-
[show abstract]
[hide abstract]
ABSTRACT: Little is known about mechanisms through which OmpR/EnvZ and CpxA/CpxR regulate the expression of OmpC and OmpF, and thereby regulate bacterial responses to antibiotics exposure. In this study we investigated the relationships among OmpC, OmpF and TCSs using Escherichia coli strains with the deletion of ompR, envZ, cpxA and cpxR genes and antibiotics nalidixic acid and chlortetracycline. Significant changes at expression levels of OmpC and OmpF were detected in the strains exposed to the antibiotics. Outer membrane proteins that were altered in response to the changes of OmpC and OmpF expression levels were identified using proteomics approaches and the networks involved in the regulation of protein changes in response to the antibiotic exposure were constructed. It was found that AtpB was upregulated and downregulated in parallel with the reduced and elevated expression of OmpC in response to chlortetracycline and nalidixic acid exposure, respectively and the change of AtpB was regulated by CpxR. These findings were validated by the detection of OmpC, OmpF and CpxR changes in ΔatpD and ΔphoE. Therefore, this study provides novel insight into the regulation network of TCSs and OM proteins involved in the differential responses of bacteria to different classes of antibiotics.
Journal of proteomics 08/2012; 75(18):5898-910. · 5.07 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Outbreaks of infectious diseases in cultured large yellow croaker have resulted in great economic losses. However, information regarding its immune defense is limited. In the present study, an approach by the combination of differential proteomics with EST resource was applied for investigation of a profile of serum immune response by large yellow croaker to Aeromonas hydrophila challenge after immunization and for characterizing of one of the targeted immune molecules. Of the twelve altered proteins involved in the response, eight were identified by MS, in which three were randomly selected for antiserum preparation and were further confirmed by Western blotting. Furthermore, three beta(2)m clones, one of the altered molecules, were obtained from a previously constructed Kidney Smart cDNA library of this fish, and were compared for their identity, which contributed to the identification of beta(2)m cDNA diversity. Meanwhile, the up-regulated beta(2)m in response to the bacterial immunization and challenge was further confirmed by Western blotting. Our results indicate that beta(2)m is involved in the immune response of large yellow croaker to the challenge by A. hydrophila after immunization, which suggests an efficient approach for characterizing of targeted molecules at both the gene and protein levels.
Fish & Shellfish Immunology 10/2009; 28(1):151-8. · 3.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Bacterium is still one of the major causes of life-threatening microbial infections. The most effective way to control bacterial infections is probably vaccine prevention. However, development of bacterial vaccine, especially polyvalent vaccines that could be used to fight against a variety of bacterial serotypes and species, is challenging due to the difficulty in identifying broad cross-protective antigens for different serotypes and species of pathogenic bacteria. In the present study, we developed a new approach to identify polyvalent vaccine candidates from outer membrane (OM) proteins of Vibrio alginolyticus with the ability to fight against infections caused by different genera and families of Gram-negative bacteria. This approach combined heterogeneous antiserum-based immunoproteomics with bacterial immunization challenging method. Four of the 35 protein spots resolved in a 2-DE gel of V. alginolyticus sarcosine-insoluble fraction could be recognized not only by homogeneous antiserum, but also by heterogeneous antisera obtained from other bacterial species, genera and families. The genes encoding the four OM proteins were then cloned and expressed in E. coli BL21. The expressed recombinant proteins were used as broadly cross-protective immunogens to immunize carps for investigation of their cross-protective spectra, activities and protective abilities in carps. The carps immunized with OmpA (VA0764) and Pal (VA1061) have abilities to fight against infections not only caused by V. alginolyticus, but also by Aeromonas hydrophila and Pseudomonas fluorescens. This study provides a novel approach for the identification of broadly cross-protective antigens, and possibly polyvalent vaccines against a variety of microbial infections.
Journal of Proteome Research 08/2009; 8(9):4342-9. · 5.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The available Escherichia coli genome sequences offer an opportunity to further expand our understanding of this bacterium. In the current study, we present a rapid method for the isolation of bacterial alkaline proteins using acid incubation, purification and protein array by 2-DE, followed by protein identification using MS. Fifty-seven proteins were randomly chosen, in which 55 were identified by a database searching of MS data. The searching results showed that most of these alkaline proteins were involved in special functions within the cell, suggesting that alkaline proteome is an ideal fraction for an understanding of their special functions. Furthermore, alkaline proteomes were compared between the period of majority live bacteria (18-h culture), the period of similar amount of live and dead bacteria (30-h culture) and the period of majority dead bacteria (48-h culture). Six proteins were identified as differentially expressed targets, in which putative transcriptional regulator and superoxide dismutase genes were cloned and expressed for antiserum preparations. The antisera were applied for the confirmation of results obtained from 2-DE. The presented data clearly reveal that alkaline proteome analysis by 2-DE with MS plays an important role in the understanding of protein functions within the cell, and six alkaline proteins are determined as key ones in an overgrowth-mediated growth cycle of E. coli.
PROTEOMICS 11/2006; 6(19):5212-20. · 4.51 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Photobacterium damsela is a marine pathogen to both fish and human beings. The bacterium can shift between the ambient seawater and hosts, suggesting the existence of proteins rapidly responding to salt concentration. In the current study, proteomic methodologies were applied to screen the outer membrane proteins (OMPs) related to salt stress. OmpW and OmpV were determined in the response in this bacterium as OmpC and OmpF did in E. coli. Furthermore, the two genes were overexpressed in E. coli Top10F and complemented in V. paraheamolyticus mutants. The ability in salt-tolerance was elevated in the E. coli overexpressed OmpW and reduced in the cells overexpressed OmpV. These V. paraheamolyticus mutants could recover their response to environmental salt concentration when they were complemented by P. damsela OmpW and OmpV. These findings indicate that OmpW and OmpV are required for environmental salt regulation in P. damsela, in which OmpW and OmpV, respectively, elevate and reduce the ability in salinity-tolerance.
Journal of Proteome Research 10/2006; 5(9):2250-7. · 5.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: An unknown protein reacted with anti-human IgA, namely, IgA-like protein, has been reported in shrimp, but information regarding its identification is not available. In the present study, an affinity proteomic strategy was applied to identify the IgA-like protein of shrimp Litopenaeus vannamei. The protein of 75 kDa was isolated and confirmed by affinity chromatography and Western blotting with goat anti-human IgA, respectively, and then identified as hemocyanin, a member of IgSF, by mass spectrometry. Moreover, our results showed that human IgA and L. vannamei hemocyanin could separately react with goat anti-human IgA or rabbit anti-shrimp affinity hemocyanin (a-hemocyanin). Further evidences indicated that the recombinant protein of the Ig-like conserved domain could react with anti-human IgA. Interestingly, our results indicated that L. vannamei hemocyanin could aggregate with eight species of shrimp pathogenic bacteria and four types of animal erythrocytes directly. These results indicate that L. vannamei hemocyanin, an IgA-like protein, has dual function of reaction with anti-human IgA as an antigen and of activity binding to bacteria and animal erythrocytes as an agglutinin, suggesting its characteristic role as an IgSF molecule. In addition, our approach suggests that affinity proteomics based on heterogeneous antibody can speed up the identification of Fossman antigens.
Journal of Proteome Research 05/2006; 5(4):815-21. · 5.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The elucidation of the molecular details of antibiotic resistance will lead to improvements in extending the efficacy of current antimicrobials. In the current study, proteomic methodologies were applied to characterize functional outer membrane proteins (Omps) of E. coli K-12 responded to tetracycline and ampicillin resistance for understanding of universal pathways that form barriers for antimicrobial agents. For this purpose, E. coli K-12 expressional outer membrane proteome was characterized and identified with the use of 2-DE and MALDI-TOF/MS methods. Then, differential Omps due to tetracycline or ampcilin resistance were determined by comparison between tetracycline minimum inhibitory concentration (MIC)10, ampicillin MIC10, control0 and control10, showing 9 proteins with 11 spots for tetracycline and 8 protein with 9 spots for ampicillin, showing a difference in only 1 protein (decreased LamB in tetracyclin) between the two antibiotics. Among the proteins, 3 were known as antibiotic-resistant proteins, including TolC, OmpC and YhiU, while FimD precursor, LamB, Tsx, YfiO, OmpW, NlpB were first reported here to be antibiotic-resistance-related proteins. Our findings will be helpful for further understanding of antibiotic-resistant mechanism(s). This study also shows that the combination of Omp purification methods certainly contributes the sensitivity of Omp detection.
PROTEOMICS 02/2006; 6(2):462-73. · 4.51 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The ability of osmoregulation is crucial to marine pathogens that always face the change of osmotic pressure when they shift between natural marine water-bodies and hosts. Previous studies indicated that the expressional patterns of outer membrane proteins (OMPs) changed when Gram-negative bacteria were transferred in different environments. In the present study, proteomic methodologies were used to investigate the expressional pattern of OMPs of Vibrio alginolyticus, a universal marine pathogen, at different Na(+) concentrations. OmpW, OmpV, and Omp TolC were determined to be osmotic stress responsive proteins. Of the three proteins, importantly, OmpV and OmpW showed distinctly reverse changes to each other, indicating that the two proteins might be the two components varied with changed NaCl concentrations. In addition, our results suggest that closely related species of bacteria with available whole genomic databases should be applied after item microorganism species was used when proteins from a bacterium with unavailable whole genomic information were identified by PMF. Therefore, our results not only expand our knowledge on osmotic stress responsive proteins, but also provide valuable information for strategies on screening of these proteins.
PROTEOMICS 09/2005; 5(12):3142-52. · 4.51 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The expression of p62 autoantigen and the frequency of p62 autoantibody have been reported in hepatocellular carcinoma (HCC) and many types of malignant tumors, respectively, but information regarding to the expression of p62 in other cancer tissues and the association of autoantibody to p62 with tumor behaviors is not available. In the current study, the expression of p62 in tissues and the appearance of p62 autoantibody in sera were detected by immunohistochemical staining and ELISA in four clinical types of digestive system cancers including gastric cancer, esophageal cancer, large intestine cancer and HCC, respectively. Interestingly, the immunohistochemistry staining of p62 has been shown in all of digestive canal tissues (stomach, esophagus, large intestine) including tissues with cancers, beside cancers and from non-malignant patients, whereas the frequencies were 62.5% and 0% in tissues with cancer and beside cancer in patients with HCC, respectively. Importantly, we found that the p62 expression and the frequency of autoantibody to p62 were associated to cell differentiation and tumor metastasis, respectively. These results suggest that the expression of p62 in tissues and the appearance of autoantibody to p62 in sera might be related to cell malignant manifestations. Moreover, p62 autoantibody is a significant marker for the prognosis of cancers and the evaluation of clinical treatments.
Clinical Immunology 09/2005; 116(2):118-23. · 4.05 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In the present study, antigenic cross-reactivity of OMPs was investigated in several species of bacterial pathogens. Heterogeneous mouse or fish antisera were used to ascertain OMPs with cross-reactivity and cluster analysis was performed to analyze the distribution of cross-antigenic OMPs in diverse bacterial strains. We interestingly found that eleven and seven bands could be reacted with four kinds of heterogeneous mouse and fish antisera, respectively, and the phenograms constructed could provide ideal targeted bacteria for candidate genes of polyvalent vaccines. Importantly, there were significant differences in reaction with bacteria between mouse and fish antisera, but commonly antigenic bands still existed between them. Our results suggest that the cross-reactivity of OMPs exists commonly in Gram-negative bacteria, which may be a promising choice for the development of polyvalent OMP vaccines. Meanwhile, cluster analysis will help to understand the relation of cross-antigenic OMPs among Gram-negative bacteria.
International Immunopharmacology 08/2005; 5(7-8):1151-63. · 2.38 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Osteoporosis is a chronic condition chiefly affecting postmenopausal women, in whom the skeleton loses a significant percentage of its mineralized mass and mechanical resiliency, thereby becoming prone to fracture. Although the effect of the loss of estrogen on bone metabolism has been documented, its mechanism is still poorly understood. In the present proteomic study, we characterized the effect of estrogen deficiency on protein expression in rat bones. Using two-dimensional gel electrophoresis, mass spectrometry and rat protein database, we successfully identified three distinctly changed proteins named thioredoxin peroxidase 1, myosin light polypeptide 2 and ubiquitin-conjugating enzyme E2-17 kD, among which ubiquitin-conjugating enzyme E2-17 kD has been documented to be an estrogen-related protein, but the other two are first reported to be osteoporosis-related proteins in the current study. These results provide valuable experimental evidences for the elucidation of the molecular mechanism of osteoporosis related to the loss of estrogen.
Life Sciences 06/2005; 76(25):2893-901. · 2.53 Impact Factor
-
Cuiling Yu,
Meiling Dong,
Xiaokun Wu,
Shengguo Li,
Shengfeng Huang,
Jing Su,
Jianwen Wei,
Yang Shen,
Chunyan Mou,
Xiaojin Xie,
Jianghai Lin,
Shaochun Yuan,
Xuesong Yu,
Yanhong Yu,
Jingchun Du,
Shicui Zhang, Xuanxian Peng,
Mengqing Xiang,
Anlong Xu
[show abstract]
[hide abstract]
ABSTRACT: In seeking evidence of the existence of adaptive immune system (AIS) in ancient chordate, cDNA clones of six libraries from a protochordate, the Chinese amphioxus, were sequenced. Although the key molecules such as TCR, MHC, Ig, and RAG in AIS have not been identified from our database, we demonstrated in this study the extensive molecular evidence for the presence of genes homologous to many genes that are involved in AIS directly or indirectly, including some of which may represent the putative precursors of vertebrate AIS-related genes. The comparative analyses of these genes in different model organisms revealed the different fates of these genes during evolution. Their gene expression pattern suggested that the primitive digestive system is the pivotal place of the origin and evolution of the AIS. Our studies support the general statement that AIS appears after the jawless/jawed vertebrate split. However our study further reveals the fact that AIS is in its twilight in amphioxus and the evolution of the molecules in amphioxus are waiting for recruitment by the emergence of AIS.
The Journal of Immunology 04/2005; 174(6):3493-500. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: p62 is a cancer-associated antigen binding to mRNA encoding insulin-like growth factor II that was isolated by immunoscreening a cDNA expression library with autoantibodies from patients with hepatocellular carcinoma (HCC). In the present study, multiple methods including flow cytometry, confocal laser-scanning microscope, electron microscope were used to characterize the effect of ATRA on BGC-823 cells, which presented two phenotypes of differentiation and apoptosis in cells treated with 1.0 and 50 microM ATRA, respectively. Interestingly, we found that p62 was cytoplasmic in location, but it significantly decreased in cytoplasm and appeared in nucleus of cells when the cells were treated with 50 microM all-trans retinoic acid (ATRA) for 5 days. Furthermore, proteomics approach on differential nucleus proteins showed that the up-regulation and/or down-regulation of cell cycle proteins and IGF binding proteins were involved in the apoptosis of BGC-823 cells induced by ATRA. These results suggest that there is a significant association between expression and distribution of p62 and the growth arrest of tumor cells, in which p62 is associated with cell apoptosis induced by ATRA.
The International Journal of Biochemistry & Cell Biology 04/2005; 37(3):616-27. · 4.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The distribution of the cancer-associated protein p62 in human gastric carcinoma (BGC-823) cells was examined by confocal laser scanning microscopy and electron microscope immunocytochemistry. In control cells p62 was cytoplasmic in location and concentrated in the cytoplasmic matrix, but when cell growth was inhibited by treatment with 50 microM all-trans-retinoic acid (ATRA) for 5 days, p62 expression decreased and the protein was translocated from cytoplasm to nucleus. Ultrastructural localization using gold particles showed that p62 was bond mainly to a linear structure in nucleus. The speculation that p62 binds Insulin-like growth factor (IGF)-II mRNA indicates its probable involvement in the posttranscriptional IGF-II mRNA processing and p62 could play a role in tumorgenesis by regulating the expression of IGF-II. Further studies will be needed to confirm this view.
Cell Biology International 03/2005; 29(2):127-31. · 1.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Proteome analysis by two-dimensional gel electrophoresis (2-DE) together with mass spectrometry was applied to screen acute phase response (APR)-related proteins with low molecular weight in loach skin following injury. Furthermore, Western blotting and function tests were applied to confirm the results obtained from the proteomic study. Fifteen APR-related proteins with sixteen spots (PLA with two spots) on a 2-DE map were identified in this study. Furthermore, six were known acute phase proteins including galactose-binding lectin (GBL), lysozyme, C3, CD59, double PLA and 50s ribosomal protein; while ATP kinase, zinc finger protein 183, alpha-neurotoxin homology, angiostatin, serine/threonine kinase, metalloproteinase inhibitor, regulator of G-protein 4, cryptdin-9 and disintegrin trigranin were found by our lab to be APR-related proteins. In addition, our results suggest that proteomes with low molecular weight can be characterized by 2-DE with a Tris-tricine system followed by mass spectrometry.
PROTEOMICS 01/2005; 4(12):3989-97. · 4.51 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Vibrio parahaemolyticus, a universal marine pathogen with available genome sequences, could be used as a bacterial model to clarify the various physiological phenomena of its native and host environments. In the present study, proteomic methodologies were applied to investigate the expression pattern of outer membrane proteins (OMPs) of V. parahaemolyticus at different NaCl concentrations. OmpW, OmpV, elongation factor TU and polar flagellin were determined to be osmoregulation-sensitive OMPs, among which OmpW and OmpV were reported to vary with changed NaCl concentrations in the pattern of osmolarity regulation. Therefore, our results not only expand our knowledge on osmoregulation-related proteins, but also provide a valuable strategy for the screening of salt-sensitive proteins.
Research in Microbiology 01/2005; 155(10):835-42. · 2.76 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Diseases caused by microorganisms can be controlled by vaccines, which require neutralizing antigens. Therefore, it is very important to identify highly efficient immunogens for immune prevention. By combining immunoproteomics and bacterial challenge after immunization, we developed a rapid method for screening protected antigens of pathogenic bacteria in aquaculture. Our approach may be divided into three consecutive steps. First, dominant immunogens of outer membrane proteins are screened by immunoproteomics. Second, proteins with the ability to induce production of neutralizing antibodies are identified from the immunogens by virulent bacterium challenge following vaccination. Third, vaccine candidates are determined by evaluation of neutralizing abilities. Information on the candidates has been obtained for further gene cloning by mass spectrometry. Our results indicate that highly efficient protected antigens were identified from the outer membrane proteome of Aeromonas hydrophila, in which an immunogen showed 71.4% protective ability with multivalent functions to A. hydrophila and Aeromonas sobria. In summary, we have developed a high-throughout, accurate, rapid and highly efficient method which will play an active role in immune prevention for microbiological diseases.
PROTEOMICS 11/2004; 4(10):3203-13. · 4.51 Impact Factor
-
Jinquan Li,
Qinxi Li,
Changchuan Xie,
Huamin Zhou,
Yuqian Wang,
Na Zhang,
Hanjuan Shao,
Siu Chiu Chan, Xuanxian Peng,
Sheng-Cai Lin,
Jiahuai Han
[show abstract]
[hide abstract]
ABSTRACT: Tumor necrosis factor (TNF)-alpha induces caspase-independent cell death in the fibrosarcoma cell line L929. This cell death has a necrotic phenotype and is dependent on production of reactive oxygen species (ROS) in the mitochondria. To identify genes involved in this TNF-induced, ROS-dependent cell death pathway, we utilized retrovirus insertion-mediated random mutagenesis to generate TNF-resistant L929 cell lines and we subsequently identified genes whose mutations are responsible for the TNF-resistant phenotype. In one such resistant line, beta-actin was disrupted by viral insertion, and subsequent reconstitution of beta-actin expression levels in the mutant line Actin(mut) restored its sensitivity to TNF. Resistance to TNF in Actin(mut) cells is signal specific since the sensitivity to other death stimuli is either unchanged or even increased. Comparable NF-kappaB activation and p38 phosphorylation in TNF-treated wild-type and Actin(mut) cells also indicates that reduced expression of actin only selectively blocked some of the TNF-induced cellular changes. Actin cleavage involved in apoptosis does not occur in TNF-treated L929 cell death, as in HeLa cells. Consistent over-expression of a caspase-cleaved product, a 15 kDa actin fragment, had no effect on TNF-induced necrosis of L929 cell. By contrast, TNF-induced mitochondria clustering and ROS production were dramatically reduced in Actin(mut) cells, indicating that actin-deficiency-mediated TNF resistance is most likely due to impaired mitochondrial responses to TNF stimulation. Our findings suggest that a full complement of actin is required for transduction of a cell death signal to mitochondria in TNF-treated L929 cells.
Journal of Cell Science 10/2004; 117(Pt 20):4673-80. · 6.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Human gastric carcinoma BGC-823 cells underwent morphological differentiation and cell cycle arrest in vitro when treated with 5mM hexamethylene bisacetamide (HMBA) for 48h. To further understand the mechanism of HMBA-induced differentiation, proteomic methodologies were applied to screen and identify altered proteins involved in the commitment of BGC-823 cells to differentiate. Five distinct altered proteins were acquired by two-dimensional (2-D) PAGE and were consequently identified as ras-related protein rab-35 (Rab-35), splice truncated isoform of transmembrane protease, serine 3 (serine TADG-12), regulator of G-protein signaling 1 (RGS1), ret finger protein-like 1 (RFPL1) and F-actin capping protein alpha-3 subunit (GSG3) by analysis of mass spectrograph. Of the five proteins, serine TADG-12 down-regulated under the detectable level after HMBA treatment, Rab-35, RGS1 and RFPL1 sharply up-regulated within the HMBA-induced BGC-823 cells, and GSG3, appearing in both treated and untreated cells, remarkably increased within BGC-823 cells after HMBA stimulation. Our results implicate that the molecular mechanism of BGC-823 cell differentiation in response to HMBA may involved in complex processes including a signaling network linking vesicle transport, actin cytoskeleton remodeling except for morphology differentiation, cell cycle G1 arrest.
The International Journal of Biochemistry & Cell Biology 09/2004; 36(8):1613-23. · 4.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Shigella spp. are one of the most important etiological factors for people who are living in developing countries and travelers to tropical countries. High priority has been given by the World Health Organization to the development of vaccines to control Shigellosis caused by these bacteria. However, information regarding to profile of immunogenic proteins of Shigella is not available now. In the present study, sub-immunoproteomics was applied to screen novel immunogenic proteins which could be reacted with antisera produced by challenge of a whole bacterium. Our results indicated that 13 immunogens were identified, in which seven proteins and six proteins from outer membrane and soluble proteome, respectively. Of the 13 proteins, 12 showed to be novel immunogens. These results suggest that immunoproteomics can greatly improve the chances of identification and result in discovery of novel immunogenic proteins.
Vaccine 08/2004; 22(21-22):2750-6. · 3.77 Impact Factor
