Transcellobiosylation Reactions Catalyzed by Different Exoglucanase Components of a Trichoderma viride Cellulase in Aqueous Organic Solvent

The contribution of three exoglucanases from a commercial Trichoderma viride cellulase to transcellobiosylation, and the tolerance of these enzymes to acetonitrile co-solvent were studied. The enzymatic reactions were performed with p-nitrophenyl-β-d-cellobioside as the starting substrate. Among these enzymes, the least anionic exoglucanase (Exo I) showed the highest transcellobiosylation activity and acetonitrile tolerance. Exo I retained considerable activity even in 30% MeCN/water and produced p-nitrophenyl-β-d-cellotetraoside at about 1.5% conversion from the initial substrate in 30% MeCN/water. The residual activity of Exo I after incubation in MeCN/water mixture was almost identical to that of the crude cellulase and a considerable amount of the transcellobiosylation properties of the crude cellulase seemed to be attributable to this Exo I component.     

Circular permutation of ligand‐binding module improves dynamic range of genetically encoded FRET‐based nanosensor

Quantitative measurement of small molecules with high spatiotemporal resolution provides a solid basis for correct understanding and accurate modeling of metabolic regulation. A promising approach toward this goal is the FLIP (fluorescent indicator protein) nanosensor based on bacterial periplasmic binding proteins (PBPs) and fluorescence resonance energy transfer (FRET) between the yellow and cyan variants of green fluorescent protein (GFP). Each FLIP has a PBP module that specifically binds its ligand to induce a conformation change, leading to a change in FRET between the two GFP variant modules attached to the N‐ and C‐termini of the PBP. The larger is the dynamic range the more reliable is the measurement. Thus, we attempted to expand the dynamic range of FLIP by introducing a circular permutation with a hinge loop deletion to the PBP module. All the six circularly permutated PBPs tested, including structurally distinct Type I and Type II PBPs, showed larger dynamic ranges than their respective native forms when used for FLIP. Notably, the circular permutation made three PBPs, which totally failed to show FRET change when used as their native forms, fully capable of functioning as a ligand binding module of FLIP. These FLIPs were successfully used for the determination of amino acid concentration in complex solutions as well as real‐time measurement of amino acid influx in living yeast cells. Thus, the circular permutation strategy would not only improve the performance of each nanosensor but also expand the repertoire of metabolites that can be measured by the FLIP nanosensor technology.     

Pleiotropic roles of polyglycerolphosphate synthase of lipoteichoic acid in growth of Staphylococcus aureus cells

Lipoteichoic acid (LTA) is one of two anionic polymers on the surface of the gram-positive bacterium Staphylococcus aureus. LTA is critical for the bacterium-host cell interaction and has recently been shown to be required for cell growth and division. To determine additional biological roles of LTA, we found it necessary to identify permissive conditions for the growth of an LTA-deficient mutant. We found that an LTA-deficient S. aureus ΔltaS mutant could grow at 30°C but not at 37°C. Even at the permissive temperature, ΔltaS mutant cells had aberrant cell division and separation, decreased autolysis, and reduced levels of peptidoglycan hydrolases. Upshift of ΔltaS mutant cells to a nonpermissive temperature caused an inability to exclude Sytox green dye. A high-osmolarity growth medium remarkably rescued the colony-forming ability of the ΔltaS mutant at 37°C, indicating that LTA synthesis is required for growth under low-osmolarity conditions. In addition, the ΔltaS mutation was found to be synthetically lethal with the ΔtagO mutation, which disrupts the synthesis of the other anionic polymer, wall teichoic acid (WTA), at 30°C, suggesting that LTA and WTA compensate for one another in an essential function.     

Connective tissue growth factor promotes articular damage by increased osteoclastogenesis in patients with rheumatoid arthritis

Introduction

A protein analysis using a mass spectrometry indicated that there are serum proteins showing significant quantitative changes after the administration of infliximab. Among them, connective tissue growth factor (CTGF) seems to be related to the pathogenesis of rheumatoid arthritis (RA). Therefore, this study was conducted to investigate how CTGF is associated with the disease progression of RA.

Methods

Serum samples were collected from RA patients in active or inactive disease stages, and before or after treatments with infliximab. CTGF production was evaluated by ELISA, RT-PCR, indirect immunofluorescence microscopy, and immunoblotting. Osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase (TRAP) staining, a bone resorption assay and osteoclasts specific catalytic enzymes productions.

Results

The serum concentrations of CTGF in RA were greater than in normal healthy controls and disease controls. Interestingly, those were significantly higher in active RA patients compared to inactive RA patients. Furthermore, the CTGF levels significantly were decreased by infliximab concomitant with the disease amelioration. In addition, tumour necrosis factor (TNF)α can induce the CTGF production from synovial fibroblasts even though TNFα can oppositely inhibit the production of CTGF from chondrocytes. CTGF promoted the induction of the quantitative and qualitative activities of osteoclasts in combination with M-CSF and receptor activator of NF-κB ligand (RANKL). In addition, we newly found integrin αVβ3 on the osteoclasts as a CTGF receptor.

Conclusions

These results indicate that aberrant CTGF production induced by TNFα plays a central role for the abnormal osteoclastic activation in RA patients. Restoration of aberrant CTGF production may contribute to the inhibition of articular destruction in infliximab treatment.     

A physical study on the thermal ice ridge in a closed deep lake: Lake Kuttara, Hokkaido, Japan

An ice-ridge line more than 2,000 m long was discovered in a closed, deep lake—Lake Kuttara, Hokkaido, Japan—in the ice-covered season of 1995. To clarify when and how the ice-ridge line built up, meteorology and ice and water temperatures were monitored, and the density, thermal conductivity, and thickness of the covered ice were measured from January through March 1995. The covered ice on 4 and 5 March 1995, which corresponded to the period with the thickest lake ice, consisted of snow ice 0.05- to 0.07-m thick and candle ice 0.18- to 0.21-m thick. Numerical simulations for the ice thickness and temperature were carried out using a one-dimensional, unsteady, three-layer model. The simulated results, which were reasonable to the observations, indicate that due to the ice contraction and expansion, the ice-ridge line started to construct on 2 February and was completed the next day.     

Partial biopterin deficiency disturbs postnatal development of the dopaminergic system in the brain

Postnatal development of dopaminergic system is closely related to the development of psychomotor function. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of dopamine and requires tetrahydrobiopterin (BH4) as a cofactor. To clarify the effect of partial BH4 deficiency on postnatal development of the dopaminergic system, we examined two lines of mutant mice lacking a BH4-biosynthesizing enzyme, including sepiapterin reductase knock-out (Spr(-/-)) mice and genetically rescued 6-pyruvoyltetrahydropterin synthase knock-out (DPS-Pts(-/-)) mice. We found that biopterin contents in the brains of these knock-out mice were moderately decreased from postnatal day 0 (P0) and remained constant up to P21. In contrast, the effects of BH4 deficiency on dopamine and TH protein levels were more manifested during the postnatal development. Both of dopamine and TH protein levels were greatly increased from P0 to P21 in wild-type mice but not in those mutant mice. Serotonin levels in those mutant mice were also severely suppressed after P7. Moreover, striatal TH immunoreactivity in Spr(-/-) mice showed a drop in the late developmental stage, when those mice exhibited hind-limb clasping behavior, a type of motor dysfunction. Our results demonstrate a critical role of biopterin in the augmentation of TH protein in the postnatal period. The developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to high dependence of dopaminergic development on BH4 availability.     

ATP9B, a P4-ATPase (a putative aminophospholipid translocase), localizes to the trans-Golgi network in a CDC50 protein-independent manner

Type IV P-type ATPases (P4-ATPases) are putative phospholipid flippases that translocate phospholipids from the exoplasmic (lumenal) to the cytoplasmic leaflet of lipid bilayers and are believed to function in complex with CDC50 proteins. In Saccharomyces cerevisiae, five P4-ATPases are localized to specific cellular compartments and are required for vesicle-mediated protein transport from these compartments, suggesting a role for phospholipid translocation in vesicular transport. The human genome encodes 14 P4-ATPases and three CDC50 proteins. However, the subcellular localization of human P4-ATPases and their interactions with CDC50 proteins are poorly understood. Here, we show that class 5 (ATP10A, ATP10B, and ATP10D) and class 6 (ATP11A, ATP11B, and ATP11C) P4-ATPases require CDC50 proteins, primarily CDC50A, for their exit from the endoplasmic reticulum (ER) and final subcellular localization. In contrast, class 2 P4-ATPases (ATP9A and ATP9B) are able to exit the ER in the absence of exogenous CDC50 expression: ATP9B, but not ATP11B, was able to exit the ER despite depletion of CDC50 proteins by RNAi. Although ATP9A and ATP9B show a high overall sequence similarity, ATP9A localizes to endosomes and the trans-Golgi network (TGN), whereas ATP9B localizes exclusively to the TGN. A chimeric ATP9 protein in which the N-terminal cytoplasmic region of ATP9A was replaced with the corresponding region of ATP9B was localized exclusively to the Golgi. These results indicate that ATP9B is able to exit the ER and localize to the TGN independently of CDC50 proteins and that this protein contains a Golgi localization signal in its N-terminal cytoplasmic region.     

Adipocyte/macrophage fatty acid binding proteins control integrated metabolic responses in obesity and diabetes

Fatty acid binding proteins (FABPs) are cytosolic fatty acid chaperones whose biological role and mechanisms of action are not well understood. Here, we developed mice with targeted mutations in two related adipocyte FABPs, aP2 and mal1, to resolve their role in systemic lipid, glucose, and energy metabolism. Mice lacking aP2 and mal1 exhibited a striking phenotype with strong protection from diet-induced obesity, insulin resistance, type 2 diabetes, and fatty liver disease. These mice have altered cellular and systemic lipid transport and composition, leading to enhanced insulin receptor signaling, enhanced muscle AMP-activated kinase (AMP-K) activity, and dramatically reduced liver stearoyl-CoA desaturase-1 (SCD-1) activity underlying their phenotype. Taken together with the previously reported strong protection against atherosclerosis, these results demonstrate that adipocyte/macrophage FABPs have a robust impact on multiple components of metabolic syndrome, integrating metabolic and inflammatory responses in mice and constituting a powerful target for the treatment of these diseases.