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PubMed2004

The influence of protein tyrosine phosphatase-1B on Na,K-ATPase activity in lens.

The abnormal sodium content of many cataracts suggests Na,K-ATPase is vital for maintenance of eye lens transparency. Since tyrosine phosphorylation is considered a possible regulatory mechanism for Na,K-ATPase, experiments were conducted to test the influence of protein tyrosine phosphatase-1B (PTP-1B) on Na,K-ATPase activity. Membrane material was isolated separately from porcine lens epithelium and fiber cells. Tyrosine phosphoproteins, Na,K-ATPase alpha1 polypeptide and PTP-1B were examined by Western blot. Na,K-ATPase activity was determined by measuring ATP hydrolysis in the presence or absence of ouabain. Western blot analysis revealed tyrosine phosphorylation of multiple membrane proteins in both lens cell types, the differentiated fiber cells and non-differentiated epithelium. When membrane material was subjected to immunoprecipitation using an antibody directed against Na,K-ATPase alpha1, a colocalized phosphotyrosine band was detected in lens fibers but not epithelium. Incubation with PTP-1B caused a approximately 50% increase of Na,K-ATPase activity in fiber membrane material. Na,K-ATPase activity in lens epithelium membrane material was not significantly altered by PTP-1B treatment even though PTP-1B was demonstrated to cause dephosphorylation of multiple membrane proteins in the epithelium as well as fibers. While endogenous PTP-1B was detected in both cell types, endogenous tyrosine phosphatase activity was low in both epithelium and fiber membrane material. The results illustrate endogenous tyrosine phosphorylation of Na,K-ATPase alpha1 polypeptide in fibers. Na,K-ATPase alpha1 in lens fibers may be a potential target for PTP-1B.

Adenosine Triphosphate↗
PubMed2001

Centrosomal association of histone macroH2A1.2 in embryonic stem cells and somatic cells.

The histone 2A variant macroH2A1.2 is expressed in female and male mammals and is implicated in X-chromosome inactivation and autosomal gene silencing. In undifferentiated and early differentiating murine embryonic stem (ES) cells a cytosolic pool of macroH2A1.2 has recently been reported and found to be associated with the centrosome. Here, we show that the centrosomal association of macroH2A1.2 is a widespread phenomenon and is not restricted to undifferentiated and early differentiating ES cells. By indirect immunofluorescence we detect macroH2A1.2 protein in a juxtanuclear structure that duplicates once per cell cycle and colocalizes with centrosomal gamma-tubulin in both XX and XY ES cells prior to and throughout their differentiation. MacroH2A1.2 localization to the centrosome is also observed in female and male somatic cells, both in interphase and in mitosis. Biochemical analysis demonstrates that the association between macroH2A1.2 and the centrosome in somatic cells is stable, as macroH2A1.2 copurifies with centrosomes isolated from human lymphoblasts. Therefore, in addition to a nuclear pool of macroH2A1.2 a fraction of the histone is associated with the centrosome in various cell types and throughout ES cell differentiation.

Animals↗
PubMed1993

Double-label analyses of the coexistence of somatostatin with GABA and glycine in amacrine cells of the larval tiger salamander retina.

To investigate the possible GABAergic nature of somatostatin-immunoreactive neurons of the larval tiger salamander retina, somatostatin immunocytochemistry was combined with either gamma-aminobutyric acid (GABA) immunocytochemistry or autoradiography of GABA high-affinity uptake. A total of 1,062 somatostatin cells were visualized in these studies. Double-label immunocytochemistry revealed that 96.3% of somatostatin-immunoreactive cells expressed GABA immunoreactivity. Double-label studies combining somatostatin immunocytochemistry with autoradiography of GABA high-affinity uptake revealed a slightly lower percentage (93%) of colocalization. Double-labelled cells were identified as Type 1, Type 2 and displaced amacrine cells. The small percentage of somatostatin-immunoreactive cells that did not co-label for GABA were identified as Type 1 amacrine cells. An analysis of retinal sections processed for double-label immunocytochemistry revealed that approximately 5% of GABA-immunoreactive cells in the amacrine and ganglion cell layers co-label for somatostatin. Somatostatin immunocytochemistry was combined with autoradiography of glycine high-affinity uptake to examine whether tiger salamander somatostatin-amacrine cells express this glycine marker. A total of 100 somatostatin-immunoreactive amacrine cells were visualized in double-label preparations. None of these cells were observed to exhibit glycine high-affinity uptake.

Animals↗
PubMed2004

Regulation of nestin expression after cortical ablation in adult rat brain.

During embryogenesis, transient expression of nestin in proliferating neuroepithelial stem cells signals the commitment of progenitor cells to differentiate. Although adult mammalian brain contains very little nestin, significant upregulation of nestin has been reported following cerebral injury, leading to speculation that nestin may be involved in brain repair. In this study, we assessed the temporal profile of nestin expression following ablation injury of the sensory barrel cortex and investigated the influence of contralateral whisker stimulation on nestin expression. Since the adult mammalian brain contains proliferating neuronal progenitor cells that can be labeled with bromodeoxyuridine (BrdU), we also determined the association of nestin reexpression with BrdU-labeled cells. Nestin reexpression was detected predominantly in the ipsilateral cortex 3 days post-ablation. There was no significant nestin upregulation in the subcortical region. Nestin reexpression was most marked surrounding the lesion, but also extended throughout the entire lateral cortex. Nestin in the ipsilateral cortex subsided by day 7, although perilesional nestin expression was still apparent 28 days post-injury. Western blot analysis of nestin expression 3 days post-ablation confirmed a significant two-fold increase in nestin expression (p<0.05). Double immunofluorescence labeling demonstrated that the majority of nestin expression occurred in astrocytes. We were unable to detect any colocalization with neuronal makers. However, BrdU-labeled cells, which were readily detected in the subventricular zone prior to injury, were readily detected in the perilesional area 3 days post-ablation, concomitant with nestin in this area. Confocal microscopy detected several BrdU-positive cells expressing nestin. Taken together, the data support a potential role for nestin reexpression in brain repair.

Animals↗
PubMed2005

PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.

Animals↗
PubMed2006

Morphology and synaptic input of substance P receptor-immunoreactive interneurons in control and epileptic human hippocampus.

Substance P (SP) is known to be a peptide that facilitates epileptic activity of principal cells in the hippocampus. Paradoxically, in other models, it was found to be protective against seizures by activating substance P receptor (SPR)-expressing interneurons. Thus, these cells appear to play an important role in the generation and regulation of epileptic seizures. The number, distribution, morphological features and input characteristics of SPR-immunoreactive cells were analyzed in surgically removed hippocampi of 28 temporal lobe epileptic patients and eight control hippocampi in order to examine their changes in epileptic tissues. SPR is expressed in a subset of inhibitory cells in the control human hippocampus, they are multipolar interneurons with smooth dendrites, present in all hippocampal subfields. This cell population is considerably different from SPR-positive cells of the rat hippocampus. The CA1 (cornu Ammonis subfield 1) region was chosen for the detailed morphological analysis of the SPR-immunoreactive cells because of its extreme vulnerability in epilepsy. The presence of various neurochemical markers identifies functionally distinct interneuron types, such as those responsible for perisomatic, dendritic or interneuron-selective inhibition. We found considerable colocalization of SPR with calbindin but not with parvalbumin, calretinin, cholecystokinin and somatostatin, therefore we suppose that SPR-positive cells participate mainly in dendritic inhibition. In the non-sclerotic CA1 region they are mainly preserved, whereas their number is decreased in the sclerotic cases. In the epileptic samples their morphology is considerably altered, they possessed more dendritic branches, which often became beaded. Analyses of synaptic coverage revealed that the ratio of symmetric synaptic input of SPR-immunoreactive cells has increased in epileptic samples. Our results suggest that SPR-positive cells are preserved while principal cells are present in the CA1 region, but show reactive changes in epilepsy including intense branching and growth of their dendritic arborization.

Adult↗
PubMed2005

Expression of epithin in mouse preimplantation development: its functional role in compaction.

The preimplantation development of mammalian embryo after fertilization encompasses a series of events including cleavage, compaction, and differentiation into blastocyst. These events are likely to be associated with substantial changes in embryonic gene expression. In the present study, we explored the expression patterns and function of epithin, a mouse type II transmembrane serine protease, during preimplantation embryo development. RT-PCR analysis showed that epithin mRNAs were detectable during the cleavage stages from a 1-cell zygote to the blastocyst. Immunocytochemical studies revealed that epithin protein was expressed at blastomere contacts of the compacted 8-cell and later embryonic stages. Epithin colocalized with E-cadherin at the membrane contacts of the compacted morula-stage embryo as revealed by double-staining immunocytochemistry and confocal microscopy, respectively. Post-transcriptional epithin gene silencing by RNA interference (RNAi) resulted in the blockade of 8-cell in vitro-stage embryo compaction and subsequent embryonic deaths after several rounds of cell division. These results strongly suggest that epithin plays an important role in the compaction processes that elicit the signal for the differentiation into trophectoderm and inner cell mass.

Animals↗
PubMed2004

Localization of orexin-A-like immunoreactivity in prolactin cells in the bullfrog (Rana catesbeiana) pituitary.

Immunohistochemical techniques were employed to investigate the distribution of orexin-A-like immunoreactivity in the bullfrog (Rana catesbeiana) pituitary. Orexin-A-immunoreactive cells were scattered throughout the pars distalis. We found that these cells corresponded to the cells immunostained with antiserum against bullfrog prolactin (fPRL). Immunoelectron microscopic analysis indicated that an orexin-A-like substance coexisted with fPRL within secretory granules. Western blot analysis of bullfrog pituitary extract revealed that anti-human orexin-A antiserum labeled two separate bands which were not labeled with anti-fPRL antiserum. The present study has, for the first time, provided evidence of the intragranular colocalization of orexin-A-like and PRL immunoreactivities in the bullfrog pituitary.

Animals↗
PubMed2003

Dendritic cells in neointima formation after rat carotid balloon injury: coordinated expression withanti-apoptotic Bcl-2 and HSP47 in arterial repair.

OBJECTIVES: We sought to evaluate: 1) the contribution of dendritic cells (DCs); and 2) the impact of B-cell lymphoma 2 protein (Bcl-2), a central anti-apoptotic protooncogene, and of heat shock protein 47 (HSP47), indicating subsequent collagen deposition, in neointima formation after angioplasty. BACKGROUND: The origin of neointimal cells and the factors that promote their accumulation are still unclear. Previous studies reported intimal presence of DCs and suggested cells of primarily extravascular origin to contribute to arterial repair. METHODS: Sprague-Dawley rats underwent carotid balloon angioplasty. At different times after angioplasty, tissue sections were analyzed by immunohistochemistry using OX-62 and S100 as DC markers and antibodies against Bcl-2 and HSP47, supplemented by electron microscopic analysis of cell type and apoptosis. RESULTS: Four days after injury, DCs adhered along the internal elastic lamina and demonstrated intense Bcl-2 and HSP47 expression, consistent with low apoptosis. With ongoing neointima enlargement, luminal DCs remained prevalent and were colocalized with Bcl-2 and HSP47, while signaling decreased to basal regions. Media showed no DCs and only low Bcl-2 and HSP47 immunoreactivity. Adventitia transiently revealed a structural separation between day 4 and 7. Whereas the inner layer demonstrated sparse cellularity, apoptosis and no DC, Bcl-2, and HSP47 labeling, the outer layer was characterized by high myofibroblast density with strong Bcl-2 and HSP47 expression but absence of DCs. CONCLUSIONS: We identify DCs as novel components in early neointima formation, promoted by coordinated anti-apoptotic Bcl-2 and HSP47 expression. Despite intense adventitial remodeling, there is no evidence of adventitial cell transmigration.

Angioplasty, Balloon↗
PubMed2002

Two kinesin-like Kin I family proteins in fission yeast regulate the establishment of metaphase and the onset of anaphase A.

BACKGROUND: Metaphase is thought to be a force-equilibrium state of "tug of war," in which poleward forces are pulling kinetochores and counteracting the cohesive forces between the centromeres. Unlike conventional kinesins, members of the Kin I family are microtubule-depolymerizing enzymes, which are expected to be molecules that could generate poleward forces. RESULTS: We have characterized mitotic roles of two Kin I homologs, Klp5 and Klp6, in fission yeast. Klp5 and Klp6 colocalize to the mitotic kinetochores and the spindle midzone. These two proteins form a heterocomplex, but not a homocomplex. Albeit not essential, both proteins are required for accurate chromosome segregation and normal morphology of interphase microtubules. Time-lapse live analysis using GFP-alpha-tubulin indicates that these mutants spend a much longer time (2-fold) in mitosis before the initiation of anaphase B. Further observation using kinetochore and centromere markers shows that, in these mutants, sister centromeres move back and forth between the two poles, indicating that entry into anaphase A is delayed. This is supported by live image analysis showing that Cut2 securin is retained during the prolonged mitosis. Furthermore, the mitotic extension is dependent upon the Mad2 spindle checkpoint. CONCLUSIONS: We discuss two models of Kin I function in fission yeast. One proposes that Klp5 and Klp6 are required for efficient capturing of kinetochores by the spindles, while the other proposes that they are required to generate tension upon kinetochore capturing. Kin I, therefore, plays a fundamental role in the establishment of metaphase, probably by generating poleward forces at the kinetochores.

Anaphase↗
PubMed2005

The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin.

Mixed lineage leukemia (MLL) fusion proteins are derived from translocations at 11q23 that occur in aggressive subtypes of leukemia. As a consequence, MLL is joined to different unrelated proteins to form oncogenic transcription factors. Here we demonstrate a direct interaction between several nuclear MLL fusion partners and present evidence for a role of these proteins in histone binding. In two-hybrid studies, ENL interacted with AF4 and AF5q31 as well as with a fragment of AF10. A structure-function analysis revealed that the AF4/AF5q31/AF10 binding domain in ENL coincided with the C-terminus that is essential for transformation by MLL-ENL. The ENL/AF4 association was corroborated by GST-pulldown experiments and by mutual coprecipitation. Both proteins colocalized in vivo in a nuclear speckled pattern. Moreover, AF4 and ENL coeluted on sizing columns together with the known ENL binding partner Polycomb3, suggesting the presence of a multiprotein complex. The overexpression of ENL alone activated a reporter construct and a mutational screen indicated the conserved YEATS domain as essential for this function. Overlay and pulldown-assays finally showed a specific and YEATS domain-dependent association of ENL with histones H3 and H1. In summary, our studies support a common role for nuclear MLL fusion partners in chromatin biology.

Animals↗
PubMed2003

Identification of phospholipid scramblase 1 as a novel interacting molecule with beta -secretase (beta -site amyloid precursor protein (APP) cleaving enzyme (BACE)).

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) is an integral membrane aspartic proteinase responsible for beta-site processing of APP, and its cytoplasmic region composed of 24 amino acid residues has been shown to be involved in the endosomal localization of BACE. With the yeast two-hybrid screening, we found that the cytoplasmic domain of phospholipid scramblase 1 (PLSCR1), a type II integral membrane protein, interacts with the cytoplasmic region of BACE. In cultured cells, BACE and PLSCR1 were colocalized in the Golgi area and in endosomal compartments, whereas they were co-redistributed in late endosome-derived multivesicular bodies when treated with U18666A, suggesting that both proteins share a common trafficking pathway in cells. Co-immunoprecipitation analysis showed that both proteins form a protein complex at an endogenous expression level in the human neuroblastoma SH-SY5Ycells, and the dileucine residue of the BACE tail is also revealed to be essential for the physical interaction with PLSCR1 in vitro and in vivo. Moreover, both BACE and PLSCR1 were localized in a low buoyant lipid microdomain in SH-SY5Y cells. The dileucine-defective BACE mutant was also fractionated into the lipid microdomain, but much less stably than wild-type BACE. Taken together, our current study suggests the functional involvement of PLSCR1 in the intracellular distribution of BACE and/or recruitment of BACE into the detergent-insoluble lipid raft.

Amyloid Precursor Protein Secretases↗
PubMed2001

Differentiation- and stress-dependent nuclear cytoplasmic redistribution of myopodin, a novel actin-bundling protein.

We report the cloning and functional characterization of myopodin, the second member of the synaptopodin gene family. Myopodin shows no significant homology to any known protein except synaptopodin. Northern blot analysis resulted in a 3.6-kb transcript for mouse skeletal and heart muscle. Western blots showed an 80-kD signal for skeletal and a 95-kD signal for heart muscle. Myopodin contains one PPXY motif and multiple PXXP motifs. Myopodin colocalizes with alpha-actinin and is found at the Z-disc as shown by immunogold electron microscopy. In myoblasts, myopodin shows preferential nuclear localization. During myotube differentiation, myopodin binds to stress fibers in a punctuated pattern before incorporation into the Z-disc. Myopodin can directly bind to actin and contains a novel actin binding site in the center of the protein. Myopodin has actin-bundling activity as shown by formation of latrunculin-A-sensitive cytosolic actin bundles and nuclear actin loops in transfected cells expressing green fluorescent protein-myopodin. Under stress conditions, myopodin accumulates in the nucleus and is depleted from the cytoplasm. Nuclear export of myopodin is sensitive to leptomycin B, despite the absence of a classical nuclear export sequence. We propose a dual role for myopodin as a structural protein also participating in signaling pathways between the Z-disc and the nucleus.

Actins↗
PubMed2003

APP processing is regulated by cytoplasmic phosphorylation.

Amyloid-beta peptide (Abeta) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the beta-secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by alpha-secretase. The production of Abeta is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase Abeta generation.

Alzheimer Disease↗
PubMed1999

Nuclear accumulation of S-adenosylhomocysteine hydrolase in transcriptionally active cells during development of Xenopus laevis.

The oocyte nuclear antigen of the monoclonal antibody 32-5B6 of Xenopus laevis is subject to regulated nuclear translocation during embryogenesis. It is distributed in the cytoplasm during oocyte maturation, where it remains during cleavage and blastula stages, before it gradually reaccumulates in the nuclei during gastrulation. We have now identified this antigen to be the enzyme S-adenosylhomocysteine hydrolase (SAHH). SAHH is the only enzyme that cleaves S-adenosylhomocysteine, a reaction product and an inhibitor of all S-adenosylmethionine-dependent methylation reactions. We have compared the spatial and temporal patterns of nuclear localization of SAHH and of nuclear methyltransferase activities during embryogenesis and in tissue culture cells. Nuclear localization of Xenopus SAHH did not temporally correlate with DNA methylation. However, we found that SAHH nuclear localization coincides with high rates of mRNA synthesis, a subpopulation colocalizes with RNA polymerase II, and inhibitors of SAHH reduce both methylation and synthesis of poly(A)(+) RNA. We therefore propose that accumulation of SAHH in the nucleus may be required for efficient cap methylation in transcriptionally active cells. Mutation analysis revealed that the C terminus and the N terminus are both required for efficient nuclear translocation in tissue culture cells, indicating that more than one interacting domain contributes to nuclear accumulation of Xenopus SAHH.

Adenosylhomocysteinase↗
PubMed2005

Bisecting GlcNAc mediates the binding of annexin V to Hsp47.

The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : beta-D-mannoside beta-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GlcNAc structure. In this study, we reported on a search for potential target glycoproteins for annexin V in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5 microM, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.

Acetylglucosamine↗
PubMed1999

Induction of argininosuccinate synthetase in rat brain glial cells after striatal microinjection of immunostimulants.

The enzyme argininosuccinate synthetase (ASS) initiates the metabolic pathway leading from L-citrulline to L-arginine, the only physiological substrate of all isoforms of nitric oxide synthases. The presence of ASS in glial cells in vivo was investigated by immunohistochemical methods in a model of rat brain inflammation. Phosphate-buffered saline or a mixture of bacterial lipopolysaccharide and interferon-gamma was injected into the left striatum, and animals were killed 24 hours later. Ipsilateral and contralateral sides of brain sections were incubated with an antiserum against ASS or antibodies against cell-specific markers. In the three areas examined, striatum, corpus callosum, and cortex, a strong induction of ASS immunoreactivity was observed in glial cells after injection of immunostimulants. A detailed quantitative analysis of double-stained sections revealed that ASS was almost exclusively expressed in reactive, ED1-positive microglial cells/brain macrophages in immunostimulant- or sham-injected ipsilateral sides of the sections. Furthermore, ASS/ED1 costaining was observed in perivascular cells. Colocalization of ASS with astroglial marker glial fibrillary acidic protein was given only occasionally after immunostimulation. ASS-positive neurons were detected in control and experimental animals; staining intensity was comparable in both cases. The results suggest that neurons express ASS constitutively, whereas the enzyme is induced in glial cells in response to proinflammatory stimuli. This finding is the first demonstration of an induction of a pathway auxiliary to generation of nitric oxide in brain in response to immunostimulants and provides new insight into neural arginine metabolism.

Adjuvants, Immunologic↗
PubMed2003

Immunohistochemical characterization of hepatic malondialdehyde and 4-hydroxynonenal modified proteins during early stages of ethanol-induced liver injury.

BACKGROUND: Chronic ethanol consumption is associated with hepatic lipid peroxidation and the deposition or retention of aldehyde-adducted proteins postulated to be involved in alcohol-induced liver injury. The purpose of this study was to characterize hepatocellular formation of aldehyde-protein adducts during early stages of alcohol-induced liver injury. METHODS: Female Sprague Dawley(R) rats were subjected to the intragastric administration of a low-carbohydrate/high-fat total enteral nutrition diet or a total enteral nutrition diet containing ethanol for a period of 36 days. Indexes of hepatic responses to ethanol were evaluated in terms of changes in plasma alanine aminotransferase activity, hepatic histopathologic analysis, and induction of cytochrome P-4502E1 (CYP2E1). Immunohistochemical methods were used to detect hepatic proteins modified with malondialdehyde (MDA) or 4-hydroxynonenal (4-HNE) for subsequent quantitative image analysis. RESULTS: After 36 days of treatment, rats receiving the alcohol-containing diet displayed hepatic histopathologies characterized by marked micro- and macrosteatosis associated with only minor inflammation and necrosis. Alcohol administration resulted in a 3-fold elevation of plasma alanine aminotransferase activity and 3-fold increases (p < 0.01) in hepatic CYP2E1 apoprotein and activity. Quantitative immunohistochemical analysis revealed significant (p < 0.01) 5-fold increases in MDA- and 4-HNE modified proteins in liver sections prepared from rats treated with alcohol. The MDA- or 4-HNE modified proteins were contained in hepatocytes displaying intact morphology and were colocalized primarily with microvesicular deposits of lipid. Aldehyde-modified proteins were not prevalent in parenchymal or nonparenchymal cells associated with foci of necrosis or inflammation. CONCLUSIONS: These results suggest that alcohol-induced lipid peroxidation is an early event during alcohol-mediated liver injury and may be a sensitizing event resulting in the production of bioactive aldehydes that have the potential to initiate or propagate ensuing proinflammatory or profibrogenic cellular events.

Aldehydes↗