Rat Liver Protein Research Articles

Seasonal variation of xanthine oxidoreductase activity in the digestive gland cells of the mussel Mytilus galloprovincialis: A biochemical, histochemical and immunochemical study

The activity and the tissue distribution of the oxygen radical producing enzyme xanthine oxidoreductase (XOR) were measured in the digestive gland of the common marine mussel Mytilus galloprovincialis Lmk along an annual cycle. No xanthine oxidase (XOX) activity could be measured, the enzyme only displaying xanthine dehydrogenase (XDH) activity in all the cases. This is interpreted as a mechanism to avoid the harmful effects of the oxygen radicals that would be produced by XOX during periods following anoxic conditions at low tide. The highest XDH activities coincided with the late spring/early summer months, the activity maxima being recorded from May to July. Histochemically XOR activity was very pronounced in duct and stomach epithelial cells as well as in the surrounding connective tissue and hemolymph vessels, the activity increasing towards the summer months. These seasonal variations in XDH or XOR activities are possibly linked to hormonal changes governing the reproductive cycle and to changes in food availability. The localization of the protein in the connective tissue lining the hemolymph vessels was confirmed immunohistochemically using a polyclonal antibody against rat liver protein that cross-reacted specifically with a polypeptide of 150 kDa of molecular mass in homogenates of the digestive gland. This polypeptide was linked to cytosolic fractions isolated by differential centrifugation from mussel digestive glands. In paraffin sections the antibody labeled the digestive cells of digestive tubules, as well as the connective tissue surrounding the hemolymph vessels, gonadal follicles, digestive epithelia and certain protozoan parasites. Taken together our results suggest that in the digestive gland of bivalve molluscs XOR is involved in the metabolism of purines and in the scavenging of oxygen free radicals.

Seasonal variation of xanthine oxidoreductase activity in the digestive gland cells of the mussel Mytilus galloprovincialis: A biochemical, histochemical and immunochemical study

The activity and the tissue distribution of the oxygen radical producing enzyme xanthine oxidoreductase (XOR) were measured in the digestive gland of the common marine mussel Mytilus galloprovincialis Lmk along an annual cycle. No xanthine oxidase (XOX) activity could be measured, the enzyme only displaying xanthine dehydrogenase (XDH) activity in all the cases. This is interpreted as a mechanism to avoid the harmful effects of the oxygen radicals that would be produced by XOX during periods following anoxic conditions at low tide. The highest XDH activities coincided with the late spring/early summer months, the activity maxima being recorded from May to July. Histochemically XOR activity was very pronounced in duct and stomach epithelial cells as well as in the surrounding connective tissue and hemolymph vessels, the activity increasing towards the summer months. These seasonal variations in XDH or XOR activities are possibly linked to hormonal changes governing the reproductive cycle and to changes in food availability. The localization of the protein in the connective tissue lining the hemolymph vessels was confirmed immunohistochemically using a polyclonal antibody against rat liver protein that cross-reacted specifically with a polypeptide of 150 kDa of molecular mass in homogenates of the digestive gland. This polypeptide was linked to cytosolic fractions isolated by differential centrifugation from mussel digestive glands. In paraffin sections the antibody labeled the digestive cells of digestive tubules, as well as the connective tissue surrounding the hemolymph vessels, gonadal follicles, digestive epithelia and certain protozoan parasites. Taken together our results suggest that in the digestive gland of bivalve molluscs XOR is involved in the metabolism of purines and in the scavenging of oxygen free radicals.

Overlapping Distribution of the 130- and 110-kDa Myosin I Isoforms on Rat Liver Membranes

The biochemical and mechanochemical properties and localization of myosin I suggest the involvement of these small members of the myosin superfamily in some aspects of intracellular motility in higher cells. We have determined by quantitative immunoblotting with isoform-specific antibodies that the 130-kDa myosin I (myr 1 gene product) and 110-kDa myosin I (myr 2 gene product) account for 0.5 and 0.4%, respectively, of total rat liver protein. Immunoblot analyses reveal that the 130- and 110-kDa myosins I are found in several purified subcellular fractions from rat liver. The membrane-associated 130-kDa myosin I is found at the highest concentration in the plasma membrane (28 ng/μg plasma membrane protein) followed by the endoplasmic reticulum-like mitochondria-associated membrane fraction (MAM; 10 ng/μg MAM protein), whereas the 110-kDa myosin I is found at the highest concentration in Golgi (50 ng/μg Golgi protein) followed by plasma membrane (20 ng/μg) and MAM (7 ng/μg). Our analyses indicate that myosin I is peripherally associated with Golgi and MAM and its presence in these fractions is not a consequence of myosin I bound to contaminating actin filaments. Although found in relatively low concentrations in microsomes, because of the abundance of microsomes, in liver most of the membrane-associated myosin I is associated with microsomes. Neither myosin I isoform is detected in purified mitochondria. This is the first quantitative analysis addressing the cellular distribution of these mammalian class I myosins.

Diphenylacetaldehyde-generated excited states promote damage to isolated rat liver mitochondrial DNA, phospholipids, and proteins

This work studies damage to rat liver mitochondrial protein, lipid, and DNA caused by electronically excited states generated by cytochrome c-catalyzed diphenylacetaldehyde enol oxidation to triplet benzophenone. The extension of lipid peroxidation was estimated by production of thiobarbituric acid–reactive substances and by formation of Schiff bases with membrane proteins, evaluated by SDS-polyacrylamide gel electrophoresis. Concomitant with DPAA-driven mitochondrial permeabilization, extensive mtDNA fragmentation occurred and DNA adducts with aldehydes—products of fatty acid oxidation—were observed. The degree of lipid peroxidation and mtDNA alterations were significantly decreased by butylated hydroxytoluene, a potent peroxidation chain breaker. The lipid peroxidation process was also partially inhibited by the bioflavonoid rutin and urate totally prevented the mitochondrial transmembrane potential collapse. In all cases, the mitochondrial damage was dependent on the presence of phosphate ions, a putative bifunctional catalyst of carbonyl enolization. These data are consistent with the notion that triplet ketones may act like alkoxyl radicals as deleterious reactive oxygen species on biologic structures. Involvement of singlet dioxygen formed by triplet-triplet energy transfer from benzophenone in the model reaction with DPAA/cytochrome c in the presence of DCP liposomes was suggested by quenching of the accompanying chemiluminescence upon addition of histidine and lycopene.

Vitamin D receptor ontogenesis in rat liver.

Vitamin D through its receptor (VDR) plays a major role in bone mineral metabolism. However, VDR is also present in a variety of cell lines as well as in numerous tissues, suggesting other functions of the hormone beyond bone metabolism and mineral homeostasis. At the liver level, it has been shown that vitamin D induces numerous changes (i.e. enzyme activity level, stimulation of some metabolic pathways and stimulation of the normal liver recovery after partial hepatectomy). However, some works did not find VDR in the liver, and also used liver tissue as a negative control of VDR gene expression. In this paper, we examined fetal, neonatal and adult rat tissues for the presence of VDR using a sensitive RT-PCR technique and immunohistochemistry. We found VDR mRNA and VDR protein in rat liver at all different periods of rat life. Thus, we suggest that some of the actions of vitamin D on liver could be mediated at the genomic level through the VDR, and that the use of this tissue as a negative control of VDR gene expression is clearly inappropriate.

Fasting-induced apoptosis in rat liver is blocked by cycloheximide

The effect of cycloheximide (CH) on the fasting-induced changes of rat liver cell and protein turnover has been investigated. Late starvation phase (3–4-day-fasting period) was characterised by a decrease in liver weight and protein and DNA content. The loss of DNA was not related to liver cell necrosis but due not only to depression of cell proliferation as shown by the drop in the labelling index but also induction of apoptosis. This type of apoptosis was documented by the increase in the apoptotic index (cells labelled by TUNEL) and transglutaminase activity as well as by DNA fragmentation. The liver cells of fasted rats appeared smaller as shown by the higher cell density and DNA/protein ratio than in controls. Females were more resistant to fasting-induced apoptosas than males. A single dose of CH, a drng primary known as inhibitor of protein synthesis, induced or enhanced apoptosis in fed and 2-days fasted male rats, respectively, without any sign of cell necrosis. On the contrary, the administration of repeated doses of CH blocked apoptosis induced by fasting. CH “froze” protein and DNA content as well as apoptotic process at the level of 2 days-fasted rats. While fasting-induced liver protein loss resulted from a marked reduction in protein synthesis with a slight decrease in degradation, repeated treatment with CH virtually blocked protein loss by abolishing protein catabolism. These data suggest a direct relationship between the catabolic side of protein turnover and the apoptotic process.

Coincident increase in periportal expression of iron proteins in the iron-loaded rat liver.

The liver is the major iron storage organ in the body and, as a result, total body iron stores closely regulate hepatocyte iron uptake, storage and release. Transferrin, transferrin receptor and ferritin facilitate these processes. Expression of the three proteins was localized by immunohistochemistry and in situ hybridization on normal, iron-loaded and iron-deficient rat livers. Gel shift assays were used to determine iron regulatory protein (IRP) binding activity. In the normal rat liver, all three proteins and mRNA were evenly distributed throughout the hepatic lobule. In iron-loaded liver, increased iron stores were found in a periportal distribution, coinciding with increased periportal protein levels of each protein. Periportal transferrin and ferritin mRNA levels were also increased. Hepatic transferrin and transferrin receptor expression was increased in iron deficiency compared with controls; however, despite no change in ferritin mRNA levels being found, ferritin protein was not detected. Hepatic IRP2 binding activity was decreased in iron loading and increased in iron deficiency. The combined findings of this study were that, in the dietary iron-loaded rat model, increased iron stores were localized to periportal hepatocytes and that these same hepatocytes also had increased ferritin, transferrin receptor and transferrin protein expression. This response suggests that additional, non-IRP control mechanisms may be involved in the regulation or stability of these proteins. In iron deficiency the inverse post-transcriptional regulation of ferritin and transferrin receptor was consistent with IRP regulation.

Protein oxidation in thyroid hormone-induced liver oxidative stress: relation to lipid peroxidation

The influence of thyroid hormone administration (daily doses of 0.1 mg of 3,3′,5-triiodothyranine (T 3)/kg for 1–3 consecutive days) on rat liver protein oxidation was investigated in relation to the calorigenic and lipid peroxidative actions of the hormone. T 3 treatment elicited a progressive enhancement in the serum levels of the hormone, the rectal temperature of the animals, and in the rate of O 2 uptake of the liver, changes that are significantly correlated and evidence the development of thyroid calorigenesis. Liver lipid peroxidation was augmented by T 3 administration as determined by the tissue content of thiobarbituric acid reactants, with a maximal effect (3.1-fold increase) being found at 2 days after treatment, whereas protein oxidation measured by the content of protein hydrazone derivatives exhibited a maximal 88% increase at 3 days. Maximal rates of lipid peroxidation occur at 1 day after the administration of T 3, whereas those of protein oxidation are attained after treatment with three daily doses of T 3, time at which the former levels off. It is concluded that T 3 administration induces a substantial enhancement in hepatic protein oxidation, in addition to lipid peroxidation, that seems to be due to the higher oxidative stress status conditioned in the liver by thyroid calorigenesis. Both processes exhibit a differential time course of changes, that may represent differences in the susceptibility of target molecules to free radical attack and/or in the efficiency of repair mechamisms.

Identification of two rat liver proteins with paraoxonase activity: biochemical evidence for the identity of paraoxonase and arylesterase

The existence of two or more enzyme forms with paraoxonase activity has been reported in sheep, rabbit, human and rat serum and recently in mouse and rat liver. In this study we describe the presence of two peaks with paraoxonase activity (M1 and M2) after non-specific affinity chromatography of rat liver microsomes on Cibacron Blue 3GA. The first peak (M1) was obtained during the washing of the column and coeluted with albumin. The second active peak (M2) was eluted with 1 M NaCl. The characterization of each peak was determined by SDS/PAGE electrophoresis and Western-blotting. A comparison of both active fractions on the basis of kinetic parameters, heat inactivation and pH stability, calcium requirement and inhibition by EDTA and several metals was performed. Our results support the fact that two proteins capable of hydrolyzing paraoxon are present in rat liver microsomes. Furthermore, during the purification to homogeneity of rat liver paraoxonase we have performed a study of its hydrolytic ability against three different substrates: paraoxon, phenylacetate and phenyl thioacetate (Paraoxonase (PON), Arylesterase (ArE), Phenyl thioacetate esterase (PTase)). The elution profile in different chromatographic steps, as well as the activity ratios from the crude extract throughout the purification process, heat inactivation and effect of inhibitors were used as identity criteria for the three hydrolytic activities. Our results show evidence for the hydrolysis of paraoxon and phenylacetate by the same protein from rat liver (paraoxonase).

Influence of parasitization by Trichinella spiralis on the levels of heat shock proteins in rat liver and muscle.

To examine levels of heat shock proteins (HSPs) in host responses to helminth infection, rats were infected with Trichinella spiralis. The kinetics of HSP25, HSP60, HSP70 and HSP90 production in the liver and muscle of infected rats was compared with that of non-infected controls. HSPs were detected using electrophoretic analysis of fixed amounts of proteins (0.02 mg) and then blotting and incubation of membranes with polyclonal anti-HSP25 antibody, or monoclonal antibody against HSP60, or HSP70 or HSP90. Quantitation of blotted separated polypeptides reactive with the specific anti-HSP antibodies was achieved using an image analyser. Enhancement of HSP25 production was observed in the liver of infected rats, whereas muscle from the same rats exhibited enhanced production of HSP25 and HSP60 one day after infection only. These data indicate that HSPs levels can be used successfully to measure stress injury brought about by helminth infection in organs and tissues of the host.

Detection of adducts of bromobenzene 3,4-oxide with rat liver microsomal protein sulfhydryl groups using specific antibodies.

The hepatotoxicity of bromobenzene (BB) has been attributed to covalent modification of cellular proteins by reactive metabolites generated during its oxidative biotransformation. Much of the net covalent binding which occurs originates via quinone metabolites, but bromobenzene 3,4-oxide (BBO), which is the reactive metabolite thought to be most significant toxicologically, also arylates protein side chains, although to a lesser extent. To facilitate the detection, isolation, and identification of rat liver proteins specifically adducted by BBO, we raised polyclonal antibodies capable of recognizing S-(p-bromophenyl)cysteine moieties (anti-BP) by immunizing rabbits with p-bromophenylmercapturic acid conjugated to keyhole limpet hemocyanin. The antiserum had a high titer, showed a high specificity for hapten in competitive ELISA with hapten analogues, and performed well in Western blot experiments using synthetically haptenized control proteins. When used for Western analysis of protein fractions from in vitro incubations of rat liver microsomes with [14C]BB, affinity-purified anti-BP recognized a limited number of bands, each of which also contained 14C. One of these bands corresponds to hydrolase B, a nonspecific esterase known to contain one free sulfhydryl group and previously shown to be a target protein for [14C]BB metabolites.

An Improved Method for the Purification and Characterization of a 54 kDa Protein in Rat Liver Which Has Recently Been Identified as a Selenium-Binding Protein

A 54kDa protein, which is induced by treatment with 3, 3', 4, 4', 5-pentachlorobiphenyl, 3-methylcholanthrene and butylated hydroxytoluene, was purified from rat liver by conventional methods involving DEAE-Sephacel column chromatography and processing using a Rotofor CellTM. This method does not require a denaturation or solubilization step and yields a single protein on SDS-PAGE. The molecular mass of the purified protein was 54.7kDa. We also characterized the 54kDa protein by immunoblot analysis and measured the binding ratio of selenium. The purified 54kDa protein was stained with antiserum which had been previously obtained, and the molecular ratio of selenium to the 54kDa protein was 0.014. Glutathione peroxidase (GPx) is a typical selenoprotein which has a selenocysteine amino acid sequence. We measured the binding ratio of selenium with commercial GPx as a positive control and obtained a value of 1.214. The binding ratio of selenium to 54kDa protein was much lower than that of GPx, and this led us to believe that there is no selenocysteine in the 54kDa protein. In this report, we have developed a convenient purification procedure and measured the purification factors and binding ratio of selenium associated with a 54kDa protein in rat liver which is homologous with a selenium-binding protein in the mouse.

Immunochemical localisation of proliferating cells in mussel digestive gland tissue.

The distribution of proliferating cells in the digestive gland of the common marine mussel, Mytilus galloprovincialis Lmk, was investigated by means of immunochemical techniques employing PC10, a commercial monoclonal antibody to the proliferating cell nuclear antigen (PCNA). Immunoblot analysis of digestive gland whole homogenates revealed a single crossreactive band of 36-37 kDa, identical to the corresponding protein of rat liver and murine melanoma cells. A band of slightly higher electrophoretic mobility (34-35 kDa) was found in fish liver. In mussel digestive gland, the samples obtained from young specimens presented a more intense signal for PCNA than in those obtained from old mussels, suggesting that the digestive gland cells of young mussels exhibit a higher proliferative activity. In paraffin sections, PC10 specifically labelled nuclei of all cell types, but only a smaller number of cells lining the different digestive epithelia. PCNA expression was more intense in digestive cells than in basophilic cells. Hemocytes circulating along the interdiverticular spaces also presented immunoreactive nuclei. Electron microscopy revealed a specific and moderate PC10 labelling in nuclei. Thus, single gold particles appeared disseminated throughout the nuclei with accumulations of particles in the sites of DNA replication. Taken together, these data reveal that the capacity to proliferate resides within all cell types in the digestive diverticula and do not support the hypothesis of the existence of one stem cell in this epithelium. As opposed to the hepatopancreas of the crab, Carcinus maenas, where mitotic figures and PCNA immunoreactivity are only observed in the embryonic cells within the distal portions of the digestive diverticula, apparently there are not discrete regions of cell proliferation in the digestive gland of mussels.

Studies on the multidrug resistance gene expression in the livers of rats associated with different susceptibility of hepatocarcinogenesis.

Inbred carcinogen-resistant DRH rat strain developed from the closed colony Donryu rats on the basis of selective markers such as poor induction of gamma-glutamyltranspeptidase and marked reduced incidences of liver tumors during 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) administration, which took more than 10 years. Previously, we observed that the Donryu rat liver was quite sensitive to both DNA-damaging and cytotoxic effects of 3'-Me-DAB, while the DRH rat liver showed tolerance to 3'-Me-DAB under the same conditions. In the present study, we examined mRNA levels related to the cytotoxic drug resistance mechanism in the livers of DRH and Donryu rats using RT-PCR. Contrary to our expectation, we observed rather similar levels of mRNAs between the two rat strains under the following conditions: i) mdr1 mRNA induction after 3'-Me-DAB administration. ii) MLP-2 mRNA reduction by 3'-Me-DAB administration, iii) MLP-2 mRNA induction after cholestasis and iv) constitutive levels of cMOAT gene expression. On the other hand, the levels of p53 mRNA and p53 protein in the Donryu rat liver were higher than those in DRH rat liver during 3'-Me-DAB administration, suggesting that the former were more sensitive to 3'-Me-DAB than DRH rat under these conditions. In conclusion, we failed to demonstrate the difference in the cytotoxic drug resistance mechanism between DRH and Donryu rats at least under the conditions examined in this study.

Immunohistochemical Detection of Glutathione S-transferase P in Postnatal Rat Liver after Administration of Dexamethasone.

The localization of glutathione S-transferase P (GST-P) in postnatal rat liver was examined by an immunohistochemical technique. Dexamethasone (DEX) was injected intraperitoneally into postnatal rats at a dose of 2 μg/g body weight daily for 4 days. GST-P was detected only in the cytoplasm of epithelial cells of the interlobular bile ducts in the livers of 8-day-old rats that did not receive DEX-administration. In contrast, dispersed-positive hepatocytes were observed in the liver lobules of rats that were administered DEX from 4 days after birth. In these rats, GST-P was found in the cytoplasm of those hepatocytes and epithelial cells of the interlobular bile ducts. GST-P was observed only in the cytoplasm of epithelial cells of the interlobular bile ducts in the livers of 13-day-old rats that did not receive DEX-administration and those that began DEX-administration at 9 days. The immunoreactivity was stronger compared to the 8-day-old rats. These results indicate that DEX promotes the appearance of hepatocytes that contain the GST-P protein in early postnatal rat liver.

Induction of a cytosolic 54 kDa protein in rat liver that is highly homologous to selenium-binding protein

We have previously shown that a 54 kDa protein in rat liver is highly homologous to selenium-binding protein (SeBP) or acetaminophen-binding protein (APBP) in mice and is highly inducible by treatment with 3,3′,4,4′,5-pentachlorobiphenyl or 3-methylcholanthrene. In this study, we examine the effect of six typical inducers, 3-methylcholanthrene (MC), isosafrole (ISO), phenobarbital (PB), dexamethasone (DEX), clofibrate (CLO), pyrazole (PYR) and butylated hydroxytoluene (BHT), on the expression level of this 54 kDa protein. Male Wistar rats were given each inducer following a predetermined schedule. Among these inducers, the 54 kDa protein was inducible by MC and BHT. The response to MC and BHT was compared with that of NAD(P)H: quinone oxidoreductase and ethoxyresorufin O-deethylase activities. The induction mechanisms and physiological role of the 54 kDa protein are discussed in the light of our results.

Identification of glutathione S-transferase isozymes and gamma-glutamylcysteine synthetase as negative acute-phase proteins in rat liver.

Because acute infection and inflammation affect drug metabolism and drug-metabolizing enzymes, the effect of the acute-phase response on the expression of glutathione S-transferase (GST) isoenzymes, glutathione synthesis, and several antioxidant enzymes was investigated. Hepatic expression of GST isozymes, positive and negative acute-phase reactants, and antioxidant enzymes were determined by Northern blotting and hybridization with gene-specific oligonucleotide probes after lipopolysaccharide treatment of rats. Lipopolysaccharide caused the expected acute-phase response as judged by the increased expression of positive and decreased expression of negative acute-phase proteins. The messenger RNA (mRNA) expression of the major hepatic rat GST isozymes A1, A2, A3, M1, and M2 was decreased 50% to 90%. Total hepatic GST activity toward 1-chloro-2,4-dinitrobenzene was also significantly decreased. mRNA expression of gamma-glutamylcysteine synthetase (GCS) large subunit and catalase was reduced by approximately 60%. GCS enzyme activity was also decreased, resulting in a 35% decrease in the hepatic content of reduced glutathione 4 days after lipopolysaccharide challenge. Mn-Superoxide dismutase expression was increased 13-fold, and thioredoxin level was elevated 3-fold after lipopolysaccharide challenge. The expression of all parameters determined returned to near control levels 7 days after treatment. Together, these data show that GSTs and GCS are negative acute-phase proteins and that decreased GCS activity results in a decrease in hepatic glutathione content. Thus, in addition to the phase I drug-metabolizing enzymes known to be decreased during the acute-phase response, some phase II enzymes involved in the elimination of xenobiotics and carcinogens are also decreased.

Growth-related expression of the ectonucleotide pyrophosphatase PC-1 in rat liver.

Plasma cell differentiation antigen-1 (PC-1) is a 5'-ectonucleotide pyrophosphatase that has been implicated in various processes including insulin- and nucleotide-mediated signaling and cell growth. We show here that the expression of both PC-1 mRNA and protein in rat liver and in hepatoma cells is strictly growth-related. Thus, the level of PC-1 in FAO hepatoma cells increased with the cell density. PC-1 was not expressed in the neonatal rat liver, but gradually appeared in the first weeks of age, to reach adult levels around the weaning period. Furthermore, PC-1 protein and mRNA largely disappeared from the liver within 24 hours following a hepatectomy of 70%, but re-appeared in the later phases (3-15 days) of the ensuing regeneration period. An equally rapid loss of PC-1 protein and mRNA could also be provoked in normal livers by the administration of the translational inhibitor, cycloheximide, but the transcriptional inhibitors, actinomycin D and alpha-amanitin, did not show these effects. Nuclear run-on assays revealed that the loss of PC-1 mRNA after hepatectomy or after the administration of cycloheximide was not caused by a decreased transcription of the PC-1 gene, suggesting that the level of PC-1 is controlled by an mRNA-stabilizing protein that is lost after hepatectomy and has a high turnover.

Cholangiocyte-specific rat liver proteins identified by establishment of a two-dimensional gel protein database.

The liver is composed of a variety of cells that form a functional unit involved in uptake, synthesis, metabolism, and secretion. Until recently, most studies examining liver function did not analyze the specific proteins expressed or functions performed by the multiple individual cell types that constitute the hepatic mass. In the last decade, novel isolation methods have been developed that allow the purification of liver cell populations highly enriched in one type of liver cell. Here, we present a detailed two-dimensional (2-D) protein map of rat bile duct epithelial cells (i.e., cholangiocytes) using a recently developed isolation procedure. In addition, we identify 27 major cholangiocyte proteins either by comparison to maps of known rat liver proteins (based on pI and Mr) or by tryptic digestion and microsequencing. Finally, we compare the relative abundance of individual proteins present in cholangiocytes to whole liver as well as hepatocyte-specific proteins. Our results show that cholangiocytes express a unique array of individual proteins. The cholangiocyte 2-D protein pattern is markedly different from that of isolated rat hepatocytes or whole rat liver, with high levels of proteins previously known to be expressed by cholangiocytes (e.g., cytokeratins, actins) as well as protein not previously demonstrated to be expressed at high levels (e.g., annexin V, selenium binding protein). We conclude that this cholangiocyte-derived, 2-D protein map will be a crucial resource for studies directed at our understanding of cholangiocyte physiology and pathobiology.

Detection of circulating antibodies to malondialdehyde-acetaldehyde adducts in ethanol-fed rats

Background & Aims: Malondialdehyde and acetaldehyde react together with proteins and form hybrid protein conjugates designated as MAA adducts, which have been detected in livers of ethanol-fed rats. The aim of this study was to examine the immune response to MAA adducts and other aldehyde adducts during long-term ethanol exposure. Methods: Rats were pair-fed for 7 months with a liquid diet containing either ethanol or isocaloric carbohydrate. Circulating antibody titers against MAA adducts and acetaldehyde adducts were measured and characterized in these animals. Results: A significant increase in antibody titers against MAA-adducted proteins was observed in the ethanol-fed animals. Competitive inhibitions of antibody binding indicated that the circulating antibodies against MAA-modified proteins in the ethanol-fed rats recognized mainly a specific, chemically defined MAA epitope. Antibody titers to reduced and nonreduced acetaldehyde adducts were very low, and no significant differences were observed between ethanol-fed and control animals. Significant plasma immunoreactivity to not only MAA-adducted but also unmodified rat liver proteins (cytosol, microsomes, and especially plasma membrane) were also observed in the ethanol-fed rats. Conclusions: Long-term ethanol feeding generates circulating antibodies not only against MAA epitopes but possibly also against unmodified, native (self) protein epitopes, suggesting that MAA adducts could trigger harmful autoimmune responses. GASTROENTEROLOGY 1998;115:686-692