By using a cell-based high throughput screening model for the CLA-1 up-regulator, Streptomyces 203909 was found to produce up-regulator of CLA-1. A novel trichostatin analogue was isolated from the rice fermentation of Streptomyces sp. CPCC 203909by a combination of various chromatographic techniques including column chromatography (CC) over silica gel, flash C18 CC, and reversed-phase HPLC. Its structure was identified as (-)-(R,2E,4Z)-7-[(4'-dimethylamino) phenyl]-4,6-dimethyl-7-oxohepta-2,4-dienoyl-L-glutamine (1) by the spectroscopic and chemical methods, and combination with the CD spectroscopy and Marfey's method. In the prelimi- nary assays, Compound 1 showed cytotoxicity against human embryonic kidney 293 cell line with IC50 value 35.3 [µmol · L(-1).
Cell Survival ; drug effects ; Fermentation ; Hep G2 Cells ; Humans ; Hydroxamic Acids ; chemistry ; isolation & purification ; metabolism ; pharmacology ; Molecular Structure ; Streptomyces ; chemistry ; metabolism

OBJECTIVETo establish a new drug screening model based on transcriptional regulation of human high density lipoprotein (HDL) receptor gene CD36 and LIMPII analogous-1 (CLA-1) for discovering up-regulator of this receptor.

METHODSThe upstream regulatory sequence of CLA-1 was obtained by polymerase chain reaction. A recombinant reporter plasmid pGL3-CLAP was constructed by inserting the regulatory sequence upstream of luciferase gene of pGL3-Basic. Human hepatoma cell line BEL-7402 was transfected with pGL3-CLAP. Samples were detected by testing luciferase activity of transfected BEL-7402 cells in microtiter wells.

RESULTSThe drug screening model was established and optimized. Significant difference was present between pGL3-CLAP and pGL3-Basic transfected BEL-7402 cells (P< 0.001), and coefficient of variation was less than 10%. After primary and secondary screening, 1 compounds and 3 fermentation extracts had up-regulating activities.

CONCLUSIONThis new drug screening model may be efficiently used to screen up-regulators of human HDL receptor expression, which might become lead compounds for new anti-atherosclerosis drugs.

CD36 Antigens ; Cholesterol Esters ; metabolism ; Drug Evaluation, Preclinical ; methods ; Gene Expression Regulation ; drug effects ; Humans ; Hypolipidemic Agents ; chemical synthesis ; pharmacology ; Lipoproteins, HDL ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA-Binding Proteins ; Receptors, Immunologic ; genetics ; Receptors, Lipoprotein ; genetics ; Receptors, Scavenger ; Scavenger Receptors, Class B ; Transcription, Genetic ; drug effects ; Up-Regulation

OBJECTIVETo develop a new high-throughput screening model for human high-density lipoprotein (HDL) receptor (CD36 and LIMPII analogous-1, CLA-1) agonists using CLA-1-expressing insect cells.

METHODSWith the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA (cDNA) of CLA-1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV) into donor vector pFastBac1 and recombinant pFastBac1-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacmid DNA. Recombinant bacmid-CLA-1 was transfected into Spodoptera frugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA-1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized.

RESULTSWestern blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries.

CONCLUSIONHuman HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.

Animals ; Baculoviridae ; genetics ; metabolism ; Biological Assay ; Carbocyanines ; metabolism ; Cell Line, Tumor ; Cholesterol, HDL ; metabolism ; DNA, Complementary ; genetics ; metabolism ; Fluorescent Dyes ; metabolism ; Gene Expression ; Humans ; Lipoproteins, HDL ; agonists ; genetics ; metabolism ; Lipoproteins, LDL ; metabolism ; Receptors, Lipoprotein ; agonists ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Scavenger Receptors, Class B ; agonists ; genetics ; metabolism ; Spodoptera ; genetics ; metabolism

To investigate the anti-cardiac hypertrophic mechanism of statins, thirty-eight male Wistar rats were randomly allocated to four groups. Rats in model group received nitric oxide synthase inhibitor, N-nitro-L-arginine (L-NNA) 15 mg/(kg.d) by peritoneal injection. Rats in simvastatin treatment groups were given simultaneously L-NNA as those in model group and simvastatin 5 or 30 mg/(kg.d) intragastrically respectively. Rats in control group received the same volume of normal sodium. Left ventricular function, left ventricular mass index (LVMI), the content of brain natriuretic peptide (BNP) in plasma and myocardium, myocardial hydroxyproline and heme oxygenase activity were determined after 6 weeks. The results showed that rats in model group developed significant cardiac hypertrophy associated with reduced left ventricular function compared with the control group. However, compared with the model group, L-NNA-induced cardiac hypertrophy of rats was significantly relieved in simvastatin treatment groups, associated with improved left ventricular function, decreased LVMI, lower BNP levels in plasma and myocardium, lower content of myocardial hydroxyproline, and increased myocardial heme oxygenase (HO) activity. In cultured rat neonatal cardiomyocytes, simvastatin (30 or 100 mumol/L) significantly increased heme oxygenase-1 (HO-1) mRNA expression, HO activity as well as the production of CO in cardiomyocytes. Cultured with zinc protoporphyrin, a HO inhibitor, or simvastatin alone did not change [(3)H]leucine uptake of cardiomyocytes. However, cocultured with simvastatin significantly inhibited the cardiomyocyte [(3)H]leucine uptake induced by angiotensin II in a concentration-dependent manner. Cotreatment with zinc protoporphyrin significantly abolished the suppressive effect of simvastatin on cardiomyocyte [(3)H]leucine uptake. These data suggest that the activation of HO-1/CO pathway may be one of the important mechanisms by which statins inhibit cardiac hypertrophy caused by hypertension.
Angiotensins ; antagonists & inhibitors ; pharmacology ; Animals ; Carbon Monoxide ; metabolism ; Cardiomegaly ; etiology ; prevention & control ; Cell Enlargement ; drug effects ; Heme Oxygenase-1 ; metabolism ; Hypertension ; complications ; drug therapy ; Male ; Myocytes, Cardiac ; cytology ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Simvastatin ; pharmacology ; therapeutic use

OBJECTIVETo establish fluorescence resonance energy transfer (FRET) assay method of detecting proteolytic activity of non-structural protein 3-4A (NS3-4A) serine protease of hepatitis C virus (HCV) for high throughput screening inhibitors against HCV in vitro.

METHODSHCV recombinant plasmid pMAL~c2/NS3-4A was transformed into the E.coli strain K12TB1. Maltose-binding-protein (MBP) NS3-4A fusion protein expression was induced by adding isopropyl-β-D-thiogalacto-pyranoside (IPTG) and purified by affinity chromatography. The proteolytic activity of MBP-NS3-4A protease was analyzed by FRET with the special protease substrate. The reaction system in this model was optimized, and the reliability of the model was evaluated.

RESULTSHigh throughput screening model for HCV NS3-4A protease inhibitors was established, and the best concentrations of enzyme and substrate were optimized. In the model, the Km value of protease was 4.74 μmol/L, Z factor was up to 0.80, and coefficient of variation (CV) was 1.91%. BILN 2061, one of the known HCV protease inhibitors, was measured with the Ki of 0.30 nmol/L.

CONCLUSIONThe assay model using FRET method for HCV NS3 4A serine protease is stable and reliable, and the model is suitable for high throughput screening for HCV NS3 4A protease inhibitors.

Antiviral Agents ; pharmacology ; Drug Evaluation, Preclinical ; Fluorescence Resonance Energy Transfer ; Hepacivirus ; enzymology ; High-Throughput Screening Assays ; methods ; Protease Inhibitors ; pharmacology ; Viral Nonstructural Proteins ; antagonists & inhibitors ; genetics

OBJECTIVETo evaluate the efficacy and safety of live combined Bifidobacterium, Lactobacillus and Enterococcus capsules in treatment of irritable bowel syndrome.

METHODSEighty-five patients [male 32, female 53; age (45.31+/-11.72) years] were given live combined Bifidobacterium, Lactobacillus and Enterococcus capsules 1260 mg/d t.i.d.x4 weeks. Syndrome scales were used to evaluate the efficacy in gastrointestinal syndrome. Fecal flora was also measured before and after the treatment. Six bacteria were cultured and the colony forming units were counted in stool. SPSS was used for data analysis.

RESULTSSeventy-four patients finished the follow-up. No side-effect was found. For treatment of irritable bowel syndrome, the effective rate of live combined Bifidobacterium, Lactobacillus and Enterococcus capsules was 56.8% in the second week, 74.3% in the fourth week and 73.0% in the sixth week. Single symptom was improved, especially in abdominal pain and stool character. The probiotica containing live combined Bifidobacterium, Lactobacillus and Enterococcus could increase bifidobacterium count (P<0.01) and lactobacillus count (P<0.05); decrease bacteroides count (P<0.05) and enterococci count (P<0.01); No obvious changes were observed in clostridium difficile colonitis and enterobacteriaceae (P>0.05).

CONCLUSIONThe result of the study indicated that the administration of live combined Bifidobacterium, Lactobacillus and Enterococcus improved the symptom of irritable bowel syndrome and that there was a gradual increase of this effect. Thereafter conditions remained stable for 2 weeks. That improvement may be associated with alterations in gastrointestinal flora.

Adult ; Bifidobacterium ; Enterococcus ; Female ; Humans ; Intestines ; microbiology ; Irritable Bowel Syndrome ; drug therapy ; Lactobacillus ; Male ; Middle Aged ; Probiotics ; therapeutic use

OBJECTIVEThe purpose of this study was to investigate the clinical value of radiolabeled tracer method, methylene blue method and combination of these two methods in detection of sentinel lymph node (SLN), and to evaluate the accuracy of SLN in predicting the cervical lymph nodes status in laryngeal carcinoma patients with clinically negative neck lymph nodes (cN0 ).

METHODSForty-one patients with cN0 laryngeal neoplasms underwent SLN detection using both of radiolabeled tracer and methylene blue. SLN imaging was performed with laryngoscope-guided injection of radioactive isotope 99Tc(m)-sulfur colloid (SC) into the laryngeal carcinoma before surgery, then all these patients underwent intraoperative lymphatic mapping with a handheld gamma-detecting probe. After mapping of SLN, methylene blue was subsequently injected at the same spots around the tumor in order to identify SLN during surgery. The results of SLN detection by isotope tracer, dye and combination of both methods were compared.

RESULTSThe SLN detection rates by radiolabeled tracer, methylene blue and combined method were 87.8%, 70.7% and 92.7%, respectively (P < 0.01). The number of detected SLN was significantly different between radiolabeled tracer method and combined method (P < 0.05), and also between blue dye method and combined method (P < 0.01). However, no statistically significant difference was found between methylene blue method and radiolabeled tracer method (P > 0.05). Nine patients were found to have lymph node metastasis by final pathological examination. The sensitivity, accuracy and negative predictive values of SLN detection by the combined method using radiolabeled tracer and methylene blue were 88.9%, 97.4% and 96.7%, respectively.

CONCLUSIONThe combined method using radiolabeled tracer and methylene blue can improve the accuracy of sentinel lymph node detection. Furthermore, sentinel lymph node detection can accurately predict the cervical lymph node status in cN0 laryngeal carcinoma.

Adolescent ; Adult ; Aged ; Carcinoma, Squamous Cell ; diagnostic imaging ; pathology ; Female ; Humans ; Laryngeal Neoplasms ; diagnostic imaging ; pathology ; Larynx ; Lymph Nodes ; diagnostic imaging ; Lymphatic Metastasis ; Male ; Methylene Blue ; Middle Aged ; Neoplasm Staging ; Radiopharmaceuticals ; Technetium Tc 99m Sulfur Colloid ; Tomography, Emission-Computed, Single-Photon ; methods ; Tomography, X-Ray Computed ; Young Adult

OBJECTIVETo identify the side effect of all-trans retinoic acid (ATRA), and improve early therapeutic response in patients with acute promyelocytic leukemia (APL).

METHODThe first case of Sweet's syndrome (SS) developed in a APL patient treated with ATRA was reported in mainland of China, and reviewed correlative literature.

RESULTSOnly 14 cases of SS associated with ATRA therapy in APL have been reported in the literature, including the present case. The median age was 49.5 years (9 -84) and 10 were women and 4 men. Of them, SS was restricted to the skin in 10 case, the other 4 muscle, fascia, kidney, and lung were involved. SS appeared after a median of 18 days of ATRA therapy (6 - 34 days). The median WBC count was 7.05 (0.80 - 23.00) x 10(9)/L. Four patients continued with the ATRA therapy without interruption, 13 patients treated with steroids and 12 responded. One patient improved without any treatment. Two cases of SS developed retinoic acid syndromes after ATRA therapy.

CONCLUSIONSweet's syndrome is a rare adverse effect of ATRA, and has similar features with inflammatory or infective dermatosis. The corticosteroids treatment could improve the systemic and cutaneous symptoms. When ATRA therapy was restarted after SS subsided, no recurrence of rashes was observed.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Female ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; Male ; Middle Aged ; Sweet Syndrome ; chemically induced ; Tretinoin ; adverse effects ; therapeutic use

Scavenger receptor CD36 could bind and endocytose oxLDL into macrophages which were then differentiated into foam cells that constitute the atherosclerotic lesion core, and was considered to be a potential target to treat atherosclerosis. In the establishment of the compound library of berberine (BBR, 1) analogues, we discovered that 13-hexylberberine (2) showed an antagonistic activity against CD36. Taking 2 as the lead compound, 21 derivatives were synthesized and their antagonistic activities were evaluated via an ELISA-like high-throughput screening (HTS) model. The primary structure-activity relationships were studied. It was indicated that the introduction of suitable groups at the 2- and 3-position of the aromatic ring A or at the 9-position of the aromatic ring D could enhance the activity. Among the 21 studied compounds, 7g bearing a benzyloxyl group at the 9-position provided a highest CD36 antagonistic activity with the IC50 value of 7.7 micromol L(-1). Besides, its antagonistic activity was further verified with Sf9 insect cell HTS model. So berberine analogues are a new family of CD36 receptor antagonists and worthy to be studied further.
Animals ; Berberine ; analogs & derivatives ; chemical synthesis ; chemistry ; pharmacology ; CD36 Antigens ; metabolism ; Cell Line ; Cell Survival ; drug effects ; Enzyme-Linked Immunosorbent Assay ; High-Throughput Screening Assays ; Receptors, Scavenger ; antagonists & inhibitors ; Spodoptera ; cytology ; virology ; Structure-Activity Relationship

Objective To investigate the clinical value of sentinel lymph node (SLN) detection in laryngeal and hypopharyngeal carcinoma patients with clinically negative neckk (cNO) by lymphoscintigraphy method and blue dye. Methods Forty patients with cNO laryngeal neoplasms and ten patients with cNO hypopharyngeal carcinoma scheduled for tumor resection and neck dissection, were eligible for the study.single photon emission computed tomography (SPECT)/CT lymphoscintigraphy was performed with injection of radioactivity isotope ~(99)Tc~m labeled sulfur colloid(~(99)Tc~m-SC). Methylthioninium was injected into the same paints as ~(99)Tc~m-SC during surgery, and the patients underwent lymphatic mapping with a handheld gamma-detecting probe. All removed lymph nodes were examined by routine histopathology. Results Thirty-five patients with laryngeal carcinoma and six patients with hypopharyngeal carcinoma detected SLN by radiolabeled tracer method, the detection rate of SLN was 82.0%.Tnenty-nine patients with laryngeal carcinoma and 4 patients with hypopharyngeal carcinoma detected SLN by blue dye method, the detection rate of SLN was 66.0%. There were significant difference between two groups (χ~2=2.769, P<0.05), and the number of SLN were respectively 96 and 83 by radiolabeled tracer method and blue dye (χ~2=-2.098,P<0.05), The sensitivity of SLN detection were respectively 83.3% and 66.7%. Twelve (24.0%) patients had lymph node metastasis. Conclusions Either lymphoscintigraphy or blue dye mapping can be used to detect the SLN in cNO laryngeal and hypopharyngeal carcinoma. The lymphoscintigraphy not only preoperatively can locate the accuracy of SLN detection, but also has higher detection rate and sensitivity than dye method.