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.

Cholesteryl ester transfer protein deficiency causes slow egg embryonation of Schistosoma japonicum.

In our attempt to discover a potential cause for accumulation of cholesteryl ester transfer protein (CETP) deficiency in Eastern Asia, we studied the association of CETP deficiency with pathogenesis of Schistosoma japonicum, a life-threatening parasite peculiar to this region. The eggs of S. japonicum showed slow embryonation when cultured in CETP-deficient human plasma. Restoration of CETP to the deficient plasma rescued it, while inhibition of CETP in normal plasma did not cause slow embryonation of the cultured eggs. The egg embryonation was also retarded in the liver but not in the intestine of wild-type mice in comparison to the CETP-transgenic mice. The granulomatous lesion around the parasite eggs in the liver was less in the wild-type than in the CETP-transgenic mice. Thus, CETP deficiency may act against Schistosomiasis japonica by retarding egg embryonation, a potential cause of liver granulomatosis. It does not seem directly due to the lack of CETP activity in plasma but to abnormal lipoprotein generated by chronic CETP deficiency.

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.

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.

Human cholesteryl ester transfer protein measured by enzyme-linked immunosorbent assay with two monoclonal antibodies against rabbit cholesteryl ester transfer protein: plasma cholesteryl ester transfer protein and lipoproteins among Japanese hypercholesterolemic patients.

Plasma cholesteryl ester transfer protein (CETP) concentrations were measured in Japanese subjects by an ELISA with two different monoclonal antibodies that were raised against rabbit CETP and cross-reacted against human CETP. Among 63 patients who consecutively underwent coronary angiography, the plasma CETP of 37 patients with luminal stenosis > or = 50% in their coronary arteries was not significantly different from that of the 26 patients with luminal stenosis < 50%. No other lipoprotein-related measurement except HDL-cholesterol differentiated the two groups. Among 40 hypercholesterolemic patients, no lipoprotein-related measurement other than LDL-cholesterol was found to positive correlate with the CETP. Before and after the treatment of 23 patients with simvastatin 5 mg a day for 4 weeks, plasma CETP markedly decreased in those whose pretreatment CETP was > or = 3 mg/L; no change was observed for those with lower pretreatment CETP. In the former group, negative correlation between CETP and HDL-cholesterol was demonstrated only in the posttreatment plasma.

Cholesteryl ester transfer protein reaction between plasma lipoproteins.

The rate of the non-directional transfer of cholesteryl ester and triglyceride by human cholesteryl ester transfer protein (CETP) was measured between human plasma lipoproteins by monitoring fluorescence spectrum of pyrene-labeled lipid. The transfer rates between high density lipoproteins (HDLs) and between low density lipoproteins (LDLs) were both directly proportional to the substrate lipid concentration within the physiological range of the lipoprotein concentration. Higher preference of cholesteryl ester transfer to triglyceride was demonstrated with HDL than LDL. Although the highly selective binding of CETP to HDL was observed in the electrophoretic analysis, the transfer rate was only moderately higher with HDL for cholesteryl ester and not so at all for triglyceride. In addition, the rate of cholesteryl ester transfer between LDLs was uninfluenced by the presence of a small amount of HDL that is just sufficient to absorb all the CETP in the reaction mixture. The results indicated the preferential transfer of cholesteryl ester over triglyceride by CETP in the interaction with HDL in non-directional lipid transfer reaction among lipoproteins. However, the apparent binding of CETP to HDL does not seem to play an essential role in this type of lipid transfer by CETP.

Presence of NF-kappaB-like and IkappaB-like immunoreactivities in postsynaptic densities.

The distributions of IkappaB and NF-kappa B 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 IkappaB or NF-kappa B. Both IkappaB alpha and NF-kappa B 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-kappa B to the kappa B DNA elements. These results suggest that the NF-kappa B/IkappaB system or a similar mechanism may play a role in signal transmission from synapses to the nucleus.

Excitable membranes and synaptic transmission: postsynaptic mechanisms. Localization of alpha-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.

NMDA receptor subunits epsilon 1 (NR2A) and epsilon 2 (NR2B) are substrates for Fyn in the postsynaptic density fraction isolated from the rat brain.

Fyn, protein tyrosine kinase, and its substrates were highly concentrated in the postsynaptic density (PSD) fraction prepared from the rat forebrain. There were a number of Fyn substrates unique to the PSD fraction. One of the major substrates in the PSD fraction was found to be a concanavalin A-binding glycoprotein, PSD-gp180, which is the N-methyl-D-aspartate (NMDA) receptor subunit epsilon 2 (NR2B). Western blotting and immunoprecipitation supported the phosphorylation of epsilon 2 by Fyn. NMDA receptor subunit epsilon 1 (NR2A) was also a substrate for Fyn. These results suggest that Fyn is involved in the modulation of synaptic efficacy through the phosphorylation of synapse-specific substrates such as the NMDA receptor/channel.

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.

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

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), alpha 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 approximately 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 degrees 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 degrees C, by which the content of CaMKII alpha in the PSD fraction was increased to twofold. These results suggest that most of the CaMKII alpha subunit associated with the PSD fraction (mPSDp) is translocated from cytosol after decapitation. We also showed similar translocation of CaMKII beta/beta'.

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.

Calpain may produce a Ca(2+)-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.

Antibody specific for the Thr-286-autophosphorylated alpha subunit of Ca2+/calmodulin-dependent protein kinase II.

We report the production of an antibody specific for Ca2+/calmodulin-dependent protein kinase II (CaM-KII) autophosphorylated only at Thr-286 of the alpha subunit. Peptide Y-66 [sequence MHRQETVDC (Met-281 to Cys-289 of alpha subunit of CaM-KII)] was synthesized and phosphorylated by the CaM-KII endogenous to synaptic cytoskeleton (postsynaptic density-enriched fraction); the phosphorylated amino acid residue threonine corresponds to Thr-286 in the kinase alpha subunit. The phosphorylated Y-66 peptide was separated from the unphosphorylated peptide by HPLC and used as an immunogen after being coupled to hemocyanin. The antibodies that reacted with hemocyanin and unphosphorylated Y-66 peptide were adsorbed, and then IgG was purified. ELISA proved that the IgG obtained reacted specifically with phosphorylated Y-66 peptide. Immunoblot analysis showed that the antibody reacted specifically to the autophosphorylated CaM-KII both in purified and synaptic cytoskeleton-associated form. Appearance of CaM-KII subunits immunoreactive to anti-phosphorylated Y-66 antibody paralleled the generation of Ca(2+)-independent kinase activity. Immunocytochemical experiments clearly showed expression of the Thr-286- or Thr-287-autophosphorylated form of CaM-KII in cultured hippocampal cells treated with N-methyl-D-aspartate. Thus, this antibody could be extremely useful for studying the biological functions of CaM-KII.

Stimulation by glia maturation factor of Ca(2+)-dependent phosphorylation of M(r) 100 k protein in rat glioblasts.

When quiescent rat glioblasts were stimulated by glia maturation factor (GMF), their intrinsic Ca(2+)-dependent phosphorylation of proteins, especially that of M(r) 100 k protein, increased. The phosphorylation of M(r) 100 k protein in the homogenate started rising 13 h (S phase) after GMF stimulation and reached the maximal level (8-fold greater than the control) at 26 h. Phosphorylation was also detected in intact cells by the use of [(32)P]orthophosphate. Calmodulin augmented and W-7 (calmodulin inhibitor) slightly inhibited the phosphorylation, suggesting that Ca(2+)/calmodulin-dependent protein kinase may partly be involved in phosphorylation of the M(r) 100 k protein. Subcellular fractionation experiments revealed that both M(r) 100 k protein and its kinase were localized exclusively in the cytosol. We also found marked phosphorylation of M(r) 100 k protein in neural tumor cell lines, mouse neuroblastoma (Neuro2a and NAs-1) and glioma (C6 and 354A). Since the peptide maps of (32)P-labeled peptides obtained by chemical cleavage from M(r) 100 k protein of the cells were identical to those of glioblasts, the M(r) 100 k proteins, regardless of cell origin, may be closely related in structure. Growth inhibitors, W-7 (50 ?M), puromucin (2 ?M), spongoadenosine (50 ?M), diphenylhydantoin (0.3 mM), ?-sialosyl cholesterol (20 ?g/ml) and protein kinase inhibitor, K252a (50 nM), lowered the phosphorylation of the M(r) 100 k protein in the cell homogenate derived from glioblasts pretreated with the drugs for 24 h. M(r) 100 k protein of glioblasts and C6 cells was immunoprecipitated by anti-elongation factor-2 (EF-2) antiserum indicating an identity or similarity in structure between the protein and EF-2. These findings provide a possibility that cell growth may be brought about through a phosphorylation of M(r) 100 k protein as one of the signal transduction processes subsequent to a mitogen stimulation.

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.

Inhibition by diphenylhydantoin of growth and morphological differentiation of glial cells: Implication of calcium.

The effects of diphenylhydantoin (DPH) and other anticonvulsants on the growth of glial cells as well as neuroblastoma cell lines was studied. The DPH inhibition of the DNA synthesis was most marked in rat fetal glioblasts induced by glia maturation factor (GMF) among the cell lines studied, and that in C6 cells. The IC(50) of DPH for glioblasts and C6 cells were about 0.2 and 0.4 mM, respectively. The inhibitory effects of DPH and valproate on DNA synthesis was specific for glial cells, and the DNA synthesis of such neuronal cell lines as Neuro2a, NAs-1, and PC12 was unaffected at pharmacological concentrations. Diazepam inhibited the DNA synthesis of all cell types examined in the contrary to DPH, which preferentially inhibited glial cells. Phenobarbital showed no effect, and thiopental inhibition was <30% of the DNA synthesis in control condition with all cell lines used at the concentration of 0.4 mM. Both DPH and diazepam suppressed the extrusion induced by GMF of the glioblast processes, and the neuroblastoma cell neurites that had extended in the presence of dibutylyl cAMP disappeared by the exposure to DPH, suggesting that these drugs affected the cytoskeletal rearrangement of both glial and neuronal cells during morphological differentiation.

Ca2+-dependent phosphorylation by endogenous kinases of Mr 95 K and 50 K-55 K proteins in PC12 pheochromocytoma cells.

Endogenous protein phosphorylation of PC12 cells was investigated with the homogenate as well as intact cells. In the case of the homogenate, the major proteins that were phosphorylated in the presence of Ca2+ were found to be of Mr 95 K and Mr 50 K-55 K. Ca2+/calmodulin-dependent protein kinase appeared to be responsible for phosphorylation of Mr 50 K-55 K proteins and partly of Mr 95 K protein. The apparent Km's for Ca2+ of Mr 95 K and 50 K-55 K protein phosphorylation were 2.2 x 10(-7) M and around 1.5 x 10(-6) M, respectively. Since several cell lines of neuroblastoma exhibited Mr 95 K protein phosphorylation of similar type, the protein phosphorylation may be a common process shared by neuronal cells. Depolarization of intact PC12 cells by high K+ concentrations induced Mr 95 K protein phosphorylation. The results suggest that a physiological increase by excitation in the intracellular Ca2+ concentration triggers phosphorylation of Mr 95 K protein in neuronal cells and this phosphorylation may play a role in the regulation of transmitter release.

In vitro and in vivo growth characteristics of a new ascites-type neuroblastoma cell from mouse C1300 neuroblastoma.

A clonal ascited type cell, NAs-1, was obtained in culture from a mouse neuroblastoma C1300. The cells were adapted to anchorage-independently grow in the flask by the in vitro-in vivo alternate passage technique, and retained the ability of growing and producing ascites fluid when intraperitoneally injected into mice. Although the majority of growing cells in culture medium showed a small and round cell shape without any neuronal process, occasionally non-specific attachment onto the flask surface was observed, but devoid of the extrusion of processes. Karyotype analysis showed a homogeneous chromosome number, 40, with a marker chromosome [t(13:16)] and a minichromosome. Catecholamines, norepinephrine and dopamine, were found in the cell extracts and the contents of dopamine was particularly high as shown in another catecholaminergic neuroblastoma cell, N1E-115. Neuron specific enolase (?-subunit) was also detected. The treatment of the cells by dibutyryl cyclic AMP, prostaglandin E(1), or BL191 (phosphodiesterase inhibitor) induced the biochemical differentiation in terms of catecholamine and cyclic AMP contents, but failed to promote typical morphological differentiations including the extension of process or the significant promotion of adherence onto the flask surface.