Glutathione transferases with vanadium-binding activity isolated from the vanadium-rich ascidian Ascidia sydneiensis samea

Some ascidians accumulate vanadium in vanadocytes, which are vanadium-containing blood cells, at high levels and with high selectivity. However, the mechanism and physiological significance of vanadium accumulation remain unknown. In this study, we isolated novel proteins with a striking homology to glutathione transferases (GSTs), designated AsGST-I and AsGST-II, from the digestive system of the vanadium-accumulating ascidian Ascidia sydneiensis samea, in which the digestive system is thought to be involved in vanadium uptake. Analysis of recombinant AsGST-I confirmed that AsGST-I has GST activity and forms a dimer, as do other GSTs. In addition, AsGST-I was revealed to have vanadium-binding activity, which has never been reported for GSTs isolated from other organisms. AsGST-I bound about 16 vanadium atoms as either V(IV) or V(V) per dimer, and the apparent dissociation constants for V(IV) and V(V) were 1.8 x 10(-4) M and 1.2 x 10(-4) M, respectively. Western blot analysis revealed that AsGSTs were expressed in the digestive system at exceptionally high levels, although they were localized in almost all organs and tissues examined. Considering these results, we postulate that AsGSTs play important roles in vanadium accumulation in the ascidian digestive system.     

Gliding locomotion of Siberian flying squirrels in low-canopy forests: the role of energy-inefficient short-distance glides

Short glides of less than 20 m seem energy inefficient for the Siberian flying squirrel Pteromys volans as with the northern flying squirrel Glaucomys sabrinus. However, Siberian flying squirrels in low-canopy forests frequently use short glides. Therefore, we sought to clarify the gliding patterns of Siberian flying squirrels for energy-efficient gliding transport in low-canopy forests (mean tree height, 15.3 m) in Hokkaido, Japan, based on records of 66 glides and 35 launch and landing trees. Mean launch height, landing height, and horizontal glide distance were 14.4, 2.7, and 21.4 m, respectively. For short distances, horizontal glide distance was strongly correlated with launch heights but not with launch tree height. For glides of more than 20 m, horizontal glide distance was significantly correlated with both launch height and launch tree height. The mean heights of launch and landing trees for short glides were 15.6 and 19.5 m, respectively. For long glides, these heights were 22.7 and 19.2 m. For short glides, mean launch tree height did not differ from overall mean tree height. However, for long glides, the mean launch tree height was greater than the overall mean tree height. Also, for short glides, the height of the landing tree was greater than that of the launch tree. Launch trees used for long glides were as high as the landing trees used in short glides. From these results, we conclude that Siberian flying squirrels in low-canopy forests save energy by gliding initially from a tree with sufficient height to permit a glide to a taller tree. This taller tree then permits long-distance glides that are energetically more efficient.     

Effect of acute exercise on glycogen synthase in muscle from obese and diabetic subjects

Insulin stimulates glycogen synthase (GS) through dephosphorylation of serine residues, and this effect is impaired in skeletal muscle from insulin-resistant [obese and type 2 diabetic (T2DM)] subjects. Exercise also increases GS activity, yet it is not known whether the ability of exercise to affect GS is impaired in insulin-resistant subjects. The objective of this study was to examine the effect of acute exercise on GS phosphorylation and enzyme kinetic properties in muscle from insulin-resistant individuals. Lean normal glucose-tolerant (NGT), obese NGT, and obese T2DM subjects performed 40 min of moderate-intensity cycle exercise (70% of Vo(2max)). GS kinetic properties and phosphorylation were measured in vastus lateralis muscle before exercise, immediately after exercise, and 3.5 h postexercise. In lean subjects, GS fractional activity increased twofold after 40 min of exercise, and it remained elevated after the 3.5-h rest period. Importantly, exercise also decreased GS K(m) for UDP-glucose from ≈0.5 to ≈0.2 mM. In lean subjects, exercise caused significant dephosphorylation of GS by 50-70% (Ser(641), Ser(645), and Ser(645,649,653,657)), and phosphorylation of these sites remained decreased after 3.5 h; Ser? phosphorylation was not regulated by exercise. In obese NGT and T2DM subjects, exercise increased GS fractional activity, decreased K(m) for UDP-glucose, and decreased GS phosphorylation as effectively as in lean NGT subjects. We conclude that the molecular regulatory process by which exercise promotes glycogen synthesis in muscle is preserved in insulin-resistant subjects.     

Thr649Ala-AS160 knock-in mutation does not impair contraction/AICAR-induced glucose transport in mouse muscle

AS160 and its closely related protein TBC1D1 have emerged as key mediators for both insulin- and contraction-stimulated muscle glucose uptake through regulating GLUT4 trafficking. Insulin increases AS160 phosphorylation at multiple Akt/PKB consensus sites, including Thr(649), and promotes its binding to 14-3-3 proteins through phospho-Thr(649). We recently provided genetic evidence that AS160-Thr(649) phosphorylation/14-3-3 binding plays a key role in mediating insulin-stimulated glucose uptake in muscle. Contraction has also been proposed to increase phosphorylation of AS160 and TBC1D1 via AMPK, which could be detected by a generic phospho-Akt substrate (PAS) antibody. Here, analysis of AS160 immunoprecipitates from muscle extracts with site-specific phospho-antibodies revealed that contraction and AICAR caused no increase but rather a slight decrease in phosphorylation of the major PAS recognition site AS160-Thr(649). In line with this, contraction failed to enhance 14-3-3 binding to AS160. Consistent with previous reports, we also observed that in situ contraction stimulated the signal intensity of PAS antibody immunoreactive protein of ～150-160 kDa in muscle extracts. Using a TBC1D1 deletion mutant mouse, we showed that TBC1D1 protein accounted for the majority of the PAS antibody immunoreactive signals of ～150-160 kDa in extracts of contracted muscles. Consistent with the proposed role of AS160-Thr(649) phosphorylation/14-3-3 binding in mediating glucose uptake, AS160-Thr(649)Ala knock-in mice displayed normal glucose uptake upon contraction and AICAR in isolated muscles. We conclude that the previously reported PAS antibody immunoreactive band ～150-160 kDa, which were increased upon contraction, does not represent AS160 but TBC1D1, and that AS160-Thr(649)Ala substitution impairs insulin- but neither contraction- nor AICAR-stimulated glucose uptake in mouse skeletal muscle.     

Dehydroepiandrosterone reduces preadipocyte proliferation via androgen receptor

Several studies have suggested that both testosterone and dehydroepiandrosterone (DHEA) have weight-reducing and antidiabetic effects, especially in rodent studies; however, the precise mechanism of their action remains unclear. Here, we investigated the effect of DHEA on cell growth in adipose tissue. The appearance of senescence-associated β-galactosidase in stromal vascular fraction (SVF) isolated from Otsuka Long-Evans Tokushima fatty rats, an animal model of inherent obese type 2 diabetes, was prevented by DHEA administration. Next, the effects of DHEA and testosterone were compared in vivo and in vitro to evaluate whether these hormones influence cell growth in adipose tissue. Both DHEA and testosterone reduced body weight and epididymal fat weight equivalently when administered for 4 wk. To assess the effect of DHEA and testosterone on cell growth in adipose tissue, 5-bromo-2'-deoxyuridine (BrdU) uptake by SVF was measured. Quantification analysis of BrdU uptake by examining DNA isolated from each SVF revealed that treatment with DHEA and testosterone reduced cell replication. These results indicated that DHEA- and testosterone-induced decreased adiposity was associated with reduced SVF growth. Incubation with DHEA and testosterone equally decreased BrdU uptake by 3T3-L1 preadipocytes. Pretreatment with the androgen receptor (AR) inhibitor flutamide, but not the estrogen receptor inhibitor fulvestrant, abolished these effects. Knockdown of AR with siRNA also inhibited DHEA-induced decreases in BrdU uptake. These results suggest that DHEA-induced growth suppression of preadipocytes is mediated via AR. Therefore, both DHEA and testosterone similarly decrease adipocyte growth possibly via a common mechanism.     

The AMPK-related kinase SIK2 is regulated by cAMP via phosphorylation at Ser358 in adipocytes

SIK2 (salt-inducible kinase 2) is a member of the AMPK (AMP-activated protein kinase) family of kinases and is highly expressed in adipocytes. We investigated the regulation of SIK2 in adipocytes in response to cellular stimuli with relevance for adipocyte function and/or AMPK signalling. None of the treatments, including insulin, cAMP inducers or AICAR (5-amino-4-imidazolecarboxamide riboside), affected SIK2 activity towards peptide or protein substrates in vitro. However, stimulation with the cAMP-elevating agent forskolin and the β-adrenergic receptor agonist CL 316,243 resulted in a PKA (protein kinase A)-dependent phosphorylation and 14-3-3 binding of SIK2. Phosphopeptide mapping of SIK2 revealed several sites phosphorylated in response to cAMP induction, including Ser(358). Site-directed mutagenesis demonstrated that phosphorylation of Ser(358), but not the previously reported PKA site Ser(587), was required for 14-3-3 binding. Immunocytochemistry illustrated that the localization of exogenously expressed SIK2 in HEK (human embryonic kidney)-293 cells was exclusively cytosolic and remained unchanged after cAMP elevation. Fractionation of adipocytes, however, revealed a significant increase of wild-type, but not Ser358Ala, HA (haemagglutinin)-SIK2 in the cytosol and a concomitant decrease in a particulate fraction after CL 316,243 treatment. This supports a phosphorylation-dependent relocalization in adipocytes. We hypothesize that regulation of SIK2 by cAMP could play a role for the critical effects of this second messenger on lipid metabolism in adipocytes.     

REACH Coarse-Grained Simulation of a Cellulose Fiber

A molecular level understanding of the structure, dynamics and mechanics of cellulose fibers can aid in understanding the recalcitrance of biomass to hydrolysis in cellulosic biofuel production. Here, a residue-scale REACH (Realistic Extension Algorithm via Covariance Hessian) coarse-grained force field was derived from all-atom molecular dynamics (MD) simulations of the crystalline Iβ cellulose fibril. REACH maps the atomistic covariance matrix onto coarse-grained elastic force constants. The REACH force field was found to reproduce the positional fluctuations and low-frequency vibrational spectra from the all-atom model, allowing elastic properties of the cellulose fibril to be characterized using the coarse-grained force field with a speedup of >20 relative to atomistic MD on systems of the same size. The calculated longitudinal/transversal Young's modulus and the velocity of sound are in agreement with experiment. The persistence length of a 36-chain cellulose microcrystal was estimated to be ～380 μm. Finally, the normal-mode analysis with the REACH force field suggests that intrinsic dynamics might facilitate the deconstruction of the cellulose fibril from the hydrophobic surface.     

Proteomic analysis of ganglioside‐associated membrane molecules: Substantial basis for molecular clustering

Ganglioside GD3 is specifically expressed in human melanomas, and plays a role in the enhancement of malignant phenotypes of melanoma cells. To analyze the mechanisms by which GD3 enhances malignant properties and signals in melanomas, it is essential to clarify how GD3 interacts with membrane molecules on the cell membrane. In this study, we performed proteomics analysis of glycolipid‐enriched microdomains (GEM) with current sucrose density gradient ultracentrifugation of Triton X‐100 extracts and MS. We also examined GD3‐associated molecules using enzyme‐mediated activation of radical sources (EMARS) reaction combined with MS. Comparison of molecules identified as residents in GEM/rafts and those detected by EMARS reaction using an anti‐GD3 antibody revealed that a relatively low number of molecules is recruited around GD3, while a number of membrane and secreted molecules was defined in GEM/rafts. These results suggested that EMARS reaction is useful to identify actually interacting molecules with gangliosides such as GD3 on the cell membrane, and many other microdomains than GD3‐associating rafts exist. Representative examples of GD3‐associated molecules such as neogenin and MCAM were shown.     

Introgression of Oncorhynchus masou subsp. (Biwa salmon) genome into lake-run O. m. ishikawae (Amago salmon) introduced into Lake Biwa, Japan

An investigation to determine the extent of introgression between Biwa salmon Oncorhynchus masou subsp. and lake-run Amago salmon O. m. ishikawae from Lake Biwa was conducted using mtDNA sequence and amplified fragment length polymorphism (AFLP) analysis. Hatchery-reared Amago salmon from the Samegai Trout Farm, the main source of stock released into the inflowing rivers of Lake Biwa, were also investigated. The extent of mtDNA introgression between these two subspecies was quite low: an anomalous haplotype was detected in just 1 of 55 Biwa salmon and 2 of 63 lake-run Amago salmon. Many of the mtDNA haplotypes found in lake-run Amago salmon were common to those in hatchery-reared Amago salmon. In the AFLP analysis, the nuclear genome of Amago salmon was hardly detected in Biwa salmon, but the Biwa salmon genome comprised 13.4% of the total amplified fragments from lake-run Amago salmon, being detected in different proportions in 28 of the 63 investigated individuals of the latter subspecies. These results suggest that introgression has occurred between Biwa salmon and lake-run Amago salmon descending from hatchery-reared Amago salmon from the Samegai Trout Farm.