Shoji Odani

A new form of amyloid protein associated with chronic hemodialysis was identified as β2-microglobulin

Amyloid fibrils were isolated from amyloid-laden tissue obtained from a chronic hemodialysis patient with carpal tunnel syndrome. After solubilization in guanidine HCl, a significant amount of the protein was located in a homogeneous low molecular weight fraction. The protein was found to be identical to beta 2-microglobulin, with regard to its molecular weight of 11,000, amino acid composition and 16 amino-terminal amino acids: Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-. These results demonstrate that the amyloid associated with chronic hemodialysis contains as major component a new form of amyloid fibril protein that is homologous to beta 2-microglobulin.

The coxsackievirus-adenovirus receptor protein as a cell adhesion molecule in the developing mouse brain

In an attempt to elucidate the molecular mechanisms underlying neuro-network formation in the developing brain, we analyzed 130 proteolytic cleavage peptides of membrane proteins purified from newborn mouse brains. We describe here the characterization of a membrane protein with an apparent molecular mass of 46 kDa, a member of the immunoglobulin superfamily of which the cDNA sequence was recently reported, encoding the mouse homologue of the human coxsackievirus and adenovirus receptor (mCAR). Western and Northern blot analyses demonstrated the abundant expression of mCAR in the mouse brain, the highest level being observed in the newborn mouse brain, and its expression was detected in embryos as early as at 10. 5 days post-coitus (dpc), but decreased rapidly after birth. On in situ hybridization, mCAR mRNA expression was observed throughout the newborn mouse brain. In primary neurons from the hippocampi of mouse embryos the expression of mCAR was observed throughout the cells including those in growth cones on immunohistochemistry. In order to determine whether or not mCAR is involved in cell adhesion, aggregation assays were carried out. C6 cells transfected with mCAR cDNA aggregated homophilically, which was inhibited by specific antibodies against the extracellular domain of mCAR. In addition to its action as a virus receptor, mCAR may function naturally as an adhesion molecule involved in neuro-network formation in the developing nervous system.

Isolation and immunohistochemical localization of a cerebellar protein

Our previous report described that a protein called spot 35 is found in the cerebellar cytosol of adult rats by two-dimensional gel electrophoresis. In this paper we isolated this protein from the soluble fraction of bovine and rat cerebella and then prepared an antiserum against the bovine protein. This protein shows pI around 5.3 and Mr around 27 kdalton. Determination of the amino acid composition of this protein shows high glutamic acid, aspartic acid and leucine contents. Using the antiserum we examined the immunohistochemical localization of this protein by the peroxidase-antiperoxidase method. Purkinje cells, their dendrites and axons were immunohistochemically stained in the cerebella of adult rats, rabbits and humans. Other cells, such as granule cells and glial cells, and myelin did not react to the antiserum.

Evidence for megalin-mediated proximal tubular uptake of L-FABP, a carrier of potentially nephrotoxic molecules.

Liver-type fatty acid binding protein (L-FABP) binds with high affinity to hydrophobic molecules including free fatty acid, bile acid and bilirubin, which are potentially nephrotoxic, and is involved in their metabolism mainly in hepatocytes. L-FABP is released into the circulation, and patients with liver damage have an elevated plasma L-FABP level. L-FABP is also present in renal tubules; however, the precise localization of L-FABP and its potential role in the renal tubules are not known. In this study, we examined the cellular and subcellular localization of L-FABP in the rat kidney and tried to determine from where the L-FABP in kidney tissues had originated. Immunohistochemical studies of kidney sections localized L-FABP in the lysosomes of proximal tubule cells (PTC). In rats with carbon tetrachloride (CCl4)-induced acute liver injury, we detected high levels of L-FABP in the circulation and in the kidney compared with those in the control rat by immunoblotting, while reverse transcription-polymerase chain reaction showed that the level of L-FABP mRNA expression in the kidney of CCl4-treated rats was low and did not differ from that in the control rat. When 35S-L-FABP was intravenously administered to rats, the kidneys took up 35S-L-FABP more preferentially than the liver and heart, and histoautoradiography of kidney sections revealed that 35S-L-FABP was internalized via the apical domains of PTC. Quartz-crystal microbalance analysis revealed that L-FABP bound to megalin, a multiligand endocytotic receptor on PTC, in a Ca2+-dependent manner. Degradation assays using megalin-expressing rat yolk sac tumor-derived L2 cells demonstrated that megalin mediated the cellular uptake and catabolism of 125I-L-FABP. In conclusion, circulatory L-FABP was found to be filtered by glomeruli and internalized by PTC probably via megalin-mediated endocytosis. These results suggest a novel renal uptake pathway for L-FABP, a carrier of hydrophobic molecules, some of which may exert nephrotoxic effects.

A complex of rab3A, SNAP-25, VAMP/synaptobrevin-2 and syntaxins in brain presynaptic terminals.

Two monoclonal antibodies (SPM‐1 and SPM‐2) immunoprecipitate brain N‐type calcium channels. On immunoaffinity chromatography of digitonin extracts of bovine brain membranes on SPM‐1‐ and SPM‐2‐Sepharose, proteins of 36 (syntaxins A and B), 28 and 19 kDa are specifically retained by both columns. Here we show that the 19 and 28 kDa bands contain VAMP/synaptobrevin‐2, and rab3A/smg25A and SNAP‐25, respectively. Since SPM‐1 and SPM‐2 recognize only syntaxins and the 28 kDa band (rab3A/smg25A and SNAP‐25), respectively, the results indicate that all these proteins form a complex. Our results suggest tight linkage between the components involved in neurotransmitter release.

A close structural relationship of rat liver Z-protein to cellular retinoid binding proteins and peripheral nerve myellin P2 protein

Abstract Striking sequence homologies were found among the major cytosolic lipid and dye binding protein (Z-protein), cellular retinoid binding proteins and peripheral nerve myelin P2 protein. In addition, the presence of a number of cytosolic lipid binding (or exchange) proteins having similar molecular weights and amino acid compositions suggests a new family of intracellular proteins with high affinities to lipids, possibly diverged from a common ancestor. In view of regulatory roles of these proteins in lipid metabolism and transport, myelin P2 protein may also possess a similar physiological function(s).

Homozygous deletions and point mutations of the Ikaros gene in γ-ray-induced mouse thymic lymphomas

Our previous genome-wide analysis of allelic loss for thymic lymphomas that were induced by γ-irradiation in F1 hybrid mice between BALB/c and MSM strains suggested the centromeric region on chromosome 11 as a site harboring a tumor suppressor gene. Interestingly, to this region the mouse Ikaros gene was mapped which was postulated to participate in oncogenic process from the study of Ikaros knockout mice. Here we show fine allelic loss mapping in the vicinity of Ikaros in 191 lymphomas, indicating that the critical region of allelic loss was centered at the Ikaros locus. PCR analysis revealed that nine lymphomas failed to give PCR-amplification for either of two exon primer pairs, indicative of homozygous deletion. Six and five mutations were detected in the N-terminal zinc finger domain and the activation domain of Ikaros, respectively, and six of the eleven were frameshift or nonsense mutations that resulted in truncation of Ikaros protein. The results strongly suggest a direct role for Ikaros in development of mouse thymic lymphomas. This provides the experimental basis for further analysis of Ikaros mutations in human cancer.

Histidine-rich glycoprotein is evolutionarily related to the cystatin superfamily: Presence of two cystatin domains in the N-terminal region

A new member of the cystatin superfamily is introduced. Human plasma histidine‐rich glycoprotein (HRG) was found to contain 2 cystatin‐like sequences in tandem in the N‐terminal region. Domain 1 (residues 1–112) was most homologous to domain 1 of the heavy chain of human kininogen and domain 2 (residues 113–225) was most homologous to human cystatin S as well as other cystatins and domain 3 of the heavy chain of kininogen, suggesting that the cystatin domains of HRG may represent a hitherto unknown binary form (or intermediate molecule) composed of 2 cystatin domains, and evolutionarily intermediate between the cystatin and the kininogen families.

Primary structure of rat liver Z-protein: A low-Mr cytosol protein that binds sterols, fatty acids and other small molecules

In the cytosol of animal tissues, Z-protein [I] (fatty acid-binding protein [2] or squaleneand sterolcarrier protein [3]) binds free fatty acids, their CoA derivatives, bilirubin, organic anions and other small molecules [4]. It appears to play a significant role as a carrier protein in reversing the inhibitory effect of palmitoyl CoA on acetyl CoA carboxylase [5] and on adenine nucleotide transport of isolated mitochondria [6]. A possible role of Z-protein in fatty acid metabolism has been suggested by the findings that it stimulated microsomal acyl CoA:glycerophosphate acyltransferase [7] and mitochondrial P-oxidation of fatty acids [8]. homogenates of rat livers in 0.1 M Trls-HCl (pH 7.4) with 1 mM EDTA by centrifugation at 105 000 X g for 60 min. The supematant obtained from fractionation with 70% saturated ammonium sulfate was dialyzed against 30 mM Tris-HCl (pH 8.5), concentrated by ultrafiltration (Amicon UM-10) and applied to a column of Sephadex G-75. The Z-protein fraction binding indocyanine green (Daiichi Seiyaku) was applied to a DEAEcellulose column equilibrated with 30 mM Tris-HCl (pH 8.5).

NY-ESO-1 expression and its serum immunoreactivity in esophageal cancer

PurposeNY-ESO-1, a member of the cancer/testis antigen (CTA) family, elicits humoral and cellular immune responses in patients with advanced cancer. Unresectable or metastatic esophageal carcinoma patients do not benefit from the present multimodality treatment regimens in terms of survival. The objectives of this study were to analyze the antibody response to NY-ESO-1 antigen in patients with esophageal cancer and to determine the potential of NY-ESO-1 for use in tumor-specific immunotherapy.MethodsSerum from 69 patients with esophageal cancer was investigated for antibody production against NY-ESO-1 by Western blot analysis. Also analyzed by immunohistochemistry were 56 tissue samples from these patients for NY-ESO-1 protein expression.ResultsNY-ESO-1 protein expression was found in 18 of 56 (32%) esophageal carcinomas. Serum immunoreactivity specific for NY-ESO-1 was found in 9 patients (13%) of whom 8 were in the advanced stage (stages III and IV). There was no relationship between clinicopathologic features and serum immunoreactivity for NY-ESO-1. NY-ESO-1 protein expression was detected in three of five antibody-positive patients whose tissue was available for analysis. Survival analysis showed no significant difference between antibody-positive and antibody-negative patient groups.ConclusionsA humoral immune response to NY-ESO-1 antigen was established in patients with advanced esophageal cancer. NY-ESO-1 is a good candidate for vaccine-based immunotherapy for advanced esophageal carcinoma.