No abstract

Missense mutations in the human presenilin-1 (PS1) gene, which is found on chromosome 14, cause early-onset familial Alzheimer's disease (FAD). FAD-linked PS1 variants alter proteolytic processing of the amyloid precursor protein and cause an increase in vulnerability to apoptosis induced by various cell stresses. However, the mechanisms responsible for these phenomena are not clear. Here we report that mutations in PS1 affect the unfolded-protein response (UPR), which responds to the increased amount of unfolded proteins that accumulate in the endoplasmic reticulum (ER) under conditions that cause ER stress. PS1 mutations also lead to decreased expression of GRP78/Bip, a molecular chaperone, present in the ER, that can enable protein folding. Interestingly, GRP78 levels are reduced in the brains of Alzheimer's disease patients. The downregulation of UPR signalling by PS1 mutations is caused by disturbed function of IRE1, which is the proximal sensor of conditions in the ER lumen. Overexpression of GRP78 in neuroblastoma cells bearing PS1 mutants almost completely restores resistance to ER stress to the level of cells expressing wild-type PS1. These results show that mutations in PS1 may increase vulnerability to ER stress by altering the UPR signalling pathway.

The potential use of circularly polarized luminescence for object identification in a sensor application is demonstrated. New luminescence probes using pyrene derivatives as sensor luminophores were developed. (R,R)-Im Py and (S,S)-Im Py contain two chiral imidazole moieties at 1,6-positions through ethynyl spacers (angle between spacers ca. 180°). The probe molecules spontaneously self-assemble into chiral stacks (P or M helicity) upon coordination to metal ions with tetrahedral coordination (Zn ). The chiral probes display neither circular dichroism (CD) nor circularly polarized luminescence (CPL) without metal ions. However, (R,R)-Im Py and (S,S)-Im Py exhibit intense chiroptical activity (CD and CPL) upon self-assembly with Zn ions. (R,R)-Im Py and (S,S)-Im Py with chemical stimuli-responsibility allow sensing using the CPL signal as detection output, enabling us to discriminate between a signal from the target analyte and that from non-target species.

Mouse embryonic stem (ES) cells can proliferate indefinitely in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), or differentiate into all three germ layers upon removal of this factor. To determine cellular factors associated with self-renewal of undifferentiated ES cells, we used polymerase chain reaction-assisted cDNA subtraction to screen genes that are expressed in undifferentiated ES cells and down-regulated after incubating these cells in a differentiation medium without LIF for 48 h. The mRNA expression of a tetraspanin transmembrane protein, CD9, was high in undifferentiated ES cells and decreased shortly after cell differentiation. An immunohistochemical analysis confirmed that plasma membrane-associated CD9 was expressed in undifferentiated ES cells but low in the differentiated cells. Addition of LIF to differentiating ES cells reinduced mRNA expression of CD9, and CD9 expression was accompanied with a reappearance of undifferentiated ES cells. Furthermore, activation of STAT3 induced the expression of CD9, indicating the LIF/STAT3 pathway is critical for maintaining CD9 expression. Finally, addition of anti-CD9 antibody blocked ES cell colony formation and reduced cell viability. These results indicate that CD9 may play a role in LIF-mediated maintenance of undifferentiated ES cells. INTRODUCTIONMouse embryonic stem (ES) cells, which originally derived from inner cell mass of an early embryo named blastocyst, are able to sustain their pluripotency in in vitro cell culture (Evans and Kaufman, 1981;Martin, 1981). Undifferentiated mouse ES cells can be maintained for a long time in media containing the cytokine leukemia inhibitory factor (LIF) (Smith et al., 1988;Williams et al., 1988). Pluripotency of such cultured ES cells has been demonstrated in both in vivo and in vitro experiments. When injected into blastocysts, ES cells participate in embryonic development involving all three germ layers producing chimeric mice (Bradley et al., 1984). ES cells also form teratomas containing various mature tissues when injected into immunocompromised mice (Evans and Kaufman, 1981;Martin, 1981). In vitro, mouse ES cells start to differentiate to every possible lineage upon removal of LIF from the culture medium. The mechanism by which LIF maintains ES cells in undifferentiated state is not completely understood. The transcription factor STAT3 is a downstream target of LIF and its receptor interaction Matsuda et al., 1999). It has been shown that the activity of STAT3 is necessary and sufficient for LIF-induced self-renewal of mouse ES cells (Matsuda et al., 1999). It remains unclear, however, which genes are regulated by the STAT3 transcription factor in mouse ES cells and play actual roles in the maintenance of stem cells. Indeed, there is no generally accepted mechanism by which stem cells are maintained as undifferentiated cells. Human ES cell cultures have been recently established using mouse fibroblasts as feeder cells (Shamblott et al., 1998;Thomson et al., 1998). In contrast ...

The establishment of an animal model with a missense mutation of presenilin-1 (PS1) is an initial step toward understanding the molecular pathogenesis of familial Alzheimer's disease (FAD) and developing therapeutic strategies for the disease. We previously described a Japanese family with FAD caused by the I213T mutation of PS1, in which typical signs and symptoms of Alzheimer's disease were observed at the age of 45 +/- 4.2 years [Hardy, J. (1997) Trends. Neurosci., 20, 154-159; Kamino, K et al. (1996) Neurosci. Lett., 208, 195-198]. Here, we report the establishment of 'knock-in' mice with the I213T PS1 missense mutation. Northern blot and reverse transcription polymerase chain reaction (RT-PCR) analyses showed that the mutated PS1 allele was expressed at the same level as the endogenous PS1 allele, demonstrating that the PS1 missense mutation was successfully introduced into the mouse PS1 locus, and therefore that the situation mimics that in FAD patients bearing PS1 missense mutations. Amyloid beta (Abeta) 42(43) peptide, but not Abeta40 peptide, accumulated in 'knock-in' mice at the age of 16-20 weeks. A clear gene-dosage effect on the increase of Abeta42(43) was observed in 'knock-in' mice: the percentage increase of Abeta42(43) in mice with mutations in both alleles was twice as high as that in mice with a single allele. These results indicate that the level of the mutated PS1 gene expression is likely to be critically involved in the production of highly amyloidogenic Abeta42(43), and confirm that PS1 mutation has an important effect on amyloid precursor protein (APP) processing, in proportion to the level of the expression of the mutant gene.

To clarify the fate of glycosylphosphatidylinositol (GPI) in mammals, we developed GPI-anchored enhanced green fluorescent protein (EGFP-GPI) and transgenic mice carrying this fusion construct. When it was introduced to culture cells, the EGFP-GPI protein was correctly sorted to plasma membranes and microsomes depending on GPI biosynthesis. Transgenic mice carrying EGFP-GPI were found to show a broad transgene expression. Histologically, a prominent polarized localization of EGFP-GPI protein was observed in various epithelia, the nervous system and liver and secreted from some exocrine glands, as well as non-polarized presence in non-epithelial tissues, demonstrating a tissue-inherent manner of GPI sorting.z 1999 Federation of European Biochemical Societies.

A cDNA encoding a DNA methyltransferase, with a predicted polypeptide of 1556 amino acid residues containing all motifs conserved in this enzyme family, was isolated from tobacco plants, and the corresponding gene was designated as NtMET1. RNA blot analysis indicated NtMET1 transcripts to accumulate in dividing tissues of tobacco plants, and they could be detected during the S phase in synchronized dividing BY2 cells. In situ hybridization revealed the transcripts to be localized exclusively in actively proliferating tissues around axillary apical meristem. In order to ascertain physiological roles, transgenic tobacco plants that had the antisense construct were made and examined for phenotypes. Methylation levels of genomic DNA from transgenic plants significantly decreased in comparison with wild-type levels, and distinct phenotypic changes including small leaves, short internodes and abnormal flower morphology were noted. Microscopic observation revealed that leaf structure differed between transgenic and wild-type plants. These results suggest that NtMET1 functions during DNA replication, and that DNA methylation plays an important role in plant morphogenesis.

We compared the effects of quantitative control in closed system versus electrical conductivity (EC)-based control in open system of nutrients on the growth and yield of tomatoes in rockwool cultivation. Quantitative control supplied all nutrients once a day based on criteria for three application rates (1.0 ×, 1.25 ×, and 1.5 × the standard). These criteria were previously developed in the deep flow technique (DFT) to estimate their nutrient requirements, which used the three-day average amount of water absorbed by tomato plants. We compared the quantitative control in a closed system with two EC treatments in an open system, in which we supplied nutrient solution at high and low EC ranges. The 1.0 × quantity standard reduced the nutrient supply to 30-37% of the levels used in the low-EC treatment. N, P, and K concentrations in the 1.0 × substrate solution remained low and stable for two months after transplanting. The 1.0 × treatment had 27% lower total leaf dry weight and 23% higher total fruit yield than the low-EC treatment. However, greatly reduced growth and nitrogen content of upper leaf and slightly lower soluble solids content in the fruits suggest that the 1.0 × standard might provide insufficient nutrients during late growth stages. Excessive nutrient supplies, which were much lower than the low-EC treatment, increased substrate salinity owing to the accumulation of nutrients during the latter half of the growth period in the 1.25 × and 1.5 × treatments. The stable EC and nutrient concentrations in the 1.0 × substrate solution over time suggest that the three-day adjustment interval would be short enough. The quantity criteria developed for the DFT system could generally be applied to long-term rockwool culture, but the optimum range of nutrient supply was found to be from 1.0 × to 1.25 × the DFT standard in the later growth stages.