Clinical and Radiological Characterisation of Patients with Mucopolysaccharidosis in a Genetic Clinic

Abstract The mucopolysaccharidoses (MPS) are a relatively uncommon group of inherited metabolic disorders. MPSs should be suspected in a child with coarse facies, organomegaly, recurrent respiratory tract infections, developmental delay, and hernias. Early diagnosis and treatment can greatly improve the quality of life in these children. In this study we studied 46 MPS patients diagnosed on enzyme and/or DNA testing and we found that the MPS II was the most common type followed by MPS I and MPS IVA. While the mean age of onset of symptoms was 12 months, the mean age at diagnosis was 4.5 years, a significant delay. One of major presenting features was recurrent respiratory problems, more prevalent in MPS II cases. Many patients also had short stature and contractures. Increasing awareness among physicians is of paramount importance for the early diagnosis and optimal treatment and prevention by prenatal testing and counselling.

Novel carbohydrate-triazole derivatives as potential α-glucosidase inhibitors / 中国天然药物

A series of novel pyrano[2, 3-d]trizaole compounds were synthesized and their α-glucosidase inhibitory activities were evaluated by in vitro enzyme assay. The experimental data demonstrated that compound 10f showed up to 10-fold higher inhibition (IC74.0 ± 1.3 μmol·L) than acarbose. The molecular docking revealed that compound 10f could bind to α-glucosidase via the hydrophobic, π-π stacking, and hydrogen bonding interactions. The results may benefit further structural modifications to find new and potent α-glucosidase inhibitors.

Design, Docking And Synthesis Of Novel Bromo Isatin Incorporated Isoxazole Derivatives As Vegfr-2 Inhibitors

Objective: To design, synthesize, in vitro Vascular Endothelial Growth Factor Receptor (VEGFR-2) assay, antiproliferative activity an Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) studies of some novel bromoisatin incorporated isoxazole derivatives. Methods: Designed compounds were synthesized by the condensation of different 3-aryl-5-methylisoxazole-4-carbohydrazides (5a-h) with 5-bromoisatin to give the target molecules. To predict the affinity and activity of the ligand molecule the docking program GOLD 3.1 was employed to generate different bioactive binding poses of designing molecules at the active site of protein VEGFR-2. All the synthesized compounds were characterized based on the spectral and elemental analysis data. Antiproliferative activity performed against Human Umbilical vein endothelial cells (HUVEC cell line). Results: All the synthesized compounds showed the characteristic peaks in FTIR,1H, C[13]NMR and Mass spectral analysis. In molecular docking, all the synthesized compounds (6a-j) exhibited high fitness scores with minimum three bonding interaction with the active site VEGFR-2 kinase. In in-vitro, VEGFR-2 kinase assay, compounds 6a, 6b, 6d and 6e exhibited more than 70% inhibition at a single dose concentration of 5μM. In antiproliferative assay against HUVEC cell lines, compounds 6d and 6e exhibited potent activity with IC50 values in nanomolar concentrations. ADMET results of 6a, 6b, 6d and 6e are quite promising with least hepatotoxicity and good bioavailability. Conclusion: The derivatives were synthesized in quantitative yields. New derivatives posses antiproliferative activity, least hepatotoxicity and good bioavailability.

Recent advances in enzyme inhibitor screening based on mass spectrometry / 药学学报

Enzymes play crucial functional roles in all biological processes. Enzymatic inhibitors can regulate enzyme activity and may become the starting point for drug discovery. Mass spectrometry (MS) has the advantage for rapid qualitative and quantitative analyses of compounds and enzyme reactions, emerging as an important analytical tool in enzyme inhibitor screening assay for drug discovery. This review will highlight recent advances in the inhibitor screening assay using MS and related techniques, including frontier affinity chromatography, immobilized enzyme beads, ultrafiltration, surface plasmon resonance, capillary electrophoresis and microfluidic chips. The existing MS methods for screening enzyme inhibitor were divided into two types: affinity screening and activity screening.

Insecticide susceptibility status and resistance mechanism of Anopheles cracens Sallum and Peyton and Anopheles maculatus Theobald (Family: Culicidae) from knowlesi malaria endemic areas in Peninsular Malaysia

Objective: To assess the insecticide susceptibility status of Anopheles cracens (An. cracens) and Anopheles maculatus (An. maculatus) from knowlesi malaria endemic areas in Peninsular Malaysia towards DDT, malathion and deltamethrin and to determine the resistance mechanism involved. Methods: Adult and larval mosquitos were collected for surveillance. Susceptibility status of Anopheles was determined using the standard WHO adult bioassay, larval bioassay and biochemical enzyme assay. Results: WHO adult bioassay results indicated An. cracens collected from Kampung Sungai Ular, Pahang was resistant towards 4% DDT, while An. maculatus collected from Kampung Sokor, Kelantan and Kampung Sungai Lui, Selangor exhibited resistance towards 4% DDT. However, the enzyme activity profiles varied according to strains and species. The resistance ratio of larval bioassay, showed that all strains and species tested were susceptible to malathion and temephos. Conclusions: Since only a few anopheline strains exhibited low level of insecticide resistance towards malathion, DDT and temephos. These insecticides are still considered effective for vector control program towards An. cracens and An. maculatus.

Insecticide susceptibility status and resistance mechanism of Anopheles cracens Sallum and Peyton and Anopheles maculatus Theobald (Family: Culicidae) from knowlesi malaria endemic areas in Peninsular Malaysia

Objective: To assess the insecticide susceptibility status of Anopheles cracens (An. cracens) and Anopheles maculatus (An. maculatus) from knowlesi malaria endemic areas in Peninsular Malaysia towards DDT, malathion and deltamethrin and to determine the resistance mechanism involved. Methods: Adult and larval mosquitos were collected for surveillance. Susceptibility status of Anopheles was determined using the standard WHO adult bioassay, larval bioassay and biochemical enzyme assay. Results: WHO adult bioassay results indicated An. cracens collected from Kampung Sungai Ular, Pahang was resistant towards 4% DDT, while An. maculatus collected from Kampung Sokor, Kelantan and Kampung Sungai Lui, Selangor exhibited resistance towards 4% DDT. However, the enzyme activity profiles varied according to strains and species. The resistance ratio of larval bioassay, showed that all strains and species tested were susceptible to malathion and temephos. Conclusions: Since only a few anopheline strains exhibited low level of insecticide resistance towards malathion, DDT and temephos. These insecticides are still considered effective for vector control program towards An. cracens and An. maculatus.

Validation of a Multiplex Tandem Mass Spectrometry Method for the Detection of Selected Lysosomal Storage Diseases in Dried Blood Spots

Abstract Background: Interest in screening methods for lysosomal storage diseases (LSDs) has increased in recent years, since early diagnosis and treatment are essential to prevent or attenuate the onset of symptoms and the complications of these diseases. In the current work, we evaluated the performance of tandem mass spectrometry (MS/MS) for the detection of some LSDs, aiming the future use of this methodology for the screening of these disorders. Methods: Standard curves and quality control dried blood spots were assayed to evaluate the precision, linearity, and accuracy. A total of 150 controls were grouped according to age and subjected to measurement of lysosomal enzymes deficient in Niemann-Pick A/B, Krabbe, Gaucher, Fabry, Pompe, and Mucopolysaccharidosis type I diseases. Samples from 59 affected patients with a diagnosis of LSDs previously confirmed by fluorimetric methods were analyzed. Results: Data from standard calibration demonstrated good linearity and accuracy and the intra- and interassay precisions varied from 1.17% to 11.60% and 5.39% to 31.24%, respectively. Except for galactocerebrosidase and ?-l-iduronidase, enzyme activities were significantly higher in newborns compared to children and adult controls. Affected patients presented enzymatic activities significantly lower compared to all control participants. Conclusion: Our results show that MS/MS is a promising methodology, suitable for the screening of LSDs, but accurate diagnoses will depend on its correlation with other biochemical and/or molecular analyses.

Cloning, expression, purification and assay of sorbitol dehydrogenase from "Feicheng" peach fruit (Prunus persica)

A gene encoding NAD+-dependent sorbitol dehydrogenase (SDH) in peach fruit was cloned and expressed in Escherichia coli. Recombinant SDH protein with 6×His-tagged was localized exclusively in the cytoplasmic soluble fraction of E. coli when the strains were grown for 4-5 h at 37 ºC. Highly pure protein was isolated by Ni2+-resin chromatography with 150 mM imidazole in 50 mM Tris, pH 8.0, by elution. In order to ensure that the recombinant SDH could be used for further study, the fluorescence and ultraviolet spectrum of the recombinant SDH were detected. Recombinant SDH was confirmed to be capable of oxidizing sorbitol by enzymatic activity assay. The activity of the recombinant SDH was 2.73 U mg-1min-1, which was similar with that directly extracted from peach fruits. The activities of SDH extracted from the fruits in different periods (30, 60, 90 days after flowing) were 7.75, 5.95, 3.26 U mg-1min-1, respectively.

Use of Tandem Mass Spectrometry for Newborn Screening of 6 Lysosomal Storage Disorders in a Korean Population / 대한진단검사의학회지

BACKGROUND: We evaluated the performance of multiplex tandem mass spectrometry (MS/MS) in newborn screening for detection of 6 lysosomal storage disorders (LSDs), namely, Niemann-Pick A/B, Krabbe, Gaucher, Fabry, and Pompe diseases and Hurler syndrome. METHODS: We revised the conditions and procedures of multiplex enzyme assay for the MS/MS analysis and determined the precision of our enzyme assay and the effects of sample amounts and incubation time on the results. We also measured the degree of correlation between the enzyme activities in the dried blood spots (DBSs) and those in the leukocytes. DBSs of 211 normal newborns and 13 newborns with various LSDs were analyzed using our revised methods. RESULTS: The intra- and inter-assay precisions were 2.9-18.7% and 8.1-18.1%, respectively. The amount of product obtained was proportional to the DBS eluate volume, but a slight flattening was observed in the product vs. sample volume curve at higher sample volumes. For each enzyme assay, the amount of product obtained increased linearly with the incubation period (range, 0-24 hr). Passing and Bablok regression analysis revealed that the enzyme activities in the DBSs and those in the leukocytes were favorably correlated. The enzyme activities measured in the DBSs were consistently lower in patients with LSDs than in normal newborns. CONCLUSIONS: The performance of our revised techniques for MS/MS detection and enzyme assays was of the generally acceptable standard. To our knowledge, this is the first report on the use of MS/MS for newborn screening of LSDs in an Asian population.

Development of an inhibitive enzyme assay for copper.

In this work the development of an inhibitive assay for copper using the molybdenum-reducing enzyme assay is presented. The enzyme is assayed using 12-molybdophosphoric acid at pH 5.0 as an electron acceptor substrate and NADH as the electron donor substrate. The enzyme converts the yellowish solution into a deep blue solution. The assay is based on the ability of copper to inhibit the molybdenum-reducing enzyme from the molybdate-reducing Serratia sp. Strain DRY5. Other heavy metals tested did not inhibit the enzyme at 10 mg l-1. The best model with high regression coefficient to measure copper inhibition is one-phase binding. The calculated IC50 (concentration causing 50% inhibition) is 0.099 mg l-1 and the regression coefficient is 0.98. The comparative LC50, EC50 and IC50 data for copper in different toxicity tests show that the IC50 value for copper in this study is lower than those for immobilized urease, bromelain, Rainbow trout, R. meliloti, Baker’s Yeast dehydrogenase activity, Spirillum volutans, P. fluorescens, Aeromonas hydrophilia and synthetic activated sludge assays. However, the IC50 value is higher than those for Ulva pertusa and papain assays, but within the reported range for Daphnia magna and Microtox™ assays.

Funcriohal analysis of novel mutations P459H and R483W in 21-hydroxylase gene / 中华内分泌代谢杂志

Mutations P459H and R483W detected in CYP21A2 gene in two Chinese patients with simple virilizing 21-hydroxylase deficiency were studied.Plasmid vectors containing P459H and R483W were constructed and transfected into COS-7 cells.The converting rate of progesterone to 11-desoxycortisone was calculated.P459H reduce 21-hydroxylase activity to 6.8%,while the residual enzyme activity of R483W was only 2.9%.

Cloning and Expression of the Full-length cDNAs Encoding Human Class Ⅰ Alcohol Dehydrogenases / 现代生物医学进展

Background & Objective:Background &Objective: The class Ⅰ Alcohol Dehydrogenases (ADH) play a key role in hepatic alcohol catabolism. Human ADH is encoded by at least seven genes, and three class Ⅰ ADH genes-ADH1, ADH2 and ADH3, which encode the α, β, and γ subunit respectively, had been isolated and mapped on chromosome 4q21-q25. This experiment tends to clone the human class Ⅰ ADH and investigate its role in the hepatic alcohol catabolism. Methods: A pair of primers were designed and the full-length cDNAs encoding human Class Ⅰ ADH were cloned at one time. Class Ⅰ ADH cDNAs were amplified with RT-PCR from total RNA extracted from fetal human liver and kidney, and cloned into pGEM-T vector. To identify cDNA segments, a pair of differential primers was designed. By using them, a portion of the ADHs which encodes the segment from -4 to 296 was cloned. These cDNA segments then were detected directly when being digested with Kpn Ⅰ and Pst Ⅰ, respectively. Then all the full-length cDNAs were subcloned in the plasmid pTYB11 and expressed in E. Coli. Stably. Alcohol Dehydrogenase activity of catalyzing alcohol were monitored at 340 nm. Results: Here we had successfully the human class Ⅰ ADH cloned and the full-length cDNAs expressed in E.col.I stably. The relative activity of recombinant enzymes metabolizing ethanol was 0.81 ～1.31 U/mg,0.09 ～0.15 U/mg and 0.76～1.11 U/mg, respectively. Conclusions: In the paper, the full-length cDNAs encoding human class Ⅰ AD H were successfully cloned and expressed and the recombinant enzymes showed the activities similar to the ones isolated from liver.

Cloning and Expression of the Full-length cDNAs Encoding Human Class Ⅰ Alcohol Dehydrogenases / 现代生物医学进展

Background & Objective:Background &Objective: The class Ⅰ Alcohol Dehydrogenases (ADH) play a key role in hepatic alcohol catabolism. Human ADH is encoded by at least seven genes, and three class Ⅰ ADH genes-ADH1, ADH2 and ADH3, which encode the α, β, and γ subunit respectively, had been isolated and mapped on chromosome 4q21-q25. This experiment tends to clone the human class Ⅰ ADH and investigate its role in the hepatic alcohol catabolism. Methods: A pair of primers were designed and the full-length cDNAs encoding human Class Ⅰ ADH were cloned at one time. Class Ⅰ ADH cDNAs were amplified with RT-PCR from total RNA extracted from fetal human liver and kidney, and cloned into pGEM-T vector. To identify cDNA segments, a pair of differential primers was designed. By using them, a portion of the ADHs which encodes the segment from -4 to 296 was cloned. These cDNA segments then were detected directly when being digested with Kpn Ⅰ and Pst Ⅰ, respectively. Then all the full-length cDNAs were subcloned in the plasmid pTYB11 and expressed in E. Coli. Stably. Alcohol Dehydrogenase activity of catalyzing alcohol were monitored at 340 nm. Results: Here we had successfully the human class Ⅰ ADH cloned and the full-length cDNAs expressed in E.col.I stably. The relative activity of recombinant enzymes metabolizing ethanol was 0.81 ～1.31 U/mg,0.09 ～0.15 U/mg and 0.76～1.11 U/mg, respectively. Conclusions: In the paper, the full-length cDNAs encoding human class Ⅰ AD H were successfully cloned and expressed and the recombinant enzymes showed the activities similar to the ones isolated from liver.

Cloning and Expression of the Full-length cDNAs Encoding Human Class Ⅰ Alcohol Dehydrogenases / 现代生物医学进展

Background & Objective:Background &Objective: The class Ⅰ Alcohol Dehydrogenases (ADH) play a key role in hepatic alcohol catabolism. Human ADH is encoded by at least seven genes, and three class Ⅰ ADH genes-ADH1, ADH2 and ADH3, which encode the α, β, and γ subunit respectively, had been isolated and mapped on chromosome 4q21-q25. This experiment tends to clone the human class Ⅰ ADH and investigate its role in the hepatic alcohol catabolism. Methods: A pair of primers were designed and the full-length cDNAs encoding human Class Ⅰ ADH were cloned at one time. Class Ⅰ ADH cDNAs were amplified with RT-PCR from total RNA extracted from fetal human liver and kidney, and cloned into pGEM-T vector. To identify cDNA segments, a pair of differential primers was designed. By using them, a portion of the ADHs which encodes the segment from -4 to 296 was cloned. These cDNA segments then were detected directly when being digested with Kpn Ⅰ and Pst Ⅰ, respectively. Then all the full-length cDNAs were subcloned in the plasmid pTYB11 and expressed in E. Coli. Stably. Alcohol Dehydrogenase activity of catalyzing alcohol were monitored at 340 nm. Results: Here we had successfully the human class Ⅰ ADH cloned and the full-length cDNAs expressed in E.col.I stably. The relative activity of recombinant enzymes metabolizing ethanol was 0.81 ～1.31 U/mg,0.09 ～0.15 U/mg and 0.76～1.11 U/mg, respectively. Conclusions: In the paper, the full-length cDNAs encoding human class Ⅰ AD H were successfully cloned and expressed and the recombinant enzymes showed the activities similar to the ones isolated from liver.

Zellweger Syndrome : First Confirmed Neonatal Case in Korea

We describe below a case of Zellweger syndrome case with facial dysmorphism, profound hypotonia, and hepatomegaly. He died at the age of 2 months. Zellweger syndrome is a disease marked by the absence of hepatic and renal peroxisomes. Because peroxisomes have many vital anabolic and catabolic functions within the cell, their absence results in profound cellular dysfunction. A biochemical study of plasma revealed elevation of very long chains of fatty acids and pipecolic acid, consistent with peroxisomal disorder. The cultured skin fibroblasts showed a marked decrease in plasmalogen synthesis enzyme : dihydroxyacetonephosphate acyl transferase(DHAP-AT) The clinical characteristics and biochemical findings led to the diagnosis of Zellweger syndrome. The pattern of inheritance is autosomal recessive, hence genetic counseling can help the families. In infantile hypotonia patients with unknown cause, peroxisomal disorder should be included in the differential diagnosis. We report the first confirmed case of Zellweger syndrome by enzyme assay in Korea.

Comparison of enzyme and DNA analysis in a Tay-Sachs disease carrier screening program

Tay-Sachs disease (GM2 gangliosidosis, type 1; TSD) is an autosomal recessive GM2 gangliosidosis resulting from the deficient activity of the lysosomal hydrolase beta-hexosaminidase A (Hex A). With a carrier frequency estimated at 1 in 25, it is a common lysosomal disorder in the Ashkenazi Jewish population. Tay-Sachs disease has provided the prototype for the prevention of severe recessive genetic diseases. Molecular analysis of the Hex A gene (HEXA) of Ashkenazi Jewish individuals affected with Tay-Sachs disease revealed that three common mutations cause the infantile and adult onset forms of the disease; a four base insertion in exon 11, a splice junction mutation in intron 12 and a point mutation in exon 7 (G269S). A study was undertaken to determine whether mutation analysis would be useful in TSD screening programs in identifying carriers and clarifying the status of individuals whose enzyme assays are inconclusive. Ashkenazi Jewish individuals who had been diagnosed as carriers, inconclusives by enzyme assay and non-carriers with low normal enzyme levels in the Mount Sinai Tay-Sachs Disease Prevention Program were examined for the presence of the three mutations using polymerase chain reaction (PCR) and allele specific oligonucleotide (ASO) hybridization. The insertion mutation was present in 29 of 34 carriers and 2 of 36 inconclusive individuals, the splice junction mutation was found in 4 of 34 carriers and the G269S mutation was found in 1 of 34 carriers. Of the 313 non-carrier individuals with normal enzyme activity in the lower normal range, one was positive for the splice junction mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

APPLICATION OF CELL ENZYME IMMUNOASSAY FOR DETECTION AND ANALYSIS OF MONOCLONAL ANTIBODIES TO HUMAN COLORECTAL ADENOCARCINOMA / 中国免疫学杂志

A solid-phase cell enzyme-linked immunoassay is described for screeningand analyzing monoclonal antibodies against the cell surface antigensof human colorectal adenocarcinoma.Horse-radish peroxidase conjugatedProtein A was used to detect the binding of mouse monoclonal antibodiesto human colorectal adenocarcinoma.HR_(8348) cells which had been cultured inand then fixed to the wells of microtiter plates by using glutaraldehyde.Thismethod was found to be specific,reproducible and practical,and especiallyto be advantageous for the large scale screening and analyzing of monoclo-nal antibodies with a panel of cell types.