小麦木聚糖酶抑制蛋白的分离纯化及其性质研究

【目的】从成熟的小麦籽粒中纯化小麦木聚糖酶抑制蛋白,并研究其对木聚糖酶的抑制作用,从而探明小麦中木聚糖酶抑制蛋白对外加木聚糖酶的影响。【方法】采用硫酸铵盐析、Macro-prep CM阳离子交换层析、Macro-prep DEAE阴离子交换层析以及Macro-prep 25S阳离子交换层析等方法,从小偃22小麦中分离纯化木聚糖酶抑制蛋白,通过SDS-PAGE电泳检测木聚糖酶抑制蛋白的纯度和分子质量,并进一步研究了不同温度和反应时间对木聚糖酶抑制蛋白抑制活力的影响。【结果】小麦木聚糖酶抑制蛋白分子质量约为29ku,N端氨基酸序列为SVSSVVS,经NCBI BLAST比对,该抑制蛋白N端序列与其他木聚糖酶抑制蛋白无同源性,但与大麦几丁质酶的N端序列高度同源,却无几丁质酶活性;该蛋白对来自黑曲霉的GH11家族木聚糖酶有强烈的抑制活性,但对GH10家族的木聚糖酶不敏感;该蛋白与木聚糖酶结合的最适温度为30℃,最佳时间为30min;该抑制蛋白的半失活温度为74℃,具有较好的热稳定性。【结论】所获得的小麦木聚糖酶抑制蛋白可能是一种新型的XIP型木聚糖酶抑制蛋白。

重组textlinin-1抑制活性的性质研究

目的：对毕赤酵母表达的 textlinin-1（Q8008）进行分离纯化，并对其抑制plasmin活性的性质进行研究。方法构建表达 Q8008 pachia pastoris工程菌，经发酵、纯化得到纯度达95％以上的 Q8008。考察不同的温度、pH 缓冲液和金属离子等条件对 Q8008抑制plasmin活性的影响。结果 Q8008在中性条件下比较稳定，其最适pH 值为7.0，最适温度为40℃，在30～50℃相对酶活力在90.0％以上；有2个金属离子对 Q8008活性测定方法的影响产生了显著性差异，Zn2＋起激活作用，Co2＋起抑制作用，其余金属离子均无显著性差异。结论 Q8008在中性 pH 下抑制活性稳定；在50℃以下，受温度变化影响较小；金属离子对其抑制活性测定有一定影响。

XIP木聚糖酶抑制蛋白对不同家族木聚糖酶的抑制作用

研究利用毕赤酵母GS115表达的小麦木聚糖酶抑制蛋白,探索了该XIP抑制蛋白对GH10和GH11家族木聚糖酶的抑制作用。结果显示,该抑制蛋白分子量约为37 k Da,高于氨基酸序列计算所得分子量30.2 k Da,可能是由于该蛋白发生了糖基化作用。该蛋白分别作用于黑曲霉GH10木聚糖酶和哈茨木霉GH11木聚糖酶,得到最适抑制比例分别为1.2??1和8??1,最适抑制时间分别是50 min和30 min,最适抑制p H值均为6.0,最适作用温度均是40℃。

手性质子泵抑制剂艾司奥美拉唑的研究进展

艾司奥美拉唑是第一个上市的手性质子泵抑制剂(PPI),其经CYP2C19与CYP3A4代谢。同其他PPI相比,艾司奥美拉唑在强、弱代谢者中的AUC比值变异度最低;且AUC高于消旋体奥美拉唑,具有较强的抑酸效果;在治疗胃食管返流病、幽门螺旋杆菌感染、胃溃疡以及消化道出血中疗效较好,不良反应少。作为手性PPI,艾司奥美拉唑与奥美拉唑具有相似的优点,又具有自己独特的药动学特征,弥补了不足,在临床上有着广阔的应用前景。

Overexpression of lentivirus-mediated glial cell line-derived neurotrophic factor in bone marrow stromal cells and its neuroprotection for the PC12 cells damaged by lactacystin

Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor （GDNF）, and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfecting it into bone marrow stromal cells （BMSCs）. Methods pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. Results Virus stock of GDNF was harvested with the titer of 5.6×10^5 TU/mL. After tmnsduction, GDNF-BMSCs successfully secreted GDNF to supematant with nigher concentration （800 pg/mL） than BMSCs did （less than 100 pg/mL）. The supematant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin （10 μmol/L）. Conclusion Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.

Cantharidin and Its Analogues:Anticancer and Ser/Thr Protein Phosphatase Inhibitory Activities

This paper mainly describes the anticancer activities and Ser/Thr protein phosphatase inhibitory activities of cantharidin and its analogues.

Corrosion inhibition effect of N, N′-bis(2-pyridylmethylidene)-1,2-diiminoethane on AZ91D magnesium alloy in acidic media

The inhibition effect of electrochemical noise, EIS and surface analysis to evaluate N'-bis (2-pyridylmethylidene)- 1,2-diiminoethane (BPIE) Schiff base against AZ91D alloy corrosion in 0.01 mol/L HCl was investigated by different electrochemical methods. Potentiodynamic polarization curves revealed that the BPIE acts as a mixed-type corrosion inhibitor. Electrochemical impedance spectroscopy (EIS) measurements confirmed the corrosion inhibition effect of the BPIE. As the inhibitor concentration increased, the charge transfer resistance increased and the double layer capacitance decreased due to more inhibitor adsorption on the surface. The results obtained by analysis of electrochemical noise (EN) data in time and frequency domains are in good agreement with EIS and polarization results. Moreover, scanning electron microscopy (SEM), X-ray diffraction (XRD) and energy dispersive X-ray (EDX) were used to investigate the corrosion inhibition of the BPIE. SEM images showed that the corrosion damage of the alloy surface reduced in the presence of BPIE. The intensity of the XRD peaks corresponding to magnesium-rich α phase increased in the presence of BPIE, indicating lower corrosion of alloy sample. Also, EDX analysis approved the corrosion inhibition performance of the BPIE. The studied Schiff base compound acts by physical adsorption on the alloy surface and its adsorption obeys the Langmuir isotherm.

Management of acute nonvariceal upper gastrointestinal bleeding:Current policies and future perspectives

Acute upper gastrointestinal bleeding(UGIB) is a gastroenterological emergency with a mortality of 6%-13%.The vast majority of these bleeds are due to peptic ulcers.Nonsteroidal anti-inflammatory drugs and Helicobacter pylori are the main risk factors for peptic ulcer disease.Endoscopy has become the mainstay for diagnosis and treatment of acute UGIB,and is recommended within 24 h of presentation.Proton pump inhibitor(PPI) administration before endoscopy can downstage the bleeding lesion and reduce the need for endoscopic therapy,but has no effect on rebleeding,mortality and need for surgery.Endoscopic therapy should be undertaken for ulcers with high-risk stigmata,to reduce the risk of rebleeding.This can be done with a variety of modalities.High-dose PPI administration after endoscopy can prevent rebleeding and reduce the need for further intervention and mortality,particularly in patients with high-risk stigmata.

Management of non-variceal upper gastrointestinal tract hemorrhage:Controversies and areas of uncertainty

Upper gastrointestinal tract hemorrhage （UGIH） remains a common presentation requiring urgent evaluation and treatment. Accurate assessment, appropriate intervention and apt clinical skills are needed for proper management from time of presentation to discharge. The advent of pharmacologic acid suppression, endoscopic hemostatic techniques, and recognition of Helicobacter pylori as an etiologic agent in peptic ulcer disease （PUD） has revolutionized the treatment of UGIH. Despite this, acute UGIH still carries considerable rates of morbidity and mortality. This review aims to discuss current areas of uncertainty and controversy in the management of UGIH. Neoadjuvant proton pump inhibitor （PPI） therapy has become standard empiric treatment for UGIH given that PUD is the leading cause of non variceal UGIH, and PPIs are extremely effective at promoting ulcer healing. Howeve, neoadjuvant PPI administration has not been shown to affect hard clinical outcomes such as rebleeding or mortality. The optimal timing of upper endoscopy in UGIH is often debated. Upon completion of volume resuscitation and hemodynamic stabilization, upper endoscopy should be performed within 24 h in all patientswith evidence of UGIH for both diagnostic and therapeutic purposes. With rising healthcare cost paramount in today＇s medical landscape, the ability to appropriately triage UGIH patients is of increasing value. Upper en- doscopy in conjunction with the clinical scenario allows for accurate decision making concerning early discharge home in low risk lesions or admission for further moni toring and treatment in higher risk lesions. Concomitant pharmacotherapy with non steroidal antiinflammatory drugs （NSAIDs） and antiplatelet agents, such as clopidogrel, has a major impact on the etiology, severib/, and potential treatment of UGIH. Long term PPI use in patients taking chronic NSAIDs or clopidogrel is discussed thoroughly in this review.

Purification and Characterization of Angiotensin I Converting Enzyme Inhibition Peptides from Sandworm Sipunculus nudus

Three angiotensin I converting enzyme(ACE) inhibition peptides were isolated from sandworm Sipunculus nudus protein hydrolysate prepared using protamex. Consecutive purification methods, including size exclusion chromatography and reverse-phase high performance liquid chromatography(RP-HPLC), were used to isolate the ACE inhibition peptides. The amino acid sequences of the peptides were identified as Ile-Asn-Asp, Val-Glu-Pro-Gly and Leu-Ala-Asp-Glu-Phe. The IC_(50) values of the purified peptides for ACE inhibition activity were 34.72 μmol L^(-1), 20.55 μmol L^(-1) and 22.77 μmol L^(-1), respectively. These results suggested that S. nudus proteins contain specific peptides that can be released by enzymatic hydrolysis. This study may provide an experimental basis for further systematic research, rational development and clinical utilization of sandworm resources.

Proton pump inhibitor-induced hypomagnesemia: A new challenge

Proton pump inhibitors （PPIs） are commonly used in clinical practice for the prevention and treatment of peptic ulcer, gastritis, esophagitis and gastroesophageal refux. Hypomagnesemia has recently been recognized as a side effect of PPIs. Low magnesium levels may cause symptoms from several systems, some of which being potentially serious, such as tetany, seizures and arrhythmias. It seems that PPIs affect the gastrointesti-nal absorption of magnesium. Clinicians should be vigi-lant in order to timely consider and prevent or reverse hypomagnesemia in patients who take PPIs, especially if they are prone to this electrolyte disorder.

Double layered self-expanding metal stents for malignant esophageal obstruction, especially across the gastroesophageal junction

AIM: To evaluate the clinical outcomes of double-layered self-expanding metal stents (SEMS) for treatment of malignant esophageal obstruction according to whether SEMS crosses the gastroesophageal junction (GEJ). METHODS: Forty eight patients who underwent the SEMS insertion for malignant esophageal obstruction were enrolled. Patients were classified as GEJ group (SEMS across GEJ, 18 patients) and non-GEJ group (SEMS above GEJ, 30 patients) according to SEMS position. Double layered (outer uncovered and inner covered stent) esophageal stents were placed. RESULTS: The SEMS insertion and the clinical improvement were achieved in all patients in both groups. Stent malfunction occurred in seven patients in the GEJ group and nine patients in the non-GEJ group. Tumor overgrowth occurred in five and eight patients, respectively, food impaction occurred in one patient in each group, and stent migration occurred in one and no patient, respectively. There were no significant differences between the two groups. Reflux esophagitis occurred more frequently in the GEJ group (eight vs five patients, P = 0.036) and was controlled by proton pump inhibitor. Aspiration pneumonia occurred in zero and five patients, respectively, and tracheoesophageal fistula occurred in zero and two patients, respectively. CONCLUSION: Double-layered SEMS are a feasible and effective treatment when placed across the GEJ for malignant esophageal obstruction. Double-layered SEMS provide acceptable complications, especially migration, although reflux esophagitis is more common in the GEJ group.

Head-to-head comparison of H_2-receptor antagonists and proton pump inhibitors in the treatment of erosive esophagitis: A meta-analysis

AIM: To systematically evaluate the efficacy of H2-receptor antagonists (H2RAs) and proton pump inhibitors in healing erosive esophagitis (EE).METHODS: A meta-analysis was performed. A literature search was conducted in PubMed, Medline, Embase, and Cochrane databases to include randomized controlled head-to-head comparative trials evaluating the efficacy of H2RAs or proton pump inhibitors in healing EE. Relative risk (RR) and 95% confidence interval (CI) were calculated under a random-effects model.RESULTS: RRs of cumulative healing rates for each comparison at 8 wk were: high dose vs standard dose H2RAs,1.17 (95%CI, 1.02-1.33); standard dose proton pump inhibitors vsstandard dose H2RAs, 1.59 (95%CI, 1.44-1.75);standard dose other proton pump inhibitors vs standard dose omeprazole, 1.06 (95%CI, 0.98-1.06). Proton pump inhibitors produced consistently greater healing rates than H2RAs of all doses across all grades of esophagitis, including patients refractory to H2RAs. Healing rates achieved with standard dose omeprazole were similar to those with other proton pump inhibitors in all grades of esophagitis.CONCLUSION: H2RAS are less effective for treating patients with erosive esophagitis, especially in those with severe forms of esophagitis. Standard dose proton pump inhibitors are significantly more effective than H2RAs in healing esophagitis of all grades. Proton pump inhibitors given at the recommended dose are equally effective for healing esophagitis.

Reduction of gastrointestinal motility by unilateral thyroparathyroidectomy plus subdiaphragmatic vagotomy in rats

AIM: To investigate whether the combined methods of unilateral thyroparathyroidectomy (TPX) and subdiaphragmatic vagotomy (VAX) can be adapted for rats and used as a reliable method to produce a rat model of long-term reduction of gastrointestinal (GI) motor function. METHODS: Male Sprague-Dawley rats were randomly divided into 3 groups, normal, sham-operated and unilateral TPX plus VAX. The TPX plus VAX rats received VAX 7 d after application of TPX, and dietary intake and fecal output were then measured daily for 1 wk.After completion of the experiments, gastric emptying and small bowel transit were measured in vivo, and the contractile responses of colonic strips to excitatory and inhibitory neurotransmitters were estimated using isometric force transducers in vitro. RESULTS: In comparison with normal and sham-operated rats, rats which received unilateral TPX plus VAX showed a significant decrease in body weight and in fecal pellet number and weight throughout the entire week. Application of TPX plus VAX to rats markedly delayed gastric emptying and small bowel transit. In TPX plus VAX rats, the longitudinal muscles of the proximal colon showed a significant reduction in contractile responses to acetylcholine (5 × 10-6 mol/L), and a dramatic attenuation of contractile responses was also observed in both the longitudinal and circular muscles of the distal colon. However, the spontaneous contractility of the colonic strips from TPX plus VAX rats was not significantly affected by treatment with N-nitro-Larginine-methyl ester (0.1 mol/L). CONCLUSION: The results indicate that unilateral TPX plus VAX reduced the motor function of the GI tract in rats, and the reduced gut motility is likely mediated, at least in part, by inhibition of the excitatory neurotransmitter system.

Impaired suppressive activities of human MUTYH variant proteins against oxidative mutagenesis

AIM:To investigate the suppressive activity of MUTYH variant proteins against mutations caused by oxidative lesion,8-hydroxyguanine(8OHG),in human cells.METHODS:p.R154H,p.M255V,p.L360P,and p.P377L MUTYH variants,which were previously found in patients with colorectal polyposis and cancer,were selected for use in this study.Human H1299 cancer cell lines inducibly expressing wild-type(WT) MUTYH(type 2) or one of the 4 above-mentioned MUTYH variants were established using the piggyBac transposon vector system,enabling the genomic integration of the transposon sequence for MUTYH expression.MUTYH expression was examined after cumate induction using Western blotting analysis and immunofluorescence analysis.The intracellular localization of MUTYH variants tagged with FLAG was also immunofluorescently examined.Next,the mutation frequency in the supF of the shuttle plasmid pMY189 containing a single 8OHG residue at position 159 of the supF was compared between empty vector cells and cells expressing WT MUTYH or one of the 4 MUTYH variants using a supF forward mutation assay.RESULTS:The successful establishment of human cell lines inducibly expressing WT MUTYH or one of the 4 MUTYH variants was concluded based on the detection of MUTYH expression in these cell lines after treatment with cumate.All of the MUTYH variants and WT MUTYH were localized in the nucleus,and nuclear localization was also observed for FLAG-tagged MUTYH.The mutation frequency of supF was 2.2 × 10-2 in the 8OHG-containing pMY189 plasmid and 2.5 × 10-4 in WT pMY189 in empty vector cells,which was an 86-fold increase with the introduction of 8OHG.The mutation frequency(4.7 × 10-3) of supF in the 8OHG-containing pMY189 plasmid in cells overexpressing WT MUTYH was significantly lower than in the empty vector cells(P < 0.01).However,the mutation frequencies of the supF in the 8OHG-containing pMY189 plasmid in cells overexpressing the p.R154H,p.M255V,p.L360P,or p.P377L MUTYH variant were 1.84 × 10-2,1.55 × 10-2,1.91 × 10-2,and 1.96 × 10-2,respectively,meaning that no significant difference was observed in the mutation frequency between the empty vector cells and cells overexpressing MUTYH mutants.CONCLUSION:The suppressive activities of p.R154H,p.M255V,p.L360P,and p.P377L MUTYH variants against mutations caused by 8OHG are thought to be severely impaired in human cells.

Construction of Free Amino Acids Composition Data Base for Food

Significant amounts of free amino acids exist in commercially sold vegetables and fruits. Despite of the fact, only a little information is available about the free amino acid contents in foods. To utilize information of free amino acids in food, we have carried out the experiments to quantitate the free amino acids by derivatized with NBD-F （4-fluoro-7-nitrobenzofurazan） and analyzed on reversed-phase UHPLC （ultra-high pressure liquid chromatography） equipped with ultraviolet visible detector. Almost all of food extracts contained free amino acids including GABA （T-amino butyrate）. Contents of free amino acids vary considerably depending upon vegetables and fruits. Principal free amino acids found in vegetables and fruits were asparagine, glutamine, arginine and GABA, which are involved in important metabolic pathways in human. About 140 species of vegetables and fruits were subjected for the data base. All of the plants and fruits we examined exhibit significant amount of free amino acids, those are clearly distinct from data bases obtained after acid hydrolysis treated food samples. Since glutamate and GABA act as excitatory and inhibitory neurotransmitters in CNS, respectively; free amino acids in vegetables and fruits that we eat daily, should be an important source for the cellular metabolic activities.

Antidepressant Drugs Modulate Differentially Anti-inflammatory Lithium＇s Property： An in Vitro and in Vivo Study

Li （lithium）, a mood stabilizer has anti-inflammatory effect. However, in clinical practice, Li can be administered together with other antidepressants drugs, such as FLX （fluoxetine）, IMI （imipramine）, NOR （nortriptyline） and ESC （escitalopram）. As interaction between Li and these antidepressant drugs on inflammatory modulation has not been investigated yet, we performed an in vitro protocol using a non-human macrophage cell line. Oxidative and inflammatory markers, as well as cell cycle analysis and cytokine gene expressions were compared among treatments. An IR （inflammatory ratio） was calculated based on the following oxidative-inflammatory variables： nitric oxide, superoxide anion, reactive oxygen molecules, cytokines IL-113, IL-6, TNF-ct and IL-10. The in vitro calculated IR data were validated through an in vivo analysis of 154 human subjects with similar IR. Li and control cells presented similar IR values. FLX, NOR and IMI increased slightly IR values indicating some proinflammatory effect, whereas ESC decreased IR values indicating some anti-inflammatory effect. However, cells exposed to Li ＋ ESC triggered a proinflammatory response on macrophages. Thus, IR comparison results suggest that the Li anti-inflammatory effect is not universal and could be influenced by both basal macrophage-inflammatory state and interaction of other psychiatric drugs. These results could be useful to understand some inconsistencies observed in human studies involving Li and other psychiatric drugs.

Up-regulation of Raf kinase inhibitor protein enhances chemosensitivity of cervical cancer cell

Objective： The purpose of the study is to investigate the effects of up-regulation of Raf kinase inhibitor protein （RKIP） on the chemosensitivity of cervical cancer Hela cells. Methods： Eukaryotic expression plasmid pcDNA3.1（±）-ssRKIP containing human overall length RKIPcDNA was transfected into cervical cancer Hela cell by lipofectin assay, establishing a stable cell line containing a target gene by G418. Expression of RKIP in Hela cells was measured by Western blot analysis. After treatment with cisplatin of different concentrations and intervals of time, the effect of RKIP on the proliferation of Hela cells was evaluated by MTT method. The flow cytometry was used to investigate whether the RKIP could inhibit apoptosis in Hela cells induced by cisplatin. Results： The expression of RKIP in Hela cells transfected with pcDNA3.1-ssRKIP was increased obviously. After different concentrations of cisplatin treatment cells for 24, 48 and 72 h, the growth inhibition rate in Hela cells transfected with pcDNA3.1-ssRKIP was significantly higher than in control cells （P 〈 0.05）. With 5 pg/mL cisplatin treatment for 24 h, pcDNA3.1-ssRKIP-transfected Hela cells had an obviously higher percentage of apoptosis （23.2 ± 0.24）% than non-transfected cells （12.4 ± 0.31）% and empty vector-transfected cells （13.4 ± 0.47）%. Without treatment of cisplatin, the percentage of apoptosis for Hela cells transfected with pcDNA3.1-ssRKIP was （5.7 ± 0.12）%, which was still higher than those of the non-transfected cells （2.9 ± 0.21）% and empty vector-transfected cells （3 ± 0.08）%. Conclusion： Higher expres- sion of RKIP gene can improve chemosensitivitv of cervical cancer Hela cells to cisplatin.

Effect of germination on the nutritional quality of Pearl millet

Changes in proximate composition, trypsin inhibitor activity, phytic acid, tannins, in vitro protein digestibility and amino acids content of Pearl millet were investigated after germination for 5 days. Germination significantly increased protein content of pearl millet, with a parallel decrease in lipid and carbohydrates. Trypsin inhibitor activity and the phytic acid content showed significant decrease whereas tannin content increased after 5 days germination. In vitro protein digestibility （IVPD） was significantly decreased with germination, suggesting that tannins may be responsible for enzymes inhibition. Amino acid analysis revealed significant increase in essential amino acids and none essential amino acids.

Macro Model Study for Nonlinear Spring of Tense Torsion Bar in Gap-Closing Type Electrostatic Micromirror

Nonlinear spring characteristics of the tense torsion bar in the gap-closing type electrostatic micromirror are examined. The macro model is introduced for the experimental study. The tension applied in the torsion bar is well controlled using the electromagnetic attraction. This controllability is suited for clearing the nonlinear nature. The tension is confirmed to have the effect to widen the controllable angle range of the mirror suppressing the pull-in. The pull-in angle is observed to increases to about 74% of the mechanical limit angle at the tension of 0,96 N. This is significantly larger than 44% of the case with the linear spring. The larger resonant frequency is maintained. The hardening of the spring can keep the balance with the electrostatic force over the limit of the linear spring. The observed features are explained reasonably with the combination of twisting and bending displacements of the torsion bar.