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Enzymatic
Terms modified by Enzymatic Selected AbstractsSanfilippo B in an elderly female psychiatric patient: a rare but relevant diagnosis in presenile dementiaACTA PSYCHIATRICA SCANDINAVICA, Issue 2 2010W. M. A. Verhoeven Verhoeven WMA, Csepán R, Marcelis CLM, Lefeber DJ, Egger JIM, Tuinier S. Sanfilippo B in an elderly female psychiatric patient: a rare but relevant diagnosis in presenile dementia. Objective:, Sanfilippo B is a rare autosomal recessive mucopolysaccharidosis (MPS IIIB) caused by a deficiency of N -acetyl-,-D-glucosaminidase (NAGLU). Method:, A mild mentally retarded elderly female patient is described with a slowly progressive dementia who had given birth to a daughter who developed normally. Results:, Metabolic screening revealed an enhanced concentration of heparan sulfate in urine. Enzymatic assay demonstrated deficiency of N -acetyl-,-D-glucosaminidase. Mutations in the NAGLU gene were found. One mentally retarded and hospitalized elder brother was also found to have MPS IIIB, whereas a second brother, who had died earlier, is suspected to have had the same metabolic disorder. Prior to the development of dementia, both the patient and her brother showed autistic like features, signs of ideomotor apraxia and weakness in verbal comprehension. Conclusion:, Screening for metabolic disorders, in particular MPSes, should always be considered in patients with a history of mental deficit and dementia or progressive functional decline. [source] Diastereoselective Synthesis of -Hydroxy Sulfoxides: Enzymatic and Biomimetic ApproachesEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 2 2007Stefano Colonna Abstract Stereoselectivities of up to 98,% have been found in the enzymatic synthesis of ,-hydroxy sulfoxides catalyzed by cyclohexanone monooxygenase (CHMO). The diastereoselectivity of the "one-pot" preparation of the title compounds in the presence of bovine serum albumin has also been investigated. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] Novel Model Sulfur Compounds as Mechanistic Probes for Enzymatic and Biomimetic OxidationsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 1 2005Alicia B. Peñéñory Abstract To test for the intermediacy of sulfide radical cations in biomimetic and enzymatic oxidations, the sulfides PhSCH3 (1a), PhSCH2Ph (1b), PhSCHPh2 (1c), PhSCPh3 (1d), CH3SCHPh2 (2), PhSCH2CH=CH2 (3), PhSCH2CH=CHPh (4) and CH3SCH2CH=CHPh (5) were studied, and their results were compared to those obtained for the corresponding chemical electron transfer (CET) and photoinduced electron transfer (PET) oxidations. The radical cations generated from 3,5 by CET in the presence of cerium(IV) ammonium nitrate (CAN) yielded only fragmentation products from the alkyl cations and the thiyl radicals (RS·), whereas 2·+ afforded both fragmentation and mainly ,-deprotonation products. Photochemical treatment of the sulfides 1a and 1b with C(NO2)4 gave only the corresponding sulfoxides, while fragmentation was the main pathway for the photoreactions of 1c, 2 and 5, and for 1d only this latter process was observed. These results support our selection of the sulfides RSCHPh2, RSCH2CH=CHPh (R = Me, Ph) and PhSCPh3 as models for the biomimetic and enzymatic studies. As evidenced by the sulfoxides and sulfones detected as unique products both in protic and in aprotic solvents, it is proposed that the mechanism of the biomimetic sulfoxidations of sulfides 1c and 2,5 by TPPFeIIICl is direct oxygen transfer. Three enzymes , Coprinus cinereus peroxidase (CiP), horseradish peroxidase (HRP) and chloroperoxidase (CPO) , were studied in the oxidation of sulfides 1a, 2, 4 and 5. The use of a racemic alkyl hydroperoxide in the CiP enzymatic oxidation of sulfides 5 and 2 yielded the corresponding sulfoxides (23 and 29%) and the aldehyde or benzophenone (5%), respectively. These results suggest the involvement of an ET process for the CiP-catalysed oxidation. Fragmentation products were observed in the enzymatic oxidation of sulfide 4 with HRP, which confirms the previously proposed ET mechanism. On the other hand, the CPO-enzymatic oxidation of sulfide 5 yielded only the corresponding sulfoxide, as would be expected for a direct oxygen-transfer or oxene mechanism. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005) [source] Effective High-Pressure Cleavage of Sterically Hindered Steroid EstersHELVETICA CHIMICA ACTA, Issue 6 2004Wojciech Kroszczy A simple and effective method to deprotect of sterically hindered steroid esters is described. Deprotection was carried out in MeOH in the presence of a catalytic amount of Et3N under high-pressure conditions. Enzymatic, anionite, and high-pressure methods are compared. [source] Enzymatic and immunochemical evaluation of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in testes and epididymal spermatozoa of rats of different agesINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 2 2002Federica Tramer Selenium (Se) and selenoproteins such as glutathione peroxidases are necessary for the proper development and fertilizing capacity of sperm cells. Phospholipid hydroperoxide glutathione peroxidase (PHGPx, E.C. 1.11.1.12) is a monomeric seleno-enzyme present in different mammalian tissues in soluble and bound form. Its function, like the other glutathione peroxidases, was originally viewed as a protective role against hydroperoxides, but direct and indirect evidence indicates that it has additional regulatory roles. PHGPx is present in testis cells and sperm cells, and its appearance is hormone regulated. We present here biochemical data, which clearly indicate that the enzyme specific activity in rat is age-dependent during the life-span monitored (from 36 to 365 days), with a maximum at 3 months of age in the testis germ cells and at 6 months of age in the isolated epididymal sperm cells. Western blotting and immunocytochemical analysis by means of anti-PHGPx antibodies show the different distribution and the strong binding of PHGPx in the testes and sperm cell subcellular compartments (nucleus, acrosome, mitochondria and residual bodies) of rats of different age. The presence of the protein exhibits in the testis cells a pattern different from that of the catalytic activity, with a maximum at 6 months of age. The subcellular distribution of PHGPx is qualitatively, but not quantitatively, unchanged during ageing. These different behaviours are compared and discussed. [source] Analysis of exotoxins produced by atypical isolates of Aeromonas salmonicida, by enzymatic and serological methodsJOURNAL OF FISH DISEASES, Issue 1 2003B K Gudmundsdóttir Abstract In this study, exotoxins produced by 62 Aeromonas salmonicida strains and the bacterium Haemophilus piscium were analysed. Enzymatic assays, zymograms and serological detection were used to monitor secretion by bacterial strains of the previously described exotoxins P1, GCAT and AsaP1 and also the extracellular P2 metallo-gelatinase and a serine caseinase, which is different from the P1 protease and has not yet been characterized. Based on the results, the strains were divided into five groups. One comprised the type strains for A. salmonicida ssp. masoucida, H. piscium and 36% of the atypical isolates, and another, a type strain for A. salmonicida ssp. smithia together with 14% of the atypical isolates. A second type strain of A. salmonicida ssp. smithia was grouped with 8% of the atypical isolates. The largest group contained the type strains for A. salmonicida ssp. achromogenes and 38% of the atypical isolates. The type strains for A. salmonicida ssp. salmonicida were in the last group with all the four typical strains and 4% of the atypical isolates. The combination of zymogram and serological detection used is recommended as the most reliable method for characterizing A. salmonicida strains according to their exotoxin secretion. [source] Evaluation of Bitterness in Enzymatic Hydrolysates of Soy Protein Isolate by Taste Dilution AnalysisJOURNAL OF FOOD SCIENCE, Issue 1 2008W.H. Seo ABSTRACT:, Although enzymatic hydrolysates of soy protein isolate (SPI) have physiological functionality, partially hydrolyzed SPI exhibits bitter taste depending on proteases and degree of hydrolysis (DH). To determine proteolysis conditions for SPI, it is important to evaluate bitterness during enzymatic hydrolysis. Taste dilution analysis (TDA) has been developed for the screening technique of taste-active compounds in foods. The objectives of the present study were to evaluate bitterness of enzyme-hydrolyzed SPI by TDA and to compare bitterness of SPI hydrolysates with respect to kinds of proteases and DH. SPI was hydrolyzed at 50 °C and pH 6.8 to 7.1 to obtain various DH with commercial proteases (flavourzyme, alcalase, neutrase, protamex, papain, and bromelain) at E/S ratios of 0.5%, 1%, and 2%. The DH of enzymatic hydrolysates was measured by trinitrobenzenesulfonic acid method. The bitterness of enzymatic hydrolysates was evaluated by TDA, which is based on threshold detection in serially diluted samples. Taste dilution (TD) factor was defined as the dilution at which a taste difference between the diluted sample and 2 blanks could be detected. As DH increased, the bitterness increased for all proteases evaluated. Alcalase showed the highest TD factor at the same DH, followed by neutrase. Flavourzyme showed the lowest TD factor at the entire DH ranges. At the DH of 10%, TD factor of hydrolysate by flavourzyme was 0 whereas those by protamex and alcalase were 4 and 16, respectively. These results suggest that TDA could be applied for the alternative of bitterness evaluation to the hedonic scale sensory evaluation. [source] Lecithin Associated Off-Aromas in Fermented MilkJOURNAL OF FOOD SCIENCE, Issue 4 2001O. Suriyaphan ABSTRACT Soy, rice and hydrogenated soy lecithins were added to milk with lactic acid fermentation to characterize and elucidate formation of volatile off-aromas. Sensory panelists detected off-aromas in fermented milk containing unmodified soy or rice lecithin. Instrumental aroma analysis revealed that off-flavor compounds included (E,E)-2,4,nonadienal and (E,Z)- and (E,E)-2,4,decadienal. Formation of 2,4,decadienals occurred within the first 4 h of lactic acid fermentation and reached maximum levels within 14 h of incubation. Enzymatic assays confirmed that washed cells of Lactococcus produced H2O2. Hydrogenated soy lecithin was suitable to use in cultured dairy products, but use of other soy or rice lecithin resulted in off-flavor formation due to oxidation of polyunsaturated fatty acids. [source] Synthesis of Polycaprolactone Using Free/Supported Enzymatic and Non-Enzymatic CatalystsMACROMOLECULAR RAPID COMMUNICATIONS, Issue 24 2004María Laura Foresti Abstract Summary: Polymerization of caprolactone using lipases from Candida antarctica B, Rhizomucor meihei, Candida rugosa, and Pseudomonas fluorescens is highly effective, with 97% conversion into polycaprolactone. Poly(propylene)-supported Candida rugosa lipase achieves higher conversion values (85,92%) than free lipase (75%). Acidic and basic non-conventional catalysis with butanol yields 50,85% conversion. Simple UV/visible techniques gave the same results for measuring conversion than other studies. Applications are opened for the non-conventional catalysts. Mechanism of the polymerization of caprolactone polymerization using a basic catalyst. [source] Examination of the dehiscence zone in soybean pods and isolation of a dehiscence-related endopolygalacturonase genePLANT CELL & ENVIRONMENT, Issue 4 2002L. C. Christiansen Abstract Microscopic examination of cross sections of dorsal and ventral sutures of soybean pods (Glycine max cv. TGx1835-2E) at two different stages of maturity revealed that the dehiscence zone of soybean pods is functionally equivalent to the dehiscence zone known from crucifers. Enzymatic assays demonstrated the presence of endo-1,4- , -glucanases and endopolygalacturonases, the activity of which accumulated in the dehiscence zone and peaked during maturation. A single partial cDNA encoding an endopolygalacturonase was isolated by polymerase chain reaction and this clone was used to isolate the complete gene encoding the endopolygalacturonase in question. Approximately 1·2 kb of 5, upstream sequence was cloned in the plant transformation vector pCAMBIA1301 in front of the uidA (GUS) gene and transformed into Arabidopsis thaliana. Expression analysis of the soybean endopolygalacturonase transcript revealed that the endopolygalacturonase is primarily found in dehiscence-related tissue and is presumably involved in the breakdown of the middle lamella prior to dehiscence. This result was corroborated by GUS stainings of the transgenic Arabidopsis lines [source] Enzymatic and structural analysis of the I47A mutation contributing to the reduced susceptibility to HIV protease inhibitor lopinavirPROTEIN SCIENCE, Issue 9 2008Klára Grantz, ková Abstract Lopinavir (LPV) is a second-generation HIV protease inhibitor (PI) designed to overcome resistance development in patients undergoing long-term antiviral therapy. The mutation of isoleucine at position 47 of the HIV protease (PR) to alanine is associated with a high level of resistance to LPV. In this study, we show that recombinant PR containing a single I47A substitution has the inhibition constant (Ki) value for lopinavir by two orders of magnitude higher than for the wild-type PR. The addition of the I47A substitution to the background of a multiply mutated PR species from an AIDS patient showed a three-order-of-magnitude increase in Ki in vitro relative to the patient PR without the I47A mutation. The crystal structure of I47A PR in complex with LPV showed the loss of van der Waals interactions in the S2/S2, subsites. This is caused by the loss of three side-chain methyl groups due to the I47A substitution and by structural changes in the A47 main chain that lead to structural changes in the flap antiparallel ,-strand. Furthermore, we analyzed possible interaction of the I47A mutation with secondary mutations V32I and I54V. We show that both mutations in combination with I47A synergistically increase the relative resistance to LPV in vitro. The crystal structure of the I47A/I54V PR double mutant in complex with LPV shows that the I54V mutation leads to a compaction of the flap, and molecular modeling suggests that the introduction of the I54V mutation indirectly affects the strain of the bound inhibitor in the PR binding cleft. [source] Nitroxide tempo, a small molecule, induces apoptosis in prostate carcinoma cells and suppresses tumor growth in athymic miceCANCER, Issue 6 2005Simeng Suy Ph.D. Abstract BACKGROUND In previous studies, nitroxide tempo (2, 2, 6, 6-tetramethyl-piperidine-1-oxyl), a small molecule, induced cell death in cancer cells. The current study examined the antineoplastic properties of tempo in the human hormone-dependent/hormone-independent prostate carcinoma models (LNCaP, DU-145, and PC-3). METHODS The apoptotic effects of tempo were examined by the flow cytometric analysis of cells labeled with fluorescein isothiocyanate-conjugated annexin-V, and by electron microscopy. Enzymatic assays were performed to measure the activities of 2 cysteine proteases, i.e., caspase-9 and caspase-3, in tempo-treated cells. The effects of tempo on cell proliferation and on cell cycle distribution profiles were measured by the flow cytometric assay using immunofluorescent staining of incorporated 5'-bromo-2'-deoxyuridine (BrdU) coupled with 7-amino-actinomycin D (7-AAD) staining of total DNA. The number of proliferating cells was also determined independently by enzyme-linked immunosorbent assay using chemiluminescent detection of incorporated BrdU. Subcutaneous growth of human prostate carcinoma in athymic mice was monitored after intratumoral administration of tempo into tumor-bearing mice. In addition, cell viability assays were performed to compare the cytotoxic effect of a combination of doxorubicin or mitoxantrone and tempo with single agents. RESULTS Tempo treatment of prostate carcinoma cells caused a significant increase in the number of apoptotic cells compared with control groups (tempo, 2.5 mM, 24 hours: DU-145, approximately 3.4-fold; PC-3, approximately 6,7-fold; tempo 1 mM, 24 hours: LNCaP, approximately 12-fold). Tempo-induced loss of cell viability was blocked partially or completely after pretreatment of cells with actinomycin-D or cycloheximide, suggesting a de novo macromolecule synthesis-dependent mechanism of cell death. Electron microscopy revealed aggregation and marginalization of chromatin in the nuclei of a large number of tempo-treated LNCaP cells. Tempo treatment of LNCaP cells resulted in enhanced activities of caspase-9 (tempo, 5 mM, 15 hours: approximately 2-fold) and caspase-3 (tempo, 2.5 mM, 24 hours: approximately 12-fold). Tempo treatment also led to an enhanced number of cells in G2/M phase of the cell cycle (tempo, 5.0 mM, 24 hours: DU-145, approximately 1.6-fold; PC-3, approximately 1.5-fold; LNCaP, approximately 5.3-fold), and decreased BrdU incorporation indicative of a decline in the number of proliferating cells (tempo, 2.5 mM, 24 or 48 hours; DU-145, approximately 2,3-fold; PC-3, approximately 1.2-fold; LNCaP, approximately 5,10-fold). Administration of tempo into LNCaP tumor-bearing mice resulted in a significant inhibition of tumor growth (percent initial tumor volume [Day 30, n = 4]: vehicle, 845.35 ± 272.83; tempo, 9.72 ± 9.72; tempo vs. vehicle, P < 0.02). In hormone-refractory prostate carcinoma cells, a combination of relatively low doses of tempo and doxorubicin or mitoxantrone caused enhanced cytotoxicity as compared with single agents. CONCLUSIONS These data demonstrated that nitroxide tempo induced apoptosis and activated a caspase-mediated signaling pathway in prostate carcinoma cells. Tempo treatment also caused cell cycle arrest in G2/M phase and decreased the number of proliferating cells (S phase). Tempo treatment of tumor-bearing mice led to inhibition of tumor growth, suggesting that tempo is a novel member of the small-molecule family of antineoplastic agents. Cancer 2005. © 2005 American Cancer Society. [source] Realization of the Synthesis of ,,,-Disubstituted Carbamylacetates and Cyanoacetates by Either Enzymatic or Chemical Functional Group Transformation, Depending upon the Substrate Specificity of Rhodococcus Amidase.CHEMINFORM, Issue 9 2005Masahiro Yokoyama Abstract For Abstract see ChemInform Abstract in Full Text. [source] Changes in expression and activity levels of ecto-5,-nucleotidase/CD73 along the mouse female estrous cycleACTA PHYSIOLOGICA, Issue 2 2010E. Aliagas Abstract Aim:, Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, hormone synthesis and maintenance of fluid composition. Moreover, adenosine is a key molecule for sperm capacitation. Extracellular nucleotide and nucleoside levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family is the most abundant and effective to hydrolyse ATP and ADP to AMP. In the female reproductive tract three members of this family have been recently identified: NTPDase1, NTPDase2 and NTPDase3 (Histochem. Cell Biol.131, 2009, 615). The purpose of the present study was to characterize in this system the expression profile of ecto-5,-nucleotidase (CD73), the enzyme generating adenosine from AMP. Methods:, Immunological techniques and in situ enzymatic assays were used to characterize the ecto-5,-nucleotidase expression in the mouse female reproductive tract along the four stages of the estrous cycle, that were determined by vaginal smear examination. Results:, Ecto-5,-nucleotidase was abundantly detected in the corpora lutea of the ovaries, as well as in several epithelia, such as that of oviducts, uterus and endometrial glands. Marked changes in endometrial ecto-5,-nucleotidase expression and activity along the estrous cycle are described, these being maximum at estrus phase, coinciding with optimal female sexual receptivity. Conclusion:, The adenosine generated thereby, besides other functions, might contribute to sperm capacitation, thus significantly influencing fertility. [source] Recent developments and applications of EMMA in enzymatic and derivatization reactionsELECTROPHORESIS, Issue 1 2010Jie Zhang Abstract This review covers the time period of 2007 until mid-2009 and describes new developments in the field of electrophoretically mediated microanalysis. The review is subdivided in two parts dealing with (i) enzymatic and (ii) derivatization or chemical reactions. A compilation of the relevant literature is given for each part. [source] Electroenzymatic Synthesis of Chiral SulfoxidesENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 2 2006C. Kohlmann Abstract Chloroperoxidase (CPO) from Caldariomyces fumago (E.C.,1.11.1.10) is able to enantioselectively oxidize various sulfides to the corresponding (R)-enantiomer of the sulfoxides. For these oxidations the enzyme requires an oxidant. Most commonly, tert -butyl hydroperoxide (TBHP) and hydrogen peroxide are used. As it is known that these oxidants inactivate the enzyme, the enzymatic reaction was combined with the electrochemical in situ generation of hydrogen peroxide. As substrates for this combination of an enzymatic and an electrochemical reaction methyl p-tolyl sulfide, 1-methoxy-4-(methylthio)benzene and N-MOC- L -methionine methyl ester were used to carry out batch experiments. [source] Transcript and activity levels of different Pleurotus ostreatus peroxidases are differentially affected by Mn2+ENVIRONMENTAL MICROBIOLOGY, Issue 5 2001Roni Cohen The white-rot fungus Pleurotus ostreatus produces both manganese-dependent peroxidase (MnP) and versatile peroxidase (VP) in non-manganese-amended peptone medium (PM). We studied the effect of Mn2+ supplementation on MnPs and VPs in P. ostreatus by analysing the enzymatic and transcript abundance profiles of the peroxidases, as well as the lignin mineralization rate. The fungus was grown in PM under solid-state conditions using perlite as an inert solid support. Mn2+ amendment resulted in a 1.7-fold increase in [14C]-lignin mineralization relative to unamended medium. Anion-exchange chromatography was used to resolve the fungal peroxidase's enzymatic activity profile. Five peaks (P1,P5) of VP and one peak (P6) of MnP activity were detected in unamended medium. In Mn2+ -amended medium, a reduction in the activity of the VPs was observed. On the other hand, a sharp increase in the MnP activity level of peak P6 was detected. The P6 isoenzyme was purified and showed manganese-dependent peroxidation of phenolic substrates. Internal sequence analysis of the purified enzyme revealed 100% identity with the deduced amino acid sequence of P. ostreatus MnP3 (GenBank AB016519). The effect of Mn2+ on the relative abundance of gene transcripts of three VPs and one MnP from P. ostreatus was monitored using reverse transcription,polymerase chain reaction (RT,PCR) with oligonucleotide primer sets synthesized on the basis of non-conserved sequences of the different peroxidases. The reduction in VP gene transcript abundance and the increase in mnp3 transcript level were collinear with the changes observed in the enzyme activity profiles. These results indicate that the activity of peroxidases is regulated at the transcriptional level. We suggest that the expression of MnP and VP may be differentially regulated by the presence of Mn2+. [source] Attenuating effects of natural organic matter on microcystin toxicity in zebra fish (Danio rerio) embryos,benefits and costs of microcystin detoxicationENVIRONMENTAL TOXICOLOGY, Issue 1 2006Jimena Cazenave Abstract To contribute to the understanding of joined factors in the environment, impact of pure microcystins (-RR and -LF) on zebra fish (Danio rerio) embryos were investigated individually and in combination with a natural organic matter (NOM). The applied NOM was a reverse osmosis isolate from Lake Schwarzer See (i.e., Black Lake, BL-NOM). Teratogenic effects were evaluated through changes in embryonic development within 48 h of exposure. Detoxication activities were assessed by the activities of phase II biotransformation enzymes, soluble and microsomal glutathione S -transferase (s, mGST). Oxidative stress was assessed by determining both the production of hydrogen peroxide and by analyzing the activities of the antioxidative enzymes, guajacol peroxidase (POD), catalase (CAT), glutathione peroxidase (GPx), and the glutathione restoring enzyme glutathione reductase (GR). Energetic costs were evaluated by determining contents of fat, carbohydrates, and proteins in both exposed and control embryos. BL-NOM attenuated toxic effects of MC-LF and MC-RR verified by less pronounced teratological effects within 24 h, in particular, as well as less rise in the activity of s-GST, when compared with embryos exposed to either pure toxins or in combination with organic matter. BL-NOM also diminished oxidative effects caused by MC-LF; however, it failed to attenuate oxidative stress caused by MC-RR. Content of lipids was significantly reduced in exposed embryos following a trend similar to that obtained with teratological and enzymatic assays confirming the attenuating effect of BL-NOM. Physiological responses to microcystins and NOM required energetic costs, which were compensated to the expense of the energy resources of the yolk, which in turn might affect the normal development of embryos. © 2006 Wiley Periodicals, Inc. Environ Toxicol 21: 22,32, 2006. [source] Transformation of 2,4,6-trinitrotoluene in soil in the presence of the earthworm Eisenia andrei,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 6 2000Agnès Y. Renoux Abstract The ability of the earthworm Eisenia andrei to metabolize 2,4,6-trinitrotoluene (TNT) was studied in experiments with TNT-spiked soils, dermal contact tests, and with an in vitro assay. Lethality of TNT in a forest sandy soil was first determined (14-d LC50 = 143 mg/kg). Then TNT at lethal and sublethal concentrations was applied to the same soil and was monitored along with its metabolites in extracts of soil and earthworm tissue for up to 14 d postapplication. High performance liquid chromatography-ultra violet analyses indicated that TNT was transformed in the presence of E. andrei by a reductive pathway to 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4-diamino-6-nitrotoluene (2,4-DANT), and traces of 2,6-di-amino-4-nitrotoluene (2,6-DANT) in earthworm tissues. This transformation could be explained by either a metabolic mechanism within the earthworm or by the enhancement of an earthworm-associated microbial activity or both. The TNT concentrations decreased from the spiked soils. However, the monoamino-dinitrotoluene (2-ADNT and 4-ADNT) concentrations increased with exposure duration and were dependent on the initial TNT soil concentrations. This was also observed to a lesser extent in the TNT-spiked soils with no earthworms present. The biotransformation of TNT into 2-ADNT, 4-ADNT, and 2,4-DANT and the presence of these metabolites in E. andrei after dermal contact on TNT-spiked filter paper showed that dermal uptake can be a significant exposure route for TNT. In vitro experiments showed that earthworm homogenate could metabolize TNT and form 2-ADNT and 4-ADNT at room temperature and at 37°C. This effect was inhibited by heat inactivation prior to incubation or by incubation at 4°C, suggesting that the biotransformation of TNT in the presence of E. andrei may be enzymatic in nature. [source] Juvenile-onset neuronal ceroid lipofuscinosis with infantile CLN1 mutation and palmitoyl-protein thioesterase deficiencyEUROPEAN JOURNAL OF NEUROLOGY, Issue 4 2007R. Kälviäinen Accurate diagnosis, especially in progressive hereditary diseases, is essential for the treatment and genetic counseling of the patient and the family. Neuronal ceroid lipofuscinoses (NCL) are amongst the most common groups of neurodegenerative diseases. Infantile, juvenile, and adult-onset types with multiple genotype,phenotype associations have been described. A fluorimetric enzyme assay for palmitoyl protein thioesterase (PPT) from leukocytes and fibroblasts has been previously developed to confirm the diagnosis of infantile NCL. We describe a patient with juvenile-onset NCL phenotype with a new CLN1 mutation and deficient PPT activity. Over 40 different mutations have been found in patients with PPT deficiency, indicating that screening for known mutations is not an efficient way to diagnose this disorder. Therefore, PPT enzyme analysis should precede mutation analysis in suspected PPT deficiency, particularly in patients with granular osmiophilic deposits (GROD) or in patients who have negative ultrastructural data. The use of enzyme assay led to the diagnosis of this patient with juvenile-onset Finnish variant NCL with PPT deficiency, and we expect that greater awareness of the utility of the enzymatic assay may lead to identification of other similar cases awaiting a definitive diagnosis. [source] Enantioselective One-Pot Two-Step Synthesis of Hydrophobic Allylic Alcohols in Aqueous Medium through the Combination of a Wittig Reaction and an Enzymatic Ketone ReductionEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 31 2007Marina Kraußer Abstract A one-pot two-step process for the enantioselective synthesis of hydrophobic allylic alcohols was developed, which comprises ketone formation by the Wittig reaction and their enzymatic in situ biotransformation into the desired target products. By means of this combined Wittig reaction and bioreduction, the allylic alcohols were prepared with conversions of up to 90,%, and with excellent enantioselectivities of >99,% ee. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] Substrate Channelling in a Creatine Kinase System of Rat Skeletal Muscle Under Various pH ConditionsEXPERIMENTAL PHYSIOLOGY, Issue 1 2003M. Gregor The aim of this study was to evaluate myofibrillar creatine kinase (CK) activity and to quantify the substrate channelling of ATP between CK and myosin ATPase under different pH conditions within the integrity of myofibrils. A pure myofibrillar fraction was prepared using differential centrifugation. The homogeneity of the preparation and the purity of the fraction were confirmed microscopically and by enzymatic assays for contaminant enzyme activities. The specific activity of myofibrillar CK reached 584 ± 33 nmol PCr min,1 mg,1 at pH 6.75. Two methods were used to detect CK activity: (1) measurement of direct ATP production, and (2) measurement of PCr consumption. This method of evaluation has been tested in experiments with isolated creatine kinase. No discrepancy in CK activity between the methods was observed in the pH range tested (6.0-7.5). However, the same procedures resulted in a significant discrepancy between the amounts of reacted PCr and produced ATP within the pure myofibrillar fraction. This discrepancy represents the portion of ATP produced by the CK reaction, which is preferentially channelled to the myosin ATPase before diffusing into the bulk solution. The maximum evaluated difference reached 42.3 % at pH 6.95. The substrate channelling between myofibrillar-bound CK and myosin ATPase was evaluated under various pH levels within the physiological range and it reached a maximum value in a slightly acidic environment. These results suggest that ATP/ADP flux control by the CK system is more important at lower pH, corresponding to the physiological state of muscle fatigue. [source] Thermally induced conformational changes in horseradish peroxidaseFEBS JOURNAL, Issue 1 2001David G. Pina Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV and visible CD studies, together with enzymatic assays, were carried out to monitor the thermal denaturation of horseradish peroxidase isoenzyme c (HRPc) at pH 3.0. The spectral parameters were complementary to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while that in the Soret region, as well as changes in intrinsic tryptophan fluorescence emission, corresponded to changes in the tertiary structure of the enzyme. The results, supported by data about changes in enzymatic activity with temperature, show that thermally induced transitions for peroxidase are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of HRPc denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated. [source] Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24FEBS JOURNAL, Issue 20 2000Toshiyuki Sakaki Human 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D3, 1,,25(OH)2D3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D3 and 1,,25(OH)2D3, whereas rat CYP24 showed almost no C-23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. & Inouye, K. (1999) Eur. J. Biochem. 262, 43,48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C-23 hydroxylation pathway from 25(OH)D3 via 23S,25(OH)2D3, 23S,25,26(OH)3D3 and 25(OH)D3 -26,23-lactol to 25(OH)D3 -26,23-lactone in addition to the C-24 hydroxylation pathway from 25(OH)D3 via 24R,25(OH)2D3, 24-oxo-25(OH)D3, 24-oxo-23S,25(OH)2D3 to 24,25,26,27-tetranor-23(OH)D3. On 1,,25(OH)2D3 metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D3 and 1,,25(OH)2D3. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH-adrenodoxin reductase in E. coli. Addition of 25(OH)D3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs. [source] Mass spectrometry study of ecto-5,-nucleotidase from bull seminal plasmaFEBS JOURNAL, Issue 16 2000Carlo Fini The structure of ecto-5,-nucleotidase from bull seminal plasma, containing a glycosyl-phosphatidylinositol anchor, was studied using mass spectrometry. MALDI-MS analysis of intact protein indicated a mass of 65 568.2 Da for the monomeric form, and it also showed a heterogeneous population of glycoforms with the glycosidic moiety accounting for ,,6000 Da. MALDI-MS analysis showed that Asn53, Asn311, Asn333 and Asn403 were four sites of N -glycosylation. GC-MS analysis provided information on the glycosidic structures linked to the four asparagines. Asn53, Asn311 and Asn333 were linked to high-mannose saccharide chains, whereas the glycan chains linked to Asn403 contained a heterogeneous mixture of oligosaccharides, the high-mannose type structure being the most abundant and hybrid or complex type glycans being minor components. By combining enzymatic and/or chemical hydrolysis with GC-MS analysis, detailed characterization of the glycosyl-phpsphatidylinositol anchor was obtained. MALDI spectral analysis indicated that the glycosyl-phosphatidylinositol core contained EtN(P)Man3GlcNH2 -myo-inositol(P)-glycerol, principally modified by stearoyl and palmitoyl residues or by stearoyl and myristoyl residues to a minor extent. Moreover, 1-palmitoylglycerol and 1-stearoylglycerol outweighed 2-palmitoylglycerol and 2-stearoylglycerol. The combination of chemical and enzymatic digestions of the protein with the mass spectral analysis yielded a complete pattern of S,S bridges. The protein does not contain free thiols and its eight cysteines are linked by intramolecular disulfide bonds, the pairs being: Cys51,Cys57, Cys353,Cys358, Cys365,Cys387 and Cys476,Cys479. This work resolves details of the structure of ecto-5,-nucleotidase, with particular regard to the localization and composition of the glycidic moiety, number and localization of the disulfide bridges and characterization of the glycosyl-phosphatidylinositol anchor. [source] Low Leptin Levels in Migraine: A Case Control StudyHEADACHE, Issue 7 2008Baburhan Guldiken MD Background., Obesity has been shown to be a risk factor for transformation of episodic migraine to chronic form, and adipocytokines have been implicated to modulate some of the cytokins such as interleukin-6 and tumor necrosis factor, which also act in the neurogenic inflammation in migraine. The aim of the study was to assess leptin levels, one of the adipocytokines, in headache-free period of migraine patients and investigate its relation to vascular risk factors. Material and Methods., Sixty-one patients with episodic migraine headaches and 64 control subjects were enrolled in the study. Demographic data and anthropometric measurements were obtained from all participants; body mass index and fat mass values were calculated. Glucose and lipid parameters were measured by oxidase technique and cholesterol esterase enzymatic assays, and leptin levels were measured by ELISA in serum samples obtained after an overnight fasting. Results., Leptin levels were found significantly lower in migraineurs than controls (40.1 ± 21.2 ng/mL, 48.5 ± 24.5 ng/mL; P < .05). Although body mass index did not differ between 2 groups, fat mass, and fat percentages were significantly lower in migraine patients (19.4 ± 8.8 kg, 26.0 ± 8.7 kg; P < .001 and 28 ± 9%, 34 ± 5%; P < .001, respectively). Conclusion., Migraine patients have low leptin levels and fat mass which may be related to the pathogenesis of migraine. The importance and impact of our findings on the prevalence, characteristics, and treatment of migraine needs to be investigated in further detailed studies. [source] Evidence for Antinociceptive Activity of Botulinum Toxin Type A in Pain ManagementHEADACHE, Issue 2003K. Roger Aoki PhD The neurotoxin, botulinum toxin type A, has been used successfully, in some patients, as an analgesic for myofascial pain syndromes, migraine, and other headache types. The toxin inhibits the release of the neurotransmitter, acetylcholine, at the neuromuscular junction thereby inhibiting striated muscle contractions. In the majority of pain syndromes where botulinum toxin type A is effective, inhibiting muscle spasms is an important component of its activity. Even so, the reduction of pain often occurs before the decrease in muscle contractions suggesting that botulinum toxin type A has a more complex mechanism of action than initially hypothesized. Current data points to an antinociceptive effect of botulinum toxin type A that is separate from its neuromuscular activity. The common biochemical mechanism, however, remains the same between botulinum toxin type A's effect on the motor nerve or the sensory nerve: enzymatic blockade of neurotransmitter release. The antinociceptive effect of the toxin was reported to block substance P release using in vitro culture systems.1 The current investigation evaluated the in vivo mechanism of action for the antinociceptive action of botulinum toxin type A. In these studies, botulinum toxin type A was found to block the release of glutamate. Furthermore, Fos, a product of the immediate early gene, c- fos, expressed with neuronal stimuli was prevented upon peripheral exposure to the toxin. These findings suggest that botulinum toxin type A blocks peripheral sensitization and, indirectly, reduces central sensitization. The recent hypothesis that migraine involves both peripheral and central sensitization may help explain how botulinum toxin type A inhibits migraine pain by acting on these two pathways. Further research is needed to determine whether the antinociceptive mechanism mediated by botulinum toxin type A affects the neuronal signaling pathways that are activated during migraine. [source] A Novel Synthesis of Highly Substituted Perhydropyrrolizines, Perhydroindolizines, and Pyrrolidines: Inhibition of the Peptidyl-Prolyl cis/trans Isomerase (PPIase) Pin1HELVETICA CHIMICA ACTA, Issue 2 2007Romain Siegrist Abstract In this paper, we describe the synthesis and biological evaluation of highly substituted perhydropyrrolizines that inhibit the peptidyl-prolyl cis/trans isomerase (PPIase) Pin1, an oncogenic target. The enzyme selectively catalyzes the cis/trans isomerization of peptide bonds between a phosphorylated serine or threonine, and proline, thereby inducing a conformational change. Such structural modifications play an important role in many cellular events, such as cell-cycle progression, transcriptional regulation, RNA processing, as well as cell proliferation and differentiation. Based on computer modeling (Fig.,2), the new perhydropyrrolizinone derivatives (,)- 1a,b, decorated with two substituents, were selected and synthesized (Schemes,1,3). While enzymatic assays showed no biological activity, 15N,1H-HSQC-NMR spectroscopy revealed that (,)- 1a,b bind to the WW recognition domain of Pin1, apparently in a mode that does not inhibit PPIase activity. To enforce complexation into the larger active site rather than into the tighter WW domain of Pin1 and to enhance the overall binding affinity, we designed a perhydropyrrolizine scaffold substituted with additional aromatic residues (Fig.,5). A novel, straightforward synthesis towards this class of compounds was developed (Schemes,4 and 5), and the racemic compounds (±)- 22a,22d were found to inhibit Pin1 with Ki values (Ki,=,inhibition constant) in the micromolar range (Table,2). To further enhance the potency of these inhibitors, the optically pure ligands (+)- 22a and (+)- 33b,c were prepared (Schemes,6 and 7) and shown to inhibit Pin1 with Ki values down to the single-digit micromolar range. According to 15N,1H-HSQC-NMR spectroscopy and enzymatic activity assays, binding occurs at both the WW domain and the active site of Pin1. Furthermore, the new synthetic protocol towards perhydropyrrolizines was extended to the preparation of highly substituted perhydroindolizine ((±)- 43; Scheme,8) and pyrrolidine ((±)- 48a,b; Scheme,9) derivatives, illustrating a new, potentially general access to these highly substituted heterocycles. [source] Emodin reverses CCl4 induced hepatic cytochrome P450 (CYP) enzymatic and ultrastructural changes: The in vivo evidenceHEPATOLOGY RESEARCH, Issue 3 2009Monika Bhadauria Aim:, The curative effect of emodin (1,3,8-trihydroxy-6-methyl anthraquinone), an active compound of the plant species Ventilago maderaspatana Gaertn, was evaluated against carbon tetrachloride (CCl4) induced hepatic cytochrome P450 (CYP) enzymatic and ultrastructural alterations in rats. Methods:, Female rats were administered CCl4 (1.5 mL/kg, ip) followed by varying doses of emodin (20, 30 and 40 mg/kg, oral po) after 24 h of CCl4 administration. Animals were euthanized after 24 h of last administration to determine liver function tests in serum, hepatic light microscopic and ultrastructural changes, activity of CYP enzymes, microsomal lipid peroxidation and protein contents, hexobarbitone induced sleep time and bromosulphalein retention. Results:, The CCl4 induced-toxic effects were observed with sharp elevation in the release of serum transaminases, alkaline phosphatase, lactate dehydrogenase and ,-glutamyl transpeptidase. An initial study for an optimum dose of emodin among different dose levels revealed that a 30 mg/kg dose was effective in restoring all the enzymatic variables and liver histoarchitecture in a dose dependent manner. Exposure to CCl4 diminished the activities of CYP enzymes (i.e. aniline hydroxylase and amidopyrine-N-demethylase and microsomal protein contents with concomitant increase in microsomal lipid peroxidation). Emodin at 30 mg/kg effectively reversed the CCl4 induced hepatotoxic events, which was consistent with ultrastructural observations. Hexobarbitone-induced sleep time and plasma bromosulphalein retention also improved liver functions after emodin therapy. Conclusion:, By reversal CYP activity and ultrastructural changes, emodin shows a strong hepatoprotective abilities. [source] Molecular analysis of ARSA and PSAP genes in twenty-one Italian patients with metachromatic leukodystrophy: identification and functional characterization of 11 novel ARSA alleles,HUMAN MUTATION, Issue 11 2008Serena Grossi Abstract Metachromatic leukodystrophy (MLD), the demyelinating disorder resulting from impaired sulfatide catabolism, is caused by allelic mutations of the Arylsulfatase A (ARSA) locus except for extremely rare cases of Saposin-B (Sap-B) deficiency. We characterized twenty-one unrelated Italian patients among which seventeen were due to ARSA activity deficiency and 4 others resulted from Saposin-B defect. Overall, we found 20 different mutant ARSA alleles and 2 different Sap-B alleles. The eleven new ARSA alleles (c.53C>A; c.88G>C; c.372G>A; c.409_411delCCC; c.634G>C; [c.650G>A;c.1108C>T]; c.845A>G; c.906G>C; c.919G>T; c.1102-3C>G; c.1126T>A) were functionally characterized and the novel amino acid changes were also modelled into the three-dimensional structure. The present study is aimed at providing a broader picture of the molecular basis of MLD in the Italian population. It also emphasizes the importance of a comprehensive evaluation in MLD diagnosis including biochemical, enzymatic and molecular investigations. © 2008 Wiley-Liss, Inc. [source] |