代谢性谷氨酸受体配体(s)-4C3HPG对大鼠脑缺血耐受性诱导的影响

目的：观察侧脑室注射代谢型谷氨酸受体1／5亚型（mGluR1/5)配体（s)-4C3HPG对海马脑缺血耐受（BIT）诱导的影响,以探讨mGLUR1/5在BIT诱导中的作用。方法：采用大鼠四血管闭塞全脑缺血模型（4－vessel occlusion,4VO),应用硫堇染色和GFAP免疫组化法。36只大鼠椎动脉凝闭后分为sham组、单纯缺血组、BIT组和（s)-4C3HPG组,其中（s)-4C3HPG组又按所给药物剂量不同,分为0．2、0．04和0．008mg三个亚组。所有动物均在手术后或末次缺血后7d处死取材观察。结果：（1）单纯8min缺血可使海马CA1区组织学分级升高、锥体神经元密度降低和胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP)阳性表达增加（P＜0．05vs sham）.(2)BIT组未见单纯缺血组的上述变化,表明CIP可防止后续8min缺血造成的神经元损伤。（3）CIP的这种保护作用可被（s)-4C3HPG阻断,表现为海马CA1区组织学分级升高和锥体神经元密度降低（P＜0．05 vs sham)。这种变化与（s)-4C3HPG的剂量呈现明显的相关性,即剂量越大,上述改变越明显。结论：（s)-4C3HPG可阻断CIP诱导BIT的作用,提示mGluR1/5参与BIT的诱导。     

Inhibition of established subcutaneous and metastatic murine tumors by intramuscular electroporation of the interleukin-12 gene

In vivo electroporation (EP) of the murine interleukin-12 (IL-12) gene in an expression plasmid (pIL-12) was evaluated for antitumor activity. EP transfer of pIL-12 into mouse quadriceps muscles elicited significant levels of serum IL-12 and interferon-gamma. Intramuscular EP of pIL-12 resulted in complete regression or substantial inhibition of 38C13 B-cell lymphoma, whereas pIL-12 delivered by gene gun or intramuscular injection without EP showed little therapeutic effect. Impressive antitumor activity by intramuscular EP was also demonstrated in animals with advanced malignant disease. At day 14 after 38C13 tumor inoculation, all animals were found to carry large tumors and to have metastases; without treatment, most died within a week. A single intramuscular EP of pIL-12 resulted in regression of 50% of large subcutaneous tumors and significantly prolonged the lifespan of these animals. Moreover, animals that were previously cured of 38C13 tumors by in vivo EP treatment significantly suppressed tumor growth when challenged 60 days later. In vivo EP of the IL-12 gene was also effective in suppressing subcutaneous and lung metastatic tumors of CT-26 colon adenocarcinoma and B16F1 melanoma cells. Together, these results show that intramuscular electrotransfer of the IL-12 gene may represent a simple and effective strategy for cancer treatment.     

Lipoproteins produced by ApoE-/- astrocytes infected with adenovirus expressing human ApoE

We have developed an astrocyte cell culture system that is attractive for the study of apoE structure and its impact on astrocyte lipoproteins and neuronal function. Primary astrocytes from apoE-/- mice were infected with adenovirus expressing apoE3 or apoE4 and the nascent lipoproteins secreted were characterized. The nascent apoE-containing astrocyte particles were predominantly the size of plasma high density lipoprotein (HDL). ApoE4, in contrast to apoE3, appeared to be distributed in two distinct lipoprotein peaks and the apoE4-containing lipoproteins contained significantly more radiolabeled triglyceride. On electron micrographs the astrocyte particles were both discoidal and spherical in shape with a prevalence of stacked discs in apoE3 particles, but single discs and larger spheres in apoE4 particles. The apoE4 discs were significantly wider than apoE3 discs. These properties of the astrocyte lipoproteins are similar to those obtained from apoE isoform transgenic mice. Astrocyte lipoproteins containing apoE3, but not apoE4, stimulated neurite outgrowth in Neuro-2a cells. These studies suggest that the isoform-specific effects of apoE lipoproteins may involve differences in particle size and composition. Finally we demonstrate the usefulness of this system by expressing a truncated apoE3 (delta202-299) mutant and show preliminary data indicating that a liver X receptor agonist promotes HDL output by the astrocytes without an increase in apoE in the media. This cell culture system is more flexible and allows for more rapid expression of apoE mutants.     

Depolarization-induced ERK phosphorylation depends on the cytosolic Ca2+ level rather than on the Ca2+ channel subtype of chromaffin cells

The contribution of Ca2+ entry through different voltage-activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-alpha (PKC-alpha), but not on Ca2+/calmodulin-dependent protein kinase nor cyclic AMP-dependent protein kinase. Blockade of L-type Ca2+ channels by 3 microm furnidipine, or blockade of N channels by 1 micromomega-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 micromomega-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.     

Differential influence of increased polyol pathway on protein kinase C expressions between endoneurial and epineurial tissues in diabetic mice

To explore the relationship between polyol pathway and protein kinase C (PKC), we examined PKC activities and expressions of PKC isoforms separately in endoneurial and vessel-rich epineurial tissues in diabetic mice transgenic for human aldose reductase (Tg). Tg and littermate control mice (Lm) were made diabetic by streptozotocin at 8 weeks of age and treated orally with aldose reductase inhibitor (ARI) (fidarestat 3-5 mg/kg/day) or placebo for 12 weeks. At the end, compared with non-diabetic state, sorbitol contents were increased 6.4-fold in endoneurium and 5.1-fold in epineurium in diabetic Tg, whereas the increase was detected only in endoneurium in diabetic Lm. Endoneurial PKC activity was significantly reduced in diabetic Tg. By contrast, epineurial PKC activity was increased in both diabetic Lm and diabetic Tg and there was no significant difference between the two groups. These changes were all corrected by ARI treatment. Consistent with the changes of PKC activities, diabetic Tg showed decreased expression of PKC alpha in endoneurium, whereas there was an increased expression of PKC beta II in epineurium in both diabetic Tg and diabetic Lm. These findings suggest the presence of dichotomous metabolic pathway between neural and vascular tissues in the polyol-PKC-related pathogenesis of diabetic neuropathy.     

The role of phosphatidylinositol 3-kinase in neural cell adhesion molecule-mediated neuronal differentiation and survival

The neural cell adhesion molecule, NCAM, is known to stimulate neurite outgrowth from primary neurones and PC12 cells presumably through signalling pathways involving the fibroblast growth factor receptor (FGFR), protein kinase A (PKA), protein kinase C (PKC), the Ras-mitogen activated protein kinase (MAPK) pathway and an increase in intracellular Ca2+ levels. Stimulation of neurones with the synthetic NCAM-ligand, C3, induces neurite outgrowth through signalling pathways similar to the pathways activated through physiological, homophilic NCAM-stimulation. We present here data indicating that phosphatidylinositol 3-kinase (PI3K) is required for NCAM-mediated neurite outgrowth from PC12-E2 cells and from cerebellar and dopaminergic neurones in primary culture, and that the thr/ser kinase Akt/protein kinase B (PKB) is phosphorylated downstream of PI3K after stimulation with C3. Moreover, we present data indicating a survival-promoting effect of NCAM-stimulation by C3 on cerebellar and dopaminergic neurones induced to undergo apoptosis. This protective effect of C3 included an inhibition of both DNA-fragmentation and caspase-3 activation. The survival-promoting effect of NCAM-stimulation was also shown to be dependent on PI3K.     

Pro-apoptotic protein kinase C delta is associated with intranuclear inclusions in a transgenic model of Huntington's disease

In order to investigate any effect of truncated mutant huntingtin (tNhtt) aggregation on protein kinase C (PKC) signaling in Huntington's disease (HD), we studied a possible association of PKC isoforms with the aggregates using cellular and transgenic models of HD. In this report we describe an association of mutant tNhtt with at least three PKC isoforms (alpha, delta, zeta), as revealed by co-immunoprecipitation assays and immunocytochemistry in a cellular model of HD (Neuro2a cells expressing tNhtt-150Q-EGFP), as well as a specific association of PKC delta with intranuclear aggregates in a transgenic model (R6/2 mice). Immunoblot analysis of isolated nuclear fractions shows an elevation of nuclear PKC delta in transgenic brain tissue. The observed elevation has a strong similarity with the apoptotic translocation of PKC delta detected in experiments with the mouse neuroblastoma Neuro2a cells. Using a Neuro2a cell line expressing tNhtt with the nuclear localization signal, we demonstrate the association of PKC delta with intranuclear aggregates and present evidence that accumulation of PKC delta in cell nuclei does not depend on mutant htt nuclear translocation. Our results suggest that the association of PKC delta with intranuclear htt-aggregates may affect its apoptotic function in a transgenic model of HD.     

An immobilized metal ion affinity adsorption and scintillation proximity assay for receptor-stimulated phosphoinositide hydrolysis

A novel approach to measuring receptor-stimulated phosphoinositide hydrolysis was developed based on the principles of immobilized metal ion affinity chromatography (IMAC) and scintillation proximity assay (SPA). Hard Lewis metal ions, such as Zr(4+), Ga(3+), Al(3+), Fe(3+), Lu(3+), and Sc(3+), were immobilized on SPA beads via metal chelate and utilized as affinity ligands to entrap inositol phosphates. [3H]Inositol phosphates bound to IMAC-SPA beads through the strong interaction of their phosphate group with the immobilized metal ions. The binding brought [3H]inositol phosphates in close proximity to the scintillant embedded in the SPA beads, thereby allowing the radioactivity to be quantified. Quantification of [3H]inositol phosphate production in cells preincubated with [3H]inositol provided a highly sensitive measurement of phosphoinositide hydrolysis. The utility of this approach was demonstrated in measuring the response mediated by the G-protein-coupled neurokinin NK1 receptor and the tyrosine kinase-linked platelet-derived growth factor (PDGF) receptor. Substance P stimulated phosphoinositide hydrolysis concentration-dependently in CHO cells expressing NK1 receptors with a maximal 12-fold increase in inositol phosphate production. Similarly, PDGF-BB stimulated a 5-fold increase in phosphoinositide hydrolysis in quiescent Swiss 3T3 cells. This new approach is highly sensitive, fast, simple, easily performed on 96-well plates, and amenable for high-throughput screening.