Ramaswamy K. Iyer

Helicobacter pylori Induces Macrophage Apoptosis by Activation of Arginase II

Helicobacter pylori infection induces innate immune responses in macrophages, contributing to mucosal inflammation and damage. Macrophage apoptosis is important in the pathogenesis of mucosal infections but has not been studied with H. pylori. NO derived from inducible NO synthase (iNOS) can activate macrophage apoptosis. Arginase competes with iNOS by converting l-arginine to l-ornithine. Since we reported that H. pylori induces iNOS in macrophages, we now determined whether this bacterium induces arginase and the effect of this activation on apoptosis. NF-κB-dependent induction of arginase II, but not arginase I, was observed in RAW 264.7 macrophages cocultured with H. pylori. The time course of apoptosis matched those of both arginase and iNOS activities. Surprisingly, apoptosis was blocked by the arginase inhibitors Nω-hydroxy-l-arginine or Nω-hydroxy-nor-l-arginine, but not by the iNOS inhibitor N-iminoethyl-l-lysine. These findings were confirmed in peritoneal macrophages from iNOS-deficient mice and were not dependent on bacterial-macrophage contact. Ornithine decarboxylase (ODC), which metabolizes l-ornithine to polyamines, was also induced in H. pylori-stimulated macrophages. Apoptosis was abolished by inhibition of ODC and was restored by the polyamines spermidine and spermine. We also demonstrate that arginase II expression is up-regulated in both murine and human H. pylori gastritis tissues, indicating the likely in vivo relevance of our findings. Therefore, we describe arginase- and ODC-dependent macrophage apoptosis, which implicates polyamines in the pathophysiology of H. pylori infection.

Mouse Model for Human Arginase Deficiency

ABSTRACT 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.

The pathogenesis of ataxia-telangiectasia. Learning from a Rosetta Stone

ConclusionMany issues in A-T research remain unresolved. However, certain common denominators are becoming clear that were not obvious just a few years ago, i.e., the relationship between double-strand break rejoining, radiosensitivity, and immunodeficiency. The downstream pathways that are activated by ATM phosphorylating p53, c-abl, chk2, Brca1, nibrin, RPA, and other proteins that contribute to cell-cycles checkpoints and DNA repair complexes, are helping to understand the cancer susceptibility of A-T patients, and perhaps of A-T heterozygotes. The identification and categorization of mutations in the ATM, NBS and Mre11 genes now allow more definitive diagnoses. Developmental studies are rapidly identifying early sites of pathogenesis that can perhaps be targeted for neural stem cell implantation therapy. If over-whelmed oxidative stress responses underlie the neurological degeneration of A-T patients, maintaining adequate therapeutic levels of free-radical scavengers, such as Vitamin E, alpha-lipoic acid, aspirin, and Coenzyme Q10, may be of some therapeutic benefit.

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.

Expression of arginase isozymes in mouse brain

The two forms of arginase (AI and AII) in man, identical in enzymatic function, are encoded in separate genes and are expressed differentially in various tissues. AI is expressed predominantly in the liver cytosol and is thought to function primarily to detoxify ammonia as part of the urea cycle. AII, in contrast, is predominantly mitochondrial, is more widely expressed, and is thought to function primarily to produce ornithine. Ornithine is a precursor in the synthesis of proline, glutamate, and polyamines. This study was undertaken to explore the cellular and regional distribution of AI and AII expression in brain using in situ hybridization and immunohistochemistry. AI and AII were detected only in neurons and not in glial cells. AI presented stronger expression than AII, but AII was generally coexpressed with AI in most cells studied. Expression was particularly high in the cerebral cortex, cerebellum, pons, medulla, and spinal cord neurons. Glutamic acid decarboxylase 65 and glutamic acid decarboxylase 67, postulated to be related to the risk of glutamate excitotoxic and/or γ‐aminobutyric acid inhibitoxic injury, were similarly ubiquitous in their expression and generally paralleled arginase expression patterns, especially in cerebral cortex, hippocampus, cerebellum, pons, medulla, and spinal cord. This study showed that AI is expressed in the mouse brain, and more strongly than AII, and sheds light on the anatomic basis for the arginine→ornithine→glutamate→GABA pathway. J. Neurosci. Res. 66:406–422, 2001.

The tetraspan protein epithelial membrane protein-2 interacts with beta1 integrins and regulates adhesion.

The growth arrest-specific-3 (GAS3)/PMP22 proteins are members of the four-transmembrane (tetraspan) superfamily. Although the function of these proteins is poorly understood, GAS3/PMP22 proteins have been implicated in the control of growth and progression of certain cancers. Epithelial membrane protein-2 (EMP2), a GAS3/PMP22 family member, was recently identified as a putative tumor suppressor gene. Here, we addressed the normal function of EMP2 by testing the prediction that it influences integrin-related cell functions. We observed that EMP2 associates with the β1 integrin subunit. Co-immunoprecipitation and immunodepletion experiments indicated that ∼60% of β1integrins and EMP2 can be isolated in common protein complexes. Whereas this association between EMP2 and β1 integrin may be direct or indirect, it has features of integrin heterodimer selectivity. Thus, by laser confocal microscopy, EMP2 colocalized with α6β1 but not α5β1 integrin. Increased expression of EMP2 also influenced the integrin heterodimer repertoire present on the plasma membrane. EMP2 specifically increased the surface expression of the α6β1 integrin while decreasing that of the α5β1 protein. Reciprocally, reduction in EMP2 expression using a specific ribozyme decreased surface expression of α6β1 integrin. Accordingly, these EMP2-mediated changes resulted in a dramatic alteration in cellular adhesion to extracellular matrix proteins. This study demonstrates for the first time the interaction of a GAS3/PMP22 family member with an integrin protein and suggests that such interactions and their functional consequences are a physiologic role of GAS3/PMP22 proteins.

New observations on maternal age effect on germline de novo mutations

Germline mutations are the source of evolution and contribute substantially to many health-related processes. Here we use whole-genome deep sequencing data from 693 parents–offspring trios to examine the de novo point mutations (DNMs) in the offspring. Our estimate for the mutation rate per base pair per generation is 1.05 × 10−8, well within the range of previous studies. We show that maternal age has a small but significant correlation with the total number of DNMs in the offspring after controlling for paternal age (0.51 additional mutations per year, 95% CI: 0.29, 0.73), which was not detectable in the smaller and younger parental cohorts of earlier studies. Furthermore, while the total number of DNMs increases at a constant rate for paternal age, the contribution from the mother increases at an accelerated rate with age.These observations have implications related to the incidence of de novo mutations relating to maternal age.

Utility of whole-genome sequencing for detection of newborn screening disorders in a population cohort of 1,696 neonates

Purpose:To assess the potential of whole-genome sequencing (WGS) to replicate and augment results from conventional blood-based newborn screening (NBS).Methods:Research-generated WGS data from an ancestrally diverse cohort of 1,696 infants and both parents of each infant were analyzed for variants in 163 genes involved in disorders included or under discussion for inclusion in US NBS programs. WGS results were compared with results from state NBS and related follow-up testing.Results:NBS genes are generally well covered by WGS. There is a median of one (range: 0–6) database-annotated pathogenic variant in the NBS genes per infant. Results of WGS and NBS in detecting 28 state-screened disorders and four hemoglobin traits were concordant for 88.6% of true positives (n = 35) and 98.9% of true negatives (n = 45,757). Of the five infants affected with a state-screened disorder, WGS identified two whereas NBS detected four. WGS yielded fewer false positives than NBS (0.037 vs. 0.17%) but more results of uncertain significance (0.90 vs. 0.013%).Conclusion:WGS may help rule in and rule out NBS disorders, pinpoint molecular diagnoses, and detect conditions not amenable to current NBS assays.Genet Med 18 3, 221–230.

A Novel Method for Creating Artificial Mutant Samples for Performance Evaluation and Quality Control in Clinical Molecular Genetics

The lack of readily available, patient-derived materials for molecular genetic testing of many heterozygous or rare disorders creates a major impediment for laboratory proficiency and quality control procedures. The paucity of clinically derived mutation-positive samples could be surmounted if it were possible to construct artificial samples containing mutations of interest that would sufficiently resemble natural human samples. Such samples could then function as acceptable and realistic performance evaluation challenges and quality control reagents for recipient laboratories. Using the cystic fibrosis gene (CFTR) as a prototype, we have devised and executed experiments designed to generate unique DNA samples that could be used for these purposes. We used site-directed mutagenesis to generate mutations of interest in plasmid DNA derived from common bacterial artificial chromosome sources containing the cystic fibrosis transmembrane conductance receptor gene. CFTR mutations G85E and 1078delT were chosen to represent mutations in the original American College of Medical Genetics-recommended population-screening panel of 25 mutations. DNA samples containing predetermined concentrations and ratios of wild-type and mutated plasmids, bacterial artificial chromosomes of interest, and nonhuman genomic carrier DNA were characterized and tested in-house and in a group of nine pilot testing laboratories using a variety of technical platforms. The results indicate that these constructs, containing CFTR mutations in heterozygous and homozygous states, can serve as valid and accessible materials for quality assurance, including performance evaluation, proficiency testing, and assay quality control.

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.