The mechanism of gamma-secretase activities through high molecular weight complex formation of presenilins is conserved in Drosophila melanogaster and mammals

Mutations in presenilin 1 (PS1) and PS2 genes contribute to the pathogenesis of early onset familial Alzheimer's disease by increasing secretion of the pathologically relevant Abeta42 polypeptides. PS genes are also implicated in Notch signaling through proteolytic processing of the Notch receptor in Caenorhabditis elegans, Drosophila melanogaster, and mammals. Here we show that Drosophila PS (Psn) protein undergoes endoproteolytic cleavage and forms a stable high molecular weight (HMW) complex in Drosophila S2 or mouse neuro2a (N2a) cells in a similar manner to mammalian PS. The loss-of-function recessive point mutations located in the C-terminal region of Psn, that cause an early pupal-lethal phenotype resembling Notch mutant in vivo, disrupted the HMW complex formation, and abolished gamma-secretase activities in cultured cells. The overexpression of Psn in mouse embryonic fibroblasts lacking PS1 and PS2 genes rescued the Notch processing. Moreover, disruption of the expression of Psn by double-stranded RNA-mediated interference completely abolished the gamma-secretase activity in S2 cells. Surprisingly, gamma-secretase activity dependent on wild-type Psn was associated with a drastic overproduction of Abeta1-42 from human betaAPP in N2a cells, but not in S2 cells. Our data suggest that the mechanism of gamma-secretase activities through formation of HMW PS complex, as well as its abolition by loss-of-function mutations located in the C terminus, are highly conserved features in Drosophila and mammals.     

Polyhistidine tract expansions in HOXA1 result in intranuclear aggregation and increased cell death

HOXA1 gene is part of a cluster of homeotic selector genes that regulates the anteroposterior patterning of mammals during embryonic development. HOXA1 encodes two alternatively spliced mRNAs with two isoforms, A and B, the former contains the homeodomain and expressed in early embryonic development. HOXA1 contains a string of 10 histidine repeats. However, individuals heterozygous for 7, 9, 11, and 12 histidine repeat variants were present among the Japanese population, notably in some autism cases. To determine the biological implications of the different polyhistidine repeat lengths, we expressed these variants in COS-7 and a human neuroblastoma cell line (SK-N-SH). Expression of expanded variants of HOXA1 isoform A, containing 11 and 12 polyhistidine, resulted in early and great degree of protein aggregation in the nucleus. This aggregation resulted in accelerated cell death in cells expressing 11 and 12 expanded variants compared to those transfected with 7 and 10 polyhistidine variants. Furthermore, we showed that these aggregates were ubiquitinated and were inhibited by a histidine-modifying compound, DEPC. These data suggest that HOXA1 protein with polyhistidine tract expansions misfold, aggregate, and have a toxic effect on cell.     

Glutathione content is correlated with the sensitivity of lines of PC12 cells to cisplatin without a corresponding change in the accumulation of platinum

A study of the involvement of glutathione (GSH) in cellular resistance to cisplatin was performed using methylmercury-resistant sublines (PC12/TM series) of the PC12 line of rat pheochromocytoma cells. The seven clonal sublines of PC12 cells (PC12/TM, PC12/TM2, PC12/TM5, PC12/TM11, PC12/TM15, PC12/TM23, PC12/TM26) used in the study had intracellular levels of GSH that ranged from 8.7–39.9 nmol/mg protein. The intracellular level of GSH was significantly correlated (p < 0.01, r = 0.87) with the sensitivity to cisplatin of PC12 cells and the seven sublines. Among the seven sublines, PC12/TM cells contained the highest concentration of GSH and were the most resistant to cisplatin. Treatment of PC12/TM cells with L-buthionine-SR-sulfoximine, which reduced the level of GSH to that in the parental PC12 cells, significantly reduced the resistance of the cells to cisplatin. The amount of platinum accumulated by resistant PC12/TM cells after treatment with cisplatin was higher than that by sensitive PC12 cells. These results suggest that the intracellular level of GSH might be directly involved in the resistance to cisplatin of these cell lines. However, a high intracellular concentration of GSH does not appear to contribute to a decrease in the accumulation of cisplatin in these cells.     

Suppressed proliferative response of spleen T cells from metallothionein null mice

To investigate the role of metal-binding protein, metallothionein (MT), in lymphocyte activation, the mitogen-induced proliferation of freshly isolated spleen cells was compared among MT-I, II null, and control 129/Sv mice. Spleen cells from MT null mice exhibited a markedly reduced proliferation compared with control cells when stimulated by concanavalin A or anti-CD3(epsilon) mAb, but not by lipopolysaccharide, indicating that only the response of T cells to mitogens was suppressed in MT null mice. Flow cytometric analysis of unstimulated spleen cells demonstrated no significant difference in the relative percentages of either B220+ and CD3+ cells or CD4+ and CD8+ cells between the two strains of mice. The production of interleukin (IL)-2 by MT null spleen cells after the stimulation by anti-CD3(epsilon) mAb was lower than that of control spleen cells, especially within 24 hr after the stimulation. The addition of IL-2 recovered the proliferation of MT null spleen cells to the control level. The reduced proliferative response to mitogenic stimulation of MT null T cells was confirmed by using purified splenic T cells. These results suggest that the MT expressed at basal level in the splenocytes plays an important role in T cell mitogen-induced proliferative response, probably by positively regulating the production of IL-2.     

Involvement of Oxidative Stress in Paraquat-Induced Metallothionein Synthesis Under Glutathione Depletion

The inhibition of glutathione (GSH) synthesis by -buthionine-SR-sulfoximine (BSO) causes aggravation of hepatotoxicity of paraquat (PQ), an oxidative-stress inducing substance, in mice. On the other hand, synthesis of metallothionein (MT), a cysteine-rich protein having radical scavenging activity, is induced by PQ, and the induction by PQ is significantly enhanced by pretreatment of mice with BSO. The purpose of present study is to examine whether generation of reactive oxygens is involved in the induction of MT synthesis by PQ under inhibition of GSH synthesis. Administration of PQ to BSO-pretreated mice increased hepatic lipid peroxidation and frequency of DNA single strand breakage followed by manifestation of the liver injury and induction of MT synthesis. Both vitamin E and deferoxamine prevented MT induction as well as lipid peroxidation in the liver of mice caused by administration of BSO and PQ. In cultured colon 26 cells, both cytotoxicity and the increase in MT mRNA level caused by PQ were significantly enhanced by pretreatment with BSO. Facilitation of PQ-induced reactive oxygen generation was also observed by BSO treatment. These results suggest that reactive oxygens generated by PQ under inhibition of GSH synthesis may stimulate MT synthesis. GSH depletion markedly increased reactive oxygen generation induced by PQ, probably due to the reduced cellular capability to remove the radical species produced.     

Plant regeneration via somatic embryogenesis from protoplasts of six plant species related to Citrus

Protoplasts isolated from embryogenic callus of Fortunella polyandra (Ridl.), Atalantia bilocularis (Pieree ex Guill.), Hesperethusa crenulata (Roxb.), Glycosmis pentaphylla (Retz.) Corr., Triphasia trifolia (Burm. f.) P. Wils. and Murraya koenigii (L.) Spreng. were cultured in MT (Murashige and Tucker 1969) basal medium containing 5% sucrose supplemented with 0.0, 0.001, 0.01, 0.1 or 1.0 mg l^–1 BA and 0.6 M sorbitol. The highest plating efficiencies for all species were obtained on MT basal medium containing 5% sucrose supplemented with 0.001 mg l^–1 BA. F. polyandra produced higher percentages of globular somatic embryo development, while A. bilocularis consistently showed a lower percentage of globular somatic embryo development in all 5 concentrations of BA. MT basal medium containing 5% sucrose and supplemented with 0.001 mg l^–1 BA was found to be a suitable medium for development of globular somatic embryos derived from protoplasts to form heart-shaped somatic embryos with cotyledon-like structures. The highest percentages of shoot formation for all 6 species were obtained using 0.1 mg l^–1 GA₃. A complete protoplast-to-plant system was developed for F. polyandra, A. bilocularis and T. trifolia, which could facilitate the transfer of nuclear and cytoplasmic genes from these species into cultivated Citrus through protoplast fusion.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA₃ gibberellin A₃     

Characterization of C-4-methylated sterols by pyridine-induced solvent shifts in proton magnetic resonance spectroscopy

The proton magnetic resonance (PMR) spectra were measured in deuterochloroform (CDCl₃) and pyridine solutions for some 4-desmethyl, 4α-methy1, 4β-methyl, and 4,4-dimethyl sterols related to 5α-cholestane series as well as for their C-3-oxo derivatives. The influence of pyridine, relative to CDCl₃, on methyl group chemical shifts was discussed. The technique utilizing pyridine-induced solvent shifts in PMR spectroscopy was found useful in characterizing the individual classes of sterols.