葛根素对D-半乳糖诱导大鼠抑制蛋白非酶糖基化及增强胰岛素敏感性的作用

目的观察葛根素对D-半乳糖(D-gal)诱导大鼠体内蛋白非酶糖基化反应、胰岛素抵抗、糖耐量和醛糖还原酶(AR)活性的作用。方法除空白对照组外,其余大鼠均采用腹腔注射(ip)D-gal150mg/kg,1次/d×56d致病,D-gal处理大鼠2周后,将大鼠按体重均衡随机分成:模型对照、葛根素和氨基胍3组,然后继续ipD-gal处理同时灌胃给予葛根素和氨基胍(每10ml300mg/kg)或蒸馏水,1次/d×42d;实验结束时,观察葛根素和氨基胍对D-半乳糖诱导大鼠体内晚期糖基化终末产物(AGEs)、糖化血红蛋白(HbA1c)、果糖胺(FRA)、糖耐量、血糖和胰岛素含量,并计算胰岛素敏感指数(ISI),以及醛糖还原酶(AR)活性的作用。结果与空白对照组比较,D-gal处理大鼠出现血糖升高、糖耐量减退(IGT)、糖基化产物(包括HbA1c,FRA和AGEs)和胰岛素含量增高,ISI降低,以及红细胞内AR活性增强(P<0.01);葛根素和氨基胍均能改善D-gal诱导大鼠IGT和降低空腹血糖、糖基化产物和胰岛素含量(P<0.01～0.05),提高ISI(P<0.01),并明显抑制AR活性(P<0.01～0.05)。结论D-gal能诱导大鼠体内糖基化反应、高胰岛素血症、胰岛素抵抗和醛糖还原酶活性增强;而葛根素和氨基胍能抑制蛋白非酶糖基化反应和醛糖还原酶活性,提高胰岛素敏感性,改善高胰岛素血症,提示葛根素可用于治疗糖尿病某些慢性并发症。     

Membrane protein antigens in multiple sclerosis

Rabbits were immunized with white matter (WM) membrane fractions isolated from autopsy brain specimens of three patients with multiple sclerosis (MS), and three controls. All the rabbits developed high serum antibody titers to the MS and control WM fractions, as tested by enzyme immunoassay. Antibodies against WM membrane components were analyzed further by immunoprecipitation of radiolabeled WM proteins and subsequent polyacrylamide gradient gel electrophoresis. Antigenic membrane components with molecular weights of 138 000, 111 000, 86 500, 79 600, 69 000, 63 000, 58 000, 53 400, 45 700, 24 500 and 22 300 were found in both MS and control WM. Although there may have been some quantitative differences in these immunogenic proteins of MS and normal WM, no multiple sclerosis-specific membrane antigen could be demonstrated.The hyperimmune anti-WM sera did not precipitate ³⁵S-labeled polypeptides from cells infected with herpes simplex type 1, adeno type 5, measles, mumps, rubella, respiratory syncytial, parainfluenza type 2 or cytomegaloviruses, which suggests that the MS brain WM membrane proteins do not share common antigenic determinants with the viral polypeptides.     

A microelectrode for continuous recording of volume fluxes in isolated perfused tubule segments

Manufacture, properties and use of a micro enzyme electrode for continuous monitoring of volume fluxes in the isolated tubule preparation is described. The specific electrode is a galactose-oxidase enzyme electrode, which can be used to detect changes in raffinose concentrations. The electrode's response to raffinose is almost linear over concentrations from 0–12 mmol/l. The electrode equally responds to galactose as to raffinose but is insensitive to other sugars, to pH changes (from 6.0–8.0), CO₂ (from 1–10%) and electrolytes tested. Reducing O₂ from 100 to 10% and to 1%, leads to a reduction of the reading by 10% and 30%, respectively. The reading is almost doubled when the temperature is increased from 20–40° C. Furthermore, reducing agents such as uric acid and ascorbic acid interfere with the reading. If these substances and raffinose are omitted from the perfusate for isolated perfused proximal mouse tubules, the reading is identical in perfusate and collected fluid, indicating that the tubular epithelium does not produce substances in sufficient amounts to interfere with the electrode reading. After addition of 6 mmol/l raffinose to the perfusate the raffinose concentration in the collected fluid of 0.76±0.05 mm segments of straight proximal mouse tubules (perfusion rate = 3.4±0.45 nl/min) is 10.2±0.3 mmol/l, indicating a volume reabsorption of 1.5±0.3 nl/min. Peritubular application of acetazolamide reduces the volume reabsorption by 42±4%.Supported by Österr. Forschungsrat, Proj. No. 4366     

Galactose Metabolism in a Patient with Hereditary Galactokinase Deficiency

Abstract. The ability of a galactokinase deficient patient to metabolize galactose, galactitol and galactonate was quantitated. In galactokinase deficiency, conversion of galactose to CO₂ is minimal. Apparently the defect is extensive, involving all tissues. Galactitol and galactonate, injected intravenously, were not metabolized. The administration of C-1 and C-2 labelled galactose resulted in ¹⁴CO₂ excretory patterns similar to that observed in uridyltransferase deficient mutants. The different fates of C-1 and C-2 observed in this galactokinase deficient patient give support to the existence of a direct oxidative pathway for galactose. Galactonate, although present in urine during the period of observation following injection of radioactive galactose failed to become labelled.     

肿瘤浸润性淋巴细胞结合偶联半乳精抗CD_3单抗趋肝性初探

目的探讨结合偶联半乳糖（Gal）的大鼠抗小鼠CD3单克隆抗体（Mouse-anti-rst—CD3monocloalantibodyAntiCD3-McAb）肿瘤浸润性淋巴细胞的（TIL）趋肝性，方法本实验把大鼠抗小鼠CD3单克隆抗体和半乳糖（Gal）偶联在一起，与3H-TdR标记TIL结合后，从尾静脉注入小鼠体内，分别在注射后不同时间抠眼取血0．5ml，然后处死，切除肝、脾、肺组织，分别称重后进行放射性强度测定。结果结果显示：注射Gal-anti-CD3-McAb-TIL小鼠肝脏组织比注射单纯TIL的小鼠肝脏组织具有较高的放射性强度（P<0.001），且该放射性持续较长一段时间，而脾、肺、血组织内放射性强度无明显差异（P>0．5P>0.5P>0．2）。结论该结果提示Gal-anti-CD3-McAb-TIL较单纯TIL具有更良好的趋肝性，本实验为肝脏恶性肿瘤生物导向治疗和靶向性基因治疗提供了新的方法。     

Increased serum urate in galactosemia patients after a galactose load: A possible role of nucleotide deficiency in galactosemic liver injury

Summary Five galactosemic and 5 normal children received an oral load of galactose under standardized conditions. The maximal blood galactose level after 1.5 hours was 12.6±2.0 (S.D.) mmol/l in individuals with a deficiency of uridylyl transferase (EC2.7.7.12) as compared to 5.8±1.2 (S.D.) mmol/l in the controls. The concentration of serum urate in galactosemics increased to 155% of the fasting level (P<0.005); no rise was detectable in the controls. The elimination of urate with the urine was augmented by the same amount in both groups. Our studies provide evidence for an increased catabolism of hepatic nucleotides. This may lead to a deficiency of nucleotides which is proposed as a cause of galactosemic liver injury.This work was supported by grants from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Ke 200/3, and Forschergruppe Leberkrankheiten, Freiburg.     

Differential receptor targeting of liver cells using 99mTc-neoglycosylated human serum albumins

Neolactosyl human serum albumin (LSA) targets asialoglycoprotein receptor and shows high liver uptake due to accumulation in hepatocytes. Although neomannosyl human serum albumin (MSA) also shows high liver uptake, it has been reported to be taken up by Kupffer cells and endothelial cells. We compared the biological properties of LSA and MSA. ^99mTc-LSA and ^99mTc-MSA biodistribution in mice were investigated after intravenous injection. In vivo localization of rhodaminisothiocyanate (RITC)-LSA and fluoresceineisothiocyanate (FITC)-MSA were investigated in mouse liver. Excretion routes of ^99mTc-LSA and ^99mTc-MSA metabolites were examined. Both ^99mTc-LSA and ^99mTc-MSA showed high liver uptakes. RITC-LSA was taken up by hepatocytes whereas FITC-MSA was taken up by Kupffer cells and endothelial cells. ^99mTc-MSA showed higher spleen and kidney uptakes than ^99mTc-LSA. ^99mTc-LSA metabolites excreted in urine and feces accounted for 44.4 and 50.0% of ^99mTc-LSA injected, respectively, while ^99mTc-MSA metabolites accounted for 51.5 and 10.3%, respectively. In conclusion, LSA is specifically taken up by hepatcytes while MSA by Kupffer cells and endothelial cells. After taken up by the liver, LSA is metabolized by the hepatocytes and then excreted through both the hepatobiliary tract and kidney, whereas MSA is metabolized by Kupffer cells and endoghelial cells and then excreted mainly through the kidney.     

Thermal treatment of galactose-branched polyelectrolyte microcapsules to improve drug delivery with reserved targetability

A novel multilayered drug delivery system by LbL assembly of galactosylated polyelectrolyte, which is possible to have the potential in hepatic targeting by the presence of galactose residues at the microcapsule's surface, is designed. Thermal treatment was performed on the capsules and a dramatic thermal shrinkage up to 60% decrease of capsule diameter above 50 °C was observed. This thermal behavior was then used to manipulate drug loading capacity and release rate. Heating after drug loading could seal the capsule shell, enhancing the loading capacity and reducing the release rate significantly. Excellent affinity between galactose-binding lectin and heated galactose-containing microcapsules were observed, indicating a stable targeting potential even after high temperature elevating up to 90 °C.     

Production, purification, and characterization of a novel galactose oxidase from Fusarium acuminatum

Extra-cellular production of a novel galactose oxidase from Fusarium acuminatum using submerged fermentation was studied. Glucose (1.0% w/v) was used as the sole carbon source. Maximum galactose oxidase production (approximately 4.0 U/ml) was obtained when fermentation was carried out at 25 degrees C, with orbital shaking (100 rpm) and an initial medium of pH 7.0, for 96 h, using a 2% (v/v) inoculum made from a homogenized four-day-old liquid culture, in the presence of copper, manganese, and magnesium. The enzyme was purified by one-step affinity chromatography, with a recovery of 42% of the initial activity. The purified enzyme ran as a single band of 66 kDa in SDS-PAGE. Optimal pH and temperature for the enzyme activity were 8.0 and 30 degrees C, respectively. The enzyme was thermoinactivated at temperatures above 60 degrees C. The purified enzyme was active toward various substrates, including galactose, dihydroxyacetone, guar gum, lactose, melibiose, methyl-galactopyranoside, and raffinose. SDS was an inhibitor but EDTA, Tween 80, NH(4)(+), Na(+), Mg(2+), K(+), and glycerol were not. The Michaelis-Menten constant (K(m)) for galactose was estimated to be 16.2 mM, while maximal velocity (V(max)) was 0.27 micromol of H(2)O(2) . ml(-1) . min(-1).