A novel enzymatic reaction for converting DNA to CO-DNA

We have found previously that DNA from both the chick cerebrum and cardiac muscle has a modified structure. We named this novel DNA, CO-DNA. CO-DNA is a form of DNA in which a carbonyl group is attached to C-1 of the 2-deoxyribose and to the nitrogenous base. Therefore, 3-deoxyglucosone is the sugar constituent for CO-DNA. We found previously that the modification of the sugar moiety in DNA occurs around embryonic day 12 in the chick embryo. In this study, we isolated enzymes for the conversion of DNA to CO-DNA from chick cerebra. In our reaction system, uniformly labeled 14C-glucose was used as substrate. During incubation, the radioactivity was incorporated into DNA. From the analysis of 14C-labeled deoxynucleoside, the radioactive sugar was confirmed to be 3-deoxyglucosone. We propose a series of reactions involved in the conversion of DNA to CO-DNA: (1) DNA-enzyme complex is formed during preincubation, (2) 14C-glucose is transformed to 14C-3-deoxyhexonic acid, (3) 14C-3-deoxyhexonic acid is subsequently transformed into the sugar-phosphate, which is a mixture of phosphorylated 14C-3-deoxyhexonic acid and phosphorylated 14C-3-deoxyglucosone, (4) 2-deoxyribose in DNA is replaced with 14C-3-deoxyglucosone through its intermediate phosphorylated form, and (5) DNA is finally converted to CO-DNA.     

Mutation analysis in spinal muscular atrophy using allele-specific polymerase chain reaction

Polymerase chain reaction (PCR), followed by restriction digestion is universally used for molecular diagnosis of spinal muscular atrophy (SMA). In the present study, we have used a modified strategy based on amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) to develop a rapid and reliable method for mutation detection and prenatal diagnosis in SMA patients. The telomeric (SMN1) and centromeric (SMN2) copies of exon 7 of the survival motor neuron (SMN) gene were amplified by ARMS-PCR, using primers specific to SMN1 and SMN2 nucleotide sequence with the exonic mismatch G (for SMN1) and A (for SMN2) at the 3' end. The PCR products were analyzed on agarose gels. All the patients who had homozygous deletion of exon 7 of SMN1 gene by conventional PCR-restriction fragment length polymorphism (PCR-RFLP) method showed the same deletion status by ARMS-PCR. This procedure showed a 100% concordance between PCR-RFLP and ARMS-PCR methods for the detection of SMN1/SMN2 status in patients with SMA. An artifact due to incomplete digestion is not a problem while using ARMS-PCR. The modified protocol is specific, rapid and highly reliable for use in prenatal diagnosis as well.     

Multiple equilibria in complex chemical reaction networks: extensions to entrapped species models

In two earlier papers, means were provided to decide the capacity of complex chemical reaction networks, taken with mass-action kinetics, to admit multiple equilibria in the context of the isothermal homogeneous continuous flow stirred tank reactor (CFSTR). In such a reactor, all species are deemed to be in the outflow, a fact which has an important bearing on the nature of the governing equations. In contrast, one can imagine CFSTR-like models of the cell in which certain large molecules (e.g., enzymes) remain entrapped within the cell, whereas smaller ones (e.g., metabolites) are free to diffuse through the cell boundary. Although such models bear a strong physical resemblance to the classical CFSTR picture, there are substantive differences in the corresponding mathematics. Without a presumption of mass-action kinetics, this research is intended to indicate a general way in which results about uniqueness of equilibria in the classical CFSTR context extend to entrapped species models.     

A novel control of enzymatic enantioselectivity through the racemic temperature influenced by reaction media

The influence of reaction media on the racemic temperature (T_r) in the lipase-catalyzed resolution of ketoprofen vinyl ester was investigated. An effective approach to the control of the enzymatic enantioselectivity and the prediction of the increasing tendency was developed based on the T_r influenced by reaction media. The T_r for the resolution catalyzed by Candida rugosa lipase (CRL) was found at 29 °C in aqueous and S-ketoprofen was obtained predominantly at 40 °C. However, CRL showed R-selectivity at 40 °C in diisopropyl ether because the T_r was changed to 56 °C. CRL, lipase from AYS Amano^® and Mucor javanicus lipase were further applied for the investigation of the enzymatic enantioselectivity in dioxane, DIPE, isooctane and their mixed media with water. The effects of the reaction medium on T_r could be related to the solvent hydrophobicity, the lipase conformational flexibility and the interaction between the enantiomers and the lipase.     

A method to analyze allele-specific methylation

We have developed a method to analyze the methylation patterns of individual alleles of a gene. The target gene must have alleles identifiable by restriction fragment length polymorphism analysis. The method involves separation of the alleles after digestion by restriction enzyme digestion and electrophoresis, followed by recovery from the gel on ion-exchange paper. Methylation analysis can be done on the separate alleles by Southern blot after digestion by methylation-sensitive enzymes. As an example, we studied human c-Ha-ras-1 and showed that the methylation patterns of different alleles are stable and inherited. The method can be applied to the study of inheritance and methylation in genes where alleles can be identified by restriction fragment length polymorphism.     

Atomic motion in enzymatic reaction coordinates

Atomic excursions of reactants in enzymatic catalytic sites can be estimated from high-resolution crystal structures of enzyme complexes with substrates, transition state analog inhibitors and products. Transition state structures, defined from kinetic isotope effect studies, are compared to crystallographic structures to validate the properties of the transition state analog. Atomic excursions in enzymatic catalytic sites can differ from those in solution and define the role of the enzymatic catalyst in directing atomic motion.     

Use of multiplex allele-specific polymerase chain reaction (MAS-PCR) to detect multidrug-resistant tuberculosis in Panama

The frequency of individual genetic mutations conferring drug resistance (DR) to Mycobacterium tuberculosis has not been studied previously in Central America, the place of origin of many immigrants to the United States. The current gold standard for detecting multidrug-resistant tuberculosis (MDR-TB) is phenotypic drug susceptibility testing (DST), which is resource-intensive and slow, leading to increased MDR-TB transmission in the community. We evaluated multiplex allele-specific polymerase chain reaction (MAS-PCR) as a rapid molecular tool to detect MDR-TB in Panama. Based on DST, 67 MDR-TB and 31 drug-sensitive clinical isolates were identified and cultured from an archived collection. Primers were designed to target five mutation hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and MAS-PCR was performed. Whole-genome sequencing confirmed DR mutations identified by MAS-PCR, and provided frequencies of genetic mutations. DNA sequencing revealed 70.1% of MDR strains to have point mutations at codon 315 of the katG gene, 19.4% within mabA-inhA promoter, and 98.5% at three hotspots within rpoB. MAS-PCR detected each of these mutations, yielding 82.8% sensitivity and 100% specificity for isoniazid resistance, and 98.4% sensitivity and 100% specificity for rifampin resistance relative to DST. The frequency of individual DR mutations among MDR strains in Panama parallels that of other TB-endemic countries. The performance of MAS-PCR suggests that it may be a relatively inexpensive and technically feasible method for rapid detection of MDR-TB in developing countries.     

Low reaction temperature increases the selectivity in an enzymatic reaction due to substrate solvation effects

Immobilized a-chymotrypsin was used as catalyst for studying temperature effects on acyl transfer reactions (acyl-donor: Bz-TyrOEt) in a water-immiscible organic solvent. The solubility of the two nucleophiles, Phe-NH and water, decreased with decreasing temperature. The relative decrease for the amide was larger (2.4-fold) than for water. Therefore the thermodynamic activity (estimated by the relative saturation) increased more for this substrate and hence the selectivity in the reaction was increased.     

Apolipoprotein E genotyping using the polymerase chain reaction and allele-specific oligonucleotide probes

A rapid procedure for determining apolipoprotein E genotype from genomic DNA has been developed. In this procedure, DNA is amplified by the polymerase chain reaction, and allele-specific oligonucleotide probes are used to detect the cysteine-arginine interchanges at residues 112 and 158 that distinguish the three common isoforms of apolipoprotein E. The method was tested with 68 subjects, representing the six common phenotypes, and yielded results consistent with the known phenotype.     

Competitive inhibition of glutamate dehydrogenase reaction

Irrespective of their pyridine nucleotide specificity, all glutamate dehydrogenases share a common chemical mechanism that involves an enzyme bound 'iminoglutarate' intermediate. Three compounds, structurally related to this intermediate, were tested for the inhibition of purified NADP-glutamate dehydrogenases from two Aspergilli, as also the bovine liver NAD(P)-glutamate dehydrogenase. 2-Methyleneglutarate, closely resembling iminoglutarate, was a potent competitive inhibitor of the glutamate dehydrogenase reaction. This is the first report of a non-aromatic structure with a better glutamate dehydrogenase inhibitory potency than aryl carboxylic acids such as isophthalate. A suitably located 2-methylene group to mimic the iminium ion could be exploited to design inhibitors of other amino acid dehydrogenases.     

Application of an allele-specific polymerase chain reaction to the direct determination of ABO blood group genotypes.

The allele-specific polymerase chain reaction (ASPCR) procedure has proven a powerful tool for the detection and analysis of known genetic polymorphisms. Here, we present a novel application of the ASPCR technique to determine the ABO genotypes of individuals without the need of family analysis. The method introduces a new strategy for primer design that permits the identification of the different ABO genotypes according to the molecular size of allele-specific amplification products. Four primer sets, each specific for a different set of ABO alleles, are mixed in one reaction and the amplification products are resolved on a polyacrylamide gel. Forty-one individuals belonging to various families, whose ABO phenotypes were previously determined serologically, were typed with this new variation of the ASPCR technique. A 100% correlation between the serology and the ASPCR data was found. The Mendelian segregation of ABO alleles was also demonstrated in families. The method is rapid, simple, reproducible, and specific. Potential applications include gene mapping, genetic disease diagnosis, HLA typing, paternity testing, and forensic science.     

Apolipoprotein E genotyping using the polymerase chain reaction and allele-specific oligonucleotide primers

A method for apolipoprotein (apo) E genotyping was developed using the polymerase chain reaction (PCR) with allele-specific oligonucleotide primers (ASP). Synthetic oligonucleotides with base-pair mismatches at the 3' terminus were used as primers to amplify the apoE gene in subjects previously phenotyped using isoelectric focusing (IEF). Complementary primer-allele combinations were specifically amplified by PCR, together with a control pair of primers specific to the human prothrombin gene. Identification of genotype by PCR using ASP was consistent with the phenotypes that were determined by IEF for 14 healthy normolipidemic subjects. These results were achieved using DNA isolated from buccal epithelial cells obtained from a mouthwash or DNA extracted from leukocytes. Genotype identification required analysis of the PCR products on an ethidium-stained agarose gel, yielding results 3 h after DNA extraction. In comparison with other current methods, PCR using ASP is suggested as a rapid and simple noninvasive technique for determining population apoE allelic distribution.     

Reduction of benzoquinones to hydroquinones via spontaneous reaction with glutathione and enzymatic reaction by S-glutathionyl-hydroquinone reductases

S-Glutathionyl-hydroquinone reductases (GS-HQRs) are a new class of glutathione transferases, widely present in bacteria, halobacteria, fungi, and plants. They catalyze glutathione (GSH)-dependent reduction of GS-trichloro-p-hydroquinone to trichloro-p-hydroquinone. Since GS-trichloro-p-hydroquinone is uncommon in nature, the extensive presence of GS-HQRs suggests they use common GS-hydroquinones. Here we demonstrate that several benzoquinones spontaneously reacted with GSH to form GS-hydroquinones via Michael addition, and four GS-HQRs from yeast and bacteria reduced the GS-hydroquinones to the corresponding hydroquinones. The spontaneous and enzymatic reactions led to the reduction of benzoquinones to hydroquinones with the concomitant oxidation of GSH to oxidized glutathione (GS-SG). The enzymes did not use GS-benzoquinones or other thiol-hydroquinones, for example, S-cysteinyl-hydroquinone, as substrates. Apparent kinetic parameters showed the enzymes preferred hydrophobic, bulky substrates, such as GS-menadiol. The broad substrate range and their wide distribution suggest two potential physiological roles: channeling GS-hydroquinones back to hydroquinones and reducing benzoquinones via spontaneous formation of GS-hydroquinones and then enzymatic reduction to hydroquinones. The functions are likely important in metabolic pathways with quinone intermediates.     

Ultrafast enzymatic reaction dynamics in protochlorophyllide oxidoreductase

The reaction catalyzed by the light-driven enzyme protochlorophyllide oxidoreductase (POR) has been initiated with a 50-fs laser pulse. We show that the catalytic mechanism, involving proton and hydride transfers, proceeds with time constants of 3 ps and 400 ps. It is known that catalysis by POR involves thermally excited protein dynamics; our results show that these molecular motions occur on an ultrafast timescale.     

Exploring multiplicity conditions in enzymatic reaction networks

In this work, a novel algorithmic approach to detect multiplicity of steady states in enzymatic reaction networks is presented. The method exploits the structural properties of networks derived from the Chemical Reaction Network Theory. In first instance, the space of parameters is divided in different regions according to the qualitative behavior induced by the parameters in the long term dynamics of the network. Once the regions are identified, a condition for the appearance of multiplicities is checked in the different regions by solving a given optimization problem. In this way, the method allows the characterization of the whole parameter space of biochemical networks in terms of the appearance or not of multistability. The approach is illustrated through a well‐known case of enzymatic catalysis with substrate inhibition. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The enzymatic reaction of chlorotrifluoroethylene with glutathione

The nephrotoxic gas chlorotrifluoroethylene is a substrate for glutathione S-transferase activity in rat hepatic cytosolic and microsomal fractions. The rates of reaction, determined by measuring glutathione disappearance, were 5–15 or 35–70 nmol/min/mg of cytosolic or microsomal protein, respectively. Glutathione disappearance was completely abolished by heat-denaturing the subcellular fractions. A product of the cytosolcatalyzed reaction between chlorotrifluoroethylene and glutathione was isolated and shown by amino acid analysis and ¹H- and ¹⁹F-NMR to be S-(2-chloro-1,1,2-trifluoroethyl)glutathione. This appears to be the first demonstration of a glutathione S-transferase-catalyzed addition reaction with a halogenated olefin, and this reaction may be of toxicological significance.