Glutathione-dependent binding of a photoaffinity analog of agosterol A to the C-terminal half of human multidrug resistance protein

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD(0)), which is connected to a P-glycoprotein-like core region (DeltaMRP) by a cytoplasmic linker domain zero (L(0)). It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD(0) and L(0) are not known. We synthesized [(125)I]11-azidophenyl agosterol A ([(125)I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C(932-1531)) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C(4). Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L(0) is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L(0) of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.     

Light-induced changes in resistivity of spinach chloroplasts toward modification with diazonium-1,2,4-triazole

Spinach chloroplasts in the light and in the dark were treated with several reagents for protein modification to see the effect of light on their resistivity toward modification. The reagents were p-diazobenzenesulfonic acid, diazonium-1-H-tetrazole, sodium-2,4,6-trinitrobenzenesulfonate, sodium-β-naphtoquinone-4,6-disulfonate and diazonium-1,2,4-triazole. No difference in the absorption spectrum was found between chloroplasts treated with these reagents in the light and those treated in the dark. However, these light- and dark-treated samples when solubilized with a nonionic detergent showed a difference in turbidity. Diazonium-1,2,4-triazole was the most suitable of the above reagents, and the solubilized sample of chloroplasts treated with diazonium-1,2,4-triazole in the light showed a turbidity which was about 2-fold higher than that of the same sample treated in the dark. This increase in turbidity was interpreted as being due to a change in the resistivity toward chemical modification of chloroplasts caused by illumination. In the presence of 3-(p-chlorophenyl)-1,1-dimethylurea, pentachlorophenol and 2-methylthio-4,6-bis-isopropylamino-s-triazine, which are inhibitors of the Hill reaction, the light-induced increase of turbidity was suppressed by 72, 78 and 62%, respectively. The addition of ATP caused a much greater increase of turbidity both in the light and in the dark. It was thus found that light and ATP induce a configurational change of chloroplasts or a conformational change of chloroplast proteins inside.     

Polymorphism of dopamine receptor D4 exon I corresponding region in chicken

In stockbreeding, there are indications that behavioral traits of livestock have an effect on breeding and production. If the variation in individual behavior is related to that in neurotransmitter-related genes such as in humans, it would be possible to breed pedigrees composed of individuals having behavioral traits that are useful to production and breeding using selection based on genotypes. In this study, we investigated the exon I region of dopamine receptor D4 (DRD4), in which variation is related to psychiatric disorder in humans, in major poultry species namely Japanese quail (Coturnix japonica), chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus) and helmeted guinea fowl (Numida meleagris). Furthermore, we investigated Japanese cormorant (Phalacrocorax capillatus) and Japanese jungle crow (Corvus macrorhynchos) as an out-group. In these species of birds, the repeat of proline was identified in the region corresponding to the human polymorphic region. The repeat number was 9 in Japanese quail, ring-necked pheasant and Japanese cormorant; 12 in helmeted guinea fowl; and 3 in Japanese jungle crow. However, no polymorphism was found in these species. In contrast, polymorphism was observed in chicken and two alleles with 8 and 9 repeats were identified. Although 9 repeats (allele 9) were predominant in most chicken breeds, Black Minorca had only 8 repeats (allele 8). Intra-breed polymorphism was found in 6 out of 12 breeds, and two alleles (alleles 8 and 9) were detected in these breeds. This polymorphism, which is the first to be reported on a neurotransmitter-related gene in birds, would contribute significant information for elucidation of differences in behavioral traits in chicken breeds.     

Phenolic composition and radical scavenging activity of sweetpotato-derived shochu distillery by-products treated with koji

Phenolic composition and radical scavenging activity in the shochu distillery by-products of sweetpotato (Ipomoea batatas L.) treated with koji (Aspergillus awamori mut.) and cellulase (Cellulosin T2) were investigated to develop new uses. Koji and Cellulosin T2 treatment of shochu distillery by-products from sweetpotatoes, rice, and barley increased phenolic content. Caffeic acid was identified as a dominant phenolic component in the shochu distillery by-products of the sweetpotato. Adding koji and/or Cellulosin T2 to the shochu distillery by-product indicated that koji was involved in caffeic acid production. Caffeic acid was not detected in raw or steamed roots of "Koganesengan", the material of sweetpotato for shochu production, suggesting that it is produced during shochu fermentation. The phenolic content and radical scavenging activity the shochu distillery by-product treated with koji and Cellulosin T2 were superior to those of commercial vinegar. These results suggest that koji treatment of sweetpotato-derived shochu distillery by-products has potential for food materials with physiological functions. Further koji treatment of sweetpotato shochu-distillery by-products may be applicable to mass production of caffeic acid.     

Molecular phylogeny of monocotyledons inferred from combined analysis of plastid<Emphasis Type="Italic"> matK</Emphasis> and<Emphasis Type="Italic"> rbcL</Emphasis> gene sequences

Using matK and rbcL sequences (3,269 bp in total) from 113 genera of 45 families, we conducted a combined analysis to contribute to the understanding of major evolutionary relationships in the monocotyledons. Trees resulting from the parsimony analysis are similar to those generated by earlier single or multiple gene analyses, but their strict consensus tree provides much better resolution of relationships among major clades. We find that Acorus (Acorales) is a sister group to the rest of the monocots, which receives 100% bootstrap support. A clade comprising Alismatales is diverged as the next branch, followed successively by Petrosaviaceae, the Dioscoreales–Pandanales clade, Liliales, Asparagales and commelinoids. All of these clades are strongly supported (with more than 90% bootstrap support). The sister-group relationship is also strongly supported between Alismatales and the remaining monocots (except for Acorus) (100%), between Petrosaviaceae and the remaining monocots (except for Acorus and Alismatales) (100%), between the clade comprising Dioscoreales and Pandanales and the clade comprising Liliales, Asparagales and commelinoids (87%), and between Liliales and the Asparagales–commelinoids clade (89%). Only the sister-group relationship between Asparagales and commelinoids is weakly supported (68%). Results also support the inclusion of Petrosaviaceae in its own order Petrosaviales, Nartheciaceae in Dioscoreales and Hanguanaceae in Commelinales.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s10265-003-0133-3     

Design and regulation of the AAA+ microtubule motor dynein

Dyneins are highly complex molecular motors that transport their attached cargo towards the minus end of microtubules. These enzymes are required for many essential motile activities within the cytoplasm and also power eukaryotic cilia and flagella. Each dynein contains one or more heavy chain motor units that consist of an N-terminal stem domain that is involved in cargo attachment, and six AAA+ domains (AAA1-6) plus a C-terminal globular segment that are arranged in a heptameric ring. At least one AAA+ domain (AAA1) is capable of ATP binding and hydrolysis, and the available data suggest that one or more additional domains also may bind nucleotide. The ATP-sensitive microtubule binding site is located at the tip of a 10nm coiled coil stalk that emanates from between AAA4 and AAA5. The function of this motor both in the cytoplasm and the flagellum must be tightly regulated in order to result in useful work. Consequently, dyneins also contain a series of additional components that serve to define the cargo-binding properties of the enzyme and which act as sensors to transmit regulatory inputs to the motor units. Here we describe the two basic dynein designs and detail the various regulatory systems that impinge on this motor within the eukaryotic flagellum.     

Involvement of c-Jun N-terminal kinase in amyloid precursor protein-mediated neuronal cell death

Amyloid precursor protein (APP), the precursor of Abeta, has been shown to function as a cell surface receptor that mediates neuronal cell death by anti-APP antibody. The c-Jun N-terminal kinase (JNK) can mediate various neurotoxic signals, including Abeta neurotoxicity. However, the relationship of APP-mediated neurotoxicity to JNK is not clear, partly because APP cytotoxicity is Abeta independent. Here we examined whether JNK is involved in APP-mediated neuronal cell death and found that: (i) neuronal cell death by antibody-bound APP was inhibited by dominant-negative JNK, JIP-1b and SP600125, the specific inhibitor of JNK, but not by SB203580 or PD98059; (ii) constitutively active (ca) JNK caused neuronal cell death and (iii) the pharmacological profile of caJNK-mediated cell death closely coincided with that of APP-mediated cell death. Pertussis toxin (PTX) suppressed APP-mediated cell death but not caJNK-induced cell death, which was suppressed by Humanin, a newly identified neuroprotective factor which inhibits APP-mediated cytotoxicity. In the presence of PTX, the PTX-resistant mutant of Galphao, but not that of Galphai, recovered the cytotoxic action of APP. These findings demonstrate that JNK is involved in APP-mediated neuronal cell death as a downstream signal transducer of Go.     

Identification and characterization of an intermediate in the alkali degradation of (6-4) photoproduct-containing DNA

The (6-4) photoproduct formed by ultraviolet light is known as an alkali-labile DNA lesion. Strand breaks occur at (6-4) photoproducts when UV-irradiated DNA is treated with hot alkali. We have analyzed the degradation reaction of this photoproduct under alkaline conditions using synthetic oligonucleotides. A tetramer, d(GT(6-4)TC), was prepared, and its degradation in 50 mm KOH at 60 degrees C was monitored by high performance liquid chromatography. A single peak with a UV absorption spectrum similar to that of the starting material was detected after the reaction, and this compound was regarded as an intermediate before the strand break. The formation of this intermediate was compared with intermediates from the degradation of other alkali-labile lesions such as the abasic site, thymine glycol, and 5,6-dihydrothymine. The results strongly suggested that the first step of the alkali degradation of the (6-4) photoproduct was the hydrolysis between the N3 and C4 positions of the 5'-pyrimidine component. Analyses by NMR spectroscopy and mass spectrometry supported the chemical structure of this product. Assays of the complex formation with XPC.HR23B and the translesion synthesis by DNA polymerase eta revealed that the biochemical properties are indistinguishable between the intact and hydrolyzed photoproducts.