Fatal perforating intestinal ulceration attributable to flunixin meglumine overdose in rats.
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Anesthesia↗
Biomedical subjects
R T Miller
Publications and source records attributed to R T Miller.
Induction of inducible nitric-oxide synthase by the heterotrimeric G protein Galpha13.
While the functions of several G protein alpha subunits such as alpha(s( and alpha(q) are relatively well understood, the action of others such as alpha13 remain largely undefined. Because of recent interest in regulation of nitric-oxide synthase (NOS) by G protein-coupled signaling systems and findings that receptors for two proinflammatory substances, thrombin and thromboxane couple to alpha13, we studied the effect of alpha13 on NOS activity in a renal epithelial cell line. We found that stable overexpression of alpha13 or its GTPase-deficient mutant, alpha13Q226L, in a continuous renal epithelial cell line (MCT) increased NOS activity. The increased NOS activity was due to increased expression of the macrophage-inducible form of NOS (iNOS). iNOS protein and activity were not increased in similar cells expressing an activated alpha(s) (alpha(s)Q227L) or were minimally increased in cells expressing activated alpha(i1) (alpha-i1Q204L) and alpha(q) (alpha(q)Q209L), members of the three other G protein alpha chain families. Transient co-expression of alpha13 or alpha13Q226L increased the activity of an iNOS promoter-CAT construct demonstrating that alpha13 increases iNOS expression through transcription. Consequently, alpha13 induces iNOS through a novel mechanism that is distinct from that of other G protein alpha chains and that may mediate the actions of G protein-dependent proinflammatory agents.
Animals↗
Transcription of the murine iNOS gene is inhibited by docosahexaenoic acid, a major constituent of fetal and neonatal sera as well as fish oils.
Macrophage activation is deficient in the fetus and neonate when the serum concentrations of docosahexaenoic acid (DHA) are 150 microM, or 10-50-fold higher than in the adult. We now show that DHA inhibits production of nitric oxide (NO) by macrophages stimulated in vitro by IFNgamma plus LPS, or by IFNgamma plus TNFalpha. The half-maximal inhibitory activity of DHA was approximately 25 microM. There were strict biochemical requirements of the fatty acid for inhibition. Polyenoic fatty acids with 22 carbons were more inhibitory than those with 20 carbons. Among 22-carbon fatty acids, those with a greater number of double bonds and a double bond in the n-3 position were more inhibitory. DHA was the most inhibitory of the polyenoic acids we tested. Inducible nitric oxide synthase (iNOS) is the enzyme responsible for the production of NO by macrophages. NO production is initiated after new iNOS enzyme is synthesized following transcription of the iNOS gene. In macrophages stimulated by IFNgamma plus LPS, DHA inhibited accumulation of iNOS mRNA, as measured by Northern blotting, and iNOS transcription, as measured by nuclear run-on assays. We transfected RAW 264.7 macrophages with a construct containing the iNOS promoter fused to the chloramphenicol acetyl transferase gene. DHA inhibited activation of this promoter by IFN gamma plus LPS. By inhibiting iNOS transcription in the fetus and neonate, DHA may contribute to their increased susceptibility to infection.
Adult↗
The peroxisome proliferations WY-14,643 and methylclofenapate induce hepatocyte ploidy alterations and ploidy-specific DNA synthesis in F344 rats.
WY-14,643 (WY) and methylclofenapate (MCP) are peroxisome proliferators (PP) and hepatocarcinogens in rats. MCP causes hepatic polyploidization and preferentially induces replicative DNA synthesis in binucleate tetraploid hepatocytes (2 X 2N) in young Alpk:AP rats. To compare the effect of WY and MCP on hepatocyte ploidy and ploidy-specific DNA synthesis, male F344 rats were fed WY (0.1% in diet) or gavaged with MCP (25 mg/kg/day in corn oil) for 2, 5, or 10 days. Four rats per treatment group (including corn oil and diet control groups) were euthanized and the livers perfused at each time point. To identify cells undergoing DNA synthesis, all animals received BrdU by continuous infusion for 2 or 5 days prior to euthanasia. Hepatocyte ploidy and DNA synthesis were determined using one- or two-parameter flow cytometry. Averages +/- SEM for adult male F344 rats as a percentage of total hepatocytes for each ploidy subclass are 2N = 3.4 +/- 0.7%, 4N = 69.9 +/- 1.9%, 2 X 2N = 14.4 +/- 2.4%, 8N = 2.2 +/- 0.4%, and 2 X 4N = 9.6 +/- 0.9%. Significant alterations were not induced in the proportions of 2 X 2N or 4N ploidy subclasses by WY or MCP at any time point. However, WY caused increases in 8N hepatocytes at 2, 5, and 10 days (2 days, 5.2% vs 2.2% for controls; 5 days, 7.0% vs 3.1% for controls; 10 days, 6.4% vs 3.6% for controls) as did MCP at 5 and 10 days (5 days, 6.3% vs 2.5% for controls; 10 days, 5.3% vs 2.9% for controls). In addition, a majority of BrdU-containing hepatocytes were 4N following 5 and 10 days of WY and MCP [34.3% (WY) and 16.8% (MCP) vs 1.8% and 1.1% for controls, respectively, for 2 X 2N (5 days) as a percentage of total hepatocytes]. Hepatocytes with intermediary DNA content (between tetraploid and octaploid) from MCP- and WY-treated rats were predominantly mononuclear, the percentage of binucleate hepatocytes being similar to or less than the percentage of binucleate cells within the total tetraploid hepatocyte population. These data suggest that polyploidization is induced by PP and induction of S-phase by WY and MCP occurs primarily in 4N hepatocytes in mature F344 rats and not within 2 X 2N hepatocytes. Identification of a ploidy subpopulation at risk for tumor development in rodents is essential for clarifying the role of cell replication in risk assessment studies of PP.
Animals↗
Effects of intracerebroventricular administration of 5-(glutathion-S-yl)-alpha-methyldopamine on brain dopamine, serotonin, and norepinephrine concentrations in male Sprague-Dawley rats.
alpha-Methyldopamine (alpha-MeDA) is a metabolite of the serotonergic neurotoxicants 3,4-(+/-)-(methylenedioxy)amphetamine (MDA) and 3,4-(+/-)-(methylenedioxy)methamphetamine (MDMA). alpha-MeDA readily oxidizes, and in the presence of glutathione (GSH) it forms 5-(glutathion-S-yl)-alpha-methyldopamine [5-(glutathion-S-yl)-alpha-MeDA]. Since GSH conjugates of many polyphenols are biologically (re)active, we investigated the role of 5-(glutathion-S-yl)-alpha-MeDA in the acute and long-term neurochemical changes observed after administration of MDA. Intracerebroventricular (icv) administration of 5-(glutathion-S-yl)-alpha-MeDA (720 nmol) to male Sprague-Dawley rats produced behavioral changes similar to those reported after subcutaneous administration of MDA. Thus, animals became hyperactive and aggressive and displayed forepaw treading and Straub tails, behaviors usually seen after administration of serotonin (5-HT) releasers, and consistent with a role for 5-(glutathion-S-yl)-alpha-MeDA in some of the behavioral alterations seen after administration of MDA and MDMA. In addition to the behavioral changes, 5-(glutathion-S-yl)-alpha-MeDA also caused short-term alterations in the dopaminergic, serotonergic, and noradrenergic systems. An increase in dopamine synthesis appears to be a prerequisite for the long-term depletion of brain 5-HT following MDMA administration. However, although 5-(glutathion-S-yl)-alpha-MeDA reproduced some of the effects of MDA on the dopaminergic system and was capable of causing acute increases in 5-HT turnover, a single icv injection of 5-(glutathion-S-yl)-alpha-MeDA did not result in long-term serotonergic toxicity. Thus, although acute stimulation of dopamine turnover may be necessary for long-term serotonergic toxicity, such changes are not sufficient to produce these effects. The effects of a multiple dosing schedule of 5-(glutathion-S-yl)-alpha-MeDA will therefore require investigation before we can define a role for this metabolite in MDA and MDMA mediated neurotoxicity. MDA also produces a pressor response that is related to its ability to release neuronal norepinephrine stores, and 5-(glutathion-S-yl)-alpha-MeDA caused comparable depletions of brain norepinephrine concentrations, indicating that both compounds produce similar effects on the noradrenergic system.
Animals↗
Effect on the expression of c-met, c-myc and PPAR-alpha in liver and liver tumors from rats chronically exposed to the hepatocarcinogenic peroxisome proliferator WY-14,643.
The induction of rodent hepatic tumors by peroxisome proliferators (PP) appears to depend on focal growth of hepatocytes. Expression of the oncogenes c-met and c-myc is altered following regenerative stimuli in rat liver, suggesting involvement of their protein products in hepatocyte replication. In addition, increases in c-myc and c-met mRNA expression are observed in multiple types of human and rodent tumors, including hepatocellular carcinoma. A study was designed to test the hypothesis that development of PP-induced hepatic neoplasms occurs as a result of overexpression of c-met or c-myc. Male F344 rats were exposed to WY-14,643 for 22 or 78 weeks (1000 p.p.m. in the diet). Messenger RNA was extracted from liver tumors (78 weeks) and surrounding non-lesion liver of exposed rats and non-lesion liver from age-matched control rats. Levels of mRNA expression were compared using Northern analysis. Significant increases in c-met (approximately 6-fold) and c-myc (approximately 7-fold) mRNA levels were observed in liver tumors compared with liver from control rats. A slight but non-significant increase in mRNA for both of these genes was observed in tumors compared with surrounding non-lesion liver tissue (approximately 2-fold). Increases in mRNA expression of c-met (approximately 3-fold) and c-myc (approximately 5-fold) were also detected in non-lesion liver from WY-14,463-exposed animals compared with non-lesion liver from naive rats. PP exposure in rats increased c-met and c-myc expression in liver and liver tumors, but in a manner which does not correspond to the rapid proliferation of hepatocytes present in tumors. To determine the potential involvement of the PP-activated receptor in PP-induced hepatocarcinogenesis, tumors were also examined for PP-activated receptor expression relative to surrounding liver and liver from naive rats. PP-activated receptor-alpha mRNA levels were significantly increased (approximately 6-fold) in tumors compared with naive liver, but only slightly increased over surrounding non-lesion liver tissue. These results suggest that modulation of c-met, c-myc and PP-activated receptor-alpha are not major determinants of PP-induced hepatocarcinogenesis.
Animals↗
Protein fold recognition by sequence threading: tools and assessment techniques.
Protein fold recognition has been approached by threading an amino acid sequence onto a library of folds, calculating a sequence-structure compatibility score, and ranking these scores. Due to imperfections in the empirically derived pairwise potential functions and the necessarily heuristic approach to the sequence-structure alignment problem, the method benefits from the assessment of threaded models to evaluate the most probable structures among the list of possible folds. THREADER and ANALYST, software tools available through the Internet, facilitate the alignment and assessment steps of a threading prediction. No process has been found to be universally reliable for the detection of folds related to the structure of a known input sequence, but several useful steps and approaches are discussed.
Algorithms↗
Evidence for DNA damage in amyotrophic lateral sclerosis.
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8-Hydroxy-2'-Deoxyguanosine↗
Differential regulation of Ca2+ release-activated Ca2+ influx by heterotrimeric G proteins.
The least understood aspect of the agonist-induced Ca2+ signal is the activation and regulation of the Ca2+ release-activated Ca2+ influx (CRAC) across the plasma membrane. To explore the possible role of heterotrimeric G proteins in the various regulatory mechanisms of CRAC, continuous renal epithelial cell lines stably expressing alpha 13 and the constitutively active alpha qQ209L were isolated and used to measure CRAC activity by the Mn2+ quench technique. Release of intracellular Ca2+ by agonist stimulation or thapsigargin was required for activation of CRAC in all cells. Although the size of the internal stores was similar in all cells, CRAC was 2-3-fold higher in alpha 13- and alpha qQ209L-expressing cells. However, the channel was differentially regulated in the two cell types. Incubation at low [Ca2+]i, inhibition of the NOS pathway, or inhibition of tyrosine kinase inhibited CRAC activity in alpha 13 but not alpha qQ209L cells. Treatment with okadaic acid prevented inhibition of the channel by low [Ca2+]i and the protein kinase inhibitors in alpha 13 cells. These results suggest that expression of alpha qQ209L dominantly activates CRAC by stabilizing a phosphorylated state, whereas expression of alpha 13 makes CRAC activation completely dependent on phosphorylation by several kinases. G proteins may also modulate CRAC activity independently of the phosphorylation/dephosphorylation state of the pathway to increase maximal CRAC activity. Furthermore, our results suggest a general mechanism for regulation of CRAC that depends on coupling of receptors to specific G proteins.
Animals↗
Gemfibrozil-induced peroxisome proliferation and hepatomegaly in male F344 rats.
Gemfibrozil is a widely used hypolipidemic drug in humans that causes peroxisome proliferation and hepatocarcinogenesis in rodents. The induction of hepatomegaly and hepatic peroxisome proliferation (measured as peroxisomal acyl CoA oxidase activity), was determined and compared to another peroxisome proliferator, WY-14,643 (0.1% in the diet) in male F344 rats. In a 21-day study, dietary no-observable-effect and lowest-observable-effect levels of gemfibrozil for both hepatomegaly and peroxisome proliferation were 0.002% and 0.005%, respectively. In a 42-day study, dietary concentrations of 0.9-2.0% gemfibrozil induced a similar magnitude of hepatomegaly to WY-14,643 (2.3-fold) but a higher level of peroxisome proliferation (16-18-fold) than the maximum induction for WY-14,643 (13-fold). The plateau in magnitude of gemfibrozil-induced peroxisome proliferation across the 0.9-2.0% dietary concentrations was associated with a plateau in serum concentration of gemfibrozil (approximately 20 micrograms/ml), similar to concentrations reported in human subjects receiving oral gemfibrozil. These results indicate that maximal induction of peroxisome proliferation by gemfibrozil can exceed that of a more potent compound such as WY-14,643, and further suggest that maximal induction of peroxisome proliferation can be limited by steady-state serum concentrations. Moreover, the reported lack of hepatic responses to gemfibrozil in humans is unlikely to be the result of inefficacy or unavailability of this drug, compared to other peroxisome proliferators, in rodents.
Animals↗
Overexpression of csk inhibits acid-induced activation of NHE-3.
Opossum kidney OKP cells express an apical membrane Na+/H+ antiporter that is encoded by NHE-3 (for Na+/H+ exchanger 3) and is similar in many respects to the renal proximal tubule apical membrane Na+/H+ antiporter. Chronic incubation of OKP cells in acid medium for 24 hr increases Na+/H(+)-antiporter activity and NHE-3 mRNA abundance. The increase in Na+/H(+)-antiporter activity was not prevented by H7, a protein kinase C/protein kinase A inhibitor, but was prevented by herbimycin A, a tyrosine kinase inhibitor. Incubation of cells in acid medium increased c-src activity, and this was inhibited by herbimycin A. To determine the role of the src family of nonreceptor protein-tyrosine kinases, Csk (for carboxyl-terminal src kinase), a physiologic inhibitor of these kinases, was overexpressed in OKP cells. In three clones overexpressing csk, acid-induced increases in Na+/H(+)-antiporter activity and NHE-3 mRNA abundance were inhibited. In these clones, inhibition of acid activation of Na+/H(+)-antiporter activity paralleled inhibition of acid activation of c-src. Neither herbimycin A nor overexpression of csk inhibited dexamethasone-induced increases in Na+/H(+)-antiporter activity. These studies show that decreases in pH activate c-src and that the src family nonreceptor protein-tyrosine kinases play a key role in acid activation of NHE-3.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Successful protein fold recognition by optimal sequence threading validated by rigorous blind testing.
Analysis of the results of the recent protein structure prediction experiment for our method shows that we achieved a high level of success. Of the 18 available prediction targets of known structure, the assessors have identified 11 chains which either entirely match a previously known fold, or which partially match a substantial region of a known fold. Of these 11 chains, we made predictions for 9, and correctly assigned the folds in 5 cases. We have also identified a further 2 chains which also partially match known folds, and both of these were correctly predicted. The success rate for our method under blind testing is therefore 7 out of 11 chains. A further 2 folds could have easily been recognized but failed due to either overzealous filtering of potential matches, or to simple human error on our part. One of the two targets for which we did not submit a prediction, prosubtilisin, would not have been recognized by our usual criteria, but even in this case, it is possible that a correct prediction could have been made by considering a combination of pairwise energy and solvation energy Z-scores. Inspection of the threading alignments for the (alpha beta)8 barrels provides clues as to how fold recognition by threading works, in that these folds are recognized by parts rather than as a whole. The prospects for developing sequence threading technology further is discussed.
Algorithms↗
Biomechanical analysis of experimental diffuse axonal injury.
The purpose of this paper is to present results from methodologies used in our laboratory that are targeted toward identifying specific brain injury thresholds. Results from studying one form of brain injury, diffuse axonal injury, are presented in this report. Physical models, or surrogates, of the skull-brain complex are used to estimate the relationship between inertial loading and brain deformation. A porcine model of diffuse axonal injury, developed with information from these physical models and earlier in vitro tissue modeling studies, is used to correlate histologic and radiologic evidence of axonal injury to predicted regions of injury from the experimental and theoretical analysis. These results form the basis for developing improved diffuse brain injury tolerance levels, as well as identifying new means of diagnostic and treatment techniques for diffuse axonal injury.
Animals↗
TGF alpha differentially expressed in liver foci induced by diethylnitrosamine initiation and peroxisome proliferator promotion.
Transforming growth factor alpha (TGF alpha) is a mitogenic growth factor for hepatocytes that may play a role in the development of liver cancer in rodents and humans. Epidermal growth factor receptor (EGFR) is the receptor for TGF alpha; its expression is also altered in mitogenic and carcinogenic processes. Homogeneous basophilic foci (HBF), the precursor lesion to hepatocellular carcinomas in peroxisome proliferator (PP)-treated rats, have labeling indices much greater than surrounding liver (approximately 5- to 20-fold) and other types of foci (approximately 2-fold greater than eosinophilic foci; EF). To test the hypothesis that PP-induced HBF over-express TGF alpha and/or EGFR, male F344 rats were treated with the PP WY-14,643 for 22 weeks (1000 p.p.m. in the diet) with (DEN-WY) and without (WY) prior diethylnitrosamine initiation (150 mg/kg body wt i.p.). Serial paraffin sections of liver were stained for TGF alpha or EGFR and with hematoxylin and eosin. DEN-WY and WY abrogated the small amount of centrilobular TGF alpha staining observed in livers from control rats. Increased staining for TGF alpha was not observed in HBF induced by WY (0/22) or DEN-WY (0/101). Additionally, increased EGFR expression was not observed in HBF induced by WY (0/22) or DEN-WY (0/19) or in EF induced by DEN-WY (0/30). An unexpectedly large proportion of EF induced by DEN-WY (6/38) and DEN-control (3/11) were TGF alpha-positive. None of the tumors induced by WY (0/13) or DEN-WY (0/11) over-expressed TGF alpha. All 13 WY-induced tumors also lacked increased expression of EGFR. TGF alpha over-expression noted in a significant proportion of EF was associated only with those regimens including DEN initiation. In conclusion, TGF alpha or EGFR over-expression is not associated with early appearing, rapidly proliferating HBF or tumors induced by PP.
Animals↗
G alpha q and G alpha 13 regulate NHE-1 and intracellular calcium in epithelial cells.
To understand the mechanisms by which G protein-coupled signaling systems regulate NHE-1 in epithelial cells, we expressed G alpha q and G alpha 13 in a renal epithelial cell line. We studied two signaling systems that have been implicated in NHE-1 regulation [intracellular Ca (Cai) and phospholipase C activity] and measured NHE-1 activity, mRNA, and antigen. Expression of alpha qWT and alpha qQ209L (a GTPase-deficient mutant) increased basal Cai and altered the kinetics of the bradykinin-induced Cai signal. The initial bradykinin-induced spike in Cai was prolonged and the plateau was higher in cells expressing alpha qWT and alpha qQ209L than in control cells. Cells expressing alpha 13WT also had a higher basal Cai and plateau after stimulation by bradykinin, but Ca release from intracellular stores was similar to that in control cells. Expression of all three alpha-chains increased NHE-1 activity, antigen, and mRNA. The alpha qQ209L had the greatest effect increasing activity by approximately twofold. The alpha 13WT increased NHE-1 activity by approximately 1.5-fold, and alpha qWT increased activity 1.2-fold. These studies demonstrate that alpha q and alpha 13 alter regulation of Cai but by different mechanisms. The Ca signal or another signal generated by alpha q and alpha 13 regulate(s) NHE-1 at the levels of activity, antigen, and mRNA.
Animals↗
Multiple mechanisms for the detection of mirror symmetry.
Symmetry detection was investigated for static and dynamic noise targets consisting of a field of approximately 0.3 million random dots on which was imposed a bilateral symmetry. The minimum duration for detection was 40 ms for static and 80 ms for dynamic symmetry. The exponents of the psychometric functions averaged about 4 for both static and dynamic tasks, as opposed to the value of 1 expected for such suprathreshold tasks, implying that there is some neural mechanism performing full temporal integration of the symmetry information up to durations of a second or more. Static symmetry was perceivable when information around the symmetry axis was masked up to 3 deg away from the symmetry axis, revealing extrafoveal symmetry detection in approximately 300 ms exposures. The static data were fitted with a model consisting of three mechanisms with Gaussian spatial profiles and mutual inhibition (two mechanisms were sufficient for the dynamic data). The profile of the widest mechanism was 20 times wider for static than for dynamic symmetry.
Dominance, Cerebral↗
Peroxisome proliferators: potential role of altered hepatocyte growth and differentiation in tumor development.
Continued, exposure-dependent proliferation of hepatocytes in basophilic proliferative lesions appears to be critical to the mechanism of peroxisome proliferator carcinogenesis in rodents. Identification of the growth regulatory pathway(s) involved in the exaggerated hepatocellular proliferation observed in these lesions is proceeding. So far, regulatory pathways involving cyclophilin and IGFII/M6P receptor have been implicated, while no evidence for involvement of HGF-R, TGF alpha, or PPAR is available. Clearly, this work is preliminary and additional information is needed. Exposure/dose response relationships for hepatocellular proliferation in the basophilic proliferative lesions, as well as species differences in regulation of hepatocellular proliferation, will be important information for development of realistic assessments of cancer risk in humans exposed to peroxisome proliferators. Given the traditional reliance on theoretical models of carcinogenesis that assume carcinogen-DNA interaction and mutation, utilization of newer information on hepatocellular growth and differentiation is expected to significantly change the estimated human risk of peroxisome proliferator-induced cancer.
Animals↗
Expression of G alpha i2 mimics several aspects of LPS priming in a murine macrophage-like cell line.
Priming of macrophages with low concentrations of lipopolysaccharide (LPS) enhances the ability of substances that act through heterotrimeric G proteins to stimulate immune cell functions. Although LPS-induced alterations in the expression and functions of G proteins of the alpha i family have been reported in hematopoietic cells, their effects on subsequent steps in LPS priming of macrophages have not been defined. To study the role of G alpha i2 in priming of macrophages by LPS, we expressed a mutant, activated form of alpha i2 (alpha i2Q205L) in P388D1 cells, and compared its effects on PAF-dependent C alpha signalling and arachidonic acid release to those in cells treated with LPS. In control P388D1 cells, treatment with LPS (100 ng/ml) for 1 hr increased the amount of alpha i2 protein 2-fold. Both LPS treatment and expression of alpha i2Q205L increased the rate of PAF-induced C alpha influx across the cell membrane and arachidonic acid release, although neither altered release of C alpha from intracellular stores by PAF. Expression of alpha i2Q205L is sufficient to mimic the effects of LPS on the PAF-induced C alpha i signal and enhanced arachidonic acid release. Consequently, although increasing the expression of alpha i2 may not be the sole mechanism by which LPS enhances signalling by PAF, increased alpha i2 expression can account for the alterations in PAF-induced C alpha i regulation, and arachidonic acid release in LPS-primed P388D1 cells.
Animals↗