Subchronic administration of para-chlorophenylalanine enhances serotonin-stimulated phosphoinositide hydrolysis in rat hippocampal slices

Summary Serotonin (5-HT) caused a dose-dependent accumulation of inositol monophosphate (IP-1) in rat hippocampal slices (maximal effect +172%, EC₅₀=630 nM) in the presence of LiCl. The 5-HT response was blocked potently by a non-selective 5-HT receptor antagonist metergoline and a 5-HT_1C/ 5-HT₂ receptor antagonist ritanserin. The 5-HT₂ receptor antagonist ketanserin and spiperone were, however, less potent at inhibiting the 5-HT response, m-Chlorophenylpiperazine (mCPP), a 5-HT_1C receptor agonist but a 5-HT₂ antagonist stimulated directly PI turnover, yet mCPP inhibited 5-HT-stimulated PI response. These findings indicate that both 5HT_1C and 5-HT₂ receptors are involved with a complicated interaction of each receptor in 5-HT-stimulated PI hydrolysis in rat hippocampus. 10-Day treatment with para-chlorophenylalanine (PCPA), a 5-HT synthesis inhibitor, resulted in a significant increase (maximal effect +225%, EC₅₀=580 nM) in 5-HT-stimulated IP-1 accumulation with no substantial change in EC₅₀ value, which suggest that subchronic treatment with PCPA enhances the 5-HT-mediated PI hydrolysis in rat hippocampus.     

Hydrolysis of single-stranded RNA in aqueous solutions—effect on quantitative hybridizations

Single stranded RNA is non-enzymatically hydrolysed in aqueous solutions at neutral pH at elevated temperatures. This hydrolysis causes practical problems in different hybridization procedures. Synthetic cRNA transcribed from the human p53 oncogene was found to be partially destroyed after 6 hours and completely destroyed after overnight incubation at 60 °C. At lower temperatures the cRNA was preserved intact in spite of overnight incubation, but at higher temperatures (80 °C) the degradation occurred in less than 2 hours. The effect of the hydrolysis on hybridization results was studied by measuring in solution the hybridization kinetics of the cRNAs of another human oncogene, N-myc. It is obvious that conditions generally used for DNA hybridizations cannot be utilized for RNA in quantitative studies.     

低密度脂蛋白胆固醇的直接测定法

目的　建立直接测定血清低密度脂蛋白胆固醇 (LDL C)的方法。方法　基于选择性水解法的原理 ,通过对多聚体和表面活性剂的筛选和测定条件的优化实验确定了方法。结果　本法反应达终点的时间为 180s ,线性达 11 7mmol/L ,批内CV <1 6 4% ,日间CV <2 6 5 % ,总CV <3 38% ,回收率平均为 99 7%。和日本一化试剂比较 :Y =0 94X 0 0 32 ,r =0 975 ,n =5 2。本法的特异性好 ,加入LDL C达 3 9mmol/L ,VLDL Trig达 19 5mmol/L ,抗坏血酸 <2 84mmol/L、血红蛋白<5g/L和胆红素 <340 μmol/L时无显著干扰。 结论　本法的总误差 <7 40 % ,达到NCEP提出的 <12 %的分析目标 ,标本无需预处理 ,适合全自动分析     

Hydrolytic Stability of Methacrylamide and Methacrylate in Gelatin Methacryloyl and Decoupling of Gelatin Methacrylamide from Gelatin Methacryloyl through Hydrolysis

Gelatin methacryloyl (GelMA; GM) is a promising nature‐derived photocurable material that can mimic the extracellular matrix because GelMA features tailorable mechanical properties, proteolytic degradation, and good cell adhesion. GelMA contains not only methacrylamide but also methacrylate. However, the hydrolytic stability of methacrylamide and methacrylate groups of GelMA in aqueous solutions has not been scrutinized. Here, the structural change of GelMA through hydrolysis is investigated for the first time. The structural change of hydrolyzed GelMA is quantitatively identified using colorimetric and ¹H NMR methods. The methacrylate groups decompose markedly at high pH solutions, but the methacrylamide groups remain stable. Further, pure gelatin methacrylamide is successfully decoupled from GelMA for a better understanding of GelMA structure and future use for biomedical applications.     

二次通用旋转试验设计优化栀子苷的酶解工艺

目的 研究苦杏仁酶水解栀子苷制备栀子苷元的工艺条件。方法 以产物得率为指标,通过二次通用旋转试验设计考察缓冲液pH值、反应温度、反应时间和酶加入量4个主要因素对工艺的影响,得出苦杏仁酶水解栀子苷的最合适工艺条件;采用理化性质及NMR谱鉴定产物结构,HPLC峰面积归一化法检测产物纯度。结果 酶加入量、缓冲液pH值和反应时间对产物得率有显著影响,所确定的最合适工艺条件为缓冲液pH值5.25,反应温度50.5 ℃,反应时间90 min,酶加入量1∶10;产物栀子苷元得率＞34.5%,质量分数为99.6%。结论 用苦杏仁酶水解栀子苷制备栀子苷元的方法是可行的。     

Agacutase体外水解牛纤维蛋白原机制的研究(英文)

Agacutase是自尖吻蝮蛇蛇毒中分离出的新的具有止血功能的蛇毒类凝血酶,它能够将纤维蛋白原转化为纤维蛋白单体。通过SDS-PAGE和ELISA方法,我们研究了Agacutase体外水解牛纤维蛋白原的分子机制。实验结果显示,Agacutase仅仅水解牛纤维蛋白原的α亚基并释放血纤肽A,而对牛纤维蛋白原的β和/或γ亚基没有影响。研究表明Agacutase属于血纤肽A类(FP-A类型)的类凝血酶。     

Characterisation of a novel conjugate of ibuprofen with 3‐hydroxybutyric acid oligomers

Objectives A conjugate of ibuprofen with 3‐hydroxybutyric acid oligomers has been evaluated as a novel drug delivery model system. Methods This paper focuses on the synthesis and the characterisation of the physicochemical properties of this conjugate, and on hydrolysis studies in aqueous buffers and simulated intestinal fluid. We also describe the development of an analytical method (HPLC) for hydrolysis studies of this compound. Key findings The conjugate had high stability in aqueous solutions of pH 6–8 and underwent slow enzymatic hydrolysis. Conclusions This conjugate is not well suited for oral administration but might be considered a candidate for development of prodrug preparations for parenteral or topical sustained release.     

Heparin–Paclitaxel Conjugates Using Mixed Anhydride as Intermediate: Synthesis,Influence of Polymer Structure on Drug Release,Anticoagulant Activity and <Emphasis Type="BoldItalic">In Vitro</Emphasis> Efficiency

Purpose  The heparin–paclitaxel conjugates using amino acid as linker (HD2), with low anticoagulant activity, the similar anticancer activity as paclitaxel, offer great potential for further investigation. Methods  Two types of heparin–paclitaxel conjugates (HD) have been developed, in which O-acetylated heparin as carrier conjugates with paclitaxel by direct ester bond (HD1) and by inserting different amino acids as spacers, including valine, leucine, and phenylalanine (HD2a, HD2b, and HD2c), respectively. Specifically, mixed anhydride groups of carrier as activating intermediates mediate the synthesis of prodrugs. The HD conjugates are characterized by ¹H NMR, FT-IR and GPC. The percentage weight of drug and hydrolysis rate for HD are detected by UV and HPLC. The anticoagulant activity and cell cycle of MCF-7 of HD are measured by APTT and FCM, respectively. Results   HD2 conjugates show better solubility and faster hydrolysis rates than those of HD1. Meanwhile, the anticoagulant activity of HD is reduced and FCM analysis show that MCF-7 cells treated with HD are arrested in the G2/M phase of cell cycle. Conclusions  Amino acids as linkers between paclitaxel and carrier are appropriate to facilitate the release of paclitaxel from carrier. Mixed anhydrides mediate the synthesis of prodrugs and HD2 conjugates are expected to further investigate in vivo experiment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hyperthyroidism modifies ecto-nucleotidase activities in synaptosomes from hippocampus and cerebral cortex of rats in different phases of development

Here we investigate the possible effects of the hyperthyroidism on the hydrolysis of the ATP to adenosine in the synaptosomes of hippocampus, cerebral cortex and blood serum of rats in different developmental phases. Manifestations of hyperthyroidism include anxiety, nervousness, tachycardia, physical hyperactivity and weight loss amongst others. The thyroid hormones modulate a number of physiological functions in central nervous system, including development, function, expression of adenosine A(1) receptors and transport of neuromodulator adenosine. Thus, hyperthyroidism was induced in male Wistar rats (5-, 60-, 150- and 330-day old) by daily injections of L-thyroxine (T4) for 14 days. Nucleotide hydrolysis was decreased by about 14-52% in both hippocampus and cerebral cortex in 5 to 60-day-old rats. These changes were also observed in rat blood serum. In addition, in 11-month-old rats, inhibition of ADP and AMP hydrolysis persisted in the hippocampus, whereas, in cerebral cortex, an increase in AMP hydrolysis was detected. Thus, hyperthyroidism affects the extracellular nucleotides balance and adenosine production, interfering in neurotransmitter release, development and others physiological processes in different systems.     

Epicatechin gallate and epigallocatechin gallate are potent inhibitors of human arylacetamide deacetylase

Recently, in addition to carboxylesterases (CESs), we found that arylacetamide deacetylase (AADAC) plays an important role in the metabolism of some clinical drugs. In this study, we screened for food-related natural compounds that could specifically inhibit human AADAC, CES1, or CES2. AADAC, CES1, and CES2 activities in human liver microsomes were measured using phenacetin, fenofibrate, and procaine as specific substrates, respectively. In total, 43 natural compounds were screened for their inhibitory effects on each of these enzymes. Curcumin and quercetin showed strong inhibitory effects against all three enzymes, whereas epicatechin, epicatechin gallate (ECg), and epigallocatechin gallate (EGCg) specifically inhibited AADAC. In particular, ECg and EGCg showed strong inhibitory effects on AADAC (IC₅₀ values: 3.0 ± 0.5 and 2.2 ± 0.2 μM, respectively). ECg and EGCg also strongly inhibited AADAC-mediated rifampicin hydrolase activity in human liver microsomes with IC₅₀ values of 2.2 ± 1.4 and 1.7 ± 0.4 μM, respectively, whereas it weakly inhibited p-nitrophenyl acetate hydrolase activity, which is catalyzed by AADAC, CES1, and CES2. Our results indicate that ECg and EGCg are potent inhibitors of AADAC.