SPECTROPHOTOMETRIC STUDY OF α-ADRENOCEPTORS AFFECTING MICROCIRCULATION OF RAT SKIN

1. To determine the α-adrenergic receptor subtypes that affect the microcirculation of skin, the relative absorption (RA) spectra of the skin on the backs of rats were measured using reflectance spectrophotometric methods. We injected α-adrenergic agonists, noradrenaline (NA), phenylephrine (PE) and clonidine (CL), intravenously and determined changes in the RA value at 569 nm, one of the isosbestic points of the oxyhaemoglobin and deoxyhaemoglobin absorption. 2. NA reduced the RA value and the reduction was inhibited significantly by pretreatment with phenoxybenzamine (PBZ; P<0.01). These findings suggested that the haemoglobin content in the skin tissue decreased as a result of vasoconstriction through α-adrenoceptors. 3. NA, PE and CL produced dose-dependent reductions in RA. CL and NA produced virtually equipotent reductions except at the highest dose used. PE produced smaller effects. The potency of these drugs in terms of changes in RA did not correlate with their potency in terms of rises in systemic blood pressure (NA ≤ PE ≥ CL). 4. Yohimbine (YO) inhibited the NA-induced reduction in RA to a greater degree than bunazosin (BU). Midaglizole, a specific α₂-adrenergic antagonist, significantly and dose dependently inhibited the NA-induced reduction in RA. 5. Although BU inhibited NA-induced reduction in RA only slightly, the effect was significant (P<0.05). BU significantly inhibited PE-induced reduction (P<0.01), but did not inhibit CL-induced reduction. 6. These observations suggest that the microcirculation of the skin of the rat is affected mainly by α₂-adrenoceptor mediated vasoconstriction. However, α₁-adrenoceptor mediated vasoconstriction also has some effect.     

蝙蝠葛碱对血小板聚集及花生四烯酸代谢的影响

蝙蝠葛碱(Dau) 抑制AA及ADP诱导的大鼠血小板聚集,也能抑制AA,ADP及Adr诱导的人血小板聚集。这种抑制作用与Dau剂量呈依赖关系。Dau抑制大鼠洗涤血小板对[1-¹⁴C]AA经环氧酶途径的代谢,TXB₂与HHT的形成均呈剂量依赖性减少。当Dau浓度达到0.1 mmol/L时亦能抑制12-HETE的形成。Dau对AA代谢的上述影响可能是其抑制血小板聚集的机理之一。     

AMINO ACID COMPOSITION AND SEQUENCE OF DERMORPHIN,A NOVEL OPIATE-LIKE PEPTIDE FROM THE SKIN OF PHYLLOMEDUSA SAUVAGEI

Dermorphin, a heptapeptide with very potent opiate-like activity, has been isolated from methanol extracts of the skin of the South American frog Phyllomedusa sauvagei. The amino acid sequence of the peptide is: H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂. Dermorphin presents striking differences from the known enkephalins; it offers a surprising example of a peptide from Vertebrata containing a D-amino acid residue in its sequence.     

EFFECTS OF ARGININOSUCCINIC ACID ON NITRIC OXIDE-MEDIATED RELAXATIONS IN RAT AORTA AND ANOCOCCYGEUS MUSCLE

1. Argininosuccinic acid (ASA), a naturally occurring NG derivative of arginine, and the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) were compared for their ability to reduce responses to nitric oxide (NO) derived from endothelial cells (aorta) and nitrergic nerves (anococcygeus muscle). 2. In isolated rings of rat aorta, endothelium-dependent relaxation responses to acetylcholine were abolished by L-NAME (0.1 mmol/L) and were reduced by ASA (0.1 and 0.3 mmol/L). Relaxations induced by sodium nitroprusside (SNP) were not affected by L-NAME but were reduced by ASA. 3. In rat isolated anococcygeus muscles, relaxations elicited by nitrergic nerve stimulation at 1 Hz were abolished by L-NAME (0.1 mmol/L) but were only slightly reduced by ASA (1 mmol/L). The effect of ASA was not sustained. L-Arginine (1 mmol/L) prevented the effect of L-NAME but not that of ASA. Neither ASA or L-NAME inhibited SNP-induced relaxation in the anococcygeus muscle. 4. The results suggest that ASA inhibits NOS but this does not totally account for its effects in reducing NO-mediated relaxations produced by the endothelium-dependent vasodilator acetylcholine in rat aortic rings and stimulation of nitrergic nerves in the rat anococcygeus muscle.     

醋酸棉酚对幼龄大鼠睾丸间质细胞分泌活性及细胞膜表面LH/hCG受体发育的影响

醋酸棉酚有抗雄性生育作用,但对睾丸间质细胞的作用尚未取得一致意见。部分研究者观察到醋酸棉酚对睾丸间质细胞有抑制作用。本室也曾证明,服醋酸棉酚后,青春前期大鼠,睾丸间质细胞的生长发育及其类固醇激素合成酶的活性均受抑制。已有报告,睾丸间质细胞表面具有分子量为20万的LH/hCG受体,该受体的数量及活性决定了间质细胞睾酮分泌活性及对促激素的反应。此受体在大鼠五周龄前逐步发育完善,而在4周龄时已表现出对外源性LH/hCG敏感的反应性。本实验以幼龄大鼠为对象,用放射免疫测定及放射受体分析法研究了醋酸棉酚对睾丸间质细胞的影响。     

甘草酸的提取、分离和纯化

以晶种法和大孔树脂法从甘草和甘草浸膏中提取分离甘草酸,粗产品采用活性炭,大孔树脂或过氧化氢纯化。甘草酸单铵盐收率较一般方法高,达65～75%,纯度90%以上,操作也较简便。     

PURIFICATION AND CHARACTERIZATION OF AN ACID PROTEASE FROM MONASCUS KAOLIANG*

An acid protease from Monascus kaoliang was purified by consecutive applications of fractional acetone precipitation, batchwise CM-cellulose method and DEAE-cellulose column chromatography. The preparation was homogeneous on disc polyacrylamide gel electrophoresis at pH4.5 and 7.5. The yield was about 30% with overall increase in specific activity of about 6-fold. The molecular weight as determined by SDS gel electrophoresis was about 34,000. The enzyme was a glycoprotease as indicated by specific carbohydrate staining on gels. It possessed the nature of an acid protease with a pH optimum at 3.0 toward heat-denatured casein and was stable over the range of pH 3.0 to 6.0. Reducing agents and thiol poisons had no effect on this enzyme, suggesting that free sulfhydryl groups were not required for enzyme activity. Diisopropyl fluorophosphate did not inactivate this protease, indicating the probable absence of serine residue in the active site. The enzyme was inactivated by reaction with the carboxy-group specific reagent, 1,2-epoxy-3-(p-nitrophenoxy) propane (EPNP). Pepstatin, a specific inhibitor for pepsin, was shown to inhibit this enzyme strongly. However, biacetyl (2,3-butadione) had little effect on this protease, although it inactivated pepsin to an 85% activity loss. Also, p-bromophenacyl bromide, another specific inhibitor of pepsin, failed to inactivate this acid protease.     

PURIFICATION AND PROPERTIES OF CRYSTALLINE HUMAN CERULOPLASMIN

Crystalline human ceruloplasmin was prepared from a Cohn's fraction F-IV-1 of normal human pooled plasma, by procedures based on acetone fractionation and chromatography on DEAE-sephadex A-50. Needle-shaped crystals of the human ceruloplasmin showed a single component on ultracentrifugation, electrophoresis and immunoelectrophoresis and had a sedimentation constant of 7.04 S and an electrophoretic mobility of 5.2 × 10^–5 cm² volt^-1 sec^-1. Light scattering of the ceruloplasmin gave a molecular weight of 160,000. Absorption coefficient at 610 nm was E^1%_{1 cm} 0.66, at 280 nm, 14.7 and the ratio E_{610 nm}/E_{280 nm}, 0.045. This protein provided 0.31% copper and contained serine, aspartic acid and valine residues as an amino terminal amino acid and showed an oxidase activity of 62 arbitrary units.     

INHIBITION BY ETHACRYNIC ACID OF NO-MEDIATED RELAXATIONS OF THE RAT ANOCOCCYGEUS MUSCLE

1. The effects of ethacrynic acid were studied on relaxations elicited by nitric oxide (NO), the NO-donors sodium nitroprusside (SNP) and glyceryl trinitrate (GTN), nitrergic nerve stimulation and the NO-independent agent papaverine in isolated preparations of rat anococcygeus muscles. 2. Ethacrynic acid (100 μmol/L) produced complete relaxation of partially contracted anococcygeus muscles, but the tone recovered after the ethacrynic acid was washed out. Following exposure to ethacrynic acid, the relaxant responses to NO, SNP, GTN and nitrergic nerve stimulation were abolished or markedly reduced; however, the response to papaverine was only slightly reduced. 3. The presence of 3 mmol/L l-cysteine during the period of exposure to ethacrynic acid prevented the inhibition of the relaxing effects of SNP, GTN and nitrergic nerve stimulation almost completely, but did not affect the slight reduction in responses to papaverine. 4. The addition of l-cysteine (3 mmol/L) after incubation with ethacrynic acid did not significantly affect the inhibited responses to SNP and GTN; however, the inhibited responses to nitrergic nerve stimulation were slightly but significantly increased. 5. The results suggest that endogenous sulphydryl groups are required for the actions of NO, NO-donating drugs and the nitrergic transmitter in the rat anococcygeus muscle and possibly for the synthesis or release of the nitrergic transmitter.     

没食子酸丙酯对大鼠肝线粒体肿胀和脂质过氧化物形成的作用

The extract of Radix Paeonia rubra, a common traditional Chinese drug was shown to inhibit platelet aggragation and oxidative phosphorylation while increasing fluidity of rat liver mitochondria. This paper reports the effects of propyl gallic acid, one component isolated from Radix Paeonia, on rat liver mitochondrial swelling and malondialdehyde (MDA) formation.Experiments on the formation MD and swelling of rat liver mitochondria induced by Fe²⁺ (5 μM)and vit C (0.1 mM) with or without the drug was carried out in vitro. In order to study the protectiye effects of drug against the damage of the mitochondria. The results show-that propyl gallic acid inhibited the swelling of mitochondrta andMDA formation. Fe²⁺ and vit C were also shown to reduce ANS fluorescence in mitochondrial suspension. It is inferred that the cross reaction of MDA with biological macromolecules and the decrease of surface potential of mitochondrial membrane are factors that induced damage to the function of mitochondria. This compound was shown to be unable to inhibit MDA damage to the mitochondria. However, the anti-oxidative and antiswelling action of the drug on mitochondria may be associated with the Pharmacological effects of Radix Paeonia rubra.     

没食子酸丙酯对大鼠肝线粒体肿胀和脂质过氧化物形成的作用

近年来,许多文献指出,线粒体内在氧代谢的过程中,伴有O_2~-和OH~-自由基的生成。它们对生物大分子和膜结构均有不同方式的损伤作用。学者们考虑到向体内补充抗氧化剂,清除过多的自由基或终止体内自由基反应,可防止线粒体肿胀和抑制膜的脂质过氧化反应、     

AROMATIC l-AMINO ACID DECARBOXYLASE: HISTOCHEMICAL LOCALIZATION IN RAT KIDNEY AND LACK OF EFFECT OF DIETARY POTASSIUM OR SODIUM LOADING ON ENZYME DISTRIBUTION

Utilizing a mono-specific antiserum produced in rabbits to hog kidney aromatic L-amino acid decarboxylase (AADC), the enzyme was localized in rat kidney by immunoperoxidase staining. AADC was located predominantly in the proximal convoluted tubules; there was also weak staining in the distal convoluted tubules and collecting ducts. An increase in dietary potassium or sodium intake produced no change in density or distribution of AADC staining in kidney. An assay of AADC enzyme activity showed no difference in cortex or medulla with chronic potassium loading. A change in distribution or activity of renal AADC does not explain the postulated dopaminergic modulation of renal function that occurs with potassium or sodium loading.     

DA—201树脂与XAD—8树脂纯化甘草酸效果的HPLC考察

以 HPLC 为检测手段,比较了 DA-201(国产)与 Amberlite XAD-8(美国)大孔吸附树脂在纯化甘草酸方面的效果,结果表明 DA-201优于 Amberlite XAD-8。并采用 DA-201树脂,对从甘草浸膏制备甘草酸新工艺进行了优化研究。二次过柱洗脱物甘草酸含量可达75%左右,回收率在66%以上。新工艺的产品含量和收率较目前的生产方法高。     

FRACTIONATION AND PROPERTIES OF AMINOPEPTIDASE B DURING PURIFICATION AND STORAGE

Purified preparations of rat liver aminopeptidase B were found to display heterogeneity which was demonstrated in isoelectric focusing and in electrophoresis on polyacrylamide columns. Most of the heterogeneity was generated during the purification of the enzyme. Acid treatment at pH 5.1 and ammonium sulphate fractionations caused considerable heterogeneity and the presence of thiols reduced it. Ampholine ampholytes themselves did not cause the generation of the heterogeneity but isoelectric focusing rendered its demonstration possible. A special type of heterogeneity was observed when aged preparations of the enzyme were found to undergo aggregation. The aggregate forms moved slowly or not at all in electrophoresis on polyacrylamide. The pIs of the main forms appearing in isoelectric focusing were around 5 and 7. The aggregate forms had a pI below 4. The enzymic properties of the different forms, whether appearing in isoelectric focusing, during ageing, or in purification were essentially similar, indicating that the heterogeneity involved changes only in those amino acid residues which were not responsible for substrate binding or catalysis.     

AMINO ACID COMPOSITION AND SEQUENCE ANALYSIS OF SAUVAGINE,A NEW ACTIVE PEPTIDE FROM THE SKIN OF PHYLLOMEDUSA SAWAGEI

The complete amino acid sequence of sauvagine, a new active polypeptide from the skin of Phyllomedusa sauvagei, a frog of Central and South America, has been determined by automated liquid-phase procedure after specific removal of the N-terminal pyrrolidonecarboxylic acid, and specific cleavages at the single methionine and at the two arginine residues. The proposed sequence is: Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Leu-Leu-Arg-Lys-Met-Ile-GIu-Ile-Glu-Lys-Gln-Glu-Lys-Glu-Lys-Gln-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Leu-Asp-Thr-Ile-NH₂. Sauvagine possesses a number of pharmacological actions on diuresis, the cardiovascular system and endocrine glands; it can be considered the prototype of a new family of amphibian peptides, in addition to the tachykinins, bradykinins, dermorphins, caerulein-like and bombesin-like peptides.     

PURIFICATION AND PROPERTIES OF NON-PRECIPITATING ANTIBODIES TO COBROTOXIN*

The heterogeneity of precipitating and non-precipitating antibodies to cobrotoxin was demonstrated by their elution pattern on cobrotoxin-Sepharose and chromatography on DEAE-cellulose column. Gel filtration patterns on a Sepharose 6B column revealed that the soluble complexes formed from non-precipitating antibody and HNB-cobrotoxin at a different molar ratio all emerged in the void volume, indicating that the molecular weight of the soluble complex is around 4,000,000 or larger. Unreacted free non-precipitating antibody coincided with the peak of IgG and was proved to be free from HNB-cobrotoxin. The molar ratio of antibody to antigen for the soluble complex was found to be 0.79 to 0.97 indicating that 1.58–1.94 molecules of non-precipitating antibody are bound to HNB-cobrotoxin instead of three molecules as in the case of precipitating antibody.     

山莨菪碱对洗涤大鼠血小板代谢花生四烯酸的影响

作者建立了一个用大鼠洗涤血小板研究药物对外源性及内源性AA代谢影响的方法。采用HPLC测定血小板AA代谢物HHT及12-HETE,观察了底物(AA)浓度、孵育时间、A23187加量等对血小板代谢AA的影响。并用此法研究了654-2对洗涤血小板AA代谢的影响。654-2显著减少血小板从内源性AA形成HHT及12-HETE,且作用随剂量增加而增强,但不影响血小板对外源性AA的代谢。上述结果表明,654-2是通过抑制AA释放而减少AA代谢物的形成。     

甘油激酶的纯化与性质的研究

由嗜热脂肪芽胞杆菌(Bacillus stearothermophilus SIPI1.687)的粗提取液,经液-液双相抽提、DEAE-Toyopearl 650 M 离子交换柱层析及 SIPI-Ⅱ蓝色(?)料配基层析等分离提纯,可得甘油激酶(81U/mg),PAGE 鉴定为单带。用 Sephacryl 400凝胶过滤测得分子量为209,000道尔顿,SDS-PAGE 测定结果表明该酶由四个分子量均为50,000道尔顿的相似亚基组成。酶作用最佳 pH 9.4,pH 5.8～11.2时稳定;最佳温度为50℃,有较好的热稳定性。以甘油为底物时米氏常数为6.25×10~(-5)M。     

PURIFICATION AND DETERMINATION OF THE AMINO ACID SEQUENCE OF EQUINE SERUM BUTYRYLCHOLINESTERASE

Butyrylcholinesterase (Eq-BChE) from equine (horse) serum has been used extensively to demonstrate the efficacy of cholinesterase as a single pretreatment drug (bioscavenger) for organophosphate toxicity in rodents and nonhuman primates. It has the longest mean retention time in animals among all ChE preparations examined. The authors describe the complete amino acid sequence of Eq-BChE. Like other mammalian BChEs, it is a glycosylated protein composed of four identical subunits. The apparent molecular weight of the subunit containing 574 amino acids and the estimated carbohydrate content of 26% is 84,551 Da. It has eight carbohydrate-modified sites at positions 57, 106, 241, 256, 341, 455, 481, and 486 as N-linked Asn residues. The active site charged triad aminoacids are Ser198, Glu325, and His438. The N-terminal aminoacids are GluGluAspIle, consistent with this being the mature form of the enzyme. In addition, a monomeric form of this enzyme that corresponds to 4% of the total BChE activity was isolated and purified. Comparison of Eq-BChE with other species shows that there is a high degree of primary sequence identity. There is a 90.1% identity with the human serum BChE sequence, although the equine enzyme has one less N-glycosylation site and Cys residue. If conserved amino acid substitutions are included then the proteins share 93.4% homology.