Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin.

Several lines of evidence suggest that aberrant Notch signaling contributes to the development of several types of cancer. Activation of Notch receptor is executed through intramembrane proteolysis by γ-secretase, which is a multimeric membrane-embedded protease comprised of presenilin, nicastrin (NCT), anterior pharynx defective 1 and PEN-2. In this study, we report the neutralization of the γ-secretase activity by a novel monoclonal antibody A5226A against the extracellular domain of NCT, generated by using a recombinant budded baculovirus as an immunogen. This antibody recognized fully glycosylated mature NCT in the active γ-secretase complex on the cell surface, and inhibited the γ-secretase activity by competing with the substrate binding in vitro. Moreover, A5226A abolished the γ-secretase activity-dependent growth of cancer cells in a xenograft model. Our data provide compelling evidence that NCT is a molecular target for the mechanism-based inhibition of γ-secretase, and that targeting NCT might be a novel therapeutic strategy against cancer caused by aberrant γ-secretase activity and Notch signaling.

The first proline of PALP motif at the C terminus of presenilins is obligatory for stabilization, complex formation, and gamma-secretase activities of presenilins.

Mutations in presenilin (PS) genes cause early-onset familial Alzheimer's disease by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). PS is an evolutionarily conserved multipass transmembrane protein, and all known PS proteins contain a proline-alanine-leucine-proline (PALP) motif starting at proline (P) 414 (amino acid numbering based on human PS2) at the C terminus. Furthermore, missense mutations that replace the first proline of PALP with leucine (P414L) lead to a loss-of-function of PS in Drosophila melanogaster and Caenorhabditis elegans. To elucidate the roles of the PALP motif in PS structure and function, we analyzed neuro2a as well as PS1/2 null fibroblast cell lines transfected with human PS harboring mutations at the PALP motif. P414L mutation in PS2 (and its equivalent in PS1) abrogated stabilization, high molecular weight complex formation, and entry to Golgi/trans-Golgi network of PS proteins, resulting in failure of Abeta42 overproduction on familial Alzheimer's disease mutant basis as well as of site-3 cleavage of Notch. These data suggest that the first proline of the PALP motif plays a crucial role in the stabilization and formation of the high molecular weight complex of PS, the latter being the active form with intramembrane proteolytic activities.

Molecular cloning and expression of the novel splice variants of K(+) channel-interacting protein 2.

Two cDNAs encoding the splice variants of K(+) channel-interacting protein 2 (KChIP2) recently reported as human KChIP2 have been identified from rat, mouse, and human heart by RT-PCR. A longer variant, KChIP2L encodes a protein of 270 amino acids, which has a 50-amino-acid insertion in N-terminus in comparison with a shorter one, KChIP2S. Interestingly, both KChIP2S and KChIP2L (KChIP2S/L) but not the original KChIP2 were expressed in human heart and umbilical vein endothelial cells (HUVECs). KChIP2S transcripts but not KChIP2L were predominantly expressed in rat, mouse, and human heart and HUVECs, whereas both transcripts were expressed at low levels in other tissues such as brain, aorta, and kidney. Using chimeric proteins of green fluorescence protein (GFP) fused to the N-terminus of KChIP2S/L, the interactions between Kv4.3 and KChIP2S/L were analyzed in native and Kv4.3-expressed HEK293 cells. Specific localization of GFP-fused KChIP2S/L proteins on or near cell membrane was observed only in Kv4.3-expressed HEK293 cells.

Water stress enhances beta-amylase activity in cucumber cotyledons.

Cotyledons detached from 4-d-old cucumber (Cucumis sativus L.) seedlings were subjected to water stress (air-drying or PEG-treatment) to examine the effects of the stress on carbohydrate metabolism. Amylolytic activity in the cotyledon was increased about 6-fold by water stress within 1 d. The substrate specificity and the action pattern indicated that beta-amylase is responsible for the activity. Activities of azocaseinase, malate dehydrogenase and triose-phosphate isomerase were not affected by water stress, indicating that the effect of the stress on beta-amylase is rather specific. Cycloheximide-treatment strongly reduced the enhancement of beta-amylase activity. The hypocotyl of cucumber seedlings also exhibited an increase in the enzyme activity when subjected to water stress. The major free sugars in cucumber cotyledons were glucose, fructose, maltose, and sucrose; sucrose being the most abundant. Sucrose content in excised, unstressed cotyledons increased markedly during the incubation. Changes in other free sugars were small compared with that of sucrose. Starch also accumulated in unstressed cotyledons. In stressed cotyledons more sucrose and less starch accumulated than in unstressed ones. Such results were discussed in relation to the enhancement of beta-amylase activity.

Development of beta-1,3-glucanase activity in germinated tomato seeds.

Laminarin-hydrolysing activity developed in the endosperm of tomato (Lycopersicon esculentum) seeds following germination. The enzyme was basic (pI>10) and the apparent molecular mass was estimated to be 35 kDa by SDS-PAGE. It was specific for linear beta-1,3-glucan substrates. Laminarin was hydrolysed by the enzyme to yield a mixture of oligoglucosides, indicating that the enzyme had an endo-action pattern. Thus, the enzyme was identified as beta-1,3- endoglucanase (EC 3.2.1.39). The activity of the enzyme developed in the endosperm after radicle protrusion (germination) had occurred and the enzyme activity was localized exclusively in the micropylar region of the endosperm where the radicle had penetrated. When the lateral endosperm region, where no induction of the enzyme occurred, was wounded (cut or punctured), there was a marked enhancement of beta-1,3-glucanase activity. Thus the post-germinative beta-1, 3-glucanase activity in the micropylar endosperm portion might be brought about by wounding resulting from endosperm rupture by radicle penetration.

C terminus of presenilin is required for overproduction of amyloidogenic Abeta42 through stabilization and endoproteolysis of presenilin.

Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). To identify a PS subdomain responsible for overproduction of Abeta42, we analyzed neuro2a cell lines expressing modified forms of PS2 that harbor an N141I FAD mutation. Deletion or addition of amino acids at the C terminus and Ile448 substitution in PS2 with the N141I FAD mutation abrogated the increase in Abeta42 secretion, and Abeta42 overproduction was dependent on the stabilization and endoproteolysis of PS2. The same C-terminal modifications in PS1 produced similar effects. Hence, we suggest that the C terminus of PS plays a crucial role in the overproduction of Abeta42 through stabilization of endoproteolytic PS derivatives and that these derivatives may be the pathologically active species of PS that cause FAD.

Controversial issues surrounding the case of HIV infections and AIDS through the use of unheated commercial blood products.

It has been reported in the recent years that not a small number of Japanese patients, including 1,792 hemophiliacs, have contracted HIV infections through the use of commercially available unheated blood products, such as factor VIII or IX concentrates. Many of them subsequently developed AIDS, and died shortly thereafter. It was later found that this tragedy was caused by misconducts and/or negligence of at least several persons responsible for the safety of the aforementioned blood products. Subsequently, a former chief of the Office of Biologics at the Ministry of Health and Welfare was arrested and indicted for the failure to prevent the spread of HIV infections throughout the country. His negligence allegedly killed two patients. No other person in the government, however, was indicted. There are controversies for and against the legal action. It has been questioned whether only one former senior medical officer of the government should and/or can assume full responsibility for the entire spectrum of the fiasco. It has also been claimed that the tragedy was caused by unfortunate combination of various factors in the government, industries, and medical communities, and that indictment of only one former senior medical officer is nothing but oversimplification of this very complicated issue. In order to increase the safety of blood products as well as to prevent the recurrence of the aforementioned tragedy, thorough investigation as well as adequate discussion on this case is highly desirable. Reformation of the current system should be made mandatory.

Molecular cloning of a cDNA encoding a (1-->4)-beta-mannan endohydrolase from the seeds of germinated tomato (Lycopersicon esculentum).

Mannose-containing polysaccharides are widely distributed in cell walls of higher plants. During endosperm mobilization in germinated tomato seeds (1-->4)-beta-mannan endohydrolases (EC 3.2.1.78) participate in the enzymic depolymerization of these cell wall polysaccharides. A cDNA encoding a (1-->4)-beta-mannanase from the endosperm of germinated tomato (Lycopersicon esculentum Mill.) seeds has been isolated and characterized. The amino acid sequence deduced from the 5'-region of the cDNA exactly matches the sequence of the 65 NH2-terminal amino acids determined directly from the purified enzyme. The mature enzyme consists of 346 amino acid residues, it has a calculated M(r) of 38,950 and an isoelectric point of 5.3. Overall, the enzyme exhibits only 28-30% sequence identity with fungal (1-->4)-beta-mannanases, but more highly conserved regions, which may represent catalytic and substrate-binding domains, can be identified. Based on classification of the tomato (1-->4)-beta-mannanase as a member of the family 5 group of glycosyl hydrolases, Glu-148 and Glu-265 would be expected to be the catalytic acid and the catalytic nucleophile, respectively. Southern hybridization analyses indicate that the enzyme is derived from a family of about four genes. Expression of the genes, as determined by the presence of mRNA transcripts in Northern hybridization analyses, occurs in the endosperm of germinated seeds; no transcripts are detected in hypocotyls, cotyledons, roots or leaves.

An Endo-[beta]-Mannanase Develops Exclusively in the Micropylar Endosperm of Tomato Seeds Prior to Radicle Emergence.

A galactomannan-hydrolyzing enzyme that develops pregerminatively in the micropylar region of the endosperm of the tomato (Lycopersicon esculentum [L.] Mill.) seed was characterized. The enzyme was endo-[beta]-mannanase (EC 3.2.1.78), since it hydrolyzed galactomannan into oligosaccharides with no release of galactose and mannose. The mobility of this pregerminative enzyme in sodium dodecyl sulfate and native polyacrylamide gel electrophoresis was not identical to that of any of the three endo-[beta]-mannanases that develop in the same tissue (endosperm) after germination (H. Nonogaki, M. Nomaguchi, Y. Morohashi [1995] Physiol Plant 94: 328-334). There were also some differences in the products of galactomannan hydrolysis between the pregerminative and the postgerminative enzymes, indicating that the action pattern is different between the two types of enzymes. The pregerminative enzyme began to develop in the micropylar region of the endosperm at about 18 h postimbibition and increased up to the time immediately before radicle protrusion (24 h postimbibition). This enzyme was not present in the lateral part of the endosperm at any stage before or after germination. It is proposed that the enzyme develops prior to germination specifically at the micropylar region of the endosperm.

Phytochrome Control of the Development of Ascorbate Oxidase Activity in Mustard (Sinapis alba L.) Cotyledons.

The activity of ascorbate oxidase (AOX) in mustard (Sinapis alba L.) cotyledons was markedly increased by irradiation with continuous far-red light. The involvement of phytochrome in this light-mediated response was demonstrated by red/far-red reversibility experiments. To determine immunochemically the contents of AOX in cotyledons, the antibody against the enzyme was raised in a rabbit. However, the antiserum was not monospecific to AOX; it also recognized glycoproteins. To remove antibodies that are specific to a carbohydrate moiety of glycoproteins, the anti-AOX antiserum was applied to a horseradish peroxidase-conjugated Sepharose column. By using the antibodies that were not retained in the column, the changes in the content of AOX were followed. Western immunoblot profiles revealed that the content of AOX protein in cotyledons notably increased after continuous far-red light treatment. Pulse-labeling experiments indicated that the synthesis of AOX protein occurred in the cotyledons. These results are in good agreement with the hypothesis that phytochrome-mediated increase in AOX activity is accompanied by the synthesis of the enzyme.

Hospitals are facing financial crisis.

Hospitals in Japan are facing serious financial crisis, with many establishments going bankrupt one after the other from rising costs of salaries and general expenses. The future is dark for the hospital business. Presented in this article are what the author considers indispensible measures for resetting the hospitals on their feet.