Big mitogen-activated protein kinase 1 (BMK1)/extracellular signal regulated kinase 5 (ERK5) is involved in platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell migration

Big mitogen-activated protein kinase 1 (BMK1), also known as extracellular signal-regulated kinase 5 (ERK5), is a newly identified member of the mitogen-activated protein (MAP) kinase family. Recently, several studies have suggested that BMK1 plays an important role in the pathogenesis of cardiovascular disease. To clarify the pathophysiological significance of BMK1 in the process of vascular remodeling, we explored the molecular mechanisms of BMK1 activation in vascular smooth muscle cells (VSMCs). From the results of co-immunoprecipitation and immunoblotting analyses, it was found that platelet-derived growth factor (PDGF), a known potent mitogen, activated BMK1 and triggered the Gab1-SHP-2 interaction in rat aortic smooth muscle cells (RASMCs). The abrogation of SHP-2 phosphatase activity by transfection of the SHP-2-C/S mutant suppressed PDGF-stimulated BMK1 activation. Infection with an adenoviral vector expressing dominant-negative MEK5alpha, which can suppress PDGF-stimulated BMK1 activation to the control level, inhibited PDGF-induced RASMC migration. Moreover, we observed an increase of BMK1 activation in injured mouse femoral arteries. From these findings, it is suggested that BMK1 activation leads to VSMC migration induced by PDGF via Gab1-SHP-2 interaction, and that BMK1-mediated VSMC migration may play a role in the pathogenesis of vascular remodeling.     

Role of Ca2+ on Endotoxin-Sensitivity by Galactosamine Challenge: Lipid Peroxide Formation and Hepatotoxicity in Zymosan-Primed Mice

Abstract: This study was investigated to clarify the role of intracellular Ca²⁺ following endotoxin treatment (1 mg/kg, intraperitoneally) to D-galactosamine-sensitized mice (400 mg/kg, intraperitoneally), and to observe lipid peroxide levels, an index of hepatotoxicity, in endotoxin/galactosamine (Ga1N)-challenged mice under activation of macrophages, especially Kupffer cells, by zymosan. The liver lipid peroxide level and serum glutamic pyruvic transminase activity in mice 18 hr after administration of endotoxin/Ga1N were markedly higher than those in mice treated only with endotoxin. In spite of an increase in lipid peroxide formation, there was little or no effect of Ga1N administration on xanthine oxidase and superoxide dismutase activities in mice given endotoxin. However, the injection of verapamil (10 mg/kg, subcutaneously) markedly decreased lipid peroxide levels in liver of endotoxin/Ga1N-injected mice. In the mice given a Ca²⁺-deficient diet, lipid peroxide level in liver after endotoxin/Ga1N injection was markedly decreased compared to that in mice fed a normal diet. Administration of dexamethasone (200 μg/kg, intraperitoneally) in mice 1 hr before treatment with endotoxin/Ga1N did not induce lipid peroxide formation. Administration of endotoxin to Ga1N-treated mice resulted in a higher level of liver cytosolic free Ca²⁺ ([Ca²⁺]_i) than that in endotoxin-treated mice. On the other hand, Ca²⁺-ATPase activity in liver plasma membrane in the endotoxin/Ga1N-treated mice was markedly decreased as compared with endotoxin alone. On the contrary, the Ca²⁺-ATPase activity in liver mitochondria was higher in endotoxaemic mice treated with Ga1N than in mice given endotoxin alone. State 3 respiration and respiratory control index, which are parameters of mitochondrial function, were decreased more in the liver of mice treated with endotoxin/Ga1N than in the endotoxin-treated group. These findings suggest that [Ca²⁺]_i may participate in the lipid peroxide formation which results from endotoxin/Ga1N-induced hepatotoxicity under conditions of zymosan-activated macrophages, and that the increases of endotoxin-sensitivity caused by Ga1N challenge may greatly contribute to Ca²⁺-mobilization in the hepatocyte.     

Immunohistochemical detection of imidazolone, a novel advanced glycation end product, in kidneys and aortas of diabetic patients.

To investigate the role of the Maillard reaction in the pathogenesis of diabetic complications, we produced several clones of monoclonal antibodies against advanced glycation end products (AGEs) by immunizing mice with AGE-modified keyhole limpet hemocyanin, and found that one clone (AG-1) of the anti-AGE antibodies reacted specifically with imidazolones A and B, novel AGEs. Thus, the imidazolones, which are the reaction products of the guanidino group of arginine with 3-deoxyglucosone (3-DG), a reactive intermediate of the Maillard reaction, were found to be common epitopes of AGE-modified proteins produced in vitro. We determined the erythrocyte levels of imidazolone in diabetic patients using ELISA with the monoclonal anti-imidazolone antibody. The imidazolone levels in the erythrocytes of diabetic patients were found to be significantly increased as compared with those of healthy subjects. Then we studied the localization of imidazolone in the kidneys and aortas obtained from diabetic patients by immunohistochemistry using the antibody. Specific imidazolone immunoreactivity was detected in nodular lesions and expanded mesangial matrix of glomeruli, and renal arteries in an advanced stage of diabetic nephropathy, as well as in atherosclerotic lesions of aortas. This study first demonstrates the localization of imidazolone in the characteristic lesions of diabetic nephropathy and atherosclerosis. These results, taken together with a recent demonstration of increased serum 3-DG levels in diabetes, strongly suggest that imidazolone produced by 3-DG may contribute to the progression of long-term diabetic complications such as nephropathy and atherosclerosis.     

Case Report: Gianotti-Crosti Syndrome Associated with Human Herpesvirus-6 Infection

We report a case of Gianotti-Crosti syndrome associated with human herpesvirus-6 (HHV-6) infection. An eight-month-old girl developed monomorphous papules on her cheeks, buttocks, and extremities after the subsidence of exanthema subitum. Viral antibody analysis confirmed primary HHV-6 infection. HHV-6 may be added to the list of causative agents of Gianotti-Crosti syndrome.     

The inclusion of an omental flap in pancreatoduodenectomy

A technique for reducing the morbidity and mortality of pancreatoduodenectomy by using an omental flap to protect the anastomoses and splanchnic vessels exposed during dissection is described herein.     

Two cases of HTLV-I associated myelopathy (HAM) after cardiac operation

Sixty-one and 51-year-old males had progressive walk disturbance after cardiac operations at the intervals of 1 and 2 years. They had received blood transfusion at their operations. Physical examination revealed spastic paraparesis, sensory disturbance of the lower and rectobladder disorder. High titers of anti-Human T-lymphotrophic Virus type-I (HTLV-I) antibody were found in sera and CNF in both cases. They were diagnosed as HTLV-I associated myelopathy (HAM). Because they lived in Aichi Prefecture where the virus is non-epidemic, they were possibly infected through the blood transfusion at their operations. For prevention of HAM, the anti-HTLV-I antibody of all donor blood should be checked before transfusion.     

Structural changes and cell viability of cultured epithelium after freezing storage

Numerous clinical reports have shown the utility of cultured epithelial grafting in the field of plastic and reconstructive surgery. Recently, freezing storage of the cultured epithelium has been tried and has successfully grafted after thawing. It is clinically convenient if it is possible for cultured epithelium to keep its normal structure and viability. However, few papers have described the structural changes in cultured epithelium after freezing storage. In the present study, the morphological changes and cell viability of cultured mucosal epithelial sheets after freezing were studied in comparison with cultured epidermal sheets. Furthermore, we discuss the effect of storage temperature and cryoprotectants.As a result, there were some structural changes such as vacuolar degeneration in the cultured mucosal sheets using dimethyl sulphoxide (DMSO) as a cryoprotectant. Such changes were more clearly observed at −80°C than at −196°C with DMSO. However, little morphological change was observed in both epithelial sheets cultured with glycerin. The cell viability analysed by flow cytometry showed that more than 62% of the cells kept their viability after freezing storage. These results suggest that the optimum conditions of freezing for cultured epithelium were −196°C storage by slow cooling methods with glycerin as a cryoprotectant.     

Disease associations and altered immune function in CD45 138G variant carriers

The CD45 antigen is a haemopoietic cell specific tyrosine phosphatase essential for antigen receptor mediated signalling in lymphocytes. Expression of different patterns of alternatively spliced CD45 isoforms is associated with distinct functions. We recently identified a polymorphism in exon 6 (A138G) of the gene encoding CD45 (PTPRC) that results in altered CD45 splicing. The 138G allele is present at a high frequency among Japanese (23.7%), with 5.1% individuals homozygous for the G allele. In this study we show that the A138G polymorphism is the cause of altered CD45 isoform expression, promoting splicing towards low molecular weight CD45 isoforms. We further report that the frequency of A138G heterozygotes is significantly reduced in number in cohorts of patients with autoimmune Graves' disease or hepatitis B infection, whereas G138G homozygotes are absent from a cohort of Hashimoto's thyroiditis patients. We also show that 138G individuals exhibit altered cytokine production in vitro and an increased proportion of memory T cells. These data suggest that the 138G variant allele strongly influences these diseases by modulation of immune mechanisms and may have achieved its high frequency as a result of a natural selection probably related to pathogen resistance.     

Synergism between interleukin-11 and bone morphogenetic protein-2 in the healing of segmental bone defects in a rabbit model.

Recombinant human interleukin-11 (rHuIL-11) and recombinant human bone morphogenetic protein-2 (rHuBMP-2) have been shown to act synergistically in the induction of osteoblast differentiation. To determine whether these two proteins can be used clinically in fracture healing and reconstructive surgery, we investigated whether rHuIL-11 and rHuBMP-2 act synergistically to heal segmental bone defects in a rabbit model. A 1.5-cm segmental defect was created in the right ulnar diaphysis of 20 Japanese white rabbits. Polylactic-co-glycolic acid (PLGA)-coated gelatin sponges (PGS) permeated with rHuBMP-2 (n = 8), rHuIL-11 plus rHuBMP-2 (n = 8), or rHuIL-11 (n = 4) were implanted into the bone defects. Radiographs were scored by two independent observers for bone formation and union rates after 2, 3, 4, and 8 weeks. Bone formation was higher in rabbits implanted with rHuBMP-2 plus rHuIL-11 than in those implanted with rHuBMP-2 alone, reaching statistical significance after 4 weeks. At early time points, the union rate in rabbits implanted with rHuBMP-2 plus rHuIL-11 was higher than in rabbits implanted with rHuBMP-2. At 2, 4, and 8 weeks, new bone volume was significantly higher in rabbits administered rHuIL-11 plus rHuBMP-2 than in those given rHuBMP-2 alone. In contrast, mechanical testing after 8 weeks showed that bone strength in the two groups of rabbits was equivalent. These findings show that rHuIL-11 and rHuBMP-2 act synergistically to accelerate bone formation without affecting bone strength. Treatment with a combination of rHuIL-11 and rHuBMP-2 may thus be of great benefit in fracture healing and for patients undergoing reconstructive surgery.