The tumor multidrug resistance reversal effect of NPB304, a novel taxane, was studied. MTT assay was used to determine the IC50 of chemotherapy drugs. Western blotting assay was applied to analyze the expression of P-glycoprotein (P-gp). The effect of compounds on the P-gp function and P-gp ATPase activity was determined by rhodamine 123 (Rh123) accumulation assay and analysis kit, respectively. Molecular docking was employed to predict the binding force between compounds and P-gp. Transmembrane transport of NPB304 was analyzed using MDCK II and MDR1-MDCK II cell model. NPB304 displayed multidrug resistance reversal effect on KBV cells and MCF-7/paclitaxel cells, NPB304 collaborative with P-glycoprotein (P-gp) inhibitors verapamil enhanced the reversal activity, specifically, 10 μmol x L(-1) verapamil in combination with paclitaxel reversed resistance by 56.5-fold, while combined with NPB304 increased the reversal fold; NPB304 synergistically increased Rh123 accumulation in the resistant cells when combined with verapamil, and NPB304 at 0-1 μmol x L(-1) enhanced the ATPase activity activated by verapamil was observed. NPB304 existed the hydrophobic interactions with the TM regions of P-gp, and the binding force between NPB304 and the A chain of the TM region was stronger. P-gp ATPase activity assay demonstrated NPB304 at lower concentrations (0-1.5 μmol x L(-1)) could activate the P-gp ATPase, playing a role on inhibition of P-gp function. However, NPB304 did not have an obvious feature of P-gp substrate. NPB304 exerted itself and synergy with verapamil activity on reversing tumor resistance via inhibiting the P-gp function.
ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antineoplastic Agents ; pharmacology ; Biological Transport ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Drug Synergism ; Humans ; MCF-7 Cells ; Rhodamine 123 ; Taxoids ; pharmacology ; Verapamil ; pharmacology

To improve the reliability and credibility of genotyping hepatitis E virus (HEV) and to explore the possibility of unifying standards of HEV genotyping by designing HEV universal primers for amplification of a long genomic fragment of different HEV genotypes. A set of universal primers (HEVuPrimer) was designed based on conserved regions determined by alignment analysis of 82 HEV strains with complete genome in GenBank. HEVuPrimer was compared with a set of previously used primers (MXJ primers) for their sequence-matching to different HEV strains and applied to amplify HEV genomic fragments from HEV reference strains with known different genotypes and clinical serum samples with anti-HEV-IgM by RT-nPCR. HEV genotyping based on the fragments amplified with HEVuPrimer was compared and validated with that based on HEV full genome and fragments obtained with MXJ primers. HEV genotyping by the phylogenetic analysis supplemented with the percent of nucleotide identity of the HEVuPrimer-determined fragments showed good correspondence with that based on HEV full-length genome. In addition, HEVuPrimer was much better than MXJ primers in matching sequences of HEV strains available from GenBank, and was able to amplify all the reference HEV strains with different genotypes. Among 124 samples with anti-HEV-IgM, 60 were positive for HEV RNA determined by a 644bp amplicon of RT-nPCR with the HEVuPrimenr. All the positive isolates belonged to HEV genotype 4 with nucleotide homology of 80.0%-99.9%, and could be further divided into 4 subgenotypes. Moreover, a novel subtype was identified with 6 HEV strains isolated very recently. The RT-nPCR using the HEVuPrimer and phylogenetic analysis of the amplified region provided strong evidences for its feasibility in HEV genetic classification. Our data have new implication for the consensus of genotype classification of HEV.
DNA Primers ; genetics ; Genome, Viral ; genetics ; Genotype ; Hepatitis E virus ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods

OBJECTIVETo evaluate the in vitro anti-hypertrophic effect of total Glycosides of Ranunculus Japonius (TGRJ).

METHODSNeonatal rat cardiomyocytes were cultured and hypertrophy was induced by administrating isoproterenol (ISO, 10 µmol/L) or angiotensin 2 (Ang 2, 1 µmol/L) for 48 hours. In the treatment groups, cells were pretreated with TGRJ (0.3 g/L) for 30 minutes prior to hypertrophic stimuli. The anti-hypertrophic effects of TGRJ were examined by measuring cell size, total protein content, and protein synthesis. Intracellular free Ca(2+) concentration ([Ca(2+)]i) was evaluated using fluorescence dye Fura-2/AM. Sacroplasmic/endoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and beta-myosin heavy chain (β-MHC) protein expression levels were measured by Western blotting . SERCA2a activity was assayed by p-nitrophenal phosphate disodium salt hexahydrate method.

RESULTSIncreased cell size, total protein content, and protein synthesis following ISO or Ang 2 stimulation were significantly inhibited by pretreatment with TGRJ (all P<0.05). This anti-hypertrophic effect of TGRJ was confirmed by its suppressing effect on elevated expression of the three hypertrophic related genetic markers, ANP, BNP, and β-MHC. In addition, TGRJ inhibited ISO or Ang 2 induced up-regulation of [Ca(2+)]i under chronic but not acute conditions. And ISO or Ang 2 induced down-regulation of SERCA2a expression and activity was also effectively rectified by TGRJ pretreatment.

CONCLUSIONSThe results of present study suggested that TGRJ could prevent ISO or Ang 2 induced cardiac hypertrophy through improving chronic [Ca(2+)]i disorder, might via normalizing SERCA2a expression and activity.

Animals ; Animals, Newborn ; Calcium ; metabolism ; Cells, Cultured ; Glycosides ; analysis ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Ranunculus ; chemistry ; Rats

AIMTo study the microbial transformation of sinenxan A.

METHODSChoose two strains of Fungi (Mucor spinosus AS 3.3450 and Cunninghamella echinulata AS 3.3400) and a strain of bacterium (Proteus vulgaris AS 1.1208) to transform the substrate.

RESULTSThree products were obtained and identified as 10-deacetylsinenxan A1, 6 alpha-hydroxy-10-deacetylsinenxan A2 and 9 alpha-hydroxy-10-deacetylsinenxan A3 respectively.

CONCLUSIONSinenxan A is facile to be transformed by microorganisms, the 10-acetyl group of which is an active group.

Acetates ; isolation & purification ; metabolism ; Biotransformation ; Culture Techniques ; Cunninghamella ; metabolism ; Diterpenes ; isolation & purification ; metabolism ; Mucor ; metabolism ; Plants, Medicinal ; chemistry ; Proteus vulgaris ; metabolism ; Taxus ; chemistry

BACKGROUNDDespite 100 years of research, the continued absence of well-established risk factors impedes the diagnosis and treatment of interstitial cystitis/painful bladder syndrome (IC/PBS). We aimed to identify risk factors in patients with lower urinary tract symptoms (LUTS) without urinary tract infection or benign prostate hyperplasia in China.

METHODSA total of 397 outpatients with LUTS presenting for care to urology clinics in several hospitals throughout China were surveyed using a standardized questionnaire and validated outcome measures. The definitions for painful bladder syndrome based on the O'Leary-Sant interstitial cystitis symptom and problem indices were used. The prevalence of possible risk factors was analyzed using the Fisher's exact test and Pearson chi-square test, and multivariate predictive models were developed using binary Logistic regression methods.

RESULTSOf those multi-centre patients surveyed, including 174 women and 223 men, 41% (162/397) met criteria for painful bladder syndrome. There was a significant difference between women and men (55% (95/174) vs. 30% (67/223), P < 0.001). Women with IC/PBS were more likely than those without IC/PBS to report a history of gynecological infections (odds ratio (OR): 2.85; 95% confidence interval (CI): 1.32 - 6.16, P = 0.007), intake of stimulatory foods (OR: 3.52; 95%CI: 1.50 - 8.30; P = 0.004), irritable bowel (OR: 3.46; 95%CI: 1.22 - 9.80; P = 0.014) and/or anorectal disease (OR: 2.68; 95%CI: 1.12 - 6.40, P = 0.023). After adjusting for confounding factors, bladder pain was significantly associated with stimulatory foods (OR: 3.85; 95%CI: 1.58 - 9.36, P = 0.003) and anorectal disease (OR: 2.76; 95%CI: 1.09 - 7.04, P = 0.03) in women. Caffeine beverage intake (OR: 3.54; 95%CI: 1.54 - 8.12, P = 0.003) was identified the only modifiable association noted in multivariate analysis of men.

CONCLUSIONSWe found that stimulatory foods, anorectal disease and caffeine beverages are potential risk factors for IC/PBS. Further studies are necessary to determine their role in the pathogenesis of this disorder.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; China ; Cystitis, Interstitial ; epidemiology ; etiology ; Female ; Humans ; Male ; Middle Aged ; Multivariate Analysis ; Prevalence ; Prostatic Hyperplasia ; complications ; Risk Factors ; Sex Characteristics ; Surveys and Questionnaires ; Urination Disorders ; complications

OBJECTIVETo explore the categories of drugs causing hepatotoxicity and analyze the clinical and histological features of the corresponding drug-induced liver injury (DILI), in order to gain insights into potential diagnostic factors for DILI.

METHODSA total of 138 DILI patients treated at our hospital from April 2008 to April 2010 were retrospectively analyzed. The responsible drug for each DILI case was recorded. The Roussel Uclaf Causality Assessment Method (RUCAM) had been used to diagnose DILI. Only cases that had scored as highly probable or probable (more than or equal to 6 points by RUCAM) were included in this study. The patients' general condition, clinical manifestations, and serum biochemical and immunological parameters were assessed. Sixty-six of the patients underwent liver biopsy, and were assessed for liver pathological changes. Clinical and laboratory test data were collected and used to classify the total 138 cases as hepatocellular injury, cholestatic, or mixed hepatocellular-cholestatic types.

RESULTSWithin our patient population, the leading cause of DILI was Chinese herb medicine, accounting for 53.62% of cases. Antibiotics were implicated in 7.97% of cases, and dietary supplement in 6.52% of cases. Correlation between the clinical features and histological injury pattern was stronger at the time of biopsy (more than or equal to 3 days after laboratory results) (kappa = 0.63, P less than 0.05) than at the onset of DILI (kappa = 0.25, P less than 0.05). All modified hepatic activity index (HAI) necroinflammatory scores and fibrosis scores were more severe in the cholestatic and mixed injury types than in the hepatocellular injury type (P less than 0.01 and P less than 0.05, respectively).

CONCLUSIONChinese herbal medicine, dietary supplements and antibiotics were the main causes of DILI in our patient population. The clinical and histological features correlated well, especially at later stages of DILI. The degree of inflammation and fibrosis was significantly higher in cholestatic and mixed hepatocellular-cholestatic injury types than in the hepatocellular injury type. Assessment of both clinical and pathological features may represent a more accurate diagnostic method for DILI.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Anti-Bacterial Agents ; adverse effects ; Anti-Infective Agents ; adverse effects ; Chemical and Drug Induced Liver Injury ; pathology ; Drugs, Chinese Herbal ; adverse effects ; Female ; Humans ; Liver ; pathology ; Male ; Middle Aged ; Young Adult

OBJECTIVETo study the protective effects of naja naja atra venom (NNAV) in a rat model of diabetic nephropathy (DN).

METHODSThe rat diabetes model was induced by intraperitoneal injection of streptozotocin (STZ). Thirty-two model rats were randomly divided into one DN group (n=8) and three treatment groups (n=8 each) that received NNAV at doses of 30, 90, or 270 μg/(kg·day) via oral gavage, another eight rats as normal controls. After 12 weeks, all rats were sacrificed and the changes in serum and urine biological index levels were determined by colorimetric assay. Microalbumin (mALB), N-acetyl-β- glucosaminidase (NAG) and cystatin C (CysC) concentrations were measured by ELISA. Renal tissues were sliced for pathological and immunohistochemical observations.

RESULTSComparied with the DN group, serum glucose was decreased by 31.04%, total cholesterol 21.96%, triglyceride 23.78%, serum creatinine 19.83%, blood urea nitrogen 31.28%, urinary protein excretion 45.42%, mALB 10.42%, NAG 20.65%, CysC 19.57%, whereas albumin increased by 5.55%, high-density lipoprotein-cholesterol 59.09%, creatinine clearance 19.05% in the treatment group by NNAV administration at dose of 90 μg/(kg·day). NNAV also reduced the levels of malondialdehyde in serum (22.56%) and kidney tissue (9.79%), and increased superoxide dismutase concentration in serum (15%) and decreased it in renal tissue (8.85%). In addition, under light microscopy kidney structure was improved and glomerular hypertrophy decreased by 8.29%. As shown by immunohistochemistry, NNAV inhibited transforming growth factor-β1 by 6.70% and nuclear actor-κB by 5.15%.

CONCLUSIONNNAV improves biological indexes in DN, and it may exert renoprotective effects in rats with STZ-induced diabetes.

Animals ; Body Weight ; Diabetes Mellitus, Experimental ; complications ; Diabetic Nephropathies ; drug therapy ; pathology ; Dose-Response Relationship, Drug ; Elapid Venoms ; administration & dosage ; pharmacology ; Elapidae ; physiology ; Kidney ; drug effects ; pathology ; Male ; Malondialdehyde ; Organ Size ; Rats ; Rats, Wistar ; Superoxide Dismutase

Animals ; Antidepressive Agents ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Hindlimb Suspension ; Male ; Medicine, Chinese Traditional ; Mice ; Swimming

Glutathione transferases (GSTs) are a family of multifunctional proteins that mainly catalyze the conjugation of intracellular glutathione (GSH) to a wide variety of endogenous and exogenous electrophilic compounds. GSTs play important roles in stress tolerance and in the detoxification metabolism in organisms. A novel GST gene, Pc gstB, was cloned from penicillin producing fungus Penicillium chrysogenum using RT-PCR. The open reading frame (ORF) of Pc gstB was 651 bp and encoded a peptide of 216 residues. The deduced amino acids sequence had conserved GST domain and showed 65% identity to the characterized Aspergillus fumigutus gstB. The entire ORF of Pc gstB was inserted into vector pTrc99A and transformed into Escherichia coli DH5alpha. Recombinant PcGstB was overexpressed and its GST activity toward substrate 1-chloro-2,4-dinitrobenzene (CDNB) was validated.
Catalysis ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fungal Proteins ; genetics ; metabolism ; Genes, Bacterial ; genetics ; Glutathione Transferase ; genetics ; metabolism ; Open Reading Frames ; Penicillium chrysogenum ; enzymology ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, Protein

OBJECTIVETo discuss the expression and significance of caspase-3 gene in the apoptotic muscle cells in gamma-radiation-induced muscle cell lines.

METHODSThe caspase-3 mRNA in the control and gamma-radiation induced apoptotic muscle cells was analysed by RT-PCR.

RESULTSThe expression of caspase-3 gene transcript was higher in 103Pd radioactive stent dog bile duct than in general stent dog bile duct, and apoptotic muscle cells were higher in 103Pd radioactive stent dog bile duct than in general stent dog bile duct.

CONCLUSIONSThe high level expression of caspase-3 gene may help to understand the muscle cells sensitivity to gamma-radiation apoptosis. 103Pd radioactive stent may increase the expression of caspase-3 gene in dog bile duct and prevent the billiary narrow when dog bile duct was injured by balloon.

Animals ; Apoptosis ; radiation effects ; Apoptosis Regulatory Proteins ; Bile Ducts ; enzymology ; radiation effects ; Caspase 3 ; Caspases ; genetics ; radiation effects ; Dogs ; Myocytes, Smooth Muscle ; cytology ; radiation effects ; Palladium ; administration & dosage ; RNA, Messenger ; genetics ; radiation effects ; Radioisotopes ; administration & dosage ; Reverse Transcriptase Polymerase Chain Reaction ; Stents