Kuniko Okumura-Noji

Rapid translocation of cytosolic Ca2+/calmodulin-dependent protein kinase II into postsynaptic density after decapitation.

Abstract: The postsynaptic density (PSD) fraction prepared from rat forebrains frozen with liquid nitrogen immediately after dissection (within 30 s after decapitation) contained major postsynaptic density protein (mPSDp), α subunit of Ca2+/calmodulin‐dependent protein kinase II (CaMKII) at a level of merely 2.7% of the total protein. The content of the protein in the fraction was increased to ∼10% by placing the forebrains on ice for a few minutes. Accumulation, but to a lesser extent, of the protein after placement was also observed in the particulate, synaptosome, and synaptic plasma membrane fractions with its concomitant decrease in the cytosolic fraction. The distribution change may be translocation of the protein, because the amounts of the losses of the protein in the cytosolic fraction were balanced by the gains in the particulate fractions. By translocation, CaMKII became Triton X‐100 insoluble and partially inactivated. The amount of CaMKII transferred from the cytosol to particulate fractions at 0°C was about the same as that contained in the conventional PSD fraction. Furthermore, the thickness of the PSD was increased by the treatment of the forebrains at 37°C, by which the content of CaMKIIα in the PSD fraction was increased to twofold. These results suggest that most of the CaMKII α subunit associated with the PSD fraction (mPSDp) is translocated from cytosol after decapitation. We also showed similar translocation of CaMKIIβ/β′.

Cholesteryl ester transfer protein and atherosclerosis in Japanese subjects: a study based on coronary angiography

We undertook a cross-sectional analysis on CETP and atherosclerosis among Japanese subjects, by means of CETP mass assay, its gene polymorphism and coronary angiogram. The 110 consecutive patients who underwent coronary angiography were enrolled into the study except for those over 70 years and taking lipid-lowering drugs. Association was analyzed among plasma lipid and lipoproteins, CETP mass, its gene polymorphisms and the finding in coronary angiography. Four CETP-deficiency heterozygotes were identified and excluded from the analysis. CETP mass level showed neither significant correlation with the coronary score (CS) (r=0.06, P=0.52) nor the difference between the groups eventually diagnosed as coronary heart disease (CHD) positive and CHD negative (2.36+/-0.57 vs. 2.24+/-0.21, P=0.24). CETP mass correlated with the total and LDL cholesterol (r=0.43, P<0.001; r=0.36, P<0.001, respectively) but not with HDL cholesterol (r=0.08, P=0.40). While I405V polymorphism had no impact on CETP mass, HDL cholesterol or CS, CETP mass was low with TaqIB polymorphism (B1B1>B2B2, P<0.05) only in the low CS group (<4). Among the lipid and lipoprotein, HDL cholesterol had a greater impact than LDL cholesterol on coronary atherosclerosis. We concluded that CETP mass in plasma does not correlate with coronary atherosclerosis as whole in the non-CETP-deficient. However, the B2B2 genotype in CETP TaqIB polymorphism, only when it decreases the CETP level, may act as a protective factor against atherosclerosis. It should also be noted that CETP mass in general correlates to total and LDL cholesterol, so that it would be an indirect atherogenic parameter.

Presence of NF-κB-like and IκB-like immunoreactivities in postsynaptic densities

THE distributions of IκB and NF-κB immunoreactivities were examined immunohistochemically in the rat brain by the electron microscopy. Antibodies were raised against synthetic peptides with the sequences specific to the human MAD-3 type IκB or NF-κB. Both IκBa and NF-κB immunoreactivities were localized in the dendrites including the spines and, particularly, the postsynaptic densities (PSDs) of the hippocampus and the cerebral cortex. The PSD fraction prepared from the rat brain contained an activity that inhibited the binding of NF-κB to the κB DNA elements. These results suggest that the NF-κB/IκB system or a similar mechanism may play a role in signal transmission from synapses to the nucleus.

ERK2-TYPE MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) AND ITS SUBSTRATES IN POSTSYNAPTIC DENSITY FRACTIONS FROM THE RAT BRAIN

Mitogen-activated protein kinase (MAPK) and MAPK kinase (MAPKK) were detected by Western blotting in the synaptic fraction prepared from the rat brain. There were two bands immunoreactive to the anti-MAPK antiserum in the soluble, P2, synaptosome, and synaptic plasma membrane fractions. These immunoreactive bands possibly corresponded to extracellular signal-regulated kinase (ERK) 1 and 2 (Boulton et al., 1991b), respectively. Only ERK2 was detected in the postsynaptic density (PSD) fraction. We then surveyed MAPK substrates in the synaptic fractions using purified Xenopus MAPK (ERK2-type MAPK), and found a number of MAPK substrates unique to the PSD fraction. Thus, ERK2 is present in the synapse, especially at the postsynaptic site, and it may play a role(s) in synaptic function via the phosphorylation of synapse-specific substrates. Developmental changes in ERK2 also supported its role in the synapse.

Apolipoprotein-mediated cellular cholesterol/phospholipid efflux and plasma high density lipoprotein level in mice

Helical apolipoprotein(apo)s generate pre-beta-high density lipoprotein (HDL) by removing cellular cholesterol and phospholipid upon the interaction with cells. To investigate its physiological relevance, we studied the effect of an in vitro inhibitor of this reaction, probucol, in mice on the cell-apo interaction and plasma HDL levels. Plasma HDL severely dropped in a few days with probucol-containing chow while low density protein decreased more mildly over a few weeks. The peritoneal macrophages were assayed for apoA-I binding, apoA-I-mediated release of cellular cholesterol and phospholipid and the reduction by apoA-I of the ACAT-available intracellular cholesterol pool. All of these parameters were strongly suppressed in the probucol-fed mice. In contrast, the mRNA levels of the potential regulatory proteins of the HDL level such as apoA-I, apoE, LCAT, PLTP, SRB1 and ABC1 did not change with probucol. The fractional clearance rate of plasma HDL-cholesteryl ester was uninfluenced by probucol, but that of the HDL-apoprotein was slightly increased. No measurable CETP activity was detected either in the control or probucol-fed mice plasma. The change in these functional parameters is consistent with that observed in the Tangier disease patients. We thus concluded that generation of HDL by apo-cell interaction is a major source of plasma HDL in mice.

Calpain may produce a CA2+-independent form of kinase C in long-term potentiation

Both an enhancement of Ca(2+)-independent kinase activity in the supernatant fraction and enhanced breakdown of type beta kinase C (PKC-beta) were observed in the hippocampus after induction of tetanus-induced long-term potentiation (LTP) in the hippocampal CA1 region of rat. The enhanced activity was inhibited by the PKC-specific inhibitor, PKC19-36. Both phenomena were also observed simultaneously in the in vitro model system in which hippocampal homogenate was treated with CaCl2, and both enhancements were inhibited by the addition of calpain inhibitors, leupeptin and benzyloxycarbonyl-Leu-Met-H. The results suggest that Ca(2+)-independent kinase activity enhanced in the supernatant fraction during LTP derives from the catalytic fragment of PKC-beta released by calpain.

Excitable membranes and synaptic transmission: postsynaptic mechanisms.: Localization of α-internexin in the postsynaptic density of the rat brain

The synaptic localization of alpha-internexin, a brain-specific intermediate filament protein, was investigated immunohistochemically in the rat brain. The specificity of the antibody used in this study was confirmed by Western blotting and the antibody specifically reacted with alpha-internexin in the neurofilament preparation and in the postsynaptic density (PSD) fraction. The alpha-internexin immunoreactivity was distributed in neurons, especially in the somata and dendrites, throughout the cerebral cortex. Immunoelectron microscopic examination showed the immunoreactivity in the PSD, while neurofilament M was not in the PSD. Thus alpha-internexin and neurofilament M are differentially localized in neuronal cells. Alpha-internexin content in the PSD fraction was relatively high even before the period of synaptogenesis and the content in the fraction was unchanged between young and adult rats (2-6 weeks old). These results suggest a role of alpha-internexin for early development and organization of the PSD.

Occurrence of a transcription factor, cAMP response element-binding protein (CREB), in the postsynaptic sites of the brain

The postsynaptic density (PSD) fraction prepared from the rat forebrain contained a transcription factor, cAMP response element-binding protein (CREB). The occurrence of CREB in the PSD was confirmed by immunoelectron microscopic examination. CREB in the PSD fraction was phosphorylated both by protein kinase A and Ca2+/calmodulin-dependent protein kinase II (CaMKII) endogenous to the fraction, and dissociated from the PSD after phosphorylation, especially under CaMKII-activated conditions. The fraction containing CREB that was released from PSD after phosphorylation possessed cAMP response element (CRE)-binding activity. Thus, PSD anchors functionally active CREB. These results suggest that CREB anchored to the PSD is liberated by phosphorylation upon specific synaptic stimulation, translocates into the nucleus, and then triggers synaptic activity-dependent changes in gene expression.

Characterization of protein kinase C activities in postsynaptic density fractions prepared from cerebral cortex, hippocampus, and cerebellum

Protein kinase C (PKC) activities, especially, substrates and PKC isozymes, associated with postsynaptic density (PSD) fractions isolated from rat cerebral cortex, hippocampus, or cerebellum were investigated. The 17k M(r) major substrate for PKC was associated with PSD fractions prepared from cerebral cortex and hippocampus, and several substrates including 18k M(r) protein were associated with PSD fraction isolated from cerebellum. The content of 17k M(r) substrate was extremely low in the PSD fraction prepared from cerebellum. PKCs-beta and gamma were associated with PSD fractions and PKC-alpha was virtually absent in the fraction prepared from the three different regions of the brain. All of PKCs-alpha, beta, and gamma were associated with synaptosome fractions. The 36k M(r) bands immunoreactive with anti-PKC-beta antibody, probably degradation products of native PKC-beta, were detected in both the PSD and synaptosome fractions from the three regions, and the ratio of the degradation fragments to native PKC molecule was higher in PSD fractions than in synaptosome fractions. The results suggest postsynaptic roles of PKCs-beta and gamma and involvement of proteolytic activation of PKC-beta in the postsynaptic signal processing.

Induction of astroglial growth inhibition and differentiation by sialosyl cholesterol

Normal rat astroblasts in culture were exposed to 11 sialosyl or cholesterol derivatives at concentrations lower than 20 microM. Synthesized sialosyl cholesterols (alpha- and beta-D-N-acetyl neuraminyl cholesterols) and cholesterol sulfate showed a marked growth inhibitory action. Sialosyl cholesterol uniquely evoked an astroglia-like stellation resembling that induced by glia maturation factor (GMF) as well as a suppression of GMF-induced mitogenesis of astroblasts. The minimal incubation period of sialosyl cholesterol for the initiation of growth inhibition was as short as one hour. The inhibitory effect retained an irreversibility even after removal of the drug. Cytosolic protein with 58 kDa Mr in size was specifically phosphorylated by sialosyl cholesterol through a certain protein kinase dependent on neither Ca2+ nor cyclic AMP. The competition experiment of sialosyl cholesterol action revealed that sialosyl and cholesterol moieties were indispensable for the phenomena. These results most likely imply that sialosyl cholesterol alters the membrane microenvironment to affect the affinity of growth factor receptor, protein kinase activity, and/or cytoskeletal anchorages.