Cholinergic and serotonergic alterations in the rat hippocampus following trimethyltin exposure and fetal neural transplantation

The M2 receptor (M2-mAChR) is quantitatively the dominant muscarinic subtype in animal bladders. The alterations in its protein quantity and biosynthesis during diabetic cystopathy were investigated. Three-month-old male Wistar rats were divided into two groups: (1) 2-week-old diabetics; and (2) normoglycemic control rats. Diabetes was induced by single intravenous injection of 60 mg/kg streptozotocin. The amount of M2 receptor protein in the rat bladder body tissue was measured by Western immunoblotting using monoclonal antibodies. For determination of M2 muscarinic receptor mRNA in the bladder tissue, the method of Northern blotting was employed. The results of the Western immunoblotting showed that the amount of M2-mAChR protein in the diabetic bladder was significantly increased by 40.0 +/- 6.2% when compared with the control bladder (P < 0.05, n = 8). The Northern blotting demonstrated a 69.3 +/- 8.5% increase of the M2-mAChR mRNA in the diabetic bladder (P < 0.05, n = 8). The findings of the present study demonstrated an up-regulation of M2-mAChR biosynthesis in the diabetic urinary bladder. This phenomenon could lead to increased reactivity to acetylcholine and thus results in detrusor instability.     

Administration of an aldose reductase inhibitor, ONO-2235, to streptozotocin-diabetic mice restores reductions of DRG neuronal attachment to extracellular matrix proteins in vitro

Attachments of cultured dorsal root ganglion (DRG) neurons to the extracellular matrix (ECM) proteins (type I and IV collagens, laminin and fibronectin) and the adhesion ligand arginine-glycine-aspartic acid (RGD) were impaired in mice 2 weeks after the induction of diabetes by streptozotocin (STZ). However, administration of the aldose reductase inhibitor, ONO-2235, to the STZ-diabetic mice for 1 week restored DRG neuronal attachment to the ECM proteins and RGD to a level close to normal mice. These results suggest that activation of the aldose reductase and subsequent metabolic disorders in diabetic animals may play an important role in detrimental alterations of the neuronal cell-surface receptors for the ECM proteins.     

Role of hyperglycaemia in the pathogenesis of hypotension observed in type-1 diabetic rats

The role of hyperglycaemia in the pathogenesis of hypotension in diabetic disorders was investigated using the changes in cardiac M₂-muscarinic receptor (M₂-mAChR) gene expression in type-1-like diabetic rats and cultured cardiomyocytes. Blood pressure was markedly decreased in diabetic rats following the intravenous injection of streptozotocin (STZ) for 8 weeks. Also, the baroreflex sensitivity (ΔHR/ΔBP), as measured by the changes in heart rate (ΔHR) and mean blood pressure (ΔBP) 1 min after the intravenous injection of phenylephrine (10 μg/kg), was significantly increased. Arecaidine propargyl ester (APE), a M₂-mAChR agonist produced a marked reduction in heart rate in these diabetic rats. Normalization of plasma glucose in diabetic rats using insulin (0.5 IU) or phlorizin (1 mg/kg) injection attenuated the blood pressure reduction and reversed the mRNA and protein levels of cardiac M₂-mAChR. A high concentration of glucose (20 mmol/l) directly influenced the increase in gene expression of M₂-mAChR in the H9c2 cardiac cell line. Hyperglycaemia induced an increase in cardiac M₂-mAChR gene expression, suggesting a role in the pathogenesis of hypotension in diabetic disorders.     

Sorbitol metabolism in inner medullary collecting duct cells of diabetic rats

Intracellular accumulation of sorbitol, generated fromd-glucose via the aldose reductase pathway, is thought to play an important role in diabetic complications such as lens cataracts and neuropathy. In order to elucidate the effect of diabetes on the renal inner medulla, another sorbitol-rich tissue, male Wistar rats were treated with a single dose of streptozotocin (60 mg/kg body weight, i.p.). Six wecks later total inner medullary tissue (IM) or isolated inner medullary collecting duct (IMCD) cells were prepared. In diabetic IM tissue, sorbitol content was 1.8-fold higher than in control IM tissue (134±17 vs. 74±22 mol/g tissue protein). Sorbitol production in both normal and diabetic IMCD cells was strongly dependent on extracellulard-glucose concentration. In normal cells, for example, sorbitol production was 90±9 mol sorbitol/g protein x h at 45 mMd-glucose compared to 13±1 mol/g protein x h at 5 mM. At identicald-glucose concentrations sorbitol synthesis in diabetic IMCD cells was, however, always significantly higher than in control cells (122% of control at 15 mM and 126% of control at 45 mM). In addition, aldose reductase activity in diabetic IM was found to be augmented. The maximal velocity was 4.2 times higher (97±22 U/g protein vs. 23±7 U/g protein) while theK _m of the enzyme remained unchanged. Membrane permeability for sorbitol or the response to changes in extracellular osmolarity was not significantly different in diabetic IMCD cells and normal cells with correspondingly high intracellular sorbitol concentrations. Similarly the kinetic parameters ofd-glucose uptake were not altered by streptozotocin treatment. These results suggest that increased medullary sorbitol content in diabetic rats is a result of increased sorbitol synthesis due to a higher extracellulard-glucose concentration and augmented aldose reductase activity in face of an unaltered sorbitol permeability of the plasma membrane.     

Activation of muscarinic M-1 cholinoceptors by curcumin to increase glucose uptake into skeletal muscle isolated from Wistar rats

Curcumin, an active principle contained in rhizome of Curcuma longa, has been mentioned to show merit for diabetes through its anti-oxidative and anti-inflammatory properties. In the present study, we found that curcumin caused a concentration-dependent increase of glucose uptake into skeletal muscle isolated from Wistar rats. This action was inhibited by pirenzepine at concentration enough to block muscarinic M-1 cholinoceptor (M₁-mAChR). In radioligand binding assay, the binding of [³H]-pirenzepine was also displaced by curcumin in a concentration-dependent manner. In the presence of inhibitors for PLC–PI3K pathway, either U73122 (phospholipase C inhibitor) or LY294002 (phosphoinositide 3-kinase inhibitor), curcumin-stimulated glucose uptake into skeletal muscle was markedly reduced. In Western blotting analysis, the membrane protein level of glucose transporter 4 (GLUT4) increased by curcumin was also reversed by blockade of M₁-mAChR or PLC–PI3K pathway in a same manner. In conclusion, the obtained results suggest that curcumin can activate M₁-mAChR at concentrations lower than to scavenge free radicals for increase of glucose uptake into skeletal muscle through PLC-PI3-kinase pathway.     

Polyol Pathway Mediates Enhanced Degradation of Extracellular Matrix Via p38 MAPK Activation in Intervertebral Disc of Diabetic Rats

The aim of this study was to determine the significance of diabetes on degradation of intervertebral disc (IVD) extracellular matrix. Diabetic rats showed a significant increase in glucose and sorbitol contents in the IVD. The levels of aldose reductase, p38 and metalloproteinases, and degradation of metalloproteinase-derived aggrecan and type II collagen were increased, while tissue inhibitors of metalloproteinases levels were decreased in the IVD of diabetic rats. These changes were markedly affected by inhibition of aldose reductase or p38. Diabetes might contribute to enhanced matrix degradation in the IVD and the polyol pathway might mediate this process via p38 activation.     

N-hexacosanol prevents diabetes-induced rat ileal dysfunction without qualitative alteration of the muscarinic receptor system

We evaluated the effects of N-hexacosanol, a cyclohexenonic long-chain fatty alcohol, on muscarinic receptors in diabetic rat ileal dysfunction. Eight-week-old male SD rats were divided into four groups. After induction of diabetes (streptozotocin 50 mg/kg, i.p.), three groups were maintained for eight weeks with treatment by N-hexacosanol (0, 2 or 8 mg/kg, s.c. every day). Ileum function was investigated by organ bath studies using carbachol and KCl, and the expression levels of muscarinic M(2) and M(3) receptors were investigated by real-time polymerase chain reaction. Various concentrations of subtype-selective muscarinic antagonists, i.e., atropine (non-selective), pirenzepine (M(1) selective), methoctramine (M(2) selective), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, M(1)/M(3) selective), were used in this study. In the presence and absence of these antagonists, contractile response curves to increasing concentrations of carbachol were investigated. Treatment with N-hexacosanol did not alter the diabetic status of the rats, but did significantly prevent the carbachol-induced hypercontractility in diabetic rat ileum. Estimation of the pA(2) values for atropine, pirenzepine, methoctramine, and 4-DAMP indicated that the carbacholinduced contractile response in the ileum is mainly mediated through the muscarinic M(3) receptor subtype in all groups. Furthermore, N-hexacosanol significantly prevented the diabetes-induced up-regulation of intestinal muscarinic M(2) and M(3) receptor mRNAs in streptozotocin-diabetic rats. Our data indicated that N-hexacosanol exerts preventive effects with respect to carbachol-induced hypercontractility in the diabetic rat ileum without qualitative alteration of the muscarinic receptor system.     

重组腺相关病毒介导的VLDLR基因对2型糖尿病大鼠脂代谢紊乱的作用

目的： 探索增加极低密度脂蛋白受体 (VLDLR) 基因表达对2型糖尿病大鼠脂代谢紊乱和动脉粥样斑块的影响。方法: 构建携带人VLDLR基因的重组腺相关病毒载体（rAAV-VLDLR）。高脂高糖饮食8周后尾静脉注射小剂量链脲佐菌素(STZ)造2型糖尿病动物模型,VLDLR治疗组大鼠注射rAAV-VLDLR,糖尿病对照组注射rAAV-0。RT-PCR及Western blotting分别检测大鼠骨骼肌、心脏、主动脉、肝脏、脂肪VLDLR mRNA及蛋白表达水平。检测VLDLR基因对血糖、胰岛素、稳态模型胰岛素抵抗指数（HOMA-IR）、甘油三酯（TG）、胆固醇（TC）、脂蛋白脂酶（LPL）活性及主动脉粥样斑块的影响。结果: rAAV-0组大鼠骨骼肌、主动脉、脂肪VLDLR mRNA及蛋白水平低于正常组,VLDLR治疗组VLDLR mRNA及蛋白水平较rAAV-0组有不同程度增加。VLDLR治疗后4、8周的TG及4、8、12周的TC明显降低（P＜0.05）,8周HOMA-IR明显降低(P＜0.05),LPL活性明显增强(P＜0.05),主动脉内膜损伤减轻。结论: 增加VLDLR基因表达对改善2型糖尿病大鼠脂代谢紊乱效果显著,并可在此基础上减轻主动脉粥样斑块程度。     

Decrease of muscarinic M2 cholinoceptor gene expression in the heart of aged rat

It is well known that the baroreflex activity decreases with aging. However, the mechanisms of this change are still not clear. Thus, we investigated one of the parameters to see whether aging alters gene expression of muscarinic receptors in the heart of Wistar rat aged between 2 months (adult) and 24 months (aged). The mRNA level determination by Northern blot analysis for muscarinic M2 cholinoceptors in aged rat was decreased as compared to that in 2-month-old rats. Quantification of receptor protein using selective antibodies indicated that the level of muscarinic M2 cholinoceptor in the heart of 24-month-old rats was lower than that in 2-month-old animals. These results indicate the decrease of muscarinic M2 cholinoceptor in heart with aging that may contribute as one of the parameters for dysfunction in baroreflex activity.     

糖尿病大鼠肾组织中PTEN/AKT/mTOR通路对自噬的调控作用

目的:观察糖尿病大鼠肾组织中PTEN和自噬水平的变化,探讨糖尿病肾病中PTEN/AKT/m TOR通路对自噬的调控机制。方法:将SD大鼠随机分为正常对照组(NC组)和糖尿病组(DM组),每组各8只。用链脲佐菌素复制DM大鼠模型。于成模后10周处死大鼠后测定相应生化指标和肾脏指数,免疫组化观察肾小管上皮细胞PTEN蛋白的表达部位;Western blotting法检测肾组织LC3、PTEN及PTEN/AKT/m TOR通路的变化;realtime PCR检测肾组织PTEN的mRNA表达水平。结果:DM组的血糖、24 h尿蛋白量和肾脏指数均显著高于NC组(P0.05)。与NC组相比,DM组大鼠肾组织的LC3I和LC3II水平明显降低(P0.05)。PTEN主要分布于肾小管上皮细胞中,DM组PTEN蛋白的表达水平明显低于NC组(P0.05)。DM大鼠肾组织中,PTEN/AKT/m TOR通路的活性增高。结论:糖尿病大鼠肾脏组织中细胞自噬水平下降,而参与自噬调控的PTEN/AKT/m TOR通路活性升高,提示其自噬水平的变化可能受到PTEN/AKT/m TOR通路的调节。     

成纤维细胞生长因子1非促分裂突变体对2型糖尿病大鼠血管功能的保护作用

目的:研究成纤维细胞生长因子1非促分裂突变体(n FGF1)对链脲佐菌素和高脂饮食诱导的2型糖尿病大鼠主动脉血管功能的保护作用并探讨其机制。方法:将5周龄左右(200±20)g雄性SD大鼠30只随机分为正常对照组、2型糖尿病模型组和2型糖尿病模型+n FGF1给药组,每组10只。给药组予以0.5 mg/kg n FGF1腹腔注射4周(隔天给药),对照组和糖尿病模型组则给予等量的生理盐水。监测各组大鼠的血糖变化情况,检测大鼠主动脉舒张功能变化,检测动脉组织超氧化物歧化酶(SOD)水平,检测环氧化酶2(COX-2)、磷酸化细胞外信号调节激酶(p-ERK)和内皮型一氧化氮合酶(e NOS)的蛋白表达水平,检测血清中葡萄糖、胆固醇和甘油三酯水平,研究n FGF1对2型糖尿病大鼠主动脉血管功能的调节作用。结果:n FGF1可明显降低2型糖尿病大鼠血清中葡萄糖、胆固醇和甘油三酯水平,显著增强主动脉SOD活性及e NOS蛋白表达水平,并明显下调COX-2和p-ERK的蛋白水平。结论:n FGF1可以有效保护2型糖尿病大鼠主动脉血管功能,其机制可能与降低血糖和血脂,减轻炎症和氧化应激反应,以及上调e NOS信号通路有关。     

银杏叶提取物对2型糖尿病大鼠肝脏糖皮质激素受体表达的影响

目的： 观察银杏叶提取物（EGB）对2型糖尿病大鼠肝脏糖皮质激素受体(GR)表达的影响,并探讨其机制。方法：雄性SD大鼠30只随机均分成3组：正常对照组、糖尿病对照组、EGB干预组。后两组采用高脂饮食4周,按30 mg/kg 腹腔注射链脲佐菌素（STZ） 诱导2 型糖尿病大鼠模型, EGB干预组给予50 mg·kg-1·d-1EGB连续灌胃12周。治疗结束取血生化检测血糖、血脂水平,放免法检测血胰岛素水平,HE染色光镜下观察肝脏组织的形态学改变,以RT-PCR法检测肝组织GR mRNA的表达水平,免疫组化法检测肝组织GR蛋白表达水平。结果：EGB可降低糖尿病大鼠血糖、血脂、血胰岛素水平,减轻肝细胞脂肪变性、坏死、炎细胞浸润,使肝组织GR mRNA的表达与蛋白表达明显减低。结论：EGB具有抑制2型糖尿病大鼠肝脏GR基因mRNA表达与蛋白表达的作用,这可能是其降低2型糖尿病大鼠血糖、血脂水平,改善肝内脂肪沉积的重要机制之一。     

Defective sarcoplasmic reticular calcium transport in diabetic cardiomyopathy

The effects of insulin and thyroid hormone treatments on cardiac sarcoplasmic reticular function were investigated in chronic streptozotocin-induced diabetes in rats. ATP-dependent Ca2+ transport and Ca2+-stimulated ATPase activities were depressed significantly in microsomal samples from diabetic rats in comparison with control (P less than 0.05). This defect was seen at various times of incubation (1-20 min) and different concentrations of free Ca2+ (10(-7) to 10(-5) M Ca2+) and was accompanied by changes in the protein composition and phospholipid contents of the microsomal fraction. The defect in calcium transport in microsomal vesicles was not evident until 28 days after streptozotocin (65 mg/kg iv) injection, whereas increases in plasma glucose levels due to insulin-deficiency occurred within 3 days. All changes in function and composition of the sarcoplasmic reticulum were reversed by insulin administration to the diabetic rats. Although the plasma level of thyroid hormone was decreased in the diabetic rat, thyroid hormone treatment did not restore microsomal calcium transport in the diabetic animals. The results of this study provide some evidence that the depression in cardiac sarcoplasmic reticular calcium accumulation during diabetes is a consequence of insulin deficiency and associated chronic metabolic changes but the hypothyroid condition that accompanies experimental diabetes does not appear to play any role in this defect.     

白藜芦醇对糖尿病大鼠心功能障碍及酸性鞘磷脂酶-神经酰胺通路的影响

目的:观察白藜芦醇(RSV)对糖尿病大鼠心功能障碍的影响并探讨其与酸性鞘磷脂酶-神经酰胺通路的关系。方法:通过一次性腹腔注射小剂量链脲佐菌素(STZ;30 mg/kg)联合高脂饮食饲养12周构建2型糖尿病(T2DM)大鼠模型。实验分为正常对照(control)组、T2DM组、T2DM+RSV组(灌胃给予糖尿病大鼠白藜芦醇100 mg·kg~(-1)·d~(-1))和RSV组(正常大鼠灌胃给予同等容量RSV),共灌胃12周,每周称量体重并调整给药剂量。实验结束后,釆用小动物M型超声检测模型大鼠心脏功能、形态和结构的变化;颈动脉插管检测血流动力学变化;生化方法检测血清中糖、脂水平以及心脏组织中超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;油红O染色和天狼星红染色分别观察心脏组织中脂肪堆积和心肌纤维化情况;高效液相质谱法检测心脏组织中神经酰胺(ceramide)含量;Western blot检测酸性鞘磷脂酶(ASMase)和过氧化物酶增殖体激活受体γ辅激活因子1α(PGC~(-1)α)的蛋白表达。结果:T2DM组大鼠空腹血糖、甘油三酯、胆固醇及低密度脂蛋白胆固醇水平均较control组显著升高,心功能显著降低(P0.05);T2DM组大鼠心脏组织中脂肪堆积、MDA含量显著增加(P0.05),SOD活性、ATP水平和PGC~(-1)α蛋白表达均显著降低(P0.05),且出现明显心肌纤维化;给予白藜芦醇治疗12周可显著改善以上指标的异常变化,同时,显著下调糖尿病大鼠升高的心肌ASMase蛋白和ceramide水平。结论:白藜芦醇可显著改善糖尿病诱导的大鼠心功能障碍和心肌纤维化,其机制可能与抑制ASMase-ceramide通路有关。     

糖尿病大鼠肾组织Smurf2表达及其与SnoN蛋白降解的关系

目的:观察Smad泛素化调节因子2(Smurf2)和核转录共抑制因子SnoN在糖尿病(DM)大鼠肾组织的动态表达,并初步探讨Smurf2在DM大鼠肾组织SnoN蛋白表达变化中的作用。方法:链脲佐菌素(STZ)尾静脉注射复制DM大鼠模型,随机分为DM2、4、8、12、16和24周组,每组均设鼠龄匹配的正常对照组(n=6)。生化方法测血糖、血肌酐(Scr)及24h尿蛋白量,计算肾脏指数;HE染色观察胰腺和肾组织病理学改变;免疫荧光染色检测胰腺组织胰岛素、肾组织Smurf2和SnoN的表达;Westernblotting检测肾皮质Smurf2、SnoN、转化生长因子β1(TGF-β1)和磷酸化Smad2(p-Smad2)蛋白的表达;RT-PCR检测肾皮质Smurf2和SnoN的mRNA。结果:(1)DM各组血糖、血Scr、24h尿蛋白量和肾脏指数均高于正常对照组,正常胰腺组织胰岛素表达强,DM大鼠胰岛素表达则明显减弱;(2)Smurf2和SnoN蛋白均主要表达于肾小管上皮细胞,从DM2周始各DM组Smurf2、TGF-β1和p-Smad2蛋白表达均多于正常对照组,DM4周起各DM组SnoN蛋白均少于正常对照组,DM各组SnoNmRNA与正常组相比无显著差异,Smurf2mRNA随病程进展逐渐增多;(3)Smurf2和SnoN蛋白的表达量呈显著负相关(r=-0.88,P0.01)。结论:在糖尿病肾病发病过程中SnoN蛋白的表达减少可能与Smurf2介导其泛素化降解有关。     

Polymorphisms of sorbitol dehydrogenase (SDH) gene and susceptibility to diabetic retinopathy

The polyol pathway consists of two enzymes aldose reductase (AR) and sorbitol dehydrogenase (SDH); the former is the first enzyme in the polyol pathway, that catalyzes the reduction of glucose to sorbitol, the latter is the second one, that converts sorbitol to fructose using by NAD(+) as a cofactor. We along with others have recently found that SDH activity, the second step in the polyol pathway, might make a greater contribution to the etiology of diabetic retinopathy than does the first step involving AR. In this paper, we propose a novel hypothesis that polymorphisms of SDH gene may be correlated with SDH gene expression levels in diabetic retinas, thus being a valuable genetic marker for diabetic retinopathy.     

阿托伐他汀对糖尿病大鼠急性心肌梗死后心功能及HGF/c-Met信号通路的影响*

 目的： 糖尿病患者急性心肌梗死（AMI）后的心室重构及心功能恶化较非糖尿病者更为明显。本研究旨在观察阿托伐他汀对糖尿病大鼠AMI后心肌细胞凋亡、心室重构及心功能的影响,并探讨其作用是否与肝细胞生长因子及其受体 (HGF/c-Met)信号通路有关。方法: 70只雄性SD大鼠经链脲霉素 (STZ, 65 mg/kg)腹腔注射,诱导糖尿病大鼠模型。8周后对糖尿病大鼠结扎左冠状动脉前降支构建AMI大鼠模型,术后存活32只大鼠随机分为2组：AMI对照组(n=16)和阿托伐他汀干预组(n=16, 阿托伐他汀20 mg·kg-1·d-1),并在糖尿病大鼠中设假手术组(n=11),术后24 h予以灌胃给药。2周后比较各组大鼠心功能、心肌组织病理改变、心肌细胞凋亡、HGF和c-Met mRNA及蛋白表达差异。结果: (1) AMI对照组心功能显著低于假手术组（P<0.05）,胶原容积分数、心肌细胞凋亡指数、HGF及c-Met mRNA及蛋白表达均显著高于假手术组（P<0.05）;(2) 阿托伐他汀干预组的胶原容积分数和心肌细胞凋亡指数显著低于AMI对照组（P<0.05）,心功能、HGF及c-Met mRNA及蛋白表达均显著高于AMI对照组（P<0.05）。结论: 阿托伐他汀对糖尿病大鼠AMI后心肌细胞凋亡、心室重构及心功能具有显著改善作用;HGF/c-Met信号通路在AMI后会激活,阿托伐他汀的上述作用机制可能与其进一步增强HGF/c-Met信号通路有关。     

Increased nerve polyol levels in experimental diabetes and their reversal by Sorbinil.

Sciatic nerve glucose, sorbitol, fructose and myo-inositol levels were measured over a 24-week period in rats made diabetic using a single intraperitoneal injection of streptozotocin at 45 mg kg-1 body weight. In addition, the effect of the aldose reductase inhibitor Sorbinil (Pfizer Ltd) at 25 mg kg-1 day-1 in reversing the accumulation of nerve polyols following 8 weeks of diabetes was investigated. Following induction of diabetes 47, 471 and 456% increases and a 43% decrease in sciatic nerve glucose, sorbitol, fructose and myo-inositol concentrations (mumol g-1 wet weight) respectively, were observed by day 14. Over the remainder of the experimental course untreated diabetic control animals demonstrated relatively consistent elevations in sciatic nerve glucose, sorbitol and fructose. Although a significant, progressive reduction in sciatic nerve myo-inositol to 30% of onset values was observed over the first 84 days of the study, this was followed by a spontaneous partial recovery (31%) over the remainder of the experimental course. However, sciatic nerve myo-inositol levels at the end of the study were still significantly lower than onset values (P less than 0.01). Sorbinil treatment, initiated after 8 weeks of diabetes, and without effect on sciatic nerve glucose levels, normalized sorbitol concentrations following 4, 8, or 12 weeks of treatment but only partially reversed the accumulation of fructose by 368, 161 and 199%, compared to age-matched non-diabetic control values, at the above times, respectively. Mean myo-inositol levels were progressively increased following Sorbinil treatment over the experimental period, although the increase was only significant, compared to results from untreated diabetic animals, at weeks 4 and 16.(ABSTRACT TRUNCATED AT 250 WORDS)