Molecular cloning,expression, and functional characterization of a 2Cys-peroxiredoxin in Chinese cabbage

A cDNA (C2C-Prx) corresponding to a 2Cys-peroxiredoxin (2Cys-Prx) was isolated from a leaf cDNA library of Chinese cabbage. The predicted amino acid sequence of C2C-Prx has 2 conserved cysteines and several peptide domains present in most of the 2Cys-Prx subfamily members. It shows the highest sequence homology to the 2Cys-Prx enzymes of spinach (88%) and Arabidopsis (86%). Southern analysis using the cDNA insert of C2C-Prx revealed that it consists of a small multigene family in Chinese cabbage genome. RNA blot analysis showed that the gene was predominantly expressed in the leaf tissue of Chinese cabbage seedlings, but the mRNA was generally expressed in most tissues of mature plant, except roots. The expression of C2C-Prx was slightly induced by treatment with H₂O₂ (100M) or Fe³⁺/O₂/DTT oxidation system, but not by ABA (50 M) or GA₃ (10 M). The C2C-Prx is encoded as a preprotein of 273 amino acids containing a putative chloroplast-targeting signal of 65 amino acids at its N-terminus. The N-terminally truncated recombinant protein (C2C-Prx) migrates as a dimer in a non-reducing SDS-polyacrylamide gel and as a monomer in a reducing condition. The C2C-Prx shows no immuno cross-reactivity to antiserum of the yeast thiol-specific antioxidant protein, and vice versa. The C2C-Prx prevents the inactivation of glutamine synthetase and the DNA cleavage in the metal-catalyzed oxidation system. In the yeast thioredoxin system containing thioredoxin reductase, thioredoxin, and NADPH, the C2C-Prx exhibits peroxidase activity on H₂O₂.     

NMDA受体与中枢神经系统发育

中枢神经系统兴奋性氨基酸离子型受体－ＮＭＤＡ受体,是由ＮＭＤＡＲ１和ＮＭＤＡＲ２两个亚单位共同构成的受体通道复合体。ＮＭＤＡ受本激活后可引起神经元细胞对Ｎａ＾＋,Ｋ＾＋和Ｃａ＾２＋通透性增强,产生兴奋性突触后电位,在中枢神经发育的过程中,ＮＭＤＡ受体通过不同亚型的选择性表达,改变自身的结构和功能,进而影响ＮＭＤＡ受体介导的Ｃａ＾２＋内流,调节神经元内Ｃａ＾２＋依赖的第二信使系统,最终实现对中枢神经     

γ—氨基丁酸受体的反应动力学及其功能意义

γ－氨基丁酸（ＧＡＢＡ）受体的反应动力学（激活,失敏和失活）是否在快速抑制性突触传递中起作用及怎样发挥作用是一个重要问题。随着分子生物学的发展,膜片钳技术及快速施药系统的应用,对ＧＡＢＡ受体的反应动力学的结构基础,单通道水平的发生机制及其人功能意义等开始形成较全面的认识,揭示了其反应动力学在调节抑制性突触后电流（ＩＰＳＣ）中的关键作用及在快速抑制性突触传递中的重要意义。     

Protein kinase C-mediated phosphorylation of the leukemia-associated HOXA9 protein impairs its DNA binding ability and induces myeloid differentiation

HOXA9 expression is a common feature of acute myeloid leukemia, and high-level expression is correlated with poor prognosis. Moreover, HOXA9 overexpression immortalizes murine marrow progenitors that are arrested at a promyelocytic stage of differentiation when cultured and causes leukemia in recipient mice following transplantation of HOXA9 expressing bone marrow. The molecular mechanisms underlying the physiologic functions and transforming properties of HOXA9 are poorly understood. This study demonstrates that HOXA9 is phosphorylated by protein kinase C (PKC) and casein kinase II and that PKC mediates phosphorylation of purified HOXA9 on S204 as well as on T205, within a highly conserved consensus sequence, in the N-terminal region of the homeodomain. S204 in the endogenous HOXA9 protein was phosphorylated in PLB985 myeloid cells, as well as in HOXA9-immortalized murine marrow cells. This phosphorylation was enhanced by phorbol ester, a known inducer of PKC, and was inhibited by a specific PKC inhibitor. PKC-mediated phosphorylation of S204 decreased HOXA9 DNA binding affinity in vitro and the ability of the endogenous HOXA9 to form cooperative DNA binding complexes with PBX. PKC inhibition significantly reduced the phorbol-ester induced differentiation of the PLB985 hematopoietic cell line as well as HOXA9-immortalized murine bone marrow cells. These data suggest that phorbol ester-induced myeloid differentiation is in part due to PKC-mediated phosphorylation of HOXA9, which decreases the DNA binding of the homeoprotein.     

Brk activates rac1 and promotes cell migration and invasion by phosphorylating paxillin

Brk (for breast tumor kinase) is a nonreceptor tyrosine kinase containing SH3, SH2, and tyrosine kinase catalytic domains. Brk was originally identified from a human metastatic breast tumor, and its overexpression is frequently observed in breast cancer and several other cancer types. However, the molecular mechanism by which this kinase participates in tumorigenesis remains poorly characterized. In the present study, we not only identified paxillin as the binding partner and substrate of Brk but also discovered a novel signaling pathway by which Brk mediates epidermal growth factor (EGF)-induced paxillin phosphorylation. We show that EGF stimulation activates the catalytic activity of Brk, which in turn phosphorylates paxillin at Y31 and Y118. These phosphorylation events promote the activation of small GTPase Rac1 via the function of CrkII. Through this pathway, Brk is capable of promoting cell motility and invasion and functions as a mediator of EGF-induced migration and invasion. In accordance with these functional roles, Brk translocates to membrane ruffles, where it colocalizes with paxillin during cell migration. Together, our findings identify novel signaling and biological roles of Brk and indicate the first potential link between Brk and metastatic malignancy.     

Telomere shortening exposes functions for the mouse Werner and Bloom syndrome genes

The Werner and Bloom syndromes are caused by loss-of-function mutations in WRN and BLM, respectively, which encode the RecQ family DNA helicases WRN and BLM, respectively. Persons with Werner syndrome displays premature aging of the skin, vasculature, reproductive system, and bone, and those with Bloom syndrome display more limited features of aging, including premature menopause; both syndromes involve genome instability and increased cancer. The proteins participate in recombinational repair of stalled replication forks or DNA breaks, but the precise functions of the proteins that prevent rapid aging are unknown. Accumulating evidence points to telomeres as targets of WRN and BLM, but the importance in vivo of the proteins in telomere biology has not been tested. We show that Wrn and Blm mutations each accentuate pathology in later-generation mice lacking the telomerase RNA template Terc, including acceleration of phenotypes characteristic of latest-generation Terc mutants. Furthermore, pathology not observed in Terc mutants but similar to that observed in Werner syndrome and Bloom syndrome, such as bone loss, was observed. The pathology was accompanied by enhanced telomere dysfunction, including end-to-end chromosome fusions and greater loss of telomere repeat DNA compared with Terc mutants. These findings indicate that telomere dysfunction may contribute to the pathogenesis of Werner syndrome and Bloom syndrome.     

Spectroscopic properties, catalytic activities and mechanism studies of [(TpPh)Co(X)(CH3OH)m] . nCH3OH: bicarbonate dehydration in the presence of inhibitors

Two inhibitor-containing 'half-sandwich' cobalt(II) complexes [(TpPh)Co(X)(CH3OH)m] x nCH3OH ((TpPh) = hydrotris (3-phenylpyrazolyl)borate; 1: X- = N3-, m = 1, n = 2; 2: X- = NCS-, m = 0, n = 0) have been synthesized and used as the catalysts in the bicarbonate dehydration reaction. The structures of 1 and 2 were determined by X-ray diffraction analysis, which shows that N3- and NCS- coordinate to the Co(II) ions of 1 and 2, respectively, with the Co-N bond lengths of 1.992(6) A and 1.901(3) A. The coordination geometries of the Co(II) complexes in solution are five-coordinated trigonal bipyramid as revealed by the spectroscopic measurements. The dehydration kinetic measurements of HCO3- are performed by the stopped-flow techniques at pH < 7.9. The apparent dehydration rate constant k(obs) varies linearly with Co(II) complex and H+ concentrations, respectively, and the catalytic activity of 2 is lower than that of 1. The aqua Co(II) complex must be the reactive catalytic species in the catalyzed dehydration reaction and the rate-determining step is the substitution of the labile water molecule by HCO3-. The k(obs) values increase with increasing reaction temperature, and the large negative entropy of activation also indicates the associative activation mode. The inhibition ability of NCS- is stronger than that of N3-, which can be rationalized by the decreases in the Co-N(N3-/NCS-) bond lengths and effective atomic charges of the Co(II) ions based on the X-ray crystallographic data and theoretical calculations in this work.     

X-ray photoelectron spectroscopy and infrared spectroscopy study of maleimide-activated supports for immobilization of oligodeoxyribonucleotides

Surface-tethered nucleic acids are widely applied in solid-phase assays in which complementary strands must be detected against a complex mixture of other sequences. In response to such needs, numerous methods have been developed for immobilizing nucleic acids on solid supports. Often, detailed analysis of associated chemical transformations and of potential side reactions is difficult to obtain. Combined use of planar and high surface area powder supports allows characterization using surface as well as bulk diagnostic techniques. This approach is followed in the present study in which X-ray photoelectron spectroscopy (XPS), transmission infrared spectroscopy (FTIR) and reactivity titrations are used to investigate siliceous supports modified with an aminosilane precursor followed by a maleimide-bearing crosslinker for attachment of nucleic acids. The supports retain maleimide activity for approximately a day when stored under buffer, but deactivation is accelerated under basic conditions or by incomplete conversion of the precursor aminosilane monolayer. Reactions involving the olefinic bond of the imide as well as its carbonyl groups are observed and analyzed. Attachment of sulfhydryl-terminated oligodeoxyribonucleotides is highly site specific, and immobilized strands exhibit excellent hybridization activity. Quantitative use of XPS for label-free determination of DNA coverage based on calibration against reference materials is also described.