The first synthesis of natural disulfide bruguiesulfurol and biological evaluation of its derivatives as a novel scaffold for PTP1B inhibitors

Bruguiesulfurol (1), a cyclic 4-hydroxy-dithiosulfonate isolated from mangrove plant Bruguiera gymnorrhiza, was concisely synthesized for the first time in four steps, and a series of its synthetic derivatives were evaluated for in vitro inhibitory effects on PTP1B and related PTPs. Some derivatives were found to have improved pharmacological profile compared with hit 1. Among them, 5a showed the potent selectivity towards PTP1B over other PTPs, including TCPTP, and 7j exhibited the strongest PTP1B inhibitory activity with an IC₅₀ value of 4.54 μM.     

Design and synthesis of 2-N-substituted indazolone derivatives as non-carboxylic acid glycogen synthase activators

Glycogen synthase (GS) catalyzes the transfer of glucose residues from UDP-glucose to a glycogen polymer chain, a critical step for glucose storage. Patients with type 2 diabetes normally exhibit low glycogen levels and decreased muscle glucose uptake is the major defect in whole body glucose disposal. Therefore, activating GS may provide a potential approach for the treatment of type 2 diabetes. In order to identify non-carboxylic acids GS activators, we designed and synthesized a series of 2-N-alkyl- and 2-N-aryl-indazolone derivatives and studied their activity in activating human GS.     

Characterization of the Electrogenic Sodium Channel from Rat Brain Membranes Using Neurotoxin-Dependent 22Na Uptake

The sodium channel was studied in osmotically-sensitive membrane preparations from rat brain and in innervated and chronically denervated rat soleus and extensor digitorum longus muscles. These experiments were undertaken in order to define a set of parameters for sodium channel function at the subcellular level to be used as a measure of retention of channel integrity upon subsequent isolation of the channel. Various neurotoxins and drugs were employed to control the permeability of the brain membranes to ²²Na and the sodium-conductance properties of the muscles. Batrachotoxin (ED₅₀ = 0.2 μM), veratridine (ED₅₀ = 1 μM), or grayanotoxin I (ED₅₀ = 30 μM) stimulated ²²Na uptake in brain membranes is inhibited in an apparently uncompetitive manner by the sodium channel blocking agents tetrodotoxin and saxitoxin in a simple competitive manner by Ca²⁺ and in a partial or allosteric competitive manner by lidocaine and procaine. This ²²Na uptake assay, which can be equated to a measure of equilibrium toxin binding, shows dependence on the concentration of the membranes and is sensitive to pH, temperature, ionic strength, and the ionic composition of the media. Parallel biophysical studies on sodium channels in rat muscle show that the properties of the sodium channel are similarly affected by these agents.     

Direct Regulation of Na+-Dependent myo-Inositol Transport by Sugars in Retinal Pigment Epithelium: Role of Phorbol Ester and Staurosporin

An Na⁺-dependent active process for myo-inositol (MI) uptake, sharing a common carrier system with glucose and sensitive to phlorizin, was previously established in primary cultures of bovine retinal pigment epithelial (RPE) cells (26, 32). The present report further examines the nature of glucose-induced inhibition of MI transport in primary cultures of RPE cells. RPE cells were grown in supplemented Dulbecco's modification of Eagle's medium (DMEM) containing 5 mM D-glucose (basic growth media) or 40 mM D-glucose or its nonmetabolizable analogue, α-methyl-D-glucoside (αMG); 1–5 mM nonradioactive MI, pyruvate, or lactate; or 0.2–20 µM phorbol 12-myristate 13-acetate (TPA) or straurosporin (modified growth media), for up to 4 weeks. The capacity of RPE cells to accumulate ³H-MI (ratios of intracellular transported radioactive MI, [MI]_i, to external free MI concentration, [MI]_i/[MI]₀) decreased by up to 41% or 34% when cells were grown for 10 days or longer with 40 mM D-glucose or 40 mM αMG, respectively, compared to cells grown in basic growth media. The rate of uptake of ³H-MI also was reduced to 63 ± 15% or 48 ± 8% of the control values when cells were fed 1 or 5 mM nonradioactive MI, respectively. In addition, cellular capacity to bind to [³H]phlorizin was reduced to 52 ± 7%, 61 ± 5%, or 38 ± 6% of the controls when RPE cells were fed 40 mM D-glucose, 40 mM αMG, or 5 mM nonradioactive MI, respectively. Growth media containing either pyruvate or lactate, the glucose metabolites, did not suppress the ability of RPE cells to accumulate MI. An 18 ± 8% reduction in [³H]thymidine incorporation into DNA occurred when cells were grown in 40 mM glucose for 12–14 days, compared to cells grown with 5 mM glucose. Chronic treatment (12–14 days) of the cells with phorbol ester, an activator of protein kinase C, caused up to twofold increase in MI uptake, [³H]phlorizin binding, cell number, and DNA synthesis. However, when the rates of MI uptake into cells grown in basic growth media or TPA-treated media were normalized to cell number, no significant difference in MI uptake was found between the treated and untreated cells. Addition of staurosporin, a protein kinase C inhibitor, together with TPA, in the growth media reversed the phorbol-induced increase of MI uptake. In contrast to its chronic effect, a 60-min incubation (acute effect) of cells in the presence of TPA, with or without inclusion of stauropsorin, did not alter the uptake of ³H-MI into RPE cells, regardless of glucose levels in the growth media. These studies indicated that glucose itself, and not glucose metabolites, regulated uptake of MI into primary cultures of RPE cells. In addition, glucose-induced down-regulation of MI uptake was not mediated through the protein kinase C pathway, but the staurosporin-inhibited, TPA-stimulated protein kinase C was partly responsible for growth and proliferation of RPE cells.     

强化控糖后加用盐酸吡格列酮治疗2型糖尿病的临床疗效观察

目的观察强化控糖后加用盐酸吡格列酮治疗2型糖尿病的临床疗效。方法 60例使用口服降糖药物治疗的血糖控制不佳的2型糖尿病患者,入院后先进行胰岛素泵强化控糖治疗,患者血糖达到目标值后（FPG〈7.0 mmol/L,2 h PG〈10.0 mmol/L）,改为三餐前门冬胰岛素联合睡前甘精胰岛素继续强化治疗,1周后按1：1的比例随机分为两组,一组继续使用三餐前门冬胰岛素联合睡前甘精胰岛素治疗（对照组）,一组在三餐前门冬胰岛素联合睡前甘精胰岛素的基础上加用盐酸吡格列酮30 mg/日（治疗组）。1～4周我院住院治疗,5～12周门诊随访,若出现FPG及2 h PG明显下降或低血糖反应,则减少胰岛素用量。观察加用盐酸吡格列酮治疗前以及治疗12周时FPG、2 h PG、HbAlc、胰岛素用量、甘油三酯（TG）、总胆固醇（TC）、高密度脂蛋白胆固醇（HDL-C）、低密度脂蛋白胆固醇（LDL-C）、体重指数的变化情况。结果 （1）治疗组胰岛素用量明显下降,与加用盐酸吡格列酮治疗前有明显差异（P〈0.05）;对照组的胰岛素用量治疗前后无明显差异（P〉0.05）;（2）治疗组与对照组加用盐酸吡格列酮治疗前后FPG、2hPG均无明显差异（P〉0.05）。（3）治疗组HbAlc有所下降,与加用盐酸吡格列酮治疗前有明显差异（P〈0.05）,对照组HbAlc也有所下降,差异明显（P〈0.05）,但治疗后两组比较无明显差异（P〉0.05）。（4）加用盐酸吡格列酮后,治疗组TG下降,HDL-C升高,与同组治疗前相比,差异明显（P〈0.05）,对照组与治疗前相比差异不明显,组间比较差异有显著性（P〈0.05）。结论在强化控糖血糖达标后,加用盐酸吡格列酮继续治疗,可以明显降低患者胰岛素的使用剂量,提高患者的依从性,并且能改善血脂代谢紊乱,对于减少心血管并发症的发生有一定益处。     

Purification and Properties of Penicillin Acylase from Kluyvera citrophila

Penicillin acylase (EC 3.5.1.11) of Kluyvera citrophila KY7844 was purified approximately 120-fold by DEAE-cellulose chromatography, hydroxyapatite chromatography and isoelectro-focusing fractionation. The purified enzyme, with an approximate molecular weight of 63,000, appeared to be homogeneous in disc electrophoretic analysis, and showed isoelectric point (Ip) 8.12 and 13.0 units/mg of specific activity for cephalexin hydrolysis. The Michaelis constant (Km) for cephalexin and for 7-[1-(1H)-tetrazolylacetamido]-desacetoxycephalosporanic acid ((1H) T-7ADCA) was 1.4 mM and 3.6 mM, respectively. This enzyme was capable of producing (1H) T-7ADCA in 80% yield from 1-(1H)-tetrazolylacetate methylester and 7-aminodesacetoxycephalosporanic acid.     

Isolation of a Novel Auxin,Methyl 4-Chloroindoleacetate from Immature Seeds of Pisum sativum

Human casein was separated by gel filtration on a column of Sephadex G–200 with 0.1 m Tris buffer (pH 8.5) containing 1.0 m NaCl. The effluent which increased in turbidity at 25°C was centrifuged at 25,000 × g for 30 min and the precipitate was obtained as Fraction 6. After centrifugation, the effluent was separated into 5 elution fractions.

Disc gel electrophoretic patterns of each fraction showed occurrence of secondary bands other than major bands especially in Fractions 3, 4 and 5. The casein solutions unheated and heated at 100°C for 5 and 10 min were kept at 5°C for 5 days. No marked changes of electrophoretic pattern were observed among these casein solutions. However, when a casein solution heated at 100°C for 5 min was chroma to graphed under the same condition, secondary bands also appeared.     

Improved Hypolipidemic Effects of Xanthan Gum-Galactomannan Mixtures in Rats

This study describes the effects of mixtures of xanthan gum and galactomannan, guar gum, or locust bean gum, on the lipids in plasma and liver in non-diabetic and diabetic rats. Non-diabetic rats were fed cholesterol-free diets with 3% guar gum, locust bean gum, or xanthan gum (3G, 3L, and 3X), or a mixture of xanthan gum and guar gum or locust bean gum (1:2, w/w) (2G1X, 2L1X) for 2 weeks. Rats fed diets not containing these polysaccharides were used as controls. The total cholesterol in plasma and the triacylglycerol in liver were significantly lowered in rats fed the 2G1X diet. The 3G, 3X, 3L, and 2L1X diets showed no significant effect on the total cholesterol and triacylglycerol in plasma and liver. In the streptozotocin-induced (STZ) diabetic rats, the total cholesterol in plasma was lowered in rats fed the 3G, 3X or 2G1X diet for 4 weeks, and the 2G1X diet was more effective than the 3G and 3X diets. The triacylglycerol in plasma in STZ diabetic rats was also significantly lowered by the 2G1X diet. These results showed that a mixture of xanthan gum and guar gum has an improved hypolipidemic effect on non-diabetic and STZ diabetic rats. The effects of the 2G1X diet on the diabetic symptoms in STZ diabetic rats, suppression of food and water intakes, decrease in glucose in urine, and lowering of plasma glucose, were also observed.     

Palmitate Contributes to Insulin Resistance through Downregulation of the Src-Mediated Phosphorylation of Akt in C2C12 Myotubes

The mechanisms of free fatty acid (FFA)-induced peripheral insulin resistance remain elusive. This study aimed to investigate the effect of palmitate, a saturated fatty acid, on glucose metabolism in C2C12 myotubes, and to explore the underlying mechanisms. In it, palmitate decreased insulin-stimulated glucose uptake and consumption in a dose-dependent manner, and it reduced the insulin-stimulated phosphorylation of Akt at Thr308 and Ser473, but had no effect on the protein expression of PI3K-p85 or the activity of PI3K. Additionally, it inhibited the insulin-stimulated phosphorylation of Src at Tyr416, causing a reduction in the Src-mediated phosphorylation of Akt. Inhibition of Src by PP2 resulted in decreases in insulin-stimulated glucose uptake and phosphorylation of Src at Tyr416 and Akt at Thr308 and Ser473. The findings indicate that palmitate contributes to insulin resistance by inhibiting the Src-mediated phosphorylation of Akt in C2C12 myotubes, and this provides insight into the molecular mechanisms of FFA-induced insulin resistance.