Exposure to oxidized low-density lipoprotein reduces activable Ras protein in vascular endothelial cells

Oxidized low-density lipoprotein (ox-LDL) has been shown to alter the migratory and proliferative activities of the vascular endothelial cells (EC) in response to serum and growth factors. The mechanism underlying the antiproliferative effect of ox-LDL on vascular EC has not been fully elucidated. In this report, we show that exposure of vascular EC to ox-LDL results in a marked reduction of the membrane-associated Ras protein. Further study shows that in ox-LDL-treated EC, reduction of the membrane-associated Ras protein is correlated with a reduced amount of active Ras (Ras-guanosine triphosphate), indicating that the Ras signaling pathway is attenuated. The attenuation of the Ras signaling pathway in ox-LDL-treated EC may thus be responsible for the retarded response to the mitogenic stimulation of serum and growth factors.     

A salvianolic acid B-rich fraction of Salvia miltiorrhiza induces neointimal cell apoptosis in rabbit angioplasty model

Apoptosis has been suggested to participate in stabilizing cell number in restenosis. Salvia miltiorrhiza (SM) Bunge which is a Chinese herb widely used for the treatment of cardiovascular disorders contains a potent antioxidant, Salvianolic acid B. To determine whether the antioxidant affects vascular apoptosis, the present study examined the frequency of apoptotic cell death in atherosclerotic plaques and in restenotic lesions of cholesterol-fed rabbits. New Zealand White rabbits were treated with a normal diet (normal), a 2% cholesterol diet (HC), a 2% cholesterol diet and endothelial denudation (HC-ED), a 2% cholesterol diet with 5% water-soluble extract of SM (4.8 g/Kg B.W./day) and endothelial denudation (HC-ED-SM), or with a 2% cholesterol diet containing probucol (0.6 g/kg B.W./day) and endothelial denudation (HC-ED-probucol). Apoptosis and associated cell types were examined in serial paraffin sections by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunohistochemistry. The expression of p53, an apoptosis-related protein, was also examined. Apoptosis was mainly detected in the neointima of the three groups with endothelial denudation. The percentage of apoptotic cells in SM-treated group (68.5+/-5.9%) was significantly higher than that of normal (0%), HC (1.9+/-1.2%), HC-ED (46.1+/-5.4%), and probucol-treated (32.8+/-3.9%) groups. The SM treatment markedly reduced the thickness of the neointima which was mainly composed of smooth muscle cells with few macrophages. In accordance with the apoptotic cell counts, positive immunoreactivity for p53 was observed in restenotic lesions from HC-ED, SM-treated and probucol-treated groups but not in the intima of the other two groups. These results suggest that the treatment with salvianolic acid B-rich fraction of SM induces apoptosis in neointima which in turn may help prevent the neointimal thickening.     

Monoubiquitin carries a novel internalization signal that is appended to activated receptors

Ubiquitin modification of signal transducing receptors at the plasma membrane is necessary for rapid receptor internalization and downregulation. We have investigated whether ubiquitylation alters a receptor cytoplasmic tail to reveal a previously masked internalization signal, or whether ubiquitin itself carries an internalization signal. Using an alpha-factor receptor-ubiquitin chimeric protein, we demonstrate that monoubiquitin can mediate internalization of an activated receptor that lacks all cytoplasmic tail sequences. Furthermore, fusion of ubiquitin in-frame to the stable plasma membrane protein Pma1p stimulates endocytosis of this protein. Ubiquitin does not carry a functional tyrosine- or di-leucine-based internalization signal. Instead, the three-dimensional structure of the folded ubiquitin polypeptide carries an internalization signal that consists of two surface patches surrounding the critical residues Phe4 and Ile44. We conclude that ubiquitin functions as a novel regulated internalization signal that can be appended to a plasma membrane protein to trigger downregulation.     

Expression of SpC3, the sea urchin complement component,in response to lipopolysaccharide

The homologue of the vertebrate complement component C3 that is expressed in the coelomocytes of the purple sea urchin, Strongylocentrotus purpuratus, designated SpC3, was investigated for changes in response to immune challenge or injury. Immunoquiescent animals were used in this study because they have reduced or no detectable SpC3 in their coelomocytes or coelomic fluid (CF). Animals were injected with lipopolysaccharide (LPS) or sterile sea water (SSW, injury control). Changes in the amounts of SpC3 in coelomic fluid and in coelomocytes were then followed over time by Western blots and ELISA. Changes in mRNA from the SpC3 gene (Sp064) were also followed by RT-PCR. Although all animals responded to injury with increased levels of SpC3 in the coelomic fluid, those challenged with LPS had greater amounts of SpC3 in both CF and coelomocytes than those receiving SSW. In most of the animals receiving LPS, initial increases in SpC3 were observed within 1 h post-injection, while the earliest response in the animals receiving SSW was 6 h. The appearance of SpC3 in the coelomocytes was delayed compared to its appearance in CF, and was first detected several days after challenge. Changes in mRNA from the Sp064 gene paralleled the appearance of SpC3 in the coelomic fluid. Increases in the number of coelomocytes per milliliter of CF and in the percentage of coelomocytes that were SpC3+ also occurred after challenge with LPS or in response to injury, with a slightly greater increase in response to LPS. Although the changes in SpC3 were not as great as those identified previously for human C3 expressed in macrophages, the kinetics of the response are similar to that of acute-phase reactants in mammals.     

The hPLIC proteins may provide a link between the ubiquitination machinery and the proteasome

Although there is a binding site on the proteasome for the polyubiquitin chains attached to degradation substrates by the ubiquitination machinery, it is currently unclear whether in vivo the activities of the ubiquitination machinery and the proteasome are coupled. Here we show that two human homologs of the yeast ubiquitin-like Dsk2 protein, hPLIC-1 and hPLIC-2, physically associate with both proteasomes and ubiquitin ligases in large complexes. Overexpression of hPLIC proteins interferes with the in vivo degradation of two unrelated ubiquitin-dependent proteasome substrates, p53 and IkappaBalpha, but not a ubiquitin-independent substrate. Our findings raise the possibility that the hPLIC proteins, and possibly related ubiquitin-like family members, may functionally link the ubiquitination machinery to the proteasome to affect in vivo protein degradation.     

Gastric mucosal damage induced by arecoline seizure in rats

In an attempt to know the relation of seizure and gastric mucosal damage, we challenged arecoline (ACL) centrally to induce seizure and investigated gastric hemorrhagic injury in acid-irrigated stomachs of rats. The protective effects of several drugs also were evaluated. After deprivation of food for 24 h, rats were received laparotomy under diethylether-anesthesia. Both pylorus sphincters and carotid esophagus were ligated. The forestomach was equipped with a cannula for gastric irrigation. After recovery from anesthesia (approximately 1 h), the stomach was irrigated for 2 h with an acid solution containing 100 mM HCl and 54 mM NaCl or the same volume of normal saline. Intracerebroventricular (i.c.v.) ACL (0, 1, 3 or 10 mg/kg dissolved in 10 microl of CSF) was challenged to rats immediately after gastric irrigation. The seizure in rats was produced by ACL in a dose-related manner. The ulcerogenic parameters such as decrease of gastric mucosal glutathione levels and increase of histamine concentrations and lipid peroxide generations as well as the raise of luminal hemoglobin contents and exacerbated mucosal lesions were obtained depending on the doses of ACL challenged. These ulcerogenic parameters produced in ACL (10 mg/kg, i.c.v.) seizure rats were markedly ameliorated by gastric vagotomy or central anticholinergics. Intraperitoneal ketotifen, zinc sulfate, diphenhydramine or cimetidine also produced significant (p<0.05) inhibitions of these ulcerogenic parameters in ACL seizure rats. In conclusion, central ACL seizure may produce gastric oxidative stress and hemorrhagic lesions via vagal nervous activation and histamine release in acid-irrigated stomachs of rats.