Objective To observe the development of hematopoietic stem cell and the apoptosis of ICM(intermediate cell mass) in folic acid deficient zebrafish embryos and investigate the mechanism by which folic acid deficiency induces abnormal hematopoiesis.Method The folic acid deficient zebrafish model was induced by both using the dihydrofolate reductase antagonism methotrexate(MTX) and knock-down dihydrofolate reductase gene.The development of embryos was observed under microscope.The blood cells were detected by O-dianisidine staining.Whole-mount in situ hybridization and real-time PCR were performed to examine the expression of FLK-1,GATA1and GATA2.Apoptosis in intermediate cell mass(ICM) was examined by TUNEL(terminal-deoxynucleotidyl transferase mediated nick end labeling) method.Results The abnormal developments of ICMs were observed both in MTX treated embryos and DHFR knock-down embryos.O-dianisidine staining revealed that folic acid deficiency resulted in the decreasing number of blood cells.In folic acid deficient embryos,the expression of FLK-1、GATA1and GATA2 was reduced and the apoptosis in ICMs was increased.Conclusion The abnormal hematopoiesis in zebrafish induced by folic acid deficiency is related with the increasing apoptosis in ICMs and decreasing expressions of FLK-1,GATA1and GATA2.

An investigation on the chemical constituents of the 90% EtOH extract of Perovskia atriplicifolia led to the isolation of fifteen compounds from the EtOAc fraction. Based on the detailed spectral analysis (MS, 1D and 2D NMR), as well as comparison with the literatures, the structures of compounds 1-15 were determined as cirsimaritin (1), salvigenin (2), syringaldehyde (3), vinyl caffeate (4), 2α, 3α-dihydroxyolean-12-en-28-oicacid (5), 2α, 3α-dihydroxyurs-12-en-28-oicacid (6), niga-ichigoside F1 (2α, 3β, 19α, 23- tetrahydroxyurs - 12-en-28-oicacid- O-β-D- glucopyranoside, 7), sericoside (8), 4-epi-niga-ichigoside F1 (2α, 3β, 19α, 24-tetrahydroxyurs-12-en-28-oicacid O-β-D-glucopyranoside, 9), 2α, 3β, 24-trihydroxyolean-12-en-28-oicacid O-β-D-glucopyranosyl-(1 --> 2) - β-D-glucopyranoside (10), pruvuloside A (11), asteryunnanoside A [2α, 3β, 23-trihydroxyolean-12-en-28-oicacid O-β-D-glucopyranosyl-(1 --> 2)-β- D- glucopyranoside,12], rosmarinic acid methyl ester (13), β-sitosterol (14), and daucosterol (15), respectively. Compounds 1-13 were isolated from the Perovskia genus for the first time. All the compounds were obtained from P. atriplicifolia for the first time.
Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Lamiaceae ; chemistry ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization

Objective The aim of this phase Ⅱ study was to determine the efficacy and safety of combined bortezomib and thalidomide (VT) regime as initial treatment for newly diagnosed multiple myeloma (MM) in China. Methods Thirty-four patients were enrolled in this study and received VT regime up to 21-day cycles. Bortezomib (1.3 mg/m2) was administered intravenously on days 1, 4, 8, and 11, while oral thalidomide ( 100 mg/day) was given from days 1 to 21. The primary end point was clinical response.The secondary end point was safety. Results Among the 34 patients, 20 were male, 14 were female, with a median age of 59 years, and 15 in international stage system (ISS) Ⅲ ,18 in ISS Ⅱ , 1 in ISS Ⅰ . Among them, 28 completed 2 cycles' treatment and achieved an overall response rate (ORR) of 92.9%; 26 were able to complete the planned 8 cycles of therapy. After 8 cycles, the ORR was 100% ( complete response 30. 8%, near-complete response 23.1%, partial response 42. 3%, minimal response 3.8% ). After followed up with a median time of 12 months, the estimated rate without progress of disease was 62%, and the estimated continous remission rate of 12 months was 62%. The median survival time was not achieved. The most common adverse events were mild to moderate ( grades 1, 2). The main toxicities were hematologic (53. 3% ), gastrointestinal ( 40. 0% ), peripheral neuropathy ( 38.0% ), fatigue ( 36. 6% ) and fever (32. 0% ). Conclusions VT regime provides a very high ORR and complete response rate in the treatment of newly diagnosed MM patients. No patients experienced deep venous thrombosis. In conclusion,bortezomib in combination with thalidomide is a very effective regimen for newly diagnosed MM patients and the toxicities are manageable.

Objective: To investigate the efifcacy of supplemental walking training in patients with coronary artery disease (CAD) combining heart failure (HF) under routine medication.
Methods: A total of 80 patients with CAD combining HF were randomly divided into 2 groups: Exercise group,n=40 including 25 male and 15 female at (61.2 ± 9.8) years of age, the patients received additional six-minute walking exercise training based on routine medication and Control group,n=40 including 26 male and 14 female at (58.1 ± 10.9) years of age, the patients received routine medication. All patients were treated for 3 months. Plasma levels of BNP and general conditions at before and after the treatment were compared between 2 groups.
Results:①After 3 months treatment, both groups had signiifcantly improved LVESD, LVEDD, LVEF, plasma levels of BNP and six-minute walk test (except LVESD and LVEDD in Control group), allP<0.05.②Compared with Control group, Exercise group showed obviously improved LVEF, plasma level of BNP and six-minute walk test, allP<0.05.
Conclusion: Walking training may increase the exercise tolerance, which is beneifcial to recover the cardiac function in patients with CAD combining HF in addition to routine medication .

Objective To examine the expressions of FHIT gene and HER_2 in non-small cell lung cancer(NSCLC)and the relationship between the expressions and the clinicopathological features of NSCLC, and to investigate the relationship between FHIT protein and HER_2 expression. Methods FHIT protein and HER_2 expressions in 50 lung cancer cases and 21 adjacent non-cancer tissues were performed by immunohistochemistry. And the positive rates of FHIT and HER_2 were measured. Results The positive rates of FHIT protein were 30.00% in lung cancer tissues. The expression levels of FHIT were significantly lower in lung cancer tissues than that in non-cancer lung tissues (P

In order to provide effective tool for further studying of the function of HCMV genome, developing of molecular vaccine and diagnostic reagents. Extraction of HCMV mRNA from HF cell infected by HCMV AD169 strain for 96h was reverse transcripted into cDNA, then was cloned into EcoR I-digested lambda gt11. HCMV AD169 strain cDNA expressing library has been constructed after packaging. The volume and the recombination rate of the prime cDNA expressing libraries was 3.6?10 6 and 96%, 168 positive clones of HCMV were screened by immune blotting with anti-HCMV mouse convalescent sera, 34 positive clones were obtained by dot nucleic acid hybridization with DIG-labled HCMV pp65 gene probe. 2 positive clones were amplified by HCMV pp65 all length primer. The PCR product has been tested by southern-blotting.The PCR product was sequenced and was taken as homology comparison by DNASIS software,and the homology is 98%.To lay the foundation of furher cloning,expressing the pp65 gene,further studying of the function of the pp65 prodct.

Objective To investigate the expression of Akt and phosphoryled Akt (p-Akt1) protein in pancreatic carcinoma and to determine the clinical significance. Methods In 74 cases of pancreatic carcinoma and 10 cases of normal pancreatic tissue samples, the expression of Akt and p-Akt1 were detected by immunohistochemical method, and the its relationship with clinicopathologic characteristics and prognosis were analyzed. Results The positive expression rate of Akt and p-Akt1 in pancreatic carcinoma were 87.8% and 83.8% respectively, while there was no expression of Akt and p-Akt1 in normal pancreatic tissue, and the difference was statistically significant (p < 0.05). There was a positive correlation between the expression of Akt and p-Akt1 in pancreatic carcinoma (r =0.274, P =0. 018). The expression of Akt and p-Akt1 was not significantly associated with the age, sex, location, size, pathology stages, lymph nodes metastasis, clinical stages and nerve invasion of the tumor (P >0.05). But the higher expression of p-Akt1 was associated with T stages and TNM staging (p =0. 002). Patients with high intensity of Akt and p-Akt1 expression showed a significantly longer median survival time [(16.0 ± 5.7) month and (23.0 ± 5.5) month, respectively]than those with low intensity expression [(9.3 ± 0.2) month and (11.1 ± 1.8) month (P = 0. 007 and P = 0.004) respectively]. Conclusions p-Akt1 expression is a significant positive prognostic factor for pancreaticcarcinoma and detection of p-Akt1 expression may be of clinical value.

OBJECTIVETo establish ELISAs based on rLipL32/1-LipL21-OmpL1/2 fusion antigen of Leptospira interrogans for detecting specific IgG and IgM in serum of patients with leptospirosis.

METHODSMicroscope agglutination test(MAT) was performed to detect serum specimens from leptospirosis patients and to determine titers of rabbbit antiserum agaist rLipL32/1-LipL21-OmpL1/2 to reference standard strains of L. interrogans. By using rLipL32/1-LipL21-OmpL1/2, rLipL32/1, rLipL21 and rOmpL1/2 as the coated antigens, ELISAs for detecting specific serum IgM and IgG were established. The established ELISAs were applied to MAT-positive serum specimens from 107 patients with leptospirosis.

RESULTThe results of MAT confirmed that 66% (71/107) of the patients were infected with L.interrogans serogroup Icterohaemorrhagiae, and the rLipL32/1-LipL21-OmpL1/2 antiserum were able to agglutinate all 15 reference standard L.interrogans strains with 1 : 20approximate, equals1 : 160 titers. The positive rates of ELISAs using rLipL32/1-LipL21-OmpL1/2, rLipL32/1, rLipL21 or rOmpL1/2 as the antigen were 89.7%, 75.7%, 85.1% and 79.4% for detecting IgM, respectively, while 99.1%, 99.1%, 94.4% and 86.0% for detecting IgG, respectively. The positive detection rate of rLipL32/1-LipL21-OmpL1/2-IgM-ELISA was higher than those of the other three IgM detection ELISAs (P<0.05). The positive detection rate of rLipL32/1-LipL21-OmpL1/2-IgG-ELISA was higher than that of rOmpL1/2-IgG-ELISA (P<0.05), while there was no significant differnce with that of rLipL21-IgG-ELISA and rLipL32/1-IgG-ELISA (P>0.05).

CONCLUSIONThe ELISAs using rLipL32/1-LipL21-OmpL1/2 as the antigen can be applied as a sensitive,specific and universal serological method for diagnosis of leptospirosis.rLipL32/1-LipL21-OmpL1/2-IgM-ELISA shows a definite value for early diagnosis of leptospirosis compared with the other ELISAs used in this study.

Agglutination Tests ; Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; genetics ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Leptospira interrogans ; genetics ; immunology ; Leptospirosis ; diagnosis ; immunology ; Lipoproteins ; genetics ; metabolism ; Rabbits ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Sensitivity and Specificity ; Serologic Tests ; methods

Objective To study the calcium metabolism in tacrolimus(FK506)induced rats nephrotoxicity and the preventive effect of calcium channel blocker.Methods Twenty-four Spragueinduced or FK506-induced nephropathy model.Blood creatinine,blood electrolytes,renal tissue histopathology(HE stain)and the change of ultrastructural organization in renal cells by transmission electron microscope were observed.Results The blood creatinine levels of both CsA and FK506 groups [(36.00±2.61)and(34.17±4.54)μmol/L] were significantly higher than those of the FK506+Dilgroup and control group(all P＜0.05).The blood calcium levels of both CsA and FK506 groups (2.00±0.04 and 2.05±0.04 mmol/L) were significantly lower than those of the FK506+Dil group and control group(all P＜0.05).The blood creatinine and calcium levels of FK506+Dil group were not significantly different with those of control group(P＞O.05).Histopathology examination showed cloudy swelling and vacuolization of the renal tubular epithelial cells and intra-cellular mitochondria swelling and vacuolization in the CsA and FK506 groups.However,the pathological changes of the FK506+Dil group were remarkably milder in comparison with the CsA and FK506 groups.Concluum channel blocker,Dil,could prevent the FK506-induced nephrotoxicity.

OBJECTIVETo study the effect of methotrexate (MTX), a folic acid antagonist which can lead to folic acid deficient, on the cardiac development and on the expressions of BMP2b and HAS2 in zebrafish.

METHODSThe zebrafish embryos at 6-48 hrs post fertilization (hpf) were treated with various concentrations of MTX (0.5 x 10(-3), 1.0 x 10(-3) and 2.0 x 10(-3) M). At 48 hpf, the percentage of cardiac malformation and heart rate were recorded. The zebrafish embryos at 6-10 hpf treated with 1.5 x 10(-3) M MTX were used as the MTX treatment group. At 24 and 48 hpf the cardiac morphology was observed under a microscope. The expressions of BMP2b and HAS2 in zebrafish were detected by in situ antisense RNA hybridization and real-time PCR.

RESULTS6-12 hpf, the early embryonic developmental stage, was a sensitive period that MTX affected cardiac formation of zebrafish. The retardant cardiac development and the evidently abnormal cardiac morphology was found in the MTX treatment group. The results of in situ antisense RNA hybridization showed that the expressions of BMP2b and HAS2 in the zebrafish heart were reduced in the MTX treatment group at 36 and 48 hpf. The real-time PCR results demonstrated that the BMP2b expression decreased at 12, 24, 36 and 48 hpf, and that the HAS2 expression decreased at 24, 36 and 48 hpf in the treatment group compared with the control group without MTX treatment.

CONCLUSIONSThe inhibition of folic acid function may affect cardiac development of early embryos, resulting in a retardant development and a morphological abnormality of the heart in zebrafish, possibly by down-regulating the expressions of BMP2b and HAS2.

Abnormalities, Drug-Induced ; etiology ; Animals ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; genetics ; Down-Regulation ; Folic Acid Antagonists ; toxicity ; Gene Expression Regulation ; drug effects ; Glucuronosyltransferase ; genetics ; Heart Defects, Congenital ; chemically induced ; Hyaluronan Synthases ; Methotrexate ; toxicity ; Polymerase Chain Reaction ; Zebrafish ; Zebrafish Proteins ; genetics