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Biomedical subjects

R L Levine

Publications and source records attributed to R L Levine.

At least 55 records · Page 3Linked to original sources
PubMed2000

Mutational analysis of the CTNNB1 and APC genes in uterine endometrioid carcinoma.

Despite recent studies, the molecular genetic events responsible for the development of uterine endometrioid carcinoma (UEC) remain incompletely characterized. Mutations in the beta-catenin (CTNNB1) gene have been recently reported in a small percentage of UECs and in the endometrioid variant of ovarian carcinoma suggesting that the Wnt signal transduction pathway is involved in the development of female genital tract tumors with endometrioid morphology. The Wnt pathway is a critical pathway in the development of colorectal cancer (CRC) with mutations occurring in the beta-catenin (CTNNB1) or adenomatous polyposis coli (APC) genes in 10 to 15% and 85% of cases, respectively. Because UEC and CRC share other molecular genetic alterations and histologic features and previous studies of UEC have not reported an analysis of the APC gene, we chose to further elucidate the role of the Wnt pathway in UEC. To this end, we analyzed 32 cases of UEC for mutations of the CTNNB1 and APC genes. Mutations of CTNNB1 were present in six of 32 (18%) cases: four grade 1 carcinomas, one grade 2, and one grade 3 carcinoma. Five missense mutations were identified, three involving Ser/Thr phosphorylation sites and two adjacent to a Ser phosphorylation site. One case contained a deletion encompassing codons 34 to 37, which includes a Ser phosphorylation site. No mutations resulting in truncation of the APC protein were found. Our results support a role for the Wnt signaling pathway via mutation of CTNNB1, but not APC, in the development of a subset of UECs.

Blotting, Western↗
PubMed2000

Protein oxidation.

The oxidative modification of proteins by reactive species, especially reactive oxygen species, is implicated in the etiology or progression of a panoply of disorders and diseases. These reactive species form through a large number of physiological and non-physiological reactions. An increase in the rate of their production or a decrease in their rate of scavenging will increase the oxidative modification of cellular molecules, including proteins. For the most part, oxidatively modified proteins are not repaired and must be removed by proteolytic degradation, and a decrease in the efficiency of proteolysis will cause an increase in the cellular content of oxidatively modified proteins. The level of these modified molecules can be quantitated by measurement of the protein carbonyl content, which has been shown to increase in a variety of diseases and processes, most notably during aging. Accumulation of modified proteins disrupts cellular function either by loss of catalytic and structural integrity or by interruption of regulatory pathways.

Antioxidants↗
PubMed1999

Modification of the ADP-ribosyltransferase and NAD glycohydrolase activities of a mammalian transferase (ADP-ribosyltransferase 5) by auto-ADP-ribosylation.

Mono-ADP-ribosylation, a post-translational modification in which the ADP-ribose moiety of NAD is transferred to an acceptor protein, is catalyzed by a family of amino acid-specific ADP-ribosyltransferases. ADP-ribosyltransferase 5 (ART5), a murine transferase originally isolated from Yac-1 lymphoma cells, differed in properties from previously identified eukaryotic transferases in that it exhibited significant NAD glycohydrolase (NADase) activity. To investigate the mechanism of regulation of transferase and NADase activities, ART5 was synthesized as a FLAG fusion protein in Escherichia coli. Agmatine was used as the ADP-ribose acceptor to quantify transferase activity. ART5 was found to be primarily an NADase at 10 microM NAD, whereas at higher NAD concentrations (1 mM), after some delay, transferase activity increased, whereas NADase activity fell. This change in catalytic activity was correlated with auto-ADP-ribosylation and occurred in a time- and NAD concentration-dependent manner. Based on the change in mobility of auto-ADP-ribosylated ART5 by SDS-polyacrylamide gel electrophoresis, the modification appeared to be stoichiometric and resulted in the addition of at least two ADP-ribose moieties. Auto-ADP-ribosylated ART5 isolated after incubation with NAD was primarily a transferase. These findings suggest that auto-ADP-ribosylation of ART5 was stoichiometric, resulted in at least two modifications and converted ART5 from an NADase to a transferase, and could be one mechanism for regulating enzyme activity.

ADP Ribose Transferases↗
PubMed1999

Loss of the ataxia-telangiectasia gene product causes oxidative damage in target organs.

Ataxia-telangiectasia (A-T) is characterized by a markedly increased sensitivity to ionizing radiation, increased incidence of cancer, and neurodegeneration, especially of the cerebellar Purkinje cells. Ionizing radiation oxidizes macromolecules and causes tissue damage through the generation of reactive oxygen species (ROS). We therefore hypothesized that A-T is due to oxidative damage resulting from loss of function of the A-T gene product. To assess this hypothesis, we employed an animal model of A-T, the mouse with a disrupted Atm gene. We show that organs which develop pathologic changes in the Atm-deficient mice are targets of oxidative damage, and that cerebellar Purkinje cells are particularly affected. These observations provide a mechanistic basis for the A-T phenotype and lay a rational foundation for therapeutic intervention.

Animals↗
PubMed1999

Thiols mediate superoxide-dependent NADH modification of glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is covalently modified by NAD in the presence of nitric oxide (NO) and dithiothreitol. Replacement of NAD with NADH in the presence of SIN-1 (3-morpholinosydnonimine) and dithiothreitol increased modification 25-fold. We now demonstrate that in contrast to NO-mediated attachment of NAD, covalent attachment of NADH to GAPDH proceeds in the presence of low molecular weight thiols, independent of NO. Removal of oxygen and transition metal ions inhibited modification, consistent with a role for reactive oxygen species; inhibition by superoxide dismutase, stimulation by xanthine oxidase/hypoxanthine, and the lack of an effect of catalase supported the hypothesis that superoxide, generated from thiol oxidation, was involved. Electrospray mass spectrometry showed covalent linkage of the NADH molecule to GAPDH. Characterization of the product of phosphodiesterase cleavage demonstrated that linkage to GAPDH occurred through the nicotinamide of NADH. Lys-C digestion of GAPDH, followed by peptide isolation by high performance liquid chromatography, matrix-assisted laser desorption ionization time-of-flight analysis, and Edman sequencing, demonstrated that NADH attachment occurred at Cys-149, the active-site thiol. This thiol linkage was stable to HgCl2. Thus, linkage of GAPDH to NADH, in contrast to NAD, occurs in the presence of thiol, is independent of NO, and is mediated by superoxide.

Animals↗
PubMed1999

Peroxynitrite-mediated modification of proteins at physiological carbon dioxide concentration: pH dependence of carbonyl formation, tyrosine nitration, and methionine oxidation.

The ability of peroxynitrite to modify amino acid residues in glutamine synthetase (GS) and BSA is greatly influenced by pH and CO2. At physiological concentrations of CO2 (1.3 mM), the generation of carbonyl groups (0.2-0.4 equivalents/subunit) is little affected by pH over the range of 7.2-9.0, but, in the absence of CO2, carbonyl formation increases (from 0.1- 1.2 equivalents/subunit) as the pH is raised from 7.2 to 10.5. This increase is attributable, in part but not entirely, to the increase in peroxynitrite (PN) stability with increasing pH. Of several amino acid polymers tested, only those containing lysine residues yielded carbonyl derivatives. In contrast, the nitration of tyrosine residues of both GS and BSA at pH 7.5 almost completely depends on the presence of CO2. However, the pH profiles of tyrosine nitration in GS and BSA are not the same. With both proteins, nitration decreases approximately 65% with increasing pH over the range of 7.2-8.4, but, then in the case of GS only, there is a 3.4-fold increase in the level of nitration over the range pH 8.4-8.8. The oxidation of methionine residues in both proteins and in the tripeptide Ala-Met-Ala was inhibited by CO2 at both high and low pH values. These results emphasize the importance of controlling the pH and CO2 concentrations in studies involving PN and indicate that PN is not likely to contribute appreciably to carbonyl formation or oxidation of methionine residues of proteins at physiological pH and CO2 concentrations.

Carbon Dioxide↗
PubMed1999

Modification of cysteine residues in vitro and in vivo affects the immunogenicity and antigenicity of major histocompatibility complex class I-restricted viral determinants.

In studying the subdominant status of two cysteine-containing influenza virus nuclear protein (NP) determinants (NP39-47 and NP218-226) restricted by H-2Kd, we found that the antigenicity of synthetic peptides was enhanced 10-100-fold by treatment with reducing agents, despite the fact that the affinity for Kd was not enhanced. Reducing agents also markedly enhanced the immunogenicity of cysteine-containing peptides, as measured by propagation of long-term T cell lines in vitro. Similar enhancing effects were obtained by substituting cysteine with alanine or serine in the synthetic peptides, demonstrating that sulfhydryl modification of cysteine is responsible for the impaired antigenicity and immunogenicity of NP39-47 and NP218-226. We found similar effects for two widely studied, cysteine-containing peptides from lymphocytic choriomeningitis virus. The major modifications of cysteine-containing synthetic peptides are cysteinylation and dimerization occurring through cysteine residues. We demonstrate that both of these modifications occur in cells synthesizing a cytosolic NP218-226 minigene product and, further, that T cells specific for cysteinylated NP218-226 are induced by influenza virus infection in mice, demonstrating that this modification occurs in vivo. These findings demonstrate that posttranslational modifications affect the immunogenicity and antigenicity of cysteine-containing viral peptides and that this must be considered in studying the status of such peptides in immunodominance hierarchies.

Amino Acid Sequence↗
PubMed1999

Methionine residues may protect proteins from critical oxidative damage.

Cysteine and methionine are the two sulfur-containing residues normally found in proteins. Cysteine residues function in the catalytic cycle of many enzymes, and they form disulfide bonds which contribute to protein structure. In contrast, the key functions of methionine residues are not known. We propose that methionine residues constitute an important antioxidant defense mechanism. A variety of oxidants react readily with methionine to form methionine sulfoxide, and surface exposed methionine residues create an extremely high concentration of reactant, providing for efficient scavenging of oxidants. The effect of hydrogen peroxide exposure upon glutamine synthetase from Escherichia coli was studied as an in vitro model system. Eight of the sixteen methionine residues could be oxidized with little effect on activity. The oxidizable methionine residues were found to be relatively surface exposed while the intact residues were generally buried within the core of the protein. Further, the susceptible residues were physically arranged in an array which guarded the entrance to the active site. Methionine sulfoxide can be reduced back to methionine by the enzyme methionine sulfoxide reductase, providing a catalytic amplification of the antioxidant potential of each methionine residue. Given the importance of oxidative stress during aging, the potential function of methionine residues as antioxidants during aging should be investigated experimentally.

Antioxidants↗
PubMed1999

Molecular cloning and characterization of a mitochondrial selenocysteine-containing thioredoxin reductase from rat liver.

A thioredoxin reductase (TrxR), named here TrxR2, that did not react with antibodies to the previously identified TrxR (now named TrxR1) was purified from rat liver. Like TrxR1, TrxR2 was a dimeric enzyme containing selenocysteine (Secys) as the COOH-terminal penultimate residue. A cDNA encoding TrxR2 was cloned from rat liver; the open reading frame predicts a polypeptide of 526 amino acids with a COOH-terminal Gly-Cys-Secys-Gly motif provided that an in-frame TGA codon encodes Secys. The 3'-untranslated region of the cDNA contains a canonical Secys insertion sequence element. The deduced amino acid sequence of TrxR2 shows 54% identity to that of TrxR1 and contained 36 additional residues upstream of the experimentally determined NH2-terminal sequence. The sequence of this 36-residue region is typical of that of a mitochondrial leader peptide. Immunoblot analysis confirmed that TrxR2 is localized almost exclusively in mitochondria, whereas TrxR1 is a cytosolic protein. Unlike TrxR1, which was expressed at a level of 0.6 to 1.6 microgram/milligram of total soluble protein in all rat tissues examined, TrxR2 was relatively abundant (0.3 to 0.6 microgram/mg) only in liver, kidney, adrenal gland, and heart. The specific localization of TrxR2 in mitochondria, together with the previous identification of mitochondria-specific thioredoxin and thioredoxin-dependent peroxidase, suggest that these three proteins provide a primary line of defense against H2O2 produced by the mitochondrial respiratory chain.

Amino Acid Sequence↗
PubMed1999

Laparoscopy in morbidly obese patients.

STUDY OBJECTIVE: To assess the safety and efficacy of different insufflation methods in morbidly obese women undergoing laparoscopy. DESIGN: Retrospective analysis of 13 years' experience (Canadian Task Force classification II-2). SETTING: University-affiliated hospital. PATIENTS: One hundred thirty-eight morbidly obese women (weight >250 lbs, body mass index >36). The heaviest patient weighed 400 lbs and had a body mass index of 66. INTERVENTION: Laparoscopic tubal sterilizations and diagnostic laparoscopies performed on an outpatient basis by residents under faculty supervision. MEASUREMENTS AND MAIN RESULTS: Of 138 patients, 36 underwent standard transumbilical insufflation with 5 failures, 83 had transuterine insufflation with 3 failures, 12 had subcostal insufflation with 1 failure, and 7 had open laparoscopy with 2 failures. CONCLUSION: The insufflation failure rate was significantly high for transumbilical insufflation and open laparoscopy, and not for transuterine or subcostal insufflation. Morbid obesity was not a contraindication to laparoscopy. (J Am Assoc Gynecol Laparosc 6(3):307-312, 1999)

Adult↗
PubMed1999

Influence of age upon complications of carotid artery stenting. UAB Neurovascular Angioplasty Team.

PURPOSE: To examine the impact of age upon the development of neurological and major systemic complications during or after carotid artery stenting. METHODS: We reviewed the complications that occurred in patients undergoing elective carotid stent implantation between September 1994 and August 1996. The study population was then divided into 3 groups according to age: > or = 80 (group A), 75 to 79 (group B), and < or = 74 (group C) years. The rates of death, major and minor stroke, and myocardial infarction were compared among the groups, as well as with the rates reported by the major carotid endarterectomy (CEA) trials. RESULTS: During the study period, 182 patients (216 vessels) were treated with carotid stenting. There were 19 (10.4%) complications: 1 (0.5%) death, 2 (1.1%) major strokes, 15 (8.2%) minor strokes, and 1 (0.5%) myocardial infarction. Neurological complications were clearly related to increased age with rates of 25.0% in group A versus 8.6% in group C (p = 0.042). The overall per patient rate of death or major stroke was 1.6% (1.4% per vessel). CONCLUSIONS: Increasing age has a negative impact on the rate of complications in carotid stent patients. However, the majority of those complications are minor. The relative roles of medical therapy, stenting, and CEA in patients > 80 years of age must await the results of randomized trials.

Age Factors↗
PubMed1998

The levels of retinal mRNA for gefiltin, a neuronal intermediate filament protein, are regulated by the tectum during optic fiber regeneration in the goldfish.

Reorganization of the intermediate filament (IF) network during axonal regeneration is accompanied by changes in the expression of various IF proteins. An increase in expression of the neuronal IF subunit gefiltin in goldfish retinal ganglion cells (RGCs) has been linked to the unique ability of the goldfish optic nerve to regenerate following injury. Evidence suggests that the optic tectum, the target of optic fibers, may regulate the expression of gefiltin during regeneration. To address this issue we examined gefiltin mRNA levels during optic fiber regeneration in the presence or absence of the tectum. We found that gefiltin mRNA levels in the RGCs of animals that received an optic nerve crush (ONC group) began increasing by 10 days, peaked from 20 to 38 days at 5.5-fold over normal, and declined to near normal values by 115 days. In animals that had the entire tectum removed as well as an optic nerve crush (ETR group), gefiltin mRNA levels increased by 10 days, peaked at 20 days at 5.5 to 6.5-fold over normal, and although they dropped slightly thereafter, they remained elevated at 5-fold over normal for at least 115 days. When axons regenerated to the ipsilateral tectal lobe as a result of a left tectal lobe removal and left eye removal surgery (LTR/LER group), the expression pattern of gefiltin mRNA paralleled that of the ONC group. We also found that the abundance of gefiltin subunits in the retina was elevated at 30 days of regeneration in ONC and ETR animals, and that levels in the nerve were reconstituted to 80% of normal by 30 days. These results demonstrate that increases in gefiltin mRNA and protein levels during optic nerve regeneration are independent of the tectum, whereas the downregulation of gefiltin mRNA levels in the late stages of regeneration is entirely dependent upon the tectum.

Animals↗
PubMed1998

PTEN mutations and microsatellite instability in complex atypical hyperplasia, a precursor lesion to uterine endometrioid carcinoma.

The two most common types of genetic alterations yet identified in uterine endometrioid carcinoma (UEC) are PTEN mutations and microsatellite instability (MI). Furthermore, MI-positive UECs (defined as tumors with detectable alterations at two or more different microsatellite loci) are significantly more likely to contain PTEN mutations than are MI-negative UECs. To determine whether PTEN inactivation is a relatively early event in endometrial tumorigenesis, we evaluated complex atypical hyperplasia (CAH), the direct precursor to UEC, for the presence of PTEN mutations. Mutations were present in 3 of 11 (27%) CAHs with synchronous UEC and in 4 of 18 (22%) CAHs that were not associated with invasive carcinoma. One case with synchronous CAH and UEC contained a germ-line PTEN mutation. In addition, we evaluated the same series of CAHs for MI. We identified four MI-positive CAHs with synchronous UEC but did not detect the MI phenotype in any CAHs without associated invasive carcinoma. A PTEN-mutant (germ-line mutation) MI-negative CAH was synchronous with a PTEN-mutant MI-positive UEC. These results suggest that mutation of PTEN can be an early event in the pathogenesis of UEC and may precede the development of the MI phenotype in a subset of cases.

Carcinoma, Endometrioid↗
PubMed1998

Iron-dependent oxidation, ubiquitination, and degradation of iron regulatory protein 2: implications for degradation of oxidized proteins.

The ability of iron to catalyze formation of reactive oxygen species significantly contributes to its toxicity in cells and animals. Iron uptake and distribution is regulated tightly in mammalian cells, in part by iron regulatory protein 2 (IRP2), a protein that is degraded efficiently by the proteasome in iron-replete cells. Here, we demonstrate that IRP2 is oxidized and ubiquitinated in cells before degradation. Moreover, iron-dependent oxidation converts IRP2 into a substrate for ubiquitination in vitro. A regulatory pathway is described in which excess iron is sensed by its ability to catalyze site-specific oxidations in IRP2, oxidized IRP2 is ubiquitinated, and ubiquitinated IRP2 subsequently is degraded by the proteasome. Selective targeting and removal of oxidatively modified proteins may contribute to the turnover of many proteins that are degraded by the proteasome.

Animals↗