Thermolabile Enzyme Research Articles

Ischaemic stroke with multi-focal venous and arterial thrombosis due to hyperhomocysteinemia: anabolic androgenic steroid use and MTHFR c.667 C > T variant – a case report

BackgroundSeverely elevated serum homocysteine is a rare cause of ischaemic stroke and extra-cranial arterial and venous thrombosis. Several factors can lead to mild elevation of homocysteine including dietary folate and B12 deficiency, and genetic variants of the methylenetetrahydrofolate reductase (MTHFR) enzyme. The use of Anabolic androgenic steroid (AAS) is under-reported, but increasingly linked to ischaemic stroke and can raise homocysteine levels.Case ReportWe present a case of a man in his 40s with a large left middle cerebral artery (MCA) territory ischaemic stroke and combined multifocal, extracranial venous, and arterial thrombosis. His past medical history was significant for Crohn’s disease and covert use of AAS. A young stroke screen was negative except for a severely elevated total homocysteine concentration, folate and B12 deficiencies. Further tests revealed he was homozygous for the methylenetetrahydrofolate reductase enzyme thermolabile variant (MTHFR c.667 C > T). The etiology of this stroke was a hypercoagulable state induced by raised plasma homocysteine. Raised homocysteine in this case was likely multifactorial and related to chronic AAS use in combination with the homozygous MTHFR c.677 C > T thermolabile variant, folate deficiency and, vitamin B12 deficiency.ConclusionIn summary, hyperhomocysteinemia is an important potential cause of ischaemic stroke and may result from genetic, dietary, and social factors. Anabolic androgenic steroid use is an important risk factor for clinicians to consider, particularly in cases of young stroke with elevated serum homocysteine. Testing for MFTHR variants in stroke patients with raised homocysteine may be useful to guide secondary stroke prevention through adequate vitamin supplementation. Further studies looking into primary and secondary stroke prevention in the high-risk MTHFR variant cohort are necessary.

Normal Thermostability of p.Ser113Leu and p.Arg631Cys Variants of Mitochondrial Carnitine Palmitoyltransferase II (CPT II) in Human Muscle Homogenate.

Previous fibroblast and recombinant enzyme studies showed a markedly thermolabile p.Ser113Leu variant compared to the wild-type (WT) in muscle carnitine palmitoyltransferase II (CPT II) deficiency. Additionally, it has been shown that cardiolipin (CLP) stimulated or inhibited the p.Ser113Leu recombinant variant depending on the pre-incubation temperatures. In this study, the thermolabilities of mitochondrial enzyme CPT II in muscle homogenates of patients with the p.Ser113Leu (n = 3) and p.Arg631Cys (n = 2) variants were identified to be similar to that of WT. Pre-incubation with CLP on ice stimulated the WT enzyme more than both variants. However, CLP stimulated the variants and WT at 46 °C to about 6-18-fold. The present data indicate that the thermostability of CPT II variant in muscle homogenate is similar to that of WT. This is in contrast to the increased thermolability of enzymes derived from fibroblast and that of recombinant enzymes. Hence, it can be speculated that the disruption of the compartmentation in muscle homogenate mediates a protective effect on the thermolability of the native variant. However, the exact mechanism remains unclear. However, the activating effect of CLP on CPT II in muscle homogenate seems to align with those on recombinant enzymes.

P1682: MTHFR C677T GENOTYPE IN CORRELATION WITH THROMBOSIS AND ASSOCIATION WITH FVIII COAGULANT ACTIVITY IN GREEK PAEDIATRIC POPULATION

Background: The polymorphism C>T substitution in position 677 (C677T) of the encoding gene of methylenetetrahydrofolate reductase (MTHFR), creates a thermolabile enzyme with reduced activity that may predispose to hyperhomocysteinemia, an established risk factor for arterial thrombosis and premature atherosclerosis and a probable risk factor for venous thromboembolic disease and its recurrence in the general population. There are conflicting results as to the role of the MTHFR C677T polymorphism as a risk factor for thrombosis. Although the homozygous substitution is often associated with hyperhomocysteinemia, mainly in folate deficiency, a clear association between this genetic marker and thrombosis has not yet been established. Aims: Primary aim was to evaluate the prevalence of mutant C677T MTHFR and its association with thrombosis in the Greek paediatric population. Secondary aim was to evaluate whether different MTHFR genotype is associated with evidence of activation of coagulation cascade through increased factor VIII activity. Methods: Data were retrospectively collected from children referred to our Haemostasis and Thrombosis Centre for laboratory screening of thrombophilia between 2018 and 2020. Some of them were checked due to history of thrombosis. Children were categorized according to MTHFR C677T genotype. FVIII Coagulant activity (%) was measured by One Stage Assay, away from acute thrombosis. The identification of mutations was based on polymerase chain reaction (PCR) and reverse- hybridization. Statistical analysis was performed with t-test and ANOVA. Results: A total of 688 children (boys: 49 %) of mean age 8.9± 8 years (0-18) were investigated. Twelve percent (81/688) of them had history of thrombosis, mainly venous thromboembolic disease. Children were categorized as follows: homozygous MTHFR C677T (15%), heterozygous (47%) and normal (38%). There was thrombosis history in 12% of homozygous MTHFR C677T, 11% heterozygous and 12 % of normal, fact showing that there is no association between MTHFR C677T genotype with thrombosis. Children with homozygosity MTHFR C677T, compared to children with heterozygosity and normal MTHFR C677T showed increased factor VIII values (132.8 vs 126.4 and 131.6, %, respectively), but without statistically significant difference. However, independently of MTHFR C677T genotype, mean factor VIII values were significantly increased in all children, with thrombosis history when they were compared to children without thrombosis history (161.3 vs 125.1, %, p<0.001). Interestingly, in children with thrombosis, mean factor VIII level was statistically increased in heterozygous and normal MTHFR C677T in contrast to children with MTHFR C677T homozygosity, in whom no statistically significant increase was found (166.9 vs 121.4, %-p<0.05, 160.2 vs 127.6, %-p<0.05 and 148.3 vs 130.6, %-p>0.05, respectively). Finally, it should be noticed that during the year 2020 mean factor VIII values were much increased in all children in comparison to the years 2018, 2019 and 2021 (137 vs 129, 123 and 127, %, respectively), probably reflecting the effects of unknown pandemic COVID-19 which initiated in 2020, and showing that factor VIII could be an evidence of stress condition. Summary/Conclusion: There is no probably association between MTHFR C677T genotype and thrombosis in our paediatric population. Moreover, it was not proved that different MTHFR genotypes affect factor VIII activity.

Biochemical characterization and inhibition of thermolabile hemolysin fromVibrio parahaemolyticusby phenolic compounds

Vibrio parahaemolyticus (Vp), a typical microorganism inhabiting marine ecosystems, uses pathogenic virulence molecules such as hemolysins to cause bacterial infections of both human and marine animals. The thermolabile hemolysin VpTLH lyses human erythrocytes by a phospholipase B/A2 enzymatic activity in egg-yolk lecithin. However, few studies have been characterized the biochemical properties and the use of VpTLH as a molecular target for natural compounds as an alternative to control Vp infection. Here, we evaluated the biochemical and inhibition parameters of the recombinant VpTLH using enzymatic and hemolytic assays and determined the molecular interactions by in silico docking analysis. The highest enzymatic activity was at pH 8 and 50 °C, and it was inactivated by 20 min at 60 °C with Tm = 50.9 °C. Additionally, the flavonoids quercetin, epigallocatechin gallate, and morin inhibited the VpTLH activity with IC50 values of 4.5 µM, 6.3 µM, and 9.9 µM, respectively; while phenolics acids were not effective inhibitors for this enzyme. Boltzmann and Arrhenius equation analysis indicate that VpTLH is a thermolabile enzyme. The inhibition of both enzymatic and hemolytic activities by flavonoids agrees with molecular docking, suggesting that flavonoids could interact with the active site’s amino acids. Future research is necessary to evaluate the antibacterial activity of flavonoids against Vp in vivo.

Recent Strategies and Applications for l-Asparaginase Confinement.

l-asparaginase (ASNase, EC 3.5.1.1) is an aminohydrolase enzyme with important uses in the therapeutic/pharmaceutical and food industries. Its main applications are as an anticancer drug, mostly for acute lymphoblastic leukaemia (ALL) treatment, and in acrylamide reduction when starch-rich foods are cooked at temperatures above 100 °C. Its use as a biosensor for asparagine in both industries has also been reported. However, there are certain challenges associated with ASNase applications. Depending on the ASNase source, the major challenges of its pharmaceutical application are the hypersensitivity reactions that it causes in ALL patients and its short half-life and fast plasma clearance in the blood system by native proteases. In addition, ASNase is generally unstable and it is a thermolabile enzyme, which also hinders its application in the food sector. These drawbacks have been overcome by the ASNase confinement in different (nano)materials through distinct techniques, such as physical adsorption, covalent attachment and entrapment. Overall, this review describes the most recent strategies reported for ASNase confinement in numerous (nano)materials, highlighting its improved properties, especially specificity, half-life enhancement and thermal and operational stability improvement, allowing its reuse, increased proteolysis resistance and immunogenicity elimination. The most recent applications of confined ASNase in nanomaterials are reviewed for the first time, simultaneously providing prospects in the described fields of application.

Thermal inactivation of pectin methylesterase from different potato cultivars (Solanum tuberosum L.)

Pectin methylesterase (PME) is an important enzyme that can alter the texture of potato products during processing and storage. To manage the textural properties of potato based products, low and/or high temperature blanching can be used to promote or inactivate PME, respectively. However, the many potato cultivars being used for processing may contain PMEs with a different thermal stability. Therefore, the thermal inactivation of PME extract from three different potato cultivars (Fontane, Royal and Innovator) was studied. A thermostable and thermolabile enzyme fraction were found in all three cultivars, being inactivated at temperatures from 45 °C and 57.5 °C on, respectively. Innovator may contain a lower level of thermostable PME than the other cultivars tested. The inactivation rate constant of the thermostable PME fraction was lower than the rate constant of the labile fraction at a reference temperature of 55 °C, but no significant differences between the respective rate constants of the different cultivars were found. The thermal inactivation kinetics of PME extract from Fontane were investigated in detail and represented by a first order model that allows the calculation of temperature-time combinations to reach a target residual PME activity.

Removal of N-terminal tail changes the thermostability of the low-temperature-active exo-inulinase InuAGN25

ABSTRACT Exo-inulinases are members of the glycoside hydrolase family 32 and function by hydrolyzing inulin into fructose with yields up to 90–95%. The N-terminal tail contributes to enzyme thermotolerance, which plays an important role in enzyme applications. However, the role of N-terminal amino acid residues in the thermal performance and structural properties of exo-inulinases remains to be elucidated. In this study, three and six residues of the N-terminus starting from Gln23 of the exo-inulinase InuAGN25 were deleted and expressed in Escherichia coli. After digestion with human rhinovirus 3 C protease to remove the N-terminal amino acid fusion sequence that may affect the thermolability of enzymes, wild-type RfsMInuAGN25 and its mutants RfsMutNGln23Δ3 and RfsMutNGln23Δ6 were produced. Compared with RfsMInuAGN25, thermostability of RfsMutNGln23Δ3 was enhanced while that of RfsMutNGln23Δ6 was slightly reduced. Compared with the N-terminal structures of RfsMInuAGN25 and RfsMutNGln23Δ6, RfsMutNGln23Δ3 had a higher content of (1) the helix structure, (2) salt bridges (three of which were organized in a network), (3) cation–π interactions (one of which anchored the N-terminal tail). These structural properties may account for the improved thermostability of RfsMutNGln23Δ3. The study provides a better understanding of the N-terminus–function relationships that are useful for rational design of thermostability of exo-inulinases.

The effect of convective heating and microwave heating on antioxidant enzymes in pooled mature human milk

The effects of convective and microwave heating at constant temperature (62.5, 66 and 70 °C) on the activity of antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPx) in pooled mature human milk were compared. Activities of the enzymes were determined using spectrophotometric kits. Activity of GPx decreased significantly in the first stage of heating when milk samples were warmed to pasteurisation temperature. CAT was the most thermolabile enzyme but microwave heating induced a smaller decrease in CAT activity than convective heating. SOD was most resistant to thermal pasteurisation, regardless of the heating method. SOD and GPx activity temporary increased during microwaves heating. Considering shorter pasteurisation period and lower demand for energy, it can be concluded that microwaves pasteurisation enjoys special merits. However, still there is no clear answer as to whether the microwave field itself can affect the antioxidant enzymes of human milk.

Peculiarities and biotechnological potential of environmental adaptation by Geobacillus species.

The genus Geobacillus comprises thermophilic bacilli capable of endospore formation. The members of this genus provide thermostable proteins and can be used in whole cell applications at elevated temperatures; therefore, these organisms are of biotechnological importance. While these applications have been described in previous reviews, the present paper highlights the environmental adaptations and genome diversifications of Geobacillus spp. and their applications in evolutionary-protein engineering. Despite their obligate thermophilic properties, Geobacillus spp. are widely distributed in nature. Because several isolates demonstrate remarkable properties for cell reproduction in their respective niches, they seem to exist not only as endospores but also as vegetative cells in diverse environments. This suggests their excellence in environmental adaptation via genome diversification; in fact, evidence suggests that Geobacillus spp. were derived from Bacillus spp. while diversifying their genomes via horizontal gene transfer. Moreover, when subjected to an environmental stressor, Geobacillus spp. diversify their genomes using inductive mutations and transposable elements to produce derivative cells that are adaptive to the stressor. Notably, inductive mutations in Geobacillus spp. occur more rapidly and frequently than the stress-induced mutagenesis observed in other microorganisms. Owing to this, Geobacillus spp. can efficiently generate mutant genes coding for thermostable enzyme variants from the thermolabile enzyme genes under appropriate selection pressures. This phenomenon provides a new approach to generate thermostable enzymes, termed as thermoadaptation-directed enzyme evolution, thereby expanding the biotechnological potentials of Geobacillus spp. In this review, we have discussed this approach using successful examples and major challenges yet to be addressed.

METHYLENETETRAHYDROFOLATE REDUCTASE C677T GENE POLYMORPHISM AND PROSTATE CANCER RISK

Objective: The single nucleotide polymorphism C677T of the methylenetetrahydrofolate reductase (MTHFR) gene encodes a thermolabile enzyme. This polymorphism was found to be implicated in cancer susceptibility. In this study, we analyzed the distribution of the MTHFR C677T polymorphism in two cohorts; patients and controls native of East of Algeria to explore the possible association between this polymorphism and prostate cancer susceptibility.Methods: Our examination has been conducted in 98 cases and 98 healthy controls. Genotyping was realized by polymerase chain reaction-restriction fragment length polymorphism method.Results: Compared with CC homozygous, the CT heterozygous was found to have a significantly increased risk of prostate cancer (p=0.04; odds ratio [OR]=2.01, 95% confidence interval [CI]: 1.02–3.95). However, no statistically significant difference was observed concerning the TT homozygous (p=0.74; OR=1.25, 95% CI: 0.51–3.04).Conclusion: Our results indicate that the genotype CT is a risk factor for prostate cancer in East of Algeria.

Crystal Structure of the Isocitrate Dehydrogenase 2 from Acinetobacter baumannii (AbIDH2) Reveals a Novel Dimeric Structure with Two Monomeric-IDH-Like Subunits.

Monomeric isocitrate dehydrogenases (IDHs) have a single polypeptide sizing around 85 kDa. The IDH2 from the opportunistic bacterium Acinetobacter baumannii (AbIDH2) with a mass of 83 kDa was formerly recognized as a typical monomeric IDH. However, both size exclusion chromatography and analytical ultracentrifugation analysis indicated that AbIDH2 exists as a homodimer in solution. The crystallographic study of the substrate/coenzyme-free AbIDH2 gave a dimeric structure and each subunit contained a domain I and a domain II. The dimeric assembly is mainly stabilized by hydrophobic interactions (16 hydrogen bonds and 11 salt bridges) from the dimer’s interface platform, which centered around the three parallel helices (α4, α12, and α17) and one loop from the domain II. Kinetic analysis showed that the dimeric AbIDH2 showed much lower catalytic efficiency (0.39 μM−1·s−1) as compared to the typical monomeric IDHs (~15 μM−1·s−1). Key residues crucial for dimer formation were simultaneously changed to generate the mutant mAbIDH2. The disruption of the hydrophobic forces disassociated the dimeric AbIDH2, making mAbIDH2 a monomeric enzyme. mAbIDH2 sustained specific activity (21.9 ± 2 U/mg) comparable to AbIDH2 (25.4 ± 0.7 U/mg). However, mAbIDH2 proved to be a thermolabile enzyme, indicating that the thermostable dimeric AbIDH2 may have a physiological significance for the growth and pathogenesis of A. baumannii. Phylogenetic analysis demonstrated the existence of numerous AbIDH2 homologous proteins, thus expanding the monomeric IDH protein family.

No evidence for association of MTHFR 677C>T and 1298A>C variants with placental DNA methylation

Background5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in one-carbon metabolism that ensures the availability of methyl groups for methylation reactions. Two single-nucleotide polymorphisms (SNPs) in the MTHFR gene, 677C>T and 1298A>C, result in a thermolabile enzyme with reduced function. These variants, in both the maternal and/or fetal genes, have been associated with pregnancy complications including miscarriage, neural tube defects (NTDs), and preeclampsia (PE), perhaps due to altered capacity for DNA methylation (DNAm). In this study, we assessed the association between MTHFR 677TT and 1298CC genotypes and risk of NTDs, PE, or normotensive intrauterine growth restriction (nIUGR). Additionally, we assessed whether these high-risk genotypes are associated with altered DNAm in the placenta.ResultsIn 303 placentas screened for this study, we observed no significant association between the occurrence of NTDs (N = 55), PE (early-onset: N = 28, late-onset: N = 20), or nIUGR (N = 21) and placental (fetal) MTHFR 677TT or 1298CC genotypes compared to healthy pregnancies (N = 179), though a trend of increased 677TT genotype in PE/IUGR together was observed (OR 2.53, p = 0.048). DNAm was profiled in 10 high-risk 677 (677TT + 1298AA), 10 high-risk 1298 (677CC + 1298CC), and 10 reference (677CC + 1298AA) genotype placentas. Linear modeling identified no significantly differentially methylated sites between high-risk 677 or 1298 and reference placentas at a false discovery rate < 0.05 and Δβ ≥ 0.05 using the Illumina Infinium HumanMethylation450 BeadChip. Using a differentially methylated region analysis or separating cytosine-guanine dinucleotides (CpGs) by CpG density to reduce multiple comparisons also did not identify differential methylation. Additionally, there was no consistent evidence for altered methylation of repetitive DNA between high-risk and reference placentas.ConclusionsWe conclude that large-scale, genome-wide disruption in DNAm does not occur in placentas with the high-risk MTHFR 677TT or 1298CC genotypes. Furthermore, there was no evidence for an association of the 1298CC genotype and only a tendency to higher 677TT in pregnancy complications of PE/IUGR. This may be due to small sample sizes or folate repletion in our Canadian population attenuating effects of the high-risk MTHFR variants. However, given our results and the conflicting results in the literature, investigations into alternative mechanisms that may explain the link between MTHFR variants and pregnancy complications, or in populations at risk of folate deficiencies, are warranted.

Uticaj polimorfizma C677T gena za metilen-tetrahidrofolat reduktazu na nastanak spontanih pobačaja

Introduction: Spontaneous abortion (SA) is a frequent obstetric complication. Up to 15% of pregnancies end by spontaneous abortion in the first trimester. Five percent of women have two, and 1-2% of women have more than three abortions. The risk of spontaneous abortion is influenced by variety of genetic and environmental factors. Enzyme methylenetetrahydrofolate reductase (MTHFR) is primarily involved in homocysteine remethylation to the methionine. Gene polymorphism C677T in the MTHFR gene causes enzyme thermolability which leads to hyperhomocysteinemia, that might increase the risk of developing placental thrombosis and spontaneous abortion. Aim: The aim of our study was to determine whether the MTHFR C677T polymorphism is associated with the increased risk of SA. Material and methods: This study included 157 women who had at least two unexplained spontaneous abortions and 135 healthy women with at least one child, without previous SA. Detection of MTHFR C677T polymorphism genotypes was performed by PCR-RFLPs method. Investigation of differences between genotype and allele frequencies, in the group of women with SA and the control group, was completed using the x2 test. Results: In the group of patients with recurrent spontaneous abortions, 58 of them had CC genotype (36.94%), 74 had CT genotype (47.13%), and 25 had TT genotype (15.92%). In the control group, 59 of them had CC genotype (43.07%), 55 had CT genotype (40.74%), and 21 had TT genotype (15.56%). The frequency of C allele in the group of patients was 60.51%, and T allele 39.49%; while the frequencies in the control group were 64.07% and 35.93%, respectively. The difference in the genotype (p=0.470) and allele (p=0.393) frequency between the two groups of patients was statistically insignificant. Conclusion: Our results do not show any influence of the analyzed polymorphism on SA.

MTHFR C677T gene polymorphism in Type 2 diabetes mellitus patients with and without vascular complications: A case-control study

PurposeEvidences suggest that polymorphism in genes involved in folate metabolism plays a critical role in the etiology of diabetes and diabetic complications. Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme involved in folate metabolism. MTHFRC677T gene polymorphism, which is located within the N terminal catalytic domain makes enzyme thermolabile with reduced activity. The present study was conducted to investigate whether the MTHFR C677T gene polymorphism is associated with the susceptibility to Type 2 diabetes mellitus (T2DM) in patients with and without vascular (micro and macro) complications and controls. MethodsThe study included 200 T2DM patients (n=100, with vascular complications and n=100, without complications) and 100 healthy individuals as controls. DNA was isolated from the blood samples and genotyping of MTHFR C677T gene polymorphism was performed using polymerase chain reaction followed by restriction fragment length polymorphism method. ResultsThe results indicated that the occurrence of homozygous mutant (TT) was completely absent in T2DM patients without complications and controls. The type 2 diabetes patients with vascular complications have shown only 2% of MTHFR 677TT genotype. The calculated genotype frequencies between the patients with and without vascular complications and controls did not differ significantly from frequencies predicted on the basis of the Hardy Weinberg law of population genetics. Odds ratio (OR) of the genotypes did not show significant difference between T2DM patients without complications and controls {OR/P; (Confidence Intervals)=0.516/0.21; (0.183-1.45)}. Similarly, there were no significant differences between T2DM patients with vascular complications and controls {OR/P; (Confidence Intervals)=0.335/0.02; (0.125-0.895)}. ConclusionsWe suggest that the MTHFR C677T gene polymorphism is not considered as a risk factor for the development of T2DM and its vascular complications in the south Indian population.

Influence of moderate electric field on inactivation kinetics of peroxidase and polyphenol oxidase and on phenolic compounds of sugarcane juice treated by ohmic heating

This study investigated the non-thermal effects of electricity on enzymatic inactivation kinetics of peroxidase and polyphenol oxidase and on degradation of total phenolic and total flavonoid compounds during ohmic heating of sugarcane juice. A kinetic modeling was carried out from the adjustment of various models applicable to enzyme inactivation, and the Weibull distribution model was the most suitable to describe the inactivation of peroxidase. The presence of an electric field with low intensity significantly influenced the biochemical reactions occurred during peroxidase activation at 60 °C and inactivation at 80 °C. Polyphenol oxidase, a more thermolabile enzyme, was almost totally inactivated before reaching the target temperature (holding phase) of the experiments. The maximum total phenolic and total flavonoid degradation were approximately 23 and 39%, respectively. Statistical analyses showed no differences between conventional and ohmic heating for total phenolic and total flavonoid compounds.

Acid phosphatases and ribonucleases from Dactylis glomerata seeds. I. Chromatographic and electrophoretic heterogeneity of the enzymes

Acid phosphatase and ribonuclease extracted with 0.1 M sodium acetate buffer, pH 5.1 from Dactylis glomerata seeds, and partially purified by means of 70% ethanol precipitation showed electrophoretic and Chromatographic heterogeneity. After chromatography on DEAE-cellulose acid phosphatase and ribonuclease were separated into four peaks. Nonadsorbing acid phosphatase on DEAE-cellulose (peak I) was separated into four peaks on CM-cellulose. The highest activity (11 units/mg) was found in fraction b (acid phosphatase Ib). The enzyme was activated by Mg&lt;sup&gt;2+&lt;/sup&gt;, Ca&lt;sup&gt;2+&lt;/sup&gt;, Li&lt;sup&gt;+&lt;/sup&gt;, Cs&lt;sup&gt;+&lt;/sup&gt;, K&lt;sup&gt;+&lt;/sup&gt; ions and inhibited by Cu&lt;sup&gt;2+&lt;/sup&gt;, Zu&lt;sup&gt;2+&lt;/sup&gt;, F&lt;sup&gt;-&lt;/sup&gt; and Mo&lt;sup&gt;-6&lt;/sup&gt; at optimum pH 5.0. Strong absorbing ribonuclease on DEAE-cellulose (peak IV) was further separated on G-200 Sephadex into two molecular forms: RN-asa1 and RN-ase2. Ribonuclease l, a thermolabile enzyme with specific activity 807 units/mg, showed an optimal activity at pH 4.8-5.1.

Bladder-Exstrophy-Epispadias-Complex risk and C677T polymorphism in MTHFR gene: case-control study among Indian children

Background Bladder exstrophy is a congenital anomaly in which part of the urinary bladder is present outside the body. It is rare in occurrence but the frequency is increasing very rapidly. 5, 10-methyltetrahydrofolate reductase (MTHFR) enzyme, which catalyzes the synthesis of 5-methylenetetrahydrofolate and C677T polymorphism in MTHFR, shows a significant role with a thermolabile enzyme and decreased specific MTHFR activity. The objective of the present investigation was to study the case-control association between C677T polymorphism in relation to Bladder-Exstrophy-Epispadias-Complex. Materials and methods For the present study, a total of 50 patients classified as Bladder-Exstrophy-Epispadias-Complex & cloacal exstrophy patient and 50 healthy school going children (as control) were recruited to participate in this study. Genomic DNA was extracted from peripheral blood lymphocytes by using commercially available kits. Genotypes of the MTHFR C677T polymorphisms were detected by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Results Frequency distributions of genotypes and combined genotypes were obtained. The overall distribution of the C677T genotype was found to be significantly associated with cloacal exstrophy but not with epispadias as compared to the controls. Conclusions Genotyping of 50 patient cases and 50 controls revealed a significant association between C677T polymorphism and Bladder-Exstrophy.

A Comparison of Barley Malt Amylolytic Enzyme Thermostabilities and Wort Sugars Produced during Mashing

This study was conducted primarily to determine the relationship between barley amylolytic enzyme thermolability of malts produced over several days of germination and wort sugar concentrations produced during mashing. Seeds of 4 two-row and 4 six-row North American elite barley cultivars were steeped and germinated in a micromalter. At 24 hour intervals throughout 6 days of germination, green malt was removed and kilned. Malts were assayed for activities of α- and β-amylase and limit dextrinase before and after mashing at 70°C for 30 min to determine thermostabilities. Wort sugars were assayed after mashing. α-Amylase thermostability was either unaffected or higher after the 70°C mashing indicating that it is not thermolabile for 30 min at 70°C. In contrast, malt β-amylase and limit dextrinase thermostabilities greatly decreased between days 1 and 2, indicating a strong developmental influence on this parameter. For all cultivars combined, over all days of germination, malt β-amylase and limit dextrinase thermostabilities correlated negatively and highly significantly with total wort sugars (r = −0.656, P < 0.0001; r = −0.767, P < 0.0001, respectively) and with fermentable wort sugars. Six-row cultivar β-amylase and limit dextrinase thermostabilities correlated slightly better than two-row cultivar thermostabilities with total and individual sugar concentrations (e.g., total sugars: two-row β-amylase and limit dextrinase, r = −0.645, P < 0.0001, r = −0.770, P < 0.0001, respectively; six-row β-amylase and limit dextrinase, r = −0.686, P < 0.0001, r = −0.808, P < 0.0001, respectively). In contrast, the non-fermentable maltodextins, maltotetraose through maltoheptaose, correlated positively and highly significantly with β-amylase and limit dextrinase thermostabilities. These data suggest that β-amylase and limit dextrinase thermostabilities become more limiting to starch degradation as germination proceeds. Also, these data are in direct contrast to correlations of initial activities of these enzymes and wort sugars produced by malts over 6 days of germination. β-Amylase intron III allelic variation had no effect on thermostability or activity on the day of optimal malt modification (day 5) or any other day where LSD analysis was significant, indicating that, as in past studies, North American barley germplasm, β-amylase thermostability, and activity are not influenced by intron III allelic variation.

An approach to chemoenzymatic DKR of amines in Soxhlet apparatus

The coexistence of thermolabile enzyme and metal catalyst for racemization of amines in chemoenzymatic dynamic kinetic resolution, requiring high temperature of operation, is enabled by carrying out the reaction in modified Soxhlet extraction system. Initial insights into the scope and limitations of the developed reaction system are discussed.

The MTHFR C677T polymorphism modifies age at onset in Parkinson’s disease

Hyperhomocysteinemia is a risk factor for Parkinson's disease (PD) and may result from genetic mutations or/and environmental factors. 5,10-methylenetetrahydrofolate reductase (MTHFR) is a folate-dependent enzyme that catalyzed remethylation of homocysteine (Hcy) and the MTHFR C677T polymorphism makes the MTHFR enzyme thermolabile causing hyperhomocysteinemia. In this study we analyzed whether two functional polymorphisms of MTHFR gene, A1298C and C677T, affect age of onset in PD. We enrolled 120 patients with sporadic PD. Patients were divided into three groups based on MTHFR C677T polymorphisms: (a) homozygotes wild type (CC) (b) heterozygotes (CT) and (c) homozygotes carriers of mutation (TT). MTHFR SNPs were analyzed using High-Resolution Melt analysis and ANOVA was performed to assess whether polymorphisms of MTHFR gene could influence age of onset. The MTHFR A1298C polymorphism had no effect on PD age at onset (p = 1.0) while there was a significant association with MTHFR C677T (p = 0.019 Bonferroni-adjusted post hoc) showing an earlier onset in CC as compared with TT. (p = 0.024). No differences were found for vascular load assessed with magnetic resonance imaging, pharmacological therapy and cognitive state for two MTHFR SNPs. Our results suggest a possible association of MTHFR C677T with age at onset of PD and may have important implications regarding the role of MTHFR.