HMGB in Mollusk Crassostrea ariakensis Gould: Structure,Pro-Inflammatory Cytokine Function Characterization and Anti-Infection Role of Its Antibody

Background

Crassostrea ariakensis Gould is a representative bivalve species and an economically important oyster in China, but suffers severe mortalities in recent years that are caused by rickettsia-like organism (RLO). Prevention and control of this disease is a priority for the development of oyster aquaculture. It has been proven that mammalian HMGB (high mobility group box) can be released extracellularly and acts as an important pro-inflammatory cytokine and late mediator of inflammatory reactions. In vertebrates, HMGB’s antibody (anti-HMGB) has been shown to confer significant protection against certain local and systemic inflammatory diseases. Therefore, we investigated the functions of Ca-HMGB (oyster HMGB) and anti-CaHMGB (Ca-HMGB’s antibody) in oyster RLO/LPS (RLO or LPS)-induced disease or inflammation.

Methodology/Principal Findings

Sequencing analysis revealed Ca-HMGB shares conserved structures with mammalians. Tissue-specific expression indicates that Ca-HMGB has higher relative expression in hemocytes. Significant continuous up-regulation of Ca-HMGB was detected when the hemocytes were stimulated with RLO/LPS. Recombinant Ca-HMGB protein significantly up-regulated the expression levels of some cytokines. Indirect immunofluorescence study revealed that Ca-HMGB localized both in the hemocyte nucleus and cytoplasm before RLO challenge, but mainly in the cytoplasm 12 h after challenge. Western blot analysis demonstrated Ca-HMGB was released extracellularly 4–12 h after RLO challenge. Anti-CaHMGB was added to the RLO/LPS-challenged hemocyte monolayer and real-time RT-PCR showed that administration of anti-CaHMGB dramatically reduced the rate of RLO/LPS-induced up-regulation of LITAF at 4–12 h after treatment. Flow cytometry analysis indicated that administration of anti-CaHMGB reduced RLO/LPS-induced hemocyte apoptosis and necrosis rates.

Conclusions/Significance

Ca-HMGB can be released extracellularly and its subcellular localization varies when stimulated with RLO. Ca-HMGB is involved in oyster immune reactions and functions as a pro-inflammatory cytokine. Anti-CaHMGB can significantly suppress RLO/LPS-induced inflammatory responses and hemocyte necrosis and apoptosis, suggesting that Ca-HMGB is a potential target to prevent and control RLO/LPS-induced disease or inflammation.     

Allograft Inflammatory Factor 1 Functions as a Pro-Inflammatory Cytokine in the Oyster,Crassostrea ariakensis

The oyster Crassostrea ariakensis is an economically important bivalve species in China, unfortunately it has suffered severe mortalities in recent years caused by rickettsia-like organism (RLO) infection. Prevention and control of this disease is a priority for the development of oyster aquaculture. Allograft inflammatory factor-1 (AIF-1) was identified as a modulator of the immune response during macrophage activation and a key gene in host immune defense reaction and inflammatory response. Therefore we investigated the functions of C. ariakensis AIF-1 (Ca-AIF1) and its antibody (anti-CaAIF1) in oyster RLO/LPS-induced disease and inflammation. Ca-AIF1 encodes a 149 amino acid protein containing two typical Ca²⁺ binding EF-hand motifs and shares a 48–95% amino acid sequence identity with other animal AIF-1s. Tissue-specific expression analysis indicates that Ca-AIF1 is highly expressed in hemocytes. Significant and continuous up-regulation of Ca-AIF1 is detected when hemocytes are stimulated with RLO/LPS (RLO or LPS). Treatment with recombinant Ca-AIF1 protein significantly up-regulates the expression levels of LITAF, MyD88 and TGFβ. When anti-CaAIF1 antibody is added to RLO/LPS-challenged hemocyte monolayers, a significant reduction of RLO/LPS-induced LITAF is observed at 1.5–12 h after treatment, suggesting that interference with Ca-AIF1 can suppress the inflammatory response. Furthermore, flow cytometric analysis indicated that anti-CaAIF1 administration reduces RLO/LPS-induced apoptosis and necrosis rates of hemocytes. Collectively these findings suggest that Ca-AIF1 functions as a pro-inflammatory cytokine in the oyster immune response and is a potential target for controlling RLO infection and LPS-induced inflammation.     

Proteomics of Synechocystis sp. strain PCC 6803: identification of plasma membrane proteins

Cyanobacteria are unique prokaryotes since they in addition to outer and plasma membranes contain the photosynthetic membranes (thylakoids). The plasma membranes of Synechocystis 6803, which can be completely purified by density centrifugation and polymer two-phase partitioning, have been found to be more complex than previously anticipated, i.e. they appear to be essential for assembly of the two photosystems. A proteomic approach for the characterization of cyanobacterial plasma membranes using two-dimensional gel electrophoresis and mass spectrometry analysis revealed a total of 57 different membrane proteins of which 17 are integral membrane spanning proteins. Among the 40 peripheral proteins 20 are located on the periplasmic side of the membrane, while 20 are on the cytoplasmic side. Among the proteins identified are subunits of the two photosystems as well as Vipp1, which has been suggested to be involved in vesicular transport between plasma and thylakoid membranes and is thus relevant to the possibility that plasma membranes are the initial site for photosystem biogenesis. Four subunits of the Pilus complex responsible for cell motility were also identified as well as several subunits of the TolC and TonB transport systems. Several periplasmic and ATP-binding proteins of ATP-binding cassette transporters were also identified as were two subunits of the F(0) membrane part of the ATP synthase.     

Two-dimensional electrophoresis and mass spectrometric identification of mitochondrial proteins from an SH-SY5Y neuroblastoma cell line

To probe the mitochondrial involvement in neurodegenerative processes, we have generated a high-resolution map of the mitochondrial proteome from a human neuroblastoma SH-SY5Y cell line that has been used for creating cytoplasmic hybrid cell systems. Two mitochondrial preparations were evaluated using two-dimensional (2D) gel electrophoresis and mass spectrometry; one obtained from differential centrifugation and the other by a multiple-step percoll/metrizamide gradient. The 2D gel maps prepared from these mitochondrial fractions separated over 300 distinct spots as visualized by colloidal Coomassie blue (CCB), or closer to 400 proteins with silver staining. The most abundant proteins identified in the mitochondrial fraction prepared by differential centrifugation were those of mitochondrial, cytoplasmic, and endoplasmic reticulum origin. Proteins obtained using the more intensive two-step gradient method were almost exclusively known to be associated with mitochondria. From this latter preparation, 84 of the most abundant gel spots were analyzed, out of which 61 proteins were identified. The absence of many membrane-associated proteins known to be associated with the mitochondrion and the limited number of total proteins observed in the 2D gel maps suggest that the majority of mitochondrial proteins are not being detected under these separation and staining conditions. An insoluble pellet obtained after solubilization of the mitochondrial fraction prepared with the percoll/metrizamide gradient was boiled in sodium dodecylsulfate (SDS) and separated by 1D sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This separation yielded some additional proteins, many of which are likely membrane-associated. These studies form the basis for the analysis of differential protein expression in cybrid cellular models of neurodegenerative disorders and in affected tissue from diseased states.     

Wissadula Proliferation in Jamaica II. Psyllid transmission and antibiotic therapy

Field symptoms of Wissadula proliferation (WP) in Jamaica were reproduced on W. periplocifolia (L.) C. Presl. ex Thwaites test plants, using field-collected or laboratory reared and infected Paracarsidara concolor Crawford. This psyllid naturally is associated with W. periplocifolia in the field. Diseased plants contained phloem-restricted prokaryotes, most of which resembled the phloem-restricted rickettsialike organisms (RLO) known to be associated with a small group of plant diseases. They were bound by a “cell wall” composed of a double unit membrane separated by an electron-lucent layer. A small proportion of the prokaryotes, which were bound by only one recognizable unit membrane and were ill-defined at the periphery, were difficult to distinguish from mycoplasmalike organisms (MLO). Penicillin, applied as a soil drench of 200—400 %mUg/ml caused remission of symptoms of the psyllid-infected test plants 6—17 days after first effective application, with the concomitant disappearance of all of the structures in the resulting new growth. Since MLO are not known to be penicillin-sensitive, it is proposed that the “MLO” were in fact, poorly preserved RLO and it is suggested that RLO may be the aetiological agent of WP. Achromycin also caused symptom remission in WP at a soil drench of 100—400 %mUg/ml, but was generally slightly slower-acting than penicillin, eliciting a response in 12—23 days. Both antibiotics could produce lengthy remissions; neither seemed curative since two penicillin- and two achromycin-treated plants redeveloped symptoms after cessation of treatment. WP was not transmitted by two cicadellids, Protalebra maculata Baker and Scaphytopius fuliginosus Osborn. P. concolor did not transmit disease to test plants of Abutilon hulseanum (Torr. & A. Gray) Torr, ex. Chapm. which was affected by a proliferation disease (Abutilon proliferation), symptomatologically similar to, and which occurred in the same locations, as WP. WP and AP thus may be aetiologically distinct. The putative RLO aetiology of WP provides evidence against Wissadula being an alternate host of the MLO-associated coconut lethal yellowing (CLY) disease in Jamaica, a fact reinforced by negative psyllid transmission tests to CLY-susceptible palms. The results for WP are discussed in relation to other diseases possibly caused by phloem-restricted RLO.     

Isolation and characterization of gap junctions from Drosophila melanogaster

Summary A procedure has been developed to isolate gap junction-enriched subcellular fractions from Drosophila. Crude membranes from larval homogenates were extracted with 1% N-lauroyl sarcosine in 6 M urea and the gap junctions were collected by centrifugation. The major proteins were separated by SDS PAGE and purified by electro-elution. Electron microscopy revealed structurally pleiomorphic gap junctions in the fractions which included (1) conventional, 16–18 nm-wide septalaminar, (2) collapsed, 13–15 nm-wide pentalaminar, (3) split, and (4) aggregated forms. The fractions contained five major proteins with apparent molecular weights of 18, 26, 36, 52 and 54 kD. Evidence based on (1) the degradation and aggregation behavior of the major proteins following electro-elution and reelectrophoresis, (2) immunological cross-reactivities by affinity-purified antibodies against the major proteins on immunoblots, and (3) immunofluorescent staining of presumptive gap junctions in Drosophila imaginal discs at the light-microscopic level and immunogold staining of purified gap junctions at the electron-microscopic level suggests that the major proteins are interrelated and of gap-junction origin.     

Immunoaffinity purification of plasma membrane with secondary antibody superparamagnetic beads for proteomic analysis

Plasma membrane (PM) has very important roles in cell-cell interaction and signal transduction, and it has been extensively targeted for drug design. A major prerequisite for the analysis of PM proteome is the preparation of PM with high purity. Density gradient centrifugation has been commonly employed to isolate PM, but it often occurred with contamination of internal membrane. Here we describe a method for plasma membrane purification using second antibody superparamagnetic beads that combines subcellular fractionation and immunoisolation strategies. Four methods of immunoaffinity were compared, and the variation of crude plasma membrane (CPM), superparamagnetic beads, and antibodies was studied. The optimized method and the number of CPM, beads, and antibodies suitable for proteome analysis were obtained. The PM of mouse liver was enriched 3-fold in comparison with the density gradient centrifugation method, and contamination from mitochondria was reduced 2-fold. The PM protein bands were extracted and trypsin-digested, and the resulting peptides were resolved and characterized by MALDI-TOF-TOF and ESI-Q-TOF, respectively. Mascot software was used to analyze the data against IPI-mouse protein database. Nonredundant proteins (248) were identified, of which 67% are PM or PM-related proteins. No endoplasmic reticulum (ER) or nuclear proteins were identified according to the GO annotation in the optimized method. Our protocol represents a simple, economic, and reproducible tool for the proteomic characterization of liver plasma membrane.     

Prokaryotic utilization of the twin-arginine translocation pathway: a genomic survey

The twin-arginine translocation (Tat) pathway, which has been identified in plant chloroplasts and prokaryotes, allows for the secretion of folded proteins. However, the extent to which this pathway is used among the prokaryotes is not known. By using a genomic approach, a comprehensive list of putative Tat substrates for 84 diverse prokaryotes was established. Strikingly, the results indicate that the Tat pathway is utilized to highly varying extents. Furthermore, while many prokaryotes use this pathway predominantly for the secretion of redox proteins, analyses of the predicted substrates suggest that certain bacteria and archaea secrete mainly nonredox proteins via the Tat pathway. While no correlation was observed between the number of Tat machinery components encoded by an organism and the number of predicted Tat substrates, it was noted that the composition of this machinery was specific to phylogenetic taxa.     

合浦珠母贝外套膜细胞engrailed、BMP2和Smad3的mRNA表达水平的相关性

软体动物engrailed蛋白和骨形成相关蛋白对胚胎贝壳区域边界形成可能具有重要作用,engrailed还被推测为调节基质蛋白在外套膜组织区域化表达的重要调控因子．因此,弄清调控engrailed在软体动物中特征表达的分子机制有着重要的研究意义．但是,由于贝类基因组测序尚不完整,目前也没有建立获得贝类细胞系,以致于许多预测可能参与调控的基因需要通过克隆来鉴定,而且经典的研究细胞信号通路的方法也很难得到应用．目前,在中国南海广泛养殖的合浦珠母贝中,已获知其BMP2和Smad3的cDNA全长,以该贝的基因组为模板,PCR扩增获得了一段engrailed编码区片段．经软件分析,该片段含有EH4结构域,且与其他物种engrailed蛋白具有很高的同源性．研究的贝中,特别是外套膜组织中,engrailed、BMP2和Smad3三者表达之间的相关性,将有助于我们理解贝壳形成的分子机制．贝壳缺刻后半定量PCR试验结果表明,三者均参与贝壳修复,且在贝壳缺刻后的修复过程中,engrailed和Smad3的mRNA表达变化规律非常相似,提示它们之间可能存在相互影响的联系．用地塞米松(DXM)和过氧化氢(H₂O₂)分别处理原代培养的贝外套膜组织迁出细胞,实时相对定量PCR检测engrailed、BMP2和Smad3的mRNA表达水平,统计分析结果表明,三者具有显著的相关性．上述所有结果为进一步研究贝类生物矿化的发育和信号转导机制提供了新的思路和基础．     

Identification of expressed genes in cDNA library of hemocytes from the RLO-challenged oyster, Crassostrea ariakensis Gould with special functional implication of three complement-related fragments (CaC1q1, CaC1q2 and CaC3)

A SMARTer? cDNA library of hemocyte from Rickettsia-like organism (RLO) challenged oyster, Crassostrea ariakensis Gould was constructed. Random clones (400) were selected and single-pass sequenced, resulted in 200 unique sequences containing 96 known genes and 104 unknown genes. The 96 known genes were categorized into 11 groups based on their biological process. Furthermore, we identified and characterized three complement-related fragments (CaC1q1, CaC1q2 and CaC3). Tissue distribution analysis revealed that all of three fragments were ubiquitously expressed in all tissues studied including hemocyte, gills, mantle, digestive glands, gonads and adductor muscle, while the highest level was seen in the hemocyte. Temporal expression profile in the hemocyte monolayers reveled that the mRNA expression levels of three fragments presented huge increase after the RLO incubation at 3 h and 6 h in post-challenge, respectively. And the maximal expression levels at 3 h in post-challenge are about 256, 104 and 64 times higher than the values detected in the control of CaC1q1, CaC1q2 and CaC3, respectively.     

Partial purification of mitochondrial ribosomes from broad bean and identification of proteins encoded by the mitochondrial genome

An approach towards the identification at the protein level of the ribosomal proteins encoded by the mitochondrial genome of broad bean (Vicia faba) has been developed. After Triton X-100 treatment of isolated mitochondria, a fraction enriched in mitochondrial ribosomes was obtained by successive centrifugation, first onto a sucrose cushion, and then in a sucrose gradient. Mitochondrial translation products were labelled in isolated mitochondria with [³⁵S]methionine and added to the enriched mitochondrial ribosomal proteins before separation by two-dimensional gel electrophoresis. Six spots, identified both by Coomassie blue staining and autoradiography, were analysed by protein micro-sequencing. Two of these were shown to correspond to ribosomal proteins S10 and S12. We conclude that these two proteins are encoded by the mitochondrial genome of broad bean and that the method described here can be used to identify other proteins encoded by the mitochondrial genome. Received: 4 September 1996 / Accepted: 30 November 1996     

Global Identification of Prokaryotic Glycoproteins Based on an Escherichia coli Proteome Microarray

Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an Escherichia coli proteome microarray containing 4,256 affinity-purified E.coli proteins. Twenty-three E.coli proteins that bound Wheat-Germ Agglutinin (WGA) were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB) was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA) staining, thereby increasing the number of validated E. coli glycoproteins from 3 to 18. By cataloguing glycoproteins in E.coli, our study greatly extends our understanding of protein glycosylation in prokaryotes.     

Improved methods for detecting enteric viruses in oysters.

New and improved methods for concentrating enteroviruses, reoviruses, and adenoviruses from oysters have been developed and evaluated. Viruses are efficiently adsorbed to homogenized oyster meat by adjusting the homogenate to pH 5.0 and a conductivity of less than or equal to 2,000 mg of NaCl per liter. After low-speed centrifugation, the virus-free supernatant is discarded and the viruses are eluted from the sedimented oyster solids with pH 7.5 glycine-NaCl having a conductivity of 8,000 mg of NaCl per liter. The oyster solids are removed by low-speed centrifugation and filtration, and the viruses in the filtered supernatant are concentrated to a small volume by either ultrafiltration or acid precipitation at pH 4.5. The concentrate is treated with antibiotics and inoculated into cell cultures for virus isolation and quantitation. When these methods were tested with oysters experimentally contaminated with polioviruses, reoviruses, and adenoviruses, recovery efficiencies averaged about 46%. With the exception of virus assay and quantitation, these methods are simple and inexpensive enough to be done in typical shellfish microbiology laboratories.     

Improved methods for detecting enteric viruses in oysters.

New and improved methods for concentrating enteroviruses, reoviruses, and adenoviruses from oysters have been developed and evaluated. Viruses are efficiently adsorbed to homogenized oyster meat by adjusting the homogenate to pH 5.0 and a conductivity of less than or equal to 2,000 mg of NaCl per liter. After low-speed centrifugation, the virus-free supernatant is discarded and the viruses are eluted from the sedimented oyster solids with pH 7.5 glycine-NaCl having a conductivity of 8,000 mg of NaCl per liter. The oyster solids are removed by low-speed centrifugation and filtration, and the viruses in the filtered supernatant are concentrated to a small volume by either ultrafiltration or acid precipitation at pH 4.5. The concentrate is treated with antibiotics and inoculated into cell cultures for virus isolation and quantitation. When these methods were tested with oysters experimentally contaminated with polioviruses, reoviruses, and adenoviruses, recovery efficiencies averaged about 46%. With the exception of virus assay and quantitation, these methods are simple and inexpensive enough to be done in typical shellfish microbiology laboratories.     

Histology, ultrastructure, and morphogenesis of a rickettsia-like organism causing disease in the oyster, Crassostrea ariakensis Gould

Moribund specimens of the oyster, Crassostrea ariakensis Gould, aged 2-3 years were collected from Hailing Bay in Yangxi County of Guangdong Province from February to May and November to December in the years 2001, 2002, and 2003. A massive infection by an obligate intracellular prokaryote, specifically a rickettsia-like organism (RLO), was found. Here we report investigations of this RLO in the tissues of the oyster C. ariakensis Gould and describe the histology, ultrastructure, and morphogenesis of this pathogen in C. ariakensis Gould. Light microscopic observations of stained tissues revealed cytoplasmic inclusion bodies typical of prokaryote infection in about 87% (26/30) of the oysters. Most inclusions were observed in epithelial cells and connective tissues of the gill, mantle, and digestive gland of most of the infected oysters. The shape, size, and color of inclusions from different tissues were polymorphic. Electron microscopic examination of digestive gland, gill, and mantle tissues showed that the RLOs were intracytoplasmic. RLOs were often round, dumb-bell-shaped (undergoing binary fission), or occasionally rod-shaped and ranged from approximately 0.58 to 1.20microm in size. The organisms exhibited an ultrastructure characteristic of prokaryotic bacteria-like cells, including a trilaminar cell wall, electron-dense periplasmic ribosome zone, and a DNA nucleoid. Reproductive stages, including transverse binary fission, were observed by TEM. These stages were frequently observed within membrane-bound cytoplasmic vacuoles. Hexagonal phage-like particles in the cytoplasm of RLOs were also observed.     

A proteomics approach to the identification of mammalian mitochondrial small subunit ribosomal proteins

Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins in six individual spots were subjected to in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry. The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled. Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins correspond to Escherichia coli S10 and S14. Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of one of these proteins was observed in D. melanogaster but not in C. elegans, while a homolog of the other was present in C. elegans but not in D. melanogaster. A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome. This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes.     

Separation of novel phosphoproteins of Porphyromonas gingivalis using phosphate‐affinity chromatography

Phosphorylation of serine, threonine and tyrosine is a central mechanism for regulating the structure and function of proteins in both eukaryotes and prokaryotes. However, the action of phosphorylated proteins present in Porphyromonas gingivalis, a major periodontopathogen, is not fully understood. Here, six novel phosphoproteins that possess metabolic activities were identified, namely PGN_0004, PGN_0375, PGN_0500, PGN_0724, PGN_0733 and PGN_0880, having been separated by phosphate‐affinity chromatography. The identified proteins were detectable by immunoblotting specific to phosphorylated Ser (P‐Ser), P‐Thr, and/or P‐Tyr. These results imply that novel phosphorylated proteins might play an important role for regulation of metabolism in P. gingivalis.     

Correlation of neuropathy target esterase activity with specific tritiated di-isopropyl phosphorofluoridate-labelled proteins.

Neuropathy target esterase (NTE) is a membrane-bound carboxylesterase activity that has been proposed as the target site for initiation of organophosphate-induced delayed neuropathy. This activity is identified by its resistance to treatment with Paraoxon and sensitivity to co-incubation with Paraoxon and Mipafox. Sucrose-density-gradient centrifugation of membrane-associated proteins isolated from chick-embryo brains identified three proteins, Mr 161,000, 116,500 and 103,000, that were labelled with [3H]di-isopropyl phosphorofluoridate in an NTE-like manner and that co-migrated with NTE. The 161,000-Mr and 116,500-Mr proteins were identified in both adult and embryo brain. One or both of these proteins may therefore contribute to the activity defined as NTE. In addition, a 61,000-Mr protein was identified that does not comigrate with NTE, but that was labelled with [3H]di-isopropyl phosphorofluoridate in a Paraoxon-resistant and Mipafox-sensitive manner. The effect of Mipafox on labelling, however, was reversibly blocked by co-incubation with Paraoxon. This protein, therefore, is not NTE, but has the necessary inhibitor-sensitivity to be the target site for organophosphate-induced delayed neuropathy.     

A dynamic plasma membrane proteome analysis of alcohol-induced liver cirrhosis

ABSTRACT: Alcohol-induced injury has become one of the major causes for liver cirrhosis. However, the molecular mechanisms of ethanol-induced injury are not fully understood. To this end, we performed a dynamic plasma membrane proteomic research on rat model. A rat model from hepatitis to liver cirrhosis was developed. Plasma membrane from liver tissue with liver fibrosis stage of 2 and 4 (S2 and S4) was purified by sucrose density gradient centrifugation. Its purification was verified by western blotting. Proteins from plasma membrane were separated by two-dimensional electrophoresis (2DE) and differentially expressed proteins were identified by tandem mass spectrometry. 16 consistent differentially expressed proteins from S2 to S4 were identified by mass spectrometry. The expression of differentially expressed proteins annexin A6 and annexin A3 were verified by western blotting, and annexin A3 was futher verified by immunohistochemistry. Our research suggests a possible mechanism by which ethanol alters protein expression to enhance the liver fibrosis progression. These differentially expressed proteins might be new drug targets for treating alcoholic liver cirrhosis.     

Proteomic profiling of accessory structures from the mouse sperm flagellum

The flagellum of a mammalian spermatozoon consists of an axoneme surrounded in distinct regions by accessory structures known as the fibrous sheath, outer dense fibers, and the mitochondrial sheath. Although the characterization of individual proteins has provided clues about the roles of these accessory structures, a more complete understanding of flagellar function requires the identification of all the polypeptides in these assemblies. Epididymal mouse sperm were treated with SDS to dislodge sperm heads and to extract the axoneme and membranous elements. The remaining flagellar accessory structures were purified by sucrose gradient centrifugation. Analysis of proteins from these structures by two-dimensional gel electrophoresis and colloidal Coomassie Blue staining showed a highly reproducible pattern of >200 spots. Individual spots were picked, digested with trypsin, and identified by mass spectrometry and peptide microsequencing. Approximately 50 individual proteins were identified that could be assigned to five general categories: 1) proteins previously reported to localize to the accessory structures, e.g. ODF2 in the outer dense fibers, the sperm-specific glyceraldehyde-3-phosphate dehydrogenase in the fibrous sheath, and glutathione peroxidase in the mitochondrial sheath, validating this proteomic approach; 2) proteins that had not been shown to localize to any accessory structure but would be predicted to be present, e.g. glycolytic enzymes; 3) proteins known to be part of the flagellum but not localized to a specific site, e.g. adenylate kinase; 4) proteins not expected to be part of the accessory structures based on their previously reported locations, e.g. tektins; and 5) unknown proteins for which no information is available to make a determination as to location. The unexpected presence of the tektins in the accessory structures of the flagellum was confirmed by both immunoblot and immunofluorescence analysis. This proteomic analysis identified a number of unexpected and novel proteins in the accessory structures of the mammalian flagellum.