Jin-ichi Ito

Biochemical evidence for localization of AMPA-type glutamate receptor subunits in the dendritic raft

A low density Triton-insoluble fraction with characteristic lipid composition was prepared from synaptic plasma membrane from the rat forebrain. The fraction was named dendritic raft based on its absence of the presynaptic marker synaptophysin, the presence of postsynaptic Glutamate receptor (GluR) subunits, and its resemblance to raft, caveolae-like structure. We found a differential distribution of NMDA-type and AMPA-type GluR subunits in the dendritic raft and postsynaptic density (PSD) fractions; the latter type GluR subunits were localized to the dendritic raft as well as PSD fraction, whereas the former type was mostly localized to the PSD fraction. We also found the differential distribution of the components of ras/mitogen-activated protein kinase (MAPK) pathway to the dendritic raft and PSD fractions. Dendritic raft and PSD may possibly interact at the postsynaptic sites for efficient signal processing that is required for expression of synaptic plasticity.

Differential generation of high-density lipoprotein by endogenous and exogenous apolipoproteins in cultured fetal rat astrocytes.

Abstract: Most peripheral cells generate cholesterol‐rich high‐density lipoprotein (HDL) with exogenous apolipoprotein as one of the mechanisms for the maintenance of cellular cholesterol homeostasis. Astrocytes isolated from fetal rat brain showed a unique behavior in this reaction. Consistent with previous findings, the astrocytes synthesized apolipoprotein (apo) E and generated cholesterol‐rich pre‐β‐HDL‐like lipoprotein with this apoE, and cellular cholesterol and phospholipids. When exogenous apoA‐I and E were added to the medium, they caused generation of additional HDL with cellular phospholipid. It is interesting that this additional part was very poor in cholesterol except for the generation of relatively cholesterol‐rich HDL only in the initial few hours of the incubation. The mobilization of intracellular cholesterol for this reaction was also very limited, reflecting the poor cholesterol incorporation into the HDL. Thus, the results demonstrated a unique profile of HDL generation and cholesterol efflux by apolipoproteins in rat astrocytes, with endogenous apoE producing cholesterol‐rich HDL and exogenous apolipoproteins producing cholesterol‐poor HDL. These lipoproteins may play differential roles in cholesterol transport in the CNS.

Either high-mannose-type or hybrid-type oligosaccharide is linked to the same asparagine residue in ovalbumin

After pepsin digestion, all of the carbohydrates in ovalbumin were recovered in two glycopeptides, Glu-Glu-Lys-Tyr-Asn(CHO)-Leu-Thr-Ser-Val and Glu-Gln-Lys-Tyr-Asn(CHO)-Leu-Thr-Ser-Val. Almond glycopeptidase released quantitatively oligosaccharides from the glycopeptides. The products from both glycopeptides contained both the high-mannose-type oligosaccharides and the hybrid-type oligosaccharides in the same ratio. Thus, either the high-mannose-type or the hybrid-type oligosaccharide is attached to the unique asparagine residue in the ovalbumin molecule.

Sialosyl cholesterol induces morphological and biochemical differentiations of glioblasts without intracellular cyclic AMP level rise

Synthesized sialosyl cholesterols, alpha- and beta-D-N-acetylneuraminyl cholesterols (alpha-SC and beta-SC), induced the morphological conversion of normal rat glioblasts from a flat epithelioid morphology to an astrocytic process-bearing (stellate) morphology resembling the conversion by glia maturation factor (GMF). The stellogenic effects were rapid and detectable within 1 h after drug stimulation, and irrelevant to the intracellular cyclic adenosine monophosphate (cAMP) levels. The morphological alteration was ascertained by the fluorescence-visualization of cytoskeletons: alpha-SC elicited the reorganization of GFA protein network to the formation of bundles, the destruction of stress fiber, and the redistribution as plasmalemmal constituents. alpha-SC also evoked biological differentiations represented by an elevation of glial marker proteins, S-100 protein and GFA protein. The results provide a possibility that SC incorporated into plasma membrane may cause morphologically and biochemically astrocyte-like differentiations of glioblast through the alteration of membrane characteristics, the cytoskeletal anchorages to the membrane, the affinity of receptors, and/or the postreceptor responses distinct from cAMP-production system.

An acidic fibroblast growth factor-like factor secreted into the brain cell culture medium upregulates apoE synthesis, HDL secretion and cholesterol metabolism in rat astrocytes

Production and release of apolipoprotein (apo) E and cholesterol were highly upregulated in the astrocytes prepared by 1-week secondary culture after 1-month primary culture of rat fetal brain cells (M/W cells) in comparison to the cells prepared by a conventional method of 1-week primary and 1-week secondary culture (W/W cells). Both cell preparations were mostly composed of astrocytes with small population of other glial cells, except that type-2 astrocyte-like cells accounted for 5-15% of M/W cells indicating more activated and/or matured status. The conditioned medium of the 1-month primary culture stimulated W/W cells to increase the release of apoE and cholesterol into the medium. The treatment of W/W cells by acidic fibroblast growth factor (aFGF) similarly upregulated biosyntheses and release of apoE and cholesterol. The effect of the conditioned medium was completely inhibited by pretreatment with an anti-aFGF antibody. The increase of the aFGF message was demonstrated in the brain cells after 1-month primary culture. The findings suggested that an aFGF-like trophic factor upregulates biosynthesis and secretion of apoE-high density lipoprotein (HDL) in astrocytes probably by autocrine stimulation in this culture system. Since this cytokine is highly expressed in the development or post-injury period of the brain, it putatively activates intercellular cholesterol transport to support construction or recovery of the brain.

Prostate growth factor in the extracts of benign prostatic hypertrophy. Partial purification and physicochemical characterization.

The biochemical and physicochemical properties of prostate growth factor (PGF) in the extracts of benign prostatic hypertrophy (BPH) were investigated. The PGF activity stimulating the proliferations of fibroblasts (mouse 3T3 and human BUD-8 cells) was detected predominantly in BPH prostate, and also in normal human prostates and well-differentiated adenocarcinoma prostates. No significant correlation between PGF contents and BPH tissue weight or histological differences (fibromuscular or glandular type) was detected. Gel filtration and isoelectric focusing indicated that partially purified factor(s) by ion exchange column chromatography had a multimolecular form comprising three active components (80,000, 43,000 and 10,000 daltons) and acidic isoelectric points (pH 4.0, 4.3, and 6.0). The activity was susceptible to heat treatment at 80 degrees C for 10 min, and to trypsin, but the factor was devoid of esteropeptidase activity. Subcellular fractionation located the entire activity in cytosol fraction.

Fibroblast growth factor 1 is produced prior to apolipoprotein E in the astrocytes after cryo-injury of mouse brain.

We recently reported that fibroblast growth factor 1 (FGF-1) upregulates apolipoprotein E (apoE) synthesis and its secretion as high density lipoprotein (HDL) in cultured astrocytes potentially by an autocrine or paracrine mechanism [Biochim. Biopys. Acta 1589 (2002) 261]. In order to examine pathophysiological relevance of this reaction, we studied association of the production of FGF-1 and apoE in the post-injury mouse brain. After the spot-injury of the brain by liquid nitrogen, the surface size of the wound shrunk more rapidly in the C57BL/6 wild-type mice than the apoE-knock out C57BL/6 mice. Immunohistochemical analysis of the lesions revealed that production of FGF-1 was identified in the reactive astrocytes by the day 2 after the injury in both types of mouse, prior to the production of apoE confirmed by the day 4 in the wild-type. These findings were consistent with our in-vitro observations and hypothesis that FGF-1 upregulates apoE synthesis and subsequently HDL production in the reactive astrocytes by an autocrine or paracrine manner. FGF-1 thus would exert its effect after the CNS damage through apoE secretion.

Apolipoprotein A-I increases association of cytosolic cholesterol and caveolin-1 with microtubule cytoskeletons in rat astrocytes

Apolipoprotein (apo) A‐I induces rapid translocation of protein kinase Cα and phospholipase Cγ, and slow translocation of caveolin‐1 and newly synthesized cholesterol to the cytosolic lipid‐protein particle (CLPP) fraction in rat astrocytes. In order to understand the function of CLPP, we investigated the interaction with cytoskeletons of CLPP‐related proteins such as caveolin‐1 and protein kinase Cα and of CLPP‐related lipids in rat astrocytes. Under the conditions that microtubules were depolymerized, association of cytosolic caveolin‐1 with protein kinase Cα and α‐tubulin was enhanced when the cells were treated with apoA‐I for 5 min. This association was suppressed by a scaffolding domain‐peptide of caveolin‐1. Association with the microtubule‐like filaments of cytosolic lipids, caveolin‐1 and protein kinase Cα was also increased by the apoA‐I treatment and inhibited by the scaffolding domain peptide. Paclitaxel (taxol), a compound to stabilize microtubules, suppressed the apoA‐I‐mediated intracellular translocation and release from the cells of the de novo synthesized cholesterol and phospholipid. The findings suggested that the association of CLPP with microtubules is mediated by a scaffolding domain of caveolin‐1, induced by apoA‐I and involved in regulation of intracellular cholesterol trafficking for assembly of cellular lipids to apoA‐I–high‐density lipoprotein (HDL).

mRNA expression of complement components and regulators in rat arterial smooth muscle cells

The presence of C5b‐9 complexes, some complement regulators, and abundant cytokines in atherosclerotic lesions has been reported. However, it is unclear whether these complement‐associated proteins are produced by vascular smooth muscle cells (SMCs) and how they are influenced by the cytokines. In the present study, we demonstrated, by the reverse transcription‐polymerase chain reaction method, the mRNA expression of complement components (C3, C4, and C5) and membrane regulators (decay‐accelerating factor, membrane cofactor protein, Crry, and CD59) in cultured SMCs derived from the rat carotid artery. The expression of C9 mRNA was also induced upon stimulation by interferon‐gamma (IFN‐γ), tumor necrosis factor‐alpha (TNF‐α) and/or lipopolysaccharide (LPS). Northern blot analysis showed that the mRNA expression of C3, C4, DAF and Crry was up‐regulated, but that of CD59 was down‐regulated by IFN‐7, TNF‐α and/or LPS alone or by synergy. The increase of C3 mRNA by TNF‐α or LPS and that of C4 mRNA by IFN‐γ was induced in a dose‐dependent manner. The results indicate that the arterial SMCs of rat have the ability to produce complement components and regulators, which is affected by cytokines and/or LPS. Since atherosclerosis is characterized by the intimal proliferation of SMCs, the complement system including its regulators may be involved in the pathogenesis of the disease.

Autocrine regulation of glial proliferation and differentiation: The induction of cytodifferentiation of postmitotic normal glioblast by growth-promoting factor from astrocytoma cell

A growth-promoting factor (GPF) from astrocytoma cells (GA-1) cultured in serum-free medium (N2) exerted on normal glioblasts proliferative and differentiation-promoting effects, which have been observed in glia maturation factor (GMF) stimulation. The serum-free conditioned media of GA-1 provoked DNA synthesis of glioblasts, and subsequently elicited a morphological differentiation characterized by the extrusion of processes as well as biochemical changes including an increased cellular level of glia fibrillary acidic protein (GFA protein), S-100 protein, and alpha-enolase. The transforming growth factor activity was also found in the media. Partially purified GPF had a molecular weight range of 7100-10,000 Mr and acidic isoelectric point (pH 4.6), and showed a susceptibility to heat treatment and denaturation at low and high pHs. The present results and the findings accumulated from our previous studies on gliotrophic growth factors provide a general concept of the growth and differentiation regulations of normal or neoplastic glial cells by growth factors through autocrine systems.