DNA strand breaking capacity of acrylamide and glycidamide in mammalian cells

We compared the DNA damaging potency of acrylamide (AA) and its metabolite glycidamide (GA) in the comet assay in cell systems differing with respect to species origin and cytochrome P450-depended monooxygenase (CYP2E1) expression (V79, Caco-2, primary rat hepatocytes). Only after 24 h incubation in the highest concentration of AA (6 mM) a slight but significant increase in DNA damage was observed in V79 and Caco-2 cells. In primary rat hepatocytes, however, expressing substantial amounts of CYP2E1, no induction of DNA strand breaks was found. At the end of the incubation time period (24 h), still 67 ± 19% of the CYP2E1 protein was detected by Western blotting. Direct treatment with GA resulted in a significant increase in DNA damage in V79 cells and primary rat hepatocytes at concentrations ≥100 μM (24 h). Caco-2 cells were found to be less sensitive, exhibiting an increase in DNA strand breaks at concentrations ≥300 μM GA. These data confirm the higher genotoxic potential of GA compared to AA but also indicate that high expression of CYP2E1 per se is not necessarily associated with increased genotoxicity of AA. We, therefore, investigated whether the intracellular glutathione (GSH) level might be a critical determinant for the genotoxicity of AA in cells with different CYP2E1 status. Depletion of intracellular GSH by DL-buthionine-[S,R]-sulfoxime (BSO) in rat hepatocytes and V79 cells resulted in a significant induction of DNA strand breaks after incubation with 1 mM AA. However, at higher concentrations (≥1.25 mM) a strong increase in cytotoxicity, resulting in a severe loss of viability, was observed. In summary, the DNA strand breaking effect of AA appeared not to be directly correlated with the CYP2E1 status of the cells. Depletion of GSH is associated with an increase in AA genotoxicity but seems also to lead to a substantial enhancement of cytotoxicity.     

The time-dependent transport of chromium in adult rats from the bloodstream to the urine

While chromium was proposed to be an essential trace element over 40 years ago and if essential should possess a specific transport and distribution mechanism, the details of its transport from the bloodstream to the urine have not been elucidated. However, chromium is known to be maintained in the bloodstream bound to transferrin and to be excreted in the urine bound to the oligopeptide chromodulin or a similar chromodulin-like species. Injection of ⁵¹Cr-labeled transferrin into the bloodstream resulted in a rapid and insulin-sensitive movement of chromium into the tissues as Cr transferrin; greater than 50% of the Cr is transported to the tissues within 30 min. Tissue levels of Cr are maximal 30 min after injection; decreases in tissue Cr with time are mirrored by increases in urine Cr. Approximately 50% of the ⁵¹Cr appears in the urine within 360 min of injection in the absence of added insulin; insulin treatment concurrent with injection of ⁵¹Cr-labeled transferrin results in approximately 80% of the label appearing in the urine within 180 min. The removal of ⁵¹Cr from the blood is faster than the appearance of ⁵¹Cr in the urine; the lag in time indicates that the Cr transferrin in the blood and Cr in the urine are not in direct equilibrium and that intermediates in the transport of Cr must be involved. This establishes a clear pathway of transport of Cr starting from transport by transferrin from the bloodstream into the tissues, followed by release and processing in the tissues to form chromodulin, excretion into the bloodstream, rapid clearance of chromodulin or a similar species into the urine, and ultimately excretion as this species. Insulin stimulates the processing of Cr in the tissues.     

培菲康对肝硬化血浆内毒影响的研究

目的　探讨益生菌制剂培菲康治疗肝硬化内毒素血症的作用机制。方法　采用大鼠肝硬化模型,其中治疗组予以培菲康液每天灌胃治疗,共8周,检测治疗组及对照组血浆内毒指标;并选取70例肝硬化Child-Pugh分级为B级的患者,其中治疗组口服培菲康胶囊(每次2粒,每天3次) 3周,观察治疗前后血浆内毒素变化。结果　培菲康治疗组的肝硬化大鼠及患者血浆内毒素均明显低于对照组( P<0 .0 5 )。结论　培菲康能明显降低肝硬化患者血浆内毒素水平,可作为肝硬化患者的辅助用药     

高脂喂养联合链脲佐菌素注射的糖尿病大鼠模型特征

目的观察高脂喂养联合低剂量STZ注射的SpragueDawley(SD)大鼠2型糖尿病模型的代谢特征、病理学以及胰岛分子生物学变化。方法4周龄雄性SD大鼠36只随机分为三组(1)正常对照组(Control)9只,普通饲料喂养。(2)高脂组(HighFatchow,HE)9只,高脂饲料喂养,为普通饲料中添加20%脂肪(猪油和蛋黄粉各50%)和20%蔗糖。(3)糖尿病组(DM)18只。喂养4周后腹腔注射STZ(40mg/kg)。所有大鼠做灌胃葡萄糖耐量(OGTT)试验。放免法测定血清胰岛素,免疫组化染色观察胰岛β细胞的形态学特点,彩色图像分析系统测定胰岛素表达量,RT-PCR测定胰腺β细胞胰岛素mRNA表达水平。结果糖尿病大鼠空腹血糖(FBG)、胰岛素水平(FINS)显著高于Control组和HE组大鼠(P<0.01),空腹血清甘油三酯(TG)和游离脂肪酸(FFA)水平显著高于Control组(P<0.05);胰岛β细胞吸光度(A)显著低于高脂组大鼠(P<0.05),降低11.6%。胰岛素免疫反应阳性区占胰岛百分比显著低于Control组和HE组,分别下降31.9%(P<0.05)和43.1%(P<0.01)。胰岛素mRNA表达水平显著低于HE组(P<0.05)。STZ注射后48h(基线值)大鼠FBG水平的分布情况为A组(FBG<10.0mmol/L)占7/18;B组(FBG10~19.9mmol/L)占5/18;C组(FBG≥20mmol/L)占6/18。STZ注射后9d的OGTT结果与基线值相比,B组OGTT值总体变化最小,A组FBG的变异最大,达到25%。结论高脂喂养联合低剂量STZ注射的糖尿病大鼠模型模拟2型糖尿病发生的主要病理生理过程,具有高血糖、高胰岛素血症以及血脂异常等基本特征。     

Role of calpains in diabetes mellitus: A mini review

Type 2 diabetes mellitus (T2DM) is characterized by defects in haepatic glucose production, insulin action and insulin secretion, which can also lead to a variety of secondary disorders. The disease can lead to death without treatment and it has been predicted that T2DM will affect 215 million people world-wide by 2010. T2DM is a multifactorial condition whose precise genetic causes and biochemical defects have not been fully elucidated but at both levels, calpains appear to play a role. Positional cloning studies mapped T2DM susceptibility to CAPN10, the gene encoding the intracellular cysteine protease, calpain 10. Further studies have shown a number of non-coding polymorphisms in CAPN10 to be functionally associated with T2DM whilst the identification of coding polymorphisms, suggested that mutant calpain 10 proteins may also contribute to the disease. The presence of both calpain 10 and its mRNA have been demonstrated in tissues from several mammalian species whilst calpain 10 appears to be associated with pathways involved in glucose metabolism, insulin secretion and insulin action. It appears that other calpains may also participate in these pathways and here we present an overview of recent studies on calpains and their putative role in T2DM. (Mol Cell Biochem 261: 161–167, 2004)     

不同时辰电针对大鼠杏仁核一氧化氮合酶表达的影响

目的观察不同时辰电针对大鼠杏仁核一氧化氮合酶(NOS)表达的影响.方法采用还原型尼克酰胺腺嘌呤二核苷酸脱氢酶(NADPH-d)法,观察不同时辰电针大鼠一侧"足三里"穴对杏仁核NOS表达的影响.结果电针对大鼠杏仁皮质内侧核群、基底外侧核群NOS的表达有上调作用,并存在时辰差异(P<0.05);电针对大鼠杏仁中央核NOS表达无明显作用(P>0.05).结论电针对大鼠杏仁核NOS表达的影响有时辰差异.     

Determination of rat serum esterase activities by an HPLC method using S-acetylthiocholine iodide and p-nitrophenyl acetate

Establishing esterase assays allows the determination and comparison of esteratic activities of tissues of one organism and between organisms. We have developed a high-performance liquid chromatography (HPLC) assay for the determination of S-acetylthiocholine (ATC) and p-nitrophenyl acetate (NPA) hydrolyzing activities of rat serum esterases based on ion pair chromatography with on-line radiochemical and ultraviolet (UV) detection. ATC is a substrate for cholinesterases, whereas NPA is cleaved by a variety of esterases and other proteins (e.g., cholinesterases, paraoxonase, carboxylesterase, albumin). Both substrates were incubated, simultaneously or separately, with rat serum to explore potential interferences between the enzymatic hydrolyses of the compounds. The ratio of the peak area of the ¹⁴C-labeled substrates to the total peak area of the substrates and their corresponding cleavage products was compared with the UV quantitation of ATC and p-nitrophenolate (NP), the cleavage product of NPA, measured at 230 and 350 nm, respectively. The peak identity of ATC and NP was confirmed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The reaction rates of the assays using one substrate or both, as well as using radiochemical or UV detection, were equal. Moreover, the correlation between rat serum volumes and reaction rates was shown for both substrates. In conclusion, one can (i) choose between the two detection methods reliably, (ii) take advantage of monitoring both substrate and product by using radiochemical detection, and (iii) combine both substrates to determine esterase activities in rat serum and probably other biological matrices.     

Effects of cathepsins B and L inhibition on postischemic protein alterations in the brain

The effects of selective inhibition of cathepsins B and L on postischemic protein alterations in the brain were investigated in a rat model of middle cerebral artery occlusion (MCAO). Cathepsin B activity increased predominantly in the subcortical region of the ischemic hemisphere where the levels of collapsing mediator response protein 2, heat shock cognate 70 kDa protein, 60 kDa heat shock protein, protein disulfide isomerase A3 and albumin, were found to be significantly elevated. Postischemic treatment with Cbz-Phe-Ser(OBzl)-CHN₂, cysteine protease inhibitor 1 (CP-1), reduced infarct volume, neurological deficits and cathepsin B activity as well as the amount of heat shock proteins and albumin found in the brain. Our data strongly suggests that the decrease in heat shock protein levels and the significant reduction of serum albumin leakage into the brain following acute treatment with CP-1 is indicative of less secondary ischemic damage, which ultimately, is related to less cerebral tissue loss and improved neurological recovery of the animals.     

Are beta-adrenergic receptors in the hippocampal CA1 region required for retrieval of contextual fear memory?

It is well established that β-adrenoceptors (β-ARs) in the hippocampal CA1 region are involved in regulating synaptic plasticity and are essential for acquisition and consolidation of spatial memory and contextual fear memory. Previous studies reported that β-ARs in the CA1 region are also involved in memory retrieval. The present study re-examined the role of hippocampal β-ARs in retrieval of conditioned contextual fear. We bilaterally infused a high dose of the β-AR antagonist propranolol (15 μg in 1 μl saline) into the CA1 region 30 min before retention test and found that propranolol produced no deficit in retrieval of either 1-day or 7-day contextual fear. We then examined if β-AR stimulation would produce a beneficial effect. The β-AR agonist isoproterenol (10 μg in 1 μl saline) was infused into the CA1 region 30 min before retention test. Surprisingly, isoproterenol did not enhance but severely disrupted retrieval of 7-day contextual fear memory, with no impact on retrieval of 1-day contextual fear memory. The present study argues against the previous conclusion that β-ARs in the CA1 region play a role in memory retrieval. β-ARs in the CA1 region may be dispensable for retrieval of conditioned contextual fear.