Biomedical subjects
Publications and source records attributed to S M Roberts.
Lactones 2 and 7 were synthesised and tested against six human tumour cell lines (Pancreas-a, BXPC-3), (Thyroid ca, KAT-4), (Thyroid ca, SW1736), (Lung-NSC, NCI-H460), (Pharynx-sq, FADU) and (Prostate, DU-145). Lactone 7 proved inactive, but lactone 2 displayed some activity against four of the six cell lines examined. Both lactones were converted into an intermediate 5 en route to Altohyrtin A.
The synthesis and purinergic receptor binding of novel adenosine A3 ligands is described. Many selective A3 receptor agonists e.g. N-(3-iodobenzyl)adenosine-5'-methyluronamide (IB-MECA) contain a 4'-ribosylalkylamide moiety. We found that this amide and other 4'-functional groups could be replaced with an isosteric isoxazole, and the target molecules retained potent binding to the recombinant human A3 receptor.
Cocaine is capable of producing hepatic necrosis in laboratory animals and humans. Studies in mice indicate that N-oxidative metabolism of cocaine is required for hepatotoxicity and have suggested that toxicity may result from the adduction of proteins by cocaine-reactive metabolites. To aid in identifying protein targets for cocaine-reactive metabolites, an antibody was raised in rabbits immunized with cocaine linked via the tropane nitrogen to a carrier protein (bovine serum albumin). Hepatic proteins from cocaine-treated mice (ICR males, 50 mg of cocaine/kg of body weight, ip) and saline-treated controls were prepared from whole liver homogenate or following subcellular fractionation, and Western blot analyses of hepatic proteins using this antibody were conducted following one- and two-dimensional SDS-PAGE. Analysis of liver homogenate from cocaine-treated mice revealed major protein targets with approximate molecular masses of 20 kDa (pI = 6.0), 44 kDa (two proteins with pI's of 5.0 and 7.0), 52-54 kDa (pI = 4.5), and 64 kDa (pI = 5.5). These specific protein targets were shown to be localized in the mitochondria and microsomes. Several minor bands of immunoreactivity were also seen in mice treated with cocaine, but not in saline-treated controls. Pretreatment of mice with the P450 inhibitor SKF 525A diminished or eliminated the formation of these cocaine-protein adducts. Liver sections from cocaine-treated mice immunostained using the antibody indicated the presence of cocaine-adducted proteins in the centrilobular and midzonal regions of the lobule, corresponding to areas of hepatocyte swelling and necrosis. This study indicates that reactive metabolites from cocaine bind to discrete proteins in specific regions of the liver, consistent with a role for protein adduction in cocaine hepatotoxicity.
The heat shock protein (Hsp) response is induced by heat shock and a large variety of different chemicals. Searching for a common denominator of these different inducers, we and others developed the notion that all inducers may generate abnormally folded, i.e. non-native, proteins, and that such non-native proteins may trigger the Hsp response. Experimentation prompted by this notion resulted, for example, in the demonstration that chemically denatured proteins, introduced in cells by microinjection, can activate the response. Based on the chemical nature of inducers and on results reported from several studies, we hypothesized that inducers of the Hsp response may be generally capable of triggering oxidation of non-protein thiols, particularly glutathione. Such oxidation is known to lead to formation of glutathione-protein mixed disulfides and protein-protein disulfides. Presumably, thiol adduction and cross-linking would affect the structure of proteins involved, resulting in unfolding of a fraction of these proteins, causing heat shock factor (Hsf) activation. To test the feasibility of this hypothesis, thirteen different inducers were selected, and it was shown that all chemical inducers as well as heat shock cause drastic oxidation of glutathione under conditions under which they induce HSE DNA-binding activity. Under the same conditions, all chemical inducers and heat shock also cause trimerization of Hsf1. For several inducers, it was also shown that they enhance thiol oxidation of proteins. Finally, in vitro experiments support the notion that activation of Hsf1 does not require oxidation of the factor itself or of its coregulators. These results are in complete agreement with the above hypothesis.
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The oxidative metabolism of cocaine to norcocaine nitroxide has been postulated to be essential for cocaine hepatotoxicity. The hepatic effects of norcocaine nitroxide have never been evaluated in vivo, however. In this study mice were administered norcocaine nitroxide i.p., and hepatotoxicity was assessed using serum alanine aminotransferase activities and microscopic examination of liver tissue. Hepatotoxicity of norcocaine nitroxide was dose-related; significant injury was detectable at doses of 20 to 30 mg/kg i.p., and severe hepatocellular necrosis was observed at doses of 40 and 50 mg/kg. Elevated serum alanine aminotransferase activities peaked between 12 and 18 hr after norcocaine nitroxide treatment. Electron microscopy revealed the presence of pronounced changes in cell morphology as early as 30 min after the norcocaine nitroxide dose. Pretreatment of mice with phenobarbital had no effect on the magnitude of hepatic injury but shifted the intralobular site of necrosis from the midzonal to the periportal region. Pretreatment with diazinon, an esterase inhibitor, increased norcocaine nitroxide-induced liver damage, whereas each of the P450 inhibitors SKF 525A, cimetidine, troleandomycin, ketaconazole and chloramphenicol significantly diminished norcocaine nitroxide hepatotoxicity. The results indicate that norcocaine nitroxide is hepatotoxic and suggest the involvement of P450 enzymes.
The hepatotoxicity of the analgesic acetaminophen is believed to be mediated by covalent binding to critical proteins. Radiolabeled 3'-hydroxyacetanilide, a regioisomer of acetaminophen, covalently binds to proteins at levels similar to those of acetaminophen, but without toxicity. Covalent binding has recently been detected by Western blot to a 50-kDa microsomal protein that comigrated with CYP2E1 and was accompanied by a loss of the CYP2E1 activity. However, radiolabel studies previously indicated that a significant amount of the radiolabel is lost during electrophoresis. In the present study, 3'-hydroxyacetanilide covalent binding was detected immunohistochemically in liver using an anti-acetaminophen antiserum. 3'-Hydroxyacetanilide (1000 mg/kg, ip) administration to mice resulted in panlobular immunostaining in liver, with the single layer of hepatocytes surrounding the central veins having the greatest intensity of staining. Staining was most intense at 1 hr and somewhat decreased at 3 and 6 hr. In contrast, immunochemical staining indicated that covalent binding of acetaminophen (250 mg/kg, ip) was confined to the centrilobular hepatocytes, the area of the ensuing necrosis. Cobaltous chloride pretreatment decreased the total intensity of the panlobular immunostaining following 3'-hydroxyacetanilide. The CYP2E1 inhibitor diallyl sulfide decreased the intensity of immunostaining in the central vein area only. Western blot analysis indicated diallyl sulfide also eliminated binding to the microsomal 50-kDa protein. These data are consistent with centrilobular binding of 3'-hydroxyacetanilide, mediated in part by CYP2E1, and panlobular binding, mediated by other P450 enzymes.
It was previously shown that a necrogenic dose of acetaminophen (APAP) induced the 25- and 70-kDa heat shock proteins (hsp25 and hsp70i) in mouse liver, whereas nonnecrogenic doses failed to alter the level of either hsp. A strong correlation between the intralobular sites of APAP arylation of protein and hsp induction suggested that APAP-induced protein denaturation may play a role in triggering hsp induction. This study was conducted to determine whether APAP arylation of protein without concurrent toxicity could cause hsp induction. APAP (250 mg/kg i.p.) hepatotoxicity was eliminated using N-acetylcysteine (NAC, 300 mg/kg i.p.) or the cytochrome P-450 inhibitor diallyl sulfide (200 mg/kg p.o.). NAC did not inhibit APAP arylation of protein when administered 1 or 3 hr after the APAP dose but decreased binding by approximately 50% when administered at the same time as the APAP dose. Even though APAP hepatotoxicity was blocked by NAC administered 0 or 1 hr after the APAP dose, NAC did not inhibit the induction of hsp25 or hsp70i, indicating that APAP arylation of protein may play a key role in triggering hsp induction. Diallyl sulfide blocked APAP arylation of protein, hepatotoxicity, and induction of both hsps. These data are consistent with the hypothesis that toxicant adduction of protein triggers hsp induction.
The transcriptional adaptor protein Gcn5 has been identified as a nuclear histone acetyltransferase (HAT). Although recombinant yeast Gcn5 efficiently acetylates free histones, it fails to acetylate histones contained in nucleosomes, indicating that additional components are required for acetylation of chromosomal histones. We report here that Gcn5 functions as a catalytic subunit in two high-molecular-mass native HAT complexes, with apparent molecular masses of 0.8 and 1.8 megadalton (MD), respectively, which acetylate nucleosomal histones. Both the 0.8- and 1.8-MD Gcn5-containing complexes cofractionate with Ada2 and are lost in gcn5delta, ada2delta, or ada3delta yeast strains, illustrating that these HAT complexes are bona fide native Ada-transcriptional adaptor complexes. Importantly, the 1.8-MD adaptor/HAT complex also contains Spt gene products that are linked to TATA-binding protein (TBP) function. This complex is lost in spt20/ada5delta and spt7delta strains and Spt3, Spt7, Spt20/Ada5, Ada2, and Gcn5 all copurify with this nucleosomal HAT complex. Therefore, the 1.8-MD adaptor/HAT complex illustrates an interaction between Ada and Spt gene products and confirms the existence of a complex containing the TBP group of Spt proteins as demonstrated by genetic and biochemical studies. We have named this novel transcription regulatory complex SAGA (Spt-Ada-Gcn5-Acetyltransferase). The function of Gcn5 as a histone acetyltransferase within the Ada and SAGA adaptor complexes indicates the importance of histone acetylation during steps in transcription activation mediated by interactions with transcription activators and general transcription factors (i.e., TBP).
The effect of cocaine on heat shock protein (hsp) induction in murine liver was examined using Western blotting and immunohistochemistry. A single dose of cocaine (50 mg/kg, intraperitoneal [i.p.]) was administered to naive, phenobarbital (PB)-induced or beta-naphthoflavone (betaNF)-induced mice, and the level of hsps in the liver analyzed 3, 6, and 24 hours after the cocaine dose. As measured by Western blotting, hsp70i levels were increased at all time points, and hsp25 levels at the 6- and 24-hour time points. Levels of hsp60, hsc70, and hsp90 remained unchanged. Pretreatment of mice with the cytochrome P-450 inhibitor SKF-525A eliminated both cocaine hepatotoxicity and the induced accumulation of hsp25 and hsp70i. Immunohistochemical localization of hsp25 and hsp70i in the liver showed that concentrations of both hsps were elevated only in cells with altered morphology. As has been observed previously, hepatic enzyme induction with PB or betaNF shifted the location of the necrotic lesion within the lobule from zone 2, as observed in naive mice of this strain, toward zone 1 (PB) or zone 3 (betaNF), respectively. Localization of induced accumulation of hsp25 and hsp70i was found to shift within the lobule in parallel with the necrotic lesion in these animals. Immunostaining of cocaine reactive metabolites bound to proteins was superimposable on the areas with hsp accumulation and cells with altered morphology. Our observations indicate a strong spatial correlation within the lobule between cocaine reactive metabolite formation, induced accumulation of hsp25 and hsp70i, and cytotoxicity (necrosis).
Amphetamine has been shown previously to increase levels of the inducible 70-kDa heat shock protein (hsp70i) in mouse liver. In the present study, the hepatic concentrations of a variety of hsps in livers of mice pretreated with amphetamine (15 mg/kg, i.p.) were evaluated, and the time course of hsp induction was examined. Amphetamine treatment caused an acute rise in core body temperature to 40 degrees C for at least 1 hr and increased hsp25 and hsp70i levels, as measured by Western blotting, at 6, 24, 48, and 72 hr with no apparent induction of other hsps (hsp60, hsc70, or hsp90). A 72-hr amphetamine pretreatment lowered the hepatotoxicity of an acute dose of acetaminophen (350 mg/kg, i.p.) or bromobenzene (0.45 ml/kg, i.p.), but had no effect on the toxicity of carbon tetrachloride (0.04 ml/kg, i.p.) or cocaine (50 mg/kg, i.p.), as measured by serum alanine aminotransferase activity and histopathological analysis. No protection from acetaminophen or bromobenzene hepatotoxicity was observed when hepatotoxicant administration was delayed until hsp levels had returned to control values (144 hr after amphetamine pretreatment). Amphetamine pretreatment did not reduce in vivo covalent binding to proteins of radiolabeled [3H]acetaminophen, [14C]bromobenzene, [14C]carbon tetrachloride, or [3H]cocaine, indicating that the protective effects were not due to inhibition of reactive metabolite formation from these toxicants. These results suggest that elevated levels of hsp25 and hsp70i provide protection against acetaminophen and bromobenzene hepatotoxicity.
Postmature infants calm less when presented with sucrose solutions than term infants. To further assess the influence of postmaturity on sucrose responsivity, several motoric responses were examined in healthy term neonates and chronically stressed postmature (> or = 288 days gestational age (GA) e.g., > 41 weeks) neonates with Clifford's Syndrome tested between 5-91 h of age. Following baseline observations, each subject was presented with 0.1 ml of a 14% sucrose solution for 10 s each min for 5 min, and observations were continued for 5 min following stimulation. During sucrose stimulation and poststimulation phases, postmature infants showed more tremors during quiet state than term infants. Across conditions, postmature infants exhibited increased finger sucking, rhythmic sucking, but less mouth opening and arm movements than healthy, term infants. Sequence analysis revealed that short-latency mouthing, repetitive movements of the upper and lower lips, followed sucrose infusion reliably in postmature infants, but not term infants. These results suggest that postmaturity is associated with: 1. Increased oral behaviors associated with arousal and self soothing; and 2. Increased sucrose reactivity, as indicated by contingent mouthing and increased tremors.
2-(2-Acetoxyethyl)cyclohexanone (4) was converted into the lactone (-)-(5) regio- and enantioselectively using 2-oxo-delta 3-4,5,5-trimethylcyclopentenyl acetyl-CoA monooxygenase, an NADPH-dependent Baeyer-Villiger monooxygenase from camphor grown Pseudomonas putida NCIMB 10007. The lactone (-)-(5) was converted into (R)-(+)-lipoic acid in six steps. In contrast cyclopentanone monooxygenase, an NADPH-dependent Baeyer-Villiger monooxygenase from cyclopentanol-grown Pseudomonas sp. NCIMB 9872 selectively oxidized the (S)-enantiomer of the ketone (4) giving better access to optically enriched, naturally occurring lipoic acid.
The Saccharomyces cerevisiae transcription factor Spt20/Ada5 was originally identified by mutations that suppress Ty insertion alleles and by mutations that suppress the toxicity caused by Gal4-VP16 overexpression. Here we present evidence for physical associations between Spt20/Ada5 and three other Spt proteins, suggesting that they exist in a complex. A related study demonstrates that this complex also contains the histone acetyltransferase, Gcn5, and Ada2. This complex has been named SAGA (Spt/Ada/Gcn5 acetyltransferase). To identify functions that genetically interact with SAGA, we have screened for mutations that cause lethality in an spt20 delta/ada5 delta mutant. Our screen identified mutations in SNF2, SIN4, and GAL11. These mutations affect two known transcription complexes: Snf/Swi, which functions in nucleosome remodeling, and Srb/mediator, which is required for regulated transcription by RNA polymerase II. Systematic analysis has demonstrated that spt20 delta/ada5 delta and spt7 delta mutations cause lethality with every snf/swi and srb/mediator mutation tested. Furthermore, a gcn5 delta mutation causes severe sickness with snf/swi mutations, but not with srb/mediator mutations. These findings suggest that SAGA has multiple activities and plays critical roles in transcription by RNA polymerase II.
Several soil ingestion studies have indicated that some children ingest substantial amounts of soil on given days. Although the EPA has assumed that 95% of children ingest 200 mg soil/day or less for exposure assessment purposes, some children have been observed to ingest up to 25-60 g soil during a single day. In light of the potential for children to ingest such large amounts of soil, an assessment was made of the possibility for soil pica episodes to result in acute intoxication from contaminant concentrations the EPA regards as representing conservative screening values (i.e., EPA soil screening levels and EPA Region III risk-based concentrations for residential soils). For a set of 13 chemicals included in the analysis, contaminant doses resulting from a one-time soil pica episode (5-50 g of soil ingested) were compared with acute dosages shown to produce toxicity in humans in clinical studies or case reports. For four of these chemicals, a soil pica episode was found to result in a contaminant dose approximating or exceeding the acute human lethal dose. For five of the remaining chemicals, the contaminant dose from a soil pica episode was well within the reported dose range in humans for toxicity other than lethality. Because both the exposure episodes and the toxicological response information are derived from observations in humans, these findings are regarded as particularly relevant for human health risk assessment. They suggest that, for some chemicals, ostensibly conservative soil criteria based on chronic exposure using current EPA methodology may not be protective of children during acute soil pica episodes.
Given the widespread interest in antioxidant nutrients, we have developed a new procedure that will permit the automated determination of plasma ascorbic acid (AA) concentration with a Roche Fara centrifugal analyzer. After the deproteinization of plasma with metaphosphoric acid, AA is oxidized to dehydroascorbic acid by AA oxidase. The product is coupled to o-phenylenediamine to produce a chromophore for which the absorbance is measured at 340 nm. The procedure allows much faster throughput than conventional HPLC methods while yielding results that correlate well and provide improved precision. Linearity extends beyond the reference range of 26.1-84.6 micromol/L, and severe hemolysis is the only interference identified.
The effect of acetaminophen (APAP) and 3'-hydroxyacetanilide (AMAP) on heat shock protein (hsp) induction in mouse liver was examined using Western blotting and immunohistochemistry. Western blots from APAP (200 mg/kg i.p.)-treated mice showed increased hsp25 levels at 6 and 24 hr and increased hsp70i levels at 3, 6 and 24 hr. No apparent induction was observed for other hsps (hsp60, hsc70, or hsp90). No increase in the levels of any of the hsps was apparent in Western blots from AMAP (1000 mg/kg i.p.)-treated mice. Immunohistochemical localization of hsp25 and hsp70i in the liver after APAP treatment showed increases in the levels of both hsps within the zone of affected cells at early time points (3 and 6 hr), but at 24 hr, elevated hsp25 levels were observed primarily in cells on the periphery of the lesions. Hepatocytes with increased hsp25 or hsp70i levels also had detectable reactive metabolite binding from APAP, as determined using immunostaining. No hepatotoxicity was observed in liver sections from AMAP treated mice, even though immunostaining indicated widespread reactive metabolite binding. Immunostaining for hsps confirmed that no increase in hsp25 or hsp70i levels occurred in response to this binding. Differences in hsp expression after APAP vs. AMAP may be due to differences in protein targets adducted by their respective reactive metabolites, in the concentrations of adducted proteins or perhaps in some other differential effect necessary for hsp upregulation.
Epoxide hydrolases were found to be constitutively expressed in dematiaceous fungi coincident with secondary metabolite pigment production in stationary or idiophase. Washed-cell preparations of two fungi, Ulocladium atrum CMC 3280 and Zopfiella karachiensis CMC 3284, exhibited affinity for 2,2-dialkylated oxiranes, for which contrasting enantioselectivities were observed, but not for aromatic styrene oxide or alicyclic cyclohexene oxide type substrates. Lyophilised preparations of soluble epoxide hydrolase activities proved to be effective catalysts for the mild hydrolysis of aliphatic epoxides.