Biomedical subjects

K Maruyama

Publications and source records attributed to K Maruyama.

At least 19 recordsLinked to original sources

PubMed1992

Amyloid beta/A4 precursor protein (APP) processing in lysosomes.

Alzheimer's disease (AD) is characterized by the formation of senile plaques, the main component of which is amyloid beta protein. The processing of the amyloid beta/A4 protein has been implicated in the pathogenesis of AD. We have reported cathepsin B as a candidate APP secretase. By using APP-transfected COS-1 cells, we determined that the putative APP secretase, lysosomal cathepsin B, is involved in the constitutive processing of the precursor molecule. Digestion experiments demonstrated that cathepsin B cleaves the APP molecule into two fragments with molecular masses 115 kDa and 9 kDa, representing presumptive proteolytic fragments of constitutive processing.

Alzheimer Disease↗

PubMed1992

Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol).

The effect of poly(ethylene glycol) (PEG) on the circulation time of liposomes in mice was examined by employing amphipathic PEGs (phosphatidylethanolamine (PE) derivatives of PEG) with average molecular weights of 1000, 2000, 5000 and 12,000. The activity of dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG) in prolonging the circulation time of egg phosphatidylcholine/cholesterol large unilamellar liposomes (ePC/CH LUVs) (200 nm) was proportional to the molecular weight of PEG, i.e., 12000 = 5000 greater than 2000 greater than 1000. On the other hand, inclusion of distearoylphosphatidylethanolamine-PEG (DSPE-PEG) or dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG) of low molecular weight such as 1000 and 2000 in distearoylphosphatidylcholine (DSPC)/CH LUVs or dipalmitoyl phosphatidylcholine (DPPC)/CH LUVs effectively increased their blood circulation time. At least 3 mol% of amphipathic PEG in liposomes was required for activity. Addition of CH, which has a bilayer-tightening effect, to DSPC/CH/DSPE-PEG2000 LUVs further increased the blood residence time. A size of less than 300 nm was essential for prolonging the residence time of amphipathic PEG-containing liposomes in blood. DSPC/CH/DSPE-PEG2000 LUVs (1:1:0.13, m/m) containing 6 mol% of PEG and 200 nm in diameter remained in the circulation for over 24 h after injection and may be clinically useful for sustained release of an entrapped drug in the bloodstream and for drug accumulation in solid tumors.

Animals↗

PubMed1992

Characterization of amyloid fibril protein from a case of cerebral amyloid angiopathy showing immunohistochemical reactivity for both beta protein and cystatin C.

We isolated and carried out a chemical analysis of the amyloid fibril protein from the leptomeningeal vessels of a case with non-hereditary cerebral amyloid angiopathy (CAA) showing dual immunohistochemical reactivity with antibodies to both beta-protein and cystatin C. A crude amyloid fibril fraction reacted only with anti-beta-protein antibody, and cystatin C immunoreactivity was observed in the first PBS supernatant. Complete amino acid sequence of this cystatin C-immunoreactive protein showed a homologous structure to that of normal cystatin C. It is concluded that cystatin C is not an intrinsic component of the amyloid fibril in this type of CAA.

Aged↗

PubMed1992

Expression of necdin, an embryonal carcinoma-derived nuclear protein, in developing mouse brain.

Necdin is a polypeptide sequence encoded by neural differentiation-specific mRNA derived from embryonal carcinoma cells. We have examined the expression of necdin and its mRNA in cultured cells and mouse brain by Northern blot analysis and immunohistochemistry. Among various established cell lines including neuroblastoma and glioma cells, only differentiated embryonal carcinoma cells (P19 and F9) expressed necdin mRNA. Necdin immunoreactivity was localized in the nuclei of differentiated neurons derived from P19 cells. Necdin mRNA was detected throughout brain regions of adult mouse; the relative abundances in the hypothalamus and midbrain were the highest, whereas those in the olfactory bulb and cerebellum were the lowest. In developing mouse brain, necdin mRNA was expressed during early periods of neuronal generation and differentiation, and the peak levels were attained during postnatal days 1-4. Necdin immunoreactivity was not detected in the neural stem cells on embryonic day 10, but was concentrated in the nuclei of brain cells, mostly neurons, at advanced stages of differentiation. The majority of differentiated neurons in the brain had necdin-immunoreactive nuclei on postnatal day 33. Thus, necdin may represent a valuable molecular marker for differentiated neurons both in vitro and in vivo.

Amino Acid Sequence↗

PubMed1992

Some negatively charged phospholipid derivatives prolong the liposome circulation in vivo.

A series of negatively charged phospholipid derivatives has been synthesized by coupling aliphatic dicarboxylic acids, HOOC(CH2)nCOOH, to dioleoylphosphatidylethanolamine (DOPE). The individual derivatives were incorporated into egg phosphatidylcholine/cholesterol liposomes (2:1, molar ratio) and injected into mice to test its effect on liposome circulation in vivo. The effectiveness of DOPE derivatives was dependent on the hydrocarbon chain length between the terminal carboxyl group and the amide bond. N-Glutaryl DOPE and N-adipyl DOPE were effective in prolonging the circulation time of liposomes. On the other hand, liposome uptake by the liver and spleen was increased by the addition of N-malonyl DOPE or N-succinyl DOPE, while it was not changed by the addition of N-pimelyl DOPE and N-suberyl DOPE. Our observation suggested that not all negatively charged phospholipids enhance liposome uptake by RES, some even reduce the uptake.

Animals↗

PubMed1992

Conformational changes of actin induced by calponin.

Calponin, an actin-linked regulatory protein in smooth muscle, caused a remarkable change in the fluorescence intensity of pyrene-labeled actin in the filamentous form. Calponin, an equimolar ratio to actin, decreased the fluorescence intensity of pyrene-labeled F-actin by some 60% to the level near monomeric actin. This change was partially reversed by Ca2+, when calmodulin was present. Thus it appears that calponin causes conformational changes in actin molecules in an actin filament so as to inhibit their interactions with myosin.

Actins↗

PubMed1992

Autophosphorylation of beta-connectin (titin 2) in vitro.

Phosphorylation of beta-connectin (titin 2), an elastic protein of chicken breast muscle, occurred in the presence of [gamma-32P] ATP, 0.2 mM CaCl2 and 25 mM phosphate buffer, pH 7.0. Addition of 3 mM MgCl2 did not affect the phosphorylation. However, Ca2+ ions were required for the phosphorylation and EGTA inhibited it even if MgCl2 were present. Myosin light chain kinase (gizzard MLCK), cAMP dependent protein kinase (A kinase), and protein kinase C (C kinase) did not phosphorylate beta-connectin in vitro under optimal conditions. Thus it appears that beta-connectin, possibly containing a domain homologous with MLCK, has an autophosphorylating action.

Adenosine Triphosphate↗

PubMed1992

Monoclonal antibodies specific for low-affinity interleukin-3 (IL-3) binding protein AIC2A: evidence that AIC2A is a component of a high-affinity IL-3 receptor.

Mouse interleukin-3 (IL-3) binds to its receptor with high and low affinities. Using anti-Aic2 antibody, two distinct cDNAs (AIC2A and AIC2B) were isolated. The AIC2A gene encodes a protein of 120 Kd that binds IL-3 with low affinity, whereas the AIC2B gene encodes a protein that is 91% identical to AIC2A at the amino acid level, but which does not bind IL-3. To study the structure of the functional high-affinity IL-3 receptor (IL-3R), we generated specific monoclonal antibodies against the AIC2A protein. We produced a soluble AIC2A protein by inserting a termination codon at the beginning of the transmembrane domain of the AIC2A cDNA. Soluble AIC2A protein expressed in COS7 cells was purified to homogeneity and three anti-AIC2A monoclonal antibody-producing hybridomas (3D1, 3D4, and 9D3) were obtained from a rat immunized with the purified soluble AIC2A protein. The antibodies were specific for the AIC2A protein and did not bind to the AIC2B protein. Using chimeric receptors between AIC2A and AIC2B, recognition sites of the antibodies were mapped. The antibodies immunoprecipitated a 120-Kd protein from IL-3-dependent PT18 cells. The N-terminal sequence of the 120-Kd protein was consistent with the predicted processing site of the signal sequence of the AIC2A protein. Staining of IL-3-dependent and IL-3-independent cell lines with the 9D3 antibody were consistent with the IL-3 binding. The 9D3 antibody inhibited both the high-affinity IL-3 binding and the low-affinity binding, as well as IL-3-dependent proliferation. These results indicate that the AIC2A protein is a binding component of a high-affinity IL-3R.

Amino Acid Sequence↗

PubMed1992

Preoperative assessment of lymph node metastases in patients with gastric cancer: evaluation of the Maruyama computer program.

The probability of survival of patients with gastric cancer depends upon depth of wall penetration by the primary tumour and metastatic lymph node burden. Radical lymphadenectomy may lead to prolonged survival but with increased morbidity. A computer program from Maruyama, National Cancer Centre, Tokyo enables evaluation of individual survival time and infiltration of lymph nodes. This analysis was applied to a German population. Computer-aided predictions were determined retrospectively using the prognostic factors of sex, age, Borrmann classification, histology, depth of wall penetration, location and diameter of the tumour. Data were collected from 222 patients at the Technical University of Munich (median age 66 years, sex ratio (M:F) 2:1), who had been operated on (72 per cent total gastrectomy, 28 per cent subtotal gastrectomy) for gastric cancer. The predicted results were compared with the postoperative pathological findings. The prediction of node metastases was highly accurate (lymph nodes 13-16, 96 per cent; 7-12, 89 per cent; 1-6, 82 per cent). These computer predictions may provide perioperative information of therapeutic value.

Aged↗

PubMed1992

Spinal cord vascular and leptomeningeal amyloid beta-protein deposition in a case with cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is characterized by the deposition of amyloid fibrils on leptomeningeal and cortical blood vessels, and the incidence of this disorder increases with age. However, this form of vascular amyloid deposition rarely involves tissues outside of the brain. A 71-year-old woman first developed some deterioration in memory, and soon afterwards suffered from recurrent episodes of subcortical hemorrhage. Histopathological examination of this case revealed typical pathology of Alzheimer's disease with an extensive appearance of beta-protein type CAA, and additionally, the spinal leptomeningeal vessels and the pia-arachnoid membranes were also affected by amyloid beta-protein deposits. The spinal cord involvement associated with CAA and Alzheimer's disease is unusual, and the present case provides additional important information on the pathogenesis of disorders with beta-protein deposition including Alzheimer's disease.

Aged↗

PubMed1992

Synthesis of bombyxin-IV, an insulin superfamily peptide from the silkworm, Bombyx mori, by stepwise and selective formation of three disulfide bridges.

We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm, Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for alpha-amino group. Three disulfide bonds were bridged step by step (A6-A11, A20-B22, and A7-B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides.

Amino Acid Sequence↗

PubMed1992

Determination of disulfide bond arrangement in bombyxin-IV, an insulin superfamily peptide from the silkworm, Bombyx mori, by combination of thermolysin digestion of natural peptide and selective synthesis of disulfide bond isomers.

The mode of disulfide linkages in bombyxin-IV, an insulin superfamily peptide consisting of A- and B-chains, was determined as A6-A11, A7-B10, and A20-B22. An intermolecular bond of A20-B22 was identified by sequencing and mass spectrometric analysis of the fragments generated by thermolysin digestion of natural bombyxin-IV. The mode of the remaining two bridges was determined by chemical and selective synthesis of three possible disulfide bond isomers of bombyxin-IV. A- and B-chains were synthesized by solid-phase method, and three disulfide bonds were bridged stepwise and in a fully controlled manner. Retention time on reversed-phase high-performance liquid chromatography (HPLC), thermolysin digests, and biological activity of the synthetic [A6-A11, A7-B10, A20-B22-cystine]-bombyxin-IV revealed that it was identical with the natural bombyxin-IV. Two other isomers with respect to disulfide bond arrangement, [A6-A7, A11-B10, A20-B22-cystine]- and [A6-B10, A7-A11, A20-B22-cystine]-bombyxin-IVs, were distinguishable from the natural one by use of HPLC, thermolysin digestion, and bioassay.

Amino Acid Sequence↗

PubMed1992

Characterization and localization of alpha-connectin (titin 1): an elastic protein isolated from rabbit skeletal muscle.

A simplified procedure to isolate alpha-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate alpha-connectin is introduced. Separation of alpha-connectin from beta-connectin is introduced. Separation of alpha-connectin from beta-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of alpha-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of alpha-connectin (MW, approximately 3 x 10(6)) were similar to those of its proteolytic product, beta-connectin (MW, approximately 2 x 10(6)). Circular dichroic spectra suggested that both alpha- and beta-connectin consist of 60% beta-sheet and 30% beta-turn. It thus appears that the whole elastic filament of connectin has a folded beta-strand structure. Proteolysis of alpha-connectin by calpain resulted in formation of beta-connectin and smaller peptides. The alpha-connectin interacted with both myosin and actin filaments similarly to beta-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with alpha-connectin (titin 1, TI) but only weakly with beta-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SM1 whose position depends on sarcomere length. It thus appears that beta-connectin extends from the edge of M-line to the above epitope region in the I-band.

Actins↗

PubMed1992

Spatial relationship of nebulin relative to other myofibrillar proteins during myogenesis in embryonic chick skeletal muscle cells in vitro.

The developmental expression of nebulin was studied in embryonic chick skeletal muscle cells in vitro by means of immunofluorescence microscopy. Initially nebulin appeared homogeneously or in a punctate form in the cytoplasm, and then it was assembled into I-Z-I-like complexes containing actin and alpha-actinin but not myosin and connectin (titin). Striated patterns of nebulin ('singlets') in myofibrils appeared simultaneously with those of alpha-actinin (Z-bands), myosin (A-bands) and connectin ('doublets'), but earlier than those of actin. After actin striations were formed as myofibrils matured, each nebulin band started to exhibit 'droplets'. The delayed development of nebulin compared to the I-Z-I brush formation and the myofibril maturation seems to indicate that this giant myofibrillar protein is unnecessary for both the initial (formation of I-Z-I-like structures) and the subsequent (regular alignment of myofibrils) phases of myofibrillogenesis.

Animals↗

PubMed1992

Localization and elasticity of connectin (titin) filaments in skinned frog muscle fibres subjected to partial depolymerization of thick filaments.

The localization and elasticity of connectin (titin) filaments in skinned fibres of frog skeletal muscle were examined for changes in the localization of connectin and in resting tension during partial depolymerization of thick filaments with a relaxing solution containing increased KCl concentrations. Immunoelectron microscopic studies revealed that deposites of antibodies against connectin at a sarcomere length of 3.0 microns remained at about 0.8 microns from the M-line, until the thick filament was depolymerized to the length of approximately 0.4 microns. On further depolymerization, the bound antibodies were found to move towards the Z-line and, on complete depolymerization, were observed to be within 0.3 microns of the Z-line; a marked decrease in resting tension accompanied this further depolymerization. These results suggest that connectin filament starts from the Z-line, extends to the M-line, and contributes to resting tension. After partial depolymerization of thick filaments, the distances between the anti-connectin deposits and the Z-line and between anti-connectin deposits and the M-line increased with sarcomere length, suggesting that connectin filaments are elastic along their entire length.

Actin Cytoskeleton↗

PubMed1992

Stabilization of porcine spleen cathepsin A with chaotropic ions and its destabilization with thiols at intralysosomal pH.

1. A correct assay for cathepsin A was developed by adding 0.1 M NaNO3 as an enzyme stabilizer to the assay system. 2. Cathepsin A was purified homogeneously from porcine spleen by DE 52 column, Sephadex G-150 column and Try-Phe-CH-Sepharose column chromatography. 3. The optimum pH of the cathepsin A activity was 4.9, which is near the value of the intralysosomal pH. 4. Chaotropic agents exerted stabilizing effects on the purified cathepsin A activity at pH 5.0, with sodium nitrate being the most effective among the agents tested. 5. Cathepsin A was inactivated rapidly and irreversibly by thiols.

Animals↗

PubMed1992

Myotonic and repetitive discharges in hypokalemic myopathy associated with glycyrrhizin-induced hypochloremia.

Electromyographic studies were performed in two patients with hypokalemic myopathy induced by the administration of glycyrrhizin, 270-273 mg per day for a period of two and eight months, respectively. Myotonic and repetitive discharges were observed when the serum chloride level fell below 90 mEq/l. Following the administration of potassium chloride, when the chloride level rose above 90 mEq/l, these discharges disappeared. These findings support the causal role of hypochloremia in myotonic discharges. A serum chloride level below 90 mEq/l appears to be critical in producing the myotonic and repetitive discharges.

Aged↗

PubMed1992

Chicken leg muscle alpha-connectin as studied by a monoclonal antibody to the 1200 kDa fragment.

Chicken leg gracilis muscle contained only alpha-connectin (ca 3000 kDa) without beta-connectin. When myofibrils were kept standing for 20 hr at 4 degrees C, alpha-connectin was degraded to beta-connectin (ca 2000 kDa) and 1200 kDa peptide. The latter was prepared from myofibrils and purified by gel filtration in the presence of SDS. A monoclonal antibody, alpha 7, to this 1200 kDa fragment was prepared. The antibody reacted with the 1200 kDa fragment and its mother molecule alpha-connectin, but not with beta-connectin. Immunoelectron microscopy using alpha 7, as well as other antibodies to chicken breast muscle beta-connectin, revealed that the 1200 kDa peptide covered the portion of alpha-connectin from the Z line to the N2 line region in the I band of chicken leg gracilis muscle sarcomeres. The results were in good agreement with those observed in rabbit skeletal muscle.

Animals↗