Comparative aspects of the function and mechanism of SUR1 and MDR1 proteins

ATP-binding cassette (ABC) superfamily proteins have divergent functions and can be classified as transporters, channels, and receptors, although their predicted secondary structures are very much alike. Prominent members include the sulfonylurea receptor (SUR1) and the multidrug transporter (MDR1). SUR1 is a subunit of the pancreatic beta-cell K(ATP) channel and plays a key role in the regulation of glucose-induced insulin secretion. SUR1 binds ATP at NBF1, and ADP at NBF2 and the two NBFs work cooperatively. The pore-forming subunit of the pancreatic beta-cell K(ATP) channel, Kir6.2, is a member of the inwardly rectifying K(+) channel family, and also binds ATP. In this article, we present a model in which the activity of the K(ATP) channel is determined by the balance of the action of ADP, which activates the channel through SUR1, and the action of ATP, which stabilizes the long closed state by binding to Kir6.2. The concentration of ATP could also affect the channel activity through binding to NBF1 of SUR1. MDR1, on the other hand, is an ATP-dependent efflux pump which extrudes cytotoxic drugs from cells before they can reach their intracellular targets, and in this way confers multidrug resistance to cancer cells. Both NBFs of MDR1 can hydrolyze nucleotides, and their ATPase activity is necessary for drug transport. The interaction of SUR1 with nucleotides is quite different from that of MDR1. Variations in the interactions with nucleotides of ABC proteins may account for the differences in their functions.     

Genetic polymorphisms and antiviral activity in the bovine MX1 gene

Bovine MX1 cDNAs consisting of 2280 bp from 11 animals of five breeds and from a cultured cell line were sequenced and compared with previously reported data. Ten nucleotide substitutions were synonymous mutations, and a single nucleotide substitution at 458 resulted in an amino acid exchange of Ile (ATT) and Met (ATG). A 13-bp deletion-insertion mutation was also found in the 3'-UTR. Based on the nucleotide substitutions found in this study, bovine MX1 cDNA was classified into 11 genotypes. A phylogenetic tree of the 11 genotypes suggested that the genotypes observed in Brahman were a great genetic distance from other genotypes. An 18-bp deletion-insertion variation at position 171 was found to be the result of alternative splicing. The 18-bp deletion-insertion is located at the boundary between exon 3 and intron 3. Permanently transfected 3T3 cell lines expressing bovine MX1 mRNA were established to analyse the antiviral potential against VSVDeltaG*-G infection. Transfected cell clones expressing bovine MX1 mRNA showed a significantly smaller number of cells infected with VSVDeltaG*-G compared with the control cells. These results indicate that the bovine MX1 protein has potent antiviral activity.     

Study of color variation in the solitary ascidian Halocynthia roretzi, collected in the Inland Sea of Japan

In the vicinity of Yashiro Island in the Inland Sea of Japan, the solitary ascidian (tunicate) Halocynthia roretzi with tunics of various colors were collected. Samples of these animals were sorted into three groups on the basis of visual observation of tunic color. The red group includes animals with dark-red, light-red, or orange tunics. The pink group includes animals with tunic colors ranging between red and white. The white group includes only animals with completely white tunics. Animals in the white group lacked color internally, with the exception of the hepatopancreas and the gonads in breeding season; the epidermis and gill basket were white. In contrast, animals of both the red group and the pink group were colored internally, with red-orange epidermis and yellow gill basket. Alloreactivity was tested by mixed-hemocyte incubation between different animals belonging to the same color group and between animals belonging to different color groups. Alloreactivity between animals of the white group was 56.3%, between animals of the pink group was 60.0%, and between animals of the red group was 69.3%. The relatively high frequency of compatible combinations among the white animals is discussed.     

Identification of two novel mutations in adenine phosphoribosyltransferase gene in patients with 2,8-dihydroxyadenine urolithiasis

Five mutations in the adenine phosphoribosyltransferase (APRT) gene have been described in Japanese patients with APRT deficiency. We investigated the APRT gene from three patients with APRT deficiency and two novel mutations, G133D and V84M, were determined.     

Structural and functional analysis of the gpsA gene product of Archaeoglobus fulgidus: a glycerol-3-phosphate dehydrogenase with an unusual NADP+ preference

NAD(+)-dependent glycerol-3-phosphate dehydrogenase (G3PDH) is generally absent in archaea, because archaea, unlike eukaryotes and eubacteria, utilize glycerol-1-phosphate instead of glycerol-3-phosphate for the biosynthesis of membrane lipids. Surprisingly, the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus comprises a G3PDH ortholog, gpsA, most likely due to horizontal gene transfer from a eubacterial organism. Biochemical characterization proved G3PDH-like activity of the recombinant gpsA gene product. However, unlike other G3PDHs, the up to 85 degrees C thermostable A. fulgidus G3PDH exerted a 15-fold preference for NADPH over NADH. The A. fulgidus G3PDH bears the hallmarks of adaptation to halotolerance and thermophilicity, because its 1.7-A crystal structure showed a high surface density for negative charges and 10 additional intramolecular salt bridges compared to a mesophilic G3PDH structure. Whereas all amino acid residues required for dihydroxyacetone phosphate binding and reductive catalysis are highly conserved, the binding site for the adenine moiety of the NAD(P) cosubstrate shows a structural variation that reflects the observed NADPH preference, for example, by a putative salt bridge between R49 and the 2'-phosphate.     

Consequences of differences in flowering date on seed production in Heloniopsis orientalis (Liliaceae)

We examined the consequences of differences in flowering date on seed production in the self-compatible herb Heloniopsis orientalis. The number of selfed seeds per fruit, as determined by microsatellite markers, did not depend on when the plant flowered, whereas the number of outcrossed seeds per fruit increased with later flowering dates. Consequently, the selfing rate decreased with later flowering dates. The number of seeds (including both selfed and outcrossed ones) per fruit and the seed?:?ovule ratio increased with later flowering dates. We also examined the effects of pollinators and plant size on seed production. The visitation rate of Diptera did not depend on the flowering season, whereas that of Hymenoptera markedly increased as the flowering season progressed. Diptera stayed longer than Hymenoptera on each plant and flower. Seed production per fruit did not depend on plant size. Thus, the change in selfing rate associated with later flowering dates resulted from the seasonal change in pollinators rather than plant size.     

Intracellular delivery of glutathione S-transferase into mammalian cells

Protein transduction domains (PTDs) derived from human immunodeficiency virus Tat protein and herpes simplex virus VP22 protein are useful for the delivery of non-membrane-permeating polar or large molecules into living cells. In the course of our study aiming at evaluating PTD, we unexpectedly found that the fluorescent-dye-labeled glutathione S-transferase (GST) from Schistosoma japonicum without known PTDs was delivered into COS7 cells. The intracellular transduction of GST was also observed in HeLa, NIH3T3, and PC12 cells, as well as in hippocampal primary neurons, indicating that a wide range of cell types is permissive for GST transduction. Furthermore, we showed that the immunosuppressive peptide VIVIT fused with GST successfully inhibits NFAT activation. These results suggest that GST is a novel PTD which may be useful in the intracellular delivery of biologically active molecules, such as small-molecule drugs, bioactive peptides, or proteins.     

Synthesis and stereostructure-activity relationship of three asymmetric center pyrethroids: 2-methyl-3-phenylcyclopropylmethyl 3-phenoxybenzyl ether and cyanohydrin ester

2-Methyl-3-phenylcyclopropylmethyl 3-phenoxybenzyl ether 2 and cyanohydrin ester 3, a couple of pyrethroids with three asymmetric centers, were synthesized. Of each of the four diastereomers of 2 and 3, only the (1R*,2R*,3R*)-2a and 3a showed significant insecticidal activities. Dual sets of enantiomers [(1R,2R,3R)-(-)-2a and (1S,2S,3S)-(+)-2a] and [(1R,2R,3R)-(-)-3a and (1S,2S,3S)-(+)-3a] were synthesized through the asymmetric cyclopropanation using the Aratani catalyst. Significant separations of insecticidal activities were observed between both the enantiomers against the tobacco cutworm (Spodoptera litura) and the common mosquito (Culex pipiens pallens); (1S,2S,3S)-(+)-2a and (+)-3a showed higher activities than their antipodes (1R,2R,3R)(-)-2a and (-)-3a. This result is the second example of such synthetic pyrethroids with three asymmetric centers.