Involvement of stromal membrane-associated protein (SMAP-1) in erythropoietic microenvironment.

Erythropoiesis is regulated by the hematopoietic microenvironment of the spleen and fetal liver in mice. We showed that established stromal cells of these organs selectively support erythropoiesis in vitro. To identify the cell surface molecule(s) on the stromal cells involved in erythropoiesis, we raised monoclonal antibodies against the stromal cells. Using one of these antibodies (11D), we cloned a new gene named smap-1 (stroma membrane-associated protein-1). The SMAP-1 protein deduced from the nucleotide sequence of the cDNA was a newly identified membrane protein with direct repeats of the KKD/E units found in MAP1A and MAP1B, which is involved in the association with microtubules. By transfection of the anti-sense smap-1 cDNA into the stromal cells, we showed that SMAP-1 may have a stimulatory effect on stroma-supported erythropoiesis. Its expression was detected in the yolk sac, fetal liver, spleen, and bone marrow, and was correlated with their erythropoietic activity.

Down-regulation of GTP cyclohydrolase I and tetrahydrobiopterin by melatonin.

Tetrahydrobiopterin (BH4) is spontaneously released and extracellularly exerts a toxic effect preferentially on catecholamine cells. Its synthesis rate is mainly determined by the activity of the enzyme GTP cyclohydrolase I (GTPCH). In the present study, role of melatonin BH4 synthesis was determined using the catecholaminergic CATH.a cells. The neurohormone dose-dependently reduced both intracellular and extracellular BH4 levels. This was due to both direct inhibition of catalytic activity of the existing GTPCH enzyme and down-regulation of GTPCH gene expression. Thus, melatonin is an effective down-regulator of BH4 synthesis and is a potential therapeutic agent with which to control BH4 level in aberrant conditions where it may rise to a toxic level.

Okadaic acid induces cycloheximide and caspase sensitive apoptosis in immature neurons.

Previous studies have shown that okadaic acid (OA) evokes tau phosphorylation and neurofibrillary changes in vivo, and in cultured neurons, that resemble Alzheimer's disease pathogenesis. In order to investigate the mechanism of OA-neurotoxicity, we treated cultured rat neurons with OA and examined nuclear morphology, phosphatidylserine (PS) externalization, alpha-fodrin cleavage, and the effects of cell death inhibitors. Our results demonstrated that cycloheximide (CHX) and the broad-spectrum caspase inhibitor, ZVAD, significantly reduced cell death in a dose-dependent manner. Nuclear fragmentation, a hallmark of apoptosis, occurred after OA treatment and was inhibited by CHX or ZVAD. PS externalization was apparent in 6-12 h in neurites and in cell bodies, and peaked at 24 h after OA treatment. Cleavage of alpha-fodrin as visualized by the appearance of 150- and 120-kDa bands appeared with a time course similar to PS externalization. These results suggest that OA induce CHX and caspase sensitive neuronal apoptosis.

Okadaic acid-induced upregulation of nitrotyrosine and heme oxygenase-1 in rat cortical neuron cultures.

Hyperphosphorylation of tau is a characteristic feature of the neurodegenerative pathology in Alzheimer's disease (AD). Okadaic acid (OA) is currently used in models of AD research to increase the phosphorylation of tau. Using immunocytochemistry and fluorescent study, we found that markers of oxidative activity such as nitrotyrosine, c-jun, 2',7'-dichlorofluorescein diacetate (DCF), and heme oxygenase-1 (HO-1) were altered in OA-treated culture. Immunoreactivity of nitrotyrosine and c-jun, and DCF-oxidation were increased in degenerating neurons, while HO-1 expression was increased in astrocyte in response to OA. The data suggest that tau phosphorylation and oxidative damage be implicated in OA-induced neurodegeneration.

Upregulation of c-Kit receptor and stem cell factor in cerebellar inhibitory synapses in response to kainic acid.

Neuronal stimulation was induced in rats by systemic administration of kainic acid (KA) to determine if such stimulation is responsible for changes in the expression patterns of c-Kit and stem cell factor (SCF) in cerebellar synapses between inhibitory interneurons and Purkinje cells. Using immunocytochemistry and immunoblotting analyses, we demonstrate that c-Kit receptor tyrosine kinase and its ligand SCF are present on the pre- and postsynaptic sides of inhibitory synapses on Purkinje cells. These proteins are upregulated during the first 48 hr after KA treatment, whereas their levels fall below that of the control by 1 week and remain as such thereafter. Expression of both c-Kit and SCF are significantly elevated in the Purkinje cell layer 24 hr after KA administration, and the Purkinje cell layer exhibits a loss of calbindin D-28K immunoreactivity. Expression of c-Kit in basket cell axon terminals is activated until 48 hr after KA treatment, suggesting the transient participation of c-Kit receptor tyrosine kinase in the maintenance of these axonal terminals. Also during the first 48 hr after KA treatment, SCF levels increase in axonal processes of Purkinje cells, and these SCF-positive axons correlate with c-Kit-positive pinceau structures. The increased expression of c-Kit and SCF in response to KA-induced neuronal stimulation may indicate that c-Kit receptor tyrosine kinase and its ligand SCF function in the inhibitory synapse between cerebellar interneurons and Purkinje cells, and that this role is most pronounced during the first 48 hr after KA treatment.