[Preparation and identification of the polyclonal antibody against human VSTM1].

AIM: To prepare and characterize the polyclonal antibody against human VSTM1. METHODS: VSTM1 has two main isoforms, VSTM1-v1, a type I transmembrane protein, and VSTM1-v2, a classical secretory protein, lacking only the transmembrane domain compared with VSTM1-v1. Two recombinant prokaryotic proteins of VSTM1-v2, Trx-His-S-VSTM1-v2 and GST-VSTM1-v2, were constructed, expressed, purified, and then used for immunization of New Zealand rabbits to prepare anti-VSTM1 antibody and coupling with CNBr-activated Sepharose 4B to purify the antibody by immunoaffinity chromatography, respectively. ELISA was performed to detect the titers of the antiserums. After purification, the antibody was identified by Western blotting and immunofluorescence cytochemistry. RESULTS: The titers of the antiserums from the two immunized rabbits were both 1:10(6);. Western blotting confirmed that the purified antibody could recognize both the overexpressed and endogenous VSTM1 specifically. Immunofluorescence cytochemistry verified that the antibody could also recognize the overexpressed VSTM1-v1 on the surface of HEK293T cells. CONCLUSION: The rabbit antibody against human VSTM1 of a high titer has been obtained, which can be used for recognizing endogenous VSTM1 in immunofluorescence cytochemistry.

Simvastatin induces estrogen receptor-alpha expression in bone, restores bone loss, and decreases ERα expression and uterine wet weight in ovariectomized rats.

We previously reported that simvastatin induces estrogen receptor-alpha (ERα) in murine bone marrow stromal cells in vitro. In this study, we investigated the effect of simvastatin on ERα expression in bone and uterus in ovariectomized (OVX) rats and evaluated bone mass, bone strength, and uterine wet weight. Three-month-old Sprague-Dawley female rats received OVX or sham operation. Six weeks later, the rats were treated orally with simvastatin (5 or 10 mg/kg/day), or intraperitoneally with 17-ß-estradiol (E(2)) or a combination of simvastatin and E(2) for 6 weeks. Uterine wet weight, bone mineral density (BMD) of lumbar vertebrae, biomechanics of lumbar vertebrae, and induction of ERα expression in the bone and uterus were analyzed. The 6-week simvastatin treatment improved lumbar vertebral BMD and boosted biomechanical performance of the vertebral body compared to the OVX control, suggesting that simvastatin can treat osteoporosis caused by estrogen deficiency. More interestingly, simvastatin could increase ERα expression and synergy with estradiol in bone while antagonizing estradiol in the uterus, along with uterus atrophy and uterine wet weight decreases. In conclusion, these data suggest that simvastatin exert opposing modulatory effects on ERα expression on bone and uterus in ovariectomized rats, inducing ERα expression and synergy with estrogen to perform anabolic effects on the bones while decreasing E2 efficacy and uterine wet weight. This finding may be helpful to explain the mechanism of statin treatment in osteoporosis caused by estrogen deficiency.

[Sensitizing effect of recombinant human PDCD5 protein on chemotherapy of acute monocytic leukemia cell line U937 and its mechanism].

This study was aimed to investigate the sensitizing effect of recombinant human PDCD5 (rhPDCD5) protein on chemotherapy of U937 cell line and its mechanism. The flow cytometry was performed to assess the changes of cell apoptosis and cell cycle influenced by rhPDCD5. Hochst 33258 staining was used to observe morphology of the apoptotic cells. The activity change of caspase-3 was detected to analyse the possible mechanisms of rhPDCD5-induced apoptosis. RT-PCR was performed to observe the expression level of drug-resistant genes. The results showed that the percentage of apoptotic cells and the activity of caspase-3 remarkably increased in U937 cells treated with rhPDCD5 combined with chemotherapeutic drug; the cell cycle arrest induced by anti-tumor drug was also enhanced when combined with rhPDCD5; meanwhile, the expression levels of drug-resistant genes were down-regulated in jointly treated U937 cells. It is concluded that the chemosensitizing mechanisms of rhPDCD5 are complex. rhPDCD5 may increase the cytotoxicity of anti-tumor drugs by promoting the caspase-3-related apoptosis, influencing cell cycle, decreasing the expression of drug-resistant genes and reversing drug-resistance.

[Outcome and significance of pulmonary pathological changes induced by a single intramuscular injection of chemokine-like factor 1 in mice].

OBJECTIVE: To investigate the significance of pathological changes in murine lung by a single intramuscular injection of chemokine-like factor 1 (CKLF1). METHODS: A total of 120 gender-matched BALB/c mice were randomly and evenly divided into treatment group and control group (60 in each). One hundred nanomilligram of pcDNA3.1-CKLF1-Myc-His, CKLF1-expressing plasmid, in 100 microl of pyrogen-free saline was injected into the anterior tibial muscle of mice followed by the delivery of electric pulses. Mice in the control group received 100 microg of pcDNA3.1-Myc-His in 100 microl of pyrogen-free saline. At the end of week 1, 4 and 8 respectively after injection of CKLF1, 20 mice were sacrificed in every group and the cellular profiles in bronchoalveolar lavage fluid (BALF) and the pulmonary pathological changes were observed. RESULTS: At the end of week 1 and 4 respectively after CKLF1 injection, the neutrophils [(35.0 +/- 5.2)% and (22.9 +/- 2.2)% respectively] and lymphocytes [(34.5 +/- 2.8)% and (22.0 +/- 2.0)% respectively] in BALF of the treatment group were higher than those of the control group [neutrophils: (6.7 +/- 2.2)% and (7.0 +/- 2.4)% respectively, lymphocytes: (5.9 +/- 1.6)% and (6.1 +/- 2.7)% respectively, all P < 0.01]. Pathological studies demonstrated shedding of bronchiolar epithelium, congestion and edema in interstitial tissue and inflammatory cell infiltration in mice at 1 week after CKLF1 injection. Week 4 after CKLF1 administration, the alveolar wall was shown significantly thickened with proliferation of neutrophils, macrophages and fibroblasts as well as remarked collagen deposition in the interstitium. At the end of week 8 after CKLF1 administration, the remarkable morphological changes of the lung gradually subsided and the structure of the lung returned to normal. CONCLUSIONS: CKLF1 causes injury of inflammation and remodeling in airway in mice. The pulmonary pathological changes induced by a single intramuscular injection of CKLF are reversible.

[Preparation and characterization of monoclonal antibodies against CMTM7].

OBJECTIVE: To obtain monoclonal antibodies against CMTM7 (CKLF-like MARVEL transmembrane domain containing 7) for further study of the structure and biological function of CMTM7. METHODS: Three polypeptides were synthesized based on the bioinformatics analysis of the CMTM7 and coupled with keyhole limpet hemocyanin (KLH). Balb/c mice were immunized with these mixed CMTM7 polypeptides. Hybridomas were generated by the fusion of the spleenocytes from these mice with Sp2/0 myeloma cells. Resulting hybridomas producing anti-CMTM7 antibodies were screened by enzymejlinked immunosorbent assay (ELISA). The specificities of these monoclonal antibodies were determined by Western blot, immunofluorescecence and immunocytochemistry (ICC). RESULTS: Two hybridoma cell lines (MC9 and 2C9) stable in secreting anti-CMTM7 monoclonal antibodies (MAbs) were generated. Both of them produced immunoglobulin G1 (IgG1) against CMTM7. 2C9 and MC9 recognized a region between amino acid residues 19-44 and 163-175 of CMTM7, respectively. MC9 antibody could be used for Western blot, immunofluorescecence and immunocytochmistry assay. However, 2C9 antibody could be only used for Western blot. Data obtained from immunofluorescence and ICC indicated that CMTM7 protein expression was upregulated in the early stage of lymphocyte activation treated with phytohemagglutinin (PHA). CONCLUSION: Monoclonal antibodies of high specificity against CMTM7 have been successfully generated, which could be utilized as a useful reagent for the analysis of biochemical, structural, and functional properties of CMTM7.

[Preparation, purification and characterization of the polyclonal antibody against human CMTM4].

AIM: To prepare, purify and characterize the polyclonal antibodies against CMTM4. METHODS: Four polypeptides named peptide 1, 2, 3, and 4 were synthesized based on the bioinformatics analysis of the three isoforms of CMTM4, CMTM4-v1, -v2, and -v3, and coupled with keyhole limpet hemocyanin (KLH) for immunization. Among them, peptide 1, 2, and 3, common for CMTM4-v1, v2, and v3, were mixed for immunization to prepare the antibody which can recognize all of the three isoforms (Ab1); while peptide 4, which is specific for CMTM4-v1, was injected separately into New Zealand rabbits to prepare the antibody specifically targeting CMTM4-v1 (Ab2). ELISA assay was used to detect the titers of the antibodies. After purification by immunoaffinity chromatography, Ab1 and Ab2 were identified by Western blot and immunohistochemistry assays. RESULTS: The titers of Ab1 and Ab2 were 1:10(5) and 1:10(6), respectively. Western blot confirmed their high specificity. Ab1 could also be used for immunohistochemistry analysis, but Ab2 could not. Immunohistochemistry analysis with Ab1 demonstrated the high expression level of CMTM4 in human testis, which was consistent with the previous result of Northern blot. Moreover, Western blot and immunohistochemistry analysis verified that Ab1 can also recognize mouse Cmtm4. CONCLUSION: Two specific antibodies against human CMTM4 were obtained, which will be helpful for further study.

[CMTM1-v17, a new potential corepressor of androgen receptor].

OBJECTIVE: To analyze the expression of CMTM1-v17 in normal prostate tissue and prostate carcinoma originated cell lines, and study its impact on the transactivation of androgen receptor and the possible mechanism. METHODS: The expression of CMTM1-v17 in normal prostate tissue was analyzed with immunohistochemistry method. In immunocytochemistry was used to analyze the expression of CMTM1-v17 in prostate carcinoma originated cell lines. Luciferase assay was used to study the impact of CMTM1-v17 on the transactivation of AR and its mechanism. RESULTS: The results of immunohistochemistry showed that CMTM1-v17 was highly expressed in prostate. In prostate cancer originated cell lines, CMTM1-v17 could also be detected in prostate cancer originated cell lines PC3, Du145 and LNCaP. And the results of luciferase implied that the relative luciferase activity of the PC3 cells transfected with 1 microg and 2 microg pCDI-CMTM1-v17 plasmids separately were 70.8 and 34.7, compared with the control set as 100. When trichostatin A, the inhibitor for histone deacetylase, was used, the repression of androgen receptor could be recovered with trichostatin A treatment,for the relative luciferase activity of the PC3 cells transfected with 1 microg and 2 microg pCDI-CMTM1-v17 plasmids and treated with 100 nmol/L trichostatin A rebound to 90.9 and 86.4. CONCLUSION: CMTM1-v17 is highly expressed in both normal prostate and prostate carcinoma originated cell lines. It may recruit histone deacetylase to inhibit the function of androgen receptor.

[Expression and significance of severe acute respiratory syndrome associated coronavirus (SARS-CoV)-X4 protein in lungs of SARS patients].

OBJECTIVE: To investigate the significance of severe acute respiratory syndrome associated coronavirus (SARS-CoV)-X4 protein expression in lungs of patients with SARS. METHODS: Pathological features of the lungs from 4 SARS patients were examined and the expression of SARS-CoV-X4 protein in the lungs was evaluated with immunohistochemical staining using specific antibodies against protein X4. RESULTS: Microscopically, all lungs from 4 cases showed edema, erythrocyte and fibrin exudates in the alveoli, hyperplasia of alveolar epithelium, necrosis, hyaline membrane formation and fibroblast foci. Immunohistochemical stains showed a strong positivity of X4 protein in denudation cells, vascular endothelial cells and also erythrocytes and neutrophils in the alveoli of the lung tissues from the 4 cases. CONCLUSIONS: Expression of SARS-CoV-X4 protein in the lungs may be involved in the pathogenesis and progression of SARS.

[Chemotactic and mitogenic effect of rat chemokine-like factor 1 on aortic smooth muscle cells].

OBJECTIVE: To study the effects of rat chemokine-like factor 1 (rCklf1) on chemotaxis and proliferation of rat aortic smooth muscle cells(ASMC). METHODS: The recombinant eukaryotic expression vector pcDNA3.1/rCklf1 was transiently transfected into 293T cells. The supernatants were harvested for chemotactic and proliferation assays. pcDNA3.1/rCklf1 was also transfected into ASMC and the proliferation of transfected cells was detected by MTT assays. RESULTS: The 10 fold diluted supernatants of pcDNA3.1/rCklf1 transfected 293T cells had chemotactic effects on rat ASMC, which could be inhibited by pertussis toxin at the concentration of 10 microg/L. Furthermore, the 10 fold diluted supernatants of rCklf1 transfected 293T cells promoted the proliferation of ASMC (1.3 fold compared with the control) and transient overexpression of rCklf1 in ASMC had promoting activities on the proliferation of rat ASMC (1.5 fold compared with the control at 72 h). CONCLUSION: rCklf1 has mitogenic and GiPCR-dependent chemotactic effects on rat ASMC,indicating that rCklf1 may be involved in the pathological process of atherosclerosis.

The protein X4 of severe acute respiratory syndrome-associated coronavirus is expressed on both virus-infected cells and lung tissue of severe acute respiratory syndrome patients and inhibits growth of Balb/c 3T3 cell line.

BACKGROUND: The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) includes sequences encoding the putative protein X4 (ORF8, ORF7a), consisting of 122 amino acids. The deduced sequence contains a probable cleaved signal peptide sequence and a C-terminal transmembrane helix, indicating that protein X4 is likely to be a type I membrane protein. This study was conducted to demonstrate whether the protein X4 was expressed and its essential function in the process of SARS-CoV infection. METHODS: The prokaryotic and eukaryotic protein X4-expressing plasmids were constructed. Recombinant soluble protein X4 was purified from E. coli using ion exchange chromatography, and the preparation was injected into chicken for rising specific polyclonal antibodies. The expression of protein X4 in SARS-CoV-infected Vero E6 cells and lung tissues from patients with SARS was performed using immunofluorescence assay and immunohistochemistry technique. The preliminary function of protein X4 was evaluated by treatment with and over-expression of protein X4 in cell lines. Western blot was employed to evaluate the expression of protein X4 in SARS-CoV particles. RESULTS: We expressed and purified soluble recombinant protein X4 from E.coli, and generated specific antibodies against protein X4. Western blot proved that the protein X4 was not assembled in the SARS-CoV particles. Indirect immunofluorescence assays revealed that the expression of protein X4 was detected at 8 hours after infection in SARS-CoV-infected Vero E6 cells. It was also detected in the lung tissues from patients with SARS. Treatment with and overexpression of protein X4 inhibited the growth of Balb/c 3T3 cells as determined by cell counting and MTT assays. CONCLUSION: The results provide the evidence of protein X4 expression following SARS-CoV infection, and may facilitate further investigation of the immunopathological mechanism of SARS.

[Preparation, identification, and analysis on tissue chips of polyclonal anti-peptide antibody to chemokine-like factor 1].

OBJECTIVE: To prepare the polyclonal anti-peptide antibody against chemokine-like factor1 (CKLF1) and apply it to the expression and functional studies of CKLF1. METHODS: CKLF1 was analyzed with bioinformatics methods. The 16 amino acids sequence peptide was selected from CKLF1 C terminal end. Antibody was raised by immunizing rabbits with the peptide conjugated to keyhole limpet hemocyanin (KLH). RESULTS: A high titer polycolonal antibody was obtained from the rabbit against the peptide. ELISA analysis proved that the titer of rabbit serum against anti-peptide of CKLF1 was up to 10(-4). Western blot analysis revealed that it could react not only with recombinant CKLF1 expressed in a cell-Free Protein Biosynthesis System and Drosophila S2 cells, but also recognize the endogenous CKLFs in the tissue array. Positive staining was detected in the normal bronchial cartilage, gastric mucosa, and gastric smooth muscle tissues. Normal rectum and well-differentiated rectal carcinoma showed strong positive staining, but the poor-differentiated rectal carcinoma samples revealed negative staining. CONCLUSION: The anti-peptide antibody can specifically recognize CKLFs and may be a useful reagent for the detection of CKLF1.