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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

Clathrin- and AP-1-coated buds are present on immature secretory granules of endocrine cells that mature into clathrin-uncoated granules. The mechanism of clathrin and adaptor protein uncoating has remained obscure. Benzyloxycarbonyl-L-leucyl-L-leucinal (ZLLal), a calpain inhibitor, reduced growth hormone (GH) secretion with intracellular accumulation, in a GH-secreting rat pituitary tumor cell. Pulse and chase demonstrated that ZLLal retarded the turnover of clathrin (Clt.H) and adaptins. ZLLal-treatment co-immunoprecipitated the increased amounts of GH with Clt.H and adaptins compared to control cells, suggesting the intracellular accumulation of immature secretory granules. Clt.H and adaptins were limited-proteolyzed by m-calpain in vitro, indicating that calpain may be involved partly in the maturation of secretory granules in endocrine cells via the process of clathrin uncoating.

The N-terminal amino acid sequence of TA02 (molecular weight 35.0 kDa, isoelectric point 5.29), which is associated with primary lung adenocarcinoma, was determined and a fragment peptide was used to generate mouse monoclonal antibodies (mAbs) against TA02. The amino acid sequence suggested that TA02 might be homologous with napsin A, a new type of aspartic proteinase. In this context, we confirmed the expression of napsin A in primary lung adenocarcinoma using reverse-transcription polymerare chain reaction (RT-PCR) and showed that the TA02 mAbs reacted with glutathione-S-transferase (GST)-napsin A fusion protein. We concluded that TA02 is the same molecule as napsin A, and showed immunohistochemically that it is distributed mainly in type II pneumocytes, alveolar macrophages, renal tubules and exocrine glands and ducts in the pancreas. In particular, type II pneumocytes and alveolar macrophages showed high expression of TA02 among human normal tissues. In primary lung adenocarcinoma, 47 out of 58 (81.0%) primary lesions were positive. All well-differentiated adenocarcinomas except those of goblet cell type showed high expression of TA02. In addition, two out of seven (28.6%) large cell carcinomas showed low expression of TA02. The other histopathological types of primary lung cancer did not express TA02 at all. A few cases of renal cell cancer, pancreatic cancer, breast cancer, thyroid cancer, colon cancer and ovarian cancer showed low expression, but the staining patterns were completely different from that of primary lung adenocarcinoma, which showed a granular staining pattern. Our novel mAbs should be valuable for immunochemical detection of TA02/napsin A.

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.

In the present study, we examined the relationship between circulating oxidized low-density lipoprotein (LDL) and the metabolic syndrome in Japanese patients. Subjects who had no histories of coronary or peripheral artery disease and were taking no medications (n=119; age 57+/-10 years; male/female, 90:29) underwent a complete history and physical examination, determination of blood chemistries and oxidized LDL levels. In stepwise regression analysis, triglycerides (p=0.0001) and HDL-cholesterol (p=0.0493, inversely) were independently correlated to oxidized LDL levels. Furthermore, a significant association (p0.0001) was found between circulating oxidized LDL levels and the accumulation of the number of the components of the metabolic syndrome. Oxidized LDL levels were one of the independent determinants of intima-media thickness of the common carotid artery, a surrogate marker of atherosclerosis. The present study reveals that circulating oxidized LDL levels are strongly associated with the metabolic syndrome. Our results suggest that elevation of oxidized LDL may be a possible molecular link between accelerated atherosclerosis and the metabolic syndrome in Japanese subjects.

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

Normal human liver cells have a limited capacity for proliferation due to telomere shortening, whereas immortalized cells prevent shortening of the 3' single strand telomeric repeat by expressing telomerases, including human telomerase reverse transcriptase (hTERT). The hTERT transcript contains three deletion sites that give rise to alternatively spliced variants (ASVs). Recently, hTERT expression was observed in cycling primary presenescent human fibroblasts, which were believed to lack hTERT expression and telomerase activity. hTERT mRNA was expressed in the synthesis (S) phase of the cell cycle. Although hTERT mRNA has eight isoforms, it is not known which of the hTERT ASVs are expressed in S phase. In order to determine the possible relationships between the cell cycle and ASV expressions, we measured the full-length isoform and ASVs of hTERT mRNA in a mortal liver cell line and immortal cell lines that were synchronized in S phase of the cell cycle. Using RT nested-PCR analysis, the full-length isoform and alpha-deletion ASV of hTERT were detected in the LI90 mortal liver cell line at points when cells in S phase represented >48% of the cell population without detectable telomerase activity. hTERT was always expressed in the HLE and Huh-7 hepatocellular carcinoma cell lines, regardless of the cell cycle. Our results suggest the possibility that telomerase is regulated in a cell cycle-dependent manner in normal liver cells.

Lipid peroxidation and oxidative stress may play a certain role in the pathogenesis of pressure-induced atherosclerosis, and alcohol related diseases. Recently, 8-isoprostane in biological fluids has been reported to be a reliable marker for lipid peroxidation and oxidative stress in vivo. In the present study, we developed an ELISA method for 8-isoprostane which has high sensitivity, intra- and inter-assay reproducibility and wide dynamic assay range. Using this method, we examined the effects of drinking and smoking habits on plasma levels of 8-isoprostane in healthy subjects. A total of 157 apparently healthy volunteers was assayed for plasma 8-isoprostane. Subjects were divided into three groups according to their alcohol consumption. Group I is non- or few-drinkers, Group II includes subjects who drink once or twice a week, and subjects of Group III intake 3 to 5 times a week or almost every day. In addition, the same population was divided into two groups, 96 non-smokers and 61 smokers. Plasma 8-isoprostane was extracted with ODS gel followed by NH2 Sep-Pak column. The 8-isoprostane fractions thus separated were assayed by a commercial ELISA kit (Cayman Chemical). The plasma 8-isoprostane was estimated to be 20.9 +/- 93 pg/ml in a total of 157 volunteers (83 male, 74 female). The plasma 8-isoprostane levels were elevated in the Group III (26.6 +/- 9.5 pg/ml) compared with Group I (20.3 +/- 6.1 pg/mL, p0.0001) and Group II (20.9 +/- 5.7 pg/ml, p0.001). Significant increase of the plasma 8-isoprostane was observed only in habitual drinkers of females, but not in those of males. On the other hand, no significant difference of the plasma 8-isoprostane levels were observed between non-smokers (21.5 +/- 7.3 pg/ml) and smokers (22.8 +/- 7.4 pg/ml, p0.05). We suppose that plasma 8-isoprostane may increase in the habitual drinkers due to the oxidization stress induced by alcohol intake, and it may become a useful marker to estimate drinking habit

There is considerable evidence to indicate that humoral factors play an important role in the development of left ventricular hypertrophy. Cardiotrophin-1 (CT-1) is a cytokine that has been shown to induce cardiac hypertrophy in a dose-dependent manner. The aim of the present study was to investigate the acute effect of CT-1 on hemodynamic parameters in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) and to study the relationship between the plasma concentration of CT-1 and its hemodynamic effect. Ten-week-old SHR and age-matched WKY were used. Blood pressure (BP), heart rate (HR) and plasma concentration of CT-1 were measured both before and for 60 min after intravenous bolus injection of human CT-1 (10 microg/kg). CT-1 injection significantly decreased BP and significantly increased HR in SHR and WKY. There were significant differences in BP and HR between the two groups at all time points after injection. The lowest BP, highest HR and maximal plasma concentrations of CT-1 were observed in both groups within 10 min after injection. However, after converting the values into the percentage change from their respective baselines, there were no significant differences between the two groups in BP or HR at any time point. There was also no significant difference between the two groups at any time point in the plasma concentration of CT-1. This study indicates that CT-1 decreases BP and increases HR in both SHR and WKY. The most obvious change occurred within 10 min after injection. However, there was no significant difference in the hypotensive effect of CT-1 on 10-week-old SHR and WKY.

The coincidence of mutated alleles of CYP2C18 and CYP2C19 was studied in 154 Japanese subjects. The mutant alleles of CYP2C18 studied were CYP2C18m1 (T204 --> A substitution in exon 2) and CYP2C18mFR (A-460 --> T substitution in the 5'-flanking region), and those of CYP2C19 were CYP2C19m1 (G689 --> A substitution in exon 5) and CYP2C19m2 (G636 --> A substitution in exon 4). They were identified by polymerase chain reaction and restriction fragment length polymorphism. The results indicate that genotypes of CYP2C18m1 and CYP2C18mFR are completely coincident with those of CYP2C19m2 and CYP2C19m1, respectively. The finding suggests that the mutations of CYP2C18 and CYP2C19 examined in the present study are very closely linked with each other (i.e. CYP2C18m1 vs CYP2C19m2 and CYP2C18mFR vs CYP2C19m1), at least in a Japanese population.

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.

With the use of five murine monoclonal antibodies (1A5, 2A4, 3F1, F5 and 3A12) and an antigen-affinity purified goat polyclonal IgG antibody, the presence of a prostate-specific antigenic domain in human prostate-specific antigen molecule was identified. The results were based upon a series of quantitative competitive inhibition assays of each 125I-labeled monoclonal antibody and polyclonal antibody binding to prostate-specific antigen by unlabeled monoclonal antibodies as inhibitors, and immunohistochemical examination of an extensive panel of human tissue specimens. A cluster of two epitopes that are spatially related or in close topographical proximity and represent a prostate-specific antigenic domain are defined by the monoclonal antibodies 1A5, 2A4, 3F1, and F5, 3A12, respectively.

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.

Cybrid clones were isolated by fusing mouse embryonal carcinoma (PCC4) cells with cytoplasts of rat myoblastic cells (L6TG X CAPr). Although some clones were similar to PCC4 (Type II), a high proportion (88%) were differentiated; the differentiated cells had a mesh-like arrangement (Type I) or were flat with many projections (Type III). Protein patterns of both Type I and Type III cells changed markedly from that of PCC4 cells. Type III cells lacked alkaline phosphatase and expressed endo A and B proteins predominantly. One Type III clone produced alpha-fetoprotein and plasminogen activator (visceral endoderm-like), while another clone consisted of trophectodermal cell-like giant cells. Therefore it was shown that introduction of the somatic cell cytoplasm induces differentiation of teratocarcinoma stem cells, suggesting a cytoplasmic element (or elements) regulating gene expression.

1. 1. Main serum α 1 -protein (α 1 P) of rainbow trout was purified and its biochemical and physico-pathological properties were studied. 2. 2. α 1 P was suggested to be a primitive protein having both properties of albumin and AFP in serum proteins of mammals according to the following results. 3. 3. Molecular weight (75,000), two kinds of molecules (pI 4.55 and 5.05) and amino acid composition. 4. 4. Dye- or ConA binding activity. 5. 5. Estrogen binding activity and inhibitory effect on lymphoblastoid-forming activity. 6. 6. Possible osmotic regulator. 7. 7. Significant elevation of blood α 1 P level in the course of hepatoma induction.

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.

Using the indirect immunoperoxidase method, we studied the localization of carbonic anhydrase-III (CA-III) in frozen sections of biopsies of human skeletal muscle which had no definite pathology. CA-III was found to be localized in Type-I muscle fibers when compared with serial sections stained with myosin ATPase and other reactions. Our finding was in accordance with the biochemical data so far reported. It was thought that CA-III could be used as a marker for abnormal Type-I muscle fibers in several neuromuscular diseases.

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.

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

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.

A daunorubicin (DNR)-resistant variant (AH66DR) of al-pha-fetoprotein (AFP)-producing rat ascites hepatoma AH66 was established. AH66DR was 169 times more resistant to DNR than AH66. A growth-inhibitory effect due to the decrease in sugar uptake following treatment of the cell lines with specific antibody against highly purified rat AFP was also observed. Pre-incubation of both lines with antibody prior to the measurement of 2-deoxy-D-glucose (2dG) uptake resulted in a 45-55% decrease in 2dG uptake compared to control cells with a 2-fold reduction of Vmax value while the Km remained unchanged. Upon pre-incubating AH66DR with antibody, an increased intracellular level of DNR concentration with reduction of efflux rate was observed. A significantly superior cytotoxic effect against AH66DR, which reached almost the same level as the cytotoxic effect of DNR against AH66, was found, as compared to various other controls when tumor cells were cultured with a mixture of antibody and DNR. Our in vitro results suggest that the DNR-resistance of the AFP-producing tumor cells may be overcome if the cells are treated with specific antibody.

Ubiquitin, which can conjugate with cellular proteins, is classified into two forms: free ubiquitin and multiubiquitin chains. The latter is active as a signal for degradation of the targeted proteins. We found both forms in human serum and, using two immunoassays, quantitated them in sera from healthy subjects and patients with some diseases. Because of putative leakage of erythrocyte ubiquitin, hemolytic serum and serum obtained after long incubation (>1–2 h) of blood at room temperature were excluded. Serum concentrations of multiubiquitin chains and free ubiquitin were substantially higher in rheumatoid arthritis and hemodialysis patients, respectively, than healthy subjects. Additionally, in acute viral hepatitis, serum multiubiquitin chain concentrations were increased in the acute phase, decreased in the recovery phase, and correlated with alanine and aspartate aminotransferase activities (r = 0.676 and 0.610, P