Z1, a novel DENV2 NS5 RdRp small molecular inhibitor, inhibits DENV2 replication and infection / 中国药理学通报

Aim To screen novel NS5 inhibitors a-gainst dengue virus ( DENV) replication. Methods His-tagged DENV2 NS5 RNA-dependent polymerase ( NS5 RdRp ) was expressed and purified in BL21 cells. The binding ability of the small molecules to NS5 polymerase was determined by SPR assay. The activity of dengue inhibition by Z1 was determined by CPE, LDH and plaque assay. RNA synthesis was as-sessed by Real-time PCR. The dsRNA synthesis and viral proteins were detected by immunofluorescence as-say. The level of viral proteins was examined by West-ern blot. The stage of DENV life cycle was evaluated by time of drug-addition assay. Results A small mo-lecular Z1 was discovered, which could bind to NS5 RdRp. Z1 inhibited DENV2 RNA replication, synthe-sis of dsRNA and protein synthesis in post-entry stage of dengue life-cycle. Cell based assay confirmed that Z1 inhibited DENV-induced cell death with EC50 val-ues of 4. 75μmol·L-1 . Conclusions The novel NS5 inhibitor Z1, inhibits DENV2 RNA replication, protein synthesis and release of progeny virus, which may be severed as an anti-DENV2 antiviral drug for further de-velopment.

Lactic acid inhibits the formation of semen-derived amyloid fibrils / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the inhibitory effect of lactic acid on semen-derived amyloid (SEVI) fibril formation.</p><p><b>METHODS</b>PAP248-286 (2 mg/mL) was incubated with 4.0, 2.0, 1.0, 0.5, 0.25, and 0.125 mg/mL of lactic acid. After incubation for different times, aliquots were drawn from each sample for Thioflavin T (ThT) and Congo red staining to monitor semen-derived amyloid fibril formation. The β sheet structure formation of PAP248-286 was measured by circular dichroism spectrum, and the morphology of amyloid fibrils incubated with or without lactic acid was observed with transmission electron microscopy (TEM). The enhancing effect of amyloid fibril incubated with lactic acid at different time points was determined using virus infection assay. PAP248-286 (2 mg/mL) was incubated with dilutions of vaginal secretion from healthy women, and amyloid fibril formation was detected with ThT and Congo red staining.</p><p><b>RESULTS</b>Lactic acid inhibited SEVI fibril formation in a dose-dependent manner in vitro. Lactic acid at 0.5 mg/mL completely inhibited 2 mg/mL SEVI fibril formation within 48 h. After incubation for 48 h, lactic acid at 1 mg/mL inhibited the formation of β-sheet structure of SEVI (2 mg/mL) and completely inhibited 2 mg/mL PAP248-286 aggregation as observed with TEM. In the presence of lactic acid, PAP248-286 lost the ability to enhance virus infection. Vaginal secretion inhibited SEVI fibril formation in a dose-dependent manner, and virtually no SEVI fibril occurred after incubation of 2 mg/mL PAP248-286 with 67% vaginal secretion.</p><p><b>CONCLUSION</b>Lactic acid inhibits SEVI fibril formation in vitro.</p>

Synergistic anti-tumor effect of obatoclax and MG-132 in esophageal cancer cell line CaES-17 / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore whether MG-132 could enhance the anti-tumor activity of obatoclax against esophageal cancer cell line CaES-17.</p><p><b>METHODS</b>MTT assay was used to determine the cytotoxicity of obatoclax and MG-132 in CaES-17 cells. The IC(50) of obatoclax and MG-132 were used to determine the molar ratio (1:2.4) of the two drugs for combined treatment of the cells. The concentrations of obatoclax and MG-132 ranged from 1/8 IC(50) to 4 IC(50) after serial dilution, and their combination index (CI) was calculated using CompuSyn software. The expression of ubiquitin and the cleavage of PARP, caspase-9, phospho-histone H3 and phospho-aurora A/B/C in the exposed cells were examined with Western blotting; the cell apoptosis was measured by flow cytometry with Annexin V staining, and the percentage of cells in each cell cycle phase was also determined by flow cytometry.</p><p><b>RESULTS</b>The CI of obatoclax and MG-132 was 0.296 for a 50% inhibition of Caes-17 cells and was 0.104 for a 95% inhibition. The cells treated with obatoclax or MG-132 alone showed increased expression of ubiquitin and cleavage of PARP and caspase-9. Compared with the cells treated with obatoclax or MG-132 alone, the cells with a combined treatment exhibited significantly increased expression of ubiquitin, cleavage of PARP and caspase-9, and expression of phospho-Histone H3 (P<0.05). The combined treatment of the cells also resulted in significantly increased expression of phospho-Aurora A/B/C compared with obatoclax treatment alone. The cells with the combined treatment showed significantly higher percentages of apoptotic cells and cells in sub-G(1) and G(2)/M phases compared with the cells treated with either of the drugs (P<0.05).</p><p><b>CONCLUSION</b>Obatoclax combined with MG-132 shows a significant synergistic anti-tumor effect against esophageal cancer CaES-17 cells by inducing apoptosis and cell cycle arrest.</p>

Efficacy of Leflunomide, Telmisartan, and Clopidogrel for Immunoglobulin A Nephropathy: A Randomized Controlled Trial / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The efficacy and safety of telmisartan combined with clopidogrel, leflunomide, or both drugs for immunoglobulin A nephropathy (IgAN) are unclear. This study was designed to evaluate the efficacy and safety of telmisartan combined with clopidogrel, leflunomide, or both drugs for IgAN.</p><p><b>METHODS</b>It is a multicenter, prospective, double-dummy randomized controlled trial. Primary IgAN patients were recruited in 13 renal units across Beijing, China, from July 2010 to June 2012. After a 4-week telmisartan (80 mg/d) wash-in, 400 patients continuing on 80 mg/d telmisartan were randomly assigned to additionally receive placebo (Group A), 50 mg/d clopidogrel (Group B), 20 mg/d leflunomide (Group C), or 50 mg/d clopidogrel and 20 mg/d leflunomide (Group D). The 24-week intervention was completed by 360 patients. The primary endpoint was change in 24-h proteinuria at 24 weeks. A linear mixed-effect model was used to analyze the changes at 4, 12, and 24 weeks. Generalized estimating equations were used to evaluate changes in hematuria grade. This trial was registered at the Chinese Clinical Trial Registry.</p><p><b>RESULTS</b>The effects of telmisartan combined with leflunomide on changes in proteinuria (0.36 [95% confidence interval (CI) 0.18-0.55] g/d, P < 0.001), in serum uric acid (76.96 [95% CI 57.44-96.49] μmol/L, P < 0.001), in serum creatinine (9.49 [95% CI 6.54-12.44] μmol/L, P < 0.001), and in estimated glomerular filtration rate (-6.72 [95% CI-9.46 to -3.98] ml·min-1·1.73 m-2, P < 0.001) were statistically significant, whereas they were not statistically significant on changes in systolic and diastolic blood pressure and weight (P > 0.05). Telmisartan combined with clopidogrel had no statistical effect on any outcome, and there was no interaction between the interventions. No obvious adverse reactions were observed.</p><p><b>CONCLUSIONS</b>Telmisartan combined with leflunomide, not clopidogrel, is safe and effective for decreasing proteinuria in certain IgAN patients.</p><p><b>TRIAL REGISTRATION</b>chictr.org.cn, ChiCTR-TRC-10000776; http://www.chictr.org.cn/showproj.aspx?proj=8760.</p>

Effects of Biejia Ruangan Tablet-containing serum on matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in cultured renal interstitial fibroblasts / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of Biejia Ruangan Tablet ([symbol in text], BRT)-containing serum on the expression of matrix metalloproteinase (MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1) in cultured renal interstitial fibroblasts.</p><p><b>METHODS</b>Different BRT-containing sera were prepared by gastric gavages to rats with the high-dose (7 g/kg), mid-dose (3.5 g/kg), and low-dose (1.75 g/kg) BRT respectively. The expression of extracellular matrix in NRK-49F cells was induced by treatment with human transforming growth factor-β1 (recombined human TGF-β1), and BRT-containing serum. Western blotting and Northern blotting were used to measure type I and III procollagen, MMP-9, and TIMP-1.</p><p><b>RESULTS</b>The high dose BRT-containing serum could decrease the type I and III procollagen gene expression which boosted by TGF-β1, at the same time cut down TIMP-1 protein and gene expression which increased by TGF-β1 (P <0.05). Treatment of cells with recombined human TGF-β1 had no significant effect on MMP-9 expression and BRT-containing serum also had no effect on MMP-9 expression.</p><p><b>CONCLUSIONS</b>High dose BRT has anti-fibrosis effects in NRK-49F cells, as indicated by its inhibition of type I and III procollagen and TIMP-1 expression.</p>

Development of peptidic MERS-CoV entry inhibitors / 药学学报

In 2012, a new SARS-like coronavirus emerged in the Middle East, namely the Middle East respiratory syndrome coronavirus (MERS-CoV). It has caused outbreaks with high mortality. During infection of target cell, MERS-CoV S protein S1 subunit binds to the cellular receptor (DPP4), and its S2 subunit HR1 and HR2 regions intact with each other to form a stable six-helix bundle to mediate the fusion between virus and target cell membranes. Hence, blocking the process of six-helix bundle formation can effectively inhibit MERS-CoV entry into the target cells. This review focuses on the recent advance in the development of peptidic entry inhibitors targeting the MERS-CoV S2 subunit.

Small molecular agents against MERS-CoV infection / 药学学报

Middle East respiratory syndrome coronavirus (MERS-CoV) has caused outbreaks of SARS-like disease with 35% case-fatality rate, mainly in the Middle East. A more severe outbreak of MERS occurred recently in the Republic of Korea, where 186 people contracted the infections, causing great concern worldwide. So far, there has been no clinically available drug for the treatment of MERS-CoV infection. The potential drugs against MERS-CoV mainly consist of monoclonal antibodies, peptides and small molecular agents. Small molecular agents have an advantage of easier synthesis, lower cost in production and relatively higher stability. There is better chance for those candidates to gain a quick development. This article reviews the progress of developing small molecular MERS-CoV agents.

Inhibition of the tubular epithelial-to-mesenchymal transition in vivo and in vitro by the Uremic Clearance Granule () / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of the Uremic Clearance Granule (UCG, ), a Chinese patent medicine, on tubular epithelial-to-mesenchymal transition (EMT) in a unilateral ureteral obstruction (UUO) model in vivo and transforming growth factor (TGF)-β1 induced EMT of HK-2 cells in vitro.</p><p><b>METHODS</b>In vivo study, 50 Sprague Dawley rats were divided into three groups: a sham operation group (n=10), a UUO group (n=20), and a UUO with UCG treatment group (n=20). The UCG was given at a dose of 4.5 g/kg body weight per day by gavage after surgery. In vitro study, HK-2 cells were cultured in 10% fetal bovine serum (FBS), 10% healthy rat serum, 10% FBS and TGF-β1 (10 ng/mL), 10% healthy rat serum and TGF-β1, or 10% rat serum containing the uremic clearance granule and TGF-β1. The expression of the epithelial marker E-cadherin and the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) in kidney tissues and HK-2 cells were investigated by Western blot analysis and immunofluorescence staining.</p><p><b>RESULTS</b>The rats of the UUO group showed obvious tubulointerstitial fibrosis, compared with the sham operation group rats. Tubulointerstitial fibrosis score was reduced by 17.5%±1.1% at day 7 and by 20.0%±1.2% at day 14 in the UCG-treated group, compared with the UUO group. The UCG could maintained expression of E-cadherin and suppressed expression of vimentin and α-SMA in kidney tissues of UUO rats at days 7 and 14, as determined by Western blot analysis and immunofluorescence staining. Rat serum containing the UCG partially inhibited TGF-β1-induced fibroblast phenotype of HK-2 cells and maintained the epithelial morphology of HK-2 cells in vitro. This occurred partially through a reduction of vimentin expression and an increase of E-cadherin expression.</p><p><b>CONCLUSION</b>These results suggest that the UCG prevents tubular EMT and may be a promising agent for treating tubulointerstitial fibrosis.</p>

Semen-derived enhancer of viral infection--a key factor in sexual transmission of HIV / 病毒学报

Semen-derived enhancer of viral infection(SEVI) is a peptide fragment (PAP248-286) from prostatic acid phosphatase(PAP), which can enhance human immunodeficiency virus infection. The mechanisms of SEVI include: (1) SEVI with several cationic amino acid residues reduced electrostatic repulsion between HIV virus and the target cells; (2) The disorder state of SEVI in the human body fluids was helpful to the interaction between virus and the target cell membranes; (3) SEVI could capture HIV particles directly and speed the velocity of virus on the surface of the target cells and improve adsorption and fusion. Currently, the substances of inhibiting SEVI activity include: EGCG from green tea, small molecule compound of aminoquinoline Surfen, ThT analogs BTA-EG6. Those compounds might block the combination of HIV and SEVI or prevent the formation of amyloid fibers, and then reduce the enhancement of SEVI. The studies on the biological characteristics and mechanisms of SEVI have a big benefit for the prevention and treatment of HIV infection.

Protective and antioxidant properties of wasp (Vespa magnifica) honeycomb extract: a potential inhibitor against acidified ethanol-induced gastric lesions / 南方医科大学学报

<p><b>OBJECTIVE</b>To examine the protective effects of wasp (Vespa magnifica) honeycomb extract (WCE) against gastric lesions in rats induced by 60% acidified ethanol, and evaluate its capacity to suppress oxidative stress in the gastric tissue.</p><p><b>METHODS</b>Wistar rats were subjected to intragastric administration of 60% acidified ethanol to induce gastric lesions following an 8-day oral pretreatment with WCE at 0, 25, 100 and 150 mg/kg or with saline. The levels of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging, myeloperoxidase (MPO) activity and total antioxidant capacity in the gastric tissues were determined.</p><p><b>RESULTS</b>Oral administration of 25, 100 and 150 mg/kg WCE prior to 60% acidified ethanol administration significantly inhibited the formation of gastric lesions (with a reduction by 44.2%-87.1%), decreased the mucosal MPO activity (by 16.4%-56.6%) and increased the total antioxidant capacity of the gastric tissue (by 0.5, 1.47 and 1.83 folds, respectively) in a dose-dependent manner. At a high concentration (above 1 mg/ml), WCE also exhibited a stronger DPPH radical scavenging activity than butylated hydroxytoluene (BHT).</p><p><b>CONCLUSION</b>The ethanol extract of wasp honeycombs can suppress the formation of acidified ethanol-induced gastric lesions by reducing free radical oxidation and neutrophils infiltration in the gastric tissue in rats.</p>

Antiviral activity of 3-hydroxyphthalic anhydride-modified ovalbumin against herpes simplex virus 2 in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the antiviral activity of 3-hydroxyphthalic anhydride-modified ovalbumin (HP-OVA) against herpes simplex virus 2 (HSV-2) in vitro.</p><p><b>METHODS</b>By chemical modification, ovalbumin (OVA) was treated with 3-hydroxyphthalic anhydride (HP) to prepare HP-OVA. The anti-HSV-2 activity against HSV-2 333 virus in vitro and the cytotoxicity of HP-OVA in African green monkey kidney cells (Vero cells) were detected by MTT colorimetric assay. The inhibitory effects of HP-OVA on 17 strains of vaginal lactobacilli were observed by microscopy.</p><p><b>RESULTS</b>Anhydride-modified ovalbumin significantly inhibited the infection by HSV-2 with an IC(50) of 23.56±8.33 µg/ml. HP-OVA showed only low cytotoxicity to the host cells with a CC(50) over 1 mg/ml. HP-OVA did not produce significant inhibitory effect on the 17 strains of vaginal lactobacilli (MIC>1 mg/ml).</p><p><b>CONCLUSION</b>Anhydride-modified protein HP-OVA exhibits potent anti-HSV-2 activity in vitro and can be a good microbicide candidate for prevention of sexually transmitted diseases.</p>

Epidermal growth factor stimulates the proliferation of human esophageal squamous cell carcinoma HKESC-1 cells by increasing COX-2 expression / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the mechanisms responsible for epidermal growth factor (EGF)-induced proliferation of human esophageal squamous cell carcinoma cells.</p><p><b>METHODS</b>(3)H-thymidine incorporation assay was used to assess the proliferation of HKESC-1 cells exposed to EGF stimulation. Enzyme immunoassay was used to measure PGE(2) release from HKESC-1 cells, and the protein levels of cyclooxygenase 1 (COX-1), COX-2, EP1 and EP2 in EGF-stimulated cells were determined by Western blotting.</p><p><b>RESULTS</b>EGF upregulated COX-2 protein expression but produced no obvious effect on COX-1 protein expression in HKESC-1 cells. As a consequence of increased COX-2, EGF further enhanced cellular PGE(2) release. EGF stimulation also resulted in increased protein expression of EP2, a subtype of PGE(2) receptors. Both the non-selective COX inhibitor indomethacin and the selective COX-2 inhibitor SC-236 completely abolished EGF-induced PGE(2) release, and suppressed the mitogenic effect of EGF.</p><p><b>CONCLUSION</b>EGF stimulates the proliferation of HKESC-1 cells by increasing COX-2 protein expression and PGE(2) release. Upregulated EP2 protein expression may further amplify the mitogenic action of PGE(2).</p>

Construction of rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries.</p><p><b>METHODS</b>Peripheral blood lymphocytes were isolated from patients with rheumatoid arthritis. The repertoires of kappa light chain (LCκ) and heavy chain variable region (VH) of the antibodies were amplified by RT-PCR. The amplified LCκ and VH genes were inserted into the vector pDGB-HC-TM separately, and the ligated libraries were transformed into competent E.coli TOPO-10 strain to construct the rheumatoid arthritis-specific antibody heavy and light chain libraries. 293T cells were co-transfected with the libraries and the full-length fully human antibody expressed on the surface of 293T cells were analyzed by flow cytometry.</p><p><b>RESULTS</b>The libraries of rheumatoid arthritis-specific antibody LCκ and heavy chain (IgG1) were constructed. The expression of full-length fully human antibody on the surface of 293T cells was confirmed by flow cytometry. With the rates of correct LCκ and heavy chain sequence insertion reaching 80% and 60%, respectively, as shown by DNA sequence analysis of the randomly selected clones, the libraries showed an expressible combinatory diversity of 6.13×10(10).</p><p><b>CONCLUSION</b>The constructed libraries provide a useful platform for screening rheumatoid arthritis-specific antibodies.</p>

Rapid construction of a mammalian cell surface display library of full-length human antibodies / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a mammalian cell surface display library of full-length human antibodies.</p><p><b>METHODS</b>The total RNA was isolated from human peripheral blood mononuclear cells (PBMCs), and the genes encoding the heavy chain variable regions and kappa light chains (VH and Cκ) of the antibodies were amplified by RT-PCR. The amplified VH and Cκ gene sequences were separately inserted into the vector pDGB-HC-TM. The ligation mixtures were transformed into competent E.coli DH5α cells to construct the antibody libraries, and the library sizes and diversity were analyzed. The library DNAs were transfected into CHO cells and the expression of the full-length human antibodies on the surface of CHO cells was analyzed by flow cytometry.</p><p><b>RESULTS</b>The heavy chain gene library constructed showed a diversity of 2.6 × 10(5), and the kappa light chain gene library had a diversity of 2.0 × 10(5). Sequence analysis of 10 clones randomly selected from the constructed heavy chain gene library and 10 from the light chain gene library showed that 8 heavy chain clones and all 10 light chain clones contained correct open reading frames. Flow cytometry demonstrated that all the 18 clones expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces was detected with the positive cells reaching 31.5.</p><p><b>CONCLUSIONS</b>A full-length human mammalian display antibody library with a combinatory diversity of 5.2 × 10(10) can be constructed in two weeks, which allows the display of full-length antibodies on mammalian cell surface.</p>

Cisplatin induces drug resistance in human esophageal squamous carcinoma cell line EC109 by decreasing CTR1 protein expression / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the mechanism of the development of cisplatin resistance in a human esophageal squamous carcinoma cell line.</p><p><b>METHODS</b>The cytotoxicity of cisplatin in the cisplatin-resistant resistant cell line EC109/CDDP and its parental cell line EC109 was measured by MTT assay. Whole-cell cisplatin accumulation and Pt-DNA adduct formation were determined by inductively coupled plasma mass spectrometry (ICP-MS). Western blotting was used to investigate the protein expression of full length PARP, cleaved PARP, and copper transporter 1 (CTR1).</p><p><b>RESULTS</b>EC109/CDDP cells was more resistant to cisplatin-induced cytotoxicity and apoptosis than EC109 cells. Compared with EC109 cells, EC109/CDDP cells exhibited less cisplatin accumulation and Pt-DNA adduct formation with also decreased CTR1 protein expression.</p><p><b>CONCLUSION</b>Cisplatin induces drug resistant phenotype by decreasing the protein level of CTR1, which controls cell accumulation and cytotoxic effect of cisplatin.</p>

Effects of UV-induced DNA damage on vector ligation and transformation into bacterial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effects of UV irradiation on DNA ligation and transformation efficiency of the expression vector into competent bacterial cells.</p><p><b>METHODS</b>The expression vector was digested with the restriction enzyme SfiI, and the purified target DNA fragments were exposed to UV light at different wavelengths. Ligation and transformation experiments with the exposed fragments were carried out and the colony number and transformation efficiency were assessed.</p><p><b>RESULTS</b>The transformation efficiency of the DNA with a 5-min exposure to 302 nm UV was 60 colonies per nanogram of the DNA, as compared with 20400 for the DNA exposed to 365 nm UV. The time course experiment showed that prolonged DNA exposure to 365 nm UV light was associated with lowered transformation efficiency. DNA exposure for 30 min caused a reduction of the transformation efficiency to lower than 50% compared to that of DNA without UV exposure. But with a 15 min exposure, the DNA maintained a transformation efficiency more than 70%, which was sufficient for most molecular biology experiments.</p><p><b>CONCLUSION</b>In construction of the expression vector, it is advisable to prevent the target DNA from UV exposure. When UV exposure is essential, we suggest that 365 nm UV be used and the exposure time controlled within 15 min.</p>

A non-infectious and quantitative cell-based bioassay for screening HIV entry inhibitors targeting HIV envelope proteins / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop an objective bioassay for quantitative detection of HIV-induced cell-cell fusion for screening HIV entry inhibitors.</p><p><b>METHODS</b>HL2/3 cells expressing HIV envelope proteins gp120/gp41, Tat, and other HIV proteins were co-cultured with HeLa-CD4-LTR-beta-gal cells expressing CD4 receptor and HIV LTR triggered reporter gene beta-galactosidase. The enzyme activities of beta-galactosidase were detected by a chromogenic substrate, chlorophenol red-beta-galactopyranoside (CPRG). Specific HIV entry inhibitors were used to validate the established detecting method.</p><p><b>RESULTS</b>No syncytium was formed by mixing HL2/3 and HeLa-CD4-LTR-beta-gal cells. However, the membrane could be fused and the Tat expressed by HL2/3 cells could bind to HIV LTR on HeLa-CD4-LTR-beta-gal cells and trigger the expression of beta-galactosidase. CPRG allowed quantitative and sensitive detection of the activity of beta-galactosidase. Further studies showed that HIV entry inhibitors could inhibit the activity of beta-galactosidase in a dose-dependent manner.</p><p><b>CONCLUSION</b>We have developed a simple, cheap, objective and quantitative non-infectious cell-cell fusion bioassay that can be used to screen for anti-HIV agents targeting the virus entry from natural and synthetic compound libraries.</p>

Construction of human full-length renal cell carcinoma patient-specific antibody library by mammalian cell surface display / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct human renal cell carcinoma patient-specific full-length antibody library using mammalian cell surface display technique.</p><p><b>METHODS</b>Peripheral blood mononuclear cells (PBMC) were isolated from patients with renal cell carcinoma. The repertoires of kappa light chain (LCkappa) and heavy chain variable region (VH) of antibody were amplified by RT-PCR. The LCkappa and VH libraries were inserted into the vector pDGB-HC-TM separately, and the ligated libraries were transformed into competent E.coli TOPO10 to construct the renal cell carcinoma patient-specific antibody heavy and light chain libraries. 293T cells were co-transfected with the libraries and the full-length human antibodies expressed on the surface of 293T cells were analyzed by flow cytometry.</p><p><b>RESULTS</b>The libraries of renal cell carcinoma-specific antibody kappa light chain (LCkappa) and heavy chain (IgG1) were constructed. The expression of the full-length human antibodies on the surface of 293T cell was confirmed by flow cytometry. The libraries showed an expressible combinatory diversity of 7.5x10(10).</p><p><b>CONCLUSION</b>The expressible antibody library provides a useful platform for screening of renal cell carcinoma-specific antibodies.</p>

Bioequivalence evaluation of orally disintegrating tablet of pentoxyverine citrate / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the bioequivalence of orally disintegrating tablets of pentoxyverine citrate (tested preparation) in healthy male volunteers.</p><p><b>METHODS</b>A single oral dose of the tested and reference preparations at 25 mg were given to 20 healthy volunteers in a randomized two-period cross-over design. Plasma pentoxyverine citrate concentrations were determined by HPLC-MS/ESI+ method. The pharmacokinetic parameters were calculated and the bioequivalence of the two preparations were evaluated using DAS program.</p><p><b>RESULTS</b>The Tmax, Cmax, AUC0 15 and AUC0infinity of tested and reference preparations were 1.62-/+0.75 h and 2.52-/+1.21 h, 62.28-/+33.06 microg/L and 59.72-/+33.25 microg/L, 234.44-/+130.01 microg.h.L(-1) and 228.77-/+129.24 microg.h.L(-1), 246.80-/+136.19 microg.h.L(-1) and 244.11-/+140.73 microg.h.L(-1), respectively. The 90% confidence interval of C(max), AUC0 15 and AUC0infinity of tested preparations were 81.4%-138.4%, 86.0%-123.3% and 86.5%-121.2%, respectively.</p><p><b>CONCLUSION</b>The tested and reference preparations are bioequivalent.</p>

A novel method for hepatitis C virus genotyping using RT-PCR reverse dot blot hybridization technique / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a rapid and specific method for hepatitis C virus ( HCV) genotyping using reverse dot blot hybridization technique and investigate the distribution of HCV genotypes and subtypes in Guangdong.</p><p><b>METHODS</b>The primers and the probes targeting the 5'untranslated region (5'UTR) and core region of HCV genotypes 1b, 2a, 3a, 3b and 6a were designed, and the RT-PCR reverse dot blot hybridization (PCR-RDH) method for HCV genotyping was established. A total of 115 patients with hepatitis C were genotyped using this method, and 38 of them were also genotyped by sequencing and phylogenetic analysis to evaluate the accuracy and specificity of the method.</p><p><b>RESULTS</b>Of the 115 patients, 111 were successfully genotyped to be 1b, 2a, 3a, 3b, 6a and mix-infection of 1b/2a at frequencies of 56.8%, 8.1 %, 3.6%, 5.4%, 25.2% and 0.9% respectively, and all the 15 healthy control samples showed negative results. The accuracy and reliability of the genotyping method of PCR-RDH was confirmed in 38 cases by amplification of HCV core and NS5B regions followed by DNA sequencing and phylogenetic analysis.</p><p><b>CONCLUSION</b>This method for HCV genotyping, with high reliability and specificity, is suitable for clinical and epidemiological investigations. The prevalence of HCV genotypes 1b and 2a decreases while 1b remains the dominant genotype in Guangdong, where the prevalence of 6a significantly increases as compared with that 10 years ago.</p>