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Enzymatic Analysis (enzymatic + analysis)
Selected AbstractsStructural and functional characterization of a putative polysaccharide deacetylase of the human parasite Encephalitozoon cuniculiPROTEIN SCIENCE, Issue 6 2009Jonathan E. Urch Abstract The microsporidian Encephalitozoon cuniculi is an intracellular eukaryotic parasite considered to be an emerging opportunistic human pathogen. The infectious stage of this parasite is a unicellular spore that is surrounded by a chitin containing endospore layer and an external proteinaceous exospore. A putative chitin deacetylase (ECU11_0510) localizes to the interface between the plasma membrane and the endospore. Chitin deacetylases are family 4 carbohydrate esterases in the CAZY classification, and several bacterial members of this family are involved in evading lysis by host glycosidases, through partial de- N -acetylation of cell wall peptidoglycan. Similarly, ECU11_0510 could be important for E. cuniculi survival in the host, by protecting the chitin layer from hydrolysis by human chitinases. Here, we describe the biochemical, structural, and glycan binding properties of the protein. Enzymatic analyses showed that the putative deacetylase is unable to deacetylate chitooligosaccharides or crystalline ,-chitin. Furthermore, carbohydrate microarray analysis revealed that the protein bound neither chitooligosaccharides nor any of a wide range of other glycans or chitin. The high resolution crystal structure revealed dramatic rearrangements in the positions of catalytic and substrate binding residues, which explain the loss of deacetylase activity, adding to the unusual structural plasticity observed in other members of this esterase family. Thus, it appears that the ECU11_0510 protein is not a carbohydrate deacetylase and may fulfill an as yet undiscovered role in the E. cuniculi parasite. [source] A rapid and inexpensive microplate assay for the enzymatic determination of glucose, fructose, sucrose, L -malate and citrate in tomato (Lycopersicon esculentum) extracts and in orange juicePHYTOCHEMICAL ANALYSIS, Issue 5 2001Joyce S. Velterop Abstract Sugars and organic acids are important determinants of taste in fruits and vegetables. Based on the enzymatic analyses originally developed by Boehringer-Mannheim, rapid and reliable microplate-based assays were devised for glucose, fructose and sucrose in tomato and orange juice, and citrate and L -malate in tomato. These microplate assays were used to compare some commercially available tomato types, and to study the compartmentation of sugars and acids between pericarp and locular tissue of tomato fruit. The microplate format allows analysis of many samples per day and the only apparatus required is a microplate reader. Copyright © 2001 John Wiley & Sons, Ltd. [source] Investigation of lysosomal storage diseases in nonimmune hydrops fetalisPRENATAL DIAGNOSIS, Issue 8 2004Maira G. Burin Abstract Objective To investigate lysosomal storage diseases (LSD) in cases of nonimmune hydrops fetalis (NIHF). Methods Thirty-three cases of NIHF were investigated, 28 in the prenatal period and 5 in hydropic newborns. In addition to a general investigation for NIHF, specific enzymatic analyses for the detection of LSD were performed. Results In our sample, we detected five patients (15%) with LSD, each patient having one of the following diseases: mucolipidosis, Niemann,Pick disease, galactosialidosis, sialidosis and mucopolysaccharidosis type IV A. Conclusion Although LSDs are rare disorders as a group, they should be considered as a possible cause of NIHF, even in the absence of consanguinity or of a previous family history. By excluding the more frequent causes of NIHF, an LSD investigation assists in clarifying the etiology of many hydropic cases, making more appropriate genetic counseling for parents possible. Copyright © 2004 John Wiley & Sons, Ltd. [source] Different adaptations of alpha-actinin isoforms to exercise training in rat skeletal musclesACTA PHYSIOLOGICA, Issue 3 2009Y. Ogura Abstract Aim:, Alpha (,)-actinins are located in the skeletal muscle Z-line and form actin,actin cross-links. Mammalian skeletal muscle has two isoforms: ,-actinin-2 and ,-actinin-3. However, the response of ,-actinin to exercise training is little understood. Therefore, the current study examined the effects of exercise training on the expression level of two ,-actinin isoforms in skeletal muscles. Methods:, Twelve male Wistar rats were assigned randomly to a control (C; n = 6) or exercise training (T; n = 6) group. After T animals were trained on an animal treadmill for 9 weeks, ,-actinin-2 and ,-actinin-3 levels in the plantaris, white and red gastrocnemius muscles were analysed. In addition, changes in the myosin heavy chain (MyHC) composition were assessed, and muscle bioenergetic enzyme activities were measured. Results:, Results show that exercise training increased ,-actinin-2 expression levels in all muscles (P < 0.05). However, no significant difference was found in ,-actinin-3 expression levels between C and T animals. Subsequent MyHC analyses of all muscle showed an MyHC shift with direction from IIb to IIa. Furthermore, enzymatic analysis revealed that exercise training improved enzyme activities related to aerobic metabolism. Conclusion:, The results of this study demonstrate that exercise training alters the expression level of ,-actinin at the isoform level. Moreover, the increase in expression levels of ,-actinin-2 is apparently related to alteration of skeletal muscle: its aerobic capacity is improved. [source] Decreased activities of mitochondrial respiratory chain complexes in non-mitochondrial respiratory chain diseasesDEVELOPMENTAL MEDICINE & CHILD NEUROLOGY, Issue 2 2006Joannie Hui MBBS The aim of this study was to illustrate the difficulties in establishing a diagnosis of mitochondrial respiratory chain (MRC) disorders based on clinical grounds in combination with intermediate activities of the MRC enzyme complexes. We reviewed retrospectively all medical and laboratory records of patients initially considered likely to have MRC disorders on clinical grounds, and subsequently diagnosed with other disorders (n=20; 11 males, 9 females). Data were retrieved from hospital records, referral letters, and results of enzymatic analysis at a reference laboratory. Clinical symptoms included developmental delay, epilepsy, hypotonia, movement disorder, spastic quadriplegia, tetany, microcephaly, visual problems, carpopedal spasms, dysmorphism, hearing loss, muscle weakness and rhabdomyolysis, and fulminant hepatitis. Blood and cerebrospinal fluid lactate levels were elevated in 13/20 and 9/20 respectively. One or more MRC complex activities (expressed as ratios relative to citrate synthase and/or complex II activity) were less than 50% of control mean activity in 11/20 patients (including patients with deficiencies of pyruvate dehydrogenase complex, pantothenate kinase, holocarboxylase synthetase, long-chain hydroxy acyl-CoA dehydrogenase, molybdenum co-factor, and neonatal haemochromatosis). One patient had a pattern suggestive of mitochondrial proliferation. We conclude that intermediate results of MRC enzymes should be interpreted with caution and clinicians should be actively looking for other underlying diagnoses. [source] The domains carrying the opposing activities in adenylyltransferase are separated by a central regulatory domainFEBS JOURNAL, Issue 11 2007Paula Clancy Adenylyltransferase is a bifunctional enzyme that controls the enzymatic activity of dodecameric glutamine synthetase in Escherichia coli by reversible adenylylation and deadenylylation. Previous studies showed that the two similar but chemically distinct reactions are carried out by separate domains within adenylyltransferase. The N-terminal domain carries the deadenylylation activity, and the C-terminal domain carries the adenylylation activity [Jaggi R, van Heeswijk WC, Westerhoff HV, Ollis DL & Vasudevan SG (1997) EMBO J16, 5562,5571]. In this study, we further map the domain junctions of adenylyltransferase on the basis of solubility and enzymatic analysis of truncation constructs, and show for the first time that adenylyltransferase has three domains: the two activity domains and a central, probably regulatory (R), domain connected by interdomain Q-linkers (N-Q1-R-Q2-C). The various constructs, which have the opposing domain and or central domain removed, all retain their activity in the absence of their respective nitrogen status indicator, i.e. PII or PII-UMP. A panel of mAbs to adenylyltransferase was used to demonstrate that the cellular nitrogen status indicators, PII and PII-UMP, probably bind in the central regulatory domain to stimulate the adenylylation and deadenylylation reactions, respectively. In the light of these results, intramolecular signaling within adenylyltransferase is discussed. [source] Digestive enzyme activity at different developmental stages of blackspot seabream, Pagellus bogaraveo (Brunnich 1768)AQUACULTURE RESEARCH, Issue 4 2008Laura Ribeiro Abstract Blackspot seabream, Pagellus bogaraveo (Brunnich), has been identified as a potential species to diversify European aquaculture production. Although rearing aspects have been widely investigated, little information exists on the nutritional requirements for this species. The aim of this study was to build up information on the activity of digestive enzymes at certain developmental stages of blackspot seabream in order to understand the nutritional needs of larvae and post larvae. Fish larvae were reared from hatching to 55 days after hatching (dah), and the feeding plan consisted in rotifers (5,35 dah), Artemia naupli (30,35 dah) metanaupli (35,45) and Gemma microdiet (45,55 dah). At 7, 11, 21, 45 and 55 days after hatching (dah), pooled samples of fish larvae were collected for analysis of trypsin, amylase, lipase, alkaline phosphatase and leucine,alanine peptidase activity. Up to 21 dah, the whole larvae body was used for enzymatic analysis, whereas in older larvae only the dissected abdominal cavity was used. Blackspot seabream body dry weight growth was exponential, increasing from 60 ,g at 5 dah to 30±9.7 mg at 55 dah. Amylase specific activity decreased significantly during development, exhibiting at 11 dah (0.6 U mg,1 protein) an average value 2.7 times lower than at 7 dah, and remaining stable between 45 and 55 dah (0.7 U mg protein,1). Trypsin specific activity remained constant until 21 dah (between 38 and 44 mU mg protein,1), which could be related to the larvae feeding regime. At later stages of development, lipase-specific activity exhibited a significant increase (P<0.05), being three times higher at 55 dah (8 U mg protein,1) than at 45 dah. The total activity of the studied digestive enzymes increased significantly during larval development (until 21 dah), whereas afterwards only lipase and leucine,alanine peptidase increased significantly between 45 and 55 dah. The pattern of digestive enzymes activity was related to organogenesis and the type of food used at different developmental stages. [source] Genetic relationship between Litopenaeus setiferus (L.) and L. schmitti (Burkenroad) determined by using 16S mitochondrial sequences and enzymatic analysisAQUACULTURE RESEARCH, Issue 12 2003L Arena Abstract Genetic differentiation and variability data of two populations of two species of shrimp (Litopenaeus setiferus (L.) and L. schmitti (Burkenroad)) have been obtained by electrophoretic analysis and by analysis of 16S mitochondrial DNA. Using eight polymorphic enzymes, the genetic distance (GD) between the two species was 0.165. The GD between L. setiferus populations was 0.0057 and between L. schmitti populations it was 0.0034. The greatest differentiation was found within, rather than between, populations, although the differentiation value between Mexican and Cuban populations varied in accordance with the geographic distance and ecological characteristic of each. We found a high similarity between these two species with a bimodal distribution of the loci with respect to genetic identity. The homology percentages for gene 16S fragments were compared with those from six different shrimp species (L. vannamei, L. stylirostris, Farfantepenaeus notialis, Metapeneopsis lamellata) and Artemia salina. Ninety-seven percent of identity was found by analysis of a 409 bp of 16S mitochondrial DNA. With these values a phylogenetic tree was made using parsimony criteria. The GDs obtained with this method confirm the classification proposed by Pérez-Farfante & Kensley (1997). [source] The importance of starch and sucrose digestion in nutritive biology of synanthropic acaridid mites: ,-Amylases and ,-glucosidases are suitable targets for inhibitor-based strategies of mite control,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2009Tomas Erban Abstract The adaptation of nine species of mites that infest stored products for starch utilization was tested by (1) enzymatic analysis using feces and whole mite extracts, (2) biotests, and (3) inhibition experiments. Acarus siro, Aleuroglyphus ovatus, and Tyroborus lini were associated with the starch-type substrates and maltose, with higher enzymatic activities observed in whole mite extracts. Lepidoglyphus destructor was associated with the same substrates but had higher activities in feces. Dermatophagoides farinae, Chortoglyphus arcuatus, and Caloglyphus redickorzevi were associated with sucrose. Tyrophagus putrescentiae and Carpoglyphus lactis had low or intermediate enzymatic activity on the tested substrates. Biotests on starch additive diets showed accelerated growth of species associated with the starch-type substrates. The inhibitor acarbose suppressed starch hydrolysis and growth of the mites. We suggest that the species with higher starch hydrolytic activity in feces were more tolerant to acarbose, and ,-amylase and ,-glucosidase of synanthropic mites are suitable targets for inhibitor-based strategies of mite control. © 2009 Wiley Periodicals, Inc. [source] | |||||||||||||