Localization of amyloid beta (Aβ1-42) protofibrils in membrane lateral compartments: Effect of cholesterol and 7-Ketocholesterol

Cholesterol plays an important role in the interaction of Alzheimer’s amyloid beta (Aβ) with cell membranes, an important event in Aβ-induced cytotoxicity. However, it is not fully understood how cholesterol influences the association of Aβ with membrane lateral compartments. We have shown that by modulating membrane fluidity, cholesterol decreased peptide localization in solid-ordered domains and increased that in liquid-ordered domains. It changed the amount of Aβ associating with liquid-disordered (Ld) phase with different tendencies depending on the composition of heterogeneous membrane systems. 7-Ketocholesterol, an oxidized derivative of cholesterol, majorly enhanced the fluidity of and Aβ interaction with Ld phase. These findings are useful for clarifying the impact of cholesterol and its oxidation in Aβ-induced toxicity.     

Molecular cloning of Rhombex-40 a transmembrane protein from the ventral medullary surface of the rat brain by differential display

Respiration-related neurons, which detect various chemicals in cerebrospinal fluid, are localized to the ventral medullary surface (VMS). We hypothesized that expression of genes involved in respiratory function is upregulated in the VMS. By differential display, we looked for genes differentially expressed in VMS neurons and cerebral cortical neurons. Seventeen clones of interest were isolated, and sequence analysis revealed that one of these clones encoded a putative transmembrane protein, rhombencephalic expression protein-40 kDa (Rhombex-40). The rat Rhombex-40 was composed of 374 amino acid residues, and the predicted secondary structure displays a signal peptide in the N-terminus and single-pass transmembrane domain in the center of the sequence. An analysis of consensus sequences identified several phosphorylation sites in the intracellular domain. Expression of rat Rhombex-40 mRNA is high in the brain, and low in lung, liver and kidney. No homologous protein sequence was found in database searches. Whereas the biological function of this protein is presently unknown, its structural features and high expression in the brain suggest that Rhombex-40 may function as a novel transmembrane molecule in neural cells of the brain.     

Divergent effects of estrogen and nicotine on Rho-kinase expression in human coronary vascular smooth muscle cells

Recent studies have demonstrated that up-regulated Rho-kinase plays an important role in the pathogenesis of coronary arteriosclerosis and vasospasm. We have shown that inflammatory stimuli, such as angiotensin II and interleukin-1beta, up-regulate Rho-kinase expression and activity in human coronary vascular smooth muscle cells, for which intracellular signal transduction mediated by protein kinase C and NF-kappaB is involved. Here, we show that estrogen down-regulates while nicotine up-regulates Rho-kinase and that nicotine counteracts the inhibitory effect of estrogen on angiotensin II-induced Rho-kinase expression. Furthermore, we demonstrated that the intracellular signal transduction of the inhibitory effect of estrogen is mediated by an estrogen receptor. These results demonstrate that inflammatory stimuli up-regulate Rho-kinase, for which estrogen (mediated by an estrogen receptor) and nicotine exert divergent inhibitory and stimulatory effects on the Rho-kinase expression, respectively, and may explain in part why the incidence of arteriosclerotic and vasospastic disorders is increased in postmenopausal women and smokers.     

Increased inactivation of nitric oxide is involved in coronary endothelial dysfunction in heart failure

Recent evidence suggests the possibility that enhanced inactivation of endothelium-derived nitric oxide (NO) by oxygen free radical (OFR) may cause endothelial dysfunction in heart failure (HF). To test this hypothesis, we examined the effect of antioxidant therapy on endothelium-dependent vasodilation of the coronary circulation in a canine model of tachycardia-induced HF. Endothelium-dependent vasodilation was less than that in controls, and OFR formation in coronary arterial and myocardial tissues was greater in HF dogs than those in controls. The immunohistochemical staining of 4-hydroxy-2-nonenal, OFR-induced lipid peroxides was detected in coronary microvessels of HF dogs. Intracoronary infusion of the cell-permeable OFR scavenger Tiron inhibited OFR formation and improved endothelium-dependent vasodilation in HF dogs but not in controls. The NO synthesis inhibitor N(G)-monomethyl-L-arginine (L-NMMA) diminished the beneficial effect of Tiron in HF dogs. Endothelium-independent vasodilation was similar between control and HF dogs, and no change in its response was noted by Tiron or Tiron plus L-NMMA in either group. In summary, antioxidant treatment with Tiron improved coronary vascular endothelium-dependent vasodilation by increasing NO activity in tachycardia-induced HF. Thus coronary endothelial dysfunction in HF may be, at least in part, due to increased inactivation of NO by OFR.     

Distribution of prosaposin in rat lymphatic tissues

Prosaposin (PSAP) is as a trophic factor and an activator protein for sphingolipid hydrolase in lysosomes. We generated a specific antibody to PSAP and examined the spatiotemporal distribution of PSAP-immunoreactive (PSAP-IR) cells in the lymphatic tissues of Wistar rats. Immunoblots of tissue homogenates separated electrophoretically showed a single band for PSAP in brain but two bands in spleen. PSAP-IR cells were distributed in both the red and white pulp of the spleen, in both the cortex and medulla of the thymus and in mesenteric lymph nodes. Many PSAP-IR cells were found in the dome portion of Peyer’s patches and the number of PSAP-IR cells increased with the age of the rat. To identify the PSAP-IR cells, double- and triple-immunostainings were performed with antibodies against PSAP, CD68 and CD1d. The large number of double- and triple-positive cells suggested that antigen-presenting cells contained much PSAP in these lymphatic tissues. Intense expression of PSAP mRNA, examined by in situ hybridisation, was observed in the red pulp and corona of the spleen. In rats, the PSAP gene generates two alternative splicing forms of mRNA: Pro+9 containing a 9-base insertion and Pro+0 without the insertion. We examined the expression patterns of the alternative splicing forms of PSAP mRNA in the spleen. The presence of both types of mRNA (Pro+9 and Pro+0) indicated that the spleen contains various types of prosaposin-producing and/or secreting cells. These findings suggest diverse functions for PSAP in the immune system.