Efficacy of enzyme replacement therapy in Fabry disease.

Enzyme replacement therapy has recently been introduced to treat Fabry disease, a rare X-linked lysosomal storage disorder. The disease occurs due to deficient activity of alpha-galactosidase A, leading to progressive accumulation of globotriaosylceramide in multiple organs and tissues. Renal, cardiac and cerebrovascular manifestations of the disease result in premature death in both hemizygous males and heterozygous females. This paper outlines the clinical signs, symptoms and diagnosis of Fabry disease, and the development of the two available enzyme replacement therapies -- agalsidase alfa and agalsidase beta. Agalsidase alfa and agalsidase beta are produced in a human cell line and in Chinese hamster ovary cells, respectively, resulting in products with the same amino acid sequence as the native human enzyme, but with different patterns of glycosylation. Correct post-translational glycosylation is important in terms of the pharmacokinetics, biodistribution, clinical efficacy and tolerability of genetically engineered protein therapeutics. Differences in glycosylation, which may affect immunogenicity and mannose-6-phosphate receptor-mediated cellular internalisation of administered enzyme, possibly account for the differences in dosing, clinical effects and safety profiles reported for agalsidase alfa and agalsidase beta.

Lack of Enantioselectivity in the SULT1A3-catalyzed Sulfoconjugation of Normetanephrine Enantiomers: An In Vitro and Computational Study.

(1R)-Normetanephrine is the natural stereoisomeric substrate for sulfotransferase 1A3 (SULT1A3)-catalyzed sulfonation. Nothing appears known on the enantioselectivity of the reaction despite its potential significance in the metabolism of adrenergic amines and in clinical biochemistry. We confronted the kinetic parameters of the sulfoconjugation of synthetic (1R)-normetanephrine and (1S)-normetanephrine by recombinant human SULT1A3 to a docking model of each normetanephrine enantiomer with SULT1A3 and the 3'-phosphoadenosine-5'-phosphosulfate cofactor on the basis of molecular modeling and molecular dynamics simulations of the stability of the complexes. The K(M) , V(max) , and k(cat) values for the sulfonation of (1R)-normetanephrine, (1S)-normetanephrine, and racemic normetanephrine were similar. In silico models were consistent with these findings as they showed that the binding modes of the two enantiomers were almost identical. In conclusion, SULT1A3 is not substrate-enantioselective toward normetanephrine, an unexpected finding explainable by a mutual adaptability between the ligands and SULT1A3 through an "induced-fit model" in the catalytic pocket. Chirality, 00:000-000, 2012.© 2012 Wiley Periodicals, Inc.

Genetic vulnerability factor for schizophrenia: association with glutamate cysteine ligase modifier gene and abnormal enzyme activity

Background: We previously reported in schizophrenia patients a decreased level of glutathione ([GSH]), the principal non-protein antioxidant and redox regulator, both in cerebrospinal-fluid and prefrontal cortex. To identify possible genetic causation, we studied genes involved in GSH metabolism. Methods: Genotyping: mass spectrometry analysis of polymerase chain reaction (PCR) amplified DNA fragments purified from peripheral blood. Gene expression: real-time PCR of total RNA isolated from fibroblast cultures derived from skin of patients (DSM-IV) and healthy controls (DIGS). Results: Case-control association study of single nucleotide polymorphisms (SNP) from the GSH key synthesizing enzyme glutamate-cysteine-ligase (GCL) modifier subunit (GCLM) was performed in two populations: Swiss (patients/controls: 40/31) and Danish (349/348). We found a strong association of SNP rs2301022 in GCLM gene (Danish: c2=3.2; P=0.001 after correction for multiple testing). Evidence for GCLM as a risk factor was confirmed in linkage study of NIMH families. Moreover, we observed a decrease in GCLM mRNA levels in patient fibroblasts, consistently with the association study. Interestingly, Dalton and collaborators reported in GCLM knock-out mice an increased feedback inhibition of GCL activity, resulting in 60% decrease of brain [GSH], a situation analogous to patients. These mice also exhibited an increased sensitivity to oxidative stress. Similarly, under oxidative stress conditions, GCL enzymatic activity was also decreased in patient fibroblasts. Conclusions: These results at the genetic and functional levels, combined with observations that GSH deficient models reveal morphological, electrophysiological, and behavioral anomalies analogous to those observed in patients, suggest that GCLM allelic variant is a vulnerability factor for schizophrenia.

SOMATIC MUTATIONS IN THE KREBS CYCLE ENZYME ISOCITRATE DEHYDROGENASE 1 (IDH1) CAUSE METAPHYSEAL ENCHONDROMATOSIS WITH D-2-HYDROXYGLUTARIC ACIDURIA

Metaphyseal chondromatosis with hydroxyglutaric aciduria (MC-HGA) is a generalized skeletal dysplasia, accompanied by urinary excretion of D-2- hydroxyglutarate (HGA), and variable cerebral involvement. By wholeexome sequencing 2 unrelated patients with MC-HGA, we have found mutations in isocitrate dehydrogenase 1 (IDH1) at codon 132, as apparent somatic mosaicism. IDH1 is a key enzyme of the Krebs cycle, which converts isocitrate into alpha-ketoglutarate (a-KG). Mutations at IDH1 Arg132 residue have originally been identified in different tumour types (isolated gliomas, leukemias, and chondrosarcomas). These mutations trans-specify the enzyme activity resulting in HGA accumulation and a-KG depletion. This induces activation of hypoxia-inducible factor 1-alpha (HIF-1a), an important regulator of chondrocyte proliferation at the growth plate. Differently from Arg132 somatic mutations found in isolated tumours, themutation in our patientsmust have occurred very early in embryogenesis to cause a generalized dysplasia with involvement of all long bones metaphyses and mutation detectability in blood. Identical mutations have subsequently been identified in chondromas excised from patients with multiple chondromatosis (Ollier disease). Tissue distribution of themutationmay explain variable cerebral involvement and the susceptibility to develop tumours in other organs. The postulated pathophysiology ofMC-HGA points out the link between Krebs cycle, hypoxia sensing and bone growth.

Bioavailability of repeated oral administration of MDL 100,240, a dual inhibitor of angiotensin-converting enzyme and neutral endopeptidase in healthy volunteers.

BACKGROUND: MDL 100,240 (pyrido[2,1-a] [2]benzazepine-4-carboxylic acid,7-[[2-(acetylthio)-1-oxo-3-phenylpropyl]amino]-1,2,3,4,6,7,8, 12b-octahydro-6-oxo, [4S-[4alpha,7alpha(R(*)),12bbeta]]-) is a molecule possessing an inhibiting ability on both angiotensin converting enzyme (ACE) and neutral endopeptidase, the enzyme responsible for atrial natriuretic peptide (ANP) degradation. Such a dual mechanism of action presents a potential clinical interest for the treatment of hypertension and congestive heart failure. OBJECTIVES: To evaluate the bioavailability of MDL 100,240 and its accumulation over repeated oral administration, using ACE inhibition as a surrogate for plasma drug level and determining its profile after oral and i.v. administration. METHODS: First, in an open, one-period, single-dose study, the ACE inhibition profile was characterised following a 12.5 mg MDL 100,240 i.v. infusion. Second, in a three-group, parallel, randomised, double-blind study, each group of four subjects received q.d., over 8 days, 2.5, 10 or 20 mg of MDL 100,240 orally. The ACE inhibition profile was determined on day 1 and day 8. Trough plasma ACE was measured on days 2, 3 and 4. The recovery of ACE activity was monitored up to 72 h after the last dose of MDL 100,240. RESULTS: ACE inhibition profile was similar on day 1 and day 8, and trough inhibition remained unchanged after the 8 days of treatment with 10 mg or 20 mg. Following repeated 2.5-mg ingestion, trough inhibition increased from 33% to 44% after the eighth dose. The oral bioavailability of MDL 100,240 was estimated at 85%, not statistically different from 100%. The accumulation ratio at steady state was estimated at 112%. Expressing the accumulation ratio in terms of half-life, a t(1/2) of 0.31 days or 7. 5 h was estimated. CONCLUSION: MDL 100,240 (oral solution) has a good bioavailability, as estimated by ACE inhibition, and no drug accumulation seems to occur over 8 days with the 10-mg and 20-mg doses, but a slight rise in the trough level is observed with the 2. 5-mg dose.

Characterization of surface functional groups present on laboratory-generated and ambient aerosol particles by means of heterogeneous titration reactions

A Knudsen flow reactor has been used to quantify surface functional groups on aerosols collected in the field. This technique is based on a heterogeneous titration reaction between a probe gas and a specific functional group on the particle surface. In the first part of this work, the reactivity of different probe gases on laboratory-generated aerosols (limonene SOA, Pb(NO3)2, Cd(NO3)2) and diesel reference soot (SRM 2975) has been studied. Five probe gases have been selected for the quantitative determination of important functional groups: N(CH3)3 (for the titration of acidic sites), NH2OH (for carbonyl functions), CF3COOH and HCl (for basic sites of different strength), and O3 (for oxidizable groups). The second part describes a field campaign that has been undertaken in several bus depots in Switzerland, where ambient fine and ultrafine particles were collected on suitable filters and quantitatively investigated using the Knudsen flow reactor. Results point to important differences in the surface reactivity of ambient particles, depending on the sampling site and season. The particle surface appears to be multi-functional, with the simultaneous presence of antagonistic functional groups which do not undergo internal chemical reactions, such as acid-base neutralization. Results also indicate that the surface of ambient particles was characterized by a high density of carbonyl functions (reactivity towards NH2OH probe in the range 0.26-6 formal molecular monolayers) and a low density of acidic sites (reactivity towards N(CH3)3 probe in the range 0.01-0.20 formal molecular monolayer). Kinetic parameters point to fast redox reactions (uptake coefficient ?0>10-3 for O3 probe) and slow acid-base reactions (?0<10-4 for N(CH3)3 probe) on the particle surface. [Authors]

Characterization of PhlG, a hydrolase that specifically degrades the antifungal compound 2,4-diacetylphloroglucinol in the biocontrol agent Pseudomonas fluorescens CHA0.

The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been elucidated thus far. The phlG gene is located upstream of the phlACBD biosynthetic operon, between the phlF and phlH genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative DeltaphlA mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a DeltaphlA DeltaphlG double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in Escherichia coli, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a kcat of 33 s(-1) and a Km of 140 microM at 30 degrees C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of phlG was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH.

Cointegrase, a naturally occurring, truncated form of IS21 transposase, catalyzes replicon fusion rather than simple insertion of IS21.

The bacterial insertion sequence IS21 contains two genes, istA and istB, which are organized as an operon. IS21 spontaneously forms tandem repeats designated (IS21)2. Plasmids carrying (IS21)2 react efficiently with other replicons, producing cointegrates via a cut-and-paste mechanism. Here we show that transposition of a single IS21 element (simple insertion) and cointegrate formation involving (IS21)2 result from two distinct non-replicative pathways, which are essentially due to two differentiated IstA proteins, transposase and cointegrase. In Escherichia coli, transposase was characterized as the full-length, 46 kDa product of the istA gene, whereas the 45 kDa cointegrase was expressed, in-frame, from a natural internal translation start of istA. The istB gene, which could be experimentally disconnected from istA, provided a helper protein that strongly stimulated the transposase and cointegrase-driven reactions. Site-directed mutagenesis was used to express either cointegrase or transposase from the istA gene. Cointegrase promoted replicon fusion at high frequencies by acting on IS21 ends which were linked by 2, 3, or 4 bp junction sequences in (IS21)2. By contrast, cointegrase poorly catalyzed simple insertion of IS21 elements. Transposase had intermediate, uniform activity in both pathways. The ability of transposase to synapse two widely spaced IS21 ends may reside in the eight N-terminal amino acid residues which are absent from cointegrase. Given the 2 or 3 bp spacing in naturally occurring IS21 tandems and the specialization of cointegrase, the fulminant spread of IS21 via cointegration can now be understood.

Formes sévères d'hypersensibilité médicamenteuse retardée [Severe delayed drug hypersensitivity reactions].

Although most delayed drug hypersensitivity reactions are mild and show rapid improvement after drug discontinuation, there are severe systemic and/or cutaneous drug reactions which may be life-threatening. These entities are discussed here, namely DRESS syndrome (Drug Reaction with Eosinophilia and Systemic Symptoms), acute generalized exanthematous pustulosis (AGEP), Stevens-Johnson syndrome and toxic epidermal necrolysis (TEN). Early detection of warning signs and symptoms may help to take appropriate measures precociously.

Sandwich enzyme immunoassay using three monoclonal antibodies against different epitopes of carcinoembryonic antigen (CEA).

Purified monoclonal antibodies (Mab) produced by 3 hybridomas and reacting with 3 different epitopes of carcinoembryonic antigen (CEA) were used in a solid phase enzyme immunoassay. Two Mabs were physically adsorbed to polystyrene balls and the third Mab was coupled to alkaline phosphatase using the bifunctional reagent N-succinimidyl-3-(2-pyridyldithio)-propionate. During a first incubation, CEA from heat-extracted serum samples was immunoadsorbed to the antibody coated balls. After washing of the balls, bound CEA was detected by a second incubation with the enzyme coupled Mab. The sensitivity of the assay was 0.6 ng per ml of serum. A total of 196 serum samples from patients with various types of carcinoma, with liver cirrhosis, or from healthy blood donors with or without smoking habits, were tested. The results obtained with the monoclonal enzyme immunoassay (M-EIA) were compared with those obtained with perchloric acid extracts of the same serum samples tested by an inhibition radioimmunoassay using conventional goat anti-CEA antiserum. There was an excellent correlation between the two assays. In particular, the new M-EIA gave good results for the detection of tumor recurrences in the follow-up of colon carcinoma patients. However, despite the use of exclusively monoclonal antibodies the new assay detected a similar percentage of slightly elevated CEA values as the conventional assay in patients with non-malignant disease, suggesting that the CEA associated with non-malignant diseases is immunologically identical to the CEA released by colon carcinoma.

Molecular identification and characterization of the Arabidopsis delta(3,5),delta(2,4)-dienoyl-coenzyme A isomerase, a peroxisomal enzyme participating in the beta-oxidation cycle of unsaturated fatty acids.

Degradation of unsaturated fatty acids through the peroxisomal beta-oxidation pathway requires the participation of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. The auxiliary enzyme delta(3,5),delta(2,4)-dienoyl-coenzyme A (CoA) isomerase has been well studied in yeast (Saccharomyces cerevisiae) and mammals, but no plant homolog had been identified and characterized at the biochemical or molecular level. A candidate gene (At5g43280) was identified in Arabidopsis (Arabidopsis thaliana) encoding a protein showing homology to the rat (Rattus norvegicus) delta(3,5),delta(2,4)-dienoyl-CoA isomerase, and possessing an enoyl-CoA hydratase/isomerase fingerprint as well as aspartic and glutamic residues shown to be important for catalytic activity of the mammalian enzyme. The protein, named AtDCI1, contains a peroxisome targeting sequence at the C terminus, and fusion of a fluorescent protein to AtDCI1 directed the chimeric protein to the peroxisome in onion (Allium cepa) cells. AtDCI1 expressed in Escherichia coli was shown to have delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vitro. Furthermore, using the synthesis of polyhydroxyalkanoate in yeast peroxisomes as an analytical tool to study the beta-oxidation cycle, expression of AtDCI1 was shown to complement the yeast mutant deficient in the delta(3,5),delta(2,4)-dienoyl-CoA isomerase, thus showing that AtDCI1 is also appropriately targeted to the peroxisome in yeast and has delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vivo. The AtDCI1 gene is expressed constitutively in several tissues, but expression is particularly induced during seed germination. Proteins showing high homology with AtDCI1 are found in gymnosperms as well as angiosperms belonging to the Monocotyledon or Dicotyledon classes.

Traitement chronique de l'hypertension arterielle par un inhibiteur de l'enzyme de conversion de l'angiotensine. [Chronic treatment of hypertension by an inhibitor of angiotensin converting enzyme]

Captopril, or SQ 14,225 an orally active inhibitor of angiotensin-converting enzyme, produced a significant blood pressure reduction in 26 hypertensives. This new drug, alone or combined with a diuretic, has normalized the blood pressure of the 22 patients on long-term treatment.