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Oxidized low-density lipoproteins (ox-LDL) such as malondialdehyde-modified LDL (MDA-LDL) play an important role in the pathogenesis of atherosclerosis. This study is aimed to establish a standard enzyme-linked immunosorbent assay (ELISA) for measuring serum MDA-LDL, and evaluate its usefulness by analyzing serum samples of diabetes mellitus (DM) patients. Serum sample stability, analytical sensitivity, intra- and inter-assay precision, dilution linearity, supplementation and recovery, and interfering substances were examined. Normal reference levels in 86 healthy subjects (33 males and 53 females; average age 34.4±7.8 years) with normal lipid profiles were also determined. MDA-LDL levels in blood and serum were unstable and gradually increased during storage. However, it could be stabilized by the addition of a reagent, which included sucrose. The detection limit of the assay was 6.3 U/l. Intra- and inter-assay imprecisions were P P P

Advanced Glycation End products (AGEs) は, メイラード反応後期生成物とも呼ばれ, 糖尿病性合併症の原因物質の一つではないかと考えられている. われわれは, 血清中AGE測定系を確立し, 糖尿病さらにその合併症進展と血中AGE量との関係等について検討を行ってきた. 今回, 腹膜透析液を用い, ゲル濾過, affinity chromatography, western blotting 等の手法により, AGEの血中存在様式について検討を行った. その結果, AGE構造の大部分は, 血清蛋白と結合していることが明らかとなった. さらに, AGE化されている蛋白成分としてIgG, アルブミン, β2-マイクログロブリンを同定することができた.

Free ubiquitin (mainly monoubiquitin) and multi-ubiquitin chains coexist in eukaryote cells and serve distinct cellular roles. However, any immunoassay systems established previously have not been proved to be applicable for measuring the former without cross-reactive responses with the latter. For this purpose, we developed a radioimmunoassay specific to monoubiquitin by employing antiserum US- I against ubiquitin. In this assay, ubiquitin-protein conjugates, prepared by a reticulocyte lysate fraction 11 and fractionated on Moro Q and Superdex 200 columns, exhibited practically no cross-reactivity. The cross-reactivity of fractionated ubiquitin-lysozyme conjugates was also analyzed as a function of their multi-ubiquitin chain size. As a result, the larger the conjugates were found to be, the weaker were the cross-reactive responses they showed, and the multi-ubiquitin chains (n > approx. 20) were substantially unreactive in the radioimmunoassay. By using the radioimmunoassay, heat-shock-induced decrease in the level of cellular free (mono)ubiquitin was detected. In addition, the standard preparation of multi-ubiquitin chains was not cross-reactive in all other five radioimmunoassays employing distinct antibodies to ubiquitin (four antisera and a monoclonal antibody). These data suggest that radioimmunoassays employing ubiquitin antibodies raised by the general methods can discriminate between monoubiquitin and multi-ubiquitin chains and quantitate cellular free ubiquitin.

This study aims to measure the oxidative status of LDL from human plasma (n=26) as assessed by biomarkers for lipid peroxidation, total hydroxyoctadecadienoic acid (tHODE), 7alpha- and 7beta-hydroxycholesterol (t7-OHCh), and 8-iso-prostaglandin F(2alpha) (t8-iso-PGF(2alpha)) after subfractionation of LDL with an anion-exchange HPLC (AE-HPLC). LDL was separated and quantified by AE-HPLC as LDL-1, LDL-2, and LDL-3 in the order of the anionic charge of the LDL particles. The concentrations of tHODE, t7-OHCh, and t8-iso-PGF(2alpha) in both plasma and LDL subfractions were assessed after reduction and saponification. In this method, the free and ester forms of hydroperoxides, ketones, and hydroxides of linoleic acid and cholesterol are measured as tHODE and t7-OHCh, respectively. It was found that tHODE significantly correlated with the proportion of LDL-2 and LDL-3 as well as with the concentration of malondialdehyde-modified LDL in plasma. Further, by the analyses of LDL subfractions, the concentrations of tHODE, t8-iso-PGF(2alpha), and t7-OHCh in LDL-3 were found to be significantly higher than those in LDL-1 and LDL-2. These results clearly indicate that the extent of oxidation increases in the order of LDL-1

We investigated the relationship between the concentration of pyridoxal-5'-phosphate (PLP) and biogenic amine in mouse brain. The production of PLP from pyridoxal (PL) by pyridoxal kinase (PLK) was inhibited by the addition of dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (5-HT), but not by that of epinephrine and N-acetyl-serotonin. DA and NE were combined with PLP by a non-enzymatic reaction, whereas 5-HT was bound only slightly with PLP. The conjugated product of PLP with DA was also detected by HPLC analysis when PLK activity was assayed using PL as a substrate in the presence of DA. In an in vivo investigation, the depletion of DA and 5-HT in mouse brain after an intraperitoneal injection of 5 mg/kg reserpine, led to slight elevation of the PLP level to 120% of the control level. By contrast, the increase in DA in the brain caused by intraperitoneal administration of 150 mg/kg L-DOPA caused the PLP concentration to decrease to 70% of the control level. However, no change in PLK activity in the brain was observed when the mice were treated with either reserpine or L-DOPA. These results suggested that the level of PLP in mouse brain was partly regulated by the concentration of biogenic amines, such as DA, NE and 5-HT, without apparent induction of PLK.

A sandwich ELISA has been developed to measure intracellular levels of multi-ubiquitin chains. The mixture of multi-ubiquitin chains, prepared in vitro by incubation of ubiquitin (plus 125I-ubiquitin) and lysozyme with ubiquitin-ligating enzymes and ATP, was partially purified and established as a standard named the multi-ubiquitin-chain reference preparation 1 (MUCRP1). The concentration of MUCRP1 was calculated from the recovered radioactivity of 125I-ubiquitin. All measurements by the ELISA were expressed in terms of MUCRP1. The ELISA showed good sensitivity (98 pg/ml), precision (intra-assays6%) and reproducibility (interassay9%). In addition, there was no substantial cross-reaction with mono-, di- and tri-ubiquitin, or mono-ubiquitinated and di-ubiquitinated lysozyme in the ELISA, and large multi-ubiquitin chains (napproximately 6) may be fully reactive. These results combined with excellent results in the recovery and dilution tests guarantee accurate measurement of multi-ubiquitin chains in cell extracts prepared with a lysis buffer (water soluble) or the buffer supplemented 8 M urea (urea soluble). The level of the water-soluble multi-ubiquitin chains in reticulocytes was lower than that of erythrocytes, but the urea-soluble chain level was higher in the reticulocytes. Heat-shock treatment of HeLa cells increased the urea-soluble multi-ubiquitin chains. These data indicate that this ELISA provides a useful and reliable approach to the study of intracellular multi-ubiquitin-conjugate turnover.

AIM: To develop a simple and accurate method for quantifying 8-isoprostane in plasma by employing a combination of two-step solid-phase extraction of samples and a commercially available ELISA kit, and by this method to examine the effects of drinking and smoking habits against the levels of plasma 8-isoprostane in healthy Japanese volunteers. METHODS: Plasma 8-isoprostane was extracted with ODS gel suspension followed by NH2 Sep-Pak column. The 8-isoprostane fractions were assayed using a commercially available ELISA kit. We measured plasma 8-isoprostane levels in 157 healthy Japanese volunteers divided into three groups (64 non-habitual drinkers, 56 moderate drinkers and 37 habitual drinkers) according to their alcohol consumption per week. Genotypes of aldehyde dehydrogenase 2 (ALDH2) were also determined to investigate the plasma 8-isoprostane levels with reference to drinking habits. In addition, the plasma 8-isoprostane levels of 96 non-smokers and 61 smokers from the same subjects were compared. RESULTS: Our method fulfilled all the requirements for use in routine clinical assays with respect to sensitivity, intra- and inter-assay reproducibility, accuracy and dynamic assay range. Significant increases of plasma 8-isoprostane levels were observed in female habitual drinkers when compared with those of non-habitual drinkers (t = 5.494, P < 0.0001) as well as moderate drinkers (t = 3.542, P < 0.005), and 8-isoprostane levels were also significantly different between ALDH2*2/1 and ALDH2*1/1 in the female habitual drinkers (t = 6.930, P < 0.0001), suggesting that excessive drinking of alcohol may increase oxidization stress, especially in females. On the contrary, no significant difference of the plasma 8-isoprostane levels was observed between non-smokers and smokers. CONCLUSION: Our present method was proved to be a simple and accurate tool for measuring plasma 8-isoprostane. However, the clinical utility of plasma 8-isoprostane for drinking and smoking habits was limited since elevated 8-isoprostane levels were observed in female heavy drinkers, and no association was found between smokers and nonsmokers.

The substrate specificity of a neutral α-glucosidase was investigated. The enzyme was active especially on malto-oligosaccharides, phenyl-α-maltoside and nigerose. The action on isomaltose and phenyl-α-glucoside was too weak. Isomalto-oligosaccharides made up of three or more glucose units were not attacked. The α-glucans, such as amylopectin, β-limit dextrin, glycogen and amylose besides soluble starch, also were hydrolyzed.The ratio of velocity of hydrolysis for maltose, nigerose, phenyl-α-maltoside, maltotriose and maltotetraose was estimated to be 100: 89: 93: 74: 80 in this order. The extraporated Km values for maltose, nigerose, phenyl-α-maltoside, maltotriose and maltotetraose were 2.1 mm, 20.0 mm, 0.33 mm, 0.28 mm and 0.15 mm, respectively. The enzyme is considered to be essentially a neutral α-glucosidase with a preferential activity on malto-oligosaccharides.

A sensitive sandwich enzyme immunoassay method for measurement of rat α-fetoprotein(AFP) was established by use of affinity purified antibodies to rat AFP. The assay system consisted of filter paper discs with immobilized antibodies and the same antibodies labeled with horseradish peroxidase(POD). The optimal conditions for the assay and for the preparation of POD labeled antibodies were determined. The assay was as sensitive as radioimmunoassay(RIA) and rat AFP at concentrations of 5 to 1280ng/ml was measurable. AFP values obtained by the present method were in good agreement with those by RIA and the correlation coefficient was 0.99.

Kinetic studies on the active site of pig serum α-glucosidase and buckwheat α-glucosidase catalyzing the hydrolyses of maltose and soluble starch were made. The kinetic features obtained by the experiments with the mixed substrates, the linearity of Lineweaver-Burk plots, and the dependence of the apparent Michaelis constants and the apparent maximal velocities on the fraction ƒ of maltose in the mixed substrate solutions, that is, ƒ=maltose/(maltose+soluble starch), were in good agreement with those expected for the single active site catalyzing the hydrolyses of both substrates. From the results it was concluded that these enzymes attacked maltose and soluble starch by the single active site mechanism.

Kinetic studies on the active site of pig serum α-glucosidase and buckwheat α-glucosidase catalyzing the hydrolyses of maltose and soluble starch were made. The kinetic features obtained by the experiments with the mixed substrates, the linearity of Lineweaver-Burk plots, and the dependence of the apparent Michaelis constants and the apparent maximal velocities on the fraction f of maltose in the mixed substrate solutions, that is, f=maltose/(maltose + soluble starch), were in good agreement with those expected for the single active site catalyzing the hydrolyses of both substrates. From the results it was concluded that these enzymes attacked maltose and soluble starch by the single active site mechanism.

Monoclonal antibodies to rat alpha-fetoprotein (AFP) were produced by hybridization of mouse myeloma cells with spleen cells from mice immunized with rat AFP. The monoclonal antibodies as well as horse anti-rat AFP antibodies were coupled via a dextran bridge to daunomycin. Both types of conjugates were tested for their antitumor activity in vitro and in vivo. They were equally cytotoxic to the rat AH66 hepatoma cell line in culture. Rats challenged with hepatoma cells were treated with the conjugates by either intraperitoneal or intravenous injection. Daunomycin conjugates with horse-anti-AFP and monoclonal mouse anti-AFP were capable of delaying the tumor development more efficiently than were the controls of antibodies or free drug, mixtures of drug with antibodies, and a conjugate of drug and normal Ig. The specific conjugates were considerably more effective when the treatments were given intravenously. The specific conjugates produced 60% long-term survival, whereas the controls only slightly delayed tumor development.

The inhibitory effects of horse antiserum against rat alpha-fetoprotein (AFP) on the uptake of 2-deoxy-D-glucose (2dG) by the AFP-producing rat ascites hepatoma AH66 cells was studied. AH66 cells cultured in medium containing 20% heat-inactivated antiserum had a 1.5-fold lower rate of sugar uptake than did AH66 cells which were cultured in medium containing 20% heat inactivated normal horse serum. The inhibition of 2dG uptake by antiserum was dependent on both the concentration and the exposure time of antiserum. Preincubation of AH66 tumor cells for 2 and 6 h with antiserum prior to the measurement of 2dG uptake resulted in a 70.1% and 58.2% decrease in 2dG uptake compared to control cells. Antiserum did not inhibit the rate of phosphorylation of 2dG by tumor cells. Kinetic constants for the uptake of 2dG in both AH66 cells treated with antiserum to AFP and in control cells were calculated from Lineweaver-Burk plots. The Km remained constant at approximately 1.2 mM, but the Vmax was twice as small for the cells treated with antiserum as for the control cells (571 vs 923 nanomoles/2 X 10(5) cells/min). These studies suggest that the inhibition of 2dG uptake by treatment with antiserum was the result of a decrease in the number of transport sites, or a decrease in the amount of carrier protein for the sugar which was present on the surface of the plasma membrane of the AH66 cells.

A neutral α-glucosidase was purified from pig serum by precipitation with ammonium sulfate, chromatographies on DEAE-cellulose and -Sephadex A–50, and gel filtration on Bio-Gel P–300 and Sephadex G–200. The purified enzyme was homogeneous in ultracentrifugal and disc electrophoretic analysis. The sedimentation coefficient (s20,w) was calculated to be 10.7 S, and the isoelectric point, 4.0. The molecular weight was estimated to be approximately 2.7 × 105 by thin-layer gel filtration and SDS-disc electrophoresis.The enzyme exhibited also glucoamylase activity. The optimal pH was found to be in the pH range of 6.0 to 7.0 for maltose and soluble starch. The ratio of velocity of hydrolysis for maltose (Km, 0.72 mg/ml), soluble starch (Km, 9.8 mg/ml) and shellfish glycogen (Km, 55.6 mg/ml) was calculated to be 100: 110: 5.15 in this order.

The anticancer drug, DNR, was conjugated to an affinity-purified horse antibody to human AFP (aAFP) via a dextran bridge. The conjugate (immunoglobulin: DNR molar ratio, 1:50) was twice as potent as free DNR in an in vitro cytotoxicity assay against an AFP-producing human yolk sac tumor. The in vivo effect of aAFP, DNR, and the conjugate was tested against the human yolk sac tumor growing in nude mice. The conjugate, at a concentration of DNR containing the equivalent amount of 20 micrograms or 70 micrograms/mouse significantly retarded tumor growth whereas free aAFP showed only a slight inhibition of tumor growth compared to the PBS-treated control. Mice which received 20 micrograms/mouse of free DNR showed a moderate retardation of tumor growth whereas those which received 70 micrograms/mouse of DNR or a mixture of DNR and aAFP showed emaciation and early death due to acute toxicity of the drug. These results suggest that the anti-body-drug conjugate accumulated preferentially on the AFP-producing tumor cells and that cytotoxicity occurred.

Daunomycin was covalently attached via a dextran bridge to specific antibodies against rat alpha-fetoprotein produced in a horse. The effect of this conjugate on an alpha-fetoprotein-producing tumor was investigated in terms of cytotoxicity and inhibition or retardation of tumor development. Under the experimental conditions used, the covalent conjugate was by both criteria more efficient than either daunomycin alone or a mixture of daunomycin and specific antibodies or a conjugate of daunomycin with horse immunoglobulin. These results show that the conjugate may be useful as a specific cytotoxic agent against alpha-fetoprotein-producing tumors.

A highly sensitive sandwich enzyme-immunoassay (EIA) for human muscle carbonic anhydrase isozyme III (CA-III) has been developed using microplate as a solid-phase and peroxidase as a labelled enzyme. The assay can detect levels as low as 2 ng/ml when 20 microliter of sample sera were used. Sera from patients with various neurological diseases were studied using this method, and elevated serum CA-III levels were found in patients with Duchenne muscular dystrophy, limb-girdle dystrophy, fascioscapulohumeral dystrophy, polymyositis and amyotrophic lateral sclerosis. The values correlated well with the results of radioimmunoassay (RIA), with a correlation coefficient of 0.92 (P less than 0.001). We feel EIA is preferable to RIA for its simple methodology.

Five rat yolk sac tumor cell lines were cloned from a yolk sac tumor line which originally arose following fetectomy. The doubling time of each of the cloned tumor lines was about 50 h. All of the cloned tumor cell lines synthesized and secreted AFP and albumin but there was a gradual decrease in the synthesis of these proteins during serial passage. The cells formed clusters which looked like vitelline ducts or the parietal yolk sac and they also had basement membranes which closely resembled Reichert's membrane. When the cloned cell lines were cultivated in the presence of 1 mM dibutyryl cyclic-AMP, myotube-like and neuron-like cells appeared. Acetylcholine esterase and creatine phosphokinase activity were present when myotube-like cells were present whereas acetylcholine esterase activity predominated when neuron-like cells were present.

The anticancer drug mitomycin C (MMC) was conjugated with an affinity-purified horse antibody to human alpha-fetoprotein (aAFP) with human serum albumin (HSA) as the intermediate drug carrier. The conjugate (aAFP:HSA:MMC molar ratio, 1:1:30) retained full antibody binding activity as determined by a competitive binding radioimmunoassay. In a cytotoxicity test in which the AFP-producing human yolk sac tumor TG-1 cells were preincubated with test materials for 2 h followed by an additional 48-h culture in fresh medium, the conjugate was 20-fold more cytotoxic than free MMC at an equivalent MMC concentration of 100 ng/ml. The in vivo antitumor effect of the conjugate was tested against the human yolk sac tumor JOG-9 growing in athymic nude mice. When the tumor-bearing mice were treated with a total of 6 injections given on 2 consecutive days and then every other day starting 8 days after SC tumor inoculation [2 (equivalent MMC) microgram/head per injection], the conjugate retarded tumor growth more effectively than free MMC and normal horse immunoglobulin conjugate.

Human of rat alpha-foetoprotein (AFP) was highly purified from ascitic fluid or serum of hepatoma bearers. The purification was carried out mainly by means of immunoadsorbent chromatography using Sepharose coupled to specific anti-AFP antibody with BrCN activation, and by Sephadex gel filtration. Horses were immunized with the purified AFP and the specific antibody was isolated from the antiserum by means of an immunoadsorbent coupled to purified AFP. The specific antibody was found to bind specifically with AFP-producing tumour cells. The antibody was applied for radio immunodetection of the tumour. 125I-labelled antibody was administered to patients or rats with hepatoma, and radioactivity localized in the tumours was scintiscanned with a scintillation camera. In this way, the location of the tumour was detected in about 50% of the hepatoma bearers. The cytotoxicity of the antibody was clearly demonstrated both in vitro and in vivo in animal experiments. The antibody was administered to patients with advanced hepatoma. Although no improvement of the disease was demonstrated, serum AFP levels decreased greatly and in some cases low AFP levels were maintained for long periods suggesting that the antibody suppressed AFP-producing hepatoma cells. No significant side effects were observed in patients who had been administered with the horse antibody.