Identification of the essential catalytic residues and selectivity-related residues of maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose. Mutations at residues D255, E286, and D380 were constructed to identify the essential catalytic residues of MTHase, while mutations at residues W218, A259, Y328, F355, and R356 were constructed to identify selectivity-related residues of the enzyme. The specific activities of the purified D255A, E286A, and D380A MTHases were only 0.15, 0.09 and 0.01%, respectively, of that of wild-type MTHase, suggesting that these three residues are essential catalytic residues. Compared with wild-type MTHase, A259S, Y328F, F355Y, and R356K MTHases had increased selectivity ratios, which were defined as the ratios of the catalytic efficiencies for glucose formation to those for trehalose formation in the hydrolysis of maltooligosaccharides and maltooligosyltrehaloses, respectively, while W218A and W218F MTHases had decreased selectivity ratios. When starch digestion was carried out at 75 degrees C and wild-type and mutant MTHases were, respectively, used with isoamylase and maltooligosyltrehalose synthase (MTSase), the ratios of initial rates of glucose formation to those of trehalose formation were inversely correlated to the peak trehalose yields.     

山东花生花叶病毒的研究

 从表现生长抑制和花叶病状的花生叶分离到一种花生花叶病毒(PMV),可通过汁液和桃蚜(Myzus Persicae)传播,它的寄主范围包括豆科和非豆科植物,和花生斑驳病毒不同。病毒的体外存活力4-5天,致死温度40°-50℃,稀释终点10^-3-14^-4;用氯仿澄清,聚乙二醇沉淀和差速离心可部分提纯。病毒颗粒长约725毫微米,沉降系数173S。在感病的心叶烟(Nicotiana glutinosa)叶细胞质中可见典型的风轮状内含体,表明PMV是马铃薯Y病毒组的一个成员。     

北京瓜类花叶病毒的调查研究

北京郊区南瓜、西葫芦花叶病是由甜瓜花叶病毒类型所致。黄瓜、参瓜、丝瓜的花叶病都是由黄瓜花叶病毒类型所引起;黄瓜花叶病毒除为害瓜类作物外,还广泛分布在郊区其他栽培作物和野生植物上。根据 CMV 各分离物在冬瓜、南瓜、蔓陀罗,心叶菸,菸上的病状反应,建议将 CMV 分离物区分为八个毒株。来自南瓜并侵染西瓜的一个甜瓜花叶病毒分离物在侵染丝瓜后不能用汁接侵染荀瓜。     

Comparison between analgesic effects of buprenorphine, carprofen, and buprenorphine with carprofen for canine ovariohysterectomy

OBJECTIVE: To compare the analgesic effects of buprenorphine, carprofen, and their combination in dogs undergoing ovariohysterectomy. STUDY DESIGN: Prospective, randomized blinded clinical study. ANIMALS: 60 dogs. METHODS: Treatments were buprenorphine 0.02 mg kg(-1), intramuscularly (IM) (group B); carprofen 4 mg kg(-1), subcutaneously (SC) (group C); or a combination of both (group CB). Anesthesia was induced with propofol and maintained with isoflurane. A Dynamic Interactive Visual Analog Scale (DIVAS, 0-100 mm) and the Glasgow Composite Pain Scale (GCMPS, 0-24) were used to evaluate comfort and sedation at baseline, 2, 4, 6, and 24 hours after extubation. Rescue analgesia was provided with buprenorphine (0.02 mg kg(-1)). Wound swelling measurements (WM) and a visual inflammation score (VIS) of the incision were made after surgery and 2, 4, 6, and 24 hours later. p < 0.05 was considered significant. RESULTS: Group C required more propofol (5.0 +/- 1.4 mg kg(-1)) compared with B (3.3 +/- 1.1 mg kg(-1)) and CB (3.2 +/- 0.7 mg kg(-1)); respectively, p = 0.0002 and 0.0001. Rescue analgesia was required in nine dogs. B had a higher GCMPS and DIVAS III score at 6 hours (2.6 +/- 2.5) and (23 +/- 22.5 mm) compared with C (1.0 +/- 1.3, 6 +/- 7.3 mm) and CB (1.5 +/- 1.4, 8 +/- 10.7 mm); respectively, p = 0.02 and 0.006. Group C had a lower sedation score at 2 hours (43 +/- 23.6 mm) compared with B (68 +/- 32.1 mm) and BC (69 +/- 22.1 mm); respectively, p = 0.03 and 0.004. Group B had a higher WM score at 2 hours (3 +/- 0.8 mm) compared with C (2 +/- 0.6 mm) p = 0.01 and at 6 hours (3 +/- 1 mm) compared with C (2 +/- 0.8 mm) and CB (2 +/- 0.8 mm); respectively, p = 0.01 and 0.008. VIS was not different between groups. CONCLUSION AND CLINICAL RELEVANCE: All treatments provided satisfactory analgesia for the first 6 hours and at 24 hours. C and CB pain score and WS were superior to B at 6 hours. No superior analgesic effect was noted when the drugs were combined.     

Inhibitory effect and mechanisms of an anthocyanins- and anthocyanidins-rich extract from purple-shoot tea on colorectal carcinoma cell proliferation

One newly bred variety of tea cultivar, purple-shoot tea, was selected to evaluate its antiproliferative effects on colorectal carcinoma cells, as well as normal colon cells. The phytochemicals and identified catechins of purple-shoot tea extract (PTE) were significantly higher than that of ordinary tea, especially the anthocyanins (surpassed by 135-fold) and anthocyanidins (surpassed by 3.5-fold). PTE inhibited the proliferation of COLO 320DM (IC(50) = 64.9 μg/mL) and HT-29 (IC(50) = 55.2 μg/mL) by blocking cell cycle progression during the G(0)/G(1) phase and inducing apoptotic death. Western blotting indicated that PTE induced cell cycle arrest by reducing the expression of cyclin E and cyclin D1 in COLO 320DM and the upregulation of p21 and p27 cyclin-dependent kinase inhibitors in HT-29. Two cells treated with PTE also indicated the cleavage of PARP, activation of caspase 3, and an increased Bax/Bcl-2 ratio. Our results showed that PTE is a potential novel dietary agent for colorectal cancer chemoprevention.     

Effect of routine cardiovascular catheterization on cardiac troponin I concentration in dogs

ObjectiveTo determine changes in cardiac troponin I concentration (cTnI) associated with cardiovascular catheterization in dogs.Animals, materials and methodscTnI was measured after transarterial coil embolization of patent ductus arteriosus (PDA), balloon valvuloplasty (BV), and pacemaker implantation (PACE). Dogs undergoing ovariohysterectomy (OHE) were used as a control, with 15 animals in each group. Blood for the cTnI assay was collected at baseline (T0), at 5 h (T5), 24 h (T24) and 10 days (T240) post-procedure. The effects of age, duration and difficulty of the procedure were evaluated.ResultsThere was no difference in cTnI concentration at T0 for any of the groups. There was a significant increase in cTnI concentration for BV and PACE, but not PDA at T5 and T24. PACE at T24 and T240 also had higher cTnI than control. Dogs with longer procedure times had significantly higher concentration of cTnI. There was no correlation between the difficulty of the procedure or peri-procedure complications and cTnI.ConclusioncTnI increased during some cardiovascular catheterization procedures, but returned to normal values at 24–240 h. Patients undergoing long catheterization procedures have increased risk for myocardial injury, but this was not related to short-term prognosis.     

Development of the activities of pancreatic and caecal enzymes in White Roman goslings

1. This study was to investigate the development of the activities of pancreatic and caecal enzymes in White Roman goslings from hatching to 28 d of age. 2. A total of 80 1-d-old goslings were used. At hatching, 3 and 7 d of age, 16 goslings (8 males and 8 females) were used. At 11, 14, 21 and 28 d of age, 8 goslings, 4 males and 4 females were selected. The activities of amylase, lipase, trypsin and chymotrypsin in each segment of the small intestine (duodenum, jejunum, ileum) and their contents and the activity of cellulase in the caecal contents were measured. 3. The specific activity (SA) of amylase in the duodenal mucosa and contents increased significantly both from 11 to 14 and 14 to 21 d of age and declined after 28 d of age. In the jejunum and ileum, there was a significant peak in the SA of amylase in the mucosa in goslings from 7 to 21 d of age. On average, the SA of lipase of mucosa and content in the small intestine was initially low but gradually increased from 14 to 21 and from 21 to 28 d of age. 4. The SA of trypsin in the contents of the duodenum and the jejunum increased both from 7 to 11 and 11 to 14 d of age and there was high activity in the ileal contents from 14 to 21 d of age. From 3 to 14 d of age, the SA of chymotrypsin in the duodenal mucosa significantly increased and peaked. There was a significant peak at 11 d of age in the SA of chymotrypsin in the duodenal or jejunal contents and in the ileal contents at 7 d. The SA of cellulase in the caecal contents increased linearly with age, reaching a plateau at 28 d of age. 5. The rate of activity development varied from hatching to 28 d of age. However, the mean SA of amylase and lipase of intestinal contents reached a peak at 21 d, trypsin and chymotrypsin at 11 d and cellulase at 28 d of age, respectively. The quantitative changes in SA of cellulase, amylase, lipase, trypsin and chymotrypsin increased by about 4-, 3-, 5-, 2- and 3-fold, respectively, in the intestinal content of goslings. Thus, development of proteases in the intestine of goslings peaked more rapidly than amylase, lipase and cellulase during the first 4 weeks.     

Expression, purification, and characterization of the maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence of IPTG induction. Wild-type MTHase was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified wild-type MTHase showed an apparent optimal pH of 5 and an optimal temperature at 85 degrees C. The enzyme was stable at pH values ranging from 3.5 to 11, and the activity was fully retained after a 2-h incubation at 45-85 degrees C. The k(cat) values of the enzyme for hydrolysis of maltooligosyltrehaloses with degree of polymerization (DP) 4-7 were 193, 1030, 1190, and 1230 s(-1), respectively, whereas the k(cat) values for glucose formation during hydrolysis of DP 4-7 maltooligosaccharides were 5.49, 17.7, 18.2, and 6.01 s(-1), respectively. The K(M) values of the enzyme for hydrolysis of DP 4-7 maltooligosyltrehaloses and those for maltooligosaccharides are similar at the same corresponding DPs. These results suggest that this MTHase could be used to produce trehalose at high temperatures.