The sfa1 gene encoded a bifunctional enzyme with the activities of both alcohol dehydrogenase and glutathione-dependent formaldehyde dehydrogenase in Saccharomyces cerevisiae.The gene disruption cassette produced by PCR using the same long oligonucleotides which comprise 19 or 22 nucleotides complementary to sequences in the templates(pUG6 and pUG66 marker plasmid)at 3' end and 45 nucleotides at 5' end that annealed to sites upstream or downstream of the genomic target sequence to be deleted.After two linear disruption cassettes with a Cre/loxP mediated marker were transformed into the cells of Saccharomyces cerevisiae YS-1,the positive transformants were checked by PCR to correct the integration of the cassette and concurrent deletion of the chromosomal target sequence.Once correctly integrated into the genome,the select marker can be efficiently rescued by transformating the plasmid pSH47 into YS-1 and inducing the Cre expression with a Cre/loxP-mediated marker removal procedure.The expression of the Cre recombinase finally resulted in the removal of the marker gene,leaving behind a single loxP site at the chromosomal locus.The diploid mutant YS-1-sfa1 was generated,which could enhance the output of ethanol with 8.0% by shaking culture in flask compared with the original strain YS-1.

OBJECTIVETo explore evolution rules of phlegm and blood stasis syndrome ( PBSS) in hyperlipidemia and atherosclerosis (AS) using NMR-based metabolic profiling and metabonomic approaches based on formulas corresponding to syndrome.

METHODSTotally 150 SD rats were divided into the normal group, the model group, the Erchen Decoction (ED) group, the Xuefu Zhuyu Decoction (XZD) group, the Lipitor group, 30 in each group. The hyperlipidemia and AS rat model was duplicated by suturing carotid artery, injecting vitamin D3, and feeding with high fat diet. ED and XZD were used as drug probes. Blood samples were withdrawn at week 2, 4, and 8 after modeling. Blood lipids, blood rheology, histopathology and metabolomics were detected and analyzed. Results Results of blood lipids and pathology showed hyperlipidemia and early AS rat models were successfully established. At week 2 after modeling, levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) significantly increased, which reached the peak at week 4 and maintained at higher levels at week 8. ED exerted obvious effect in improving TC and LDL-C levels of early models, while XZD could greatly improve levels of TC and LDL-C of late models. Rheological results showed at week 2, there was no significant difference in whole blood viscosity, plasma viscosity, or hematocrit between the model group and the normal group (P > 0.05). At week 4 partial hemorheological indicators (such as plasma viscosity) were abnormal. Till week 8 whole blood viscosity, plasma viscosity, and hematocrit were significantly abnormal (P <0. 05, P < 0.01). As time went by, whole blood viscosity, plasma viscosity, and hematocrit showed gradual increasing tendency in the ED group, while they showed gradual decreasing tendency in the XZD group. Results of metabonomics showed significant difference in spectra of metabolites between the normal group and the model group. As modeling time was prolonged, contents of acetyl glucoprotein and glucose in the model group increased in late stage, which was in. line with results of blood lipids and hemorheology. ED showed more obvious effect in early and mid-term modeling (at week 2 and 4), and increased contents of partial metabolites (such as choline, phosphatidyl choline, glycerophosphocholine), but these changes in the XZD group were consistent with those of the model group. In late modeling (at week 8) XZD showed more obvious effect in improving contents of lactic acid, acetyl glycoprotein, LDL, creatine, choline, and glucose.

CONCLUSIONSED and XZD not only showed regulatory effects on lipid disorders, but also could improve dysbolism of Chos. In formulas corresponding to syndrome, damp-phlegm was main pathogenesis of hyperlipidema and AS in early and mid stages. Blood stasis syndrome began to occur along with it progressed. Phlegm can result in blood stasis and intermingles with stasis. Phlegm turbidity runs through the whole process.

Animals ; Atherosclerosis ; metabolism ; Cholesterol ; Cholesterol, LDL ; Drugs, Chinese Herbal ; therapeutic use ; Hemorheology ; Hyperlipidemias ; Lipids ; Magnetic Resonance Imaging ; Medicine, Chinese Traditional ; Metabolome ; physiology ; Metabolomics ; Rats ; Rats, Sprague-Dawley ; Sputum ; metabolism

The expressions of StAR (steroidogenic acute regulatory protein) mRNA in testes from 7,14,23 and 37 day-old piglets were studied by tissue in situ hybridization. The results indicated that in the testes of piglets, StARmRNA was expressed in Leydig cells of pig testes. The expression level of StARmRNA was lower in 7 days piglets but higher in 14,23,37 days. The results indicated that StAR gene played an important role in steroid biosynthesis.

Objective To examine the expression of toll-like receptor 2(TLR2)in peripheral blood monocytes and explore its association with disease stages and clinical manifestations and to explore the patho- genesis of rheumatoid arthritis(RA).Methods The expression of toll-like receptor 2 in peripheral blood monocytes from 47 RA patientis(27 in active stage and 20 in stable stage)and 18 normal individuals were de- tected by flowcytometry and RT-PCR.Results The expression of toll-like receptor 2 in peripheral blood monocytes in patients with active disease was significantly increased compared to non-active patients and nor- mal individuals,The expression was found to correlate with the Disease Active Score(DAS),serum C-reactive protein(CRP)level and the erythrocyte sedimentation rate(ESR),but not correlate with rheumatoid factor (RF)and the anti-cyclic citrullinated peptide(CCP)antibody.Conclusion The expression of toll-like recep- tor 2 in peripheral blood monocytes of patients with active RA is significantly increased.And the expression is correlated with disease activity index.The innate immune system is activated in patients with active disease. And the increased expression may promote the activities of monocytes.

Objective To demonstrate high mobility group box chromosomal protein 1(HMGBI) expression in synovium and joint,and to identify the role of HMGB1 in the pathogenesis of synovitis and joint destruction in adjuvant-induced arthritis(AA).Methods AA of 15 male rats were induced in SD rats by intradermal injection of 100?l Freud's complete adjuvant in the foot pad of the left hind paw.All rats were killed at the 18th day.Synovium and joints were collected for histopathology studies and determining the expression of HMGB1 by immunohistochemistry,and serum was collected for determining the expression of HMGB1 by western blotting analysis.Results Immunostaining of specimens from normal rats showed that HMGB1 was primarily confined to the nucleus of synoviocytes with occasional cytoplasmic staining.In contrast, inflammatory synovial tissues from AA rats showed a distinctly different HMGB1 staining pattern.Nuclear HMGBI expression was accompanied by a cytoplasmic staining in many mononuclear cells.The cytoplasmic HMGB1 expression in synovium of AA rats is significantly higher than that of normal rats.Additionally,HMGBI was highly expressed in the nuclei and cytoplasm of the subchondral chondrocytes and inflammatory cells in bone erosion in AA rats(P＜0.01),while fewer positive cytoplasmic staining of HMGB1 was found in chondrocytes and fewer positive nuclear staining was found in bone cells in normal rats.HMGB1 concentration was significantly higher in serum of AA rats than that in normal rats(P＜0.001).Conclusion The cytoplasmic HMGBI expression in synovium and joints is greatly upregulated;the level of HMGB1 in serum is increased in AA rats which suggests a patbogenetic role of HMGB1 in synovitis and bone destruction of adjuvant-induced arthritis.

Objective To establish a specific fluorescence quantitative method for determining the mRNA expression of Toll-like receptor3(TLR3)in peripheral blood mononuclear cells(PBMCs).Methods Using the Beacon Designer 2.1 software,specific primers and Taqman-MGB probe were designed.The plasmid pMD18-T-TLR3 was constructed as calibrator and the amplified fragment was obtained by reverse- transcript-PCR(RT-PCR).RNA quantification based on cycle threshold values(Ct)was used to establish the standard curve.According to which,the TLR3 mRNA levels in 30 normal individuals,20 patients with primary biliary cirrhosis(PBC)and 20 ones with chronic liver cirrhosis induced by HBV were calculated automatically by software after the fluorescence of PCR product was detected continuously during amplification.Results The linear detection range of the assay for TLR3 gene and ?-actin was 10~2-10~8(r= -0.9974)and 10~3～10~8(r=-0.9984),respectively.The coefficient of variation of both intra-and inter- assay reproducibility for high concentration sample were 6.7% and 8.7%,respectively,and those for low concentration sample were 12.3% and 14.0%.The TLR3 mRNA expression level ranges from 3.46?10~2- 4.51?10~3 copies/?g RNA,4.92?10~2-1.42?10~4 copies/?g RNA and 2.58?10~2-7.17?10~3 copies/?g RNA for 30 healthy individuals,20 PBC patients and 20 ones with chronic liver cirrhosis induced by HBV, respectively.Conclusion We have successfully set up a FQ-RT-PCR method for detecting TLR3 mRNA, which may be used as an excellent tool for the clinic and basic study on the expression of TLR3 gene.

Metabonomics is a new member in the omics field following the development of genomics, traqnscriptomics and proteomics,           with its research object being the col1ection of metabolites — metabolome, the downstream products of life. Even with the development of past decade，there are still many problems to be solved in the area of experimental techniques，data analysis and results annotation. Any xenobiotics would cause the disturbance of homeostasis of organisms，resulting in the changes of metabolites. Consequently，toxicology is one of the major application areas of metabolomics，and is the earliest one. Metabolomics has been widely used in the early toxicological screening of leads， preclinical and clinical toxicology of drugs in animals and human， as well as in surrogate models such as cell lines and zebrafish. Annotation techniques for metabolic data have also been developed. In this paper，the development of metabolomic techniques and its application in toxicology are reviewed.

Objective To analyse the characteristics of orbital benign and malignant masses on diffusion weighted imaging in combination with conventional MR imaging and evaluate the diagnostic value of apparent diffusion coefficient in distinguishing benign and malignant orbital lesions.Methods Seventy- seven cases with orbital masses,including fifty-five benign lesions and twenty-two malignant tumors,who underwent conventional MRI and diffusion imaging scanning were studied with use of a 1.5 T magnetic resonance system.Quantitative ADC measurements of masses(ADC_M)and of the white matter of eontralateral temporal lobe(ADC_W)were made with two different b-values of 0 and 1000 s/mm~2.The ADC ratio(ADCR)of the lesion to the control was calculated.The receiver operating characteristic curves(ROC) were constructed using various cut points of ADC_M and ADCR for different parameters to differentiate between benign and malignant masses.The area under the ROC curve for each parameter was also calculated. Results All cases were proved by histopathology.The mean ADC_M and ADCR of benign orbital masses were(1.56?0.75)?10~(-3)mm~2/s and 1.85?0.91,respectively.The mean ADC_M and ADCR of malignant orbital masses were(1.09?0.42)?10~(-3)mm~2/s and 1.28?0.53,respectively.There were significant difference both between ADC_M and ADCR of benign and malignant masses(t=2.803,2.735,P

OBJECTIVETo evaluate the cardiopulmonary exercise capacity in patients with essential hypertension (EH) complicating with or without left ventricular hypertrophy (LVH).

METHODSGraded maximal exercise test on the bicycle ergometer with respiratory gas analysis were performed in 30 gender and age matched normotensive controls, 40 EH patients without LVH and 30 EH patients with LVH (LVMI>125 g/m2 in males and > 120 g/m2 in females). Metabolic equivalents (METs), oxygen uptake (VO2), oxygen uptake to body mass ratio (VO2/kg) and oxygen uptake to heart beat ratio (VO2/HR) at time of reaching anaerobic threshold (AT) and at maximal oxygen uptake (VO2max) were measured and compared.

RESULTSMETs and VO2/kg were significantly reduced in EH patients with or without LVH compared with controls [at AT, METs: 3.57 +/- 0.8 and 4.34 +/- 1.47 vs. 5.21 +/- 1.45; VO2/kg: 12.38 +/- 2.85 and 14.42 +/- 4.33 vs. 18.48 +/- 4.52, all P < 0.01; at VO2max, METs: 4.94 +/- 1.24 and 5.90 +/- 1.51 vs. 6.96 +/- 1.85; VO(2)/kg: (17.20 +/- 4.34) mlxmin(-1)xkg(-1) and (20.41 +/- 4.59) mlxmin(-1)xkg(-1) vs. (24.04 +/- 5.21) mlxmin(-1)xkg(-1), all P < 0.01]. METs and VO2/kg at both time points were also significantly reduced in EH patients with LVH compared EH patients without LVH (all P < 0.05). The lower VO2/kg in hypertensive patients was significantly correlated to higher LVMI (P < 0.05).

CONCLUSIONSCardiopulmonary exercise capacity was reduced in hypertensive patients, especially in hypertensive patients with LVH.

Adult ; Aged ; Case-Control Studies ; Exercise Test ; Exercise Tolerance ; Female ; Heart ; physiopathology ; Humans ; Hypertension ; physiopathology ; Hypertrophy, Left Ventricular ; physiopathology ; Lung ; physiopathology ; Male ; Middle Aged ; Respiratory Function Tests

Objective To assess the effects of gonadotropin-releasing hormone analog on glucose and lipid metabolism in girls with idiopathic central precocious puberty(ICPP).Methods A total of 26 girls (aged 6-8 years,breast stage B2) with ICPP were followed up in Department of Endocrinology,Shenzhen Children's Hospital from Jan.2008 to Jun.2011.Those girls received triptorelin therapy for 12 months.Before and the end of the 6th month,the 12th month of the treatment,body mass index(BMI) was calculated,fasting plasma glucose(FPG),fasting plasma insulin(FPI),total cholesterol,triacylglycerols,high density lipoprotein cholesterol,low density lipoprotein cholesterol,apolipoprotein A,apolipoprotein B and estradiol(E2) were measured.Insulin sensitivity was estimated by homeostasis model assessment of insulin resistance(HOMA-IR).Twenty age-matched prepuberty girls were set as controls.Results 1.Before treatment,BMI,FPG,FPI and HOMA-IR in ICPP girls had no significant difference compared with the controls.2.After 6 months treatment of triptrolin,serum E2 concentration in ICPP girls declined from(30.5 ± 9.8) ng/L at the beginning of treatment to (11.2 ± 4.6) ng/L at the end of 6th month (P ＜ 0.01) ; The end of 12 th month of the treatment,FPG and FPI had no significant difference compared with that before of the treatment,but BMI increased from(16.46 ± 1.10) kg/m2 to(18.35 ± 1.30) kg/m2,the difference was significant(P ＜0.05),HOMA-IR increased from 1.24 ±0.30 to 2.08 ±0.40,the difference was significant(P ＜0.05).3.Lipid metabolism parameters remained unchangeable after 12 months of triptrolin treatment.Conclusion Triptorelin may lead to raise of BMI and HOMA-IR in girls with ICPP at 12 months after treatment.