Degradation of amyloid β peptide by neprilysin expressed from Borna disease virus vector

Accumulation of amyloid β (Aβ40 and Aβ42) in the brain is a characteristic of Alzheimer disease (AD). Because neprilysin (NEP) is a major Aβ‐degrading enzyme, NEP delivery in the brain is a promising gene therapy for AD. Borna disease virus (BoDV) vector enables long‐term transduction of foreign genes in the central nerve system. Here, the proteolytic ability of NEP transduced by the BoDV vector was evaluated and it was found that the amounts of Aβ40 and Aβ42 decreased significantly, suggesting that NEP expressed from the BoDV vector is functional in that it degrades Aβ.

     

Sequencing and Expression of the Gene that Encodes a 20-kDa Polypeptide of the PSI Complex from Cucumber Cotyledon

A cDNA clone encoding the polypeptide from cucumber PS I thatmigrates with an apparent molecular weight of 20 kDa on SDS-polyacrylamidegels has been isolated. The 907-bp sequence of this clone hasbeen determined and contains one large open reading frame thatencodes a 22,720-Da precursor polypeptide (207 amino acid residues).The molecular weight of the mature polypeptide was predictedto be 17,037-Da (153 amino acid residues). The deduced aminoacid sequence of this protein indicates that it is routed towardsthe stromal side of the thylakoid membrane and has no membrane-spanningregions. The sequence also confirmed the identity of the proteinas the product of the psa D gene. Chemical cross-linking offerredoxin to the PS I complex identified the 20-kDa subunitas the ferredoxin-binding protein. Northern hybridization experimentsrevealed that the mRNA of approximately 1,100 nucleotides forthe 20-kDa polypeptide was present in etiolated cucumber cotyledons,and its level increased about 5-fold during greening. The 20-kDapolypeptide was not detected by immunoblotting in etiolatedcotyledons, and it accumulated only after illumination. Labelingexperiments in vivo showed the absence of incorporation of [³⁵S]Metinto the polypeptide in etiolated cotyledons. These resultssuggest that the expression of the psa D gene is controlledat the translational level. (Received April 5, 1990; Accepted June 28, 1990)     

Differentiation of reptilian neural crest cells in Vitro

An attempt was made to culture neural crest cells of the turtle embryo in vitro. Trunk neural tubes from the St. 9/10 embryos were explanted in culture dishes. The developmental potency of the turtle neural crest cells in vitro was shown to be essentially similar to that of avian neural crest cells, although they seem to be more sensitive to melanocyte-stimulating hormone (MSH) stimulation. We describe conditions under which explanted neural tube gives rise to neural crest cells that differentiate into neuronal cells and melanocytes. The potency of melanocyte differentiation was, found to vary according to the concentration of fetal bovine serum (FBS, from 5 to 20%). Melanization of neural crest cells cultured in the medium containing FBS and α-MSH was more extensive than those cultured with FBS alone, combinations of FBS and chick embryo extract, or turtle embryo extract. These culture conditions seem to be useful for the study of the developmental potency of the neural crest cells as well as for investigating local environmental factors.     

The Role of Indoleamine 2,3-Dioxygenase in Diethylnitrosamine-Induced Liver Carcinogenesis

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing intracellular enzyme of the L-kynurenine pathway, causes preneoplastic cells and tumor cells to escape the immune system by inducing immune tolerance; this mechanism might be associated with the development and progression of human malignancies. In the present study, we investigated the role of IDO in diethylnitrosamine (DEN)-induced hepatocarcinogenesis by using IDO-knockout (KO) mice. To induce hepatocellular carcinoma (HCC), hepatic adenoma, and preneoplastic hepatocellular lesions termed foci of cellular alteration (FCA), male IDO-wild-type (WT) and IDO-KO mice with a C57BL/6J background received a single intraperitoneal injection of DEN at 2 weeks of age. The mice were sacrificed to evaluate the development of FCA and hepatocellular neoplasms. HCC overexpressed IDO and L-kynurenine compared to surrounding normal tissue in the DEN-treated IDO-WT mice. The number and cell proliferative activity of FCAs, and the incidence and multiplicity of HCC were significantly greater in the IDO-WT than in the IDO-KO mice. The expression levels of the IDO protein, of L-kynurenine, and of IFN-γ, COX-2, TNF-α, and Foxp3 mRNA were also significantly increased in the DEN-induced hepatic tumors that developed in the IDO-WT mice. The mRNA expression levels of CD8, perforin and granzyme B were markedly increased in hepatic tumors developed in IDO-KO mice. Moreover, Foxp3-positive inflammatory cells had infiltrated into the livers of DEN-treated IDO-WT mice, whereas fewer cells had infiltrated into the livers of IDO-KO mice. Induction of IDO and elevation of L-kynurenine might play a critical role in both the early and late phase of liver carcinogenesis. Our findings suggest that inhibition of IDO might offer a promising strategy for the prevention of liver cancer.     

Discovery of a pimaricin analog JBIR-13, from <Emphasis Type="Italic">Streptomyces bicolor</Emphasis> NBRC 12746 as predicted by sequence analysis of type I polyketide synthase gene

Sequence analysis of ketosynthase domain amplicons from Streptomyces bicolor NBRC 12746^T revealed the presence of previously unreported type I polyketide synthases (PKS-I) genes. The clustering of these genes with the reference PKS-1 sequences suggested the possibility to produce a polyene compound similar to pimaricin. Thus, the cultured sample from NBRC 12746^T was analyzed for the production of polyene compounds. The strain produced an antifungal compound which displayed the UV absorption spectrum of tetraene macrolides. The structure determination based on the spectroscopic analysis of the purified compound resulted in the identification of a novel pimaricin analog JBIR-13 (1). This study therefore strongly suggested that a careful analysis of PKS-I genes can provide valuable information in the search of novel bioactive compounds within a class predicted from phylogenetic analysis. H. Komaki and M. Izumikawa contributed equally to this work.     

Cleavage Site Specificities of Silkworm Alkaline Proteases

The cleavage site specificities of two alkaline proteases isolated from digestive juice of the silkworm larva, Bombyx mori, were analyzed using insulin A and B chains, α-lactalbumin, and egg white lysozyme. One, named P-IIc, could cut Arg-X and Lys-X peptide bonds and another, named P-IIIa, required rather hydrophobic amino acids not only at P1 but also at P4 sites. The effects of some protease inhibitors on P-IIIa could be explained by this requirement.     

Changes in Water Content during Metamorphosis of the Housefly,Musca domestica

The synthesis of ( – )-cis-neocnidilide (1), a stereoisomer of neocnidilide (2) having inhibiting activity against mycotoxin-producing fungi, is described. ( ±)-(E)-1,3-Nonadien-5-ol was kinetically resolved to give (S)-13, which was converted to (S)-triene ester 8. The intramolecular Diels-Alder reaction of 8 afforded a mixture of 12 and 10. The dihydro derivative 14, obtained by catalytic hydrogenation of 12, was transformed via trimethylsilylketene acetal into α-bromolactone 19, which upon treatment with DBU in toluene, gave rise to ( – )-cis-neocnidilide (1).     

High temperature enhances cytotoxicity of mercury (HgCl2) on Hela S3 cells

The combined effect of mercury (HgCl₂) and high temperature on the growth and synthesis of nucleic acid and protein, and on the cell cycle of HeLa S₃ cells was investigated. The subsequent growth of the cells was dose-dependently inhibited by mercury at 37.2° and 41.2°C. The inhibitory effect of mercury on subsequent growth was enhanced at the higher temperature. IC₅₀ values for DNA and RNA synthesis but not protein synthesis, at 41.2°C, were significantly lower than those at 37.2°C (P<0.05,P<0.01, respectively). Flow cytometric analysis using synchronous cells indicated the possibility of blocking of cell cycle progression in the early part of S phase by the combined treatment. These results suggest that the cytotoxicity of mercury to cell growth was enhanced at the higher temperature and that this enhancement is related to the increased inhibitory effect of mercury on DNA and RNA synthesis and on the cell cycle at high temperatures.