Enzyme Analysis

A variety of experimental studies have demonstrated the neuroprotective effects of melatonin, based on its antioxidant activity. In a prospective randomized study, the effects of melatonin were investigated in experimental head trauma-induced oxidative stress in rabbits. The experimental study was performed on 30 rabbits. The animals were divided into three groups. Group I (sham procedure): a right parietal craniotomy was performed on each animal, and the dura mater was left intact. Group II: experimental brain trauma (EBT) was performed on each animal using a 1 cm inner diameter • 10 cm long glass tube, through which a 20 g weight (0.5 cm diameter) was dropped onto the brain at the craniotomy site, causing a contusional head trauma. Group III: the same EBT model was performed, but 2.5 mg/kg melatonin was injected intraperitoneally four times (total dose 10 mg/kg); these injections were performed 20 min before the operation, during the trauma, 1 h later and 2 h later. The rabbits were sacrificed after the EBT at 24 h after the brain trauma. The activities of the three principal antioxidant enzymes-catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px)-were determined, and the levels of malondialdehyde (MDA), a product of lipid peroxidation, and glutathione (GSH) were measured in brain homogenates. MDA levels were found to be higher in the EBT group than in the EBT+melatonin group or the sham procedure group. The SOD activity was found to be higher in the EBT group than in the sham procedure group. Enzymatic parameters (except for SOD) were significantly higher in melatonin-treated animals than in EBT animals. GSH levels in melatonin-treated animals were decreased compared with EBT animals. In conclusion, the data indicate that melatonin protects against free radical-mediated oxidative changes in brain tissue by boosting antioxidant enzymes, and in particular lowering lipid peroxidation in rabbits with EBT.

Urease (EC 3.5.1.5) was covalently attached through glutaraldehyde to partially hydrolysed nylon 6/6 tubes. The highest activity of immobilized enzyme was obtained at 65 °C and pH 6.5, while the optimum temperature for free urease was found to be 25 °C. Immobilized urease showed an improved thermal stability in comparison to free urease. It retained 76% of the original activity after 60 days when stored at 4 °C and 78% of the activity after 5 repeated uses.

Eight basidiomycetes were evaluated for their ability to decolourise a polymeric dye, Poly R-478. Decolourisation experiments were carried out in nitrogen-limited (0.25 mM) and nitrogen-rich (5.2 mM) conditions at a dye concentration of 100 mg/L. Stationary liquid cultures were inoculated with mycelium agar plugs and all fungi formed mycelium mats. The effect of nitrogen on decolourisation was also tested in shaken cultures inoculated with homogenized mycelium, where the biomass grew as mycelium beads. In static cultures, all strains apart from Polyporus sp.2 showed better decolourisation in N-rich medium. The greatest removal efficiency was achieved by the fungus Pleurotus ostreatus sp.4, attaining a maximum decolourisation of 53% in 18 days. On the other hand, in agitated cultures all strains were more efficient in decolourisation. The higher levels of removal efficiency were exhibited by Ganoderma australe in N-rich medium, obtaining decolourisation of about 93% in 18 days.

Cloning, over-expression and purification of individual enzymes. DNA manipulations were performed essentially as described by Wang and co-workers. 1 Primers for cloning galK: 5'-GAGTGTAACATATGAGTCTGAAAGAAAAAACAC-3' and 5'-CACGGATCCTCAGCACTGTCCTGCTCC-3' Primers for cloning galPUT: 5'-AGGAACGCATATGACGCAATTTAATCCCGT-3' and 5'-GACTGGATCCTTACACTCCGGATTCGC-3' The galK gene and galPUT gene were cloned from E. coli K-12 and inserted into Nde1 and BamH1 restriction sites of the pET15b vector. The resulting plasmids pET15b-galK and pET15b-galPUT, respectively, were transformed into E. coli cloning strain Novablue and then expression strain BL21 (DE3) Origami. Selected clones were characterized by restriction mapping. The expression and purification of individual enzymes from cell lysates were as described previously. 1 Enzymatic activity assay for galactokinase. 2 This radiochemical assay measures the phosphorylation of [6-3 H]-galactose; the phosphorylated product is separated from the assay mixture with the aid of an anion exchange resin. The reaction mixture (50µl) contains 10µl each of: galactose (1mM, containing 100Bq [ 3 H]-galactose-Amersham); assay buffer (50mM triethanolamine pH 7.0); ATP (1mM); co-factors (1mM DTT, 5mM MgCl 2); protein sample. Molarities quoted are the final concentrations in the assay. The reaction mixture was incubated for 20 minutes at 37 o C, diluted with Milli Q water (1ml) and applied to a column containing 0.5ml of QAE Q-25-120 Sephadex anion exchange resin. The columns were washed with Milli Q water (2ml) and the flow through was collected. The columns were then eluted with 1M NH 4 OAc (2ml) and the flow through was collected. Scintillation fluid (8ml) (Fisher High safe 8) was added to the samples collected, and the mixture was counted for 2 minutes using a liquid scintillation counter. Enzymatic activity assay for galactose-1-phosphate uridyltransferase. Galactose-1-phosphate uridyltransferase was diluted in assay buffer (50mM HEPES, 10mM

During adrenal steroidogenesis the competition between 3␤-hydroxysteroid dehydrogenase/ 5-4 isomerase (3␤HSD) and cytochrome P450 17␣-hydroxylase/17,20 lyase (CYP17A1) for 5 steroid intermediates greatly influences steroidogenic output. Cytochrome-b 5 (Cyt-b 5), a small electron transfer hemoprotein, known to augment the lyase activity of CYP17A1, has been shown to alter the steroidogenic outcome of this competition. In this study, the influence of Cyt-b 5 on 3␤HSD activity was investigated. In COS-1 cells, Cyt-b 5 was shown to significantly increase the activity of both caprine and ovine 3␤HSD towards pregnenolone, 17-OH pregnenolone and dehydroepiandrosterone in a substrate and species specific manner. Furthermore, kinetic studies revealed Cyt-b 5 to have no influence on the K m values while significantly increasing the V max values of ovine 3␤HSD for all its respective substrates. In addition, the activity of ovine 3␤HSD in microsomal preparations was significantly influenced by the addition of either purified Cyt-b 5 or anti-Cyt-b 5 IgG. The results presented in this study indicate that Cyt-b 5 augments 3␤HSD activity and represents the first documentation of such augmentation in any species.

Screening methods have been developed for detection of micro‐organisms producing thermostable dextranases. They utilize the incorporation of Blue Dextran into agar or liquid culture media for isolation of active dextranase producers growing at temperatures above 55°C. A variety of high temperature environments in sugar factories and naturally occurring thermal water samples were excellent sources of dextranase producers. A number of aerobic and anaerobic thermophilic bacteria, isolated from these sources, were found to produce thermostable dextranases. Dextranases with the greatest thermostability were found in cultures of anaerobic bacteria grown above 65°C. Temperature optima were determined for several crude enzyme preparations, four of which exhibited temperature optima in the range 65–85°C.

Liposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management. Large RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial. Screening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growt...

Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability. Hence researchers are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to t...

Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65°C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.

The adaptive significance of enzyme variation has been of central interest in population genetics. Yet, how natural selection operates on enzymes in the larger context of biochemical pathways has not been broadly explored. A basic expectation is that natural selection on metabolic phenotypes will target enzymes that control metabolic flux, but how adaptive variation is distributed among enzymes in metabolic networks is poorly understood. Here, we use population genetic methods to identify enzymes responding to adaptive selection in the pathways of central metabolism in Drosophila melanogaster and Drosophila simulans. We report polymorphism and divergence data for 17 genes that encode enzymes of 5 metabolic pathways that converge at glucose-6-phosphate (G6P). Deviations from neutral expectations were observed at five loci. Of the 10 genes that encode the enzymes of glycolysis, only aldolase (Ald) deviated from neutrality. The other 4 genes that were inconsistent with neutral evolution (glucose-6-phosphate dehydrogenase [G6pd]), phosphoglucomutase [Pgm], trehalose-6-phosphate synthetase [Tps1], and glucose-6phosphatase [G6pase] encode G6P branch point enzymes that catalyze reactions at the entry point to the pentose-phosphate, glycogenic, trehalose synthesis, and gluconeogenic pathways. We reconcile these results with population genetics theory and existing arguments on metabolic regulation and propose that the incidence of adaptive selection in this system is related to the distribution of flux control. The data suggest that adaptive evolution of G6P branch point enzymes may have special significance in metabolic adaptation.

An endoxylanase (1,4-d-xylan xylanohydrolase, EC 3.2.1.8) from the culture ®ltrates of T. lanuginosus ATCC 46882 was puri®ed to homogeneity by DEAE±Sepharose and Bio-Gel P-30 column chromatographies. The puri®ed endoxylanase had a speci®c activity of 888.8 "mol min À1 mg À1 protein and accounted for approximately 30% of the total protein secreted by this fungus. The molecular mass of native (non-denatured) and denatured endoxylanase were 26.3 and 25.7 kD as, respectively. Endoxylanase had a pI of 3.7 and was optimally active between pH 6.0±6.5 and at 75 C. The enzyme showed >50% of its original activity between pH 5.5±9.0 and at 85 C. The pH and temperature stability studies revealed that this endoxylanase was almost completely stable between pH 5.0±9.0 and up to 60 C for 5 h and at pH 10.0 up to 55 C for 5 h. Thin-layer chromatography (TLC) analysis showed that endoxylanase released mainly xylose (Xyl) and xylobiose (Xyl 2) from beechwood 4-O-methyl-d-glucuronoxylan, O-acetyl-4-Omethyl-d-glucuronoxylan and rhodymenan (a-(133)±(134)-xylan). Also, the enzyme released an acidic xylo-oligosaccharide from 4-O-methyl-d-glucuronoxylan, and an isomeric xylotetraose and an isomeric xylopentaose from rhodymenan. The enzyme hydrolysed [1-3 H]-xylo-oligosaccharides in an endofashion, but the hydrolysis of [1-3 H]-xylotriose appeared to proceed via transglycosylation, since the xylobiose was the predominant product. Endoxylanase was not active on pNPX

Alkaline endo-1,4-␤-D-glucanase was secreted by Bacillus pumilus grown in submerged culture on a combination of oat spelt xylan and corn starch as carbon sources. The enzyme was purified to homogeneity by Sephacryl S-200 and Q-Sepharose column chromatography. The protein corresponded to molecular mass and pI values of 67 kDa and 3.7, respectively. The enzyme was optimally active at pH 7.0-8.0 and 60°C and retained 50% of its optimum activity at pH 12. The most notable characteristic of the endoglucanase was its high stability up to pH 12 for 20 h at 30°C. The enzyme hydrolyzed carboxymethylcellulose (CMC) and cello-oligosaccharides but was inactive on cellobiose, cellotriose, Avicel, xylan, 4-nitrophenyl-␤-D-glucoside, 4-nitrophenyl-␤-D-cellobioside, and 4-nitrophenyl-␤-D-xyloside. Analysis of reaction mixtures by HPLC revealed that the enzyme produced almost exclusively cellotriose when acted on CMC and appeared to hydrolyze cello-oligosaccharides by successively releasing cellotriose. The use of 4-methylumbelliferyl cello-oligosaccharides and the determination of bond cleavage frequency revealed that the enzyme preferentially hydrolyzed the third glycosidic bond adjacent to the glycon. The enzyme mediated a decrease in the viscosity of CMC associated with a release of only small amounts of reducing sugar. The enzyme activity was not inhibited by metal ions, surfactants, and chelating agents used as components of laundry detergents.

INTRODUCCIÓN: En América Latina la deficiencia de glucosa 6-fosfato deshidrogenasa (d-G6PD) ha sido poco estudiada y en Colombia solo conocemos tres publicaciones antiguas. Urge conocer más la prevalencia de d-G6PD, sobre todo ahora que el tratamiento de la malaria vivax plantea aumentar la dosis diaria o total de primaquina. OBJETIVO: Medir la prevalencia de d-G6PD en poblaciones masculina sana y de enfermos con malaria por Plasmodium vivax, en Turbo (Urabá, departamento de Antioquia, Colombia). METODOLOGÍA: Encuestas de prevalencia, para evaluar la G6PD en dos poblaciones de Turbo (Antioquia): hombres sanos; hombres y mujeres con malaria vivax. Se trabajó con muestras diseñadas con criterios estadístico-epidemiológicos. La actividad enzimática se midió con el método normalizado de Beutler para valorar la G6PD en hemolizados. RESULTADOS: Entre los hombres sanos (n = 508), el intervalo de confianza 95% para el promedio (IC95%) estuvo entre 4,15 y 4,51 UI/g hemoglobina y 14,8% presen...

INTRODUCCIÓN: En América Latina la deficiencia de glucosa 6-fosfato deshidrogenasa (d-G6PD) ha sido poco estudiada y en Colombia solo conocemos tres publicaciones antiguas. Urge conocer más la prevalencia de d-G6PD, sobre todo ahora que el tratamiento de la malaria vivax plantea aumentar la dosis diaria o total de primaquina. OBJETIVO: Medir la prevalencia de d-G6PD en poblaciones masculina sana y de enfermos con malaria por Plasmodium vivax, en Turbo (Urabá, departamento de Antioquia, Colombia). METODOLOGÍA: Encuestas de prevalencia, para evaluar la G6PD en dos poblaciones de Turbo (Antioquia): hombres sanos; hombres y mujeres con malaria vivax. Se trabajó con muestras diseñadas con criterios estadístico-epidemiológicos. La actividad enzimática se midió con el método normalizado de Beutler para valorar la G6PD en hemolizados. RESULTADOS: Entre los hombres sanos (n = 508), el intervalo de confianza 95% para el promedio (IC95%) estuvo entre 4,15 y 4,51 UI/g hemoglobina y 14,8% presen...

Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability. Hence researchers are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to t...

Eighteen enzymatic loci were analysed in Aedes aegypti populations from four neighbourhoods in the city of Manaus. The analyses showed that the Downtown population was the most polymorphic (p = 55.6%) with higher observed and expected mean heterozygosities (H o = 0.152 ± 0.052; H e = 0.174 ± 0.052, respectively). The least variability was detected in the Coroado and Cidade Nova populations, both with polymorphism of 44.4%. The latter population presented the least observed heterozygosity (H o = 0.109 ± 0.037). Wright's F statistics showed that the mean value of F is was higher than that of F st (F is = 0.164 > F st = 0.048), and from analysis of molecular variance (AMOVA) it was found that 95.12% of the variability is found within populations indicating a certain intra-population differentiation possibly of the microgeographic structure resulting from some barrier in the random coupling. Although the four populations were similar genetically (D = 0.003 to 0.016), the 4.88% differentiation was significant.

Background: The relevance of regular moderate to intense exercise for ameliorating psychomotor symptoms in persons with multiple sclerosis (pwMS) is becoming increasingly evident. Over the last two decades, emerging evidence from clinical studies and animal models indicate immune regulatory mechanisms in both periphery and the central nervous system that may underlie these beneficial effects. The integrity of the blood-brain barrier as the main structural interface between periphery and brain seems to play an important role in MS. Reducing the secretion of proteolytic matrix metalloproteinases (MMP), i.e. MMP-2, as disruptors of blood-brain barrier integrity could have profound implications for MS. Methods: In this two-armed randomized controlled trial 64 participants with relapsing-remitting MS (RRMS) (EDSS 0-4.0) will be allocated to either an intervention group or a passive wait list control group. The intervention group will perform 60 min of combined functional resistance and endurance exercises 3x per week over a period of 12 weeks in a community-based and publicly available setting. Changes in serum concentration of MMP-2 will be the primary outcome. Secondary outcomes are numbers of immune cell subsets, soluble (anti-) inflammatory factors, physical capacity, cognitive performance, physical activity behavior, gait performance, and patient-reported outcomes. All outcome measures will be assessed at baseline and after week 12 with an additional blood sampling before, during and immediately after a single training session in week 6.

Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability. Hence researchers are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to t...

Zusammenfassung: Ziel der Untersuchung war, Serumparametervon klinisch unauffälligen Gazellen der Al Wabra Wildlife Preservation (AWWP), Qatar, mit Referenzbereichen von Haus- und anderen Wildwiederkäuern zu vergleichen, um einerseits Rückschlüsse auf den Ernährungszustand der Tiere zu ermöglichen und andererseits Referenzbereiche für die untersuchten Arten zu etablieren. Material und Methoden: Von 250 klinisch gesunden Tieren der Arten Sömmeringgazelle (Gazella soemmerringii), Spekegazelle (Gazella spekei), Dor-kasgazelle (Gazella dorcas), Saudigazelle (Gazella saudiya), Echtgazelle (Gazella gazella), Arabische Kropfgazelle (Gazella subgutturosa marica) und Chinkaragazelle (Gazella benetti) wurden biochemische Serumparameter und Mineralgehalte gemessen. Ergebnisse: Hinsichtlich des Ernährungszustandes war die Versorgung mit Spurenelementen (Selen, Kupfer, Zink, Eisen) in der AWWP gemäß den gemessenen Serumgehalten gewährleistet. Die Gehalte an Phosphor, Gesamtprotein und Albumin de...

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is an autosomal recessive lysosomal storage disorder resulting from a deficiency of N-acetylgalactosamine-6sulfate sulfatase (GALNS) activity. Diagnosis can be challenging and requires agreement of clinical, radiographic, and laboratory findings. A group of biochemical genetics laboratory directors and clinicians involved in the diagnosis of MPS IVA, convened by BioMarin Pharmaceutical Inc., met to develop recommendations for diagnosis. The following conclusions were reached. Due to the wide variation and subtleties of radiographic findings, imaging of multiple body regions is recommended. Urinary glycosaminoglycan analysis is particularly problematic for MPS IVA and it is strongly recommended to proceed to enzyme activity testing even if urine appears Communicated by: Maurizio Scarpa

Urease (EC 3.5.1.5) was covalently attached through glutaraldehyde to partially hydrolysed nylon 6/6 tubes. The highest activity of immobilized enzyme was obtained at 65°C and pH 6.5, while the optimum temperature for free urease was found to be 25°C. Immobilized urease showed an improved thermal stability in comparison to free urease. It retained 76% of the original activity after 60 days when stored at 4°C and 78% of the activity after 5 repeated uses.

http://link.springer.com/article/10.1007/s004490050381