Uptake of dehydroascorbic acid in high-GSH and normal-GSH dog erythrocytes

Uptake of dehydroascorbic acid (DHA) was studied in two types of dog erythrocytes with high GSH and normal GSH levels. Compared with ascorbic acid uptake, DHA produced a much greater ascorbic acid accumulation in dog erythrocytes. Both dog erythrocytes showed a concentration dependence of DHA uptake, and cellular ascorbic acid concentrations were significantly higher in high-GSH cells than in normal-GSH cells. Glucose and cytochalasin B inhibited DHA uptake. This suggests that DHA enters dog erythrocytes predominantly by the facilitated glucose transporter, particularly by the Glut 1 glucose transporter. The rate of glucose uptake was quite similar in the two types of cells. Compared with normal-GSH cells, high-GSH cells were more resistant to oxidative stress induced by high concentration of DHA. As a rapid entry of DHA inflicts on cells a heavy demand for GSH for its reduction to ascorbic acid, high-GSH cells containing a larger reserve of GSH have an advantage over normal-GSH cells in both ascorbic acid accumulation and resisting oxidative stress produced by DHA.     

Scintigraphic evaluation of myocardial uptake of thallium 201 and technetium 99m pyrophosphate utilizing a rat model of chronic doxorubicin cardiotoxicity

To evaluate the usefulness of myocardial scintigraphy as a monitoring tool for chronic doxorubicin (DXR) cardiotoxicity, a rat model was used to investigate the relationship between the myocardial uptake of thallium 201 (Tl) or rechnetium 99m pyrophosphate (^99mTc-PPi) and histological changes of the heart. Although there was no significant difference in myocardial Tl uptake between control and DXR-treated rats at an early phase after Tl injection, late-phase Tl uptake was significantly higher in the DXR-treated rats than in the control rats, indicating a slow wash-out of Tl from the myocardium. The wash-out rate calculated from scintigraphic examination of DXR-treated rats was significantly decreased with increasing degree of cardiomyopathy. Since the Tl wash-out rate was sharply decreased even in animals with minimal histological changes, it may be a possible monitoring tool for the early detection of chronic DXR cardiotoxicity. On the other hand, myocardial^99mTc-PPi images could be obtained only in rats with severe myocardial changes and hence would not useful for early detection.     

Left thoracotomy approach in reoperative off-pump coronary revascularization

Objective: Reoperative coronary bypass grafting is at high risk. Particularly in redo cases where the patent graft is running near the midline of the sternum, the graft may be exposed to injury by a median sternotomy and subsequent dissection. Whereas, off-pump bypass grafting from the left axillary artery or descending thoracic artery by a left thoracotomy approach is safe for preventing graft damage.Methods: From March 1998 to February 2002, we performed off-pump coronary artery bypass grafting by a left thoracotomy approach in 9 patients. The left axillary artery was used as the inflow vessel in 4 cases, and the descending thoracic, aorta in 5.Results: The radial artery was anastomosed proximally to the axillary artery in 4 cases and the descending thoracic aorta in one case. The saphenous vein graft was anastomosed, proximally to the descending thoracic aorta in 4 cases. Transdiaphragmatic minimally invasive bypass grafting for the right coronary artery was simultaneously performed in 3 cases. Postoperative cardiac events were ventricular arrhythmia in 6 cases and supraventricular arrhythmia in 3 cases. There was no damage to the patent grafts. Postoperative coronary angiography performed, in 8 cases revealed all the grafts to be patent without stenosis. Cardiac symptoms were not found after the operation in any of the cases.Conclusions: These procedures can prevent the injury to patent grafts caused by a median sternotomy, and will be one of the useful strategies for reoperative off-pump coronary artery bypass grafting.     

Left ventricular diastolic dysfunction as assessed by echocardiography in metabolic syndrome.

The purpose of the present study was to elucidate the cardiac structure and function in patients who have metabolic syndrome but no history of cardiovascular disease by analyzing echocardiographic findings. Echocardiographic examination was performed to screen for cardiovascular disease in 135 patients who were in their sixties. Patients were divided into metabolic syndrome (n=65, age: 65+/-2.7 years) and non-metabolic syndrome (n=70, age: 66+/-2.5 years) groups based on the criteria for metabolic syndrome proposed by the Japanese Society of Hypertension and seven other societies in 2005. The left ventricular (LV) wall thickness and dimension were measured by M-mode echocardiography. The relative wall thickness, LV mass index, and LV ejection fraction (LVEF) were calculated. LV diastolic function was assessed by the peak velocity of early rapid filling (E velocity) and the peak velocity of atrial filling (A velocity), and the ratio of E to A (E/A) was assessed by the transmitral flow. The Tei index, which reflects both LV diastolic and systolic function, was also calculated. There were no differences in relative wall thickness, LV mass index, or LVEF between the two groups. However, both the EIA and Tei index were significantly different between the metabolic syndrome (0.66+/-0.14 and 0.36+/-0.07, respectively) and non-metabolic syndrome (0.88+/-0.25 and 0.29+/-0.09) groups (p<0.001). These results indicate that patients with metabolic syndrome can have cardiac diastolic dysfunction even if they have neither LV hypertrophy nor systolic dysfunction.     

CCR1 acts downstream of NFAT2 in osteoclastogenesis and enhances cell migration.

We found that a chemokine receptor gene, CCR1, acts downstream of NFAT2 in RANKL-stimulated RAW264 and bone marrow cells. The upstream regulatory region of CCR1 showed RANKL-dependent and CsA-suppressible promoter activity. Downregulation of the expression and function of CCR1 suppressed cell migration. INTRODUCTION: We previously reported that the expression of NFAT2 induced by RANKL is a key process for progression to multinucleated cells in an in vitro osteoclastogenesis system. Identifying the target genes of NFAT2 would thus be informative about the differentiation process. We focused here on chemokine and chemokine receptor genes that act downstream of NFAT2 in RAW264 cells as well as osteoclast precursors prepared from bone marrow cells. MATERIALS AND METHODS: RAW264 mouse monocyte/macrophage line cells were cultured with or without cyclosporin A (CsA) in the presence of RANKL or glutathione S-transferase (GST). Osteoclast precursors were prepared from bone marrow cells. RANKL-inducible and CsA-suppressible genes were searched for by microarray analysis, and expression was confirmed by quantitative RT-PCR. Promoter activity was measured by luciferase gene reporter assay. Short interfering (si)RNA for CCR1 was introduced in RAW264 cells. Cell migration activity was examined using a Boyden chamber assay. RESULTS AND CONCLUSIONS: We identified the chemokine receptor gene CCR1 as a gene showing significant differential expression profiles in osteoclastogenesis in the presence versus the absence of CsA, an inhibitor of NFAT. This property was unique to CCR1 among the chemokine and chemokine receptor genes examined in both RAW264 and bone marrow cells. The upstream regulatory region was isolated from CCR1, and its RANKL-dependent and CsA-suppressible promoter activity was confirmed. The functional significance of CCR1 was assessed by monitoring the migration of cells in a transwell migration assay, and this activity was abolished when either CsA- or CCR1 siRNA-treated cells were used. Moreover, treatment with a Galpha inhibitor pertussis toxin (PTX) or methiolynated-regulated on activation, normal T cells expressed and secreted (Met-RANTES), an antagonist of CCR1, suppressed multinucleated cell formation in the bone marrow cell system. Together, these results suggest that the CCR1 signaling cascade is under the control of NFAT2 and seems to enhance the migration of differentiating osteoclasts.     

HLA typing using IL-2 activated T lymphocytes: usefulness in pediatric candidates for allogeneic bone marrow transplantation.

Following a preliminary study in healthy blood donors, we have performed serological HLA-A, B, C, DR and DQ typing using recombinant IL-2 activated T lymphocytes (IL-2.aTLs) in pediatric candidates for allogeneic bone marrow transplantation. In such patients, it is often difficult to obtain the quantity of lymphocytes required for HLA typing, particularly for class II typing using B lymphocytes, considering the timing of sampling and the volume of blood to be collected. Peripheral blood mononuclear cells (PBMCs) were activated and expanded with IL-2 until a sufficient number of IL-2.aTLs of good viability were available for the typing. In the first 10 cases, analyses of surface markers (CD2, CD20, CD25, CD36, HLA-DR and HLA-DQ, CD2/HLA-DR: two color) of IL-2.aTLs were done using flow cytometry at the time of HLA typing and indicated that IL-2.aTLs expressed HLA-DR and DQ antigens sufficient for evaluation. A small number (less than 10(6] of fresh or cryopreserved PBMCs, even those containing leukemic blast cells, were sufficient to induce and expand IL-2.aTLs for HLA typing. To date we have been able to successfully HLA-A, B, C, DR and DQ type 20/20 pediatric candidates. The HLA antigens identified on the patients' IL-2.aTLs were confirmed by a family study.