Uridine adenosine tetraphosphate induces contraction of airway smooth muscle

Contraction of airway smooth muscle (ASM) plays an important role in the regulation of air flow and is potentially involved in the pathophysiology of certain respiratory diseases. Extracellular nucleotides regulate ASM contraction via purinergic receptors, but the signaling mechanisms involved are not fully understood. Uridine adenosine tetraphosphate (Up(4)A) contains both pyrimidine and purine moieties, which are known to potentially activate P2X and P2Y receptors. Both P2X and P2Y receptors have been identified in the lung, including airway epithelial cells and ASM. We report here a study of purinergic signaling in the respiratory system, with a focus on the effect of Up(4)A on ASM contraction. Up(4)A induced contraction of rat isolated trachea and extrapulmonary bronchi as well as human intrapulmonary bronchioles. Up(4)A-induced contraction was blocked by di-inosine pentaphosphate, a P2X antagonist, but not by suramin, a nonselective P2 antagonist. Up(4)A-induced contraction was also attenuated by α,β-methylene-ATP-mediated P2X receptor desensitization. Several P2X receptors were detected at the mRNA level: P2X1, P2X4, P2X6, and P2X7, and to a lesser extent P2X3. Furthermore, the Up(4)A response was inhibited by removal of extracellular Ca(2+) and by the presence of the L-type Ca(2+) channel blocker, nifedipine, or the Rho-associated kinase inhibitor, H1152. We conclude that Up(4)A stimulates ASM contraction, and the underlying signaling mechanism appears to involve P2X (most likely P2X1) receptors, extracellular Ca(2+) entry via L-type Ca(2+) channels, and Ca(2+) sensitization through the RhoA/Rho-associated kinase pathway. This study will add to our understanding of the pathophysiological roles of extracellular nucleotides in the lung.     

番茄钙依赖性蛋白激酶基因LeCPK2在热(光)胁迫中的功能鉴定

钙依赖性蛋白激酶(calcium-dependent protein kinases,CDPKs or CPKs)作为一类钙感知蛋白在植物的生长发育和胁迫应答中起着重要的作用.LeCPK2 (GenBank accession No.:GQ205414)是我们从番茄中分离的第3个CDPK基因,前期研究表明LeCPK2可能在植物热胁迫应答中发挥作用.为了进一步研究其在热胁迫中的功能,我们通过电子克隆的方法分离了LeCPK2的启动子序列,并通过LeCPK2过表达烟草分析其在高温胁迫中的潜在的功能.生物信息学分析显示,LeCPK2启动子中包含5个热响应元件,和前期试验结果一致.野生型植株在受到热胁迫后,对光更为敏感,强光照下植株叶片发生萎蔫,而强光本身不会对未受热胁迫的健康植株造成伤害.LeCPK2转基因植株热、光胁迫后不会出现受害表型.以上研究表明,LeCPK2在植物的热胁迫应答中发挥重要作用,能够有效保护植株免受高温胁迫的损害,是一个优秀的耐热(光)基因.本研究将为揭示番茄LeCPK2遗传功能及对其开发利用奠定基础.     

An Apo-14 promoter-driven transgenic zebrafish that marks liver organogenesis

Several transgenic zebrafish lines for liver development studies had been obtained in the first decade of this century, but not any transgenic GFP zebrafish lines that mark the through liver development and organogenesis were reported. In this study, we analyzed expression pattern of endogenous Apo-14 in zebrafish embryogenesis by whole-mount in situ hybridization, and revealed its expression in liver primordium and in the following liver development. Subsequently, we isolated zebrafish Apo-14 promoter of 1763 bp 5'-flanking sequence, and developed an Apo-14 promoter-driven transgenic zebrafish Tg(Apo14: GFP). And, maternal expression and post-fertilization translocation of Apo-14 promoter-driven GFP were observed in the transgenic zebrafish line. Moreover, we traced onset expression of Apo-14 promoter-driven GFP and developmental behavior of the expressed cells in early heterozygous embryos by out-crossing the Tg(Apo14: GFP) male to the wild type female. Significantly, the Apo-14 promoter-driven GFP is initially expressed around YSL beneath the embryo body at 10 hpf when the embryos develop to tail bud prominence. In about 14-somite embryos at 16-17 hpf, a typical "salt-and-pepper" expression pattern is clearly observed in YSL around the yolk sac. Then, a green fluorescence dot begins to appear between the notochord and the yolk sac adjacent to otic vesicle at about 20 hpf, which is later demonstrated to be liver primordium that gives rise to liver. Furthermore, we investigated dynamic progression of liver organogenesis in the Tg(Apo14: GFP) zebrafish, because the Apo-14 promoter-driven GFP is sustainably expressed from hepatoblasts and liver progenitor cells in liver primordium to hepatocytes in the larval and adult liver. Additionally, we observed similar morphology between the liver progenitor cells and the GFP-positive nuclei on the YSL, suggesting that they might originate from the same progenitor cells in early embryos. Overall, the current study provides a transgenic zebrafish line that marks the through liver organogenesis.     

Identification of three duplicated Spin genes in medaka (Oryzias latipes)

Gene and genomic duplications are very important and frequent events in fish evolution, and the divergence of duplicated genes in sequences and functions is a focus of research on gene evolution. Here, we report the identification and characterization of three duplicated Spindlin (Spin) genes from medaka (Oryzias latipes): OlSpinA, OlSpinB, and OlSpinC. Molecular cloning, genomic DNA Blast analysis and phylogenetic relationship analysis demonstrated that the three duplicated OlSpin genes should belong to gene duplication. Furthermore, Western blot analysis revealed significant expression differences of the three OlSpins among different tissues and during embryogenesis in medaka, and suggested that sequence and functional divergence might have occurred in evolution among them.     

Crystal structure of C-terminal desundecapeptide nitrite reductase from Achromobacter cycloclastes

Monoclinic crystal structure of C-terminal desundecapeptide nitrite reductase (NiRc-11) from Achromobacter cycloclastes was determined at 2.6A. NiRc-11 exists as a loose trimer in the crystal. Deletion of 11 residues eliminates all intersubunit hydrogen bonds mediated by the C-terminal tail. The rigid irregular coil 105-112, which constitutes part of the sidewall of the active site pocket, undergoes conformational changes and becomes highly flexible in NiRc-11. Correspondingly, the linker segments between the two copper sites 95-100 and 135-136 are partly relaxed in conformation, which leads to disrupted active site microenvironments responsible for the activity loss and spectral change of NiRc-11. Comparison with the native structure revealed a bulky residue Met331 fastened by hydrogen bonding, which may play a direct role in keeping the right copper site geometry by protruding its side chain against the irregular coil 105-112. Sequence alignment showed that the bulky residue is conserved at position 331, indicating an equal importance of C-terminal segment in other copper-containing nitrite reductases.     

Synthesis and crystal structures of silver(I) bridged tetra-phenyl-pyrylium complexes from tetraphenyl-cyclopentadiene

Reactions of silver(I) perchlorate with tetraphenyl-cyclopentadiene (Ph₄H₂C₅) have isolated two novel silver(I) bridged tetraphenyl-pyrylium complexes: [Ag(ClO₄)(Ph₄HC₅O⁺)](ClO₄)⁻ (1) and (2), depending on moisture-content of the reactants. Structure studies using single-crystal X-ray diffraction have showed that complex 1 contains a distorted tetrahedral metal center bridging two neighboring peripheral phenyl rings of one pyrylium cation and two perchlorate anions, whereas 2 involves a three-coordinate metal ion interacting with a pair of phenyl rings and one water molecule, leaving two perchlorate anions free from coordination. For both complexes, the precursor ligand Ph₄H₂C₅ undergoes a ring-enlargement reaction, forming a six-membered pyrylium cation. The fundamentals of the synthesis, structure characterization and some properties are discussed.     

Folding of the SARS coronavirus spike glycoprotein immunological fragment (SARS_S1b): thermodynamic and kinetic investigation correlating with three-dimensional structural modeling

Spike glycoprotein of SARS coronavirus (S protein) plays a pivotal role in SARS coronavirus (SARS_CoV) infection. The immunological fragment of the S protein (Ala251-His641, SARS_S1b) is believed to be essential for SARS_CoV entering the host cell through S protein-ACE-2 interaction. We have quantitatively characterized the thermally induced and GuHCl-induced unfolding features of SARS_S1b using circular dichroism (CD), tryptophan fluorescence, and stopped-flow spectral techniques. For the thermally induced unfolding at pH 7.4, the apparent activation energy (E(app)) and transition midpoint temperature (Tm) were determined to be 16.3 +/- 0.2 kcal/mol and 52.5 +/- 0.4 degrees C, respectively. The CD spectra are not dependent on temperature, suggesting that the secondary structure of SARS_S1b has a relatively high thermal stability. GuHCl strongly affected SARS_S1b structure. Both the CD and fluorescent spectra resulted in consistent values of the transition middle concentration of the denaturant (Cm, ranging from 2.30 to 2.45 M) and the standard free energy change (deltaG(o), ranging from 2.1 to 2.5 kcal/mol) for the SARS_S1b unfolding reaction. Moreover, the kinetic features of the chemical unfolding and refolding of SARS_S1b were also characterized using a stopped-flow CD spectral technique. The obvious unfolding reaction rates and relaxation times were determined at various GuHCl concentrations, and the Cm value was obtained, which is very close to the data that resulted from CD and fluorescent spectral determinations. Secondary and three-dimensional structural predictions by homology modeling indicated that SARS_S1b folded as a globular-like structure by beta-sheets and loops; two of the four tryptophans are located on the protein surface, which is in agreement with the tryptophan fluorescence result. The three-dimensional model was also used to explain the recently published experimental results of S1-ACE-2 binding and immunizations.     

A C-type lectin associated and translocated with cortical granules during oocyte maturation and egg fertilization in fish

Oocyte maturation and egg fertilization in both vertebrates and invertebrates are marked by orchestrated cytoplasmic translocation of secretory vesicles known as cortical granules. It is thought that such redistribution of cellular content is critical for asymmetrical cell division during early development, but the mechanism and regulation of the process is poorly understood. Here we report the identification, purification and cDNA cloning of a C-type lectin from oocytes of a freshwater fish species gibel carp (Carassius auratus gibelio). The purified protein has been demonstrated to have lectin activity and to be a Ca(2+)-dependent C-type lectin by hemagglutination activity assay. Immunocytochemistry revealed that the lectin is associated with cortical granules, gradually translocated to the cell surface during oocyte maturation, and discharged to the egg envelope upon fertilization. Interestingly, the lectin becomes phosphorylated on threonine residues upon induction of exocytosis by fertilization and returns to its original state after morula stage of embryonic development, suggesting that this posttranslational modification may represent a critical molecular switch for early embryonic development.