Biomedical subjects

F Kametani

Publications and source records attributed to F Kametani.

At least 37 records · Page 2Linked to original sources

PubMed1995

Secretory cleavage of beta-amyloid precursor protein in the cerebral white matter produces amyloidogenic carboxyl-terminal fragments.

To elucidate the metabolic process generating amyloid-beta protein (A beta) from beta-amyloid precursor protein (APP) in human brain, we partially purified secretory forms and carboxyl-terminal fragments (CTFs) of APP from the white matter of a Down's syndrome brain. We obtained secretory forms of APP which lack the entire A beta sequence and CTFs which contain the full-length A beta from the cerebral white matter. Some A beta-lacking secretory APP isoforms in the white matter were derived from APP695. These results suggest that amyloidogenic CTFs can be produced by secretory cleavage of APP which is anterogradely transported through the axon in human brain.

Amyloid beta-Peptides↗

PubMed1994

Secretory cleavage site of Alzheimer amyloid precursor protein is heterogeneous in Down's syndrome brain.

A beta (beta/A4) is the major constituent of brain amyloid in Alzheimer's disease (AD), Down's syndrome (DS) and normal aged persons. This protein is presumably derived by normal proteolysis from a precursor protein (APP). In this study, C-terminal fragments of APP in a Tris/Triton soluble fraction were partially purified from DS brain by heparin-affinity and reverse phase chromatography, and analyzed by N-terminal amino acid sequencing after SDS polyacrylamide gel electrophoresis and Western blotting. We found at least six different C-terminal fragments including those with the entire A beta region. These results suggest that secretory processing of APP is heterogeneous and generates amyloidogenic C-terminal fragments.

Alzheimer Disease↗

PubMed1994

Secretory form of beta-amyloid precursor protein is much abundantly contained in the cerebral white matter in human brain.

To elucidate the metabolic process of beta-amyloid precursor proteins (APP) in cerebral gray and white matter of the human brain, we compared the content and characteristics of secretory APP between these two parts. The white matter contained much more secretory APP than the gray matter in both a control and a Down's syndrome brain, but no difference in the characteristics of the APP isoforms was detected. Our results suggest that, in the human brain, a considerable amount of APP is carried by axonal transport, during which some of the APP isoforms are processed to their secretory forms.

Aged↗

PubMed1994

Accumulation of amyloid beta-protein precursor (APP) in Purkinje cells and increase of amino-terminal fragments of APP in cerebrum and cerebellum of aged rat brain.

In aged rat brain, amyloid beta-protein precursor (APP) is accumulated in dendrites and cell bodies of Purkinje cells as full-length or truncated APP, because dendrites and cell bodies are positively stained by antibodies against both the amino- and carboxy-termini of APP. Western blot analysis of homogenates of brains of aged and young rats showed no apparent differences except for an increase in amino-terminal fragments in cerebrum and cerebellum of aged rat. These results indicate that the expression, transport or metabolism of APP in specific regions of brains may be affected by the aging process.

Aging↗

PubMed1994

Variability of beta-amyloid protein deposited lesions in Down's syndrome brains.

An immunohistochemical study was carried out on the brains of 7 adult Down's syndrome cases (ages 31 to 62) using antibodies to beta-protein, beta-amyloid protein precursor and tau-protein. Variable forms of beta-protein deposited lesions (including senile plaques and cerebrovascular amyloidosis) were observed in extensive areas of the neocortex of all cases and coexistence of both beta-protein amyloid fibrils and beta-amyloid protein precursors was also seen in some of these lesions. Moreover, 3 cases at an advanced stage showed a few plaque-like lesions with beta-protein immunoreactivity in the white matter. The following temporal morphological change is suggested for the pathogenesis of Alzheimer's disease: senile plaque undergo sequential structural changes and beta-protein amyloid deposits in the form of "early plaque" precede the development of tau-immunoreactive neurofibrillary degeneration.

Adult↗

PubMed1993

Secretory form of Alzheimer amyloid precursor protein 695 in human brain lacks beta/A4 amyloid immunoreactivity.

It is not clear how Alzheimer amyloid precursor proteins (APP) are metabolized in the brain itself. Secretory forms of APP in a phosphate buffer-soluble fraction were purified from post-mortem human brain by heparin-affinity and ion-exchange chromatography and analyzed by N-terminal amino acid sequencing and SDS polyacrylamide gel electrophoresis/immunoblotting. We found apparently similar multi-isoforms of secretory APP (at 93-97, 105-112 and 123 KDa) to those that we have described recently in cerebrospinal fluid. Antisera to the initial part of the beta/A4 sequence labelled only those bands that were found to react with antiserum to the Kunitz-type inhibitor insert of APP, suggesting that beta/A4 amyloid may be generated specifically from APP-695.

Adult↗

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

Characterization of a transthyretin-related amyloid fibril protein from cerebral amyloid angiopathy in type I familial amyloid polyneuropathy.

Recently, it has been reported that transthyretin (TTR)-immunoreactive amyloid deposition with cerebral amyloid angiopathy in central nervous system is a common pathological finding in type I familial amyloid polyneuropathy (FAP). In the present study, we performed isolation and sequence analysis of TTR-related amyloid fibril protein from the meninges of a patient with type I FAP. Purified major amyloid fibril protein had a molecular weight of 15 kDa. Complete sequence analysis revealed that this amyloid fibril protein was a variant TTR with a single amino acid substitution of methionine for valine at position 30. This variant TTR is a previously unrecognized as cerebrovascular amyloid fibril protein. Furthermore, the patients with type I FAP are well known to have the variant TTR in the serum. These suggest that cerebrovascular amyloid fibril protein in type I FAP may derive from a serum precursor.

Amino Acid Sequence↗

PubMed1992

Generation of active oxygen species in blood platelets--spin trapping analysis.

Generation of active oxygen species by bovine blood platelets was examined by the electron spin resonance (ESR) spin trapping technique with 5,5-dimethyl-1-pyroline-1-oxide (DMPO). The hydroxyl spin-trapped adduct 5,5-dimethyl-2-hydroxyl-1-pyrolidinyloxy (DMPO-OH) was formed in the presence of platelets, indicating the generation of hydroxyl radicals (.OH) by the platelets. Generation of .OH was observed even with platelets in the resting state, but was markedly enhanced when the platelets were activated with stimulants. Stronger stimulants such as the calcium ionophore ionomycin, induced greater radical generation than the weaker stimulant ADP. When the platelets were stimulated by thrombin, generation of .OH was greatest after 1.5 min, and depended on the dose of the stimulant. It was inhibited by inhibitors of platelet activation such as forskolin and phenolic antioxidants.

Adenosine Diphosphate↗

PubMed1991

"Secretase," Alzheimer amyloid protein precursor secreting enzyme is not sequence-specific.

The major pathological change in Alzheimer's disease is the deposition of amyloid beta/A4-protein (beta P) in the brain. beta P is derived from a small part of the much larger amyloid protein precursor (APP). In the normal condition, APP is cleaved in the interior of beta P, preventing the formation of beta P, by a hypothetical proteinase "secretase". To characterize this enzyme, APP and mutated APPs were expressed by cDNA transfection in COS-1 cells, a monkey kidney fibroblast derived cell line. The mutant APPs with the mutations of the proposed cleavage site (Gln686-Lys687) were processed in the same way as wild APP. The deleted mutant APP (deletion of Arg676-Asp694) was also cleaved in a similar way to wild APP. The cleavage site of this deletion mutant was located at the 12 amino acid residues from the predicted membrane spanning domain. Hence, "secretase" cleaves APP, depending not on its specific amino acid sequence, but probably on the relative conformation with plasma membrane.

Alzheimer Disease↗

PubMed1991

Alzheimer amyloid beta/A4 peptide binding sites and a possible 'APP-secretase' activity associated with rat brain cortical membranes.

We carried out ligand binding experiments on membranes from rat brain cortical grey matter using radioiodinated beta/A4 8-17, with non-specific binding determined by the addition of 10 microM unlabelled peptide. Specific, reversible binding amounted to 60-75% of total binding and showed a clear dependence on time, temperature, pH and membrane concentration. Kinetic analyses indicated a high-affinity binding site with an apparent KD of 440 pM. However, the ligand was partly degraded with loss of the Ser8, Lys16 and Leu17 residues. Excision of the two C-terminal amino acids was inhibited by EDTA, EGTA, dithiothreitol or Zn2+ but was stimulated by Ca2+ or Mn2+. These studies demonstrate high-affinity binding sites for beta/A4 8-17 (or its derivatives) in rat brain, suggesting that this region may contain a physiologically important amino acid sequence and identify a potential membrane-associated amyloid precursor protein (APP) secretase activity.

Alzheimer Disease↗

PubMed1991

Asymmetry of membrane fluidity in the lipid bilayer of blood platelets: fluorescence study with diphenylhexatriene and analogs.

Membrane fluidity of bovine platelets was examined with diphenylhexatriene (DPH), its cationic trimethylammonium derivative (TMA-DPH) and anionic propionic acid derivative (DPH-PA). After addition of these probes to platelet suspensions at 37 degrees C, the fluorescence intensity of DPH-PA reached equilibrium within 2 min, whereas those of DPH and TMA-DPH increased gradually. With increase in the fluorescence intensity of TMA-DPH, its fluorescence anisotropy decreased significantly, but the fluorescence anisotropies of DPH-PA and DPH did not change during incubation. The gradual increase of fluorescence intensity of TMA-DPH was due to its penetration into the cytoplasmic side of the platelet membrane, as shown quantitatively by monitoring decrease in its extractability with albumin. Transbilayer movement of TMA-DPH was markedly temperature-dependent, and was scarcely observed at 15 degrees C. The fluorescence intensity of TMA-DPH was much higher in platelet membranes and vesicles of extracted membrane lipids than the initial intensity in intact platelets. Moreover, the fluorescence anisotropy of TMA-DPH was much lower in the former preparations than the initial value in intact platelets. These results suggest that binding sites for TMA-DPH in the cytoplasmic side of the platelet membrane are more fluid than those in the outer leaflet of the plasma membrane. Platelet activation by ionomycin induced specific change in the fluorescence properties of TMA-DPH without causing transbilayer incorporation of the probe.

Animals↗

PubMed1991

Inhibitory effects of catechol derivatives on arachidonic acid-induced aggregation of rabbit platelets.

Pyrocatechol and other non-substituted dihydric phenols, which have strong redox power, inhibited arachidonic acid-induced aggregation of rabbit platelets at much lower concentrations than those at which these phenols inhibited stable prostaglandin endoperoxide, U46619-induced aggregation. Among non-substituted dihydric phenols, pyrocatechol was most potent. In order to clarify the physicochemical properties of the phenolic compounds which control the inhibitory potencies of dihydric phenols, we observed the inhibitory effects of 3- and 4-substituents of pyrocatechol on arachidonic acid-induced platelet aggregation. Among seven derivatives tested, the inhibitory effect of 4-C6H5-substituent was strongest and 4-COOH-substituent was weakest. Inhibitory effects of the catechol derivatives were well correlated with the quotients of their hydrophobicities and oxidation-reduction potentials. Inhibitory effects of hydroquinone and resorcinol were also on the same correlation line. These results suggest that the inhibitory effects of catechol derivatives and other dihydric phenols are controlled by two physicochemical properties: oxidation-reduction potential and hydrophobicity.

Animals↗

PubMed1990

A monoclonal antibody Hy20-54-16-3L to lambda (lambda) light chain of human immunoglobulin reacts with amyloid in Alzheimer's disease brain.

We have previously described a monoclonal antibody Hy20-54-16-3L raised against crude primary (AL) amyloid fibrils that preferentially labelled amyloid deposits in the brains of patients with Alzheimer's disease (AD) (Ishii et al., Neuropathol. Appl. Neurobiol., 12 (1986) 441-445). In the present study, we analyzed the protein from an AL-amyloid laden spleen by gel filtration and SDS-PAGE; a partial amino acid sequence of the Hy20-54-16-3L-immunoreactive band excised from Western blots was determined and found to be a lambda light chain of human immunoglobulins. Additionally we have confirmed that this monoclonal antibody reacts with amyloid in AD brain by using a peroxidase-linked immunoelectron microscope technique. It is concluded that an epitope shared with a lambda light chain is included in or associated with amyloid deposits in the AD brain.

Alzheimer Disease↗

PubMed1990

ESR analysis with long-chain alkyl spin labels in bovine blood platelets. Relationship between the increase in membrane fluidity by alcohols and phenolic compounds and their inhibitory effects on aggregation.

Four spin-labeled probes (5-doxylstearic acid (5-NS), its methyl ester (5-NMS), 16-doxylmethylstearate (16-NMS) and 4-(N,N-dimethyl-N-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-ox yl (CAT-15)) were used to monitor membrane fluidity change in bovine platelets induced by three alkyl alcohols, benzyl alcohol and two phenolic compounds. The relationship between the increase in membrane fluidity induced by these compounds and their inhibitory effects on platelet aggregation was observed. Experiments with the four probes showed that n-hexyl alcohol induced decreases in the order parameter of 5-NS and apparent rotational correlation times of the other probes at the same minimal alcohol concentration. The decreases were observed in the concentration range that inhibited aggregation. n-Amyl alcohol and n-butyl alcohol decreased the values of the parameters of the above mentioned only at higher concentrations that were dependent on their hydrophobicities. Like alkyl alcohols, benzyl alcohol and phenolic compounds decreased the values of the parameters in the concentration ranges in which these compounds inhibited platelet aggregation. The concentration of these compounds causing 50% inhibition of platelet aggregation, the IC50 values, and data on 5-NS-labeled platelets indicated that they inhibited aggregation and decreased the value of the order parameter at lower concentrations relative to their Poct values in comparison to the effective concentrations of alcohols. Phenolic compounds also decreased the values of the apparent rotational correlation times of 5-NMS and 16-NMS. These results indicate that the inhibition of platelet aggregation by alcohols and phenolic compounds is due to membrane perturbation in wide range in depths within the lipid bilayer.

Alcohols↗

PubMed1990

Inhibitory mechanism of cis-polyunsaturated fatty acids on platelet aggregation: the relation with their effects on Ca2+ mobilization, cyclic AMP levels and membrane fluidity.

The in vitro inhibitory effects of cis-polyunsaturated fatty acids, linolenic (18:2 delta 9,12), alpha-linoleic (18:3 delta 9,12,15) and eicosatrienoic (20:3 delta 11,14,17) acid, on bovine platelet aggregation and their inhibitory mechanism were investigated. These fatty acids inhibited platelet aggregation induced by ADP and thrombin to similar extent. Fluorescence analyses with fura-2-loaded platelets showed that, in the concentration ranges that inhibited aggregation, they also inhibited agonist-induced increase in cytoplasmic Ca2+. According to radioimmunoassay study, addition of these fatty acids increased cyclic AMP contents in the presence of theophylline corresponded with their inhibitory effects on aggregation. These fatty acids induced a 1.6-1.8-fold increase over basal concentration of cyclic AMP in the concentration ranges that fully inhibited aggregation. On the other hand, saturated fatty acid, stearic acid, affected neither aggregation nor cyclic AMP levels. As reported previously [1985) Biochim. Biophys. Acta 818, 391), these unsaturated fatty acids induced increase in membrane fluidity in the same concentration range. These results suggest that inhibition of platelet aggregation by cis-polyunsaturated fatty acids is due to the increase in cyclic AMP levels. This increase seems to be due to stimulation of adenylate cyclase which is mediated by membrane perturbation.

8,11,14-Eicosatrienoic Acid↗

PubMed1990

Amyloid beta-protein precursor (APP) of cultured cells: secretory and non-secretory forms of APP.

Overproduction or aberrant catabolism of the predicted amyloid beta-protein precursor (APP) is suspected as the cause of amyloid deposition in Alzheimer's disease and Down's syndrome brains. For possible in vitro experiments of amyloid formation, we have examined the expression of APP in various cultured cells. We found two types of APP producing cell lines. PC12h (rat pheochromocytoma) and HL-60 (human acute promyelocytic leukemia) cells produce a secretory form that is released into the culture medium, while Bu-17 (human glioma) cells synthesize only a non-secretory form that accumulates at the cell surface. APP immunoreactivity on the latter cells was detected at the tips of cell processes or growth cones. These observations indicate that the nonsecretory form of APP may play a role in cell contact or adhesion.

Alzheimer Disease↗

PubMed1989

Effect of intracellular Na+ on platelet aggregation and Ca2+ mobilization.

The effects of ionophores, which can carry alkali metal cations, on platelet aggregation were examined. At an alkaline extracellular pH, alkali metal cation/H+ exchanger nigericin accelerated aggregation in K+-enriched medium, whereas it rather inhibited aggregation in Na+-enriched medium, even though the intracellular pH was only slightly alkaline. The inhibitory effect of Na+ on platelet aggregation was more clearly shown with the alkali metal cation exchanger gramicidin D. The ionophore had no effect or a slightly accelerative effect on aggregation in K+-enriched medium, whereas it significantly inhibited aggregation induced by thrombin, ADP and platelet activating factor in Na+-enriched medium. Fluorescence studies on fura-2-labeled platelets revealed that in Na+-enriched medium gramicidin D inhibited agonist-induced Ca2+ mobilization both in the presence and absence of extracellular Ca2+. These results suggest that the intracellular Na+ inhibits platelet aggregation by inhibiting Ca2+ mobilization.

Animals↗