Shuichi Saheki

cDNA cloning of a neural visinin-like Ca2+-binding protein

A 21,000-dalton Ca(2+)-binding protein (Walsh, M.P., Valentine, K.A., Ngai, P.K., Carruthers, C.A., and Hollengerg, M.D. (1984) Biochem. J. 224, 117-127) was purified from the rat brain and through the use of oligonucleotide probe based on partial amino acid sequence, cDNA clones were obtained from rat brain cDNA library. The complete amino acid sequence deduced from the cDNA contains 191 residues and has a calculated molecular mass of 22,142 daltons. There are three potential Ca(2+)-binding sites like the EF hands in the sequence. It displays striking sequence homology with visinin and recoverin, retina-specific Ca(2+)-binding proteins. Northern blot analysis revealed that the protein is highly and specifically expressed in the brain.

Intestinal type alkaline phosphatase hyperphosphatasemia associated with liver cirrhosis.

Hyperphosphatasemia due to increased intestinal type serum alkaline phosphatase was noted in a 48-year-old male who had asymptomatic liver cirrhosis. The alkaline phosphatase activity in the serum was 828 U/l (our reference range in adults: 57-194 U/l), 94% of which was of the intestinal type as measured by an immunoprecipitation method. The intestinal component of alkaline phosphatase was separated into two major and some minor components using electrophoresis and isoelectrofocusing. One of the major components had similar mobility to that of a standard intestinal enzyme purified from adult intestine. The components were heat-labile and neuraminidase-resistant. Serial lectin affinity chromatography, however, indicated that sugar chain compositions of the alkaline phosphatase were different from those of the standard tissue intestinal enzyme. These results and further enzymological studies suggest that the patients serum alkaline phosphatase basically consisted of several intestine-like isoforms.

Gestational Changes in Nitric Oxide Synthase Activity in the Rat Placenta

Objective: To assess the importance of nitric oxide (NO) generated in the placenta on pregnancy, nitric oxide synthase (NOS) activities were measured in the rat placentas of different gestational ages.

Peptide structures of pyruvate kinase isozymes: 2. Origins of types M1 and M2 isozymes suggested from species-variations in their peptide maps

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) isozymes were purified: type M1 and M2 isozymes from rat, mouse and rabbit, type M1 from bull frog and type L from rat and mouse. The amino acid composition of type M1 and M2 isozymes from various species were very similar and those of type L isozymes from rat and mouse also very similar. The NH2-terminals to type M1 and M2 from rat and mouse were (Pro-Lys-Pro-), but those of the other enzymes appeared to be blocked. The COOH-terminals of type M1 and M2 from rat, mouse and rabbit were (-Val-Pro). Comparison of tryptic peptide maps of type M1 and M2 from rat, mouse and rabbit indicated that type M1 and M2 could not be produced by post-synthesis modification, but could be encoded by different type-specific mRNAs. Comparative studies on type M1 and M2 of different species showed that type M1-specific peptide spots were highly variable, whereas type M2-specific peptide spots were highly conserved. The type L isozyme showed marked species variation, indicating that this differentiated isozyme evolved more rapidly than type M2, which may be a prototype or undifferentiated isozyme.

Composition of very low density lipoproteins and in vitro effect of lipoprotein lipase

In order to clarify the relationship between composition and lipolytic responses to lipoprotein lipase (LPL), very low density lipoproteins (VLDL) from rats or humans were incubated with a commercially available LPL or with a partially purified LPL from postheparin human plasma and fatty acids released from VLDL were determined in vitro. VLDL from rats fed a diet containing 0.25% cholesterol for 6 months were rich in cholesterol and poor in triglycerides, and released less fatty acids from incubation with LPL than those from control rats. VLDL from normo-and hypertriglyceridemic human subjects were incubated with LPL. The fatty acid release poorly correlated with the apoprotein ratios of VLDL, apo C-III/C-II, B/E, and C/E with the exception of apo B/C, but it correlated well with the ratio of triglyceride/either one of the surface components including total apoproteins, free cholesterol and phospholipids in VLDL or the ratio of the triglyceride/total sum of the surface components. The correlation coefficients between fatty acid release and a ratio of triglyceride/total surface components were 0.774 (using the commercially available LPL) and 0.786 (using the partially purified human LPL). The fatty acid release increased after pretreatment of VLDL with phospholipase A2. The phospholipid content of VLDL was reduced without significant changes in other VLDL components. Thus, the responses of VLDL to LPL treatment may depend mainly upon the surface: core relationship of VLDL rather than its apoprotein composition except in rare clinical cases such as apo C-II deficiency.

In vitro degradation of very low density lipoprotein from diabetic patients by lipoprotein lipase

Fatty acid release by incubation with lipoprotein lipase (LPL) in vitro from very low density lipoproteins (VLDL) obtained from diabetic patients was low compared with that from healthy subjects, though the compositions were similar in both VLDL. Percentages of the large size VLDL decreased and those of the small size VLDL increased after the incubation with LPL. At the same time, on polyacrylamide gel disk electrophoresis, the smaller catabolic products from these VLDL appeared at a similar position to that of low density lipoproteins (LDL) and at the running front where high density lipoproteins (HDL) had migrated. The amount of the small size VLDL and the LDL-like lipoproteins produced from diabetic VLDL were less than those from normal VLDL and inversely correlated with the percent decrease of the large original size VLDL. This fact suggests that VLDL from diabetic patients are poor substrates for LPL compared with normal VLDL.

Changes in pyruvate kinase isozymes of rat small intestine during development and the synergistic effect on them of thyroid and glucocorticoid hormones.

Pyruvate kinase isozymes in rat small intestine changed markedly during the postnatal period. The activities of type M2 subunits rapidly increased before weaning, while those of type L subunits decreased slightly. The administration of hydrocortisone induced normal changes prematurely, whereas the administration of thyroxine increased their extent. On simultaneous administration, the two hormones had synergistic effects.

Glycated apolipoprotein A-I assay by combination of affinity chromatography and latex immunoagglutination

The degree of glycation of plasma apolipoprotein A-I was measured by a combination of gel filtration, boronate affinity chromatography and latex immunoagglutination. The plasma concentrations of apolipoprotein A-I determined by this combination method (y) correlated well with those determined by turbidimetric immunoassay (x) (y= 1·12x + 1·9, r = 0·964). The inter- and intra-assay coefficients of variation in the glycated apolipoprotein A-I assay were 4·1–5·0% and 4·0–4·4%, respectively. Interference from plasma glucose at concentrations up to 55·1 mmol/L was eliminated by gel filtration. Labile glycated apolipoprotein A-I did not interfere with the measurement of glycated apolipoprotein A-I. Reference values for glycated apolipoprotein A-I were determined to be 2·4–4·0% (n = 140), with no significant difference between men and women. The mean concentration of plasma glycated apolipoprotein A-I in patients with uncontrolled diabetes mellitus (5·11%) was significantly higher than in normal subjects (3·12%, P < 0·001). The method is simple, rapid and highly sensitive for determination of the glycation level of plasma apolipoprotein A-I.

Immunohistochemical detection of truncated APC protein in sporadic human colorectal adenomas and adenocarcinomas

Mutations of the APC gene frequently occur in sporadic forms of colorectal adenomas and adenocarcinomas. Phenotypically, the vast majority of these mutations result in the truncation of the APC protein. To demonstrate the defective APC gene product in human colorectal tumors, rabbit region-specific antisera raised against the APC protein of amino acid sequences between 371 and 390 (SP1) and between 1821 and 1840 (SP3) were used to exhibit the truncated APC protein. In all, 86 lesions from 67 cases of sporadic adenoma and adenocarcinoma were examined; abnormal staining patterns were distinguished in 43 lesions (50%); the incidence of abnormalities was not significantly different between adenomas and carcinomas. The majority, 75% exhibited epitopic change with the SP1-positive and SP3-negative phenotype (type P1), and 25% exhibited neither of these phenotypes (type P2). The staining pattern in all lesions was uniform, and studies of carcinomas arising in adenomas showed the same pattern of staining. These findings supported the view that the APC lesion is a very early event in colorectal carcinogenesis. Furthermore, this simple immunohistochemical approach demonstrated that different adenomas from the same patient showed different staining patterns.

Enhancement of CD3‐mediated thymocyte apoptosis by the cross‐linkage of heat‐stable antigen

Heat‐stable antigen (HSA) is a murine differentiating antigen that is expressed on both CD4−CD8− double‐negative and CD4+CD8+ double‐positive thymocytes but not CD4+ or CD8+ single‐positive thymocytes. Effects of anti‐HSA monoclonal antibody, R13, on thymocyte apoptosis induced by various stimulations were investigated by a single‐cell suspension culture system. Immobilized R13 enhanced the CD3‐mediated DNA fragmentation and killing of thymocytes but not the dexamethasone‐induced or phorbol myristate acetate‐induced killing of thymocytes. Immobilized R13 by itself could not induce thymocyte apoptosis. Soluble R13 enhanced CD3‐mediated apoptosis when HSA and T‐cell receptor (TCR)/CD3 were co‐cross‐linked by a cross‐reactive secondary antibody. Even without the cross‐reactive secondary antibody, soluble R13 enhanced CD3‐mediated apoptosis, although a greater than 100‐fold increase in the amount of R13 was needed to give a similar enhancement compared with immobilized R13. Neither R13 by itself nor R13 plus secondary antibody induced cytosolic calcium influx, whereas R13 enhanced CD3‐mediated cytosolic calcium increase. These results suggest a functional role of HSA in promoting the activation‐induced apoptosis of thymocytes and the involvement of HSA in negative selection.