Sphingolipid metabolic changes during chiral C2-ceramides induced apoptosis in human leukemia cells.

N-acetylsphingosine (C2-ceramide) is a synthetic water-soluble ceramide mimicking the activity of natural ceramides. By fixing chiral conformation on carbon numbers 2 and 3 in the ceramide structure, four chiral C2-ceramides naming d-erythro-, l-erythro-, d-threo- and l-threo C2-ceramide were synthesized. We have investigated the chiral effects of these C2-ceramides on the sphingolipid metabolism, particularly on both the sphingolipid biosynthetic pathway and on the degradation pathway. In both HL-60 and U937 cells, the chiral C2-ceramide (10 microM) showed sphingosine accumulation monitored fluoromatrically by a high performance liquid chromatographic separation of the sphingoid bases. Most importantly, in HL-60 cells, l-erythro C2-ceramide induced a 50 fold increase in sphingosine as compared to the control, while l-threo C2-ceramide exhibited a minimal 7-fold increase. In contrast, sphinganine, another sphingoid base, showed less accumulation by any chiral C2-ceramide tested under the same conditions. These results suggested that chiral C2-ceramide primarily acts on the sphingolipid degradation pathway rather than on the sphingolipid biosynthetic route. The strong G0/G1 phase arrest in the cell cycle by treatment of l-erythro C2-ceramide indicates that the blockade of the sphingolipid degradation pathway might be concomitantly involved in the dysfunction of the cell cycle. On the other hand, the fact that all chiral C2-ceramides tested failed to inhibit the activity of sphingosine kinase acting on the removal of sphingosine by producing sphingosine-l-phosphate demonstrates that chiral C2- ceramides may increase sphingosine by activating various ceramidases by which natural ceramides are divided into sphingosine and free fatty acids. However, the precise steps involved in this interaction are still unknown.

CAPD, an acceptable form of therapy in elderly ESRD patients: a comparative study.

Rapid growth in the number of dialysis patients over the age of 65 is occurring coincidentally with the overall aging of the general population. Elderly patients are often poor and physically incapacitated, needing family or social support. These patients may also be susceptible to malnutrition and have multiple complicating medical disorders in addition to end-stage renal disease (ESRD). Thus the selection of an appropriate dialysis modality is particularly critical in elderly patients. Continuous ambulatory peritoneal dialysis (CAPD) offers many advantages to elderly patients, including hemodynamic stability, steady-state chemistries, and no need to create a vascular access. However, little data are available in the literature documenting the use of CAPD in this setting. Therefore, to evaluate the efficacy of CAPD in elderly patients, we retrospectively reviewed the clinical outcomes of 23 patients 65 years of age or older at the start of CAPD (elderly group). Then for each of these patients, 23 comparison subjects younger than 65 were chosen from CAPD patients at our hospital (control group). The control group was matched for sex, CAPD duration, cause of ESRD, and initial connection device. In the elderly group, 23 patients (12 male, 11 female) with a mean age of 70 +/- 4 years (range 65-86 years) were treated with CAPD for 15 +/- 17 months. In the control group, 23 patients (12 male, 11 female) with a mean age of 41 +/- 11 (range 18-57) were treated with CAPD for 16 +/- 17 months. Diabetic nephropathy was the cause of ESRD in 35% of patients. The negative CAPD selection of patients was higher in the elderly group (61% vs 17%, p = 0.0025) as well as in the group that needed a helper (61% vs 17%, p = 0.0025). The exit-site infection and peritonitis rates were not statistically different between the two groups (0.43 vs 0.91 episodes/patient-year and 1.46 vs 2.03 episodes/patient-year). The dialysate leakage and bleeding rates were comparable (13% vs 22% and 9% vs 9%). One-year catheter survival was similar in the elderly and younger patients (87.5% vs 84.0%). Although the negative CAPD selection of patients was higher in the elderly group, outcomes were similar to those seen in younger patients. Therefore, CAPD is an acceptable form of therapy for the elderly ESRD patients, particularly if a helper can participate.

Nitric oxide as a messenger molecule for myoblast fusion.

Nitric oxide (NO) is a messenger molecule of vascular endothelial cells, macrophages, and neurons. Here, we demonstrate that the activity of NO synthase increases transiently but dramatically in chick embryonic myoblasts that are competent for fusion. This activity requires Ca2+, calmodulin, and NADPH. In addition, the increase in NO synthase activity coincides with an increase in cellular cGMP level. Furthermore, NO generated by treatment with sodium nitroprusside induces precocious myoblast fusion, while treatment with NG-monomethyl-L-arginine, a competitive inhibitor of NO synthase, or methylene blue, an inhibitor of guanylate cyclase, delays fusion. These results provide the first evidence for a strong association of NO with myoblast fusion.

Involvement of cyclic GMP in the fusion of chick embryonic myoblasts in culture.

We found that a transient rise in cGMP levels, which was closely associated with the Ca2+ influx, occurred concomitant with the onset of myoblast fusion. The Ca2+ channel blocker D600 decreased both the cell fusion and the normal rise in cGMP levels. In contrast, the Ca2+ ionophore A23187 transiently increased cGMP levels and induced precocious fusion. In addition, the cGMP analog 8-Br-cGMP induced precocious fusion as A23187 did. The guanylate cyclase inhibitor, methylene blue delayed the fusion in a dose-dependent manner without significantly affecting cell alignment, proliferation, or muscle-specific protein expression. Furthermore, methylene blue delayed the normal rise in cGMP levels, and the fusion block imposed by methylene blue was significantly recovered by 8-Br-cGMP. On the basis of our present findings, we suggest that a Ca2+ influx-dependent rise in cGMP levels is an important step in myoblast fusion.