|
Enhancing rice resistance to fungal pathogensby transformation with cell wall degrading enzyme genes from Trichoderma atroviride
LIU Mei, SUN Zong-xiu, ZHU Jie, XU Tong, HARMAN Gary E., LORITO Matteo
Journal of Zhejiang University-SCIENCE A (Applied Physics & Engineering), 2004, 5(2): 133-136.
https://doi.org/10.1631/jzus.2004.0133
Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations and transformed to rice plants. More than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had lesser effect. The expression level of endochitinase but exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the effect of endochitinase on disease resistance when the two genes co-expressed in transgenics. Resistance to Magnaporthe grisea was found in all kinds of regenerated plants including that with single gluc78. A few lines expressing either ech42 or nag70 gene were immune to the disease. Transgenic plants are being tested to further evaluate disease resistance at field level. This is the first report of multiple of expression of genes encoding CWDEs from Trichoderma atroviride that result in resistance to blast and sheath blight in rice.
|
|
Improved elastase production by Bacillus sp. EL31410 — further optimization and kinetics studies of culture medium for batch fermentation
HE Guo-qing, CHEN Qi-he, JU Xiao-jie, SHI Nai-dong
Journal of Zhejiang University-SCIENCE A (Applied Physics & Engineering), 2004, 5(2): 149-156.
https://doi.org/10.1631/jzus.2004.0149
An efficient culture medium producing a bacterial elastase with high yields was developed further following preliminary studies by means of response surface method. Central composite design (CCD) and response surface methodology were applied to optimize the medium constituents. A central composite design was used to explain the combined effect of three medium constituents, viz, glucose, K2HPO4, MgSO4·7H2O. The strain produced more elastase in the completely optimized medium, as compared with the partially optimized medium. The fitted model of the second model, as per RSM, showed that glucose was 7.4 g/100 ml, casein 1.13 g/100 ml, corn steep flour 0.616 g/100 ml, K2HPO4 0.206 g/100 ml and MgSO4·7H2O 0.034 g/100 ml. The fermentation kinetics of these two culture media in the flask experiments were analyzed. It was found that the highest elastase productivity occurred at 54 hours. Higher glucose concentration had inhibitory effect on elastase production. At the same time, we observed that the glucose consumption rate was slow in the completely optimized medium, which can explain the lag period of the highest elastase production. Some metal ions and surfactant additives also affected elastase production and cell growth.
|
|
Ex vivo expansions and transplantations of mouse bone marrow-derived hematopoietic stem/progenitor cells
WANG Jin-fu, WU Yi-fan, HARRINTONG Jenny, McNIECE Ian K.
Journal of Zhejiang University-SCIENCE A (Applied Physics & Engineering), 2004, 5(2): 157-163.
https://doi.org/10.1631/jzus.2004.0157
To examine the effects of co-culture with bone marrow mesenchymal stem cells on expansion of hematopoietic stem/progenitor cells and the capacities of rapid neutrophil engraftment and hematopoietic reconstitution of the expanded cells, we expanded mononuclear cells (MNCs) and CD34+/c-kit+ cells from mouse bone marrow and transplanted the expanded cells into the irradiated mice. MNCs were isolated from mouse bone marrow and CD34+/c-kit+ cells were selected from MNCs by using MoFlo Cell Sorter. MNCs and CD34+/c-kit+ cells were co-cultured with mouse bone marrow-derived mesenchymal stem cells (MSCs) under a two-step expansion. The expanded cells were then transplanted into sublethally irradiated BDF1 mice. Results showed that the co-culture with MSCs resulted in expansions of median total nucleated cells, CD34+ cells, GM-CFC and HPP-CFC respectively by 10.8-, 4.8-, 65.9- and 38.8-fold for the mononuclear cell culture, and respectively by 76.1-, 2.9-, 71.7- and 51.8-fold for the CD34+/c-kit+ cell culture. The expanded cells could rapidly engraft in the sublethally irradiated mice and reconstitute their hematopoiesis. Co-cultures with MSCs in conjunction with two-step expansion increased expansions of total nucleated cells, GM-CFC and HPP-CFC, which led us to conclude MSCs may create favorable environment for expansions of hematopoietic stem/progenitor cells. The availability of increased numbers of expanded cells by the co-culture with MSCs may result in more rapid engraftment of neutrophils following infusion to transplant recipients.
|
|
Cloning and GST-fused expression in E. coli of mouse β-1,4-galactosyltransferase
GONG Xing-guo, ZHONG Wen-tao, WU Wen-ying
Journal of Zhejiang University-SCIENCE A (Applied Physics & Engineering), 2004, 5(2): 164-172.
https://doi.org/10.1631/jzus.2004.0164
β-1,4-galactosyltransferase (β4Gal-T) (EC 2.4.1.38) plays a multifunctional role in many aspects of normal cell physiology. By now, several dozens of β4Gal-T genes have been cloned, separated from mouse, chick, bovine, human, etc. This paper presents the cloning and GST-fused expression of mouse β4Gal-T gene in Escherichia coli (E. coli). The target gene was cloned by PCR, followed by identification by DNA sequencing and expression in E. coli with isopropyl-β-D-thiogalactoside (IPTG) gradient concentrations, products of which were separated on SDS-PAGE showing that the target protein had the same molecular weight as that of mouse β4Gal-T. The transcriptional product of β4Gal-T gene was proved by Western hybridization analysis to be due to GST-fusion.
|
|
A pair of two-component regulatory genes ecrA1/A2 in S. coelicolor
LI Yong-quan, CEN Pei-lin, CHEN Shi-fei, WU Dan, ZHENG Jing
Journal of Zhejiang University-SCIENCE A (Applied Physics & Engineering), 2004, 5(2): 173-179.
https://doi.org/10.1631/jzus.2004.0173
Two-component genes are kinds of genetic elements involved in regulation of antibiotic production in Streptomyces coelicolor. DNA microarray analysis revealed thatecr A1/A2, which mapped at distant sites from red locus and encode respectively the kinase and regulator, expressed coordinately with genes of Red specific biosynthetic pathway. ecrA1 and ecrA2 gene-disruptive mutants were constructed using homogenotisation by reciprocal double crossover. Fermentation data showed that the undecylprodigiosin (Red) level of production was lower than that of wild-type strain. However, the change of the actinorhodin (Act) production level was not significant compared with wild type. Thus, these experiment results confirmed that the two-component system ecrA1/A2 was positive regulatory element for red gene cluster.
|
|
Degradation of chlorophenol by in-situ electrochemically generated oxidant
CONG Yan-qing, WU Zu-cheng, YE Qian, TAN Tian-en
Journal of Zhejiang University-SCIENCE A (Applied Physics & Engineering), 2004, 5(2): 180-185.
https://doi.org/10.1631/jzus.2004.0180
A novel in-situ electrochemical oxidation method was applied to the degradation of wastewater containing chlorophenol. Under oxygen sparging, the strong oxidant, hydrogen dioxide, could be in-situ generated through the reduction of oxygen on the surface of the cathode. The removal rate of chlorophenol could be increased 149% when oxygen was induced in the electrochemical cell. The promotion factor was estimated to be about 82.63% according to the pseudo-first-order reaction rate constant (min-1). Important operating parameters such as current density, sparged oxygen rate were investigated. Higher sparged oxygen rate could improve the degradation of chlorophenol. To make full use of oxygen, however, sparged oxygen rate of 0.05 m3/h was adopted in this work. Oxidation-reduction potential could remarkably affect the generation of hydrogen peroxide. It was found that the removal rate of chlorophenol was not in direct proportion to the applied current density. The optimum current density was 3.5 mA/cm2 when initial chlorophenol concentration was 100 mg/L and sparged oxygen rate was 0.05 m3/h.
|
|
Effect of polychlorinated biphenyls on spermatogenesis and testosterone secretion in adult cocks
ZHANG Cai-qiao, QIAO Hui-li
Journal of Zhejiang University-SCIENCE A (Applied Physics & Engineering), 2004, 5(2): 193-197.
https://doi.org/10.1631/jzus.2004.0193
The effects of polychlorinated biphenyls (PCBs) on reproduction of adult cocks were studied by gavaging peanut oil or PCBs (Aroclor 1254, 50 mg/kg) once a week for six consecutive weeks. Physiological parameters were recorded and gonads were removed at the end of experiment for histological examination. The results showed that there was no significant difference between the control and treatment group in body weight, respiration rate, heart rate, body temperature, and the numbers of red and white blood cells. However, there was a marked decrease in the testicular weight and serum testosterone level after PCB treatment. Morphological studies manifested severe damage of the seminiferous tubules by PCB. The number of the germ cells at the different developmental stages was decreased and condensed nuclei were observed in most of these cells. This study revealed that the reproductive function of the adult cocks is sensitive to PCBs, which inhibited mainly spermatogenesis and testosterone secretion.
|
|
Identification of genes differentially expressed in monocyte-derived dendritic cells with 1α,25-dihydroxyvitamin D3 using cDNA arrays
SHEN Qian-yun, ZHENG Shu-sen
Journal of Zhejiang University-SCIENCE A (Applied Physics & Engineering), 2004, 5(2): 222-225.
https://doi.org/10.1631/jzus.2004.0222
In order to study the molecular mechanism of the inhibitory effect of 1,25-dihydroxyvitamin D3 on dendritic cells, experiments were performed using Atlas cDNA expression arrays from Clonetech to identify the differentially expressed genes of dendritic cells by 1,25-dihydroxyvitamin D3. Analysis of cDNA arrays revealed changes in the expression of 9 genes, including those involved in DNA binding and transcription, extracellular cell signaling and communication, intracellular transducers, as well as cell adhesions. The results indicated that a multiple molecular network is involved in the inhibitory role of 1,25-dihydroxyvitamin D3 on dendritic cells. The Atlas Array technology may facilitate the elucidation of complex pharmacological process of 1,25-dihydroxyvitamin D3 on dendritic cells.
|
|
Enantioselective assay of S(+)- and R(-)-propafenone in human urine by using RP-HPLC with pre-column chiral derivatization
WU Yong-jiang, MA Ming-ming, ZENG Su
Journal of Zhejiang University-SCIENCE A (Applied Physics & Engineering), 2004, 5(2): 226-229.
https://doi.org/10.1631/jzus.2004.0226
The enantioselective assay for S(+)- and R(-)-propafenone (PPF) in human urine that developed in this work involves extraction of propafenone from human urine and using S(+)-propafenone as internal standard, chiral derivatization with 2,3,4,6-tetra-O-β-D-glucopranosyl isothiocyanate, and quantitation by an RP-HPLC system with UV detection (λ=220 nm). A baseline separation of propafenone enantiomers was achieved on a 5-μm reverse phase ODS column, with a mixture of methanol:water:glacial acetic acid (25:12:0.02,v/v) as mobile phase. There was good linear relationship from 24.9 ng/ml to 1875.0 ng/ml for both of enantiomers. The regression equations of the standard curves based on CS-PPF (or CR-PPF) versus ratio of AS-PPF/AS (or AR-PPF/AS) were y=0.0032x-0.081, (r=0.999) for S-PPF and y=0.0033x+0.0039, (r=0.998) for R-PPF, respectively. The method\'s limit of detection was 12.5 ng/ml for both enantiomers, and the method\'s limit of quantitation was 28.2±0.52 ng/ml for S-PPF, 30.4±0.53 ng/ml for R-PPF (RSD<8%, n=5). The analytical method yielded average recovery of 98.9% and 100.4% for S-PPF and R-PPF, respectively. The relative standard deviation was no more than 6.11% and 6.22% for S-PPF and R-PPF, respectively. The method enabled study of metabolism of S(+)- and R(-)-propafenone in human urine. The results from 7 volunteers administered 150 mg racemic propafenone indicated that propafenone enantiomers undergo stereoselective metabolism and that in the human body, S(+)-propafenone is metabolized more extensively than R(-)-propafenone.
|
|
Expression and significance of cyclin D1, p27kip1 protein in bronchioloalveolar carcinoma
YUAN Jian-qun, XU Jing-yao, ZHANG Jing, HE Qi-cai, ZHU Jia, SHENG Cai-xia
Journal of Zhejiang University-SCIENCE A (Applied Physics & Engineering), 2004, 5(2): 235-241.
https://doi.org/10.1631/jzus.2004.0235
Purpose: To investigate the relationship between expression of cell cycle-related protein cyclin D1, p27kip1 and the pathogenesis of bronchioloalveolar carcinoma (BAC) and the value of prediction of prognosis. Methods: Cyclin D1 and p27kip1 protein were detected by immunohistochemical En Vision method in 43 BACs. Results: The positivity of cyclin D1 in BAC was 65.1% (28/43), which was significantly higher than that in normal pulmonary tissue (0/13), P<0.01. No statistically significant association was found between cyclin D1 expression data and sex, age, tobacco-use history, histologic subtype (mucinous vs nonmucinous), stromal fibrosis, lymph node metastasis, clinical stage or postoperative survival period (P>0.05), while cyclin D1 expression was found to be negatively correlated with tumor size (P<0.05). The positivity of p27kip1 in BACs was 51.2% (22/43), significantly lower than that in normal pulmonary tissue (12/13), P<0.01. p27kip1 expression level was not associated with sex, age, tobacco-use history, tumor size or histologic subtype (P>0.05), but was negatively correlated with stromal fibrosis, lymph node metastasis and clinical stage (P<0.05); and positively associated with postoperative survival period (P<0.01). The survival rate of p27kip1 positive group was significantly higher than that of p27kip1 negative group (P<0.01). No statistically significant correlation was found between cyclin D1 and p27kip1 expression. Conclusions: Increased cyclin D1 expression and decreased p27kip1 expression are related to the pathogenesis of BAC; decreased p27kip1 expression is associated with metastasis progression; immunodetection of p27kip1 is useful for assessment of prognosis.
|
|
Distribution of H. pylori antigens in gastric mucosa and its significance
LU Xin-liang, QIAN Ke-da, TANG Xun-qiu, ZHU Yong-liang
Journal of Zhejiang University-SCIENCE A (Applied Physics & Engineering), 2004, 5(2): 242-245.
https://doi.org/10.1631/jzus.2004.0242
Objective: To investigate the distribution of H. pylori antigens in the gastric mucosa in patients with H. pylori infection, and the relationship between the distribution and gastric cancer. Methods: Of 112 patients confirmed by pathological study to have chronic superficial gastritis, precancerous changes (chronic atrophic gastritis, intestinal metaplasia or atypical hyperplasia) and gastric cancer, 28 were H. pylori negative and 84 were H. pyloripositive. H. pylori antigens in the gastric mucosa were detected by immunohistochemistry. Results: The H.pylori positive group, comprised 12 of 22 (50.0%) in the chronic superficial gastritis group, 22 of 25 (88.0%) in the precancerous changes group and 13 of 35 (37.1%) in the gastric cancer group. The positive rates of H. pylori antigens in the cytoplasm progressively increased, respectively at 0.0% (0/12), 63.6% (14/22) and 84.6% (11/13) for the same groups (χ²=19.76, P=0.000); H. pylori antigens were located in the mucus layer and above the neck of the mucosal gland in 9 of 12 (75.0%) cases with chronic superficial gastritis, at the neck of the mucosal gland and the isthmus in 12 of 22 (54.5%) cases with precancerous changes, below the isthmus in 9 of 13 (69.2%) cases with gastric cancer (χ²=25.30, P=0.000). In the H.pylori negative group, no H. pylori antigen was observed. Conclusion: With the progression of chronic superficial gastritis→precancerous changes→gastric cancer, H. pylori antigens progressively migrated from the outer part to the inner part of the cell, and from the superficial to the deep gastric mucosa.
|
|
MRS study on lentiform nucleus in idiopathic Parkinson\'s disease with unilateral symptoms
ZHENG Xu-ning, ZHU Xiong-chao, RUAN Ling-xiang, ZHANG Ling-ju, Yuan Min, SHANG De-sheng, LIU Yang
Journal of Zhejiang University-SCIENCE A (Applied Physics & Engineering), 2004, 5(2): 246-250.
https://doi.org/10.1631/jzus.2004.0246
Objective: To investigate changes in magnetic resonance spectroscopy (MRS) of lentiform nucleus during the early stage of Parkinson\'s disease. Methods: Twenty-five patients with idiopathic Parkinson disease with unilateral symptoms (IPDUS) and 25 healthy volunteers were enrolled in this study. MRS of the lentiform nucleus in each patient was taken and then concentrations of N-acetylaspartate (NAA), Creatine (Cr) and Choline (Cho) were calculated. Results: Compared to that in the control, NAA/(Cho+Cr) was significantly lower in the lentiform nucleus contralateral to symptoms and even that in the ipsilateral side in IPDUS patients (all P<0.05); while there was no difference between the two sides in the healthy volunteer (P>0.05). The ratio of NAA/(Cho+Cr) ipsilateral to the sympatomatic side of the patient was also lower than that of the control (P<0.05). Conclusions: there might be some changes with MRS on the lentiform nucleus during the early stage of idiopathic Parkinson\'s disease with unilateral symptom. MRS may be one of the reliable methods for early or even sub-clinical diagnosis.
|
21 articles
|