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Wayne W Grody

Publications and source records attributed to Wayne W Grody.

13 recordsLinked to original sources
PubMed2003

Widespread expression of arginase I in mouse tissues. Biochemical and physiological implications.

Arginase I (AI), the fifth and final enzyme of the urea cycle, detoxifies ammonia as part of the urea cycle. In previous studies from others, AI was not found in extrahepatic tissues except in primate blood cells, and its roles outside the urea cycle have not been well recognized. In this study we undertook an extensive analysis of arginase expression in postnatal mouse tissues by in situ hybridization (ISH) and RT-PCR. We also compared arginase expression patterns with those of ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT). We found that, outside of liver, AI was expressed in many tissues and cells such as the salivary gland, esophagus, stomach, pancreas, thymus, leukocytes, skin, preputial gland, uterus and sympathetic ganglia. The expression was much wider than that of arginase II, which was highly expressed only in the intestine and kidney. Several co-localization patterns of AI, ODC, and OAT have been found: (a) AI was co-localized with ODC alone in some tissues; (b) AI was co-localized with both OAT and ODC in a few tissues; (c) AI was not co-localized with OAT alone in any of the tissues examined; and (d) AI was not co-localized with either ODC or OAT in some tissues. In contrast, AII was not co-localized with either ODC or OAT alone in any of the tissues studied, and co-localization of AII with ODC and OAT was found only in the small intestine. The co-localization patterns of arginase, ODC, and OAT suggested that AI plays different roles in different tissues. The main roles of AI are regulation of arginine concentration by degrading arginine and production of ornithine for polyamine biosynthesis, but AI may not be the principal enzyme for regulating glutamate biosynthesis in tissues and cells.

Animals↗
PubMed2003

Cystic fibrosis mutations in Costa Rica.

Using polymerase chain reaction amplification of DNA in dried blood spots and a nonisotopic reverse dot blot hybridization method, we performed molecular genetic analysis for 6 and for 16 of the most common mutations of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in 24 unrelated Costa Rican individuals with cystic fibrosis (CF). While many countries and ethnic groups have been surveyed for CF mutations since the cloning of CFTR, Costa Rica has not heretofore been studied. Moreover, Costa Rica represents an especially intriguing population because of its mixed European-African-Amerindian origins and the existence of a detailed historical record of the founding Spanish families. Thus, such a study may reveal not only the population frequencies of various mutant alleles in this country, but also something about their geographic migrations and ethnic founder effects. The most common CF mutation in Caucasians, deltaF508, was found in only 11 (23%) of the CF chromosomes studied, while the G542X mutation, relatively rare in the general population but more common in southern Europe, was observed in 12 (25%). None of the other mutations tested was found in any of the subjects. We failed to detect the second mutant allele in 17 subjects and could not detect either allele in 4 subjects. The high prevalence of the G542X mutation in our cohort, which exceeds that of both the general Caucasian population and the American Hispanic population, reflects the strong genetic influence of the original Spanish founding families of Costa Rica. These results highlight important differences in Costa Rican CF genotypes as compared both to other North American and European populations and to American Hispanics, raising important implications about isolated founder effects and strategies for population screening in that country.

Adolescent↗
PubMed2003

Ethical issues raised by genetic testing with oligonucleotide microarrays.

Because genes and alterations within them determine the identity, characteristics, and inheritance of every individual, the application of genetic science to humans has long been surrounded by apprehension, controversy, and real or perceived potential for abuse. Crude eugenics practices of the past now find a theoretical rebirth and transformation through the use of modern molecular genetic technologies for mutation detection, predictive and prenatal diagnosis, and, ultimately, gene replacement. The advent of oligonucleotide microarray analysis, in which hundreds or thousands of genes and mutations can be tested in parallel, offers tremendous promise for more accurate, sensitive, and efficient genetic testing. At the same time, however, this powerful technology dramatically increases the number and scope of ethical concerns accompanying each individual test request. This article considers the evolution and implications of these concerns, from the initial ordering of a microarray test by the physician to such issues as informed consent, privacy, confidentiality, clinical utility, discrimination, stigmatization, ethnic and population impact, and reimbursement.

Confidentiality↗
PubMed2002

An electrochemical detection scheme for identification of single nucleotide polymorphisms using hairpin-forming probes.

Single nucleotide polymorphisms are implicated as having a significant role in regulating growth, development and, thereby, human health and disease. We have developed a method for identifying single nucleotide genetic alterations by combining hairpin-forming DNA probes and electrochemical detection of sandwich DNA hybridization. Incorporation of hairpin-forming competitor probes and the catalyzed reporter deposition amplification system further improves assay specificity by 7-fold and sensitivity by 100-fold. We have demonstrated that the system successfully identified the factor V Leiden mutations from human blood specimens.

DNA Probes↗
PubMed2002

Cloning and characterization of human agmatinase.

Arginine decarboxylase (ADC) and agmatinase are part of an operon in Escherichia coli, which constitutes the primary pathway of polyamine synthesis from arginine. This pathway is also known to exist in plants, but until recently, neither agmatine nor ADC, the enzyme that synthesizes it, nor agmatinase the enzyme that is responsible for conversion of agmatine to putrescine, were known to exist in man or other mammals. We describe here the cloning of the agmatinase gene and the tissue distribution of its transcription product. Human agmatinase contains 352 amino acid residues and has a calculated molecular weight of 37,688 kDa. It has 56% similarity to E. coli agmatinase and 42% similarity to human arginases I and II and shares highly conserved substrate-binding domains with these well-characterized enzymes.

Amino Acid Sequence↗
PubMed2002

Discordant pursuit asymmetry and esotropia in monozygous twins.

PURPOSE: To report the relationship between infantile esotropia and asymmetry of monocular pursuit tracking. DESIGN: Observational family case series. METHODS: A twin pair discordant for infantile esotropia was confirmed by DNA analysis to be monozygous at 99.95% probability. Digitally sampled electro-oculographic recordings of monocular horizontal pursuit eye movements were performed for the twins and their orthophoric father. RESULTS: Mean directional asymmetry for right and left eyes was 31% and 5% for the esotropic twin, 10% and 8% for the orthophoric twin, and 5% and 6% for the father. The asymmetry was statistically significant only for the deviating right eye of the esotropic twin (P = 0.0217). CONCLUSION: In this family, monocular pursuit asymmetry is probably not a heritable predisposition to infantile esotropia, but a consequence of early strabismus. Discordance of infantile esotropia and pursuit asymmetry in monozygous twins implicates environmental in addition to genetic causes.

DNA↗
PubMed2002

Arginase expression in mouse embryonic development.

We are using the model of the developing mouse embryo to elucidate the pattern of arginase expression in mammalian cells in normal animals and in arginase I (AI) deficiency during development by digoxigenin-labeled RNA in situ hybridization. Our goal is to understand the regulation of these isozymes, with the expectation that this knowledge will help patients suffering from AI deficiency. We found that AI mRNA was widely and strongly expressed in the normal developing mouse embryo; in contrast, a relatively strong AII mRNA signal was found only in the intestine. In the AI knockout mouse embryo, no AII overexpression was found. These results indicated that arginases are needed in mouse embryonic development and AI is the principal form required. The strong AI expression in the peripheral nervous system suggests that the pathogenesis of the neurological retardation in AI deficiency may be conditioned by AI deficiency in the nervous system during embryonic development.

Animals↗
PubMed2002

Expression of the liver form of arginase in erythrocytes.

Arginase I (AI) has a critical function in mammalian liver as the final enzyme in the urea cycle responsible for the disposal of ammonia from protein catabolism. AI is also expressed in various extrahepatic tissues and may play a role in regulating arginine levels and in providing ornithine for biosynthetic reactions that generate various critical intermediary metabolites such as glutamate, glutamine, GABA, agmatine, polyamines, creatine, proline, and nitric oxide. AI is expressed in red blood cells (RBCs) only in humans and certain higher primates. Macaca fascicularis has been identified as an evolutionary transition species in which RBC-AI expression is co-dominantly regulated. The M. fascicularis AI gene was analyzed to understand AI expression in erythrocytes. Erythroid progenitor cells [nucleated red blood cells (nRBCs)] isolated from cord blood were utilized to demonstrate AI expression by immunocytochemical staining using anti-AI antibody. Introduction of EGFP reporter vectors into nRBC showed that the proximal 1.2 kbp upstream of the AI gene is sufficient for AI expression. Expression of a second arginase isoform, AII, in nRBCs was discovered by cDNA profiling. This contrasts with mature fetal or adult RBCs which contain only the AI protein. In addition, an alternatively spliced AI (AI(')) variant was observed from erythroid mRNA analysis with an alternative splice acceptor site located within intron 2, causing the insertion of eight additional amino acids yet retaining significant enzymatic activity.

Alternative Splicing↗
PubMed2002

Mouse model for human arginase deficiency.

Deficiency of liver arginase (AI) causes hyperargininemia (OMIM 207800), a disorder characterized by progressive mental impairment, growth retardation, and spasticity and punctuated by sometimes fatal episodes of hyperammonemia. We constructed a knockout mouse strain carrying a nonfunctional AI gene by homologous recombination. Arginase AI knockout mice completely lacked liver arginase (AI) activity, exhibited severe symptoms of hyperammonemia, and died between postnatal days 10 and 14. During hyperammonemic crisis, plasma ammonia levels of these mice increased >10-fold compared to those for normal animals. Livers of AI-deficient animals showed hepatocyte abnormalities, including cell swelling and inclusions. Plasma amino acid analysis showed the mean arginine level in knockouts to be approximately fourfold greater than that for the wild type and threefold greater than that for heterozygotes; the mean proline level was approximately one-third and the ornithine level was one-half of the proline and ornithine levels, respectively, for wild-type or heterozygote mice--understandable biochemical consequences of arginase deficiency. Glutamic acid, citrulline, and histidine levels were about 1.5-fold higher than those seen in the phenotypically normal animals. Concentrations of the branched-chain amino acids valine, isoleucine, and leucine were 0.4 to 0.5 times the concentrations seen in phenotypically normal animals. In summary, the AI-deficient mouse duplicates several pathobiological aspects of the human condition and should prove to be a useful model for further study of the disease mechanism(s) and to explore treatment options, such as pharmaceutical administration of sodium phenylbutyrate and/or ornithine and development of gene therapy protocols.

Animals↗
PubMed2001

Molecular genetic risk screening.

Under the impetus of the Human Genome Project, new disease-associated genes are being discovered at a rapid pace. Mutations in many of these genes are present in a high enough proportion of the general population, or of particular ethnic groups, that global or targeted population screening can be contemplated. If performed early enough, identification of these mutations by molecular genetic testing can be used not merely to diagnose disease but to predict risk of future disease, either in the individual being tested or in his or her offspring. In some cases this knowledge can be the rationale for heightened surveillance and/or preventive or therapeutic interventions. Mass screening has already commenced for cystic fibrosis mutations and has been discussed for such diverse diseases as hereditary hemochromatosis, thrombophilias, familial cancer predispositions, and pharmacogenetic risk factors. However, implementation of such programs is often impeded by the complexity of the gene mutations, by incomplete penetrance, and by thorny ethical and social issues. This chapter reviews the basic criteria to be considered before embarking on population genetic risk screening, and examines multiple disease-screening examples representing a variety of modes of inheritance and technical challenges.

Cystic Fibrosis↗
PubMedDate not supplied

Standards and guidelines for CFTR mutation testing.

One mission of the ACMG Laboratory Quality Assurance (QA) Committee is to develop standards and guidelines for clinical genetics laboratories, including cytogenetics, biochemical, and molecular genetics specialties. This document was developed under the auspices of the Molecular Subcommittee of the Laboratory QA Committee by the Cystic Fibrosis (CF) Working Group. It was placed on the "fast track" to address the preanalytical, analytical, and postanalytical quality assurance practices of laboratories currently providing testing for CF. Due to the anticipated impact of the ACMG recommendation statement endorsing carrier testing of reproductive couples, it was viewed that CF testing would increase in volume and that the number of laboratories offering CF testing would also likely increase. Therefore, this document was drafted with the premise of providing useful information gained by experienced laboratory directors who have provided such testing for many years. In many instances, "tips" are given. However, these guidelines are not to be interpreted as restrictive or the only approach but to provide a helpful guide. Certainly, appropriately trained and credentialed laboratory directors have flexibility to utilize various testing platforms and design testing strategies with considerable latitude. We felt that it was essential to include technique-specific guidelines of several current technologies commonly used in laboratories providing CF testing, since three of the four technologies discussed are available commercially and are widely utilized. We take the view that these technologies will change, and thus this document will change with future review.

Cystic Fibrosis↗