A review of ERGIC-53: its structure, functions, regulation and relations with diseases.

ERGIC-53 is a type I transmembrane protein. It includes an N-terminal signal sequence, a carbohydrate recognition domain, which is calcium-dependent and pH-sensitive, a stalk region, a transmembrane domain, and a short cytoplasmic domain; ERGIC-53 mainly acts as a receptor of a limited number of glycoprotein and transports them from ER to ERGIC and Golgi, meanwhile it has a secondary glycoprotein quality control function. Recent research has revealed that UPR, heat shock and VIPL may regulate the expression of ERGIC-53. F5F8D, and some ER storage diseases have relationship with ERGIC-53.

[Effect of calmodulin antagonist O-4-ethoxyl- butyl-berbamine on the proliferation of human breast cancer cell line MCF-7 and its relevant mechanism].

OBJECTIVE: To investigate the effect of calmodulin antagonist O-(4-ethoxyl-butyl)-berbamine (EBB) on proliferation of human breast cancer cell MCF-7 and its possible mechanism. METHODS: MTT assay was used to analyze the effect of EBB on tumor cells growth. Flow cytometry was used to detect its impact on the cell cycle of MCF-7 cells. Immunofluoresce labeling technique and laser scanning confocal microscope were used to reveal the changes of the microtubule, microfilament, mitochondrion, and endoplasmic reticulum in the cells. RESULTS: The IC50 value of EBB in MCF-7 cells was (13.0 +/- 3.7) micromol/L. MCF-7 cells were arrested at S phase after EBB treatment. Meanwhile, depolymerization of the microtubule and microfilament, impairment of the mitochondrion and swelling of endoplasmic reticulum were observed. CONCLUSION: EBB arrests MCF-7 cells at S phase by inhibiting the growth of MCF-7 cells, which may be related to the changes of structures and functions of the microtubule, microfilament, mitochondrion, and endoplasmic reticulum.

Haishengsu as an adjunct therapy to conventional chemotherapy in patients with non-small cell lung cancer: a pilot randomized and placebo-controlled clinical trial.

OBJECTIVE: To investigate the effect of Haishengsu, an extract from Tegillarca granosa, on non-small cell lung cancer as an adjunct to conventional chemotherapy. DESIGNS/SETTINGS: Randomized, double-blind, placebo-controlled trial was conducted in 83 patients. The Haishengsu (n=42, 2.4mg Haishengsu in 250ml normal saline, iv, for 15 days) and the placebo group (n=41, 250ml normal saline, iv) were also treated with two cycles (28 days for each cycle) of conventional chemotherapy consisting mitomycin, vindesine and cisplatin. RESULTS: The curative effect of conventional chemotherapy was observed in 62% of Haishengsu group patients and in 39% in of the placebo group patients (P=0.04, RR 1.59, 95% CI: 1.01-2.49). Improvement in Karnofsky performance status scores was seen in 66.7% of Haishengsu group patients and in 17.1% of the placebo group patients (P<0.01, RR 3.63, 95%CI: 1.77-7.41). The ratio of patients with no or only mild gastrointestinal reaction in the Haishengsu and the placebo group was 83.3% and 39.0%, respectively (P<0.01, RR 2.13, 95% CI: 1.42-3.20). CONCLUSIONS: This study suggests that Haishengsu may be an effective adjunct therapy to the conventional chemotherapy for non-small cell lung cancer. The short-term therapeutic effect of chemotherapy may be improved and the chemotherapy-induced nausea or vomiting may be reduced by concurrent Haishengsu administration.

[The influence of Ara-C on anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells.].

OBJECTIVE: To investigate the role of Ara-C in regulating anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells. METHODS: The diabody of anti-CD3/anti-Pgp was purified by E-tag affinity chromatography. K562 and K562/A02 cells were treated with Ara-C. The expressions of B7-1 and B7-2 on K562 and K562/AO2 cells were detected by FACS. The cytotoxicity of T-lymphocytes combined with anti-CD3/anU-Pgp plus Ara-C was analyzed by CytoTox 96 nonradioactive method. RESULTS: The expressions of B7-1 and B7-2 on K562 and K562/A02 cells treated by Ara-C was significantly higher than those untreated. The effect/target ratio was from 0.39:1 to 25:1, and the killing rate of activated T cells to anti-drug-resistant leukemia cells was from (16.44 +/- 1.20)% to (60.49 +/- 2.90)%. The killing rates were increased gradually, with both the effect/target ratio and the antibody concentration increasing (P < 0.05). CONCLUSION: Ara-C may be an important adjuvant for improving anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells.

[Three-step purification of preparative-scale antiCD20 (Fab')2].

OBJECTIVE: To establish a three-step purification method of preparative-scale antiCD20 (Fab')2 using AKTA prime. METHODS: AntiCD20 (Fab')2 was extracted by hyperosmotic solution and then purified by CM sepharose FF, phenyl sepharose FF, and protein G sepharose FF. RESULTS: Around 8 mg anti-CD20 (Fab')2, whose purification was 96.678%, was purified. The antigen-binding activity of antiCD20 (Fab')2 was similar to that of antiCD20 (Fab')2 purified by protein G sepharose FF and S-100. CONCLUSION: The three-step purification method can obtain high-purity preparative-scale antiCD20 (Fab')2 in a simple way.

[Preparation and activity detection of monoclonal antibody against anti-CD3 ScFv].

OBJECTIVE: To prepare monoclonal antibody (McAb) against anti-CD3 ScFv for purifying and detecting serum anti-CD3 antibody concentration. METHODS: McAb against anti-CD3 ScFv was prepared by hybridoma technique and used to prepare affinity chromatography column, which was used to purify anti-CD3 ScFv and Diabody [CD3 x Pgp] without E-tag. The binding activities of anti-CD3 ScFv, Diabody [CD3 x Pgp] without E-tag, and Diabody [CD3 x Pgp] purified by anti-CD3 affinity chromatography column or anti-E-tag affinity chromatography column against K562/A02 cell and Jurket cells were detected by fluorescence activated cell sorting (FACS) method. ELISA was used to identify the specificity of the McAb. RESULTS: McAb against anti-CD3 ScFv specifically detected serum anti-CD3 ScFv without reacting with sera. The anti-CD3 ScFv purified by anti-CD3 affinity chromatography column and purified by anti-E-tag affinity chromatography column had the same specific binding activity with Jurkat cells. The positive binding rates of Diabody [CD3 x Pgp] without E-tag to K562/A02 and Jurkat cells were 89.87% and 83.95%, respectively. In the competitive binding experiments with K562/A02 and Jurkat cells, the binding rates of Diabody [CD3 x Pgp] without E-tag decreased to 56.30% and 43.78%, respectively. CONCLUSION: The McAb against anti-CD3 ScFv prepared in our lab can be used to purify and detect serum anti-CD3 antibody concentration.

[Construction and expression of human 4-1BBL/anti-CD20 fusion protein].

AIM: To construct and express human 4-1BBL/anti-CD20 bispecific fusion protein and identify its biological activity. METHODS: PCR and overlapping PCR were used to construct human 4-1BBL/anti-CD20 bispecific fusion protein. DNA sequencing was performed by the terminus of the fusion protein nucleotide.The product was purified by affinity chromatography and analyzed by Western blot and its antigen-binding activity was examined by FACS. RESULTS: The data of DNA sequence showed that human 4-1BBL/anti-CD20 bispecific fusion protein was correct. The fusion protein was recovered in high yield (up to 4 mg/L) after E-tag purification and predominantly(90%) as a dimer. The fusion protein could bind to Raji cells(CD20(+)) and A549 cells(4-1BB(+)), respectively. CONCLUSION: The human 4-1BBL/anti-CD20 bispecific fusion protein with high level expression was successfully obtained and could bind to Raji ceIls and A549 cells.

[Up-regulation of CD86 and cytokine expression in acute leukemia cells by Ara-C].

AIM: To observe the effects of Ara-c on the expression of CD86 molecule on acute leukemia cells and explore the possible mechanisms. METHODS: The expression of CD86 on U937, HL-60 and NB4 cell lines treated with or without Ara-C wa assayed by flow cytometry. The mRNA expression of CD86, NF-kappaB, IFN-gamma was examined by semiquantitative RT-PCR. RESULTS: UP-regulation of CD86 was observed on those cells treated by Ara-c. The level of CD86 and NF-kappaB mRNA in Ara-c treated cells was significantly enhanced. IFN-gamma was detectable 72 hours after T cell activation. CONCLUSION: Ara-C can enhance CD86 and NF-kappaB expression on acute leukemia cells, which may play critical role in T cell activation and differentiation.

[The role of extracellular domain of human 4-1BBL in regulating activities of human peripheral blood lymphocytes in vitro].

AIM: To investigate the role of the extracellular domain of human 4-1BBL (ex4-1BBL) in regulating the in vitro activities of peripheral blood lymphocytes (PBL). METHODS: Viable cells were quantified with Trypan-blue exclusion assay. CytoTox 96 Nonradioactive Cytotoxicity Assay Kit was used for measuring LDH levels of supernatant. ELISA kit was used for measuring IL-2 level. In vitro cytotoxity of PBL combined with anti-CD3/anti-Pgp bispecific diabody plus ex4-1BBL was analyzed with CytoTox 96 nonradioactive method. RESULTS: Ex4-1BBL can increase the proliferation of PBL, reduce cell death, promote IL-2 secretion, and the experimental group with ex4-1BBL showed obviously enhanced cytotoxic effect toward K562/A02 cells. CONCLUSION: Ex4-1BBL may be an important adjuvant for improving activities of PBL.

Expression of phosphatase of regeneration liver-3 in human colorectal carcinoma and its prognosis value / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the expression of phosphatase of regeneration liver-3(PRL-3) protein and its relationship with tumor invasion and metastasis in human colorectal carcinoma,and elucidate prognostic value.</p><p><b>METHODS</b>Immunohistochemistry method was applied to detect the PRL-3 expression in the primary tumor specimens and paired paratumor normal tissues from 46 colorectal carcinoma patients, the adenoma tissues from 6 patients with colorectal adenoma, all the metastatic lymph nodes from 29 cases and the metastatic liver lesions from 6 cases. The relationship between PRL-3 expression and clinicopathologic parameters was analyzed and a survival curve was achieved according to Kaplan-Meier method.</p><p><b>RESULTS</b>No or weak PRL-3 protein expression was detected in normal colorectal mucosa and colorectal adenoma. In colorectal carcinoma tissues, PRL-3 expression was confirmed in 26 of 46 cases (56.5%) of primary colorectal carcinomas (with lymph node metastasis 63.0%, without lymph node metastasis 37.0%, P=0.001), 26 of 29 (89.7%) lymph node metastases, and 5 of 6 liver metastases. The expression of PRL-3 was assembled in the cytoplasm of carcinoma cells and more intensively on the cell membrane.Analysis of the relationship between PRL-3 expression and the clinicopathologic features showed that PRL-3 expression was closely associated with tumor stage (P=0.019), lymph node metastasis (P=0.026), but no relationship with age, sex, tumor size, degree of differentiation was founded (P<0.05). The mean follow-up time was 41.4 months and results showed that patients with positive expression of PRL-3 had a significantly poorer prognosis than those with negative PRL-3 expression group(P=0.032).</p><p><b>CONCLUSIONS</b>PRL-3 protein plays a novel role in tumor progression and metastasis of colorectal carcinoma. PRL-3 can be expected to be a potential predictive biomarker for identifying the prognosis in colorectal carcinoma patients.</p>

Experience of ultralow anterior excision for rectal cancer: 508 cases analysis / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the operative techniques and postoperative effects of ultralow anterior excision for rectal cancer.</p><p><b>METHODS</b>From October 1996 to October 2006, 508 cases with rectal carcinoma at or below the peritoneal reflection with potential to preserve the anal function were divided into two groups. Of the patients, 365 cases underwent ultralow anterior excision and instrumental anastomosis, and 143 cases underwent manual colon-anal anastomosis (Parks operation).</p><p><b>RESULTS</b>In the group with anterior excision, the operations were all completed in the abdominal cavity, and avulsion of distal occlusive end occurred in 3 cases (0.9%), unsuccessful anastomosis happened in 2 cases (0.6%), unsatisfactory anastomosis with incomplete anastomosis circle turned out in 18 cases (5.6%). In the Parks operation group, the anastomosis was carried out manually at the anus and in abdominal cavity. Postoperative defecation function (times, soiling underwear, feeling of urgent defecation) in the group anterior excision was clearly better than that in the group of Parks operation (P < 0.05); difficulty of defecation (sense of residual stool, prolonging of defecation, cathartic usage) was also better in the group with anterior excision (P < 0.05). The anastomosis leakage rate was 3.5% in anterior excision group, compared to 5.6% in Parks operation group (P > 0.05). Anastomotic stenosis occurred in 77 cases (22.5%) in anterior excision group, and 40 cases (27.9%) in Parks operation group (P > 0.05). The local recurrence rate and 5-year survival rate were 11.8% and 68.8% in anterior excision group, and 10.1% and 66.8% in Parks operation group, respectively (P > 0.05).</p><p><b>CONCLUSIONS</b>Although there is no significant differences in local recurrence and 5-year survival rate between the two groups, the function and difficulty of defecation with instrumental anastomosis demonstrates clear advantages over Parks operation.</p>

[Construction and expression of diabody [CD3 x Pgp] without Etag].

AIM: To construct and express a diabody [CD3 x Pgp] without Etag and analyse its biological activity. METHODS: In this study, the diabody [CD3 x Pgp] was obtained by PCR and restriction cleavage, and expressed in E.coli 16C9. The product was purified by anti-anti-CD3 scFv affinity chromatography and verified through SDS-PAGE electrophoresis. Flow cytometry(FCM) was used to analyse the bingding properties and competitive bingding capacity. RESULTS: The sequence of diabody [CD3 x Pgp] without Etag was correct. It migrated as two bands with the expected molecular weight(25 kD and 26 kD) in SDS-PAGE. The binding rate to CD3 and Pgp antigen was 83.95% and 89.87% respectively. The competitive bingding rate to CD3 and Pgp was 43.78% and 50.25% respectively. CONCLUSION: The diabody [CD3 x Pgp] without Etag has been successfully constructed, expressed and purified. The product can bind to CD3 and Pgp antigen specifically, and its biological activity doesn't decrease.

[Design and activity determination of small molecular inhibitors of integrin alphavbeta3].

OBJECTIVE: To explore the design and activity determination of small molecular inhibitors of integrin alphavbeta3 through structure-based virtual screening. METHODS: Based on the crystal structure of integrin ctv33 extracellular segment in complex with an ARG-GLY-ASP ligand, docking procedure against the receptor binding domain was performed on 3D database. Integrin alphavbeta3-mediated cell adhesion assay was performed to assess the adhesion-inhibiting ability of the candidate compounds. Cell migration assay and capillary-structure-like formation inhibition assay were used to estimate the effects of the compounds on integrin alphavbeta3. Analysis of molecular graphics was carried out to deduce a probable binding model of compound with integrin alphavbeta3. RESULTS: From the top 1000 compounds with the best DOCK energy score, 50 compounds were selected for biological assay based on chemical and drug-like diversity. Seven of 50 compounds showed notable inhibition activity on cell adhesion, and two with half-maximum inhibition concentration (IC50) values less than 100 mol/L. The compound with best activity (1-37) showed high inhibitory activity in cell migration assay and capillary-structure-like formation inhibition assay. Molecular graphics analysis indicated that metal ion-dependent adhesion site (MIDAS) might be involved in the compound 1-37-mediated inhibition of ligand binding with integrin alphavbeta3. CONCLUSIONS: Through virtual screening combined with biological assay, a promising lead compound was discovered to inhibit integrin alphavbeta3, which embodies the rational drug design with computation aid and brings a new thought and approach to find novel inhibitors of integrin.

[Reversal of multidrug resistance in drug-resistant human breast cancer cell line MCF-7/ADR by calmodulin antagonist O-(4-ethoxyl-butyl)-berbamine].

OBJECTIVE: To investigate the reversal effect of O-(4-ethoxyl-butyl)-berbamine (EBB) on multidrug resistance (MDR) in MCF-7/ADR cell. METHODS: 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to assess the antitumor effect of EBB and determine the reversal effects of different concentrations ( < or = IC20) of EBB on MCF-7/ADR cell. Flow cytometry was applied to observe the intracellular accumulation of Rh123 and cell cycle in the presence of EBB. The expressions of MDR-related genes mdr 1 and topoisomerase II b (top II b) were evaluated by reverse transcription-polymerase chain reaction. RESULTS: The sensitivity of MCF-7/ADR to adriamycin (ADR) was enhanced up to 50. 40, 89.80, and 14.88 folds after exposure of the cells to 3 micromol/L EBB, 7.5 micromol/L EBB, and 10 micromol/L verapamil (VPL), respectively. After 2 hours of incubation with 6 micromol/L EBB, intracellular Rh123 accumulation in MCF-7/ADR cells was increased to the level comparable to that in MCF-7 cells. When 6 micromol/L EBB was added together with 2 micromol/L ADR, MCF-7/ ADR cells showed to be arrested in the G2/M phase. The declination of mdr 1 gene expression was observed when 6 micromol/L EBB, 12 micromol/L EBB, and 10 micromol/L VPL were added for 48 hours; meanwhile, the expression of top II b mRNA showed no significant change. CONCLUSION: EBB has a strong reversal effect on MDR in MCF-7/ ADR cell, which may be achieved by enhancing the arrestment of MCF-7/ADR cells at G2/M phase and increasing intracellular drug concentration.

[Cloning and expression of the extracellular domain of 4-1BBL].

RT-PCR was used to clone DNA fragment of the extracellular domain of 4-1BBL from human THP-1 cells (human monocyte), and the expression vector pAYZ4-1BBL was constructed by cloning the extracellular domain of 4-1BBL into the expression vector pAYZ. The extracellular domain of 4-1BBL was expressed in E. coli 16C9 and purified by affinity chromatography. SDS-PAGE and Western blot analysis showed that the relativae molecular weight of soluble 4-1BBL is 22kD which was consistent with the theoretically predicted value. So far as we know, it is the first time that the soluble expression of 4-1BBL in E. coli was achieved 4-1BBL induced a significant release of IL-2 in stimulated Jurkat cells after 48h incubation, especially in the presence of tumor cell. At the same time the apoptosis level of Jurkat cell reduce more than 50%. In conclusion, 4-1BBL may be useful in cancer immunotherapy.

[Specific targeting cytotoxicity to resistant leukemia cells mediated by anti-Pgp/anti-CD3 diabody].

OBJECTIVE: To study the specific targeting cytotoxicity to drug-resistant leukemia cells mediated by anti-Pgp/anti-CD3 diabody. METHODS: The diabody was purified by affinity chromatography and identified by SDS-PAGE and FACS. The effect of the anti-Pgp/anti-CD3 diabody mediated lysis of Pgp-expressing tumor cells was assayed by human leukemia nude mice xenograft model in vivo. RESULTS: The diabody was produced in E.coli in a soluble functional form and could bind both Jurkat cells (CD3(+)) and K562/A02 cells (Pgp(+)). The binding rates were 86.25% and 86.26%, respectively. It could inhibit tumor growth by 98.57% and prolonged the survival of mice bearing xenografted K562/A02 cells. CONCLUSION: The diabody was proved to be a potent agent for mediating T lymphocyte cytotoxicity to lyse Pgp expressing tumor cells in vitro and in vivo.

[Effect of calmodulin antagonist EBB on invasion of human fibrosarcoma cell HT1080].

OBJECTIVE: To investigate the potential effect of EBB, a calmodulin antagonist, on invasion of human fibrosarcoma cells HT1080. METHODS: The antitumor effect of EBB was assessed by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Activities of matrix metalloproteinase (MMP)-2 and MMP-9 were measured by Zymogrophy analysis. The mRNA levels, of MMP-2, MMP-9, and tissue inhibitor of metalloproteinases (TIMP)-1 were evaluated by reverse transcriptionpolymerase chain reaction (RT-PCR). Transwell chamber assay was applied to measure the effect of EBB on the invasion of HT1080 cells. RESULTS: Calmodulin antagonist EBB inhibited the proliferation of HT1080 cells with an IC50 of (8.2 +/- 1.2) microg/ml. EBB down-regulated the activities of MMP-2 and MMP-9, and down-regulated the mRNA levels of MMP-2 and MMP-9, while up-regulated the mRNA levels of TIMP-1. The invasive ability of HT1080 cells was decreased to (31.13 +/- 2.265)%, (59.91 +/- 2.566)%, and (71.58 +/- 0.5960)% after exposure of the cells with 2, 5, and 10 microg/ml EBB, respectively. CONCLUSION: Treatment with calmodulin antagonist EBB is effective in suppressing tumor invasion. The possible mechanism is the down-regulation of MMPs.

[Effect of monoclonal antibody against extracellular domain III of vascular endothelial growth factor receptor KDR on proliferation of vascular endothelial cells].

OBJECTIVE: To prepare a neutralizing monoclonal antibody (McAb) against vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Extracellular immunoglobulin (Ig)-like domain III of KDR (KDR III) was expressed in E. coli and purified by affinity chromatograph. Monoclonal antibody against KDR III was prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [(3)H]-TdR incorporation assay were also used to detect the activity of anti-KDR McAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on VEGF-induced mitogenesis of human endothelial cells. RESULTS: McAb Ycom1D3 against KDR III was prepared which bound specifically to both the soluble KDR III and the cell-surface expressed KDR. It effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated activation of KDR expression on human endothelial cells. Furthermore, Ycom1D3 efficiently neutralized VEGF-induced mitogenesis of human umbilical vascular endothelial cells. CONCLUSION: McAb Ycom1D3 against KDR III may suppress the action of VEGF by blocking native vascular endothelial growth factor receptor KDR. It has potential clinical applications in the treatment of cancers and other diseases where pathological angiogenesis is involved.