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M Hiraiwa

Publications and source records attributed to M Hiraiwa.

At least 19 recordsLinked to original sources
PubMed1996

Induction of MAPK phosphorylation by prosaposin and prosaptide in PC12 cells.

Prosaposin is a 66 kDa glycoprotein which has neurotrophic activity in vitro and in vivo. The neurotrophic sequence (8CEFLVKEVTKLIDNNKTEKEI29L) within prosaposin has been located to the amino terminal portion of the saposin C domain. This 22-mer peptide, prosaptide, has neurotrophic activity equivalent to prosaposin. We present binding studies using 125I-prosaposin and 125I-prosaptide which revealed a single class of specific binding sites with a Kd of 2.5 nM and 18.3 nM, respectively. Both prosaposin and prosaptide rapidly stimulated protein tyrosine phosphorylation in PC12 cells and increased phosphorylation of MAPK 20-fold especially of p44 MAPK which peaked at 5 minutes of stimulation and then rapidly declined. Treatment of PC12 cells with a mutant 22-mer prosaptide (21Asn to 21Asp) did not induce phosphorylation. These findings suggest a role for MAPK in signal transduction by prosaposin.

Amino Acid Sequence↗
PubMed1996

Patterns of growth inhibitory factor (GIF) and glial fibrillary acidic protein relative level changes differ following left middle cerebral artery occlusion in rats.

Growth inhibitory factor (GIF) has been identified as a new metallothionein-like protein, the level of which is decreased in the Alzheimer's disease brain. GIF and glial fibrillary acidic protein (GFAP) have been reported to be expressed in reactive astrocytes in the rat brain following stab wounds. Moreover, strong expression of GIF mRNA in reactive astrocytes after ventricular injection of kainic acid has been demonstrated. To clarify the biological functions of GIF and GFAP in repair of the CNS, we examined changes in their relative levels to sham control using a Western blotting technique in the rat left hemisphere following occlusion of the left middle cerebral artery, for 28 days after surgery. The GIF relative level declined to 56% of the sham-operated control value on day 7. Thereafter the GIF relative level increased and returned to the normal relative level by days 21-28. The GFAP relative level increased from day 3 and reached a maximum of 120% of the sham-operated control value on days 14-21. While GIF and GFAP were both detected in reactive astrocytes, an increase in the GFAP relative level occurred prior to an increase in GIF relative level following the ischemia. The patterns of changes in relative expression levels of GIF and GFAP were quite similar to those in our previous studies on effects of cerebral stab wounds in rats, although the changes were more rapid in the previous studies. GIF and GFAP appear to play different roles in the repair of the CNS. The present results also indicated that GIF could play an important role in CNS repair after cerebral ischemia and provide new insights into the mechanism of gliosis investigated mainly from the viewpoint of GFAP.

Animals↗
PubMed1995

Changes of growth inhibitory factor after stab wounds in rat brain.

The growth inhibitory factor (GIF) is a new metallothionein (MT)-like protein that is downregulated in Alzheimer's disease (AD) brain. The biological function of GIF has not been fully clarified yet. We have raised an antibody to the synthetic polypeptide that is specific for rat GIF. The purified antibody reacted to recombinant GIF and native rat GIF but not to MT or maltose-binding protein. Using the antibody and GIF cDNA probe, we investigated changes of GIF and GIF mRNA by Western and Northern blotting techniques in rat brains after stab wounds. The levels of GIF and GIF mRNA began to increase 4 days postoperation, reached a maximum at 14-21 days and sustained the increased level at least through 28 days. While both glial fibrillary acidic protein (GFAP) and GIF were recognized in astrocytes, the increases of these 2 proteins after stab wounds showed different patterns. The results indicated that GIF could play an important role in the repair after brain damage and also produce new insights into the mechanism of gliosis investigated mainly from the viewpoint of GFAP.

Alzheimer Disease↗
PubMed1995

Expression of growth inhibitory factor (GIF) in normal and injured rat brains.

Immunohistochemical study on growth inhibitory factor (GIF) in rat brain has revealed that a glial cell layer on the surface of cerebral cortex and the cells surrounding Purkinje cells has been reported. In addition, neurons in gray matter were weakly immunostained for GIF. In situ hybridization using digoxigenin-labeled single-strand RNA probes also demonstrated that most of the neurons and small round cells, which were presumably astrocytes, expressed GIF mRNA in the cerebral cortex of rat brain. These findings indicate that GIF is produced in neurons as well as in astrocytes. The most prominent findings in this study are, a very strong reaction of GIF and GIF mRNA in the reactive astrocytes around the site of injury induced by stab wound or kainic acid injection. These results raised the possibility that GIF may act as an acute-phase protein in reactive astrocytes and have a role in tissue repair.

Animals↗
PubMed1995

Identification of the neurotrophic factor sequence of prosaposin.

Prosaposin, recently identified as a neurotrophic factor (1), is the precursor of saposins A, B, C, and D. The neurotrophic activity of prosaposin resides in the saposin C domain. We have pinpointed the active sequence to a linear 12-mer located in the NH2-terminal sequence of saposin C (LIDNNKTEKEIL). Nanomolar concentrations of a 22-mer peptide encompassing this region stimulated neurite outgrowth and choline acetyltransferase activity, and prevented cell death in neuroblastoma cells. In primary cerebellar granule cells, the 22-mer also stimulated neurite outgroth. Studies of the neuroblastoma line NS20Y using a radiolabeled 18-mer from the neurotrophic region identified a high-affinity (Kd = 70 pM) binding site indicative of receptor-ligand interaction. The 22-mer stimulated protein phosphorylation of several proteins, some of which were tyrosine-phosphorylated after brief exposure similar to saposin C. Circular dichroism studies demonstrated that the 22-mer was converted from a random to a helical structure by addition of ganglioside GM1. The results are consistent with receptor-ligand binding by the peptide initiating a signal transduction cascade and resulting in neuronal differentiation.

Amino Acid Sequence↗
PubMed1994

Identification of prosaposin as a neurotrophic factor.

Prosaposin was identified as a neurotrophic factor stimulating neurite outgrowth in murine neuroblastoma (NS20Y) cells and choline acetyltransferase (ChAT) activity in human neuroblastoma (SK-N-MC) cells. The four naturally occurring saposins, which are derived by proteolytic processing of prosaposin, were tested for activity. Saposin C was found to be active, whereas saposins A, B, and D were inactive as neurotrophic factors. Dose-response curves demonstrated that nanomolar concentrations of prosaposin and saposin C stimulated neurite outgrowth and increased ChAT activity. Prosaposin and saposin C exerted activity by a mechanism independent of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3. Binding assays utilizing saposin C as a ligand gave two saturable binding constants, a high-affinity (Kd = 19 pM) and a low-affinity (Kd = 1 nM) constant, with 2000 and 15,000 sites per NS20Y cell, respectively. Phosphorylation stimulation experiments demonstrated that brief treatment with prosaposin or saposin C enhanced phosphorylation of a variety of proteins, some of which contained phosphorylated tyrosine(s). Since both cell lines were also stimulated by ciliary neurotrophic factor (CNTF) as well as prosaposin, inhibition was tested by utilizing an anti-gp130 monoclonal antibody, which specifically inhibited CNTF stimulation; this antibody did not inhibit prosaposin or saposin C stimulation. These results indicate that prosaposin and saposin C are neurotrophic factors which initiate signal transduction by binding to a high-affinity receptor that induces protein phosphorylation.

Animals↗
PubMed1994

Occurrence of prosaposin as a neuronal surface membrane component.

Prosaposin is a precursor of four saposins that are required for the lysosomal hydrolysis of sphingolipids by specific hydrolases. Besides its precursor role, prosaposin also exists as a secreted protein. The present investigation reveals that prosaposin also exists as an integral component of the surface membranes of neuronal cells. Subcellular fractionation studies demonstrate that the membrane-bound prosaposin occurs specifically in plasma membranes of NS20Y rat neuroblastoma cells. An immunohistochemical study of the neuroblastoma cells using rat prosaposin-specific antibodies also showed that a portion of prosaposin is located on the surface of neurites as well as on cell bodies. Similar histochemical studies with antibodies that specifically recognized human prosaposin revealed the presence of prosaposin in dendrites, axons, and cell bodies of subcortical and spinal cord neurons in both human adult brain and in fetal brain (24-wk gestation). These findings suggest an important role of prosaposin in neuronal development.

Amino Acid Sequence↗
PubMed1994

Hypometabolism and dipole localization in hemimegalencephaly: a case report.

A case of hemimegalencephaly was studied by means of neuroimaging (CT, MRI and PET) and magnetoencephalography (MEG). Hemimegalencephaly (HM) is a neuronal migration disorder. This is the first report of evaluation of HM with the use of PET and MEG from not only the morphological but also the functional point of view. PET with 11C-glucose showed a low radioactive concentration in the affected hemisphere, which suggested a metabolic deficit. MEG proved the epileptic foci existed mainly in the affected hemisphere, especially around a heterotopia and the pachygyric cortex, which was disclosed on MRI.

Brain↗
PubMed1994

The role of the conserved residue in pocket A and the polymorphic residue in pocket E of HLA-B*3501 in presentation of human minor histocompatibility peptides to T cells.

We investigated T cell recognition for human minor histocompatibility (hmH) peptides using HLA-B*3501 restricted, hmH specific cytotoxic T lymphocytes (CTL) clones. These CTL clones killed C1R cells expressing HLA-B*3501 but not C1R cells expressing chimeric antigens between HLA-B*3501 and HLA-B*5101. They also failed to kill C1R cells expressing HLA-B*3501 mutants at residue 152 (B*3501-V152E) or at residue 171 (B*3501-Y171H). The CTL clone failed to kill C1R cells expressing these mutant molecules loaded with the hmH peptides isolated from C1R-B*3501 cells although it killed a self-B cell line expressing HLA-B3501 loaded with the specific hmH peptides. The CTL clone also failed to kill T2 cells expressing the mutant molecules loaded with the specific peptides whereas it killed T2 cells expressing HLA-B*3501 loaded with the specific peptide. On the other hand, naturally occurring specific hmH peptides were isolated from purified B*3501-V152E and B*3501-Y171H molecules, indicating that both HLA-B*3501-V152E and HLA-B*3501-Y171H molecules can bind the hmH peptides. These findings indicate that both the conserved residue 171 in pocket A and the polymorphic residue 152 in pocket E are critical in recognition of the T cells but not binding of the hmH peptides. Furthermore, these results provide the possibility that the TCR recognizes a conformational structure of hmH peptides bound to HLA-B*3501 molecules.

Antigen Presentation↗
PubMed1993

Binding of cerebrosides and sulfatides to saposins A-D.

Saposins are a family of four small glycoproteins, all of which are derived from prosaposin, and are involved in the lysosomal hydrolysis of various sphingolipids. Results from this investigation demonstrate that saposins A-D bind to galactosyl- and glucosylceramide. The binding was highly dependent on the solution pH; maximum binding of glucosylceramide to all saposins occurred at pH 7. Maximum binding of galactosylceramide to saposins B and D occurred at a more basic pH (8.5). The binding of glucosylceramide to saposins was significantly inhibited by Mg2+, Ca2+, or Zn2+. Although maximum binding of sulfatide to saposins A, C, and D occurred at acidic pH, the binding to saposin B was maximum at pH 8.5. Saposin A also bound sphingomyelin or phosphatidylcholine at neutral pH. No significant binding was evident between these lipids and saposins B-D at any pH value. The existence of saposin-lipid complexes was further confirmed in selected samples by gel filtration, isoelectric focusing, and a TLC binding assay. We have also shown that galactosylceramide bound to saposins A-D was efficiently transported to a rat brain microsomal fraction. This result suggests that saposins and possibly their precursor, prosaposin, may be involved in membrane biogenesis such as the assembly of myelin and plasma membranes.

Animals↗
PubMed1993

Effect of saposins on acid sphingomyelinase.

The effect of saposins (A, B, C and D) on acid sphingomyelinase activity was determined using a crude human kidney sphingomyelinase preparation and a purified sphingomyelinase preparation from human placenta. Saposin D stimulated the activity of the crude enzyme by increasing its apparent Km and Vmax. values for sphingomyelin hydrolysis. Unlike the crude enzyme, the activity of the purified enzyme was strongly inhibited by saposin D as well as other saposins. Saposin D decreased the apparent Km and Vmax values of purified sphingomyelinase activity. The effects of saposin D on the activity of different sphingomyelinase preparations appear to depend on Triton X-100, which is present in the crude enzyme but not in the purified enzyme. When the detergent was removed from the crude preparation, the effect of saposin D changed from being stimulatory to inhibitory. Conversely, when the detergent is added to the purified enzyme, the effect of saposin D on sphingomyelinase activity changed from being inhibitory to stimulatory. While other saposins were inhibitory or had no effect on sphingomyelinase activity in the above assay system, not only saposin D but also saposins A and C exhibited a stimulatory effect upon purified sphingomyelinase activity when the substrate, sphingomyelin, was added in the form of liposomes without detergent. Saposin B was not only inhibitory in the liposome system, but also reduced the stimulatory effect of saposins A, C and D. These observations indicate that the stimulatory effect of saposins A, C and D on acid sphingomyelinase activity is greatly influenced by the physical environment of the enzyme and suggest that similar effects by saposins may be exerted in lysosomal membranes.

Detergents↗
PubMed1993

Purification and characterization of an alpha-L-fucosidase from Pomacea canaliculata.

An alpha-L-fucosidase (EC 3.2.1.51) was isolated from the hepatopancreas of Pomacea canaliculata. The enzyme was purified 285-fold from the crude enzyme extract by procedures involving first heat treatment, ammonium sulfate fractionation, second heat treatment, and chromatography on DEAE-Sepharose, hydroxylapatite, and L-fucosylamine-CH-Sepharose. When assayed by using p-nitrophenyl glycosides as substrates, the final preparation was free from other glycosidase activities and gave a single protein band which corresponded to alpha-L-fucosidase activity on disc gel electrophoresis. The molecular weight of the enzyme was estimated to be 260,000 by Sephacryl S-300 column chromatography. The enzyme has two optimum pH values, 2.5 and 5.0, and the apparent Km value and the maximum velocity for p-nitrophenyl alpha-L-fucoside at both pH were calculated to be 0.45 mM and 1.46 mumol/min/mg of protein, respectively. The enzyme was shown to hydrolyze the Fuc alpha 1-->2Gal, the Fuc alpha 1-->4GlcNAc, and the Fuc alpha 1-->6GlcNAc linkages, but hardly acts on the Fuc alpha 1-->3GlcNAc linkage in various oligosaccharides.

Ammonium Sulfate↗
PubMed1993

The effect of carbohydrate removal on stability and activity of saposin B.

Saposin B is involved in the hydrolysis of sulfatides, GM1 ganglioside, globotriaosylceramide, and several other sphingolipids and glycerolipids by lysosomal hydrolases. Saposin B is one of four small glycoproteins (saposins) derived from prosaposin. The carbohydrate chain of saposin B was removed and deglycosylated saposin B was characterized and compared with native saposin B. Deglycosylated saposin B stimulated the enzymatic hydrolysis of ganglioside GM1 by acid beta-galactosidase and sulfatide by arylsulfatase A to the same extent as native saposin B. In addition deglycosylated saposin B bound sulfatide and GM1 ganglioside identical to native saposin B. The stability of native saposin B to proteolytic digestion was unchanged by deglycosylation. Neither native saposin B nor deglycosylated saposin B were hydrolyzed by trypsin, endoproteinase Glu-C (V-8), chymotrypsin, or a mixture of acid proteases isolated from human testis. Unlike its effect on metabolic stability, the carbohydrate chain appears to affect folding of saposin B. When native and deglycosylated saposin B were reduced under denaturing conditions and refolded under identical conditions examination of the refolded products indicated that each protein was refolded in a qualitatively different way. A human mutation in saposin B-deficient metachromatic leukodystrophy, in which its glycosylation site is eliminated, has been reported. Our observations suggest that instability of the mutated saposin B is not due to the absence of a protective effect of the carbohydrate chain on proteolysis, but is likely due to aberrant folding resulting from the absence of a carbohydrate chain.

Carbohydrates↗
PubMed1993

Isolation, characterization, and proteolysis of human prosaposin, the precursor of saposins (sphingolipid activator proteins).

Prosaposin contains separate domains in tandem for four saposins, A, B, C, and D. These mature saposins are produced by limited proteolysis of prosaposin. They are involved in lysosomal hydrolysis of GM1 ganglioside, gluco- and galactocerebrosides, sulfatides, and sphingomyelin and other sphingolipids. Prosaposin also exists as a secretory protein in body fluids. In this investigation prosaposin was expressed in Spodoptera frugiperda cells (Sf9) by infection with baculovirus containing a full length cDNA coding for human prosaposin. Prosaposin was isolated and purified from spent culture medium of the recombinant Sf9 cell cultures as well as from human seminal plasma and milk. From sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of both native human prosaposins is estimated to be 66 kDa and that of recombinant prosaposin as 58 kDa. Deglycosylation of native and recombinant prosaposins yielded a protein with a molecular weight of 54 kDa and isoelectric point of 5.4. The N-terminal sequence of both native and recombinant prosaposins was identical (G-P-V-L-L-G-L-K). Like mature saposins, all prosaposins possessed stimulative activity for cerebroside beta-glucosidase (saposins A and C activity), GM1 ganglioside beta-galactosidase (saposin B activity), and sphingomyelinase (saposin D activity) but not sulfatide sulfatase (saposin B activity). Partially proteolyzed products derived from prosaposins were isolated and identified. From seminal plasma, two proteins of 48 and 29 kDa and from Sf9 culture media, two proteins of 39 and 26 kDa were characterized. N-terminal amino acid sequencing and Western blot analysis of each protein indicated that the 39-and 48-kDa proteins are cleavage products containing domains for saposins B, C, and D (trisaposins), and the 26- and 29-kDa proteins are cleavage products containing domains for saposins C and D (disaposin). These observations suggest that proteolysis of prosaposin in these tissues occurs sequentially from the N-terminal region. Proteins involved in the initial proteolysis of prosaposin were partially characterized in human testis.

Amino Acid Sequence↗
PubMed1993

Effects of cyclodextrins on the hydrolysis of ganglioside GM1 by acid beta-galactosidases.

The hydrolysis of ganglioside GM1 by acid beta-galactosidases was greatly enhanced by the inclusion of heptakis(2,6-di-O-methyl)-beta-cyclodextrin or alpha-cyclodextrin in the assay mixture. The other cyclodextrins tested were not effective. The extent of stimulation by these cyclodextrins was relatively smaller than those by taurodeoxycholate and taurochenodeoxycholate. However, it is suggested that stimulation by bile salts may be partly a reflection of the detergent effects of bile salts on GM1 and partly a reflection of the interaction between bile salts and the enzyme itself. On the other hand, the stimulation by the cyclodextrins seems to correlate to the formation of an inclusion complex between GM1 and cyclodextrin without enzyme protein interaction.

Animals↗
PubMed1993

Activation of human lysosomal sialidase.

An acid sialidase [EC 3.2.1.18], partially purified from human placenta by Con A-Sepharose adsorption and p-aminophenyl thio-beta-D-galactoside-CH-Sepharose (PATG-Sepharose) affinity chromatographies, was activated by incubation at 37 degrees C. This activation showed both time and temperature dependencies, with the most effective activation observed at 37 degrees C in the pH range between 4.3 and 5.2. The influence of various protease inhibitors on its activation was investigated. Among the protease inhibitors tested, amastatin, an inhibitor of aminopeptidase A, significantly inhibited activation. The partially purified enzyme preparation contained aminopeptidase activity, which was inhibited by amastatin. Zinc ions inhibited either the activation of sialidase or the aminopeptidase activity in the enzyme preparation. These results suggest the possibility of participation of aminopeptidase function in the activation process of sialidase.

Aminopeptidases↗
PubMed1993

[Long-term results and indications of intraurethral stents in elderly patients with prostatic hypertrophy complaining of urinary retention].

Thirty-two elderly male patients with benign prostatic hypertrophy complaining of urinary retention were treated by polyurethane intraurethral stents. All of them were unfit for prostatic surgery due to the presence of several complications, and had been indwelt with a urethral balloon catheter. Cystometry before stent insertion was performed in 23 of the 32 patients. Eleven patients showed overactive bladder, 5 patients normal bladder, and 7 patients underactive bladder. Duration of stent indwelling ranged from 2 days to 22 months (mean 6.7 months), and stent could successfully function for more than 6 months in 20 of the 32 patients (62.5%). Nine of these 20 patients (45%) could keep continence of urine, and urinary tract infections improved in 7 of these 20 patients (35%). The stent failed in 4 of the 5 patients who had had previous operations for prostatic hypertrophy. In a comparison of the findings of urodynamic study prior to insertion of stent with the results of the stent, the results were significantly better in the patients with overactive or normal bladder than in those with underactive bladder (p < 0.05, chi-square test). Bladder stone formation was found in one patient and was the only complication among the 32 patients. In conclusion, stent placement without incontinence or urinary tract infection can be successful for more than 6 months in patients who do not have an underactive bladder and who had not had a previous prostatic operation.

Aged↗