Comparison of the structural changes in two cellobiohydrolases, CcCel6A and CcCel6C, from Coprinopsis cinerea--a tweezer-like motion in the structure of CcCel6C

The basidiomycete Coprinopsis cinerea produces five cellobiohydrolases belonging to glycoside hydrolase family 6 (GH6). Among these enzymes, C. cinerea cellulase 6C (CcCel6C), but not C. cinerea cellulase 6A (CcCel6A), can efficiently hydrolyze carboxymethyl cellulose and is constitutively expressed in C. cinerea. In contrast, CcCel6A possesses a cellulose-binding domain, and is strongly induced by cellobiose. Here, we determined the crystal structures of the CcCel6A catalytic domain complexed with a Hepes buffer molecule, with cellobiose, and with p-nitrophenyl β-D-cellotrioside (pNPG3). A notable feature of the GH6 cellobiohydrolases is that the active site is enclosed by two loops to form a tunnel, and the loops have been demonstrated to open and close in response to ligand binding. The enclosed tunnel of CcCel6A-Hepes is seen as the open form, whereas the tunnels of CcCel6A-cellobiose and CcCel6A-pNPG3 adopt the closed form. pNPG3 was not hydrolyzed by CcCel6A, and bound in subsites +1 to +4. On the basis of this observation, we constructed two mutants, CcCel6A D164A and CcCel6C D102A. Neither CcCel6A D164A nor CcCel6C D102A hydrolyze phosphoric acid-swollen cellulose. We have previously determined the crystal structures of CcCel6C unbound and in complex with ligand, both of which adopt the open form. In the present study, both CcCel6A and CcCel6C mutants were identified as the closed form. However, the motion angle of CcCel6C was more than 10-fold greater than that of CcCel6A. The width of the active site cleft of CcCel6C was narrowed, owing to a tweezer-like motion.     

Genetic variants on chromosome 1q41 influence ocular axial length and high myopia

As one of the leading causes of visual impairment and blindness, myopia poses a significant public health burden in Asia. The primary determinant of myopia is an elongated ocular axial length (AL). Here we report a meta-analysis of three genome-wide association studies on AL conducted in 1,860 Chinese adults, 929 Chinese children, and 2,155 Malay adults. We identified a genetic locus on chromosome 1q41 harboring the zinc-finger 11B pseudogene ZC3H11B showing genome-wide significant association with AL variation (rs4373767, β = −0.16 mm per minor allele, P_meta = 2.69×10⁻¹⁰). The minor C allele of rs4373767 was also observed to significantly associate with decreased susceptibility to high myopia (per-allele odds ratio (OR) = 0.75, 95% CI: 0.68–0.84, P_meta = 4.38×10⁻⁷) in 1,118 highly myopic cases and 5,433 controls. ZC3H11B and two neighboring genes SLC30A10 and LYPLAL1 were expressed in the human neural retina, retinal pigment epithelium, and sclera. In an experimental myopia mouse model, we observed significant alterations to gene and protein expression in the retina and sclera of the unilateral induced myopic eyes for the murine genes ZC3H11A, SLC30A10, and LYPLAL1. This supports the likely role of genetic variants at chromosome 1q41 in influencing AL variation and high myopia.     

Impact of soil water chemistry on the apparent sex ratio of the flowering ramets of the dioecious plant Myrica gale var. tomentosa

We determined whether the apparent (M/Fl) sex ratio (male ramets/flowering ramets) and apparent reproductive ramet ratio (Fl/Li ratio; flowering ramets/living ramets) in 15 Myrica gale var. tomentosa populations varied with dissolved total nitrogen, dissolved total phosphorus, potassium, magnesium, calcium, or pH in the soil water. Our aim was to define the environmental factors affecting the M/Fl sex ratio and Fl/Li ratio of the populations. We also examined the habitat conditions of these populations by analyzing soil water chemistry and water dynamics. In 2007, 3 of the 15 populations had no females. The remaining 12 had significantly male-biased (M/Fl sex ratio?=?0.59-0.97). Although we could not explain the absence of females by the current potassium levels alone, as potassium increased, so did the M/Fl sex ratio. As nitrogen increased and potassium decreased, Fl/Li ratio decreased. Our soil water chemistry analyses suggested that the potassium supply by soil surface erosion from flooding and the inflow of anthropogenic nitrogen were the important factors influencing the M/Fl sex ratio and Fl/Li ratio. Nitrogen management would be important in one of the endangered populations where inflow of nitrogen was the highest among 15 habitats.     

Binding of phytopolyphenol piceatannol disrupts β/γ subunit interactions and rate-limiting step of steady-state rotational catalysis in Escherichia coli F1-ATPase

In observations of single molecule behavior under V(max) conditions with minimal load, the F(1) sector of the ATP synthase (F-ATPase) rotates through continuous cycles of catalytic dwells (～0.2 ms) and 120° rotation steps (～0.6 ms). We previously established that the rate-limiting transition step occurs during the catalytic dwell at the initiation of the 120° rotation. Here, we use the phytopolyphenol, piceatannol, which binds to a pocket formed by contributions from α and β stator subunits and the carboxyl-terminal region of the rotor γ subunit. Piceatannol did not interfere with the movement through the 120° rotation step, but caused increased duration of the catalytic dwell. The duration time of the intrinsic inhibited state of F(1) also became significantly longer with piceatannol. All of the beads rotated at a lower rate in the presence of saturating piceatannol, indicating that the inhibitor stays bound throughout the rotational catalytic cycle. The Arrhenius plot of the temperature dependence of the reciprocal of the duration of the catalytic dwell (catalytic rate) indicated significantly increased activation energy of the rate-limiting step to trigger the 120° rotation. The activation energy was further increased by combination of piceatannol and substitution of γ subunit Met(23) with Lys, indicating that the inhibitor and the β/γ interface mutation affect the same transition step, even though they perturb physically separated rotor-stator interactions.     

Bipartite nuclear localization signal of matrin 3 is essential for vertebrate cells

Matrin 3, a nuclear matrix protein has potential (1) to withhold promiscuously edited RNAs within the nucleus in cooperation with p54(nrb) and PSF, (2) to mediate NMDA-induced neuronal death, and (3) to modulate promoter activity of genes proximal to matrix/scaffold attachment region (MAR/SAR). We identified a bipartite nuclear localization signal (NLS) of chicken matrin 3 (cmatr3) at residues 583-602. By expressing green fluorescent protein (GFP) fused to the NLS mutant in chicken DT40 cells, we showed an essential role of the NLS for cell proliferation. Furthermore, we showed that both clusters of basic amino acids and a linker of the bipartite NLS were essential and sufficient for the nuclear import of GFP. Exogenous cmatr3 rescued the HeLa cells where human matrin 3 was suppressed by RNA interference, but cmatr3 containing deletions at either of the basic amino acid clusters or the linker could not.     

Effects of topography on status and changes in land-cover patterns,Chongqing City,China

Chongqing Municipality, located on the upper reaches of the Yangtze River, is one of China’s four largest megacities, comparable with Shanghai, Beijing, and Tianjin. In recent years, Chongqing and its surroundings have been experiencing severe environmental problems, such as urbanization, pollution, and deforestation, due to the rapid economic development of China’s inland region. Understanding the current land-cover status over a wide scale, as well as changes in land-cover over time, are necessary for improving the urban environment and implementing conservation measures in the Chongqing region. In this study, remote-sensing data from 1993 and 2001 were used to analyze land-cover changes. Due to the distinctive mountainous topography of Chongqing, digital topography data shuttle radar topography mission (SRTM)-3 were also used. The results of the analysis showed that urban area increased from 109.91 km² (5.7%) in 1993 to 166.84 km² (8.7%) in 2001. This increase came at the expense of agricultural and vegetated areas. Although irrigated paddy field and dry farm land decreased due to the development, these two land-cover categories still covered the largest agricultural area. Correlating the land-cover changes with different topography types, the results showed that forest covered a larger area and enjoyed a higher frequency of distribution at higher elevations and on steeper slopes. The increase in urban area, in contrast, was apparently associated with lower elevations and milder slopes. These results indicate that incorporating terrain characteristics into remote-sensing analysis can be a useful tool in developing conservation measures for vegetated and agricultural areas.     

Dimethyl Fumarate Protects Pancreatic Islet Cells and Non-Endocrine Tissue in L-Arginine-Induced Chronic Pancreatitis

Background

Chronic pancreatitis (CP) is a progressive disorder resulting in the destruction and fibrosis of the pancreatic parenchyma which ultimately leads to impairment of the endocrine and exocrine functions. Dimethyl Fumarate (DMF) was recently approved by FDA for treatment of patients with multiple sclerosis. DMF''s unique anti-oxidant and anti-inflammatory properties make it an interesting drug to test on other inflammatory conditions. This study was undertaken to determine the effects of DMF on islet cells and non-endocrine tissue in a rodent model of L-Arginine-induced CP.

Methods

Male Wistar rats fed daily DMF (25 mg/kg) or vehicle by oral gavage were given 5 IP injections of L-Arginine (250 mg/100 g×2, 1 hr apart). Rats were assessed with weights and intra-peritoneal glucose tolerance tests (IPGTT, 2 g/kg). Islets were isolated and assessed for islet mass and viability with flow cytometry. Non-endocrine tissue was assessed for histology, myeloperoxidase (MPO), and lipid peroxidation level (MDA). In vitro assessments included determination of heme oxygenase (HO-1) protein expression by Western blot.

Results

Weight gain was significantly reduced in untreated CP group at 6 weeks. IPGTT revealed significant impairment in untreated CP group and its restoration with DMF therapy (P <0.05). Untreated CP rats had pancreatic atrophy, severe acinar architectural damage, edema, and fatty infiltration as well as elevated MDA and MPO levels, which were significantly improved by DMF treatment. After islet isolation, the volume of non-endocrine tissue was significantly smaller in untreated CP group. Although islet counts were similar in the two groups, islet viability was significantly reduced in untreated CP group and improved with DMF treatment. In vitro incubation of human pancreatic tissue with DMF significantly increased HO-1 expression.

Conclusion

Administration of DMF attenuated L-Arginine-induced CP and islet function in rats. DMF treatment could be a possible strategy to improve clinical outcome in patients with CP.     

Elimination of epiplakin by gene targeting results in acceleration of keratinocyte migration in mice

Epiplakin (EPPK) was originally identified as a human epidermal autoantigen. To identify the function of epiplakin, we generated epiplakin "knockout" mice. These mice developed normally, with apparently normal epidermis and hair. Electron microscopy after immunostaining revealed the presence of EPPK adjacent to keratin filaments in wild-type mice, suggesting that epiplakin might associate with keratin. The appearance and localization of keratin bundles in intact epidermal keratinocytes of EPPK-/- mice were similar to those in wild-type mice. Wounds on the backs of EPPK-/- mice closed more rapidly than those on the backs of wild-type and heterozygous mice. The outgrowth of keratinocytes from skin explants from knockout mice was enhanced compared to outgrowth from explants from wild-type mice, even in the presence of mitomycin C, suggesting that the difference in keratinocyte outgrowth might be due to a difference in the speed of migration of keratinocytes. At wound edges in wild-type mice, EPPK was expressed in proliferating keratinocytes in conjunction with keratin 6. In EPPK-/- mice, no similar proliferating keratinocytes were observed, but migrating keratinocytes weakly expressed keratin 6. EPPK was coexpressed with keratin 6 in some keratinocytes in explant cultures from wild mice. We propose that EPPK might be linked functionally with keratin 6.