Mechanical unfolding of covalently linked GroES: Evidence of structural subunit intermediates

It is difficult to determine the structural stability of the individual subunits or protomers of many proteins in the cell that exist in an oligomeric or complexed state. In this study, we used single‐molecule force spectroscopy on seven subunits of covalently linked cochaperonin GroES (ESC7) to evaluate the structural stability of the subunit. A modified form of ESC7 was immobilized on a mica surface. The force‐extension profile obtained from the mechanical unfolding of this ESC7 showed a distinctive sawtooth pattern that is typical for multimodular proteins. When analyzed according to the worm‐like chain model, the contour lengths calculated from the peaks in the profile suggested that linked‐GroES subunits unfold in distinct steps after the oligomeric ring structure of ESC7 is disrupted. The evidence that structured subunits of ESC7 withstand external force to some extent even after the perturbation of the oligomeric ring structure suggests that a stable monomeric intermediate is an important component of the equilibrium unfolding reaction of GroES.     

Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

The Corynebacterium alkanolyticum xylEFGD gene cluster comprises the xylD gene that encodes an intracellular β-xylosidase next to the xylEFG operon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover for p-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s⁻¹), weak α-l-arabinofuranosidase activity (turnover for p-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s⁻¹), and high tolerance to product inhibition (K_i for xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutive tac promoter in the Corynebacterium glutamicum xylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended on cgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguous xylD gene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability of C. glutamicum to take up xylooligosaccharides, an ability that is enhanced by in the presence of a functional xylEFG-encoded xyloside ABC transporter. The finding that xylEFG imparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates.     

Glycogen debranching pathway deduced from substrate specificity of glycogen debranching enzyme

Glycoconjugate Journal - Glycogen debranching enzyme (GDE) is bifunctional in that it exhibits both 4-α-glucanotransferase and amylo-α-1,6-glucosidase activity at two distinct catalytic...     

Adaptive response in embryogenesis. III. Relationship to radiation-induced apoptosis and Trp53 gene status

We reported previously that a radiation-induced adaptive response existed in the late period of embryogenesis, and that radiation-induced apoptosis in the predigital regions was responsible for digital defects in embryonic ICR mice. To investigate the possible involvement of the Trp53 gene and radiation-induced apoptosis in radiation-induced adaptive responses in embryogenesis, the present study was conducted using Trp53 wild-type (Trp53(+/+)) and Trp53 heterozygous (Trp53(+/-)) embryonic mice of the C57BL/6 strain. The existence of a radioadaptive response in the Trp53(+/+) embryonic mice was demonstrated by irradiating the embryos with 5 or 30 cGy on embryonic day 11 prior to a challenging irradiation at 3 Gy on embryonic day 12. The two conditioning doses at 5 and 30 cGy significantly suppressed the induction of apoptosis by the challenging dose in the predigital regions of limb buds in the Trp53(+/+) embryonic mice, while no such effect was found in the Trp53(+/-) embryonic mice. These findings indicate that induction of a radioadaptive response in embryogenesis is related to Trp53 gene status and the occurrence of radiation-induced apoptosis.     

Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members

Unique among fibroblast growth factors (FGFs), FGF19, -21, and -23 act in an endocrine fashion to regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis. These FGFs require the presence of Klotho/betaKlotho in their target tissues. Here, we present the crystal structures of FGF19 alone and FGF23 in complex with sucrose octasulfate, a disaccharide chemically related to heparin. The conformation of the heparin-binding region between beta strands 10 and 12 in FGF19 and FGF23 diverges completely from the common conformation adopted by paracrine-acting FGFs. A cleft between this region and the beta1-beta2 loop, the other heparin-binding region, precludes direct interaction between heparin/heparan sulfate and backbone atoms of FGF19/23. This reduces the heparin-binding affinity of these ligands and confers endocrine function. Klotho/betaKlotho have evolved as a compensatory mechanism for the poor ability of heparin/heparan sulfate to promote binding of FGF19, -21, and -23 to their cognate receptors.     

Skp2 controls adipocyte proliferation during the development of obesity

The increase in the mass of adipose tissue during the development of obesity can arise through an increase in cell size, an increase in cell number, or both. Here we show that long term maintenance of C57BL/6 mice on a high fat diet (for approximately 25 weeks) induces an initial increase in adipocyte size followed by an increase in adipocyte number in white adipose tissue. The latter effect was found to be accompanied by up-regulation of expression of the gene for the F-box protein Skp2 as well as by downregulation of the cyclin-dependent kinase inhibitor p27(Kip1), a principal target of the SCF(Skp2) ubiquitin ligase, in white adipose tissue. Ablation of Skp2 protected mice from the development of obesity induced either by a high fat diet or by the lethal yellow agouti (A(y)) mutation, and this protective action was due to inhibition of the increase in adipocyte number without an effect on adipocyte hypertrophy. The reduction in the number of adipocyte caused by Skp2 ablation also inhibited the development of obesity-related insulin resistance in the A(y) mutant mice, although the reduced number of beta cells and reduced level of insulin secretion in Skp2-deficient mice resulted in glucose intolerance. Our observations thus indicate that Skp2 controls adipocyte proliferation during the development of obesity.     

Anterosuperior protraction of maxillae using the extraoral device,RAMPA; finite element method

Abstract

This paper presents a boosting effect against gravity by analyzing the displacement and stress distribution of craniofacial structures due to the protraction of the extraoral device, the Right Angle Maxillary Protraction Appliance(RAMPA) system, including semi-rapid maxillary expansion (sRME) using the finite element method. In addition, a patient case was illustrated and compared with the results calculated from a simulation. The results from the finite element method were obtained for 0.5?mm activation using the screw of the intraoral device, gHu-1. This study reveals that RAMPA rotates the patient’s maxilla and mandible in the forward direction and forces them to move forward and upward.     

Rapid and accurate quantitative analysis of fermentation gases by Raman spectroscopy

Raman spectroscopy was employed for the precise quantitative analysis of a gaseous mixture. The band ratio between the Raman band of the target gas and that of the external standard was calculated and found to be proportional to the pressure of the target gas. The linearity of the calibration curves was very good (higher than 0.999). The mole fractions of atmospheric nitrogen and atmospheric oxygen were determined from their partial pressures. The average molar fraction of atmospheric nitrogen was calculated to be 0.790, similar to the literature value. Those of methane, hydrogen, and carbon dioxide, which are produced by fermentation, were also determined from the Raman spectrum. The values were identical to those obtained from the measured volume of each gas. The possibility of quantifying gas molecules by Raman spectroscopy is demonstrated.     

Phosphodiesterase inhibitors. Part 5: Hybrid PDE3/4 inhibitors as dual bronchorelaxant/anti-inflammatory agents for inhaled administration

(?)-6-(7-Methoxy-2-(trifluoromethyl)pyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (KCA-1490) exhibits moderate dual PDE3/4-inhibitory activity and promises as a combined bronchodilatory/anti-inflammatory agent. N-alkylation of the pyridazinone ring markedly enhances potency against PDE4 but suppresses PDE3 inhibition. Addition of a 6-aryl-4,5-dihydropyridazin-3(2H)-one extension to the N-alkyl group facilitates both enhancement of PDE4-inhibitory activity and restoration of potent PDE3 inhibition. Both dihydropyridazinone rings, in the core and extension, can be replaced by achiral 4,4-dimethylpyrazolone subunits and the core pyrazolopyridine by isosteric bicyclic heteroaromatics. In combination, these modifications afford potent dual PDE3/4 inhibitors that suppress histamine-induced bronchoconstriction in vivo and exhibit promising anti-inflammatory activity via intratracheal administration.