A Rapid and Effective Colony PCR Procedure for Screening Transformants in Several Common Mushrooms

In the post-genomic era, gene function analysis has attracted much attention. Transformation is often needed to investigate gene function. In this study, an easy, rapid, reliable, and cost-effective colony polymerase chain reaction (PCR) method for screening mushroom transformants was developed: picking up a suitable amount of transformant's tissue (1–10 μg) to 20 μl 0.25% Lywallzyme solution, and vortexing for 10 s followed by incubation at 34 °C for 15 min. Finally, 2 μl of the suspension was used as templates to perform PCR and single target bands were successfully amplified from respective transformants of Tremella fuciformis, Pleurotus ostreatus, and Pleurotus tuber-regium. This procedure could be widely employed for screening transformants in mushroom transformation experiments.

An Enzymolysis-Assisted Agrobacterium tumefaciens-Mediated Transformation Method for the Yeast-Like Cells of Tremella fuciformis

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (≤0.1%) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per 106 YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.

An Improved Total RNA Extraction Method for White Jelly Mushroom Tremella fuciformis Rich in Polysaccharides

An improved method for extracting high quality and quantity RNA from a jelly mushroom and a dimorphic fungus—Tremella fuciformis which is especially rich in polysaccharides, is described. RNA was extracted from T. fuciformis mycelium M1332 and its parental monokaryotic yeast-like cells Y13 and Y32. The A260/280 and A260/230 ratios were both approximately 2, and the RNA integrity number was larger than 8.9. The yields of RNA were between 108 and 213 µg/g fresh wt. Downstream molecular applications including reverse transcriptional PCR and quantitative real-time PCR were also performed. This protocol is reliable and may be widely applicable for total RNA extraction from other jelly mushrooms or filamentous fungi rich in polysaccharides.

Screening of hypertension/blood stasis syndrome-related miRNA in endo-thelial cell models / 中国病理生理杂志

AIM:To screen the miRNA related to blood stasis syndrome and to explore the genetic mechanism of blood stasis syndrome in hypertension.METHODS:Human umbilical vein endothelial cell line CRL-1730 was co-cul-tured with the human sera from healthy normal controls and hypertension patients to establish a blood stasis-hypertension cell modela.The hypertension patients showed qi deficiency and blood stasis, qi stagnation and blood stasis, cold retaining and blood stasis, heat retaining and blood stasis and non-blood stasis syndromes.The endothelial cell models of blood stasis in hypertension were established.Target cells were collected for total RNA extraction.The techniques of Solexa ( high-throughput sequencing method) and digital gene expression profiling were applied for screening the target miRNA, and miRWalk software was used for online prediction the mRNA which might be the relevant target.qRT-PCR was conducted to confirm the prediction.RESULTS:SAT1-Hsa-miR-199a-5p and ATF4-Hsa-miR-1283 were found to be highly expressed in these cell models.The expression of miR-1283 and miR-199a-5p was confirmed by qRT-PCR.CONCLUSION:miR-199a-5p and miR-1283 may be the relevant miRNA for the prediction of blood stasis syndrome in hypertension.

Constructing protein-protein interaction network of hypertension with blood stasis syndrome via digital gene expression sequencing and database mining / 中西医结合学报

To construct a protein-protein interaction (PPI) network in hypertension patients with blood-stasis syndrome (BSS) by using digital gene expression (DGE) sequencing and database mining techniques.

Mathematical modeling of sign-and-symptom-oriented diagnostic procedure in traditional Chinese medicine based on the analytic hierarchy process: indications of Guizhi Decoction and its varieties in the Shanghanlun / 中西医结合学报

Objective: This study explores the applicability of a multi-attribute decision-making method in assessing Guizhi Decoction (GZD) and its varieties as noted in the Shanghanlun. Methods: A known multi-attribute decision-making method, the analytic hierarchy process (AHP), was adopted to transform the clinical challenge of selecting optional decoction for a given combination of symptoms or signs into multiple criteria decision-making problem. Results: A normative model based on the AHP was realized for indications of GZD and its varieties. The indications of sub-family GZD-f (indications of GZD itself) were exterior illness; the indications of GZD-vf1 (indications of GZD's variants which consist of fine adjustments to the ingredients or content on the basis of GZD) were exterior illness with interior excess, suggesting that GZD-vf1 consisted of most conservative variants of GZD; the indications of both GZD-vf2 (indications of Guizhi Qu Shaoyao Decoction and its derivative variants) and GZD-vf3 (indications of Guizhi Gancao Decoction and its derivative variants) showed evolution from exterior illness to interior deficiency in 2 directions. As to efficacy evaluation of the decoction, GZD and its varieties (restricted to those comparable ones) were of equal efficacy on 3 popular signs or symptoms indicating exterior illness: floating pulse, aversion to wind and spontaneous sweating, which were the common ground of indications for the GZD family. Conclusion: Modeling of diagnostic procedure based on the AHP is proved practicable to analyze the clinical judgment system of traditional Chinese medicine. Quantification research on syndrome differentiation and decoction evaluation system focused on signs and symptoms is suggested as a feasible and reliable model.

Correlation between obesity and the development of blood stasis syndrome / 中国组织工程研究

BACKGROUND: Vascular complications usually occur in diseases related to obesity such as primary hypertension or diabetes. Treating obesity with the method of promoting blood circulation to remove blood stasis gains a curative effect, so we presume that obesity may be correlated to blood stasis syndrome.OBJECTIVE: Analyzing the relationship between leptin(Lep) and insulinmetabolism of sugar and fat,endothelin(ET),blood pressure,body mass index(BMI), etc. Comparison of the differences in Lep and BMI between the blood stasis syndrome group and the non-blood stasis syndrome group was conducted to explore the correlation between obesity and the blood stasis syndrome.DESIGN: An observatory and controlled study based on patients with hypertension or diabetes.SETTING: Wards of the department of dardiovascular dedicine and the department of dndocrinology of a university of traditional Chinese medicine.PARTICIPANTS: All the patients in the present study were hospitalized at the Department of Internal Medicine of the Second Affiliated Hospital(Jinan City, Shangdong Province) of Shandong University of Traditional Chinese Medicine from September 2000 to January 2001.Among them, 126 cases with hypertension were at the Department of Cardiovascular Medicine, and 133 cases with type 2 diabetes were at the Department of Endocrinology. Inclusion criteria: ① The diagnosis of diabetes followed the diagnostic criteria for diabetes that was formulated by the WHO in 1980; ② The diagnosis of hypertension followed the diagnostic criteria in"China' s Guidebook for Hypertension Prevention and Treatment." The patients were aged between 45-76 years. Exclusion criteria: ① Secondary hypertension,diabetics; ②Complicated with severe diseases; ③ Complicated with severe disfunction of the heart,lung,liver, kidney or severe primary diseases in hematopoietic system; ④ Patients with mental disorder or pregnant women or in the state of lactation; ⑤ Patients aged <45'years old or>76 years old; ⑥ Patients who refused to participant in the study. Eighty-four cases with primary hypertension and 85 diabetics met the inclusion criteria, with a total of 169 cases including 73 male cases and 96 female cases. The cases were divided into blood stasis syndrome group and non-blood stasis syndrome group according to the clinical symptoms. There were 23 male cases and 37 female cases in the blood stasis syndrome group.METHODS: Drawing vein blood at the state of empty stomach for making plasma and serum. ET was detected with the radioimmuoassay(RIA) kit of ET by the method of RIA(the Arithmometer of Amma5500γ) . Lep and Ins were detected with the radioimmuoassay(RIA) kit of Lep and the radioimmuoassay(RIA) kit of Isu. The fasting blood sugar, total cholesterol(TC),triamid-glycerin(TG), high dense lipoprotein-cholesterol(HDL) and low dense lipoprrotein-cholesterol(LDL) were detected by the full-automatic bio-chemical analytical instrument of AMS-SaBa/8.MAIN OUTCOME MEASURES: Comparing the difference of Lep,Ins,ET between different diseases,syndromes; analyzing the relationship between Lep and Ins,BMI,blood sugar,blood lipid,blood pressure,etc.RESULTS: The detectable rate of the blood stasis syndrome in the diabetes group was higher than that in the hypertension group (P<0.05) . The ET density,Lep density,BMI,the percentage of fat of the blood stasis syndrome group were higher than those of the non- blood stasis syndrome group (P<0.05 ). The Ins density of the blood stasis syndrome group was lower than that of the non-blood stasis syndrome group(P<0.01 ). There was a positive correlation between Lep and BMI,the percentage of fat,ET,systolic pressure (SP),pulse pressure(PP) (P<0.05 or P<0. 01).CONCLUSION: There was a positive correlation between obesity and the blood stasis syndrome. Lep of patients with type 2 diabetes or hypertension was positively correlated to BMI,the percentage of fat and the level of ET.

In vitro study of atrial natriuretic peptide circadian secreted by encapsulated atrial natriuretic peptide cDNA transfection cells / 中国组织工程研究

AIM:To investigate atrial natriuretic peptide(ANP) circadian in the encapsulated human ANP(hANP) cDNA transfected cells,to alter the ANP circadian by artificial control to achieve the objective of effectively treat hypertension or congestive heart failure(CHF). METHODS:ANP cDNA was transfected into Chinese hamster ovary(CHO) cells,which were encapsulated in polycarprolactone(PCL) tubes.The longterm survival of transfected CHO cells and the levels of ANP secreted were detected.Circadian rhythm of ANP secreted by encapsulated transfected cells was also studied,which was regulated by melatonin. RESULTS:During culturing,the ANP level secreted by transfected CHO cells in 2 mL of culture medium within 24 hours could reach 210.3 ng/L in a 20 mm-long and 2 mm-diameter PCL tube.The section of ANP displayed a circadian variation:higher in daytime,but lower at night.The acrophase of circadian rhythm was 4:15 but could be shifted to 7:55 after melatonin management. CONCLUSION:ANP cDNA transfected CHO cells that encapsulated into PCL tube can secret ANP,which might be suitable for the future implantation into human body.Our research provides a new approach in the treatment of hypertension and CHF by ANP.

Effects of encapsulated plasmid recombining with hANP cDNA transfected cells on morphological and histological characteristic of experimental hypertensive rats / 生物医学工程学杂志

A technique based on release of the human atrial natriuretic peptide (hANP) from plasmid hANP cDNA transfected Chinese hamster ovary (CHO) cells encapsulated in polycaprolactone (PCL)-capsules was used for a potential therapeutic approach to hypertension or congestive heart failure (CHF). The plasmid combining with hANP cDNA was transfected into CHO cells, and then encapsulated plasmid hANP cDNA transfected CHO cells were implanted into two-kidney, one-clip (2K1C) hypertensive rats intraperitoneally. The morphological changes, histological changes were investigated after the implantation of PCL-capsules in 2K1C hypertensive rats. The results showed that the implantation of encapsulated hANP-producing cells caused a significant delay of blood pressure (BP) increase after the encapsulated cells being implanted in 2K1C hypertensive rats. These effects were reflected morphologically by an attenuation of the glomerular sclerotic lesions, tubular damage and renal arterial thickening, comparing with control group. The plasma levels of hANP in 2K1C rats implanted with the PCL-capsules containing hANP-producing cells were higher than that of the control rats. These results demonstrated the usefulness of encapsulated hANP gene transfected cells as a new tool for hANP gene delivery in studying renovascular hypertension and cardiovascular diseases, thus implying the potential of using gene transfected cells as therapeutic agents in the treatment of cardiovascular diseases.

Effect of Buyang Huanwu Decoction on expression of adhesion molecules in vascular endothelial cells of blood stasis rats / 中草药

Objective To study the effect of Buyang Huanwu Decoction (BHD) on adhesion molecules in vascular endothelial cells (VECs) of blood stasis rats. Methods Expression of ICAM-1, VCAM-1, PECAM-1, iNOS in vascular endothelial cells of blood stasis rats receiving different doses of BHD was observed by immunohistochemical method and RT-PCR.Results The expression of ICAM-1, VCAM-1, PECAM-1, iNOS was increased in model group, while was decreased by BHD. There was an increase tendency on the expression of molecules and significant dose-effect relationship along with the decrease of the BHD doses. Conclusion The increased expression of adhesion molecules in vascular endothelial cells of blood stasis rats could be reduced by BHD in a dose dependent manner.

Effect of Huangdanyishen Capsule on diabetic nephropathy of experimental rats / 中成药

AIM: To observe the efficacy of Huangdanyishen Capsule (HDC)(Radix Astragali, Radix Salviae Miltiorr hizae) on renal lesions in diabetic nephropathy(DN) rat induced by Streptozocin(STZ). METHODS: The murine model with DN induced by STZ was made from Wistar rats with insulin resistance after being fed with feedingstuff of hypersugar, hyperlipid, hypercholesterol. These rats were randomly divided into five groups, and all rats were successively administrated corresponding drugs for 10 weeks. The general state and blood sugar were observed during the period. Serum urea nitrogen and creatinine content, the excretory rate of urinary micro-protein and renal pathological features were determined after 10 weeks. RESULTS: HDC could obviously ameliorate psychic status and renal function of rats with diabetes mellitus Ⅱ, and increase DN rats' weight. HDC could reduce the content of DN rats' serum urea. nitrogen and creatinine and urinary micro-protein. The ultramicropathologic histopathologic results indicated that the glomerular basement membrane thickening, synapse displaying foul-up and amalgamation, and mitochondrial swelling were alleviated to a certain extent in HDC groups. The content of murine blood sugar in HDC groups was significantly lower than those in model group. CONCLUSION: HDC has an obvious effect on Ⅱ DN treatment and decrease the content of blood sugar at the same time.

Effects of Shenghua Decoction on the expression of adhesion molecules in vascular endothelial cells of blood-stasis rats / 中成药

AIM: To study the effects of Shenghua Decoction on the expression of adhesion molecules in vascular endothelial cells (VECs) of blood-stasis rats. METHODS: The expression of VCAM-1, ICAM-1, PECAM-1, iNOS and their corresponding mRNA in VECs were assayed by immunohistochemistry, image analysis and RT-PCR under the function of different Shenghua Decoction doses (high, middle, low). RESULTS: Immunohistochemistry and image analysis indicated: the expression of VCAM-1, ICAM-1, PECAM-1 and iNOS were higher in the model group than in the control group, Shenghua Decoction could decrease the expression of VCAM-1, ICAM-1, PECAM-1 and iNOS in the model group, moreover as the dose decreased, the expression of these molecules increase, it was of dose-effect relation. CONCLUSION: Shenghua Decoction can downregulate the expression of adhesion molecules in vascular endothelial cells of blood-stasis rats, and the dose-effect relation is obvious.

Effect of Tanshinone Ⅱ_A on vWF,TM and EPCR from patients with hypertension plus blood stasis / 中成药

AIM: To research the effect of serum from patients with blood stasis syndrome(BSS) associated with hypertension disease on vWF、TM、EPCR excreted by ECV-304 and Tanshinone Ⅱ_A's intervention. METHODS: Cultivated human umbilical vein endothelial cells(CRL-1730) were divided into four groups,according to different serum of patients with BSS associated with hypertension disease,BSS(model) group,non-blood stasis group,health group and control group.The incubation time was 24 h.Tanshinone Ⅱ_A was of different concentrations(40、20、10、5 ?g/mL),all Tanshinone Ⅱ_A groups' cells were incubated by patients' serum and Tanshinone Ⅱ_A for 24 h.vWF,TM and EPCR were assayed by enzyme-linked immunosorbent assay(ELISA). RESULTS: vWF,TM and EPCR secreted were higher in the model group,the non-blood stasis group and the health group than those in the control group;vWF,TM and EPCR secreted were higher in the model group,the non-blood stasis group than those in the health group;Thereinto,the difference was distinct between the model group and the health group(P

Effects of xijiao dihuang decoction on the expression of adhesion molecules in vascular endothelial cells of adrenine and hypothermia-treated rats / 中国病理生理杂志

AIM: To study the effects of xijiao dihuang decoction, a Chinese medicine, on the expression of adhesion molecules in vascular endothelial cells (VECs) of adrenine and hypothermia-treated rats. METHODS: The expression of VCAM-1, ICAM-1, PECAM-1, iNOS and their corresponding mRNA in VECs was assayed by immunohistochemistry analysis and RT-PCR, respectively. RESULTS: Immunohistochemistry analysis showed the expression of VCAM-1, ICAM-1, PECAM-1 and iNOS are higher in adrenine and hypothermia-treated rats than that in controls, xijiao dihuang decoction decreased the expression of VCAM-1, ICAM-1, PECAM-1 and iNOS in a dose-dependent manner in adrenine and hypothermia-treated rats. CONCLUSION: Xijiao dihuang decoction can down-regulate the expression of adhesion molecules in vascular endothelial cells of adrenine and hypothermia-treated rats.