The study of nucleoside triphosphate-binding center of DNA-dependent RNA-polymerase of phage T7 using GTP analogs

The NTP binding site of bacteriophage T7 DNA-dependent RNA polymerase was studied using GTP analogs. For four analogs the irreversible inhibition was demonstrated. The kinetic parameters for competitive (Ki) and irreversible (KI and k3) inhibition were determined. One of the analogs, 5'[2-hydroxy(4-iodoacetamido)benzoyl]guanosine, was shown to inactivate the enzyme rapidly due to the modification of SH-groups. Some suggestions on the structure of the RNA polymerase active site have been made.     

Interaction of rabbit skeletal muscle glycogen synthase I with substrate analogs--oxo-UDP hydrazones]

Inhibition of rabbit skeletal muscle glycogen synthase I was studied by using several synthetic substrate analogs: dansylhydrazone of oxo-UDP, 3-hydroxy-2-naphthoylhydrazone of oxo-UDP, salicyloylhydrazone of oxo-UDP, 1-oxyl-2,2,5,5-tetramethylpyrrolidine-3-carbonylhydrazone of oxo-UDP, N'-(dansyl)hydrazinocarbonylhydrazone of oxo-UDP and N'-(fluorenylidene-9)-hydrazinocarbonylhydrazone of oxo-UDP. All these compounds (with the exception of the nitroxyl-containing hydrazone) were characterized by a nonlinear dependence of the reverse reaction rate on the analog concentration in Dixon coordinates. The parabolic type of inhibition was due to the fact that the analogs tested except for the nitroxyl-containing hydrazone were able to interact both with the active center of the enzyme and with the FMN-binding site. The inhibition constants for oxo-UDP hydrazones were calculated for the both centers; their comparison revealed that the affinity of the analogs for the FMN-binding site increased with an increase in the radical hydrophobicity. These data suggest that the site with a high binding affinity for FMN is hydrophobic in nature. Apparently, isoalloxasine-like compounds display the highest affinity for this site.     

FoxO1在胰岛β细胞代谢灵活性受损及失代偿进程中的作用

葡萄糖及脂肪酸是胰岛β细胞的关键代谢底物,葡萄糖刺激胰岛β细胞分泌胰岛素是维持机体血糖稳态平衡的关键。胰岛素抵抗发生时,β细胞对能量代谢底物的选择失调,加速胰岛β细胞由代偿到胰岛β细胞失代偿的进程,是肥胖胰岛素抵抗最终发展为2型糖尿病的始动因素。核转录因子FoxO1属于Fox家族成员,在胰腺内广泛表达,在β细胞的代谢,发育,增殖过程中发挥着重要的调节作用。鉴于FoxO1在维持胰岛β细胞功能中的关键作用,现着重对FoxO1在胰岛β细胞代谢灵活性受损及失代偿过程发生中的作用调节进行阐述。为其作为调控胰岛β细胞功能的关键靶点提供参考。     

Effect of N-(6-aminohexyl)-5-chloronaphthalene-1-sulfamide (W-7) and its analogs on the activity of soluble guanylate cyclase and on human platelet aggregation

The effect of N-(omega-aminoalkyl) derivatives of naphthalene-1-sulfamide on the activity of soluble guanylate cyclase and on human platelet aggregation at the first (reversible) step of the guanylate cyclase reaction was studied. Low (approximately 10(-7)-10(-6) M) concentrations of the above compounds were shown to stimulate the guanylate cyclase activity; some derivatives caused simultaneous inhibition of platelet aggregation induced by ADP. Some fragments of the chemical structure of the molecules responsible for the enzyme activity regulation in the tested systems were identified. The naphthalene-1-sulfamide derivatives carrying 6-aminohexyl or 8-amino-octyl groups of the sulfamide substituent as well as chlorine atom at positions 4 or 5 of the naphthalene ring appeared to be the most potent activators of platelet guanylate cyclase and inhibitors of platelet aggregation at the reversible step of the enzymatic reaction.     

促性腺激素释放激素Buserelin 稳定性的研究

目的:研究Buserelin原料药的性质在温度、湿度、光线等条件的影响下随时间变化的规律,为该原料药的生产、包装、储存、运输及有效期的制定提供依据。方法:根据中国药典2005版二部附录XIX C药物稳定性试验指导原则及化学药物稳定性研究技术指导原则进行强光照射、高温(60℃、40℃)、高湿(RH92.5%±5%、RH75%±5%)影响因素试验,加速试验(40℃±2℃、RH75%±5%;25℃±2℃、RH60%±10%);按Buserelin原料药标准规定的质量指标及相关的检验方法对产品在试验条件下的主要质量指标进行检测。结果:强光照射、高温、高湿等影响因素对Buserelin的稳定性有明显影响,故应密封、于干燥、阴凉处保存。在加速试验中,Buserelin原料药的各项质量指标发生了小的变化,但均在质量标准规定的范围内。结论:强光照射、高温、高湿等影响因素对Buserelin的稳定性有明显影响,应在阴凉干燥处避光密封保存和运输。加速试验结果证明:在此条件下,它的各项质量指标变化均在质量标准范围内,符合Buserelin原料药质量标准规定的要求;故将其保质期暂定为两年。     

Derivatives of Benzotetrazine-1,3-dioxide Are New NO-donors, Activators of Soluble Guanylate Cyclase, and Inhibitors of Platelet Aggregation

The ability of 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides to generate nitric oxide (NO) and activate soluble guanylate cyclase was investigated. All of these compounds were found to be thiol dependent NO-donors and guanylate cyclase activators. The maximal stimulatory effect of 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides was observed at 10 M concentration and the activity increase was 4.5-, 15.0-, and 8.2-fold in the presence of 20 M dithiothreitol and 11.3-, 31.6-, and 20.5-fold, respectively, in the presence of added glutathione (100 M). The NO-dependent mechanism of benzotetrazine-1,3-dioxide nitroderivative-induced activation of soluble guanylate cyclase (in the presence of 100 M glutathione) was confirmed by the inhibition (by 78%) of 7-nitrobenzotetrazine-1,3-dioxide (10 M)-stimulated guanylate cyclase activity in the presence of the NO-scavenger-2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (Carboxy-PTIO, 50 M) and by the inhibition with 1H-[1,2,4 ]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 0.3 M) of 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides (10 M)-stimulated guanylate cyclase by 34, 69, and 39%, respectively. All compounds used inhibited ADP-induced aggregation of human platelets with IC ₅₀ of 10.0, 1.3, and 2.0 M for 5-nitro-, 7-nitro-, and 5,7-dinitrobenzotetrazine-1,3-dioxides, respectively. A clearly defined correlation was established between the ability of the compounds to generate NO, activate soluble guanylate cyclase, and inhibit platelet aggregation.     

Benzodifuroxan as an NO-dependent activator of soluble guanylate cyclase and a novel highly effective inhibitor of platelet aggregation

The ability of benzodifuroxan (BDF) to activate human platelet guanylate cyclase was investigated. The maximal stimulatory effect (1160 +/- 86%) was observed at 0.01 mM concentration. Sodium nitroprusside produced the same stimulatory effect (1220 +/- 100%) but at a higher concentration (0.1 mM). 1-H-[1,2,4,]-Oxadiazolo[4, 3-alpha]quinoxalin-1-one (ODQ), an inhibitor of NO-dependent guanylate cyclase activation, attenuated the stimulatory effect of BDF (0.01 mM) by 75% and that of sodium nitroprusside (0.1 mM) by 80%. Increasing dithiothreitol concentration in the sample from 2. 10-6 to 2.10-4 M increased the stimulatory effect of BDF 2.7-fold. The possible involvement of sulfhydryl groups of low-molecular-weight thiols and guanylate cyclase in thiol-dependent activation of the enzyme is discussed. We have also found that BDF is a highly effective inhibitor of ADP-induced human platelet aggregation with IC50 of 6.10-8 M. The effect of sodium nitroprusside was much weaker (IC50, 5.10-5 M).     

Treatment outcomes in locally advanced colorectal carcinoma

BACKGROUND: Locally advanced colorectal cancers form a distinct subgroup where contiguous organs could be involved without distant metastases and so may be amenable to curative surgical resection. It was our objective to report our experience in treating six such patients with operable locally advanced colorectal carcinomas. METHODS: We retrospectively reviewed the case notes of 47 patients who were diagnosed with colorectal cancers at M S Ramaiah Medical Teaching Hospital between the years 1996 - 2001. Six patients were identified with T4 lesions, adjacent organ involvement and with no nodal involvement. The treatments and outcomes for these patients were then reviewed. RESULTS: Two of three patients with rectal malignancies who underwent pelvic exenteration succumbed to disease recurrence within the first 18 months. One of the three patients with colonic cancers died of non malignant causes. The other two are disease free till date. CONCLUSIONS: Aggressive multivisceral resections for locally advanced colonic cancers might be appropriate. Rectal cancers when locally advanced may be considered for pelvic exenteration, but a more guarded prognosis may apply.     

Irreversible inhibition of adenylate cyclase by 5'-(p-bromomethylbenzoyl) adenosine

The effect of 5'-(p-bromomethylbenzoyl) adenosine (pBMBA) on adenylate cyclase from bovine caudate nucleus membranes was studied. Adenylyl-5'-methylenediphosphonate (but not adenosine) protected adenylate cyclase against inactivation by this compound. The degree of pBMBA-induced inhibition of adenylate cyclase increased in the presence of Mg2+. 5'-(p-fluorosulfonylbenzoyl) adenosine (pFSBA) was also a specific irreversible inhibitor of adenylate cyclase. It was demonstrated that the enzyme inactivated by pFSBA completely restored its activity under the action of dithiothreitol. The results obtained are indicative of the presence of the -SH group in the enzyme active site.     

N-Chloroacetylhydrazone of oxo-GTP irreversibly inhibits the activating function of GTP-binding protein coupled with adenylate cyclase

Guanine nucleotides are successfully used in the studies of regulatory N-proteins coupled with adenylate cyclase. In the present work N-chloroacetylhydrazones of oxo-GTP and oxo-GDP are described. After 4 hr preincubation of these nucleotides with plasma membranes from bovine brain caudate nucleus, the ability of adenylate cyclase to be activated by guanylyl-5'-methylene-diphosphonate is blocked. The degree of inhibition depends on preincubation time and increases in the presence of Mg2+. Guanylyl-5'-methylenediphosphonate protects adenylate cyclase from the action of N-chloroacetylhydrazone of oxo-GTP. These findings suggest that adenylate cyclase activation is diminished as a result of covalent modification of the Ns. N-chloroacetyl-hydrazone of oxo-GDP also causes a loss of the adenylate cyclase sensitivity to the fluoride ion and cholera toxin.