Reconstitution of CO2 Regulation of SLAC1 Anion Channel and Function of CO2-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor

Dark respiration causes an increase in leaf CO₂ concentration (Ci), and the continuing increases in atmospheric [CO₂] further increases Ci. Elevated leaf CO₂ concentration causes stomatal pores to close. Here, we demonstrate that high intracellular CO₂/HCO₃⁻ enhances currents mediated by the Arabidopsis thaliana guard cell S-type anion channel SLAC1 upon coexpression of any one of the Arabidopsis protein kinases OST1, CPK6, or CPK23 in Xenopus laevis oocytes. Split-ubiquitin screening identified the PIP2;1 aquaporin as an interactor of the βCA4 carbonic anhydrase, which was confirmed in split luciferase, bimolecular fluorescence complementation, and coimmunoprecipitation experiments. PIP2;1 exhibited CO₂ permeability. Mutation of PIP2;1 in planta alone was insufficient to impair CO₂- and abscisic acid-induced stomatal closing, likely due to redundancy. Interestingly, coexpression of βCA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1 in oocytes enabled extracellular CO₂ enhancement of SLAC1 anion channel activity. An inactive PIP2;1 point mutation was identified that abrogated water and CO₂ permeability and extracellular CO₂ regulation of SLAC1 activity. These findings identify the CO₂-permeable PIP2;1 as key interactor of βCA4 and demonstrate functional reconstitution of extracellular CO₂ signaling to ion channel regulation upon coexpression of PIP2;1, βCA4, SLAC1, and protein kinases. These data further implicate SLAC1 as a bicarbonate-responsive protein contributing to CO₂ regulation of S-type anion channels.     

Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway

Physical stimuli play critical roles in the development, regeneration, and pathology of many mesenchymal tissues, most notably bone. While mature bone cells, such as osteoblasts and osteocytes, are clearly involved in these processes, the role of their progenitors in mechanically mediated tissue responses is unknown. In this study, we investigated the effect of cyclic substrate deformation on the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs). Application of equibiaxial cyclic strain (3%, 0.25Hz) to hMSCs cultured in osteogenic media inhibited proliferation and stimulated a 2.3-fold increase in matrix mineralization over unstrained cells. The strain stimulus activated the extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase pathways, but had no effect on c-Jun N-terminal kinase phosphorylation or activity. Strain-induced mineralization was largely mediated by ERK1/2 signaling, as inhibition of ERK1/2 attenuated calcium deposition by 55%. Inhibition of the p38 pathway resulted in a more mature osteogenic phenotype, suggesting an inhibitory role for p38 signaling in the modulation of strain-induced osteogenic differentiation. These results demonstrate that mechanical signals regulate hMSC function, suggesting a critical role for physical stimulation of this specific cell population in mesenchymal tissue formation.     

EST analysis of gene expression in the tentacle of Cyanea capillata

Jellyfish, Cyanea capillata, has an important position in head patterning and ion channel evolution, in addition to containing a rich source of toxins. In the present study, 2153 expressed sequence tags (ESTs) from the tentacle cDNA library of C. capillata were analyzed. The initial ESTs consisted of 198 clusters and 818 singletons, which revealed approximately 1016 unique genes in the data set. Among these sequences, we identified several genes related to head and foot patterning, voltage-dependent anion channel gene and genes related to biological activities of venom. Five kinds of proteinase inhibitor genes were found in jellyfish for the first time, and some of them were highly expressed with unknown functions.     

A fast empirical approach to binding free energy calculations based on protein interface information

Three useful variables from the interfaces of 20 protein-protein complexes were investigated. These variables are the side-chain accessible number (N(b)), the number of hydrophilic pairs (N(pair)) and buried a polar solvent accessible surface areas (DeltaDeltaASA(apol)). An empirical model based on the three variables was developed to describe the free energy of protein associations. As the results show, the side-chain accessible numbers characterize the loss of side-chain conformational entropy of protein interactions and the effective empirical function presented here has great capability for estimating the binding free energy. It was found that the variables of interface information capture most of the significant features of protein-protein association. Also, we applied the model based on the variables as a rescoring function to docking simulations and found that it has the potential to distinguish the 'true' binding mode. It is clear that the simple and empirical scale developed here is an attractive target function for calculating binding free energy for various biological processes to rational protein design.     

Protein molecular dynamics with electrostatic force entirely determined by a single Poisson-Boltzmann calculation

The electrostatic force including the intramolecular Coulombic interactions and the electrostatic contribution of solvation effect were entirely calculated by using the finite difference Poisson-Boltzmann method (FDPB), which was incorporated into the GROMOS96 force field to complete a new finite difference stochastic dynamics procedure (FDSD). Simulations were performed on an insulin dimer. Different relative dielectric constants were successively assigned to the protein interior; a value of 17 was selected as optimal for our system. The simulation data were analyzed and compared with those obtained from 500-ps molecular dynamics (MD) simulation with explicit water and a 500-ps conventional stochastic dynamics (SD) simulation without the mean solvent force. The results indicate that the FDSD method with GROMOS96 force field is suitable to study the dynamics and structure of proteins in solution if used with the optimal protein dielectric constant.     

A new computational approach for real protein folding prediction

An effective and fast minimization approach is proposed for the prediction of protein folding, in which the 'relative entropy' is used as a minimization function and the off-lattice model is used. In this approach, we only use the information of distances between the consecutive Calpha atoms along the peptide chain and a generalized form of the contact potential for 20 types of amino acids. Tests of the algorithm are performed on the real proteins. The root mean square deviations of the structures of eight folded target proteins versus the native structures are in a reasonable range. In principle, this method is an improvement on the energy minimization approach.     

Polymorphisms in PPARD, PPARG and APM1 associated with four types of Traditional Chinese Medicine constitutions

Based on the theory of constitution of Traditional Chinese Medicine (TCM), the human population is divided into nine constitutions including one balanced constitution (Normality) and eight unbalanced constitutions (Yang-deficiency, Yin-deficiency, Phlegm-wetness, Qi-deficiency, Wetness-heat, Blood stasis, Depressed constitution, and Inherited special constitution). Different constitutions have specific metabolic features and different susceptibility to certain diseases. However, whether a genetic basis accounts for such constitution classification is yet to be determined. Here we performed a genetic study to assess the association between genetic variations of metabolic genes including PPARD, PPARG and APM1 and the constitutions. A total of 233 individuals of the Han population in China were classified into four groups, Normality, Yang-deficiency, Yin-deficiency and Phlegm-wetness with whom 23 single nucleotide polymorphisms (SNPs) in the three genes were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Biased distribution of PPARD rs2267669 and rs2076167, APM1 rs7627128 and rs1063539 in Yang-deficiency, PPARG Pro12Ala in Yin-deficiency and PPARD rs2076167, APM1 rs266729 and rs7627128 in Phlegm-wetness were observed. The frequencies of Haplotype13 (Hap13) of PPARG in Yin-deficiency, Hap25 of APM1 in Yang-deficiency and Hap2 of PPARD and Hap14 of PPARG in Phlegm-wetness, were significantly different from those in Normality, suggesting those might be group-associated haplotypes. These results suggested that single SNP and haplotypes of PPARD, PPARG and APM1 may underlie the genetic basis of the constitutions classified in TCM.     

Phylogeography of Pinus armandii and Its Relatives: Heterogeneous Contributions of Geography and Climate Changes to the Genetic Differentiation and Diversification of Chinese White Pines

Geographic barriers and Quaternary climate changes are two major forces driving the evolution, speciation, and genetic structuring of extant organisms. In this study, we used Pinus armandii and eleven other Asian white pines (subsection Strobus, subgenus Pinus) to explore the influences of geographic factors and Pleistocene climatic oscillations on species in South China, a region known to be centers of plant endemism and biodiversity hotspots. Range-wide patterns of genetic variation were investigated using chloroplast and mitochondrial DNA markers, with extensive sampling throughout the entire range of P. armandii. Both cpDNA and mtDNA revealed that P. armandii exhibits high levels of genetic diversity and significant population differentiation. Three geographically distinct subdivisions corresponding to the Qinling-Daba Mountains (QDM), Himalaya-Hengduan Mountains (HHM) and Yungui Plateau (YGP) were revealed in mainland China by cpDNA. Their break zone was located in the southeastern margin of the Qinghai-Tibetan Plateau (QTP). A series of massive mountains, induced by the QTP uplift, imposed significant geographic barriers to genetic exchange. The disjunct distribution patterns of ancestral haplotypes suggest that a large continuous population of the white pines may have existed from southwest to subtropical China. Repeated range shifts in response to the Pleistocene glaciations led to the isolation and diversification of the subtropical species. The two Taiwanese white pines share a common ancestor with the species in mainland China and obtain their chloroplasts via long-distance pollen dispersal from North Asian pines. Distinct genetic patterns were detected in populations from the Qinling-Daba Mountains, Yungui Plateau, Himalaya-Hengduan Mountains, and subtropical China, indicating significant contributions of geographic factors to the genetic differentiation in white pines. Our study depicts a clear picture of the evolutionary history of Chinese white pines and highlights the heterogeneous contributions of geography and Pleistocene climatic fluctuations to the extremely high plant species diversity and endemism in South China.     

蛋白酪氨酸激酶在类风湿性关节炎滑膜细胞中的表达和功能

克隆类风湿性关节炎（RA）滑膜细胞（FLS）中的蛋白酪氨酸激酶（PTKs）,并研究它们在RA滑膜细胞异常增殖和侵软骨中的作用。根据巳知的PTKs氨基酸序列保守区设计简并引物,采用3'快速末端扩增法（3'RACE)扩增滑膜细胞中PTKs cDNAs的3'末端,将所得序列与Genebank中序列进行比较,以鉴定其是否为巳知的PTKs或与PTKs同源的新序列,然后通过RNA点杂交方法分别观察这些PTKs在RA和骨性关节炎（OA）病人滑膜细胞中的表达水平。结果表明,从RA FLS中克隆到6种巳知PTKs的cDNAs片段,分别为血小板衍生生长因子受体A（PDGFRA）、胰岛素生长因子-1受体（IGF1R）、含discoidin结构域的受体型酪氨酸激酶（DDR2）、Lyn、Janus激酶1（JAK1）和TYK2。RNA点杂交结果显示,在4个RA病人和2个OA病人的滑膜细胞中,PDGFRA、IGF-1R和DDR2在RA滑膜细胞中的表达水平高于OA滑膜细胞,其它几处激酶在两种细胞中的表达水平相同。说明RI滑膜细胞中至少表达PDGFR、IGF1R、Lyn、DDR2、JAK1和TYK2等6种PTKs,其中PDGFRA、IGF1R和DDR2可能与RA滑膜细胞的过度增殖和对软骨的侵蚀性相关。