Hyperhomocysteinemia and impaired vasomotor function in type 2 diabetes mellitus.

BACKGROUND: Hyperhomocysteinemia has been shown to adversely affect vascular function. The aim of this study was to determine whether hyperhomocysteinemia was independently associated with changes in endothelium-dependent and -independent vasomotor functions in patients with type 2 diabetes mellitus. MATERIALS AND METHODS: Fasting homocysteine (tHcy) was measured in 123 patients with type 2 diabetes and in 61 nondiabetic controls. Endothelium-dependent and -independent vasodilation was measured using high-resolution vascular ultrasound. RESULTS: Plasma tHcy concentration was increased in the diabetic patients (11.1 +/- 3.7 micromol L(-1) vs. 9.8 +/- 2.9, P < 0.05). The prevalence of hyperhomocysteinemia (defined as tHcy > 15 micromol L(-1)) was higher in the diabetic patients (P < 0.05). Within group comparisons showed that both the abnormalities in endothelium-dependent and -independent vasodilation were significantly more severe in diabetic patients with tHcy 10-15 (P < 0.05) and tHcy > 15 micromol L(-1) (P < 0.05) than in those patients with tHcy < 10 micromol L(-1). When compared with nondiabetic controls matched for tHcy levels, impairment of endothelium-dependent and -independent vasodilation were already evident, even in patients with normal tHcy levels (P < 0.01). Despite significant univariate relationships between tHcy and endothelium-dependent (r = -0.24, P < 0.01) and -independent vasodilation (r = -0.33, P < 0.01) in patients with diabetes, only the relationship between tHcy and endothelium-independent vasodilation remained significant after adjusting for other cardiovascular risk factors in multiple regression analysis. CONCLUSIONS: Impairment of endothelium-dependent and -independent vasodilation was already present in diabetic patients with normal tHcy levels, and these abnormalities became more severe with increasing tHcy levels. Only the association between tHcy and endothelium-independent vasodilation was free of other cardiovascular risk factors.

Effect of postmenopausal hormone replacement therapy on lipoprotein and homocysteine levels in Chinese women.

Epidemiological studies have revealed that postmenopausal estrogen replacement therapy results in a marked reduction in the risk for cardiovascular diseases. In the present study, we evaluated plasma lipoprotein profile as well as homocysteine levels in 145 postmenopausal and premenopausal Chinese women living in Hong Kong. We also investigated the effect of hormone-replacement therapy (HRT) with estrogen or estrogen combined with progestin on plasma lipoprotein profile and homocysteine concentrations in those individuals. Postmenopausal women displayed significantly higher plasma levels of total cholesterol, LDL-cholesterol and apoB as well as higher plasma homocysteine levels than that of premenopausal women. HRT with either estrogen (17beta-estradiol or conjugated equine estrogen) alone or estrogen combined with progestin for 3.5-4.5 years significantly improved the lipoprotein profile in postmenopausal women by decreasing the levels of total cholesterol (12-20% reduction), LDL-cholesterol (26-29% reduction) and apoB (21-25% reduction). In women treated with 17beta-estradiol or conjugated equine estrogens their plasma levels of apoAl were significantly elevated (18% elevation) as compared to non-users. HRT also reduced plasma concentrations of homocysteine (13-15% reduction). In conclusion, we found that long-term HRT was associated with improvement in plasma lipoprotein profile and a reduction in homocysteine concentration in postmenopausal women. These results support the notion that the improvement of lipoprotein profile and a reduction in homocysteine concentration may contribute to the beneficial effect of HRT on cardiovascular risk.

Inhibition of stress-activated protein kinase in the ischemic/reperfused heart: role of magnesium tanshinoate B in preventing apoptosis.

The activation of stress-activated protein (SAP) kinase may lead to an induction of apoptosis that is responsible for part of the cardiomyocyte death in reperfusion injury. The objective of the present study was to investigate the mechanism by which magnesium tanshinoate B (MTB), a bioactive compound isolated from Danshen, prevents apoptosis in cardiomyocytes in the ischemic/reperfused heart. Isolated adult rat hearts were perfused by the Langendorff mode with medium containing MTB prior to the induction of normothermic global ischemia. At the end of the 30-min ischemic period, the heart was reperfused with the same medium with or without MTB for an additional 20 min. In the MTB-treated ischemic/reperfused heart, the number of apoptotic nuclei was reduced by 2.5-fold in comparison to that in untreated ischemic/reperfused controls [23 +/- 4 vs 57 +/- 7 (mean +/- SD) TUNEL-positive cells, respectively, N = 3-4, P < 0.001]. SAP kinase activity was elevated 1.7-fold in ischemic/reperfused rat hearts [35.6 +/- 3.8 vs 21.2 +/- 3.3 (control) (mean +/- SEM) relative densitometric units, N = 4-6, P < 0.05]. Treatment with MTB abolished this elevation in SAP kinase activity (25.0 +/- 5.2 relative densitometric units), which was also decreased by 40% in the nucleus. When the heart was subjected to ischemia alone, there was no significant change in SAP kinase activity in the presence or absence of MTB. MTB did not appear to affect the p38 mitogen-activated protein kinase activity in this model system. In conclusion, MTB was shown to have cardioprotective activity against apoptosis, probably through the inhibition of SAP kinase activity.

Lipoprotein-X stimulates monocyte chemoattractant protein-1 expression in mesangial cells via nuclear factor-kappa B.

BACKGROUND: Lipoprotein-X (Lp-X) is an abnormal lipoprotein found in the plasma of patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. The majority of patients with this disorder develop progressive glomerulosclerosis. One key event in the pathogenesis of glomerulosclerosis is the infiltration of monocytes into affected glomeruli. Mesangial cells can synthesize and secrete monocyte chemoattractant protein-1 (MCP-1), an important chemoattractant for monocytes. The objective of the present study was to examine the effect of Lp-X on MCP-1 expression in mesangial cells leading to an enhanced monocyte chemotaxis and to elucidate the mechanisms involved in this process. METHODS: Lp-X was isolated from the plasma of a patient with familial LCAT deficiency. After rat mesangial cells were incubated with Lp-X for four or six hours, the expression of MCP-1 mRNA was determined by nuclease protection assay, and MCP-1 protein was measured by Western immunoblotting analysis. Monocyte chemotaxis was determined by using a Micro Chemotaxis Chamber. RESULTS: Lp-X (50 to 100 nmol/mL) stimulated mesangial cell MCP-1 mRNA expression (137 to 220%) and MCP-1 protein levels (233 to 375%). Conditioned media collected from Lp-X-treated mesangial cells stimulated human acute monocytic leukemia (THP-1) monocyte chemotaxis (165 to 200%). The increase in MCP-1 expression in mesangial cells was associated with an elevation of intracellular diacylglycerol levels, and activation of protein kinase C (PKC) as well as nuclear factor-kappa B (NF-kappa B). CONCLUSION: These results suggest that Lp-X participates in the pathogenesis of glomerulosclerosis and subsequent renal failure in familial LCAT deficient patients by stimulating monocyte infiltration via a mechanism involving mesangial MCP-1 expression.

Homocysteine induces monocyte chemoattractant protein-1 expression by activating NF-kappaB in THP-1 macrophages.

Homocysteinemia is an independent risk factor for cardiovascular disorders. The recruitment of monocytes is an important event in atherogenesis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that stimulates monocyte migration into the intima of arterial walls. The objective of the present study was to investigate the effect of homocysteine on MCP-1 expression in macrophages and the underlying mechanism of such effect. Human monocytic cell (THP-1)-derived macrophages were incubated with homocysteine. By nuclease protection assay and ELISA, homocysteine (0.05-0.2 mM) was shown to significantly enhance the expression of MCP-1 mRNA (up to 2.6-fold) and protein (up to 4.8-fold) in these cells. Homocysteine-induced MCP-1 expression resulted in increased monocyte chemotaxis. The increase in MCP-1 expression was associated with activation of nuclear factor (NF)-kappaB due to increased phosphorylation of the inhibitory protein (IkappaB-alpha) as well as reduced expression of IkappaB-alpha mRNA in homocysteine-treated cells. In conclusion, our results demonstrate that homocysteine, at pathological concentration, stimulates MCP-1 expression in THP-1 macrophages via NF-kappaB activation.

Magnesium tanshinoate B (MTB) inhibits low density lipoprotein oxidation.

Danshen, a Chinese herbal medicine has been widely used for the treatment of cardiovascular diseases. Magnesium tanshinoate B (MTB) is an active compound purified from Danshen. The objective of this study was to investigate the effect of MTB on the susceptibility of low density lipoproteins (LDL) to oxidative modification as well as on the accumulation of lipids in THP-1 derived macrophages. Aliquots of LDL were incubated with copper sulfate in the absence or presence of MTB. The degrees of oxidative modification of LDL were assessed by examining the relative gel electrophoretic mobility, by measuring the amount of thiobarbituric acid reactive substances (TBARS), and by continuous monitoring of the formation of conjugated dienes upon the increase in absorbency at 234 nm. MTB at concentrations of 1-10 microM significantly inhibited oxidative modification of LDL. Such inhibitory effect resulted in a decrease in the uptake of LDL by THP-1 derived macrophages. Taken together, these results clearly demonstrate that MTB inhibits oxidative modification of LDL and hence prevents the uptake of LDL by cultured macrophages. Such effect may be therapeutically relevant in protecting cells from lipid peroxidation in vascular disorders.

Inhibition by ginkgolides and bilobalide of the production of nitric oxide in macrophages (THP-1) but not in endothelial cells (HUVEC).

Nitric oxide (NO) is a principal mediator in many physiological and pathological processes. NO produced by constitutive nitric oxide synthase in endothelial cells (eNOS) acts as a vasodilator, whereas excess NO production due to elevated expression of inducible nitric oxide synthase (iNOS) may produce cytotoxic effects to cells in the vascular wall. We demonstrated in our previous work that the extract of Ginkgo biloba leaves (EGb) inhibits iNOS-mediated NO production. The objective of the present study was to investigate the effects of several active EGb components on iNOS-mediated NO production in macrophages derived from a human monocytic cell line (THP-1), as well as on eNOS-mediated NO production in human umbilical vein endothelial cells (HUVEC). Ginkgolide A, ginkgolide B, or bilobalide (0.25 to 1.0 microg/mL) caused a 30-65% reduction in the levels of NO metabolites released by THP-1 macrophages after 4 hr of incubation, with a corresponding decrease in iNOS activity. Western immunoblotting analysis coupled with a nuclease protection assay and reverse transcription-polymerase chain reaction revealed a concomitant reduction in the levels of iNOS protein mass and mRNA in ginkgolide A-, ginkgolide B-, or bilobalide-treated macrophages. On the other hand, these compounds did not affect eNOS-mediated NO production or the expression of eNOS protein and mRNA in HUVEC. Taken together, these results suggest that ginkgolide A, ginkgolide B, and bilobalide may contribute to the selective inhibitory effect of EGb on iNOS expression without affecting eNOS-mediated NO production.

Homocysteine stimulates nuclear factor kappaB activity and monocyte chemoattractant protein-1 expression in vascular smooth-muscle cells: a possible role for protein kinase C.

Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that stimulates the migration of monocytes into the intima of arterial walls. Although many factors that induce MCP-1 expression have been identified, the effect of homocysteine on the expression of MCP-1 in atherogenesis and the underlying mechanisms are not entirely clear. The objective of the present study was to investigate the role of homocysteine in MCP-1 expression in human aorta vascular smooth-muscle cells (VSMCs). After VSMCs were incubated with homocysteine for various time periods, a nuclease protection assay and ELISA were performed. Homocysteine (0.05-0.2 mM) significantly increased the expression of MCP-1 mRNA (up to 2. 7-fold) and protein (up to 3.3-fold) in these cells. The increase in MCP-1 expression was associated with the activation of protein kinase C (PKC) as well as nuclear factor kappaB (NF-kappaB). Further investigation demonstrated that the activation of NF-kappaB was the result of a PKC-mediated reduction in the expression of inhibitory protein (IkappaBalpha) mRNA and protein in homocysteine-treated cells. Oxidative stress might also be involved in the activation of NF-kappaB by homocysteine in VSMCs. In conclusion, the present study has clearly demonstrated that the activation of PKC as well as superoxide production followed by activation of NF-kappaB is responsible for homocysteine-induced MCP-1 expression in VSMCs. These results suggest that homocysteine-stimulated MCP-1 expression via NF-kappaB activation may play an important role in atherogenesis.

Effect of magnesium tanshinoate B on the production of nitric oxide in endothelial cells.

Nitric oxide (NO) is a potent vasodilator which plays an important role in regulating vascular tones. Danshen, a Chinese herbal medicine has been widely used for the treatment of cardiovascular diseases. The objective of this study was to investigate the effect of magnesium tanshinoate B (MTB), a compound purified from Danshen, on the production of NO in human endothelial cell line (ECV304). After cells were incubated with MTB (1-10 microM) for 1 or 4 h, amounts of NO metabolites released by cells were quantified and cellular NOS activities were determined following the conversion of [3H]arginine to [3H]citrulline. The NOS protein expression was determined by Western immunoblotting analysis. MTB (1-10 microM) stimulated the release of NO and its metabolites from endothelial cells. Following MTB treatment, the cellular NOS activities were significantly enhanced with a concomitant increase in the levels of constitutive NOS (cNOS) protein mass (110-178%). Selective activation of cNOS by MTB may be employed therapeutically in modulating NO production in endothelial cells.

Effect of Flos carthami on stress-activated protein kinase activity in the isolated reperfused rat heart.

The apoptotic death of cardiomyocytes due to ischemia/reperfusion is one of the major complications of heart disease. Ischemia/reperfusion has been shown to lead to the activation of the stress-activated protein (SAP) kinases and the p38/reactivating kinase (p38/RK). In this study, the direct effect of an aqueous Flos carthami (FC) extract on SAP kinases was investigated. When isolated rat hearts were perfused by Langendorff mode with media containing FC extract prior to the induction of global ischemia and the subsequent reperfusion, SAP kinase activity was inhibited 95%. Untreated ischemic/reperfused hearts showed a 57% elevation in the activity of SAP kinase. The in vitro effect of these FC extracts on SAP kinase was also tested. At a concentration of 10 microg/ml, the aqueous FC extract resulted in 50% inhibition of SAP kinase activity in ischemic heart tissue. Our results showed that FC affected both the interaction of SAP kinase with c-jun as well as the phosphotransferase reaction. These results clearly demonstrate that extracts from Flos carthami exerted inhibitory effects on SAP kinase. The administration of the FC extract may lead to a modulation of the apoptotic effect of SAP kinase activation induced during ischemia/reperfusion.

Very low-density lipoprotein stimulates the expression of monocyte chemoattractant protein-1 in mesangial cells.

BACKGROUND: Elevated plasma levels of very low-density lipoprotein (VLDL) are associated with an increased risk for focal glomerulosclerosis, which is analogous to atherosclerosis. One feature of focal glomerulosclerosis is the presence of foam cells derived from the infiltration of circulating monocytes. Mesangial cells are able to express monocyte chemoattractant protein-1 (MCP-1). In this study, the ability of VLDL to stimulate MCP-1 expression in mesangial cells and consequent monocyte adhesion was investigated. METHODS: For adhesion studies, mesangial cells isolated from Sprague-Dawley rats were treated with VLDL for six hours, followed by a one-hour incubation with Tamm-Horsfall protein-1 (THP-1) cells. Mesangial MCP-1 mRNA levels were determined by reverse transcription-polymerase chain reaction. MCP-1 protein was determined by solid-phase immunoassay. RESULTS: VLDL (100 to 300 microg/mL) significantly enhanced the expression and secretion of MCP-1 (54 to 285 ng/well) in mesangial cells. Such an effect was accompanied by the increased adhesion of monocytes to mesangial cells and later the formation of foam cells from monocytes after ingesting excessive amounts of VLDL lipids. VLDL-induced MCP-1 expression and monocyte adhesion were blocked by a protein kinase C inhibitor (staurosporine), as well as a calcium channel blocker (diltiazem). CONCLUSIONS: Our results demonstrate that elevated levels of VLDL, through the action of MCP-1, may contribute to the infiltration of monocytes into the mesangium and subsequent foam cell formation. Hence, VLDLs may play a role in the pathogenesis of focal glomerulosclerosis. One of the mechanisms of such effect may be mediated through the calcium-dependent protein kinase C pathway.

Inhibitory effect of Ginkgo biloba extract on the expression of inducible nitric oxide synthase in endothelial cells.

Excessive production of nitric oxide (NO) may have cytotoxic effects through the formation of peroxynitrite with superoxide. The extract of Ginkgo biloba leaves (EGb) has been demonstrated to be a potent scavenger of free radicals. Although EGb has been shown recently to inhibit NO production in macrophages, its effect on NO production in endothelial cells is largely unknown. The objective of this study was to elucidate the mechanism by which EGb affects NO production in a human endothelial cell line (ECV304). After cells were incubated with EGb (10-100 microg/mL) for 2 or 4 hr, the amounts of NO metabolites released by the cells were quantitated, and cellular NOS activities were determined following the conversion of [3H]arginine to [3H]citrulline. NOS protein expression was determined by western immunoblotting analysis. mRNA levels were examined by reverse transcription-polymerase chain reaction (RT-PCR) analysis. EGb (50 microg/mL) caused a 30% reduction of NO metabolites released by endothelial cells. Following EGb treatment, cellular inducible NO synthase (iNOS) activity was reduced by 28% with a concomitant reduction in the levels of iNOS protein mass and mRNA. There was no change in the activity or protein mass of constitutive NO synthase in these cells. EGb inhibited NO production by attenuating the level of iNOS mRNA in ECV304 cells. Selective inhibition of iNOS by EGb may be therapeutically relevant in modulating NO production in endothelial cells.

Homocysteine stimulates the production and secretion of cholesterol in hepatic cells.

Homocysteinemia and hypercholesterolemia are important risk factors associated with the occurrence of arteriosclerotic vascular diseases. A positive correlation between plasma levels of homocysteine and cholesterol was found in homocysteinemic patients as well as in experimental animals. In the present study, the effect of homocysteine on the production and secretion of cholesterol in human hepatoma cell line HepG2 cells was investigated. When cells were incubated with 4 mM homocysteine, the amounts of total cholesterol produced as well as the cholesterol secreted by these cells were significantly increased (from 32 +/- 5 to 74 +/- 5 nmol/mg cellular protein). Further biochemical analyses revealed that the increase in cholesterol was resulted from an enhancement in the production and secretion of the unesterified cholesterol with no concomitant change in the level of cholesteryl esters. The activity of intracellular 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was markedly elevated by 131% and 190% after cells were incubated with homocysteine for 24 and 48 h. Homocysteine also stimulated the secretion of apo B100 by HepG2 cells (from 0.84 +/- 0.11 to 1.37 +/- 0.12 micrograms apolipoprotein B/mg cellular protein). Our results demonstrate that homocysteine stimulates the production and secretion of cholesterol and apolipoprotein B100 in HepG2 cells. The increase in the production of cholesterol induced by homocysteine may contribute to the pathogenesis of arteriosclerosis.

Identification of two essential phosphorylated threonine residues in the catalytic domain of Mekk1. Indirect activation by Pak3 and protein kinase C.

The 78-kDa protein kinase Mekk1 plays an important role in the stress response pathway that involves the activation of downstream kinases Sek1 and stress-activated protein kinase/c-Jun NH2-terminal kinase. Conserved serine and threonine residues located between the kinase subdomains VII and VIII of many protein kinases are phosphorylated for maximal kinase activation. Two threonine residues within this region in Mekk1 at positions 560 and 572, but not the serine at 557, were shown to be essential for catalytic activity in this study. When these threonine residues were replaced with alanine, there was a significant loss in phosphotransferase activity toward the primary substrate, Sek1, and a large decrease in autophosphorylation activity. Site-directed mutagenesis demonstrated that these threonine residues cannot be replaced with either serine or glutamic acid for preservation of phosphotransferase activity. Further examination of the Mekk1 mutants isolated from 32P-labeled transfected COS cells showed that Thr-560 and Thr-572 were indeed phosphorylated after two-dimensional tryptic-chymotryptic phosphopeptide analysis. Additional determinants in the NH2-terminal domain of Mekk1 also play a role in the regulation of Mekk1 activity. Although Pak3 and PKC can activate Mekk1 in vivo, this interaction is indirect and independent, since there was no direct phosphorylation of Mekk1 by Pak3 or PKC or of Pak3 by PKC, respectively.

Role of extracellular signal-regulated protein kinases 1 and 2 in oligodendroglial process extension.

The relationship between extracellular signal-regulated protein kinase (ERK) activation and process extension in cultured bovine oligodendrocytes (OLGs) was investigated. Process extension was induced through the exposure of cultured OLGs to phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), for various intervals. During the isolation of these OLGs from bovine brain, the original processes were lost. Therefore, any reinitiation of process extension via PMA stimulation was easily discemible through morphological monitoring. It was found that exposure of OLGs to PMA for 10 min was enough to induce OLG process extension 24-72 h later. Furthermore, this extension was still evident at least 1 week after the initial PMA stimulation, indicating that OLGs do not need continuous PKC activation to sustain process extension. Control and PMA-stimulated OLGs were also subjected to immunocytochemistry using an anti-ERK antibody selective for the mitogen-activated protein kinases p42 Erk2 (ERK2) and p44 Erk1 (ERK1) isoforms. ERK immunoreactivity in the nucleus was evident after PMA stimulation of OLGs but not in control OLGs. In parallel experiments, the control and PMA-stimulated OLGs were purified by Mono Q fractionation and subjected to ERK phosphotransferase assays using [gamma-32P]ATP and either myelin basic protein (MBP) or a synthetic peptide substrate based on the Thr97 phosphorylation site in MBP. These assays indicated that in PMA-treated OLGs, ERK activation was at least 12-fold higher than in control OLGs. Anti-ERK and anti-phosphotyrosine western blots of the assay fractions verified an enhanced phosphorylation of ERK1 and ERK2 in PMA-treated fractions relative to control fractions. When OLGs were pretreated for 15 min with the ERK kinase (MEK) inhibitor PD 098059 before PMA stimulation, they exhibited a 67% decrease in ERK activation as compared with cells treated with PMA alone. Furthermore, these MEK inhibitor-pretreated cells were still viable but showed no process extensions up to 1 week later. Therefore, we propose that a threshold level of ERK activity is required for the initiation of OLG process extension.

Neurotrophins stimulate phosphorylation of synapsin I by MAP kinase and regulate synapsin I-actin interactions.

The ability of neurotrophins to modulate the survival and differentiation of neuronal populations involves the Trk/MAP (mitogen-activated protein kinase) kinase signaling pathway. More recently, neurotrophins have also been shown to regulate synaptic transmission. The synapsins are a family of neuron-specific phosphoproteins that play a role in regulation of neurotransmitter release, in axonal elongation, and in formation and maintenance of synaptic contacts. We report here that synapsin I is a downstream effector for the neurotrophin/Trk/MAP kinase cascade. Using purified components, we show that MAP kinase stoichiometrically phosphorylated synapsin I at three sites (Ser-62, Ser-67, and Ser-549). Phosphorylation of these sites was detected in rat brain homogenates, in cultured cerebrocortical neurons, and in isolated presynaptic terminals. Brain-derived neurotrophic factor and nerve growth factor upregulated phosphorylation of synapsin I at MAP kinase-dependent sites in intact cerebrocortical neurons and PC12 cells, respectively, while KCl- induced depolarization of cultured neurons decreased the phosphorylation state at these sites. MAP kinase-dependent phosphorylation of synapsin I significantly reduced its ability to promote G-actin polymerization and to bundle actin filaments. The results suggest that MAP kinase-dependent phosphorylation of synapsin I may contribute to the modulation of synaptic plasticity by neurotrophins and by other signaling pathways that converge at the level of MAP kinase activation.

Synapsin IIa bundles actin filaments.

Synapsins are neuron-specific phosphoproteins associated with small synaptic vesicles in the presynaptic nerve terminal. Synapsin I, which has been demonstrated to bundle F-actin in vitro, has been postulated to regulate neurotransmitter release by cross-linking synaptic vesicles to the actin cytoskeleton. To investigate the possible interaction of synapsin II with actin filaments, we expressed synapsin II in Spodoptera frugiperda and High Five insect cells using a recombinant baculovirus. Purified recombinant synapsin IIa was incubated with F-actin, and bundle formation was evaluated by light scattering and electron microscopy. Synapsin IIa was found to bundle actin filaments. Dose-response curves indicated that synapsin IIa was more potent than synapsin I in bundling actin filaments. These data suggest that synapsin IIa may cross-link synaptic vesicles and actin filaments in the nerve terminal.

Networking with mitogen-activated protein kinases.

Mitogen activated protein (MAP) kinases and their target ribosomal protein S6 (RSK) kinases have been recognized as shared components in the intracellular signaling pathways of many diverse cytokines. Recent studies have extended this protein kinase cascade by identifying the major activator of vertebrate MAP kinases as a serine/threonine/tyrosine-protein kinase called MEK, which is related to yeast mating factor-regulated protein kinases encoded by the STE7 and byr1 genes. MEK, in turn, may be activated following its phosphorylation on serine by either of the kinases encoded by proto-oncogenes raf1 or mos, as well as by p78mekk, which is related to the yeast STE11 and byr2 gene products. Isoforms of all of these protein kinases may specifically combine to assemble distinct modules for intracellular signal transmission. However, the fundamental architecture of these protein kinase cascades has been highly conserved during eukaryotic evolution.

Synapsin IIa: expression in insect cells, purification, and characterization.

Synapsin IIa belongs to a family of neuron-specific phosphoproteins called synapsins, which are associated with synaptic vesicles in presynaptic nerve terminals. In order to examine the biochemical properties of synapsin IIa, and ultimately its physiological function, purified protein is required. Since attempts to purify significant quantities of synapsin IIa, an isoform of the synapsins, from mammalian brain have proven difficult, we undertook the production of recombinant synapsin IIa by utilizing the baculovirus expression system. Rat synapsin IIa cDNA was introduced into the baculovirus genome via homologous recombination, and the recombinant baculovirus was purified. Spodoptera frugiperda (Sf9) cells infected with this virus expressed synapsin IIa as 5% of the total cellular protein. The recombinant protein was extracted from the particulate fraction of the infected Sf9 cells with salt and a nonionic detergent and purified by immunoaffinity chromatography. The purified synapsin IIa was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 0.8 mol of phosphate/mol of protein. Metabolic labeling with [32P]Pi demonstrated synapsin IIa phosphorylation in infected Sf9 cells. Using a homogenate of uninfected Sf9 cells, a cAMP-dependent protein kinase activity which can phosphorylate synapsin IIa was detected. Limited proteolysis of recombinant synapsin IIa phosphorylated in vitro and in vivo resulted in identical phosphopeptide maps. Further, synapsin IIa, like synapsin I, binds with high affinity in a saturable manner to synaptic vesicles purified from rat cortex.