Structural Basis for Conformational Dynamics of GTP-bound Ras Protein

Ras family small GTPases assume two interconverting conformations, “inactive” state 1 and “active” state 2, in their GTP-bound forms. Here, to clarify the mechanism of state transition, we have carried out x-ray crystal structure analyses of a series of mutant H-Ras and M-Ras in complex with guanosine 5′-(β,γ-imido)triphosphate (GppNHp), representing various intermediate states of the transition. Crystallization of H-RasT35S-GppNHp enables us to solve the first complete tertiary structure of H-Ras state 1 possessing two surface pockets unseen in the state 2 or H-Ras-GDP structure. Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the ³¹P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions. Collectively, these results clarify the structural features distinguishing the two states and provide new insights into the molecular basis for the state transition of Ras protein.     

Application of unnatural oligonucleotides to chemical modification of gene expression

Chemical entities that specifically interact with biological molecules can reveal the biological role of their targets. Increasing knowledge of genomes and the intricate mechanisms that regulate their expression has aroused considerable interest in molecules that target nucleic acids. A wide variety of technologies have been developed based on unnatural oligonucleotides for gene silencing, gene-detection or bio-inspired chemical reactions, for example. In contrast to natural oligonucleotides, their unnatural counterparts can exhibit unique functions based on the chemical reactivity of the accessory molecule. This review focuses on the molecular design of chemically reactive accessory molecules of unnatural oligonucleotides, emphasising their application in specific recognition and reaction toward biological targets.     

Cloning of rainbow trout SLC26A1: involvement in renal sulfate secretion

The kidney plays an important role in ion regulation in both freshwater and seawater fish. However, ion transport mechanisms in the teleost kidney are poorly understood, especially at the molecular level. We have cloned a kidney-specific SLC26 sulfate/anion exchanger from rainbow trout (Oncorhynchus mykiss) that is homologous to the mammalian SLC26A1 (Sat-1). Excretion of excess plasma sulfate concentration after Na2SO4 injection corresponded to significantly higher expression of the cloned SLC26A1 mRNA. Detailed morphological observation of rainbow trout renal tubules was also performed by light microscopy and transmission electron microscopy. According to the structure of brush border and tubular system in the cytoplasm, renal tubules of rainbow trout were classified into proximal tubule first and second (PI and PII) segments and distal tubules. In situ hybridization revealed that SLC26A1 anion exchanger mRNA is specifically localized in the PI segment of kidneys from both seawater- and freshwater-adapted rainbow trout. With immunocytochemistry, Na+-K+-ATPase and vacuolar-type H+-ATPase were colocalized to the same cells and distributed in the basolateral and the apical membranes, respectively, of the cells where the SLC26A1 mRNA expressed. These findings suggest that the cloned kidney-specific SLC26A1 is located in kidney proximal tubules and is involved in excretion of excess plasma sulfate in rainbow trout.     

Involvement of Gi/o in the PAR-4-induced NO production in endothelial cells

We investigated the involvement of G(i/o) protein in NO production following the activation of proteinase-activated receptor-4 (PAR-4) in cultured bovine aortic endothelial cells. AYPGKF-NH(2) (PAR-4 activating peptide), thrombin, and ionomycin induced a concentration-dependent NO production, with the maximal production seen at 30 microM, 0.1U/ml, and 1 microM, respectively. Ionomycin elevated [Ca(2+)](i) in a concentration-dependent manner. However, AYPGKF-NH(2) and thrombin induced no [Ca(2+)](i) elevation. The loading of cells with BAPTA almost completely inhibited both the NO production and [Ca(2+)](i) elevation induced by 1 microM ionomycin, while it had no significant effect on the AYPGKF-NH(2)-induced NO production. Treatment with pertussis toxin inhibited the AYPGKF-NH(2)-induced NO production, while it had no effect on the ionomycin-induced NO production. Our findings thus demonstrate, for the first time, that PAR-4 activation induced NO production in a manner mostly independent of the Ca(2+) signal and also that G(i/o) is involved in such NO production in vascular endothelial cells.     

Application of 2-amino-6-vinylpurine as an efficient agent for conjugation of oligonucleotides

Attempts have been made to conjugate a variety of molecules with oligonucleotides to achieve useful functions. In this study, we have established a new efficient method for post-synthetic conjugation of oligonucleotides with the use of the 2-amino-6-vinylpurine nucleoside. Amino nucleophiles form the corresponding conjugates under acidic conditions, whereas thiol nucleophiles reacted efficiently under alkaline conditions. Thus, glutathione and HS-Cys-(Arg)8 without protecting groups were efficiently conjugated to the 2-amino-6-vinylpurine-bearing ODN under alkaline conditions. The use of 2-amino-6-vinylpurine as an agent for conjugation is advantageous in that it is stable during the reaction and may be applied to conjugation of ODNs with multiple functional molecules.     

Crystal structures of the state 1 conformations of the GTP-bound H-Ras protein and its oncogenic G12V and Q61L mutants

GTP-bound Ras adopts two interconverting conformations, "inactive" state 1 and "active" state 2. However, the tertiary structure of wild-type (WT) state 1 remains unsolved. Here we solve the state 1 crystal structures of H-Ras WT together with its oncogenic G12V and Q61L mutants. They assume open structures characterized by impaired interactions of both Thr-35 in switch I and Gly-60 in switch II with the γ-phosphate of GTP and possess two surface pockets of mutually different shapes unseen in state 2, a potential target for selective inhibitor development. Furthermore, they provide a structural basis for the low GTPase activity of state 1.     

Production of truncated protein by the crosslink formation of mRNA with 2'-OMe oligoribonucleotide containing 2-amino-6-vinylpurine

The development of convenient methods for controlling the protein expression is an important challenge in the postgenomic era. We applied the crosslink forming oligonucleotide (CFO) as a terminator of the ribosomal translation. In this study, we demonstrated that the improved reactivity of our CFO under physiological conditions enabled the sequence-specific introduction of a steric block for a ribosome on mRNAs. In vitro and in cell translation experiments revealed that the crosslinked mRNA can produce the truncated proteins in which the translation terminates at the desired position.     

IL-15 serves as a costimulator in determining the activity of autoreactive CD8 T cells in an experimental mouse model of graft-versus-host-like disease

To elucidate the mechanisms controlling peripheral tolerance, we established two transgenic (Tg) mouse strains expressing different levels of membrane-bound OVA (mOVA) as a skin-associated self-Ag. When we transferred autoreactive TCR-Tg CD8 T cells (OT-I cells), keratin 14 (K14)-mOVA(high) Tg mice developed autoreactive skin disease (graft-vs-host disease (GVHD)-like skin lesions) while K14-mOVA(low) Tg mice did not. OT-I cells in K14-mOVA(high) Tg mice were fully activated with full development of effector function. In contrast, OT-I cells in K14-mOVA(low) Tg mice proliferated but did not gain effector function. Exogenous IL-15 altered the functional status of OT-I cells and concomitantly induced disease in K14-mOVA(low) Tg mice. Conversely, neutralization of endogenous IL-15 activity in K14-mOVA(high) Tg mice attenuated GVHD-like skin lesions induced by OT-I cell transfer. Futhermore, K14-mOVA(high) Tg mice on IL-15 knockout or IL-15Ralpha knockout backgrounds did not develop skin lesions after adoptive transfer of OT-I cells. These results identify IL-15 as an indispensable costimulator that can determine the functional fate of autoreactive CD8 T cells and whether immunity or tolerance ensues, and they suggest that inhibition of IL-15 function may be efficacious in blocking expression of autoimmunity where a breach in peripheral tolerance is suspected.     

Distinct Na+/K+/2Cl- cotransporter localization in kidneys and gills of two euryhaline species, rainbow trout and killifish

The kidney is an organ playing an important role in ion regulation in both freshwater (FW) and seawater (SW) fish. The mechanisms of ion regulation in the fish kidney are less well studied than that of their gills, especially at the level of transporter proteins. We have found striking differences in the pattern of Na⁺/K⁺/2Cl^- cotransporter (NKCC) expression between species. In the killifish kidney, NKCC is apically localized in the distal and collecting tubules and basolaterally localized in the proximal tubules. However, in the SW killifish gill, NKCC is basolaterally co-localized with Na⁺/K⁺-ATPase, whereas in FW, NKCC immunoreactivity is primarily apical, although still colocalized within the same mitochondria-rich cell with basolateral Na⁺/K⁺-ATPase. Rainbow trout kidney has NKCC only in the apical membrane of the distal and collecting tubules in both environments, with no signal being detected in the proximal tubule. On the other hand, in the trout gill, NKCC is found basolaterally in both FW and SW environments. An important observation is that, in the gills of rainbow trout, the trailing edge of the filament possesses mostly Na⁺/K⁺-ATPase-positive but NKCC-negative mitochondria-rich cells, whereas in the region between and at the roots of the gill lamellae, most mitochondria-rich cells exhibit both Na⁺/K⁺-ATPase- and NKCC-positive immunoreactivity. These results suggest that the differential localization of transporters between the two species represents differences in function between these two euryhaline fishes with different life histories and strategies. Funding for this research was provided by NSERC Discovery Grants to G.G.G. and W.S.M., an Alberta Ingenuity Fund PDF, and a fellowship from the NSERC Research Capacity Development Grant to F.K.     

Trends in Childhood Obesity in Japan over the Last 25 Years from the National Nutrition Survey

Objective: To describe the 25‐year changes in BMI (measured in kilograms per meters squared) and the prevalence of obesity in Japanese children with special reference to urban‐rural differences. Research Methods and Procedures: We used the data sets from the cross‐sectional annual nationwide surveys (National Nutrition Survey, Japan) conducted from 1976 to 2000 and comprising 29, 052 boys and 27, 552 girls between 6 and 14 years of age. We carried out the trend analyses with the data on sex and age groups and on residential areas according to the size of the municipality (metropolitan areas, cities, and small towns). Results: The mean (age‐adjusted) BMI increased by +0.32 kg/m² per 10 years in boys and by +0.24 kg/m² per 10 years in girls, increases that were remarkable in small towns. The prevalence of obese boys and girls increased from 6.1% and 7.1%, respectively, in the time‐period 1976 to 1980, to 11.1% and 10.2% in 1996 to 2000. The increasing trend was most evident in 9‐ to 11‐year‐old children of both sexes living in small towns, whereas no changes were observed in girls in metropolitan areas. Discussion: Our data clearly show increasing trends in obesity prevalence in Japanese school children. Degrees of the increasing trends, however, differed across sex and age groups and residential areas, demonstrating a particular phenomenon that girls in metropolitan areas were unlikely to become obese. These epidemiological aspects indicate the priorities for intervention in population strategies to control obesity in children.