SDS-PAGE after micro-affinity adsorption for analysis of heterogeneous antigen polypeptides in individual sera.

A method was developed for the analysis of heterogeneity in antigen polypeptides in individual sera. Polypeptides in sera were adsorbed by polystyrene beads coated with antibody in wells of a microplate. They were dissociated with a small volume of elutant, and transferred to slots on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Polypeptides separated on gel were then immunoblotted with antibodies labeled with horseradish peroxidase. The method was applied to analyze different populations of hepatitis B surface and e antigen polypeptides in sera from carriers of hepatitis B virus. Applicability to mass-scale and high sensitivity of the method would allow surveys of heterogeneous antigen polypeptides in serum for any biological significance.

A cell-binding, immunoglobulin-like protein from human plasma. II. Cell-binding activity.

Cell adhesion to plastic surfaces coated with a new high-molecular-mass immunoglobulin-like protein from normal human plasma was studied. Mouse subdermal fibroblasts, hamster kidney cells, human umbilical vein endothelial cells, and human skin fibroblasts were found to become attached to the surface, but cancer cells derived from human stomach cancer and human breast cancer did not. The appearance of the attached cells differed from that of cells attached to surfaces coated with fibronectin or concanavalin A. The cell adhesion to the surfaces coated with the protein was inhibited by goat anti-human IgM. Furthermore, the binding of the protein to the cell surfaces was demonstrated by the indirect immunofluorescence method. It is concluded that this protein is a new cell-binding protein.

A cell-binding, immunoglobulin-like protein from human plasma. I. Isolation and subunit structure.

A protein having Mr of more than 900K (900,000) was isolated by chromatography on a column of Sepharose 4B coupled with solubilized elastin, followed by sucrose density gradient centrifugation. The protein is composed of several disulfide-linked subunits. SDS-polyacrylamide gel electrophoresis and immunoblotting indicated that two of the subunits are identical with the heavy chains of IgM and IgG, and one is immunologically related to the heavy chain of IgA. The results suggested that this protein is a new protein that belongs to the category of, or is closely related to, the immunoglobulins. As described in the succeeding paper, it has cell-binding activity (Fukamizu et al. (1986) J. Biochem. 100, 843-848). We tentatively refer to this protein as "cell-binding immunoglobulin-like protein (CIP)."

Polypeptide analysis of hepatitis virus type B surface antigen produced by a human hepatoma cell line, PLC/PRF/5.

HBs antigen purified from culture fluid of a human hepatoma cell line, PLC/PRF/5, was analyzed by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis. At least 6 polypeptide species were resolved. The polypeptide with a molecular weight of 48,000 was identified as a glycoprotein.

Heterotypic interference between influenza viruses A/Aichi/2/68 and B/Massachusetts/1/71.

Virus particles produced by MDCK cells mixedly infected with 3 PFU/cell each of A/Aichi/2/68 (H3N2) (Aichi) and B/Massachusetts/1/71 (Mass) influenza viruses exclusively possessed haemagglutinin (HA) of Mass, although approximately one-fifth of the mixed yield had coding potential for Aichi serotype. Synthesis of major viral proteins of Aichi was markedly suppressed by co-infecting Mass. By increasing the multiplicity of co-infecting Aichi to 30 PFU/cell, interference became reciprocal. Aichi interfered with replication of Mass more severely than Mass did with replication of Aichi. All the major viral proteins of both Aichi and Mass were expressed within the infected cells.

[Choice of method for statistical analysis of quantitative data obtained from toxicological studies--toxicological data].

We compared the usefulness of t-test and parametric and rank-sum tests in the statistical analysis of significant differences in the so-called "decision tree" for the quantitative data obtained from toxicity studies. The Dunnett's multiple comparison test had lower analytic power than the t-test when one of the groups showed a marked difference in variance. The Dunnett's test was less efficient with the increase in the number of groups. If one group showed a decrease in the number of animals, this test was less efficient than parametric tests, because the rank-sum tests should be chosen. The rank-sum test is required occasionally to attach the asterisks of significant difference to the mean +/- SD even in showing the same mean values. The nonparametric Dunnett's test could not be used for analysis of significant differences when the mean value for the control and treated groups showed big differences. The nonparametric Dunnett's and parametric Scheffé tests were not as efficient as the other parametric tests probably because of the vague evaluation or overlooking the effect of the test substance.

[A new decision tree method for statistical analysis of quantitative data obtained in toxicity studies on rodents].

Regarding the statistical analysis of the quantitative data obtained in control and dosage groups in toxicity studies, we tried to simplify the decision tree method. In a new decision tree presented in this article, one-way analysis of variance and Kruskal-Wallis nonparametric analysis of variance are excluded from the traditional decision tree: (1) Bartlett's test is used as a test for the equality of k variances: (2) Then, if the k sampled populations have equal variances (p > 0.05 by the Bartlett's test), Dunnett's multiple comparison test is performed: otherwise, Steel's test is used. This new method, which increases the power in some conditions, may serve as an alternative to the traditional decision tree method.

[Reoperative off-pump coronary artery bypass surgery for restenosis after percutaneous coronary intervention as a part of hybrid revascularization chronic renal failure on hemodialysis; report of a case].

60-year-old man on chronic hemodialysis had hybrid revascularization. Percutaneous coronary intervention (PCI) for right coronary artery (RCA) combined with left internal mammary artery-left anterior descending artery anastomosis through left anterior small thoracotomy were performed. But, frequent re-stenosis after PCI necessitated 4 repeated PCI. Finally, the patient agreed to re-do, and underwent reoperative off-pump coronary artery bypass through midsternotomy. Right internal mammary artery-saphenous vein lengthened composite graft was successfully anastomosed to heavily calcified RCA without heart-lung machine. Strategy of revascularization for hemodialysis patient was discussed.

["Coronary arteriographic findings in the patients of arteriosclerotic heart disease combined with cardiac arrhythmias or conduction defects" (author's transl)].

Correlation between cine-coronary arteriographic findings and various cardiac arrhythmias or conduction defects were studied in 40 patients with arteriosclerotic heart disease. These cases were divided into 3 groups according to the type of arrhythmias or conduction defects. The following conclusions were drawn. 1. In the group I of 25 patients whose arrhythmias were atrial origin, there were sinus bradycardia in 9, suins tachycardia in 5, atrial premature beats in 2 and atrial fibrillation in 9. Cine-angiographically, the degree of luminal stenosis of the main coronary arterial branches were mild to moderate in severity compared to that of the other two groups. However, it was remarkable that the sinus node artery was not visualized in 9 of 25 cases (36%). 2. The group II of 5 patients who have shown premature ventricular contractions exhibited moderate to severe degree of stenosis of coronary artery trees, especially much more often in left anterior descending coronary arteries. 3. In the group III of 10 patients who had various type of intraventricular conduction defects, the degree of stenosis of the main coronary artery branches were usually severe and also, the number of the coronary artery involved in stenosis was increased. Interestingly, atrioventricular node arteries were not opacified in 5 of 10 cases. There were 2 cases of complete A-V block where the atrioventricular node artery was not visualized at all both either from right or left coronary artery opacification. It seemed to be hard to draw a clear cut correlation between cine coronary angiographic pictures and a certain type of cardiac arrhythmias or conduction defects in such a small scale of study. However, cind coronary angiography may contribute to rule out local ischemia of the conduction tissue as a primary cause of the development of arrhythmias or conduction defects in patients with arterisclerotic heart disease.

Catalytic asymmetric synthesis of 19,20-dihydroakuammicine.

An enantiocontrolled total synthesis of 19,20-dihydroakuammicine using a catalytic asymmetric Michael addition of dimethyl malonate to cyclohexenone as the key step is described. The above catalytic asymmetric Michael addition proceeds quite efficiently in the presence of a heterobimetalic asymmetric catalyst (ALB-KO-t-Bu-MS 4A, 0.3 mol%), giving the corresponding Michael adduct in 94% yield and 99% ee.

Effect of coenzyme Q10 on hypertrophied ischemic myocardium during aortic cross clamping for 2 hr, from the aspect of energy metabolism.

In order to perform intracardiac repair safely during aortic cross clamping, we designed this study to evaluate the protective effect of coenzyme Q10 (CoQ10) on hypertrophied ischemic myocardium from the aspect of energy metabolism. Six to nine months preceding the study, aortic bandings were carried out on 14 puppies to produce left ventricular hypertrophy (LVH). These dogs with LVH were then subjected to total cardiopulmonary bypass and were evenly divided into control and CoQ10-treated groups (10 mg/kg of intravenous administration plus 1 mg/kg per hr of intracoronary injection). Myocardial ischemia was induced by aortic cross clamping for 2 hr under moderate systemic hypothermia. The results indicated that the administration of CoQ10 had a protective effect on hypertrophied ischemic myocardium, since depletion of high-energy phosphate (HEP) was uniformly prevented, and accumulation of lactate was simultaneously decreased during the 2 hr of aortic cross clamping. On the other hand, there were marked exhaustion of HEP and rapid increase in lactate following the 2 hr of ischemia in the control group, these being much more predominant in the subendocardial layer.

Collagen and elastin synthesis by desmoid tumor in vitro.

In order to characterize human desmoid tumors in vitro, the production of collagen and elastin and the expression of collagen types alpha1(I), alpha1(III) and transforming growth factor (TGF)-beta1 mRNA were investigated in six desmoid tumors; five derived from familial adenomatous polyposis patients and one from a sporadic case. The proportion of collagen production to total protein production was determined by 3H-imino acid incorporation, an indicator of collagen synthesis, using high-performance liquid chromatography (HPLC). The proportion of collagen production to total protein production was much higher in all six desmoid tumors compared with human skin fibroblasts (HSF). Quantitatively, the rate of elastin synthesis in desmoid tumor cells monitored by valine-proline peptide was also significantly higher than in HSF. Pro-alpha1(I) collagen mRNA was highly expressed in both desmoid tumors and HSF at approximately the same level, whereas pro-alpha1(III) collagen mRNA was more abundant in some of the desmoid tumors than the normal skin fibroblastic cell lines. Tumor growth factor-beta1 mRNA, which is believed to stimulate collagen synthesis, was expressed in both desmoid tumors and HSF to the same extent. These results demonstrate the increased formation of collagen and elastin in desmoid tumors in vitro and suggest that the increased synthesis of elastin rather than of collagen and TGF-beta1 may be involved in increased fibrogenesis by desmoid tumors.

Molecular heterogeneity of e antigen polypeptides in sera from carriers of hepatitis B virus.

Hepatitis B e Ag (HBeAg) was isolated from pooled sera of carriers, without abnormalities in liver function, by affinity column chromatography with mAb against HBeAg. HBeAg polypeptide with an estimated molecular size of 20,000 Da (p20e) was detected, in addition to regular HBeAg polypeptides (p17e/p18e). p20e, as well as p17e/p18e, did not bind with mAb against the carboxyl-terminal domain of the C-gene product. p20e disclosed an N-terminal sequence of MQLFHLXLII- (X unknown), whereas p17e had that of SKLXLGXLXGMDIDPXKEFG- (X's unknown). By comparing them with the amino acid sequence encoded by the precore region and C gene of hepatitis B virus DNA, p20e was deduced to possess amino acids 1 to 19 of the precore-region product at the N-terminus, which contains signal sequence and usually removed before the secretion of HBeAg. p17e had amino acids 20 to 29 of the precore-region product that continued to the C-gene product. Inasmuch as p20e was invariably detected in HBeAg preparations from carriers without evidence for liver disease, it would not have been released into the circulation from destructed hepatocytes. HBeAg polypeptide bearing an uncleaved signal sequence would help in further understanding the mechanism of HBeAg secretion.