16.0), but not for MDM2 rs937283. The risk of esophageal SCC was further elevated among patients carrying Arg/Arg or Arg/Pro and HPV16-seropositivity who were smokers (P < 0.001, OR 27.05, 95% CI 11.06–66.16) or drinkers (P < 0.001, OR 13.20, 95% CI 5.74–30.38).ConclusionHPV16 seropositivity synergized with p53 Arg/Arg or Arg/Pro and increased ESCC risk, especially in smokers or drinkers.     

Confirmation of color determination factors for Ser286 derivatives of firefly luciferase from Luciola cruciata (LUC-G)

The bioluminescence color of firefly luciferase including its mutants ranges from green to red (530–640 nm) and is affected by the species of firefly, the reaction conditions, and by the substitution of amino acids. Although there is a general agreement that the microenvironment mechanism is the dominant model for the color determination of firefly luciferase, a complete mechanism has not been shown, partially due to the lack of comprehensive data on which amino acid positions alter the bioluminescent color. In this paper, a mutant library of position serine 286 (S286) in Luciola cruciata luciferase (LUC-G) was constructed and characterized. The substitution of S286 resulted in a drastic red shift in bioluminescence color (>600 nm), and only glycine (G) and alanine (A) mutants remained yellow-green. To explain this color difference, molecular dynamics (MD) calculations of 3 S286 derivatives (S286G, S286N, and S286I), in addition to wild type (WT), were performed. The active site rigidity and active site hydrogen bonding networks were compared to WT for each derivative. The results suggested that both factors affected the active site environment and affected the difference in bioluminescence colors in LUC-G S286 derivatives.     

Different types of fruit damages of three internal apple feeders diagnosed with mitochondrial molecular markers

Three tortricid pests, Grapholita dimorpha (Komai), G. molesta (Busck), and Carposina sasakii (Matsumura), are known as internal apple feeders in Korea. To identify young larvae, this study developed two types of molecular markers from their mitochondrial DNA (mtDNA) sequences. To this end, six different loci of mtDNA were sequenced in G. dimorpha: cytochrome oxidase subunit I (460 bp), cytochrome oxidase subunit II (446 bp), cytochrome b (308 bp), NADH dehydrogenase 3 (585 bp), NADH dehydrogenase 4 (ND4, 835 bp), and 16S rRNA (1300 bp). These sequences were compared with those of G. molesta and C. sasakii in order to develop PCR–RFLP and diagnostic primers. ND4 locus was selected to be used for developing a PCR–RFLP marker. ND4-Swa I digests showed two bands for G. dimorpha, one band for G. molesta, and three bands for C. sasakii. On the other hand, species-specific diagnostic PCR primers were developed using ND4 locus. These markers were then applied to diagnose larvae infesting apples to determine species-specific fruit damage patterns, in which G. dimorpha, G. molesta, and C. sasakii showed different feeding behaviors in terms of their main feeding sites in apple fruits.     

Redesigning the active site of a carboxyl esterase from the archaeon Archaeoglobus fulgidus to improve sensitivity to organophosphorus compounds

Organophosphorus compounds (OPs) are widely used as pesticides because of their ability to inhibit the activity of acetylcholinesterase (AChE) in the nervous system. Thus, AChE is generally used as a biosensor for pesticide detection. Due to the instability of AChE a more stable enzyme would be desirable for robust applications. We investigated the sensitivity of a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus (AFEST) to seven selected OPs. The IC₅₀ of dichlorvos against AFEST (50.8 ± 2.6 nM) was 10-fold lower than that of the commercially obtained AChE, indicating that AFEST had higher sensitivity. Its sensitivity for the other OPs was lower than AChE. To enhance the sensitivity of AFEST to OPs, site-directed mutations were introduced in the cap domain of AFEST. The sensitivity of mutant N44S/S48V was enhanced toward all seven OPs compared to the wild-type and was higher than AChE for four OPs, including paraoxon (3.3 ± 0.01 nM), dichlorvos (28.0 ± 0.6 nM), profenofos (43.0 ± 1.0 nM), and diazinon (3.0 ± 0.2 nM). The half-lives of AFEST and the mutant N44S/S48V at 37 °C were over 15 d. The interactions between the enzymes and select OPs were investigated by molecular docking. The results demonstrated that AFEST and the mutant N44S/S48V have the potential to be biosensor for OP detection.     

Association between polymorphisms of matrix metalloproteinase 11 (MMP-11) and Kawasaki disease in the Korean population

AimsThis study was conducted to investigate the associations between single nucleotide polymorphisms (SNPs) of matrix metalloproteinases (MMPs) and Kawasaki disease (KD) in the Korean population.Main methodsA total 0f 101 KD patients and 306 healthy controls were examined. MMP7 (rs10502001, G/A, Arg77His), MMP11 (rs738792, T/C, Ala38Val), MMP12 (rs652438, A/G, Ile357Val) and MMP26 (rs2499953, A/G, Lys43Glu) genes were genotyped from the genomic DNA using direct sequencing. The results were then analyzed using logistic regression models, adjusting for gender as covariates.Key findingsThe four SNPs were in Hardy–Weinberg equilibrium. Only the MMP11 polymorphism (rs738792) was associated with KD. The SNP (rs738792) showed a statistically significant association with KD in the codominant (OR = 1.61, 95% CI = 1.11–2.34, P = 0.011) and dominant (OR = 1.92, 95% CI = 1.21–3.06, P = 0.006) models. However, there was no association between polymorphisms of other MMP genes and KD.SignificanceOverall, the results of this study indicate that MMP11 polymorphism may be associated with KD in the Korean population.     

Distribution of TYMS,MTHFR, p53 and MDR1 gene polymorphisms in patients with breast cancer treated with neoadjuvant chemotherapy

Purpose To investigate the role of TSER (TYMS), C677T (MTHFR), Arg72Pro (p53) and C3435T (MDR1) gene polymorphisms in breast cancer patients treated with 5-fluorouracil and cyclophosphamide-based neoadjuvant chemotherapy. Results Observed allelic frequencies were: TSER, (2) 0.54 and (3) 0.46; MTHFR C677T, (C) 0.59 and (T) 0.41; p53 Arg72Pro, (Arg) 0.73 and (Pro) 0.27; MDR1 C3435T, (C) 0.52 and (T) 0.48. MTHFR allele T and p53 allele Pro were strongly associated with toxicity due to chemotherapy (odds ratio, 7.1 (95% confidence interval, 1.4–36.1; p = 0.018) and 7.0 (95% confidence interval, 1.2–40.5; p = 0.029), respectively). Conclusion We introduced new data related to the contribution of p53 codon 72 to toxicity due to 5-fluorouracil and cyclophosphamide-based neoadjuvant chemotherapy in patients with breast cancer.