Visualization of ceramide channels in lysosomes following endogenous palmitoyl-ceramide accumulation as an initial step in the induction of necrosis

In this study, we showed that the dual addition of glucosyl ceramide synthase and ceramidase inhibitors to A549 cell culture led to the possibility of ceramide channel formation via endogenous palmitoyl-ceramide accumulation with an increase in cholesterol contents in the lysosome membrane as an initial step prior to initiation of necrotic cell death. In addition, the dual addition led to black circular structures of 10–20 nm, interpreted as stain-filled cylindrical channels on transmission electron microscopy. The formation of palmitoyl-ceramide channels in the lysosome membrane causes the liberation of cathepsin B from lysosomes for necrotic cell death. On the other hand, necrotic cell death in the dual addition was not caused by oxidative stress or cathepsin B activity, and the cell death was free from the contribution of the translation of Bax protein to the lysosome membrane.     

Effect of O-methylated and glucuronosylated flavonoids from Tamarix gallica on α-glucosidase inhibitory activity: structure–activity relationship and synergistic potential

O-Methylated and glucuronosylated flavonoids were isolated from Tamarix gallica as α-glucosidase inhibitors. Structure–activity relationship of these flavonoids suggests that catechol moiety and glucuronic acid at C-3 are factors in the increase in α-glucosidase inhibitory activity. Furthermore, rhamnetin, tamarixetin, rhamnazin, KGlcA, KGlcA-Me, QGlcA, and QGlcA-Me exhibit synergistic potential when applied with a very low concentration of acarbose to α-glucosidase from rat intestine.     

Growth inhibition of mammalian cells by eosinophil cationic protein.

Eosinophil cationic protein (ECP), one of the major components of basic granules of eosinophils, is cytotoxic to tracheal epithelium. However, the extent of this effect on other cell types has not been evaluated in vitro. In this study, we evaluated the effect of ECP on 13 mammalian cell lines. ECP inhibited the growth of several cell lines including those derived from carcinoma and leukemia in a dose-dependent manner. The IC(50) values on A431 cells, MDA-MB-453 cells, HL-60 cells and K562 cells were estimated to be approximately 1-5 microm. ECP significantly suppressed the size of colonies of A431 cells, and decreased K562 cells in G1/G0 phase. However, there was little evidence that ECP killed cells in either cell line. These effects of ECP were not enhanced by extending its N-terminus. Rhodamine B isothiocyanate-labeled ECP started to bind to A431 cells after 0.5 h and accumulated for up to 24 h, indicating that specific affinity for the cell surface may be important. The affinity of ECP for heparin was assessed and found to be reduced when tryptophan residues, one of which is located at a position in the catalytic subsite of ribonuclease in ECP, were modified. The growth-inhibitory effect was also attenuated by this modification. These results suggest that growth inhibition by ECP is dependent on cell type and is cytostatic.     

Transport of 7-ethyl-10-hydroxycamptothecin (SN-38) by breast cancer resistance protein ABCG2 in human lung cancer cells.

Overexpression of breast cancer resistance protein (BCRP) ABCG2 reportedly confers cancer cell resistance to camptothecin-based anticancer drugs, such as topotecan and 7-ethyl-10-hydroxycamptothecin (SN-38: the active metabolite of irinotecan). We have recently shown that SN-38-selected PC-6/SN2-5H human lung carcinoma cells overexpressed BCRP with the reduced intracellular accumulation of SN-38 and SN-38-glucuronide (S. Kawabata et al., Biochem. Biophys. Res. Commun. 280, 1216-1223, 2001). In the present study, we have examined whether BCRP transports SN-38 and/or SN-38-glucuronide in vitro, by using plasma membrane vesicles from the parental PC-6 and resistant PC-6/SN2-5H cells, where SN-38 and SN-38-glucuronide accumulation in membrane vesicles was measured by HPLC. Both SN-38 and SN-38-glucuronide were ATP-dependently transported into membrane vesicles prepared from PC-6/SN2-5H cells, whereas no transport activity was observed in membrane vesicles from PC-6 cells. The kinetic parameters of the transport observed in PC-6/SN2-5H vesicles were K(m) = 4.0 microM, V(max) = 714 pmol/mg/min for SN-38 and K(m) = 26 microM, V(max) = 833 pmol/mg/min for SN-38-glucuronide. These findings suggest that BCRP expressed in PC-6/SN2-5H cells transports both SN-38 and SN-38-glucuronide with a higher affinity toward SN-38.     

Essential role of mouse Dead end1 in the maintenance of spermatogonia

Dead end is a vertebrate-specific RNA-binding protein implicated in germ cell development. We have previously shown that mouse Dead end1 (DND1) is expressed in male embryonic germ cells and directly interacts with NANOS2 to cooperatively promote sexual differentiation of fetal germ cells. In addition, we have also reported that NANOS2 is expressed in self-renewing spermatogonial stem cells and is required for the maintenance of the stem cell state. However, it remains to be determined whether DND1 works with NANOS2 in the spermatogonia. Here, we show that DND1 is expressed in a subpopulation of differentiating spermatogonia and undifferentiated spermatogonia, including NANOS2-positive spermatogonia. Conditional disruption of DND1 depleted both differentiating and undifferentiated spermatogonia; however, the numbers of A_single and A_paired spermatogonia were preferentially decreased as compared with those of A_aligned spermatogonia. Finally, we found that postnatal DND1 associates with NANOS2 in vivo, independently of RNA, and interacts with some of NANOS2-target mRNAs. These data not only suggest that DND1 is a partner of NANOS2 in undifferentiated spermatogonia as well as in male embryonic germ cells, but also show that DND1 plays an essential role in the survival of differentiating spermatogonia.     

Vitamin D and phosphate regulate fibroblast growth factor-23 in K-562 cells

Female rats were treated with FSH (40 IU/kg) on the first and second diestrus days (D1 and D2) and with LH (40 IU/kg) on the proestrus (P) day to synchronize and maximize ovarian changes. Follicle area increased by 50% from D1 to P, and the estrus (E) phase showed multiple corpora lutea and massive apoptosis. Increased oxygen uptakes (42-102%) were determined in ovary slices and in isolated mitochondria in active state 3 along the proliferation phase (D1-D2-P) that returned to initial values in the E phase. Mitochondrial content and the electron transfer activities of complexes I and IV were also maximal in the P phase (20-79% higher than in D1). Production of NO by mitochondrial nitric oxide synthase (mtNOS), biochemically determined, and the mtNOS functional activity in regulating state 3 oxygen uptake were also maximal at P and 79-88% higher than at D1. The moderately increased rate of NO in the proliferative phase is associated with mitochondrial biogenesis, whereas the high rate of NO generation by mtNOS at phase P appears to trigger mitochondria-dependent apoptosis. The calculated fraction of ovary mitochondria in state 3 was at a minimal value at the P phase. Mitochondrial oxidative damage, with increased thiobarbituric acid-reactive substances and protein carbonyls, indicates progressive mitochondrial dysfunction between phases P and E. The roles of mitochondria as ATP provider, as a source of NO to signal for mitochondrial proliferation and mitochondria-dependent apoptosis, and as a source of O(2)(-) and H(2)O(2) appear well adapted to serve the proliferation-apoptosis sequence of the ovarian cycle.     

Oxygen concentration regulates NO-dependent relaxation of aortic smooth muscles

Nitric oxide (NO) functions as an endothelium-derived relaxation factor and regulates vascular resistance. Recent studies in this laboratory (Arch. Biochem. Biophys. 323, 27–32, 1995) revealed that the lifetime of NO significantly increased at physiologically low levels of oxygen concentrations and, hence, this gaseous radical strongly inhibited mitochondrial electron transport for a fairly long duration at low oxygen concentrations. The present work describes the effect of oxygen concentration on NO-induced relaxation and guanylate cyclase (GC) activity of endothelium-denuded aorta of the rat. Both NO and 2,2′-hydroxynitrosohydrazono)bis-ethanamine (NOC18), an NO donor, induced the relaxa-tion of endothelium-denuded helical segments of rat aorta which were contracted by norepinephrine. NO-dependent relaxation of arterial specimens was enhanced by lowering oxygen concentration in the medium with concomitant increase in their cGMP levels. Anoxia induced the relaxation of the aorta by some NO-enhanceable and methylene blue-insensitive mechanism. These results suggested that local concentrations of oxygen might play important roles in the regulation of NO-dependent GC activity and vascular tonus of resistance arteries.     

Hyaluronan synthesis by anaplastic large cell lymphoma with massive lymphomatous effusion. A case report

BACKGROUND: Hyaluronan (HA) synthesis is frequently observed in malignant mesothelioma cells, whereas it is rarely found in lymphoma cells. Previous studies have reported that a high HA concentration in the serum was related to poor prognosis in lymphomas, although the mechanism was not elucidated. We recently encountered a case of anaplastic large cell lymphoma with an HA-rich, massive, lymphomatous effusion. Several studies were performed to clarify the character of this unusual lymphoma and to observe whether the lymphoma cells synthesized HA. CASE: A 59-year-old female was admitted with abdominal pain. Radiologic studies revealed a pleural effusion and paraaortic lymph node swelling. A biopsied specimen was compatible with anaplastic large cell lymphoma. Detailed cytologic observations revealed that the lymphoma cells in the pleural effusion had alcian blue-positive, productive material in the prominent Golgi area and microvillous structures on the surface. Further studies found that most of the lymphoma cells had HA-binding protein and expressed CD44 antigen, a receptor for HA. In addition, the HA concentration in the supernatant of the primary culture cells was extremely high and increased time dependently. CONCLUSION: These observations suggest that the lymphoma cells synthesized and released HA. Interactions of the released HA and CD44 on the surface might play an important role in the peculiar serosal growth of lymphoma cells.