Transient expression in electroporated pea protoplasts: Elicitor responsiveness of a phenylalanine ammonia-lyase promoter

Summary High yields of viable pea protoplasts were produced from suspension cultured cells and the conditions for the optimum transient expression of the chloramphenicol acetyltransferase (CAT) gene fused to the CaMV 35S promoter after electroporation were investigated. Conditions for elicitor induction of a member of the phenylalanine ammonia-lyase (PAL) gene family in pea was also investigated using a chimeric gene carrying 480 bp of the putative promoter region of gPAL1 connected to bacterial cat gene and nos terminator. CAT activity was considerably induced by the treatment with fungal elicitor (>100 g/ml glucose equivalent) isolated from Mycosphaerella pinodes, a pea pathogen.Abbreviations CAT chloramphenicol acetyltransferase - PAL phenylalanine ammonia-lyase - CM acetylated chloramphenicol - GSH reduced glutathione - NOS nopaline synthase - ES electroporation solution - CaMV cauliflower mosaic virus - GUS -glucuronidase - CHS chalcone synthase - 2,4-D 2, 4-dichlorophenoxyacetic acid Present address: Research Institute, Takasago Perfumery Inc, 5-31-36, Kamata, Minato, Tokyo, 144, Japan Toso Inc, 4560 Oaza-Tomita, Shin-nanyo, Yamaguchi, 746, Japan Central Laboratory of Green Complex, Kasetsert University, Kamphaensaen, Nakohn Pathom, Thailand     

Temporary Suppression of the Hyperglycemic Response to 2-Deoxy-d-Glucose by Blinding

Studies were made on whether there is a time-dependency in the hyperglycemic response to intracranial injection of 2-deoxy-d-glucose (2DG) in blind rats. 2DG was given to blind rats in their subjective light and dark periods to see if the response free-runs like their circadian locomotor and feeding rhythms. The following results were obtained: (1) In control period and week 3 after blinding, 2DG caused greater hyperglycemia in the subjective light period than in the subjective dark period; (2) In weeks 4 and 6, however, 2DG caused only slight hyperglycemia, while it caused considerable hyperinsulinemia in both the subjective light and dark periods with no time-dependency. (3) In week 8, the hyperglycemic response to 2DG was completely restored while the hyperinsulinemic response was lost. These findings indicate that the subjective time-dependency in the hyperglycemic response to intracranial injection of 2DG exists until week 3 and after week 8 after blinding, however, in week 4 and 6 after blinding the subjective time-dependency appeared to disappear and the hyperglycemic response is largely suppressed in association with hyperinsulinemia. Together with a previous finding that bilateral lesions of the SCN completely abolished the response to 2DG and the fact that blind rats showed circadian rhythms of feeding and locomotive activity even in weeks 4 and 6 after blinding, these findings present the possibility that the site responding to 2DG is in the vicinity of the SCN, but is indifferent neuronal cells from those of the circadian oscillator. However, it is also possible that the blinding elicits the suppression of hyperglycemia due to 2DG through disturbing neural pathway outside the SCN which control blood glucose concentration.     

Preparation of enantiopure Wieland-Miescher ketone and derivatives by the MalphaNP acid method: substituent effect on the HPLC separation

Enantiopure Wieland-Miescher ketone (4, W-M ketone) and derivatives were prepared by the enantioresolution with 2-methoxy-2-(1-naphthyl)propionic acid (MalphaNP acid 1). Various racemic derivatives of 4 were esterified with acid (S)-(+)-1 yielding diastereomeric MalphaNP esters, which were separated by HPLC on silica gel. It was clarified that the HPLC separation of diastereomers depended on the substituent of the derivatives, leading to the working hypothesis that MalphaNP acid esters of alcohols with less polar and more bulky aliphatic substituents are more effectively separated. The best separation was obtained in the case of tert-butyldimethylsilyl (TBDMS) ether derivative (12a/12b): separation factor alpha=1.80, and resolution factor, Rs=1.30. The (1)H NMR spectra of separated MalphaNP esters showed anomalously large magnetic anisotropy effects, from which their absolute configurations were determined. Solvolysis or reduction of the separated MalphaNP esters yielded alcohols, which were converted to enantiopure W-M ketones 4. The results thus provided another route for preparation of enantiopure ketones (8aR)-(-)-4 and (8aS)-(+)-4.     

Phospholipid Flippases Lem3p-Dnf1p and Lem3p-Dnf2p Are Involved in the Sorting of the Tryptophan Permease Tat2p in Yeast

The type 4 P-type ATPases are flippases that generate phospholipid asymmetry in membranes. In budding yeast, heteromeric flippases, including Lem3p-Dnf1p and Lem3p-Dnf2p, translocate phospholipids to the cytoplasmic leaflet of membranes. Here, we report that Lem3p-Dnf1/2p are involved in transport of the tryptophan permease Tat2p to the plasma membrane. The lem3Δ mutant exhibited a tryptophan requirement due to the mislocalization of Tat2p to intracellular membranes. Tat2p was relocalized to the plasma membrane when trans-Golgi network (TGN)-to-endosome transport was inhibited. Inhibition of ubiquitination by mutations in ubiquitination machinery also rerouted Tat2p to the plasma membrane. Lem3p-Dnf1/2p are localized to endosomal/TGN membranes in addition to the plasma membrane. Endocytosis mutants, in which Lem3p-Dnf1/2p are sequestered to the plasma membrane, also exhibited the ubiquitination-dependent missorting of Tat2p. These results suggest that Tat2p is ubiquitinated at the TGN and missorted to the vacuolar pathway in the lem3Δ mutant. The NH₂-terminal cytoplasmic region of Tat2p containing ubiquitination acceptor lysines interacted with liposomes containing acidic phospholipids, including phosphatidylserine. This interaction was abrogated by alanine substitution mutations in the basic amino acids downstream of the ubiquitination sites. Interestingly, a mutant Tat2p containing these substitutions was missorted in a ubiquitination-dependent manner. We propose the following model based on these results; Tat2p is not ubiquitinated when the NH₂-terminal region is bound to membrane phospholipids, but if it dissociates from the membrane due to a low level of phosphatidylserine caused by perturbation of phospholipid asymmetry in the lem3Δ mutant, Tat2p is ubiquitinated and then transported from the TGN to the vacuole.     

Suppressors: Determinants of Specificity Produced by Plant Pathogens

Plant pathogens secrete suppressors that delay or prevent thehost defense responses, with resultant conditioning of hostcells such that they become susceptible even to avirulent ornon-pathogenic microorganisms. Suppressors have been characterizedas glycoproteins, glycopeptides, peptides and anionic and nonanionicglucans. A suppressor itself is non-toxic to plant cells and,thus, it can be distinguished from host-specific toxins producedby certain pathogens. Suppressors disturb fundamental functionsof host plasma membranes. For example, the suppressor from apea pathogen, Mycosphaerella pinodes, inhibits both the ATPaseactivity and polyphosphoinositide metabolism in pea plasma membranes,causing the temporary suppression of the signal-transductionpathway that leads to the expression of defense genes, whichencode key enzymes in the biosynthetic pathway to phytoalexin.In this review, evidence for the role of suppressors in thedetermination of plant host-parasite specificity is summarized. ³Present address: Plant Pathology Laboratory, School of Agriculture,Nagoya University, Chikusa, Nagoya, 464-01 Japan     

Flavonols and flavones as BACE-1 inhibitors: structure-activity relationship in cell-free, cell-based and in silico studies reveal novel pharmacophore features

Generation and accumulation of the amyloid beta peptide (Abeta) following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 (Beta-site APP Cleaving Enzyme-1, beta-secretase) and gamma-secretase is a main causal factor of Alzheimer's disease (AD). Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach for the treatment of AD. In this study, we discovered that natural flavonoids act as non-peptidic BACE-1 inhibitors and potently inhibit BACE-1 activity and reduce the level of secreted Abeta in primary cortical neurons. In addition, we demonstrated the calculated docking poses of flavonoids to BACE-1 and revealed the interactions of flavonoids with the BACE-1 catalytic center. We firstly revealed novel pharmacophore features of flavonoids by using cell-free, cell-based and in silico docking studies. These results contribute to the development of new BACE-1 inhibitors for the treatment of AD.     

Phosphorylation of amyloid precursor protein (APP) at Tyr687 regulates APP processing by alpha- and gamma-secretase

Abnormal proteolytic processing of amyloid precursor protein (APP) is a pathologic feature of Alzheimer’s disease. Recent studies have demonstrated that serine/threonine phosphorylation specifically at amino-acid residue Thr668 (APP695 numbering) regulates APP processing. In this study, we investigated the possibility that tyrosine phosphorylation of APP regulates APP processing. A tyrosine kinase inhibitor decreased expression of the C83 fragment which is a cleaved product of APP by α-secretase. By overexpressing APP mutant proteins, Tyr687 was found to be the major tyrosine kinase phosphorylation site. Expression of the C83 fragment was decreased in APPY687A-expressing cells relative to APP wild-type (APPWT)-expressing cells, which likely reflects the different cellular localization patterns of these two proteins. Expression of APP intracellular domain (AICD) which is a cleaved product of the C83 fragment by γ-secretase was decreased in C83Y687A-expressing cells. These results suggest that phosphorylation of APP at Tyr687 regulates APP processing by α- and γ-secretases, determining the expression level of AICD.     

Design and synthesis of regioisomerically pure unsymmetrical xanthene derivatives for staining live cells and their photochemical properties

We have demonstrated the synthesis of regioisomerically pure unsymmetrical xanthene derivatives consisting of three units which can be independently modified to control their physical properties. The photochemical properties of the synthetic unsymmetrical xanthene derivatives were investigated in solution by UV–vis absorption and fluorescence measurements, and their cell imaging properties were examined by confocal laser-scanning microscopy.