Inhibition of gastric inhibitory polypeptide signaling prevents obesity

Secretion of gastric inhibitory polypeptide (GIP), a duodenal hormone, is primarily induced by absorption of ingested fat. Here we describe a novel pathway of obesity promotion via GIP. Wild-type mice fed a high-fat diet exhibited both hypersecretion of GIP and extreme visceral and subcutaneous fat deposition with insulin resistance. In contrast, mice lacking the GIP receptor (Gipr(-/-)) fed a high-fat diet were clearly protected from both the obesity and the insulin resistance. Moreover, double-homozygous mice (Gipr(-/-), Lep(ob)/Lep(ob)) generated by crossbreeding Gipr(-/-) and obese ob/ob (Lep(ob)/Lep(ob)) mice gained less weight and had lower adiposity than Lep(ob)/Lep(ob) mice. The Gipr(-/-) mice had a lower respiratory quotient and used fat as the preferred energy substrate, and were thus resistant to obesity. Therefore, GIP directly links overnutrition to obesity and it is a potential target for anti-obesity drugs.     

Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis

During protein integration into the endoplasmic reticulum, the N-terminal domain preceding the type I signal-anchor sequence is translocated through a translocon. By fusing a streptavidin-binding peptide tag to the N terminus, we created integration intermediates of multispanning membrane proteins. In a cell-free system, N-terminal domain (N-domain) translocation was arrested by streptavidin and resumed by biotin. Even when N-domain translocation was arrested, the second hydrophobic segment mediated translocation of the downstream hydrophilic segment. In one of the defined intermediates, two hydrophilic segments and two hydrophobic segments formed a transmembrane disposition in a productive state. Both of the translocating hydrophilic segments were crosslinked with a translocon subunit, Sec61α. We conclude that two translocating hydrophilic segment in a single membrane protein can span the membrane during multispanning topogenesis flanking the translocon. Furthermore, even after six successive hydrophobic segments entered the translocon, N-domain translocation could be induced to restart from an arrested state. These observations indicate the remarkably flexible nature of the translocon.     

Inhibition of FGF10-ERK signal activation suppresses intraductal papillary neoplasm of the bile duct and its associated carcinomas

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Effects of blue light on electrical potential and turgor in pulvinar motor cells ofPhaseolus

Pulvinar motor cells ofPhaseolus vulgaris L display transient depolarization of the membrane potential and a turgor pressure decrease when exposed to a pulse of blue light. To analyze the mechanism of the transient depolarization, the effects of some factors such as anoxia, metabolic inhibitors and specific inhibitors of H⁺-ATPase have been examined. The findings have led to the conclusion that blue light inactivates the electrogenic H⁺-pumping ATPase in the plasma membrane of the motor cells. This inactivation seems to suppress ion uptake and decrease the turgor pressure of the motor cells.     

Triplet States in a Photosystem I Reaction Center Complex. Inhibition of Radical Pair Recombination by Bipyridinium Dyes and Naphthoquinones

Flash-induced absorption changes between 400 and 570 nm werestudied in a P700-chlorophyll a-protein complex from the thermophiliccyanobacterium Synechococcus sp. that lacked the bound secondaryelectron acceptors A₂ and P430. A positive peak at 520 nm, whichincreased linearly with the flash intensity and independentlyof the redox state of P700, is ascribed to a carotenoid triplet.Bleaching at 430 nm, which decayed with a half time of about10 µs, was abolished when P700 was oxidized with ferricyanideand saturated at a high flash intensity, indicative of its dependenceon the primary photochemistry of photosystem I. Several bipyridinium dyes and naphthoquinones suppressed the10 µs decay of the 430 nm signal in a way indicating thatthe 10 µs component represents the P700 triplet generatedby the back reaction between the reduced primary electron acceptorand oxidized P700 and that the added oxidants oxidize the reducedprimary acceptor so rapidly that back electron transfer to oxidizedP700 is prevented. Our results also show that the primary electronacceptor is located in a lypophilic environment in the chlorophyll-bindingsubunits of the photosystem I complexes. In a reaction centercomplex containing the secondary electron acceptors, the exogenousoxidants accept electrons only via P430. (Received February 23, 1984; Accepted May 1, 1984)     

miR-30a attenuates drug sensitivity to 5-FU by modulating cell proliferation possibly by downregulating cyclin E2 in oral squamous cell carcinoma

We aimed to determine the functional role of the miRNA, which affects drug sensitivity to 5-FU in oral squamous cell carcinoma (OSCC), using two types of 5-FU-resistant and parental OSCC cell lines. MiRNA microarray data showed that miR-30a was significantly upregulated in two resistant cell lines. Therefore, we investigated the effects and molecular mechanism of miR-30a on 5-FU sensitivity. Stable overexpression of miR-30a in parental OSCC cells decreased cell proliferation and attenuated drug sensitivity to 5-FU. Cell cycle analysis indicated that miR-30a overexpression increased the proportion of G1 phase cells and decreased the proportion of S phase cells. MiR-30a knockdown using siRNA reversed the effects of miR-30a overexpression. DNA microarray analysis using miR-30a-overexpressing cell lines and a TargetScan database search showed that cyclin E2 (CCNE2) is a target of miR-30a. A luciferase reporter assay confirmed that a miR-30a mimic interacted with the specific binding site in the 3' UTR of CCNE2. CCNE2 knockdown with siRNA in OSCC cells yielded decreased drug sensitivity to 5-FU, similar to miR-30a overexpressing cells. These findings suggest that miR-30a in OSCC may be a novel biomarker of 5-FU-resistant tumors, as well as a therapeutic target for combating resistance.     

Mutational analysis of N-glycosylation recognition sites on the biochemical properties of Aspergillus kawachii α-l-arabinofuranosidase 54

A role for N-linked oligosaccharides on the biochemical properties of recombinant α-l-arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N-linked glycosylation motifs (Asn⁸³–Thr–Thr and Asn²⁰²–Ser–Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N-linked glycosylation sites are located in the catalytic domain. Asn⁸³, Asn²⁰², and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn²⁰² may contribute to thermostability and catalysis.     

A New Method to Distinguish between Meiotic and Premeiotic Recombinational Events in DROSOPHILA MELANOGASTER

A new method is proposed to distinguish between meiotic and premeiotic exchange events in Drosophila melanogaster males associated with male recombination activities. The method was applied to data that have accumulated in this laboratory during the past five years, and it was concluded that a large fraction, perhaps the overwhelming majority, of the male recombinants were due to exchange events that took place before meiosis.     

A new method for determination of parity in optical diffraction patterns from the structures with helical symmetry

In optical diffraction patterns from the structures with helical symmetry, the reflections appear as layer-lines. The Bessel function orders (n) of the layer-lines are not easily determined, because the radial co-ordinates of the principal maxima on the layer-lines cannot be measured precisely. We developed a method to find whether n is even or odd (parity of n) by superposing two identical images of a particle back to back with the two axes aligned. The method was successfully applied to analysis of the structure of straight polyhooks isolated from the mutant SJW880 of Salmonella typhimurium.