PubMed Health⌕ Search

Biomedical subjects

L R Pohl

Publications and source records attributed to L R Pohl.

At least 37 records · Page 2Linked to original sources
PubMed1995

Processing of endoplasmic reticulum luminal antigens associated with halothane hepatitis in rat hepatocytes.

In this study we have investigated the mechanism of the processing of trifluoroacetylated liver microsomal protein antigens associated with halothane hepatitis to learn how the immune system might come in contact with these proteins to form antibodies directed against them. Rats were treated with halothane and parenchymal (PC) and non-parenchymal cells (NPC) were isolated 16 hours later. Immunoblotting of the cell lysates with antisera directed against the trifluoroacetyl hapten showed the presence of high levels of trifluoroacetylated proteins in parenchymal cells, whereas none of these proteins were detected in endothelial or Kupffer cells that were isolated by centrifugal elutriation. The half-lives of 100-, 82-, 80-, 63-, 59-, 58-, and 57-kd trifluoroacetylated and native carrier proteins of the trifluoroacetyl hapten in cultures of rat primary parenchymal cells were approximately 1 day. The turnovers of all of these trifluoroacetylated proteins, except for that of the trifluoroacetylated 100-kd protein, were inhibited by treatment of the cells with ammonium chloride, leupeptin, 4-(2-aminoethyl)-benzenesulfonyl fluoride, or 3-methyl-adenine (3-MA). These results indicate that, in liver, the major source of the formation of trifluoroacetylated antigens associated with halothane hepatitis is the parenchymal cells, It appears that most of the trifluoroacetylated antigens and possibly the native carrier protein of the trifluoroacetyl haptens are transferred from the endoplasmic reticulum (ER) to an acidic compartment of PCs, where they are enzymatically degraded. The processing of the trifluoroacetylated proteins by this pathway may be a protective mechanism that prevents these covalently altered proteins from inducing an antibody response in most patients who are administered halothane.

Animals↗
PubMed1994

Characteristics of a purified dog hepatic microsomal N,O-acyltransferase.

Dog liver microsomes have at least three different enzymes that are capable of the deacylation of amides, N-arylhydroxamic acids and carboxylesters, the acyltransfer of N-arylhydroxamic acids and the N-acetylation of arylamines. As judged by SDS-PAGE stained with silver nitrate, one of these enzymes was purified to homogeneity by sequential treatment with Triton X-100, ion-exchange column chromatography, gel filtration and chromatofocusing. The protein was a glycoprotein trimer with a subunit weight of approximately 60 kDa. It showed microheterogeneity on analytical isoelectric focusing (IEF) in polyacrylamide with pls of 5.4-5.6. Following digestion with endoglycosidase H, its subunit weight was reduced to approximately 58 kDa, and it appeared to be homogeneous on IEF with a pl of approximately 5.6. A monoclonal antibody prepared against this enzyme also reacted with the pl 6.0 carboxylesterase of rat liver microsomes, but did not react with the other two dog hepatic acyltransferases. Conversely, a polyclonal antibody raised against the rat esterase reacted with the dog enzyme. The N-terminal sequence of the enzyme was Y-P-S-L-P-P-V-V-D-T-V-Q-G-K-V-, which was homologous to the form 1 carboxylesterase of rabbit liver and the pl 6.0 carboxylesterase of rat liver. Immunohistochemical analyses showed the presence of this enzyme in the epithelium of dog liver and urinary bladder, human liver and rat liver, esophagus, forestomach, glandular stomach, small and large intestines, renal tubules, trachea and prostate and alveolar cells of lung. Since this enzyme is present in the urothelium, it may be important for the activation of urinary metabolites of carcinogenic arylamines for the initiation of bladder carcinogenesis in the dog.

Acyltransferases↗
PubMed1993

Association of anti-58 kDa endoplasmic reticulum antibodies with halothane hepatitis.

We recently showed that when rats were administered the inhalation anesthetic halothane, a 58 kDa liver endoplasmic reticulum protein became covalently trifluoroacetylated by the trifluoroacetyl chloride metabolite of halothane. Although the 58 kDa protein showed 99% identity to that of the deduced amino acid sequence of a cDNA reported to correspond to phosphatidylinositol-specific phospholipase C-alpha, it did not have phosphatidylinositol-specific phospholipase C activity. It was concluded that the reported cDNA of phosphatidylinositol-specific phospholipase C-alpha actually encoded for the 58 kDa endoplasmic reticulum protein of unknown function. Other researchers have come to the same conclusion and have shown that the 58 kDa protein has protein disulfide-isomerase and protease activities. We now report that patients with halothane hepatitis have serum antibodies that react with both purified trifluoroacetylated and native rat liver 58 kDa proteins. These results suggest that when patients are exposed to halothane a human liver orthologue of the rat liver trifluoroacetylated-58 kDa protein is formed. In certain patients, this protein may become immunogenic and lead to the formation of specific antibodies and or specific T-cells, which may react with both trifluoroacetylated and native 58 kDa proteins, and ultimately be responsible, at least in part, for the hepatitis caused by halothane.

Animals↗
PubMed1993

Halothane hepatitis patients have serum antibodies that react with protein disulfide isomerase.

Clinical and laboratory evidence suggests that the fulminant liver failure sometimes associated with the inhalation anesthetic halothane may be an immune-mediated toxicity. Most importantly, the vast majority of patients with a clinical diagnosis of halothane hepatitis have serum antibodies, which react with one or more specific liver microsomal proteins that have been covalently altered by the trifluoroacetyl chloride metabolite of halothane. The serum antibodies are specific to halothane hepatitis patients and are not seen in sera of patients with other types of liver pathology. In this study, a 57-kD trifluoroacetylated liver microsomal neoantigen associated with halothane hepatitis and native 57-kD protein were purified from liver microsomes of halothane-treated and -untreated rats, respectively. When the purified trifluoroacetylated 57-kD and native 57-kD proteins were used as test antigens in an enzyme-linked immunosorbent assay, serum antibodies from halothane hepatitis patients (n = 40) reacted with both of these proteins to a significantly greater extent than did serum antibodies from control patients (n = 32). On the basis of its apparent monomeric molecular mass, isoelectric point and NH2-terminal amino acid and tryptic peptide sequences, the 57-kD protein has been identified as rat liver protein disulfide isomerase. Antibodies raised against rat liver protein disulfide isomerase also reacted with a protein of approximately 58-kD in human liver microsomes. The results of this investigation suggest that trifluoroacetylated protein disulfide isomerase is one of the immunogens associated with halothane hepatitis. In certain patients it might lead either to specific antibodies or, possibly, to specific T cells, which could be responsible for halothane hepatitis.

Antibodies↗
PubMed1993

Immunohistochemical localization of carboxylesterase in the nasal mucosa of rats.

The enzymatic esterase activity of carboxylesterases is integral to the nasal toxicity of many esters used as industrial solvents or in polymer manufacture, including propylene glycol monomethyl ether acetate, dimethyl glutarate, dimethyl succinate, dimethyl adipate, and ethyl acrylate. Inhalation of these chemicals specifically damages the olfactory mucosa of rodents. We report the localization and differential distribution of a 59 KD carboxylesterase in nasal tissues of the rat by immunohistochemistry. Rabbit antiserum against the 59 KD rat liver microsomal carboxylesterase bound most prominently to the olfactory mucosa when applied to decalcified, paraffin-embedded sections of rat nasal turbinates. Within the olfactory mucosa, anti-carboxylesterase did not bind to sensory neurons, the target cell for ester-initiated toxicity; these cells apparently lack carboxylesterase. Instead, the antibody was preferentially bound by cells of Bowman's glands and sustentacular epithelial cells which are immediately adjacent to the olfactory nerve cells. In contrast, non-olfactory tissues (respiratory mucosa and squamous epithelium), which are more resistant to the toxicity of esters, had less carboxylesterase content. The distribution of immunoreactivity correlated well with the distribution of carboxylesterase catalytic activity described elsewhere. These findings help to link the metabolic fate of inhaled esters to the site-specific pathological findings that follow exposure to such chemicals.

Animals↗
PubMed1992

The topography of trifluoroacetylated protein antigens in liver microsomal fractions from halothane treated rats.

Sera from patients with halothane hepatitis contain immunoglobulin G (IgG) antibodies to trifluoroacetylated liver microsomal proteins of 100, 76, 59, 57 and 54 kDa, which are produced as a consequence of metabolism of halothane to trifluoroacetyl halide by cytochrome(s) P450. In the present study, the membrane topographies of the various antigens in rat liver microsomal fractions were investigated. Liver microsomal fractions from rats treated with halothane in vivo, and rat liver microsomal fractions which had been incubated with halothane in vitro, were used as the source of trifluoroacetyl antigens. The antigens were detected by immunoblotting. Whereas the 100, 76, 59 and 57 kDa antigens were solubilized from the microsomal membrane by either 0.1 M sodium carbonate or 0.1% (w/v) sodium deoxycholate, the 54 kDa antigen was not solubilized by 0.1% (w/v) sodium deoxycholate. In intact microsomal fractions, the 100, 76, 59 and 57 kDa antigens were not degraded appreciably by trypsin unless detergent was added to permeabilize the microsomal membrane. These results indicate that the 54 kDa antigen is an integral membrane protein, whereas the 100, 76, 59 and 57 kDa antigens are peripheral membrane proteins situated within the lumen of microsomal vesicles, and hence presumably located within the lumen of the endoplasmic reticulum in vivo.

Animals↗
PubMed1991

Detection of cysteine conjugate metabolite adduct formation with specific mitochondrial proteins using antibodies raised against halothane metabolite adducts.

Antibodies raised against halothane metabolite adducts cross-react with S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine metabolite adducts. Using these antibodies in immunohistochemical experiments, metabolite binding was localized to the damaged areas of the proximal tubule after treatment of male rats with TFEC. Immunoblot analysis of subcellular fractions of rat kidney tissue after in vivo treatment with TFEC revealed a high specificity for binding of metabolites to proteins of the mitochondrial fraction. These proteins may represent target molecules which play a role in cysteine conjugate induced nephrotoxicity.

Acetamides↗
PubMed1991

A metabolite of halothane covalently binds to an endoplasmic reticulum protein that is highly homologous to phosphatidylinositol-specific phospholipase C-alpha but has no activity.

When the inhalation anesthetic halothane was administered to rats, a 58 kDa protein in the liver became covalently labeled by the trifluoroacetyl chloride metabolite of halothane. The amino acid sequences of the N-terminal and of several internal peptide fragments of the protein were 99% homologous to that of the deduced amino acid sequence of a cDNA reported to correspond to phosphatidylinositol-specific phospholipase C-alpha. The purified trifluoroacetylated 58 kDa protein or native 58 kDa protein, however, did not have phosphatidylinositol-specific phospholipase C activity. We conclude that the reported cDNA of phosphatidylinositol-specific phospholipase C-alpha may encode for a microsomal protein of unknown function.

Amino Acid Sequence↗
PubMed1991

Tissue acylation by the chlorofluorocarbon substitute 2,2-dichloro-1,1,1-trifluoroethane.

Hydrochlorofluorocarbons (HCFCs) are being developed as substitutes for ozone-depleting chlorofluorocarbons (CFCs); because widespread human exposure to HCFCs may be expected, it is important to evaluate their toxicities thoroughly. Here we report studies on the bioactivation of the CFC substitute 2,2-dichloro-1,1,1-trifluoroethane (HCFC-123) to an electrophilic intermediate that reacts covalently with liver proteins. HCFC-123 and its analog halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) were studied in rats by 19F NMR spectroscopy, and we found that a trifluoroacetylated lysine adduct was formed with liver proteins. Also, the pattern of proteins immunoreactive with hapten-specific anti-trifluoroacetylprotein antibodies was identical in livers of HCFC-123- and halothane-exposed rats. Because halothane causes an idiosyncratic, and sometimes fatal, hepatitis that is associated with an immune response against several trifluoroacetylated liver proteins, the present findings raise the possibility that humans exposed to HCFC-123 or structurally related HCFCs may be at risk of developing an immunologically mediated hepatitis.

Acylation↗
PubMed1991

Characterization by NMR of the heme-myoglobin adduct formed during the reductive metabolism of BrCCl3. Covalent bonding of the proximal histidine to the ring I vinyl group.

The reductive debromination of BrCCl3 by ferrous deoxymyoglobin leads to the covalent bonding of the prosthetic heme to the protein. We have previously shown, by the use of peptide mapping and mass spectrometry, that histidine residue 93 is covalently bound to the heme moiety. In the present study the structure of the heme adduct was more completely determined by 1H and 13C NMR techniques. We have found that the ring I vinyl group of the prosthetic heme was altered by the addition of a histidine imidazole nitrogen to the alpha-carbon and a CCl2 moiety to the beta-carbon. The electronic absorption spectra of the oxidized and reduced states of the altered heme-protein indicated that the heme-iron exists in a bis-histidine-ligated form. Analysis of the crystal structure of native myoglobin suggested that for the altered heme-protein, histidine residues 97 and 64 are ligated to the heme-iron and that residue 97 has replaced the native proximal histidine residue 93. These movements, in effect a "histidine shuffle" at the active site, may be responsible for the enhanced reducing activity of the altered protein.

Heme↗
PubMed1991

Cloning and sequencing of a human liver carboxylesterase isoenzyme.

A human liver lambda gt11 library was screened with antibodies raised to a purified rat liver carboxylesterase, and several clones were isolated and sequenced. The longest cDNA contained an open reading frame of 507 amino acids that represented 92% of the sequence of a mature carboxylesterase protein. This sequence possessed many structural features that are highly conserved among rabbit and rat liver carboxylesterase proteins, including Ser, His, and Asp residues that comprise the active site, two pairs of Cys residues that may participate in disulfide bond formation, and one Asn-Xxx-Thr site for N-linked carbohydrate addition. When the clone was used to probe human liver genomic DNA that had been digested with various restriction enzymes, many hybridizing bands of differing intensities were observed. The results suggest that the carboxylesterases exist as several isoenzymes in humans, and that they are encoded by multiple genes.

Amino Acid Sequence↗
PubMed1990

Metabolism-based covalent bonding of the heme prosthetic group to its apoprotein during the reductive debromination of BrCCl3 by myoglobin.

The reductive metabolism of BrCCl3 by ferrous myoglobin leads to the alteration of the prosthetic heme to form products that can be dissociated from the protein and to those that are irreversibly bound to the protein. The major dissociable or soluble heme metabolites have recently been characterized. In this study, the irreversibly bound heme product was characterized by Edman degradation, amino acid analysis, and electronic absorption and mass spectrometry of peptides derived from the altered protein. It was found that the prosthetic heme was modified by a CCl2 moiety derived from BrCCl3 and was covalently bound to histidine residue 93, the normal proximal ligand to the heme-iron. The data are consistent with a mechanism by which the trichloromethyl radical reacts with the heme to form an intermediate that either can alkylate the proximal histidine residue or form soluble metabolites. The covalent bonding of the heme prosthetic moiety to the apoprotein likely leads to a change in the tertiary structure of the protein that may be responsible for its altered catalytic activity as well as its enhanced susceptibility to proteolysis. Similar processes may account, at least in part, for the covalent alteration of the heme prosthetic group of other hemoproteins caused by xenobiotics and endogenous substrates.

Amino Acid Sequence↗