Stabilization of proteins by low molecular weight multi-ions

A method is described to identify small molecule ligands that stabilize proteins. The procedure is based on the hypothesis that molecules of various sizes containing two to four charges should occasionally bind to unpaired charged sites on the surface of proteins and by crosslinking such residues stabilize the native state of the liganded protein. A simple turbidity assay is employed that detects inhibition of protein aggregation under selected sets of conditions. Eight test proteins were screened and in all cases specific ligands were identified that inhibited protein aggregation at millimolar to micromolar concentrations. Only small effects of these stabilizers on protein biological activities were found. In some, but not all cases, circular dichroism and fluorescence studies provided direct evidence of the binding of stabilizing ligands to the proteins suggesting multiple mechanisms of stabilization. This approach should be applicable to the development of excipients for the stabilization of pharmaceutical proteins and industrial enzymes as well as serve as starting points for second-generation inhibitors of increased affinity and specificity.     

A new test for morphine-like physical dependence (addiction liability) in rats

Summary A new test for physical dependence (addition liability) of drugs in morphine-dependent rats is described. It depends upon the abolition of spontaneous withdrawal writhing by drugs possessing morphine-like addiction liability. A quantitative estimation of addictive potency of narcotic analgesics is given and some evidence for the possible mechanism of this phenomenon of withdrawal writhing is presented.     

Determination of main low molecular weight antioxidants in urinary bladder wall using HPLC with electrochemical detector

The aim of the present work was to develop validated HPLC method using electrochemical detector for simultaneous detection of low molecular weight antioxidants (LMWA) in urinary bladder. Furthermore, the method was applied to study the distribution of LMWA in urinary bladder wall. The ascorbic acid (AA), glutathione in reduced (GSH) and oxidized (GSSG) form and uric acid (UA) were resolved by isocratic elution from C18 reversed-phase column. The bladder tissue sample preparation involved extraction with meta-phosphoric acid solution for LMWA stabilization. The AA, GSH and UA tissue peak was identified by different approaches. The obtained method validation parameters were in acceptable range: intra-day precision (<4.4%), intra-day accuracy (<8.4%), inter-day precision (<9.4%) and inter-day accuracy (<15.6%). Additionally, the method provided good linearity (r2>0.99) and recoveries (98.9-112.6%). The distribution of LMWA in urinary bladder was determined by measuring their concentration in bladder wall layers: urothelium, lamina propria, muscularis and serosa. The validated method was able to quantify the reduced form of all three LMWA in all four bladder wall layers. The LMWA concentrations were decreasing from urothelium to serosa except of UA. The developed HPLC method with electrochemical detection of LMWA is simple, fast and can be used for simultaneous quantification of LMWA in tissues, which contain low concentrations of antioxidants.     

低分子肝素在麻醉大鼠胃肠道的吸收

以血浆抗Ｘａ活性及体内抗静脉血栓形成为指标，考察了低分子肝素溶液及乳剂在麻醉大鼠胃肠道的吸收情况。     

An optimized approach for the assessment of sexual behavior in male rats

Abstract<195>The improvement and optimization of methods used to detect reproductive disorders in experimental animals are among the main challenges facing researchers in this field. The conventional method for testing male sexual behavior uses ovariectomized females rendered sexually receptive by injection of estradiol benzoate and progesterone prior to testing. The receptive females are then mated with exposed male rats during the dark phase of the cycle under dim red light followed by direct visual and momentary observation of the mating activity. The ovariectomized females may respond differently to hormonal injection (individual differences), leading to variations in the intensity of lordosis. Additionally, the data obtained by the direct visual and momentary evaluation of copulatory activity are very subjective and may produce inaccurate results. In the optimized method, the sexual cycles of female rats are determined, and only those in estrus are selected and mated with sexually experienced male rats. The mating activity is videotaped, enabling the correct observation and evaluation of the different components of mating behavior. This method fosters animal welfare by avoiding surgical intervention and enables the videotape to be kept as permanent documentation.     

Botulinum ADP-ribosyltransferase C3: a new tool to study low molecular weight GTP-binding proteins

It is well known that certain bacterial toxins, e.g. cholera and pertussis toxins, ADP-ribosylate eukaryotic regulatory proteins. They have become invaluable tools in the study of G protein-linked receptors. Less well appreciated is the fact that certain strains of Clostridium botulinum types C and D produce an ADP-ribosyltransferase, termed C3. This enzyme is structurally and functionally distinct from botulinum neurotoxins C1 and D. Its substrate is the 21 kDa GTP-binding protein rho. Klaus Aktories and Alan Hall explain why C3 is now an important tool in analysing the regulatory function of the ras-related protein rho.     

A 3.0-kDa low molecular weight heparin promotes gastric ulcer healing in rats

BACKGROUND: Previous studies have shown that intragastric administration of unfractionated heparin enhances gastric ulcer healing in rats. As the large molecule of heparin may be partially degraded in the upper gastrointestinal tract, it is likely that fragments of heparin, derived from the unfractionated parent compound, are involved in the anti-ulcer action in the stomach. Therefore, it is possible that low molecular weight heparin may have a similar ulcer healing effect. METHODS: Male Sprague-Dawley rats with acetic acid-induced gastric ulcers were given a 3.0-kDa low molecular weight heparin (0.6-6.0 mg/kg) intravenously or intragastrically once daily for 4 days. Ulcer healing, mucosal histological changes, angiogenesis and gastric mucus production both in vivo and in vitro were determined. The bleeding time was measured to indicate the anticoagulation activity. RESULTS: Both intravenous and intragastric low molecular weight heparin dose dependently accelerated gastric ulcer healing, which was accompanied by a significant increase in mucosal regeneration and proliferation, angiogenesis and mucus content in the stomach. The drug also stimulated the mucus production in MKN-28 cells. Drug administration by either route did not alter the bleeding time in rats. CONCLUSIONS: A 3.0-kDa low molecular weight heparin possesses an ulcer healing effect similar to that of unfractionated heparin in the stomach of the rat. This smaller molecular drug is superior to the unfractionated form, does not affect the coagulation activity and may show better absorption in the gastrointestinal tract.     

Induction of physical dependence upon ethanol in rats using intravenous infusion

Intravenous infusions were used to produce physical dependence upon ethanol in rats. The procedure proved to be safe, rapid, and reliable. Ethanol (30% v/v) was administered over a 7-day period. The mean daily dose ranged from 10–14 g/kg/day. Control rats were exposed to a comparable procedure except that saline, rather than ethanol, was infused. All ethanol treated rats that survived the intoxication period (n=11) showed signs of physical dependence (moderate to severe3 n=8; mild, n=3) following ethanol withdrawal. Saline treated rats (n=8) did not show any of these symptoms. The most reliable ethanol withdrawal signs observed were: spontaneous seizure (n=7), audiogenic seizure (n=7), tremors (n=6), tail stiffening (n=10) and body rigidity (n=9). These symptoms were analyzed in terms of their hour of onset and hour of maximum intensity following ethanol withdrawal. Application of the intravenous method for the study of ethanol self-administration is discussed.     

A simple,reliable method for predicting the physical dependence liability of narcotic antagonist analgesics in the rat

The rat intraperitoneal infusion procedure was used to chronically administer drugs for evaluation of the physical dependence liability of narcotic antagonist analgesics. Three methods were used to assess dependence liability: presence of withdrawal signs upon abrupt cessation of chronic infusion (primary dependence), attenuation of the withdrawal signs produced by cessation of chronic morphine infusion (morphine substitution), and production of withdrawal signs when chronically morphine-infused rats were administered the drugs (precipitated withdrawal). Butorphanol, nalbuphine and pentazocine all caused a mild withdrawal in the rat primary dependence model which agrees with the conclusions from experiments with monkey and man. None of these agents substituted for morphine in the rat and all three appeared to precipitate withdrawal. Two experimental drugs, Codorphone and TR5400, did not induce primary dependence in the rat, and in chronic-morphinized rats, they precipitated a withdrawal syndrome comparable to naloxone. Another experimental drug, TR5257, substituted for morphine. The correlation between these observations in the rat and previously published data from the monkey are excellent. It is proposed that the rat could be used as a reliable indicator of potential physical dependence liability for the narcotic antagonist analgesics.     

Studies to identify the low molecular weight bismuth-binding proteins in rat kidney

Low molecular weight bismuth-binding proteins were isolated from kidneys of rats exposed repeatedly to bismuth chloride. The proteins [(Bi,Cu)-BP] isolated by polyacrylamide gel electrophoresis yielded four peaks. The two main fractions [(Bi,Cu)-BP 2 and (Bi,Cu)-BP 3] were characterized by a molecular weight of 7500, bismuth contents of 79 and 90 μg/mg, copper contents of 14 and 23 μg/mg and zinc contents of 3.8 and 3.6 μg/mg respectively, and an A₂₅₀/A₂₈₀ ratio of 1.5. Their amino acid composition resembled that of the copper chelatin from the liver. Isoelectrofocusing separated the protein into three components of pI values of 3.8, 5.5 and 7.4. The protein differs from metallothionein by its apparent molecular weight, amino acid composition, lower SH group content and a high copper content. Traces of a low molecular weight copper-binding protein of molecular weight of about 7500, of similar electrophoretic properties, were also found in kidneys of non exposed rats. It is suggested that these proteins are related to each other. For the renal bismuth-binding proteins the term ‘renal chromochelatin’ is proposed.     

低分子肝素pH敏感混合聚合物纳米粒大鼠体内生物利用度研究

目的建立生色底物法测定大鼠血浆中低分子肝素(LMWH)含量的方法,研究p H敏感巯基壳聚糖/O-羧甲基壳聚糖(TCS/O-CMC)混合聚合物纳米粒在SD大鼠体内的相对生物利用度。方法采用生色底物法以SD大鼠为研究对象,测定LMWH注射液、LMWH溶液、p H敏感TCS纳米粒和p H敏感TCS/O-CMC混合聚合物纳米粒单剂量给药后的血药浓度,并用Win Nonlin软件计算药动学参数和生物利用度。结果 LMWH溶液、p H敏感TCS纳米粒和p H敏感TCS/O-CMC混合聚合物纳米粒的药动学参数如下:Cmax分别为(0.103±0.025)、(0.425±0.046)和(0.522±0.042)U/ml。AUC0→∞分别为(0.5±0.3)、(4.7±1.0)和(8.0±2.0)U·ml-1·h。相对生物利用度分别为2.10%、20.70%和34.98%(P<0.05)。结论 p H敏感TCS/O-CMC混合聚合物纳米粒可显著提高LMWH的口服生物利用度。     

Pharmacokinetics and organ distribution in rats of a low molecular weight heparin

Fluorescein-labelled low molecular weight heparin (OP/LMWH) pharmacokinetics and organ distribution were studied in the rat after intravenous administration at doses of 3 and 15 mg/kg. The pharmacokinetics of labelled OP/LMWH was dose-dependent like heparin, however, the anticoagulant activity such as anti-Xa was more prolonged. The rapidity of the disappearance of the fluorescence was quicker than the biological activity. The fluorescence was observed mainly in the liver and kidney as a diffuse aspect in the short time and as a globular aspect in the long time, mainly in Disse's space of the liver. These results confirm that the liver is important in the regulation of heparin metabolism and activity.     

Toxic and mutagenic effects of a low molecular weight heparin in rats

Acute, subacute, chronic toxicity and mutagenicity studies of a new low molecular weight heparin (OP/LMWH) were carried out in rats. The LD50 values resulted lower by i.m. and s.c. route than after i.p. and i.v. administration. OP/LMWH given by subcutaneous route in subacute and in chronic toxicity produced no significant adverse effects at 5 mg/kg, only marginal effects at 10 mg/kg and moderate effects at the dose of 20 mg/kg. In these studies, unfractionated heparin at 10 mg/kg by s.c. route was used for comparison. At 10 mg/kg, heparin presented effects similar to those shown by OP/LMWH at 20 mg/kg. OP/LMWH did not show any mutagenic activity when compared with mutagenic standards.     

含低相对分子质量壳聚糖凝胶对大鼠放射性皮炎的防护作用

目的:观察含低相对分子质量壳聚糖(相对分子质量<1 000)凝胶对大鼠放射性皮炎的防治作用,并初步探讨其作用机制,为研制抗放射性皮炎新制剂提供试验依据。方法:一次性⁶⁰Co 辐照60Gy造大鼠放射性皮炎模型,照射部位分别涂抹给予生理盐水(CON)、单一高相对分子质量壳聚糖凝胶(KH)、单一低相对分子质量壳寡糖(M<1 000)(KL)、含相对分子质量低于1 000的壳寡糖复配高相对分子质量壳聚糖(KH+L)、比亚芬(BYF);观察不同给药组对放射性皮炎模型大鼠的保护作用。并通过皮肤含水量测定、足肿胀测定,探讨不同凝胶抗放射性皮炎的作用机制。结果:CON组、KH组、KL组及BYF组的皮炎发生率分别为100%,50%,50%,50%,皮炎评分分别为2.65,1.25,1.38,1.25;KH+L组皮炎发生率为12.5%,皮炎评分为0.125,与生理盐水组对比差异有极其显著(P<0.01),与其他各组相比差异有显著性(P<0.05);涂药后2 h,CON组、KH组、KL组、BYF组、KH+L组的皮肤水分含量分别为19.8%,29.2%,22.3%,25.3%,30.3%;蛋清致炎,60 min后小鼠足肿胀度分别为CON组45.5%、KH组38.9%、KL组19.5%、BYF组38.9%、KH+L组12.2%。结论:单一高相对分子质量壳聚糖凝胶、单一低相对分子质量壳聚糖凝胶、比亚芬、含低相对分子质量复配壳聚糖凝胶,均具有抗放射性皮炎作用,其中复配壳聚糖凝胶效果显著,可能与其具有保湿、抗炎作用机制有关。     

低分子肝素对肾缺血—再灌注损伤的防治作用

目的　探讨低分子肝素对大鼠肾缺血—再灌注损伤的防治作用。方法　建立肾缺血—再灌注损伤大鼠模型 ,实验动物随机分为正常对照组和低分子肝素治疗组及非治疗组。观察大鼠治疗前后肾组织中P 选择素表达、组织病理学与肾功能的改变 ,以及凝血指标和凝白酶原时间 (PT)、白陶土部分凝血活酶时间 (KPTT)的变化。结果　缺血—再灌注后大鼠肾脏P 选择素表达增高 ,出现明显的组织病理学改变及肾功能损害。低分子肝素治疗组肾组织中P 选择素表达受抑 ,病理学改变与肾功能损害减轻 ,且对PT和KPTT无明显影响。结论　低分子肝素对肾缺血—再灌注损伤具有防治作用 ,且对凝血指标PT和KPTT无明显影响。     

Morphine-like physical dependence: a pharmacologic method for drug assessment using the rat

Rats maintained on various dosage regimens of morphine showed dose related taste aversions to a saccharin solution offered to them upon withdrawal from the drug. Maximal saccharin aversions occurred between 72 and 96 hr after termination of morphine injections, and gradually returned to baseline preference levels after 14 days of morphine abstinence. The results were interpreted as suggesting that the morphine treated animals associated the aversive components of the morphine withdrawal syndrome with saccharin consumption, and manifested conditioned aversions to the saccharin solution which extinguished as the withdrawal syndrome subsided. The findings were further discussed with regard to the potential use of conditioned taste aversions in determining whether test compounds are capable of causing physical dependence in rats.     

A high molecular weight form of human urinary urokinase

A very high molecular weight form of urokinase, of about 100,000 D (VHMr-UK), was isolated from human urine in trace amounts as compared to the well characterized two forms of urokinase, HMr-UK (Mr of about 50,000) and LMr-UK (Mr of about 30,000). This form was demonstrated to be a dimer of HMr-UK, in which no covalent bond is involved, on the basis of the following evidence: by SDS electrophoresis it is dissociated, to the 50,000 D form; by electrophoresis in SDS under reducing conditions it is dissociated, as HMr-UK, to two polypeptide chains of about 30,000 and 20,000 D; by short heating at pH 5 it is quantitatively converted to the 50,000 D form; the kinetic constants towards the alpha-carbobenzoxy-L-lysine-p-nitro-phenylester substrate are the same for HMr-UK and VHMr-UK.     

Mechanism of the sex-dependent effect of lithium on body weight in rats.

Two experiments are reported here. First, the effect of lithium chloride (1, 2 and 4 mEq/kg IP for 21 days) on body weight was assessed in female and male rats. Food intake was measured in the rats treated with 2 mEq/kg. All the doses tested significantly increased body weight in female rats. A linear relationship between body weight gain and lithium dose was also observed. In contrast, in male rats, the low doses of lithium (1 and 2 mEq/kg) did not affect body weight, whereas the high dose (4 mEq/kg) decreased body weight. These results confirm previous reports on a sex-dependent effect of lithium on body weight in rats. In the second experiment, body weight and food intake were assessed in female rats treated with lithium alone, or in combination with insulin or sulpiride, a D2 dopamine receptor blocker. It was found that the effect of lithium on body weight and feeding was additive to the effects of sulpiride and insulin. These findings are indirect evidence that lithium enhances body weight in rats by a different mechanism than the one described for sulpiride or insulin.     

Synthesis of arrays using low molecular weight MPEG-assisted Mitsunobu reaction

Triphenylphosphine tagged with a short poly-(ethyleneglycol)-ω-monomethyl ether chain (light MPEG, 10?16 ethylenoxy units, (M)TPP-G2) and an MPEG-tagged version of diethyl azodicarboxylate ((M)DEAD) have been used to prepare a 20 member library of esters, ethers, and sulfonamides, with cLogP's in the range of 1.4?5.7 on a 0.1 mmol scale. Removal of MPEG-tagged side products was achieved by MPEG-assisted solid-phase extraction (MSPE) on prepacked silica columns to give the products in good yield and high purity.