Liver Membranes Research Articles

Human T-leukemia and T-lymphoma express glutamate receptor AMPA GluR3, and the neurotransmitter glutamate elevates the cancer-related matrix-metalloproteinases inducer CD147/EMMPRIN, MMP-9 secretion and engraftment of T-leukemia in vivo

Glutamate is the major excitatory neurotransmitter of the nervous system. We previously found that glutamate activates normal human T-cells, inducing their adhesion and chemotaxis, via its glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype 3 (GluR3) expressed in these cells. Here, we discovered that human T-leukemia (Jurkat) and cutaneous sezary T-lymphoma (HuT-78) cells also express high levels of GluR3. Furthermore, glutamate (10 nM) elevates CD147/EMMPRIN, a cancer-associated matrix metalloproteinases (MMPs) inducer, promoting spread of many tumors. Glutamate-induced CD147 elevation in both cancerous and normal human T-cells was mimicked by AMPA (glutamate/AMPA-receptor agonist) and blocked by CNQX (glutamate/AMPA-receptor antagonist). Importantly, glutamate also increased gelatinase MMP-9 secretion by T-lymphoma. Finally, ex vivo pre-treatment of T-leukemia with glutamate enhanced their subsequent in vivo engraftment into chick embryo liver and chorioallantoic membrane. Together, these findings reveal that glutamate elevates cancer associated proteins and activity in T-cell cancers and by doing so may facilitate their growth and spread, especially to and within the nervous system. If so, glutamate receptors in T-cell malignancies should be blocked.

Copper(II) Complexes of 4, 4‐ Diaminodiphenylsulphone: Synthesis, Characterization and Biological Studies

Five complexes of copper(II) 4,4‐diaminodiphenylsulphone have been synthesized. Copper salts of counter ion (sulphate, nitrate, chloride) and different reaction media (solvents) were used for the synthesis. The complexes varied in colour and composition. The compounds were characterized by conductivity, IR, UV, NMR and mass spectroscopies. The ligand coordinated to metal ion in a monodentate and bidentate manner. All the five complexes have tetrahedral configuration. The biological activities data showed that the complexes are more active against Esherichia coli, Klebbsiella pneumoniae and Staphylococcus aureus than the free ligand (4,4‐ diaminodiphenyl sulphone). Antimalaria activities of the complexes and the ligand were investigated using mice infected with Plasmodium berghei. All the complexes exhibited lower activity than the ligand and chloroquine. The effect of administration of the complexes on alkaline phosphatase activities of kidney, liver and serum of Albino rats were investigated. The serum ALP activity showed no significant change (P> 0.05), suggesting non‐damaging effect on the plasma membrane of liver and kidney cells.

Az agrin szelektív megjelenése a hepatocellularis carcinoma erezetében: kórkeletkezési és elkülönítő diagnosztikai vonatkozások

Hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancers and is the fifth most common malignancy worldwide. HCC typically develops in the cirrhotic liver. Our preliminary results indicated that agrin, a heparan sulfate proteoglycan (HSPG) detected by us for the first time in the liver, accumulates in the basement membranes (BMs) of the cirrhotic liver and HCC. This novel finding prompted us to investigate the role of agrin in the pathogenesis and differential diagnosis of HCC. First, the previously unspecified monoclonal antibody anti-HSPG clone 7E12 was verified as anti-agrin, using mass spectrometry. Our subsequent experiments were carried out on specimens from 131 patients with chronic liver disease and 18 individuals with healthy liver, from 4 rats subjected to cirrhosis/HCC induction and 1 untreated control rat, as well as from cultured cells. In both human and rats, significantly increased expression of agrin in cirrhosis and HCC was demonstrated by immunohistochemistry (IHC), Western blot, and quantitative RT-PCR. By double immunofluorescent studies, agrin was localized to the muscular layer of blood vessel walls, the BM of bile ducts and ductular reaction, the microvessel walls of HCC, and occasionally the BM of hepatocellular tumor cells. Colocalization, gene expression, and mRNA in situ hybridization experiments suggested that the sources of agrin include vascular smooth muscle cells, epithelial cells of bile ducts and ductules, activated mesenchymal cells in the stroma of hepatocellular tumors, and occasionally tumor hepatocytes. Agrin in the BMs of bile ducts and blood vessels is thought to play an important role in the survival of bile duct epithelium and vascular endothelium, respectively. Thus, agrin may contribute to the formation of ductular reaction and HCC neovessels. As opposed to HCC neovessels that were consistently found agrin-positive, normal and cirrhotic sinusoids were always devoid of agrin, raising the possibility that agrin IHC might be useful in the differential diagnosis of benign versus malignant hepatocellular lesions. Agrin IHC was performed on 68 benign lesions (8 large regenerative nodules, 23 low-grade and 7 high-grade dysplastic nodules, 30 liver adenomas) and 29 malignant lesions (8 small HCC, 21 HCC), and was evaluated semi-quantitatively. Based on the results of IHC for agrin as well as CD34, a decision algorithm was devised that differentiated benign and malignant parenchymal lesions with a sensitivity of 93.1% and a specificity of 92.6%. Hence, we propose that agrin IHC might help distinguish between malignant hepatocellular lesions and their benign mimickers.

Nanoencapsulation of quercetin enhances its dietary efficacy in combating arsenic-induced oxidative damage in liver and brain of rats

Aims This study was performed to evaluate the therapeutic efficacy of nanocapsulated flavonoidal quercetin (QC) in combating arsenic-induced reactive oxygen species (ROS)-mediated oxidative damage in hepatocytes and brain cells in a rat model. Main methods Hepatic and neuronal cell damage in rats was made by a single injection (sc) of sodium arsenite (NaAsO 2, 13 mg/kg b. wt. in 0.5 ml of physiological saline). A single dose of 500 µl of quercetin suspension (QC) (QC 8.98 µmol/kg) or 500 µl of nanocapsulated QC (NPQC) (QC 8.98 µmol/kg) was given orally to rats at 90 min prior to the arsenite injection. Key findings Inorganic arsenic depositions (182 ± 15.6 and 110 ± 12.8 ng/g protein) were found in hepatic and neuronal mitochondrial membranes. Antioxidant levels in hepatic and neuronal cells were reduced significantly by arsenic. NPQC prevented the arsenite-induced reduction in antioxidant levels in the liver and brain. Arsenic induced a substantial decrease in liver and brain cell membrane microviscosities, and NPQC treatment resulted in a unique protection against the loss. A significant correlation between mitochondrial arsenic and its conjugated diene level was observed both in liver and brain cells for all experimental rats. Significance Arsenic-specific antidotes are used against arsenic-induced toxicity. However, the target site is poorly recognized and therefore achieving an active concentration of drug molecules can be a challenge. Thus, our objective was to formulate NPQC and to investigate its therapeutic potential in an oral route against arsenite-induced hepatic and neuronal cell damage in a rat model.

A Combination of Immobilised pH Gradients Improves Membrane Proteomics

Membrane protein analyses have been notoriously difficult due to hydrophobicity and the general low abundance of these proteins compared to their soluble cytosolic counterparts. Shotgun proteomics has become the preferred method for analyses of membrane proteins, in particular the recent development of peptide immobilized pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two-dimensional shotgun proteomics. Recently, peptide IPG-IEF has been shown to be a valuable shotgun proteomics technique through the use of acidic narrow range IPG strips, which demonstrated that small acidic p I increments are rich in peptides. In this study, we assess the utility of both broad range (BR) (p I 3-10) and narrow range (NR) (p I 3.4-4.9) IPG strips for rat liver membrane protein analyses. Furthermore, the use of these IPG strips was evaluated using label-free quantitation to demonstrate that the identification of a subset of proteins can be improved using NR IPG strips. NR IPG strips provided 2603 protein assignments on average (with 826 integral membrane proteins (IMPs)) compared to BR IPG strips, which provided 2021 protein assignments on average (with 712 IMPs). Nonredundant protein analysis demonstrated that in total from all experiments, 4195 proteins (with 1301 IMPs) could be identified with 1428 of these proteins unique to NR IPG strips with only 636 from BR IPG strips. With the use of label-free quantitation methods, 1659 proteins were used for quantitative comparison of which 319 demonstrated statistically significant increases in normalized spectral abundance factors (NSAF) in NR IPG strips compared to 364 in BR IPG strips. In particular, a selection of six highly hydrophobic transmembrane proteins was observed to increase in NSAF using NR IPG strips. These results provide evidence for the use of alternative pH gradients in combination to improve the shotgun proteomic analysis of the membrane proteome.

Disruption of the gene encoding 3β‐hydroxysterol Δ14‐reductase (Tm7sf2) in mice does not impair cholesterol biosynthesis

Tm7sf2 gene encodes 3beta-hydroxysterol Delta(14)-reductase (C14SR, DHCR14), an endoplasmic reticulum enzyme acting on Delta(14)-unsaturated sterol intermediates during the conversion of lanosterol to cholesterol. The C-terminal domain of lamin B receptor, a protein of the inner nuclear membrane mainly involved in heterochromatin organization, also possesses sterol Delta(14)-reductase activity. The subcellular localization suggests a primary role of C14SR in cholesterol biosynthesis. To investigate the role of C14SR and lamin B receptor as 3beta-hydroxysterol Delta(14)-reductases, Tm7sf2 knockout mice were generated and their biochemical characterization was performed. No Tm7sf2 mRNA was detected in the liver of knockout mice. Neither C14SR protein nor 3beta-hydroxysterol Delta(14)-reductase activity were detectable in liver microsomes of Tm7sf2((-/-)) mice, confirming the effectiveness of gene inactivation. C14SR protein and its enzymatic activity were about half of control levels in the liver of heterozygous mice. Normal cholesterol levels in liver membranes and in plasma indicated that, despite the lack of C14SR, Tm7sf2((-/-)) mice are able to perform cholesterol biosynthesis. Lamin B receptor 3beta-hydroxysterol Delta(14)-reductase activity determined in liver nuclei showed comparable values in wild-type and knockout mice. These results suggest that lamin B receptor, although residing in nuclear membranes, may contribute to cholesterol biosynthesis in Tm7sf2((-/-)) mice. Affymetrix microarray analysis of gene expression revealed that several genes involved in cell-cycle progression are downregulated in the liver of Tm7sf2((-/-)) mice, whereas genes involved in xenobiotic metabolism are upregulated.

Research Article: The effects of 2-deoxyglucose in the male zebra finch

Previous findings indicate that undirected singing performed by the male zebra finch (Taeniopygia guttata) is decreased following 2-deoxyglucose (2-DG) administration. 2-DG, a glucose analog that is not metabolized by mitochondria, is considered a metabolic stressor, causing a switch from carbohydrate to lipid metabolism, thus mimicking dietary restriction (DR). DR suppresses free radical production, thus reducing oxidative stress; reduced levels of oxidative stress are correlated with a decreased incidence of age-related disorders including cancers, diabetes and cardiovascular diseases. The present study examines the possibility of using 2-DG as a metabolic stressor in the zebra finch in order to study mechanisms of action in aging and age-related disorders. Morphology of mitochondria in liver and brain tissue and morphology of heart and liver tissue is studied. Physiological parameters, such as vocalization, body weight, food intake and corticosterone production are examined. Our results showed that 2-DG causes oncosis and a collapse of the outer membranes of liver and brain mitochondria and altered the morphological structure of the liver and heart. In addition, it was shown that corticosterone levels were significantly increased in 2-DG treated animals, while song production and food intake were significantly decreased compared to the control-treated group. The zebra finch model can be used to further study the processes of mitochondrial dysfunction/oxidative stress and might contribute to the elucidation of the underlying mechanism(s) of the processes of aging and age-related disorders.

Use of a Commercial Protease and Yeasts To Obtain CGRP-like Molecules from Saithe Protein

Different bioactive molecules, such as CGRP-like peptides, can be found in fish protein hydrolysates. Calcitonin gene-related peptide (CGRP) is a neuropeptide known to act as a potent arterial and venous vasodilator in humans. This study focuses on the industrial obtaining of CGRP-like molecules from saithe (Pollachius virens) byproduct, derived from the filleting process. Protein from P. virens was primarily hydrolyzed with Alcalase and later treated with Saccharomyces cerevisiae live cells. Treatment with Saccharomyces doubled the quantity of bioactive molecules obtained. The CGRP-like molecules were partially purified by chromatography, and the immunoreactive material was further analyzed for its CGRP-like bioactivity, using a specific radioreceptor assay. The concentration of CGRP-like molecules increased over 100-fold after purification. The bioactive molecules were able to induce cyclic AMP stimulation in rat liver membranes. Finally, partial sequencing of the bioactive peptide was performed, showing some homology with alpha-actin and myosin of several fish species.

Altered expression and distribution of aquaporin-9 in the liver of rat with obstructive extrahepatic cholestasis

Rat hepatocytes express aquaporin-9 (AQP9), a basolateral channel permeable to water, glycerol, and other small neutral solutes. Although liver AQP9 is known for mediating the uptake of sinusoidal blood glycerol, its relevance in bile secretion physiology and pathophysiology remains elusive. Here, we evaluated whether defective expression of AQP9 is associated to secretory dysfunction of rat hepatocytes following bile duct ligation (BDL). By immunoblotting, 1-day BDL resulted in a slight decrease of AQP9 protein in basolateral membranes and a simultaneous increase of AQP9 in intracellular membranes. This pattern was steadily accentuated in the subsequent days of BDL since at 7 days BDL basolateral membrane AQP9 decreased by 85% whereas intracellular AQP9 increased by 115%. However, the AQP9 immunoreactivity of the total liver membranes from day 7 of BDL rats was reduced by 49% compared with the sham counterpart. Results were confirmed by immunofluorescence and immunogold electron microscopy and consistent with biophysical studies showing considerable decrease of the basolateral membrane water and glycerol permeabilities of cholestatic hepatocytes. The AQP9 mRNA was slightly reduced only at day 7 of BDL, indicating that the dysregulation was mainly occurring at a posttranslational level. The altered expression of liver AQP9 during BDL was not dependent on insulin, a hormone known to negatively regulate AQP9 at a transcriptional level, since insulinemia was unchanged in 7-day BDL rats. Overall, these results suggest that extrahepatic cholestasis leads to downregulation of AQP9 in the hepatocyte basolateral plasma membrane and dysregulated aquaporin channels contribute to bile flow dysfunction of cholestatic hepatocyte.

Digging Deeper into the Mouse Liver Membrane Proteome: Evaluation of Different Membrane Protein Digestion Approaches with 8-plex iTRAQ Reagents

Digging Deeper into the Mouse Liver Membrane Proteome: Evaluation of Different Membrane Protein Digestion Approaches with 8-plex iTRAQ Reagents

Desperately Seeking Comprehensive Mammalian Membrane Proteomics

IPG-IEF enables the separation of chemically or enzymatically (e.g., trypsin) degraded protein products/peptides from a complex mixture solely on inherent pI. Methanol-assisted (0%, 40% & 60%) trypsin digestion of rat liver membrane proteins resulted in optimal coverage when deployed with peptide IPG-IEF based shotgun proteomics (digested proteins separated on linear 18cm 3-10 peptide IPG strips, manually separated into 24 fractions of equal width followed by peptide extraction and subsequently analyzed by ion trap LC/MS-MS). In this part of the study, peptides concentrated into three main pI regions (pI 3.5-5 5-7 & 8-9) and ~95% of all peptides were found in <2 fractions. In total, 1638 non-redundant membrane proteins were identified from all digests, with a very high level of coverage (42 identified) of the rat CYP450 protein family that are involved in drug clearance and toxicity. Of integral membrane proteins identified (60% methanolassisted digestion), 513 were predicted to contain between 1-19 transmembrane segments. Biochemical ontology indicated proteins originated mainly from doi:10.4172/jpb.s1000088

Liver membrane antibodies (LMA) recognize a 26-kD protein onthe hepatocellular surface

Sera from 82 patients with chronic inflammatory liver diseases and from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and Hashimoto's thyroiditis were studied by immunoblotting against purified liver plasma membranes (LPM) and soluble liver protein (SLP) fractions from different species after previous separation by SDS-PAGE. Eighteen of 19 sera with LMA of IgG type in immunofluorescence assay and six LMA-negative sera (three sera from patients with RA) showed antibodies of the IgG or IgM classes against a protein with a molecular weight of 26 kD which was present in LPM and SLP fractions from rats, rabbits, pigs and humans. The reaction with 26-kD liver protein did not correlate with other known autoantibody-antigen systems. All sera were negative in the 26-kD region with liver mitochondria, liver microsomes and soluble proteins of kidney (with one exception), heart and gut from the rat. The 26-kD protein was purified by affinity chromatography on immobilized anti-26-kD protein antibodies from patients, eluted from the 26-kD band of immunoblots. Studies with purified 26-kD liver protein and with SLP as antigens after separation in two-dimensional electrophoresis confirmed that patient serum and experimental rabbit antiserum react with the same protein. Eluted patient antibodies and rabbit antisera showed a linear fluorescence pattern on isolated hepatocytes from rat and rabbit. The data indicate that one of the target antigens of LMA is a species-nonspecific 26-kD protein located on the hepatocellular surface.

Plasma PCSK9 preferentially reduces liver LDL receptors in mice

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates the expression of LDL receptor (LDLR) protein. Gain-of-function mutations in PCSK9 cause hypercholesterolemia, and loss-of-function mutations result in lower plasma LDL-cholesterol. Here, we investigate the kinetics and metabolism of circulating PCSK9 relative to tissue levels of LDLRs. The administration of recombinant human PCSK9 (32 microg) to mice by a single injection reduced hepatic LDLRs by approximately 90% within 60 min, and the receptor levels returned to normal within 6 h. The half-life of the PCSK9 was estimated to be approximately 5 min. Continuous infusion of PCSK9 (32 microg/h) into wild-type mice caused a approximately 90% reduction in hepatic LDLRs within 2 h and no associated change in the level of LDLR in the adrenals. Parallel studies were performed using a catalytically inactive form of PCSK9, PCSK9(S386A), and similar results were obtained. Infusion of PCSK9(D374Y), a gain-of-function mutation, resulted in accelerated clearance of the mutant PCSK9 and a greater reduction in hepatic LDLRs. Combined, these data suggest that exogenously administrated PCSK9 in plasma preferentially reduces LDLR protein levels in liver at concentrations found in human plasma and that PCSK9's action on the LDLR is not dependent on catalytic activity in vivo.

Characterization of angiotensin II antagonism displayed by Ib, a novel nonpeptide angiotensin AT1 receptor antagonist

The pharmacologic profile of Ib, 5-n-butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one, a novel nonpeptide angiotensin AT1 receptor antagonist, was investigated by receptor-binding studies, functional in vitro assays with rabbit and rat aorta, and in vivo experiments in rats. Ib inhibited [125I] angiotensin II binding to AT1 receptors in rat liver membranes (Ki=2.5±0.5 nM) and did not interact with AT2 receptors in bovine cerebellar membranes. In functional studies with rat and rabbit aorta, Ib inhibited the contractile response to angiotensin II (pD2′ value: 7.43 and 7.29, respectively) with a significant reduction in the maximum. In pithed rats, Ib inhibited the angiotensin II induced pressor response in a dose-related manner. After intravenous administration, Ib produced a dose-dependent antihypertensive effects in spontaneously hypertensive rats and renal hypertensive rats. These results suggest that Ib is a potent angiotensin AT1 selective receptor antagonist with a mode of insurmountable antagonism.

Synthesis and AT1 affinity evaluation of benzamidophenyl analogs of known AT1 receptor ligands with similar aromatic skeleton

Taking as model compound the amido-derivative 1 described in the literature from Duncia's group as a good AngII antagonist, we have synthesized a new series of compounds (2-7) in which the principal structural variations reside in the inversion of the amidic sequence between the two phenyl ring and/or in the type of heteroaromatic substituent linked to this portion. The new compounds synthesized were evaluated for their AT1 affinity through binding assays carried out on rat liver membranes using ( 125 I)Sar1,Ile8-angiotensin II as radioligand.

Inhibition of proteolytic degradation and characterization of binding of natural angiotensins to AT1 receptors in brain and liver

Metabolism of angiotensins in receptor binding assays is a continuing problem. The suggestion that brain AT1 receptors (AT1R) differ from peripheral AT1R necessitates accurate appraisal of brain AT1R binding. The goal of this study was to establish experimental conditions that preserve 125I‐Ang II and ‐III, to characterize their binding to rat hypothalamic and liver AT1R. Negligible specific binding of 125I‐Ang III and ‐II is observed in hypothalamic membranes (45 or 60min at 24oC in 5mM EDTA, 150mM NaCl, 0.1mM bacitracin, 50mM NaPO4, pH 7.2 buffer + 10 μM PD123319) due to severe proteolytic degradation, as &lt; 5% of bound 125iodine corresponds to 125I‐Ang III or ‐II. In liver membranes, 125I‐Ang III is severely degraded, whereas 125I‐Ang II shows high affinity saturable binding to AT1R with &gt; 50% of bound 125I corresponding to 125I‐Ang II. Addition of 1 mM o‐phenanthroline &amp; phenylmethylsulfonyl fluoride, and 3 mM puromycin &amp; glutamate phosphonate to the assay buffer increased bound 125I‐Ang II and ‐III to &gt; 80%, both in liver and hypothalamic membranes revealing abundant, high‐affinity binding to AT1R. The affinity of both ligands for brain AT1R was less than for liver. 125I‐Ang III showed a smaller Bmax for AT1R in liver than 125I‐Ang II. These results indicate that both peptides bind with higher affinity for liver AT1R than brain AT1R, but that 125I‐Ang II binds to a larger population of AT1R in liver than does 125I‐Ang III.

Glucagon induces the protein expression of rat hepatocyte aquaporin‐8.

Glucagon stimulates the vesicle trafficking of aquaporin‐8 (AQP8) water channels to the canalicular membrane of rat hepatocytes, a mechanism thought to be involved in glucagon‐induced bile secretion. Our aim was to study whether glucagon is also able to stimulate the gene expression of hepatocyte AQP8. Glucagon was administered to rats at 0.2 mg/100g body wt ip for 8, 16, and 36 h. By immunoblotting, we found that 34 kDa AQP8 was increased by 79% and 107% in total liver membranes at 16 and 36 h. Hepatocyte AQP9 protein expression remained unaltered. AQP8 mRNA expression, assessed by real time PCR, was not modified over time. Immunoblotting studies in primary cultured rat hepatocytes showed that glucagon increased the total amount of AQP8 (e.g. 80% with 1μM glucagon) and confocal immunofluorescence showed a predominant AQP8 cell surface localization. The RNA polymerase II inhibitor actinomycin D did not prevent glucagon effect supporting the nontranscriptional upregulation of hepatocyte AQP8. Glucagon effect on AQP8 was mimicked by dibutyryl cAMP and supressed by the protein kinase A (PKA) inhibitors, H‐89 or PKI. In conclusion, our data suggest that glucagon induces the posttranscriptional gene expression of hepatocyte AQP8 via a cAMP‐dependent PKA pathway, a process likely relevant to the long‐term modulation of bile secretion. Supported by Grant PICT 31670 from ANPCyT

Development and Application of a Two-Phase, On-Membrane Digestion Method in the Analysis of Membrane Proteome

Analysis of membrane proteins, particularly integral membrane proteins, still presents a great challenge due to their poor water solubility and low abundance though much effort has been devoted to the solubilization and enrichment of the protein class. In this paper, a two-phase, on-membrane digestion method was developed and applied in the analysis of rat liver membrane proteome. The two-phase system was constituted by mixing n-butanol and 25 mM NH4HCO3. Comparative experiments indicated that the proteins on membranes could be digested in the two-phase system more efficiently than in both 60% methanol and 25 mM NH4HCO3 solutions under the same conditions, thereby improving the identification of the membrane proteins. When the established two-phase system and CapLC-MS/MS was used to analyze rat liver membrane proteome, a total of 411 membrane proteins were identified, more than 80% of which were transmembrane proteins with 1-12 mapped transmembrane domains (TMDs). Because of its extraction and dissolution actions, the two-phase on-membrane digestion system we developed could efficiently improve the digestion and removal of adsorbed nonmembrane proteins, and remarkably increase the number and coverage of identified membrane proteins, particularly the transmembrane proteins. Using our procedure to identify a complementary protein set from all fractions of the two-phase system could achieve a higher coverage of the membrane proteome.

Identification of plasma membrane autoantigens in autoimmune hepatitis type 1 using a proteomics tool

Autoimmune hepatitis (AIH) is a liver disease with circulating autoantibodies predominantly directed against widely held cellular components. Because AIH is a liver-specific disease, autoantibodies against plasma membrane antigens may be involved in its pathogenesis and have been reported; however, no definite identification has been described. We thus investigated the fine specificity of anti-hepatocyte plasma membrane autoantibodies in type 1 AIH (AIH-1) using a proteomic tool. A plasma membrane-enriched fraction was validated using enzymatic activity and western blot analysis experiments. Sera from AIH-1 patients (n = 65) and from 90 controls, that is, healthy blood donors (n = 40) and patients with systemic diseases (n = 20) or other liver diseases (n = 30), were studied by immunoblot performed with plasma membrane proteins resolved by either sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or 2-dimensional (2D) electrophoresis. Proteins contained in the immunoreactive spots were identified by sequences provided by ion-trap mass spectrometry. Hepatocytes probed with sera were also studied using confocal immunofluorescence and immunoelectron microscopy. The more prominent bands stained by patient sera were located at 38 kDa, 48, 50, 52 kDa, 62 kDa, 70 kDa, and a 95-kDa double band. Six proteins with known potential plasma membrane expression were identified: liver arginase (38 kDa), cytokeratins (CK) 8 and 18 (48-52 kDa), heat shock proteins (HSP) of 60, 70, 90 kDa, and valosin-containing protein (VCP) of 92 kDa. The presence of anti-membrane antibodies was confirmed by immunofluorescence and immunoelectron microscopy. Overall, our data demonstrate that liver arginase, CK 8/18, HSP 60, HSP 70, HSP 90, and VCP represent potential candidate targets on liver membrane for autoantibodies in AIH-1.

Binding kinetics and sequencing of hepatic α1‐adrenergic receptors in two marine teleosts, Mackerel (Scomber scombrus) and Anchovy (Engraulis encrasicolus)

Liver alpha(1)-adrenoceptors (ARs) are demonstrated, or at least hypothesized, in freshwater and brackish-water teleosts, whereas no data are available for marine teleosts. This study evaluates the presence of alpha(1)-ARs in the liver of two marine teleosts, the anchovy Engraulis encrasicolus and the mackerel Scomber scombrus, and examines on a broad scale the possibility that habitats posing different challenges also influence phenotypic trait selection. Binding assays were performed also on liver membranes from the carp Cyprinus carpio as a direct comparison with a freshwater species. Scatchard analysis of [(3)H]prazosin binding to purified liver membranes from anchovy, mackerel and carp resulted in K(d) values of 1.51+/-0.085, 1.26+/-0.098, and 2.61+/-0.22 nM, and B(max) values of 87.4+/-9.12, 77+/-8.29, and 115.22+/-3.31 fmol/mg protein, respectively. Thus, alpha(1)-ARs of the two marine teleosts showed higher [(3)H]prazosin affinity compared with those of the freshwater/brackish-water fish studied thus far, whereas the number of liver binding sites did not differ significantly from that of carp, eel or trout. A preliminary phylogeny based on amino acid sequence analysis indicated the presence of at least an alpha(1A)-AR in mackerel and an alpha(1D)-AR in both anchovy and mackerel. This is the first indication of alpha(1)-AR subtypes in any marine species, but further studies are needed to ascertain the physiological role of these alpha(1)-ARs in these two marine species.