不同剂量二甲双胍对老年2型糖尿病男性患者骨密度和骨代谢的影响

目的: 观察不同剂量二甲双胍对老年2型糖尿病患者骨密度和骨代谢的影响。 方法: 未经治疗的老年2型糖尿病患者102例随机分为二甲双胍低剂量组（n=34）、二甲双胍中剂量组（n=34）和二甲双胍高剂量组（n=34）。3组分别给予：二甲双胍0.5 g,每日2次;二甲双胍0.5 g,每日3次;二甲双胍0.5 g, 每日4次。3组均连续口服3个月。服药前和服药后1、3个月分别测定患者腰椎（L_2-4）与股骨颈（Neck）的骨密度（BMD）及血骨特异性碱性磷酸酶（BAP）、骨钙素（BGP）以及抗酒石酸酸性磷酸酶（TRACP）、护骨因子（OPG）、空腹血糖（FBG）和糖化血红蛋白水平（HbAlC）。结果: 3组治疗后FBG和HbAlC均明显降低,与治疗前比较差异有统计学意义（P<0.05）,但3组间同期比较无统计学差异（P>0.05）。3组治疗前、后反映骨代谢的骨抑制指标TRACP和OPG无明显变化;反映骨转换与形成的指标BGP和BAP治疗后均升高,治疗前、后有统计学差异（P<0.05）。其中,二甲双胍高剂量组和中剂量组的BGP和BAP同期高于低剂量组,组间比较有统计学差异（P<0.05）,而二甲双胍中剂量组和高剂量组的BGP和BAP同期无统计学差异（P>0.05）。3组治疗3个月后腰椎（L_2-4）与股骨颈（Neck）的BMD均明显增加,二甲双胍高剂量组和中剂量组分别与低剂量组比较有统计学差异,但二甲双胍高剂量组和中剂量组并没有明显差异。结论: 二甲双胍对老年性2型糖尿病患者在发挥降糖作用的同时,还能使患者BMD得到改善,该作用可能主要是通过促进骨形成实现的,且疗效有一定剂量相关性。     

前列腺酸性磷酸酶在胃癌中的表达及其体外诱导抗胃癌免疫应答

目的检测前列腺酸性磷酸酶(PAP)在胃癌中的表达水平,探讨PAP作为胃癌主动免疫治疗新靶点的可能性。方法分别用RT-PCR和Western blot方法检测胃癌细胞系中PAP mRNA和PAP蛋白的表达;用免疫组织化学方法检测胃癌组织中PAP的表达。利用PAP表位肽对胃癌患者的PBMCs进行体外诱导,ELISA法检测PAP特异性IFN-γ分泌水平;51Cr释放法检测CTLs的细胞毒活性。结果3种胃癌细胞(MKN-7、MKN-28和MKN-45)表达PAP mRNA,其中MKN-28和MKN-45表达PAP蛋白,胃癌组织中PAP呈阳性表达。从2/4胃癌患者PBMCs中诱导出的PAP多肽特异性CTLs可特异性杀伤HLA-A24+/PAP+的胃癌细胞MKN-45,CTLs的细胞毒活性依赖于CD8+T淋巴细胞。结论PAP有可能成为胃癌特异性免疫治疗的新靶点。     

三七总皂苷对体外培养人牙周膜细胞增殖和分化的影响

目的探讨三七总皂苷对体外培养的人牙周膜细胞(Human periodontal ligament cell,hPDLC)增殖、碱性磷酸酶(Alkaline phosphatase,ALP)活性及表达的影响。方法采用组织块法原代培养hPDLC,并经免疫组化鉴定;分别用10、1.0、0.1和0.01 mg/L浓度的三七总皂苷处理体外培养的hPDLC,设只加含10%FBS的DMEM培养基为空白对照,分别于1、3、5、7 d后,MTT法检测其对hPDLC增殖活性的影响,酶标仪上检测细胞中ALP活性,Western blot法检测细胞中ALP蛋白的表达。结果免疫组化结果显示,细胞来源于外胚间充质;MTT结果显示,0.1 mg/L浓度组在第5天和1.0、10 mg/L浓度组在第3、5天hPDLC增殖活性明显高于空白对照组(P<0.01);0.1、1.0、10 mg/L浓度组在第3、5天ALP活性明显高于空白对照组(P<0.01),1.0 mg/L浓度组APL活性明显高于其他各组(P<0.01);0.01、0.1、1.0、10 mg/L浓度组ALP蛋白表达量均明显高于空白对照组(P<0.01),以1.0 mg/L浓度组ALP表达量最高。结论三七总皂苷有促进hPDLC增殖的作用,并能增加ALP活性及其表达水平。     

Detection of alkaline phosphatase by competitive indirect ELISA using immunoglobulin in yolk (IgY) specific against bovine milk alkaline phosphatase

Immunoglobulin in yolk (IgY) (with a titer of 1.3 × 10⁶) specific against bovine milk (BM) alkaline phosphatase (ALP) was obtained by intramuscularly immunizing hens on the thigh and was used as the primary antibody to conduct competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) to determine BMALP in ALPs from BM and Escherichia coli sources. A relationship between the ELISA value and the BMALP level (0.01–10 μg/mL) in whole milk (R² = 0.9019) or in skimmed milk (R² = 0.9402) was observed. The maximal inhibition (%) of BMALP on the microtiter plate by free BMALP at 10 μg/mL whole milk (3.89 mU/μg BMALP) was about 50%, while no inhibition (%) of BMALP by free E. coli ALP at concentrations between 0.01 to 10 μg/mL (60 mU/μg E. coli ALP) was determined. At BMALP levels higher than 0.1 μg/mL, CI-ELISA was proved to be effective in differentiating between BMALP and E. coli ALP and quantifying BMALP in whole milk or skimmed milk in the presence of E. coli ALP with an activity of 0.6 U/mL. Higher inhibition (about 70%) of BMALP on the microtiter plate by free BMALP in diluted (10¹–10⁴ fold) milk samples was observed. The optimal conditions for CI-ELISA in determining BMALP (0.1–10 μg/mL) from ALPs in milk samples were using 10³-fold diluted crude IgY specific against BMALP as primary antibody and 10³-fold diluted goat anti-chicken IgG–ALP conjugate as the secondary antibody.     

Phytochemistry, traditional uses and pharmacology of Eugenia jambolana Lam. (black plum): A review

Eugenia jambolana Lam. (syn. Syzigium cumini (L.) SKEELS; S. jambolana DC; Family: Myrtaceae), commonly known as black plum or Jamun is a plant native to India. Annually the trees produce oblong or ellipsoid fruits (berries). They are green when raw and purplish black when fully ripe. The ripe fruits are sweetish sour to taste and are used to prepare health drinks, squashes, juices, jellies and wine. Studies have shown that the berries contain carbohydrates, minerals and the pharmacologically active phytochemicals like flavonoids, terpenes, and anthocyanins. Jamun is a plant with known ethnomedicinal uses. Before the discovery of insulin, Jamun was useful in the treatment of diabetes and is an integral part in the various alternative systems of medicine. Scientific studies have shown that the various extracts of Jamun possess a range of pharmacological properties such as antibacterial, antifungal, antiviral, anti-genotoxic, anti-inflammatory, anti-ulcerogenic, cardioprotective, anti-allergic, anticancer, chemopreventive, radioprotective, free radical scavenging, antioxidant, hepatoprotective, anti-diarrheal, hypoglycemic and antidiabetic effects. The present paper reviews these aspects and also addresses the lacunas in the existing knowledge.     

Kinetic behaviour and stability of Escherichia coli ATCC27257 alkaline phosphatase immobilised in soil humates

Alkaline phosphatase (EC 3.1.3.1) extracted from Escherichia coli ATCC27257 was immobilised by co‐flocculation with soil humates in the presence of Ca²⁺. The effects of time, temperature, pH and concentration of enzyme and support on immobilisation were studied. Between 58 and 92% of the added phosphatase was strongly bound to the humates, depending on the conditions of immobilisation used. Some characteristics of the humate–phosphatase complexes and of the free enzyme were compared. The enzymatic complexes showed values of K_m (2.22 mM ) and activation energy (33.4 kJ mol^?1) similar to those of the free enzyme (2.00 mM and 27.6 kJ mol^?1). The pH/activity profiles revealed no change in terms of shape or optimum pH (10.5) upon immobilisation of alkaline phosphatase. However, the immobilised enzyme showed maximal activity in the range of 80–100 °C, while the free enzyme had its highest activity at 60 °C. The thermal stability of alkaline phosphatase was enhanced by complexation to the soil humates. © 2003 Society of Chemical Industry     

Zinc deprivation inhibits extracellular matrix calcification through decreased synthesis of matrix proteins in osteoblasts

Scope: Zinc is implicated as an activator for bone formation, however, its influence on bone calcification has not been reported. This study examined how zinc regulates the bone matrix calcification in osteoblasts. Methods and Results: Two osteoblastic MC3T3‐E1 cell subclones (SC 4 and SC 24 as high and low osteogenic differentiation, respectively) were cultured in normal osteogenic (OSM), Zinc deficient (Zn–, 1 μM), or adequate (Zn+, 15 μM) media up to 20 days. Cells (SC 4) were also supplemented with (50 μg/mL) or no ascorbic acid (AA) in combination with Zinc treatment. Zn– decreased collagen synthesis and matrix accumulation. Although AA is essential for collagen formation, its supplementation could not compensate for Zinc deficiency‐induced detrimental effects on extracellular matrix mineralization. Zn– also decreased the medium and cell layer alkaline phosphatase ALP activity. This decreased ALP activity might cause the decrease of Pi accumulation in response to Zn–, as measured by von Kossa staining. Ca deposition in cell layers, measured by Alizarin red S staining, was also decreased by Zn^–. Conclusion: Our findings suggest that zinc deprivation inhibits extracellular matrix calcification in osteoblasts by decreasing the synthesis and activity of matrix proteins, type I collagen and ALP, and decreasing Ca and Pi accumulation. Therefore zinc deficiency can be considered as risk factor for poor extracellular matrix calcification.     

Substrate Selection Influences Molecular Recognition in a Screen for Lymphoid Tyrosine Phosphatase Inhibitors

Assay design is an important variable that influences the outcome of an inhibitor screen. Here, we have investigated the hypothesis that protein tyrosine phosphatase inhibitors with improved biological activity could be identified from a screen by using a biologically relevant peptide substrate, rather than traditional phosphotyrosine mimetic substrates. A 2000‐member library of drugs and drug‐like compounds was screened for inhibitors of lymphoid tyrosine phosphatase (LYP) by using both a peptide substrate (Ac‐ARLIEDNE‐pCAP‐TAREG‐NH₂, peptide 1) and a small‐molecule phosphotyrosine mimetic substrate (difluoromethyl umbelliferyl phosphate, DiFMUP). The results demonstrate that compounds that inhibited enzyme activity on the peptide substrate had greater biological activity than compounds that only inhibited enzyme activity on DiFMUP. Finally, epigallocatechin‐3,5‐digallate was identified as the most potent inhibitor of lymphoid tyrosine phosphatase activity to date, with an IC₅₀ of 50 nM and significant activity in T‐cells. Molecular docking simulations provided a first model for binding of this potent inhibitor to LYP; this will constitute the platform for ongoing lead optimization efforts.