NAC transcription factors involved in plant growth and development, as well as responses to biotic and abiotic stress. RNAi Vectors for SmNAC transcription factors of Salvia miltiorrhiza was constructed by using Gateway cloning technology, in order to further study the function of SmNAC1 transcription factor. According to Gateway cloning technology, the specific fragments of SmNAC1 containing attB adapter was amplified by PCR using ultra-fideling phusion polymerase of NEB. By the BP recombination reaction, the PCR product containing attB was transferred to an donor vector (pENTR/SD/D-TOPO). Finally, SmNACi specific gene was cloned into pK7GWIWG2D plant expression vectors by LR recombination reaction. Experimental results showed that Gateway cloning technology provide a rapid and highly efficient way to clone the interested gene.
Cloning, Molecular ; methods ; Genetic Vectors ; genetics ; Plant Proteins ; genetics ; Polymerase Chain Reaction ; RNA Interference ; Reproducibility of Results ; Salvia miltiorrhiza ; genetics ; Transcription Factors ; genetics

According to the designed specific primers of gene fragment based on the Salvia miltiorrhiza transcriptome data, a full-length cDNA sequence of SQS2 from S. miltiorrhiza f. alba was cloned by the method of reverse transcription polymerase chain reaction (RT-PCR). The SmSQS2 cDNA sequence was obtained, this sequence is named SmSQS2 and its GenBank registration number is KM244731. The full length of SmSQS2 cDNA was 1245 bp, encoding 414 amino acids including 5'UTR 115 bp and 3'UTR 237 bp. Sequence alignment and phylogenetic analysis demonstrated that SmSQS2 had relative close relationship to the SQS2 of S. miltiorrhiza. The induction of E. coli [pET28-SQS2] in different temperature, induction time, IPTG concentrations and density of inducing host bacterium (A600) were performed, Shaking the culture at 30 degrees C until the A600 is approximately 0.6 and add IPTG to final concentration of 0.2 mmol x L(-1), and then the optimal expression of SmSQS2 recombinant protein were accumulated after the induction time of 20 h. The research provided important base for the study of sterol and terpene biosynthesis of SQS2 in S. miltiorrhiza f. alba.
Cloning, Molecular ; Farnesyl-Diphosphate Farnesyltransferase ; chemistry ; genetics ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Salvia miltiorrhiza ; chemistry ; classification ; enzymology ; genetics ; Sequence Alignment

Purpose To explore the immunohistochemical features of neuroendocrine markers, CKpan and TTF-1 and their relationship to TNM stage and prognosis in small cell lung cancer (SCLC). Methods The expression of NSE, CgA, Syn, CD56, TTF-1 and CK-pan in SCLC tissue specimens were detected using immunohistochemical EnVision indirect method. Clinical data and TNM stage of 90 patients were collected and the overall survival ( OS) was followed up by telephone. Results Of 90 cases of SCLC, the vast majority were occured in the elderly men. The ratio of man to woman was 5 to 1. The median age was 64 years old. The stageⅠ+Ⅱ was 21 cases, 30 in stageⅢand 39 cases in stage IV. The positive rate of immunohistochemical staining of neuroendocrine markers for NSE, CgA, Syn and CD56 were 83. 3%, 70%, 65. 5% and 86%, respectively. The positive rate of CKpan, TTF-1 were 92. 2% and 81. 1%. Kaplan-Meier analysis indicated that the expression of TTF-1 and NSE were significantly correlated with the TNM stage and over-all survival of patients with SCLC (P<0. 05). The median OS was 8 months in positive expression of TTF-1, which was higher than those in negative expression of TTF1 (5. 5 months)(P=0. 000). The median OS was 7 months in NSE positive expression which was lower than those in negative expression of NSE (11 months)(P=0. 009). The median OS of stageⅠ+Ⅱ,ⅢandⅣwere 16 months, 9 months and 4 months with significant difference ( P=0. 000 ) . Cox multivariate analysis indicated the TTF-1 expression and TNM were independent prognostic factors for the OS of the SCLC patients. Conclusion Most of SCLC has neuroendocrine differentiation, expression CKpan and TTF-1. The expression of TTF-1 may be negative correlation but NSE positive correlation with the prognosis of SCLC patients. And the TTF-1 expression and TNM may be independent prognostic factors for the OS of the SCLC patients.

OBJECTIVETo investigate the nephrotoxic effects of methyl cantharidimide tablets on urinary protein and enzymes in Beagle dogs.

METHODBeagle dogs were randomly divided into negative control group(blank tablet), methyl cantharidimide tablets group (6.11,12.21, 24.42 mg x kg(-1)), continuously 30 days of oral adminiStration, once a day. The drug and control group were collected and determined fresh urine in 1, 2, 3 and 4 weeks of the administration; Serum urea nitrogen (BUN), creatinine (Crea), total protein (TP) and albumin (ALB) as well as sodium, potassium, chloride electrolyte were determined on 15 and 30 days of the administration; Urine albumin (mAlb), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin( NGAL), N-acetyl-beta-D-glucosaminidase (NAG), clusterin, beta2-microglobulin (beta2-MG), alpha1-microglobulin (alpha1-MG), alanine aminopeptidase( AAP) and im- munoglobulins IgG were tested on 15 and 30 days of the administration.

RESULTCompared with the control group, urine protein and white blood cells was significantly increased in each dose group. On 15 days of the administration, mAlb were higher in each dose group, KIM-1, NGAL, clusterin, NAG and AAP were significantly higher in high-dose group, while the middle and low dose group had no significant difference, as well as blood SCr and BUN no obvious abnormalities. On 30 days, mAlb, KIM-1, clusterin, NAG, AAP were increased in each dose group, appearing dose-effect relationship, beta2-MG and NGAL levels were significantly increased in high-dose group. Contents above indicators were increased with significant dose and time relationship, and serum BUN, Scr were correlated, suggesting that urine mAlb, KIM-1, clusterin, NAG and AAP indicators that can sensitively respond the changes of proteins and enzymes in urine.

CONCLUSIONMethyl cantharidimide tablets has a renal toxicity, urine mAlb, KIM-1, clusterin, NAG and AAP can be used as the early nephrotoxic biomarkers of methyl cantharidimide tablets.

Animals ; Biomarkers ; urine ; Dogs ; Female ; Kidney ; drug effects ; Kidney Diseases ; chemically induced ; Male ; Proteins ; metabolism ; Tablets ; adverse effects ; Urine ; chemistry

Objective To evaluate the knowledge,attitude and practice(KAP)related with AIDS of local floating population.Methods A total of 1 942 floating persons were sampled by multistage cluster sampling and interviewed with questionnaire in the agricultural markets,construction sites,and their habitats within 3 communities in Hongkou District.Results 82.9% of the interviewees were 20 ～49 years old.The average score was 34.77 ±3.52 (maximum score is 44)for knowledge,12.11 ±2.32(maximum score is 19)for attitude,5.50 ±0.95(maximum score is 7)for practice,respectively.The scores increased with educational level significantly.The scores of knowledge were 33.26 ±3.54,34.63 ±3.23,36.56 ±3.39 among the subjects with educational levels of primary school and below,junior high school,senior high school and above.The scores of attitude were 13.77 ±2.27,14.79 ±2.39,15.62 ±2.38,respectively.And the score of practice was 5.40 ±0.90,5.51 ±0.93,5.58 ±1.03,respectively.Conclusion In the present,the KAP relating AIDS of local floating population is poor in Hongkou District.A variety of intervention methods and operational patterns should be developed for the floating population with different educational levels and jobs.

According to the designed specific primers of gene fragment based on the Salvia miltiorrhiza transcriptome data, with the method of reverse transcription polymerase chain reaction (RT-PCR), this study cloned full-length cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase gene from Salvia miltiorrhiza bge.f.alba, this sequence is named as SmHDS and its GenBank registration number is KJ746807. SmHDS, 2 529 bp long, contains an ORF of 2 229 bp, encodes 742 amino acids, including 5' UTR 170 bp and 3' UTR 130 bp. Using bioinformatics software, having made a homology analysis of the obtained sequence, we can have a conclusion that SmHDS have a close genetic relationship with HDS of Salvia miltiorrhiza. Analysis result of prokaryotic expression revealed that in Escherichia coli, SmHDS expressed target proteins which in size are comparable with the protein predicted. Meanwhile, the 4 factors which can influence the protein expression were optimized, the 4 factors are inducing temperature, inducing time, IPTG concentrations and density of inducing host bacterium (A600). The optimal expression conditions of SmHDS were 30 degrees C until the A600 is 0.6, and add IPTG to a final concentration of 0.2 mmol x L(-1), and the induction time of 20 h. It provides theoretical basis for the further study of the function of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase in the biosynthesis of tanshinone compounds.
Cloning, Molecular ; DNA, Complementary ; genetics ; Diterpenes, Abietane ; biosynthesis ; Enzymes ; biosynthesis ; genetics ; Escherichia coli ; metabolism ; Plant Proteins ; biosynthesis ; genetics ; Salvia miltiorrhiza ; enzymology ; genetics

Objective To determine the effect of mitochondria on the protection of heat-shock proteins B1(HSPB1) from oxidative damage in rat cardiac cell.Methods HSPB1 gene-transfected rat cardiomyocytes cell line H9c2 (HSPB1 H9c2) and empty vector transfected H9c2 (control) were established,and treated by 0-1000?mol/L H_2O_2 for 2h.And then the cell morphology, mitochondrial membrane potential and endogenous reactive oxygen species (ROS) were detected. Results (1)HSPB1 inhibited the morphological changes induced by H_2O_2 markedly.(2)HSPB1 inhibited the loss of mitochondrial membrane potential induced by H_2O_2.Following the stimulation of 0,75,150,300,500,1000?mol/L H_2O_2,mitochondrial membrane potential in HSPB1 and control H9c2 cells were (10.0?0.11)vs (7.01?0.26),(9.11?0.17)vs (6.05?0.19),(7.69?0.28)vs (5.14?0.28),(6.95?0.13)vs (4.66?0.11),(6.61?0.20)vs (1.85?0.35),(6.60?0.05)vs (1.19?0.01),respectively (all P0.05).Conclusions HSPB1 protects rat cardiomyocytes cell line(H9c2) from oxidative damage,which suggests that stabilization of mitochondrial membrane potential and the decreased endogenous reactive oxygen species after oxidative stress may be involved in the protection of HSPB1 against oxidative stress in H9c2.

Objective To observe apelin and its receptor (APJ) expressions in human osteoblasts and evaluate the effect of apelin on osteoblasts.Methods The expressions of apelin and APJ in human osteoblasts were tested by RT-PCR and Western blot.After human osteoblasts were treated with apelin,cell proliferation was measured by [~3H] thymidine incorporation and cell counting.Cell function was measured by alkaline phosphatase (ALP) activity,the secreted osteocalcin level and typeⅠcollagen production .The activation of signaling cascades was tested by Western blot.Small-interfering RNA (siRNA) to blockade APJ was applied to observe effects of apelin on cell proliferation and the activation of signaling cascades.Results Both apelin and APJ were expressed in human osteoblasts.Apelin increased the proliferation and did not show the influences on ALP activity, osteocalcin secretion and type I collagen production in human osteoblasts.Apelin induced activation of phosphatidylinositol-3 kinase (PI3K) downstream effector (Akt),but not mitogen-activated protein kinase (MAPK) such as c-jun N-terminal kinase (JNK),p38 and ERK1/2 in human osteoblasts.Suppression of APJ with siRNA or LY294002 (PI3K inhibitor) abolished the apelin-induced cell proliferation and the activation of Akt.Conclusion Human osteoblasts express apelin and APJ.Apelin stimulates the proliferation of human osteoblast via APJ/PI3K/Akt pathway,but has no effect on osteoblast differentiation.

OBJECTIVETo explore the relationship between body mass index, waist circumference and blood pressure among residents in Chongqing area.

METHODSA total of 5246 residents aged 15 and over in Chongqing area were enrolled in this study by use of stratified sampling and cluster sampling methods. Data on blood pressure (SBP, DBP), pulse, height, body weight, waist and hip circumferences as well as questionnaire survey were analyzed.

RESULTSThe level of SBP and DBP and hypertension prevalence rate were significantly positively correlated with BMI (all P < 0.01). SBP, DBP levels and hypertension prevalence rate were significantly higher in people with abdomen obesity than people with normal waist circumference (all P < 0.01). BMI, waist circumference in hypertensive residents were significantly higher than non-hypertensive residents (all P < 0.01).

CONCLUSIONBlood pressure level and hypertension prevalence rate were closely related with BMI and waist circumference among residents in Chongqing area.

Adolescent ; Adult ; Aged ; Blood Pressure ; Body Mass Index ; China ; epidemiology ; Female ; Heart Rate ; Humans ; Hypertension ; epidemiology ; physiopathology ; Male ; Middle Aged ; Prevalence ; Risk Factors ; Rural Population ; Urban Population ; Waist Circumference ; Waist-Hip Ratio ; Young Adult

Case-Control Studies ; Creatine ; urine ; Female ; Humans ; Hypoxia-Ischemia, Brain ; diagnosis ; urine ; Infant, Newborn ; Lactic Acid ; urine ; Male ; Predictive Value of Tests