Insights into the catalytic and regulatory mechanisms of dihydroflavonol 4-reductase, a key enzyme of anthocyanin synthesis in Zanthoxylum bungeanum.

Accumulation of anthocyanins largely determines the fruit color, and dihydroflavonol 4-reductase (DFR) is a key enzyme involved in the formation of anthocyanins. However, the catalytic and regulatory mechanisms of DFR are unclear. In this study, the gene encoding DFR from Zanthoxylum bungeanum Maxim. was cloned and ZbDFR was analyzed in detail. The ZbDFR accepted dihydrokaempferol, dihydroquercetin and dihydromyricetin as substrates. Flavonols such as myricetin, quercetin and kaempferol significantly inhibited the activity of ZbDFR, while quercitrin and isoquercitrin slightly increased the activity. Quercetin was a competitive inhibitor at low concentrations, and it had a combined effect of competitive and noncompetitive inhibition at high concentrations, which was consistent with ZbDFR having two inhibitor binding sites. In addition, the content of different types of flavonoids in Z. bungeanum peel at green, semi-red and red stage was analyzed, and the in vivo results could be explained by the regulation of ZbDFR activity in vitro. Site-directed mutagenesis combined with enzyme activity experiments showed that Ser128, Tyr163, Phe164 and Lys167 are the key catalytic amino acid residues. The Ser128, Tyr163 and Lys167 were crucial for the hydrogen transfer reaction, and mutation of these amino acids resulted in the loss of all or most of the activity. Phe164 was found to be important for the regulation of ZbDFR by flavonols. Accordingly, ZbDFR is a node at which flavonoids regulate the synthesis of anthocyanins and proanthocyanins.

Production of 5-aminolevulinic acid from glutamate by overexpressing HemA1 and pgr7 from Arabidopsis thaliana in Escherichia coli.

The important metabolic intermediate 5-aminolevulinic acid (ALA) is useful for cancer treatment or plant growth regulation and has consequently received much attention. In this study, we introduced the HemA1 and pgr7 genes from the higher plant Arabidopsis thaliana into recombinant Escherichia coli to overproduce extracellular 5-aminolevulinic acid via the C5 pathway. In the E. coli BL21 (DE3) strain background, the ALA concentration of the strain expressing both HemA1 and pgr7 was the highest and reached 3080.62 mg/L. Among the 7 tested hosts, ALA production was the highest in E. coli Transetta (DE3). In E. coli Transetta GTR/GBP, the expression levels of zwf, gnd, pgl and RhtA were upregulated. Glutamate induced the expression of the GltJ, GltK, GltL and GltS genes that are in involved in glutamate uptake. The recombinant E. coli Transetta GTR/GBP was able to produce 7642 mg/L ALA in modified minimal medium supplemented with 10 g/L glutamate and 15 g/L glucose after 48 h of fermentation at 22 °C. The results provide persuading evidence for the efficient production of ALA from glucose and glutamate in E. coli expressing A. thaliana HemA1 and pgr7. Further optimization of the fermentation process should be done to improve the ALA production to an industrially relevant level.

Multi-enzymatic recycling of ATP and NADPH for the synthesis of 5-aminolevulinic acid using a semipermeable reaction system.

5-Aminolevulinic acid (ALA) is an important cellular metabolic intermediate that has broad agricultural and medical applications. Previously, attempts have been made to synthesize ALA by multiple enzymes in cell free systems. Here we report the development of a semi-permeable system for ALA production using stable enzymes. Glucose, sodium polyphosphate, ATP, tRNA, glutamate and NADPH were used as substrates for ALA synthesis by a total of nine enzymes: adenylate kinase, polyphosphate kinase, glucose-6-phosphate dehydrogenase, phosphogluconolactonase, 6-phosphogluconate dehydrogenase, glutamyl-tRNA synthetase and glutamate-1-semialdehyde aminotransferase from E. coli, hexokinase from yeast, as well as glutamyl-tRNA reductase and its stimulator protein glutamyl-tRNA reductase binding protein (GBP) from Arabidopsis in a semi-permeable system. After reaction for 48 h, the glutamate conversion reached about 95%. This semi-permeable system facilitated the reuse of enzymes, and was helpful for the separation and purification of the product. The ALA production could be further improved by process optimization and enzyme engineering.Abbreviations: PPK: polyphosphate kinase; ADK: adenylate kinase; ALA: 5-Aminolevulinic acid; HK: hexokinase; ZWF: glucose-6-phosphatedehydrogenase; PGL: phosphogluconolactonase; GND: 6-phosphogluconate dehydrogenase; GTS: glutamyl-tRNA synthetase; GTR: glutamyl-tRNA reductase; GBP: GTR binding protein; GSAAT: glutamate-1-semialdehyde aminotransferase.

Clinical analysis of infectious complications in 189 patients with lupus nephritis / 中国医师进修杂志

Objective To investigate the clinical and epidemiologic features of infection in patients with lupus nephritis (LN). Methods The incidence, sites and pathogen species of infection 1 year after diagnosis in 189 patients with LN were retrospectively analyzed, and the relationships between the clinical, histological types, treatment programs and infection were analyzed. Results Among 189 LN patients (16 males and 173 females), 97 patients had infection (infection group), and 92 patients had not infection (non-infection group). The incidence of infection was 51.3% (97/189), while the incidence of nosocomial infection was 39.7%(75/189). In infection group, the patients had 133 infection episodes, and the principal infection sites were lung and urinary tract system, accounting for 67.7% (90/133); the principal pathogen was bacteria, accounting for 72.9%(97/133), and the second was virus, accounting for 17.3% (23/133). Different clinical or pathological types and treatment options of LN had different infection rates, but there were no statistical differences (P>0.05). The incidence of nosocomial infection in patients with glucocorticoid + cyclophosphamide double impact was significantly higher than that in patients treated with glucocorticoid + mycophenolate mofetil: 52.9% (45/85) vs. 7.7% (2/26), and there was statistical difference (P<0.01). One year after diagnosis, the mortality in infection group was significantly higher than that in non-infection group:8.2%(8/97) vs. 1.1%(1/92), and there was statistical difference (P<0.05). Conclusions The more serious the clinical and pathological types, the higher the incidence of infection. The incidence of infection is associated with the treatment options. Infection is still the most important reason for LN patients′death.

Promoter methylation and protein expression of tumor suppressor candidate 1 gene in adolescent papillary thy-roid carcinoma / 中华实用儿科临床杂志

Objective To study the relationship between status of methylation of tumor suppressor candidate 1 gene(TUSC1) promoter and expression of its protein in adolescent papillary thyroid carcinoma(PTC). Methods Forty cases of adolescent PTC were chosen and the corresponding para carcinoma tissues were taken from July 2010 to Decem-ber 2013 in the First Affiliated Hospital of Zhengzhou University surgical specimens of the thyroid gland and were con-firmed by pathology. Male 12 cases,female 28 cases,median age 14 (10-18) years old. Tumor node metastasis (TNM) stageⅠ-Ⅱ13 cases,Ⅲ-Ⅳstage 27 cases;gradeⅠin 15 cases,gradeⅡin 25 cases;lymph node metastasis in 22 cases,18 cases were negative. Methylation-specific polymerase chain reaction (MSP) and Western blot were applied respectively to examine the methyaltion of TUSC1 gene promoter and its protein expression of 40 samples of adolescent PTC and their matched adjacent non-cancerous epithelium. Results The results of MSP revealed that there was no methylation of TUSC1 gene promoter in adjacent non-cancerous epithelium,while in the adolescent PTC,the hyper-methylation rate was 60%(24/60 cases,χ2=34. 28,P<0. 05). In additional,it was related to the TNM stage,pathological grade and lymph node metastasis (χ2=4. 862,7. 111,5. 625,all P<0. 05). The result of Western blot revealed that the positive expression rate of TUSC1 protein was 100% in adjacent non-cancerous epithelium and 30%(12/40 cases) in adolescent PTC (χ2=14. 118,P<0. 05),which was related to the TNM stage,pathological grade and lymph node metastasis (χ2=5. 215,6. 222, 5. 079,all P<0. 05). There was distinct correlation between methylation of TUSC1 gene promoter and the protein expres-sion (r=-0. 84,P<0. 05). Conclusions Methylation of promoter might be one of the important mechanisms of inactiva-tion of TUSC1 gene,and might play an important role in carcinogenesis and progression of adolescent PTC.

Efficacy analysis of endovascular graft exclusion for the treatment of 24 patients with infra-kidney abdominal aortic aneurysms / 中国医师进修杂志

Objective To investigate the efficacy and the indication and the management of perioperative complications in treatment of infra- kidney abdominal aortic aneurysm (AAA) by using endovascular graft exclusion (EVGE). Methods From April 2006 to September 2008, 24 patients with infra- kidney abdominal aortic aneurysms were diagnosed by contrast-enhanced CT or MRI scan. Vascular access was obtained through the bilateral femoral artery after arteriotomy and stent-graft was deployed into AAA of below the renal artery to occlude the left over cavity of AAA. The stent- graft was extended and anchored to the both side wall of AAA, the blood flow enter into the arteria iliaca communis through the sten't.Results Stent-graft deployment was successfully performed in all the patients. Immediate aortography after the procedure showed no leakage in 20 patients and the type Ⅰ minor leakage in 4 patients. No stent movement or organ and both lower extremities ischemia was found at the early post operative stage in all the patients. Six months after the operation, in all the 24 patients, contrast-enhanced CT scan showed the disappearance of the aneurysm and thrombosis at the level of the stent. Conclusions EVGE is simple,minimally invasive,less complication and quick recovery after operation. Thus it becomes first choice for the treatment of AAA for the elder patients.

Diagnostic value of serum brain natriuretic peptide in chronic renal failure patients with chronic heart failure / 中国基层医药

Objective To investigate the influence of renal function on serum BNP in the diagnosis of CRF with heart failure by observing the relationship between eGFR and BNP in serum and comparing cut-off values of BNP in different eGFR levels. Methods The elderly participants were enrolled in the study, including 52 patients with heart failure, and 29 patients without heart failure and 30 healthy controls. Serum BNP level was measured by ELISA.Results The level of serum BNP increased significantly in subjects with heart failure compared with those with renal dysfunction for no-heart failure patients (P ＜ 0.05) and healthy controls. BNP level was significantly higher in CRF no-heart failure patients than in control subjects. eGFR showed negative correlation with BNP in ESRD no-heart failure patients (γ= -0. 581, P ＜ 0.01). There was no correlation between eGFR and ESRD with heart failure patients (γ= - 0.081, P ＞ 0.05). The AUC of BNP in patients (eGFR 30 ～ 60 ml) was 0. 951, when cut-off value was 1 500 ng/L,the sensitivity and specificity of BNP were 96.4% and 86. 7% respectively. The AUC of BNP in patients(eGFR ＜30 ml)was 0. 860, when cut-off value was 1 850 ng/L,the sensitivity and specificity of B NP were 66.7% and 92.9%respectively. Conclusions Heart failure was major factor result in higher levels of BNP in chronic renal failure with heart failure patients. BNP could be used as a diagnostic marker for CRF with heart failure patients.

Clinical analysis to 5 cases of graves disense related to nephrosis / 中国基层医药

Objective To investigate principle and clinic pathologic characteristics of autoimmune thyroid disease(AITD) associated nephrosis. Methods By introducing 5 cases and related reference to review clinic pathologic characteristics of AITD associated nephrosis. Results AITD associated nephrosis regard proteinuria as principal, and a handful of performance is nephritic syndrome and nephritis syndrome. Pathologic characteristics are diversity. It is usually membranous nephrology, mesangial proliferative glomerulonephritis and focal and sclerosing glomerulonephritis. Conclusion Treatment of AITD associated nephrosis itself and immunosuppressive agent therapy might be useful to get remission of it.

Microscopic Identification of Medicinal Material of Herba Rabdosiae Serrae / 广州中医药大学学报

[Objective] To make a microscopic identification of medicinal material of Herba Rabdosiae Serrae (HRS) from different plant resources. [Methods] The dry leaves of the whole, the middle part and the upper-middle part from the plants of Isodon serra (Maxim.) Kudo, Isodon striatus (Benth.) Kudo, Isodon lophanthoides var. geradianus (Benth.) Hara, and Isodon lophanthoides Hara var. graciliflorus (Benth.) Hara were sliced by different methods. Structure of leaf epidermis, the transverse section of leaf and structure of leaf powder were observed under light microscope. [Results] There existed differences in epidermal cells, stomata distribution, nonglandular hair, glandular hair, small glandular hair and glandular scale of leaf epidermis from four kinds of medicinal plants of Herba Rabdosiae Serrae . And there also existed differences in epidermal cells, anticlinal wall and external-section wall of epidermal cells, nonglandular hair, glandular hair, glandular scale, stomata distribution, mesophyll, main vessels, and vascular bundle of leaf transverse section, as well as in epidermal cells, stomata distribution, glandular scale, nonglandular hair, glandular hair and ducts of leaf powder from four kinds of medicinal plants of Herba Rabdosiae Serrae. [Conclusion] Medicinal plants of different kinds of Herba Rabdosiae Serrae have different microscopic identification structure in leaf epidermis, leaf transverse section and leaf powder.

Strategy for Screening Leading Compounds From Botanicals / 中国药房

OBJECTIVE:To discuss the strategy for screening new leading compounds and discover the novel drug candidates,which have the independent intelligent property right,from botanicals METHODS:Screening the new leading componds from the single plant which plays the main therapeutic role in classical prescriptions or proved recipes were reviewed RESULTS:The results showed that the frequency of use of certain kinds of plant was higher than that of the others in treatment of certain disease,it is hypothesized that the plant may contain the active ingredient for the therapy of certain kinds of disease CONCLUSION:Screening new leading compound from Chinese materia medica is a shortcut with less risk,which may promote the development of new drugs in China

Chronic Renal Failure Complicated by Primary Hypothyroidism / 天津医药

Objective: To investigate the clinical features of chronic renal failure (CRF) complicated by primary hypothyroidism.Methods: Thyroid function, thyroid microsome antibody ( TM), and thyroid globulin antibody (TG) were determined withradioimmunoassay in 23 CRF complicated by primary hypothyroidism patients, and the serum calcium, phosphorus, potassi-um, cholesterol, glucose, urea nitrogen, hemoglobulin, and endogenous creatinine clearance rate were also assayed. Thirty-onepatients with only CRF were in control group. Results:The level of thyroid-stimulating hormone (TSH), Ch, and Ca in pa-tients with CRF complicated by primary hypothyroidism were higher than that in control group,and serum free T4 (FT4),K,and P were lower than that in control group. The others had no difference between two groups. TG and TM increased in2 cases. Conclusion:The most important criteria in diagnosing CRF complicated by primary hypothyroidism is TSH twotimes higher than normal upper limit. The others include decreased FT4, normal level of Ca, P, and K, and increased Ch,TG,and TM. The diagnosis should be performed combined with clinical manifestations.

Association between apoptosis induced in hepatoblastoma HepG2 cells by Cdk7 silencing and decreased pRb and Cdk2 phosphorylation / 中国药理学通报

Aim To evaluate the effect of Cdk7 silencing on the c ell cycle control, the phosphorylation level changes of Cdk2 and pRb in human he patoblastoma HepG2 cell culture in vitro, and to validate Cdk7 as a novel ta rget for anticancer therapeutics.Method Levels of Cdk7 and the phosphorylation levels of Cdk2 and pRb were measured by Western-blotting. DNA c ontents, cell cycle and apoptosis induced by Cdk7 silencing were analyzed by flo w cytometry and ultrastructural changes of cells were observed with transmission electron microscopy.Result The phosphorylation levels of pRb a nd Cdk2 and the levels of Cdk7 decreased in a concentration-dependent manner wh en the concentration was above 100 nmol?L -1. Indice of cells arrested in G 0/G 1 phases and apoptotic cells increased in a dosage-and time-dependent manner, the difference was significant between Cdk7 ASODN and the sense control (P