Involvement of the residues of GSKIP, AxinGID, and FRATtide in their binding with GSK3β to unravel a novel C-terminal scaffold-binding region

The specificity and regulation of GSK3β are thought to involve in the docking interactions at core kinase domain because of the particular amino acid residues. Recent X-ray diffraction studies illuminated the relative binding residues on AxinGID and FRATtide for GSK3β docking and appeared that GSK3β Val267Gly (V267G) and Tyr288Phe (Y288F) could distinguish the direct interaction between AxinGID and FRATtide. In order to explore the mode that involved the binding of GSKIP to GSK3β and compare it with that of AxinGID and FRATtide, we pinpointed the binding sites of GSKIP to GSK3β through the single-point mutation of four corresponding sites within GSK3β (residues 260–300) as scaffold-binding region I (designated SBR-I_260–300). Our data showed that these three binding proteins shared similar binding sites on GSK3β. We also found that the binding of GSK3β V267G mutant to GSKIP and AxinGID, but not that of Y288F mutant (effect on FRATtide), was affected. Further, based on the simulation data, the electron-density map of GSKIPtide bore closer similarity to the map AxinGID than to that of FRATtide. Interestingly, many C-terminal helix region point-mutants of GSK3β L359P, F362A, E366K, and L367P were able to eliminate the binding with FRATtide, but not AxinGID or GSKIP. In addition, CABYR exhibited a unique mode in binding to C-terminal helix region of GSK3β. Taken together, our data revealed that in addition to the core kinase domain, SBR-I_260–300, another novel C-terminus helix region, designated SBR-II_339–383, also appeared to participate in the recognition and specificity of GSK3β in binding to other specific proteins.     

Connective Tissue Growth Factor in Regulation of RhoA Mediated Cytoskeletal Tension Associated Osteogenesis of Mouse Adipose-Derived Stromal Cells

Background

Cytoskeletal tension is an intracellular mechanism through which cells convert a mechanical signal into a biochemical response, including production of cytokines and activation of various signaling pathways.

Methods/Principal Findings

Adipose-derived stromal cells (ASCs) were allowed to spread into large cells by seeding them at a low-density (1,250 cells/cm²), which was observed to induce osteogenesis. Conversely, ASCs seeded at a high-density (25,000 cells/cm²) featured small cells that promoted adipogenesis. RhoA and actin filaments were altered by changes in cell size. Blocking actin polymerization by Cytochalasin D influenced cytoskeletal tension and differentiation of ASCs. To understand the potential regulatory mechanisms leading to actin cytoskeletal tension, cDNA microarray was performed on large and small ASCs. Connective tissue growth factor (CTGF) was identified as a major regulator of osteogenesis associated with RhoA mediated cytoskeletal tension. Subsequently, knock-down of CTGF by siRNA in ASCs inhibited this osteogenesis.

Conclusions/Significance

We conclude that CTGF is important in the regulation of cytoskeletal tension mediated ASC osteogenic differentiation.     

Comparison of pulsed‐field gel electrophoresis and three rep‐PCR methods for evaluating the genetic relatedness of Stenotrophomonas maltophilia isolates

Aims: In this study, three facile repetitive‐sequence PCR (rep‐PCR) techniques have been compared with the pulsed‐field gel electrophoresis (PFGE) method for differentiating the genetic relatedness of clinical Stenotrophomonas maltophilia isolates. Methods and Results: The dendrograms of 20 S. maltophilia isolates were constructed based on the data obtained from PFGE and three PCR‐based methods, i.e. enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR), BOX‐PCR and repetitive extragenic palindromic‐PCR (REP‐PCR). When compared with PFGE, ERIC‐PCR displayed a much lower discriminatory power, whereas BOX‐PCR and REP‐PCR had a comparable discriminatory power for close genetic‐related isolates. Conclusion: BOX‐PCR and REP‐PCR can be convenient and effective methods for evaluating the close genetic relatedness of clinical S. maltophilia isolates. Significance and Impact of the Study: A rapid method for determining S. maltophilia’s close genetic relatedness provides a convenient tool for understanding the epidemiology of S. maltophilia.     

The PNPase,exosome and RNA helicases as the building components of evolutionarily-conserved RNA degradation machines

The structure and function of polynucleotide phosphorylase (PNPase) and the exosome, as well as their associated RNA-helicases proteins, are described in the light of recent studies. The picture raised is of an evolutionarily conserved RNA-degradation machine which exonucleolytically degrades RNA from 3′ to 5′. In prokaryotes and in eukaryotic organelles, a trimeric complex of PNPase forms a circular doughnut-shaped structure, in which the phosphorolysis catalytic sites are buried inside the barrel-shaped complex, while the RNA binding domains create a pore where RNA enters, reminiscent of the protein degrading complex, the proteasome. In some archaea and in the eukaryotes, several different proteins form a similar circle-shaped complex, the exosome, that is responsible for 3′ to 5′ exonucleolytic degradation of RNA as part of the processing, quality control, and general RNA degradation process. Both PNPase in prokaryotes and the exosome in eukaryotes are found in association with protein complexes that notably include RNA helicase.     

Diverse selective modes among orthologs/paralogs of the chalcone synthase (Chs) gene family of Arabidopsis thaliana and its relative A. halleri ssp. gemmifera

As a model system, Arabidopsis thaliana and its wild relatives have played an important role in the study of genomics and evolution in plants. In this study, we examined the genetic diversity of the chalcone synthase (Chs) gene, which encodes a key enzyme of the flavonoid pathway and is located on chromosome five, as well as two Chs-like genes on the first and fourth chromosomes of Arabidopsis. The objectives of the study are to determine if natural selection operates differentially on the paralogs of the Chs gene family in A. thaliana and Arabidopsis halleri ssp. gemmifera. The mode of selection was inferred from Tajima's D values from noncoding and coding regions, as well as from the ratio of nonsynonymous to synonymous substitutions. Both McDonald-Kreitman and HKA tests revealed the effects of selection on the allelic distribution, except for the chromosome 1 paralog in ssp. gemmifera. The Chs gene on chromosome 5 was under purifying selection in both species. Significant, negative Tajima's D values at synonymous sites and positive Fay and Wu's H values within coding region, plus reduced genetic variability in introns, indicated effects of background selection in shaping the evolution of this gene region in A. thaliana. The Chs paralog on chromosome 1 was under positive selection in A. thaliana, while interspecific introgression and balancing selection determined the fates of the paralog and resulted in high heterogeneity in ssp. gemmifera. Local adaptation differentiated populations of Japan and China at the locus. In contrast, the other Chs-paralog of chromosome 4 was shaped by purifying selection in A. thaliana, while under positive selection in ssp. gemmifera, as indicated by dn/ds>1. Moreover, these contrasting patterns of selection have likely resulted in functional divergence in Arabidopsis, as indicated by radical amino acid substitutions at the chalcone synthase/stilbene synthase motif of the Chs genes. Unlike previous studies of the evolutionary history of A. thaliana, the high levels of genetic diversity in most gene regions of Chs paralogs and nonsignificant Tajima's D in the intron sequences of the Chs gene family in A. thaliana did not reflect the effects of a recent demographic expansion.     

The role of the miR399-<Emphasis Type="Italic">PHO2</Emphasis> module in the regulation of flowering time in response to different ambient temperatures in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A moderate change in ambient temperature significantly affects plant physiology including flowering time. MiR399 and its target gene PHOSPHATE 2 (PHO2) are known to play a role in the maintenance of phosphate homeostasis. However, the regulation of flowering time by the miR399-PHO2 module has not been investigated. As we have previously identified miR399 as an ambient temperature-responsive miRNA, we further investigated whether a change in expression of the miR399-PHO2 module affects flowering time in response to ambient temperature changes. Here, we showed that miR399b-overexpressing plants and a loss-of-function allele of PHO2 (pho2) exhibited an early flowering phenotype only at normal temperature (23°C). Interestingly, their flowering time at lower temperature (16°C) was similar to that of wild-type plants, suggesting that alteration in flowering time by miR399 and its target PHO2 was seen only at normal temperature (23°C). Flowering time ratio (16°C/23°C) revealed that miR399b-overexpressing plants and pho2 mutants showed increased sensitivity to ambient temperature changes. Expression analysis indicated that expression of TWIN SISTER OF FT (TSF) was increased in miR399b-overexpressing plants and pho2 mutants at 23°C, suggesting that their early flowering phenotype is associated with TSF upregulation. Taken together, our results suggest that miR399, an ambient temperature-responsive miRNA, plays a role in ambient temperature-responsive flowering in Arabidopsis.     

Correlation between tissue oxygenation and erythrocytes elasticity

In this paper, near‐infrared spectroscopy (NIRS) and jumping optical tweezers were used to measure the tissue oxygenation and the elasticity of erythrocytes, respectively. The correlation between tissue oxygenation induced by arterial occlusion test (AOT) and the mechanical properties of individual erythrocytes from a blood sample obtained after AOT was studied. The experimental results show a linear correlation between the oxygenation signal caused by AOT and the elasticity of erythrocytes. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Synergistic effect of prostaglandin F2alpha and cyclic AMP on glucose transport in 3T3-L1 adipocytes

The combined effect of prostaglandin F2alpha (PGF2alpha) and cAMP on glucose transport in 3T3-L1 adipocytes was examined. In cells pretreated with PGF2alpha and 8-bromo cAMP for 8 h, a synergy between these two agents on glucose uptake was found. Insulin-stimulated glucose transport, on the other hand, was only slightly affected. The synergistic effect of these two agents was suppressed in the presence of cycloheximide and actinomycin D. In concord, immunoblot and Northern blot analyses revealed that GLUT1 protein and mRNA levels were both increased in cells pretreated with both PGF2alpha and 8-bromo cAMP, greater than the additive effect of each agent alone. The synergistic action of PGF2alpha with 8-bromo cAMP to enhance glucose transport was inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor. In addition, in cells depleted of diacylglycerol-sensitive PKC by prolonged treatment with 4beta-phorbol 12beta-myristate 13alpha-acetate, a PKC activator, the synergistic effects of PGF2alpha and 8-bromo cAMP on glucose transport and GLUT1 mRNA accumulation were both abolished. Taken together, these results indicate that PGF2alpha may act with cAMP in a synergistic way to increase glucose transport, probably through enhanced GLUT1 expression by a PKC-dependent mechanism.