Biomedical subjects

Y Israel

Publications and source records attributed to Y Israel.

At least 19 recordsLinked to original sources

PubMed2006

GABA receptor subtype antagonists in the nucleus accumbens shell and ventral tegmental area differentially alter feeding responses induced by deprivation, glucoprivation and lipoprivation in rats.

GABA(A) and GABA(B) receptor agonists stimulate feeding following microinjection into the nucleus accumbens shell and ventral tegmental area, effects blocked selectively and respectively by GABA(A) and GABA(B) receptor antagonists. GABA antagonists also differentially alter opioid-induced feeding responses elicited from these sites. Although GABA agonists and antagonists have been shown to modulate feeding elicited by deprivation or glucoprivation, there has been no systematic examination of feeding elicited by homeostatic challenges following GABA antagonists in these sites. Therefore, the present study examined the dose-dependent ability of GABA(A) (bicuculline, 75-150 ng) and GABA(B) (saclofen, 1.5-3 microg) antagonists administered into the nucleus accumbens shell or ventral tegmental area upon feeding responses elicited by food deprivation (24 h), 2-deoxy-D-glucose-induced glucoprivation (500 mg/kg) or mercaptoacetate-induced lipoprivation (70 mg/kg). A site-specific effect of GABA receptor antagonism was observed for deprivation-induced feeding in that both bicuculline and saclofen administered into the nucleus accumbens shell, but not the ventral tegmental area, produced short-term (1-4 h), but not long-term (24-48 h) effects upon deprivation-induced intake without meaningfully altering body weight recovery. In contrast to the relative inability of GABA receptor antagonism in both sites to alter 2-deoxy-D-glucose-induced intake, mercaptoacetate-induced intake was eliminated by saclofen and significantly reduced by bicuculline in the nucleus accumbens shell and eliminated by both bicuculline and saclofen in the ventral tegmental area. These data reinforce the findings that GABA(A) and GABA(B) receptors in the nucleus accumbens shell and ventral tegmental area are not only important in the modulation of pharmacologically induced feeding responses, but also participate in differentially mediating the short-term feeding response to food deprivation in the nucleus accumbens shell as well strongly modulating lipoprivic, but not glucoprivic feeding responses in both sites.

Animals↗

PubMed2005

NPY-induced feeding: pharmacological characterization using selective opioid antagonists and antisense probes in rats.

The ability of neuropeptide Y to potently stimulate food intake is dependent in part upon the functioning of mu and kappa opioid receptors. The combined use of selective opioid antagonists directed against mu, delta or kappa receptors and antisense probes directed against specific exons of the MOR-1, DOR-1, KOR-1 and KOR-3/ORL-1 opioid receptor genes has been successful in characterizing the precise receptor subpopulations mediating feeding elicited by opioid peptides and agonists as well as homeostatic challenges. The present study examined the dose-dependent (5-80 nmol) cerebroventricular actions of general and selective mu, delta, and kappa1 opioid receptor antagonists together with antisense probes directed against each of the four exons of the MOR-1 opioid receptor gene and each of the three exons of the DOR-1, KOR-1, and KOR-3/ORL-1 opioid receptor genes upon feeding elicited by cerebroventricular NPY (0.47 nmol, 2 ug). NPY-induced feeding was dose-dependently decreased and sometimes eliminated following pretreatment with general, mu, delta, and kappa1 opioid receptor antagonists. Moreover, NPY-induced feeding was significantly and markedly reduced by antisense probes directed against exons 1, 2, and 3 of the MOR-1 gene, exons 1 and 2 of the DOR-1 gene, exons 1, 2, and 3 of the KOR-1 gene, and exon 3 of the KOR-3/ORL-1 gene. Thus, whereas the opioid peptides, beta-endorphin and dynorphin A(1-17) elicit feeding responses that are respectively more dependent upon mu and kappa opioid receptors and their genes, the opioid mediation of NPY-induced feeding appears to involve all three major opioid receptor subtypes in a manner similar to that observed for feeding responses following glucoprivation or lipoprivation.

Animals↗

PubMed2004

Opioid receptor involvement in food deprivation-induced feeding: evaluation of selective antagonist and antisense oligodeoxynucleotide probe effects in mice and rats.

Central administration of general and selective opioid receptor subtype antagonists in the rat has revealed a substantial role for mu, a moderate role for kappa, and a minimal role for delta receptors in the mediation of deprivation-induced feeding. Antisense probes directed against the kappa opioid receptor (KOP), nociceptin opioid receptor (NOP), and delta opioid receptor (DOP) genes in rats result in reductions similar to kappa and delta antagonists, whereas antisense probes directed against the mu opioid receptor (MOP) gene produced modest reductions relative to mu antagonists, suggesting that isoforms of the MOP gene may mediate deprivation-induced feeding. Since these isoforms were initially identified in mice, the present study compared the effects of general and selective opioid receptor antagonists on deprivation-induced feeding in rats and mice and antisense probes directed against exons of the MOP, DOP, KOP, and NOP genes on deprivation-induced feeding in the mouse. Food-deprived (12 and 24 h) rats and mice displayed similar profiles of reductions in deprivation-induced feeding following general, mu, and kappa opioid antagonists. In contrast, mice, but not rats, displayed reductions in deprivation-induced intake following delta antagonism as well as DOP antisense probes, suggesting a species-specific role for the delta receptor. Antisense probes directed against the KOP and NOP genes also reduced deprivation-induced intake in mice in a manner similar to kappa antagonism. However, the significant reductions in deprivation-induced feeding following antisense probes directed against either exons 2, 4, 7, 8, or 13 of the MOP gene were modest compared with mu antagonism, suggesting a role for multiple mu-mediated mechanisms.

Animals↗

PubMed2003

Mutations in mitochondrial aldehyde dehydrogenase (ALDH2) change cofactor affinity and segregate with voluntary alcohol consumption in rats.

Genetic factors influence alcohol consumption and alcoholism. A number of groups have bred alcohol drinker and non drinker rat strains, but genetic determinants remain unknown. The University of Chile rat lines UChA (low drinkers) and UChB (high drinkers) display differences in the relative K(m) for NAD+ of mitochondrial aldehyde dehydrogenase (ALDH2) but no V(max) differences. The relative K(m) differences may be due to mitochondrial changes or to genetic differences coding for ALDH2. We investigated whether there are differences in the coding regions of ALDH2 cDNA in these lines and whether the Aldh2 genotype predicts the phenotype of alcohol consumption and the K(m) of ALDH2 for NAD+. Liver cDNA was prepared, and the Aldh2 transcript was amplified, cloned and sequenced. Genotyping was conducted by DNA amplification and restriction enzyme digestion. When compared to Aldh21 of Sprague-Dawley, 94% of the UChA (low drinker) rats (n = 61), presented a mutation that changes Gln67 to Arg in the mature enzyme (allele referred to as Aldh22). In UChB (high drinker) rats (n = 69), 58% presented the Aldh21 allele, while 42% presented the Gln67Arg change plus a second mutation that changed Glu479 to Lys (allele Aldh23). The Aldh22 allele was absent in high drinker rats. Rats of different Aldh2 genotypes displayed marked phenotypic differences in both ethanol consumption (g/kg/day; means +/- SE): (Aldh21/Aldh21) = 5.7 +/- 0.2, (Aldh22/Aldh22) = 0.9 +/- 0.2 and (Aldh23/Aldh23) = 4.6 +/- 0.2; and K(m)s for NAD+ of 43 +/- 3 microm, 132 +/- 13 microm and 41 +/- 2 microm, respectively (Aldh22 versus Aldh21 or Aldh23; P < 0.0001 for both phenotypes). Overall, the data show that alleles of Aldh2 strongly segregate with the phenotype of ethanol consumption and the relative K(m) for NAD+ of ALDH2. Bases mutated suggest that non drinker Aldh22 is ancestral with regard to the coding changes in either Aldh21 or Aldh23, variants which would allow ethanol consumption and may provide an evolutionary advantage by promoting calorie intake from fermented products along with carbohydrates.

Aldehyde Dehydrogenase↗

PubMed2002

Gene and antisense delivery in alcoholism research.

This article represents the proceedings of a symposium at the 2001 annual meeting of the Research Society on Alcoholism in Montreal, Canada. Drs. Yedy Israel and Fulton Crews were organizers and co-chairpersons. The presentations were (1) Introduction to the symposium, by Yedy Israel; (2) Gene delivery to the brain, by Fulton T. Crews; (3) Gene therapy in alcoholic liver injury, by Ronald Thurman; and (4) Antisense oligonucleotides and antisense-gene delivery, by Yedy Israel.

Alcoholism↗

PubMed2001

Eliciting the low-activity aldehyde dehydrogenase Asian phenotype by an antisense mechanism results in an aversion to ethanol.

A mutation in the gene encoding for the liver mitochondrial aldehyde dehydrogenase (ALDH2-2), present in some Asian populations, lowers or abolishes the activity of this enzyme and results in elevations in blood acetaldehyde upon ethanol consumption, a phenotype that greatly protects against alcohol abuse and alcoholism. We have determined whether the administration of antisense phosphorothioate oligonucleotides (ASOs) can mimic the low-activity ALDH2-2 Asian phenotype. Rat hepatoma cells incubated for 24 h with an antisense oligonucleotide (ASO-9) showed reductions in ALDH2 mRNA levels of 85% and ALDH2 (half-life of 22 h) activity of 55% equivalent to a >90% inhibition in ALDH2 synthesis. Glutamate dehydrogenase mRNA and activity remained unchanged. Base mismatches in the oligonucleotide rendered ASO-9 virtually inactive, confirming an antisense effect. Administration of ASO-9 (20 mg/kg/day for 4 d) to rats resulted in a 50% reduction in liver ALDH2 mRNA, a 40% inhibition in ALDH2 activity, and a fourfold (P < 0.001) increase in circulating plasma acetaldehyde levels after ethanol (1 g/kg) administration. Administration of ASO-9 to rats by osmotic pumps led to an aversion (-61%, P < 0.02) to ethanol. These studies provide a proof of principle that specific inhibition of gene expression can be used to mimic the protective effects afforded by the ALDH2-2 phenotype.

Acetaldehyde↗

PubMed2001

Selection of phage-display library peptides recognizing ethanol targets on proteins.

There is a forthcoming link between chronic alcohol consumption and proteins covalently modified by ethanol metabolites and their antibodies. To identify sensitive probes of protein-ethanol conjugates, we screened for the ethanol-altered protein domains with a phage-display combinatorial peptide library. In principle, recognition of the epitopes by the library peptides occurs through protein-protein interactions. A general screening, M13-based library with 10(9) random sequences of linear heptameric peptides was used. The peptides were displayed in five copies each, as fusion proteins with phage's minor coat protein III. They were located on one end of the surface of the phage particles. The targets were a model protein, streptavidin, and protein-ethanol conjugates (hydroxyethyl radical- or acetaldehyde-modified bovine serum albumin). They were either immobilized on a surface by direct coating or affinity captured on floating beads. An enriched library of phages with the tightest peptide binders for each target was selected and amplified in a multiple-cycle biopanning in vitro procedure. Binders were characterized by DNA sequencing of the corresponding phages and by counter-screening with positive and negative targets in either an enzyme-linked immunosorbent assay or plaque assay. We obtained the HPQ motif for streptavidin and two unique subsets of peptides that recognized each ethanol target with a selectivity of two orders of magnitude above the carrier protein and controls. The application of biopanning processes, coupled with phage-display peptide libraries on biological fluids and tissues, could provide a systematic mapping of protein--ethanol conjugates and supply a means for early diagnosis and prognosis of chronic alcohol consumption in human beings.

Amino Acid Sequence↗

PubMed2001

Alcohol biomarkers: clinical significance and biochemical basis.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Raj Lakshman and Mikihiro Tsutsumi. The presentations were (1) Sialic acid index of apolipoprotein J: A new marker for chronic alcohol consumption, by P. Ghosh and M. R. Lakshman; (2) Microheterogeneity of serum glycoproteins in alcoholics, by M. Tsutsumi and S. Takase; (3) Probing protein-ethanol adducts with combinatorial peptide libraries displayed by filamentous phage, by H. Anni, O. Nikolaeva, and Y. Israel Y; (4) Carbohydrate-deficient transferrin as a marker for heavy alcohol use: What have we learned; Where do we go from here, by R. F. Anton; (5) Sensitivity and specificity of carbohydrate-deficient transferrin in drinking experiments and different patient groups, by O. M. Lesch; (6), Transferrin variants interfere with the measurement of carbohydrate-deficient transferrin, by A. Helender, G. Eriksson, and J-O. Jeppson; and (7) Chronic ethanol on protein trafficking in liver, by P. Marmillot, M. N. Rao, and M. R. Lakshman.

Alcoholism↗

PubMed2001

Genetic selection for high and low alcohol consumption in a limited-access paradigm.

BACKGROUND: Several rat lines have been bred for their differences in alcohol consumption based on a continuous-access paradigm in which alcohol solution is available 24 hr/day. The limited-access paradigm (LAP), in which access to alcohol solution is restricted to a short period per day, however, has been used extensively to investigate the neurochemical mechanisms underlying alcohol consumption. There is evidence of possible differences in genetic determination of alcohol drinking in a continuous- versus limited-access condition. For these reasons, selective breeding for high- and low-alcohol consumption (HARF and LARF, respectively) based on a LAP was conducted. METHODS: N/Nih rats were used as the breeding stock. A within-family breeding procedure was used to develop HARF and LARF lines with 10 families per line. Access to alcohol solution was restricted to 20 min/day. Alcohol was provided as 3%, 6% and 12% w/v solutions. Average intake of alcohol during the 12% phase was used as the selection criterion. Inbreeding began in the seventh generation. RESULTS: After the sixth generation of selection, rats from the HARF line consumed an average of 1.2 g/kg, whereas rats from the LARF line consumed an average of 0.6 g/kg of alcohol during the 20-min access period. Alcohol consumption remained stable over the next eight generations of inbreeding. In the continuous-access-drinking paradigm, the HARF and LARF rats consumed an average of 5.5 to 7.0 g/kg and 1.0 to 2.0 g/kg of alcohol per day respectively. An estimated heritability of 0.25 was obtained. CONCLUSIONS: These findings indicate that alcohol drinking in the LAP is influenced by genetic factors. Differences in alcohol drinking in the LAP also generalize to continuous access drinking. These rat lines will be very useful for investigations into the genetic and neurochemical mechanisms underlying alcohol drinking.

Alcohol Drinking↗

PubMed2001

In vivo delivery of antisense oligonucleotides in pH-sensitive liposomes inhibits lipopolysaccharide-induced production of tumor necrosis factor-alpha in rats.

Kupffer cells play an important role in the pathogenesis of liver diseases. During endotoxemia and alcohol-induced liver disease, tissue injury is preceded by an excessive release of cytokines by these macrophages. Tumor necrosis factor-alpha (TNF-alpha) is one of the key cytokines associated with liver injury. Pre-exposure of animals to TNF-alpha antibodies has been shown to prevent macrophage-mediated liver injury in experimental animals. In this article, we describe a method to encapsulate in pH-sensitive liposomes and to deliver an antisense phosphorothioate oligonucleotide (TJU-2755) against TNF-alpha. We describe the efficacy of this formulation in inhibiting endotoxin-mediated production of TNF-alpha. The liposomes prepared were stable for over 4 weeks at pH 7.4, but readily released their contents when exposed to an acidic environment below pH 6, similar to the pH that exists in early endosomes. Male Sprague-Dawley rats were administered (i.v.) liposome-encapsulated TJU-2755 (1-2 mg/kg body wt.). Empty liposomes served as controls. Forty-eight hours postinjection, the animals were administered a single dose of lipopolysaccharide (50 microg/kg body wt.) and were sacrificed 90 min later. The TNF-alpha produced by excised liver incubated ex vivo and the levels of plasma TNF-alpha were determined. After a single administration of liposome-encapsulated antisense TJU-2755, a 30% reduction in TNF-alpha produced by liver slices was observed. Two daily doses of the antisense oligonucleotide inhibited TNF-alpha production by 50%. This was associated with a 65 to 70% reduction in plasma levels of TNF-alpha, compared with controls. These results indicate that oligonucleotide TJU-2755 encapsulated in pH-sensitive liposomes can be used to effectively reduce endotoxin-mediated production of TNF-alpha in macrophages in vivo and thus may be of value in attenuating or preventing macrophage-mediated liver injury.

Animals↗

PubMed2001

Protein binding of alpha-hydroxyethyl free radicals.

BACKGROUND: The auto-oxidation of ethanol is likely to proceed via the initial formation of hydroxyethyl radicals (HERs), the one-electron oxidation product. In the laboratory, HERs can be generated by the Fenton reaction (H2O2+ Fe+2) in the presence of ethanol. We report studies on the binding of HERs to serum albumin, generated under Fenton and non-Fenton conditions. METHODS: The generation of HER was determined by electron paramagnetic resonance spectroscopy. The formation of ethanol-derived protein adducts was determined by 14C-ethanol incorporation into serum albumin and by the binding of antibodies raised against HER adducts. RESULTS: We report that serum albumin, used as a model protein, is an effective trapping agent of HERs. In addition, HER radicals covalently bind to albumin to form acid stable adducts. Unexpectedly, we found that under aerobic conditions, the incubation of 50 mM ethanol and phosphate buffer (which contains iron traces) in the absence of the Fenton reagent yields HER radicals as shown by electron paramagnetic resonance spectroscopy and the formation of acid stable protein adducts that are recognized by antibodies raised against HER radical adducts. CONCLUSIONS: Proteins (serum albumin used as a model) are avid trapping agents of HER. There are minimal requirements for the generation of HER, because in the presence of oxygen and a phosphate buffer that contains traces of iron, ethanol readily generates HERs. Thus, HER production is likely to occur in many tissues. The ability of proteins to bind this ethanol radical should be valuable in the diagnosis of alcohol abuse and may be relevant to some of the chronic effects of ethanol.

Animals↗

PubMed2000

Autoimmune responses against oxidant stress and acetaldehyde-derived epitopes in human alcohol consumers.

BACKGROUND: Studies in experimental animals have indicated that chronic ethanol ingestion triggers the formation of antibodies directed against proteins modified with reactive metabolites of ethanol and products of lipid peroxidation. However, the nature and prevalence of such antibodies have not been compared previously in alcoholic patients. METHODS: Autoantibodies against adducts with acetaldehyde- (AA), malondialdehyde- (MDA), and oxidized epitopes (Ox) were examined from sera of 54 alcohol consumers with (n = 28) or without (n = 26) liver disease, and from 20 nondrinking controls. RESULTS: Anti-AA-adduct IgA and IgG antibodies were elevated in 64% and 31% of patients with biopsy-proven alcoholic liver disease (ALD, n = 28), respectively. The IgA titers were significantly higher than those from nondrinking controls (p < 0.001), or heavy drinkers without significant liver disease (p < 0.001). Anti-MDA adduct titers (IgG) were elevated in 70% of the ALD patients. These titers were significantly higher (p < 0.001) than those from nondrinking controls, or heavy drinkers without liver disease. Antibodies (IgG) against Ox epitopes occurred in 43% of ALD patients, and the titers also were significantly higher (p < 0.05) than those from nondrinking controls. The anti-AA and anti-MDA adduct titers in ALD patients correlated significantly with the combined clinical and laboratory index (CCLI) of liver disease severity (r(s) = 0.449, p < 0.05; r(s) = 0.566, p < 0.01, respectively), the highest prevalences of anti-AA-adducts (73%) and anti-MDA-adducts (76%) occurring in ALD patients with cirrhosis. CONCLUSIONS: The present results indicated that autoantibodies against several distinct types of protein modifications are generated in ALD patients showing an association with the severity of liver disease.

Acetaldehyde↗

PubMed1999

Generation of acetate and production of ethyl-lysine in the reaction of acetaldehyde plus serum albumin.

We report that incubation of acetaldehyde with bovine serum albumin results in the generation of acetate in a reaction that is directly proportional to the levels of albumin and exponentially dependent on the concentration of acetaldehyde. Both reactants need to be present for acetate to be formed. The oxidation of acetaldehyde into acetate requires that a reduced product also be generated in the reaction. It was hypothesized that, at high concentrations, acetaldehyde itself may reduce the Schiff bases formed in the reaction of a second molecule of acetaldehyde with amino groups in the protein, resulting in the generation of ethyl-lysine moieties. Incubation of acetaldehyde (240 mM) with bovine serum albumin was found to generate ethyl-lysine moieties as determined by a specific monoclonal antibody. Immunization of rabbits with products of the reaction of bovine serum albumin with acetaldehyde led to the generation of antibodies that reacted to reduced adducts formed in the reaction of acetaldehyde and proteins in the presence of sodium cyanoborohydride. However, the generation of acetate from acetaldehyde plus albumin was 60-fold greater than could be explained by the reduction of Schiff bases, as determined by the maximal incorporation of [14C]-acetaldehyde into an acid-precipitable protein fraction. Thus, other mechanisms to generate acetate also occur. The present findings provide an explanation for earlier reports that acetaldehyde adducts formed under "nonreducing" conditions generate antibodies that recognize reduced acetaldehyde protein adducts. However, the mechanism by which the bulk of acetate is generated in the reaction of acetaldehyde and bovine serum albumin remains to be elucidated.

Acetaldehyde↗

PubMed1999

Characterization of adducts of ethanol metabolites with cytochrome c.

Cytochrome c (cyt c) is found in the mitochondria of all mammalian cells where hydrogen peroxide is produced as a byproduct of the electron transport chain. In the presence of peroxide cyt c generates a ferryl heme and radicals at Tyr residues (Barr et al., 1996). These radicals can be transferred to Trp residues within the protein or to Tyr- and Trp-containing peptides (Deterding et al., 1998). We report that addition of ethanol to this system of cyt c plus peroxide results in replacement of the Tyr/Trp radicals by 1-hydroxyethyl radicals (HER), and covalent binding of up to 10 mol of ethanol per mol of cyt c. In the absence of exogenously added peroxide, ethanol incorporation to cyt c is attained also with a reconstituted system of the ethanol-inducible cytochrome P-4502E1 isozyme. Comparative studies with myoglobin and apomyoglobin suggest that the heme is necessary for ethanol adduction of the protein to occur. Structural analysis by mass spectrometry of the tryptic digestion fractions of adducted cyt c is consistent with several peptides bearing one-to-three acetaldehyde moieties on Lys residues, and three distinct Tyr/Trp-containing peptides: P[28-53], P[56-73], P[73-91] carrying one-to-two HER. The x-ray crystallographic structure of cyt c shows that the Tyr/Trp residues in the adducted peptides are in close proximity to the heme. In conclusion, our data show that ethanol metabolites alkylate cyt c under oxidative stress and point to HER-Tyr/Trp adducts as plausible markers of alcoholism.

Amino Acid Sequence↗

PubMed1999

Primary care physicians' views on screening and management of alcohol abuse: inconsistencies with national guidelines.

BACKGROUND: The effects of patients' abuse of and dependence on alcohol are well known, but screening for problem drinking by primary care physicians has been limited. The National Institute of Alcohol Abuse and Alcoholism (NIAAA) recommends that all patients be screened for alcohol use, all users be screened with the CAGE questionnaire, and all nondependent problem drinkers be counseled. We evaluated primary care physicians' screening methods for alcohol use and their management of problem drinkers to determine if they were following the NIAAA guidelines. METHODS: We mailed a questionnaire to 210 internists and family physicians to assess their alcohol screening and management methods. RESULTS: Only 64.9% of the respondents reported screening 80% to 100% of their patients for alcohol abuse or dependence during the initial visit; even less (34.4%) screened that many patients during an annual visit. Nearly all respondents (95%) reported "frequently" or "always" using quantity-frequency questions to screen for alcohol abuse, but only 35% "frequently" or "always" used the CAGE questionnaire. Only 20% of the respondents rated treatment resources as adequate for early problem drinkers, and 72% preferred not to counsel these patients themselves. A belief that a primary care physician could have a positive impact on an alcohol abuser was less likely to be held by respondents who were older, in a nonurban setting, or had more years in practice (P = .05). CONCLUSIONS: A substantial proportion of the physicians in our survey sample were not following NIAAA recommendations. Most physicians preferred not to do the counseling of nondependent problem drinkers themselves, but to refer those patients to a nurse trained in behavioral interventions.

Adult↗

PubMed1998

Tetranucleotide GGGA motif in primary RNA transcripts. Novel target site for antisense design.

Selecting effective antisense target sites on a given mRNA molecule constitutes a major problem in antisense therapeutics. By trial-and-error, only 1 in 18 (6%) of antisense oligonucleotides designed to target the primary RNA transcript of tumor necrosis factor-alpha (TNF-alpha) strongly inhibited TNF-alpha synthesis. Subsequent studies showed that the area in RNA targeted by antisense oligonucleotides could be moved effectively 10-15 bases in either direction from the original area. We observed that only molecules that incorporated a tetranucleotide motif TCCC (complementary to GGGA on RNA) yielded potent antisense oligonucleotides against TNF-alpha. A comprehensive literature survey showed that this motif is unwittingly present in 48% of the most potent antisense oligonucleotides reported in the literature. This finding was prospectively used to predict the sequences of additional antisense oligonucleotides for the rat TNF-alpha primary RNA transcript. Over 50% of antisense constructs (13 of 22) containing the TCCC motif were found to effectively inhibit TNF-alpha synthesis. Marked reductions in mRNA were also observed. This motif was found to be most effective when targeting introns in the primary RNA transcript, suggesting a nuclear localization for the antisense action. Predicting target sites based on the presence of this motif in primary RNA transcripts should be of value in the development on new antisense pharmacotherapy.

Animals↗

PubMed1998

Circulating neutrophils and liver injury in rat models of experimental alcoholic liver disease.

The present study examined the relationship between circulating neutrophils and liver injury in two widely used rat models of chronic ethanol administration. Hematological alterations, liver histopathology, and biochemical indices of liver injury were assessed in rats receiving chronic ethanol by oral liquid diet feeding (Lieber-DeCarli method) or by continuous intragastric infusion (Tsukamoto-French method). Oral administration of ethanol did not affect circulating neutrophil counts, but resulted in minimal liver injury characterized by elevated serum alanine aminotransferase (79%), increased liver mass (15%), and moderate steatosis. In contrast, rats receiving ethanol by continuous intragastric infusion showed an approximately 2-fold increase in circulating neutrophils, and a moderate degree of liver injury, indicated by a 169% elevation of serum alanine aminotransferase and a 2-fold increase in liver mass. Liver biopsies from these rats showed severe steatosis and scattered necrotic hepatocytes, and some neutrophil infiltrates. To determine whether an increase in the number of circulating neutrophils could potentiate liver injury induced by oral ethanol feeding, rats were treated with human recombinant granulocyte colony-stimulating factor at a dose of 100 microg/kg/day (s.c.) for 4 days. Treatment with granulocyte colony-stimulating factor resulted in a 6- to 9-fold increase in circulating neutrophil counts. Nevertheless, this change did not enhance the minor degree of ethanol-induced liver injury in this model. Our results indicate that, whereas neutrophil leukocytosis accompanies more severe manifestations of ethanol hepatotoxicity in rats, this condition per se does not directly induce or exacerbate ethanol-induced liver injury.

Animals↗

PubMed1998

Effect of antithyroid drugs on hydroxyl radical formation and alpha-1-proteinase inhibitor inactivation by neutrophils: therapeutic implications.

The release of proteolytic enzymes and generation of strong oxidants such as the hydroxyl radical by activated neutrophils has been proposed to play an important role in mediating toxin-induced liver injury. The antithyroid drug propylthiouracil protects against liver injury induced by many hepatotoxic agents and markedly reduces mortality in patients with alcoholic liver disease. However, the mechanism(s) by which propylthiouracil protects against liver injury is not well understood. The present studies investigate the effect of antithyroid drugs on proteolytic enzyme activity and on hydroxyl radical generation from activated neutrophils. In the presence of hydrogen peroxide and chloride, neutrophil myeloperoxidase, an enzyme from the same gene superfamily as thyroid peroxidase, generates hypochlorous acid which inactivates alpha-1-proteinase inhibitor (A1PI) present in serum. This inactivation allows neutrophil-released proteolytic enzymes to attack cells. In the present study myeloperoxidase activity was inhibited fully at therapeutic concentrations by antithyroid drugs (propylthiouracil and methimazole). Antithyroid drugs fully prevented hypochlorous acid formation, and prevented neutrophil-mediated inactivation of A1PI, with concomitant blockage of proteolytic activity. Conversely, generation of both superoxide and hydroxyl radicals by activated neutrophils was unaffected by propylthiouracil. The production of these oxygen radicals was fully inhibited by the NADPH oxidase inhibitor diphenylene iodonium chloride, however. These studies indicate that antithyroid drugs are unlikely to prevent cell injury by inhibiting hydroxyl radical generation or by scavenging hydroxyl radicals, but are likely to exert their hepatoprotective anti-inflammatory action by inhibiting neutrophil myeloperoxidase, an enzyme akin to thyroid peroxidase.

Animals↗