黄淮麦区地方小麦品种籽粒颜色相关基因Tamyb10-1等位变异检测?

Tamyb10-1基因属于MYB家族的一种转录因子,决定着小麦种皮的颜色,同时对穗发芽抗性也具有一定影响。本研究以来自我国黄淮麦区的地方小麦品种为材料,利用功能标记对参试小麦品种Tamyb10-1基因位点在3A、3B和3D染色体上的等位变异类型进行了检测。结果表明,参试材料中上述每一位点均有2种等位变异类型,由此形成了7种基因型组合,分别为Tamyb10-A1a/Tamyb10-B1a/Tamyb10-D1a、Tamyb10-A1a/Tamyb10-B1a/Tamyb10-D1b、Tamyb10-A1a/Tamyb10-B1b/Tamyb10-D1a、Tamyb10-A1b/Tamyb10-B1a/Tamyb10-D1a、Tamyb10-A1b/Tamyb10-B1b/Tamyb10-D1a、Tamyb10-A1b/Tamyb10-B1a/Tamyb10-D1b和Tamyb10-A1b/Tamyb10-B1b/Tamyb10-D1b,其分布频率分别为38.0%、15.0%、1.0%、8.0%、1.0%、33.0%和4.0%。进一步研究结果表明,种皮颜色为白色时,Tamyb10-1基因在3个位点均为野生型,而当任何一个位点发生突变时均表现为红色。由于该基因也影响穗发芽的抗性,且子粒颜色与其抗氧化能力密切相关,因此本研究对以子粒颜色性状为育种目标的优异种质资源筛选具有一定参考价值。     

Design and analysis of an engineered human interleukin-10 monomer

A monomeric form of human interleukin 10 (IL-10M1) has been engineered for detailed structure-function studies on IL-10 and its receptor complexes. Wild type IL-10 (wtIL-10) is a domain swapped dimer whose structural integrity depends on the intertwining of two peptide chains. wtIL-10 was converted to a monomeric isomer by inserting 6 amino acids into the loop connecting the swapped secondary structural elements. Characterization of IL-10M1 by mass spectroscopy, size exclusion chromatography, cross-linking, and circular dichroism shows that IL-10M1 is a stable alpha-helical monomer at physiological pH whose three-dimensional structure closely resembles one domain of wtIL-10. As previously reported, incubation of wtIL-10 with a soluble form of the IL-10Ralpha (sIL-10Ralpha) generates a complex that consists of 2 wtIL-10 molecules and 4 sIL-10Ralphas. In contrast, IL-10M1 forms a 1:1 complex with the sIL-10Ralpha. Characterization of the interaction using isothermal titration calorimetry confirmed the 1:1 stoichiometry and yielded a dissociation constant of 30 nm with an apparent binding enthalpy of -12.2 kcal/mol. Despite forming a 1:1 complex, IL-10M1 is biologically active in cellular proliferation assays. These results indicate that the 1:1 interaction between IL-10M1 and IL-10Ralpha is sufficient for recruiting the signal transducing receptor chain (IL-10Rbeta) into the signaling complex and eliciting IL-10 cellular responses.     

Purification of receptor complexes of interleukin-10 stoichiometry and the importance of deglycosylation in their crystallization.

Interleukin-10 (IL-10) is a pleiotropic immunosuppressive cytokine that has a wide range of effects in controlling inflammatory responses. Viral IL-10 (vIL-10) is a homologue of human IL-10 (hIL-10) produced by Epstein-Barr virus (EBV). Both hIL-10 and vIL-10 bind to the soluble extracellular fragment of the cytokine receptor IL-10R1 (shIL-10R1). The stoichiometry of the vIL-10 : shIL-10R1 complex has been found to be the same as hIL-10 : shIL-10R1, with two vIL-10 dimers binding to four shIL-10R1 monomers. Complexes of both hIL-10 and vIL-10 with glycosylated shIL-10R1 could not be crystallized. Controlled deglycosylation using peptide : N-glycosidase F and endo-beta-N-acetylglucosaminidase F3 resulted in the formation of crystals of both hIL-10 : shIL-10R1 and vIL-10 : shIL-10R1 complexes, indicating that the difficulty in the crystal formation was largely due to the presence of complex carbohydrate side chains. The availability of the structure of the ligand-receptor complexes should facilitate our understanding of the basis of the interaction between IL-10 and the IL-10 receptor.     

Epstein-Barr Virus IL-10 Engages IL-10R1 by a Two-step Mechanism Leading to Altered Signaling Properties

Human interleukin-10 (hIL-10) is a pleiotropic cytokine that is able to suppress or activate cellular immune responses to protect the host from invading pathogens. Epstein-Barr virus (EBV) encodes a viral IL-10 (ebvIL-10) in its genome that has retained the immunosuppressive activities of hIL-10 but lost the ability to induce immunostimulatory activities on some cells. These functional differences are at least partially due to the ～1000-fold difference in hIL-10 and ebvIL-10 binding affinity for the IL-10R1·IL-10R2 cell surface receptors. Despite weaker binding to IL-10R1, ebvIL-10 is more active than hIL-10 in inducing B-cell proliferation. To explore this counterintuitive observation further, a series of monomeric and dimeric ebvIL-10·hIL-10 chimeric proteins were produced and characterized for receptor binding and cellular proliferation on TF-1/hIL-10R1 cells that express high levels of the IL-10R1 chain. On this cell line, monomeric chimeras elicited cell proliferation in accordance with how tightly they bound to the IL-10R1 chain. In contrast, dimeric chimeras exhibiting the highest affinity for IL-10R1 exhibited reduced proliferative activity. These distinct activity profiles are correlated with kinetic analyses that reveal that the ebvIL-10 dimer is impaired in its ability to form a 1:2 ebvIL-10·IL-10R1 complex. As a result, the ebvIL-10 dimer functions like a monomer at low IL-10R1 levels, which prevents efficient signaling. At high IL-10R1 levels, the ebvIL-10 dimer is able to induce signaling responses greater than hIL-10. Thus, the ebvIL-10 dimer scaffold is essential to prevent activation of cells with low IL-10R1 levels but to maintain or enhance activity on cells with high IL-10R1 levels.     

Synthesis of (Z)-(2,3-bis-hydroxymethyl)methylenecyclopropane analogues of purine nucleosides

Synthesis of (Z)-(2,3-bis-hydroxymethyl)methylenecyclopropane analogues of nucleosides adenosine 10a, 10b, 10c and 17 is described. Epimerization of Feist's acid (11) using acetic anhydride gave cyclic anhydride 12 which was reduced in situ to give diol 13. Acetylation (compound 14) followed by addition of bromine led to dibromo derivative 15. Alkylation-elimination of adenine with 15 afforded, after deacetylation, analogue 10a. Similar treatment of 2-amino-6-chloropurine and 2,6-diaminopurine led to diacetates 16 and 18. Deprotection then gave compounds 17 and 10c. Hydrolysis of 17 furnished guanine analogue 10b. Compounds 10a, 10b or 10c were inactive against HCMV, HSV-1, HSV-2, EBV VZV and HBV. Analogues 10a and 10b were also assayed for anti-HIV activity. Compound 10a was effective in HIV-1/MT-2 culture with EC50/CC50 33/> 100 microM but 10b was inactive. Analogue 10a was not a substrate for adenosine deaminase.     

A model of the ternary complex of interleukin-10 with its soluble receptors

Background  

Interleukin-10 (IL-10) is a cytokine whose main biological function is to suppress the immune response by induction of a signal(s) leading to inhibition of synthesis of a number of cytokines and their cellular receptors. Signal transduction is initiated upon formation of a ternary complex of IL-10 with two of its receptor chains, IL-10R1 and IL-10R2, expressed on the cell membrane. The affinity of IL-10R1 toward IL-10 is very high, which allowed determination of the crystal structure of IL-10 complexed with the extracellular/soluble domain of IL-10R1, while the affinity of IL-10R2 toward either IL-10 or IL-10/sIL-10R1 complex is quite low. This so far has prevented any attempts to obtain structural information about the ternary complex of IL-10 with its receptor chains.     

The prgQ gene of the Enterococcus faecalis tetracycline resistance plasmid pCF10 encodes a peptide inhibitor, iCF10.

Conjugative transfer of the Enterococcus faecalis tetracycline resistance plasmid pCF10 is stimulated by a peptide pheromone, cCF10. Once a recipient strain acquires pCF10 and thus becomes a pheromone-responsive donor, cCF10 activity is no longer detected in culture filtrates. Here we show that pCF10 encodes a peptide inhibitor, iCF10, secreted by donor cells; this inhibitor antagonizes the cCF10 activity in culture filtrates. In order to detect and quantitate iCF10, we developed a reverse-phase high-performance liquid chromatography assay in which the inhibitor peptide elutes separately from the pheromone; this type of assay enabled us to determine that lack of pheromone activity in donor culture filtrates was due to secretion of a mixture of iCF10 and cCF10, rather than abolition of cCF10 secretion. The gene encoding iCF10, prgQ, is located on the EcoRI-C fragment of pCF10. The open reading frame comprising the prgQ gene encodes a 23-amino-acid precursor that resembles a signal peptide. This precursor is cleaved to the mature heptapeptide iCF10 during the secretion process.     

IFN-gamma-inducible protein-10 (CXCL10) is hepatoprotective during acute liver injury through the induction of CXCR2 on hepatocytes.

IFN-gamma-inducible protein-10 (IP-10/CXCL10) is a non-ELR-CXC chemokine that is present during various forms of acute and chronic liver injury. The purpose of this study was to explore the role of IP-10 during acute liver injury induced by acetaminophen (APAP). After a 400 mg/kg APAP challenge in fasted CD-1 mice, immunoreactive levels of IP-10 were dramatically elevated in the serum within 8 h. CXCR3, the receptor for IP-10, was up-regulated in the liver. Mice that received an i.v. injection of rIP-10 10 h after APAP challenge exhibited a dramatic reduction in alanine aminotransferase 8 h later. Histologic analysis confirmed that the delayed IP-10 therapy dramatically improved the appearance of the liver when examined 48 h after APAP. The therapeutic effect of IP-10 was associated with a marked increase in CXCR2 expression on hepatocytes. Neutralization of CXCR2 during IP-10 therapy resulted in an abrogation of the hepatoprotective effect of IP-10. Furthermore, IP-10 treatment of cultured hepatocytes stimulated a CXCR2-dependent proliferative response. In conclusion, IP-10 has a hepatoregenerative effect in a murine model of acute liver injury that is dependent on its up-regulation of CXCR2 on hepatocytes.     

Biphasic Modulation by ACTH-Like Peptides of Protein Synthesis in a Cell-Free System from Rat Brain

Brain protein synthesis in a cell-free system was stimulated by 10(-8) M-ACTH1-24. This stimulatory effect was completely inhibited by aurintricarboxylic acid (ATA), an inhibitor of reinitiation of new peptide chains. The N-terminal peptide sequence 4-10 exerted a biphasic modulation of cell-free protein synthesis, i.e., a stimulation at low concentrations (10(-8) and 10(-10) M) and an inhibition at a high concentration (10(-4) M). The D-isomer, ACTH4-10-7-D-phe, also showed a biphasic modulation that, however, was in a direction opposite to that shown by ACTH4-10-7-L-phe at 10(-8) M and 10(-4) M.     

Human, viral or mutant human IL-10 expressed after local adenovirus-mediated gene transfer are equally effective in ameliorating disease pathology in a rabbit knee model of antigen-induced arthritis

IL-10 is a Th2 cytokine important for inhibiting cell-mediated immunity while promoting humoral responses. Human IL-10 (hIL-10) has anti-inflammatory, immunosuppressive as well as immunostimulatory characteristics, whereas viral IL-10 (vIL-10), a homologue of hIL-10 encoded by Epstein Barr virus (EBV), lacks several immunostimulatory functions. The immunostimulatory characteristic of hIL-10 has been attributed to a single amino acid, isoleucine at position 87, which in vIL-10 is alanine. A mutant hIL-10 in which isoleucine has been substituted (mut.hIL-10) is biologically active with only immunosuppressive, but not immunostimulatory, functions, making it a potentially superior therapeutic for inflammatory diseases. To compare the efficacy of mut.hIL-10 with hIL-10 and vIL-10 in blocking the progression of rheumatoid arthritis, we used replication defective adenoviral vectors to deliver intra-articularly the gene encoding hIL-10, vIL-10 or mut.hIL-10 to antigen-induced arthritic (AIA) knee joints in rabbits. Intra-articular expression of hIL-10, vIL-10, and mut.hIL-10 resulted in significant improvement of the pathology in the treated joints to similar levels. These observed changes included a significant reduction in intra-articular leukocytosis and the degree of synovitis, as well as normalization of cartilage matrix metabolism. Our results suggest that hIL-10, vIL-10, and mut.hIL-10 are all equally therapeutic in the rabbit AIA model for treating disease pathology.     

H-41, a new minor histocompatibility locus. I. Histogenetic analysis

The B10.STA12 mouse congenic line inherited from the wild mouse parent not only the H-2w13 haplotype but also an allele at a minor H locus, which we designate H-41. This allele (H-41a) differentiates the B10.STA12 line from B10.STA10 and B10.LIB55, which carry identical H-2w13 haplotypes but a different H-41 allele (the H-41b, also present in the background strain C57BL/10Sn). The B10.STA12 and B10.STA10 lines reject each other's skin grafts and generate cytolytic T lymphocytes (CTL) after in vivo immunization and in vitro restimulation with cells of the partner strain. The B10.STA12 anti-B10.STA10 CTL react with B10.STA10, B10.LIB55, and B10.STA39 target cells and with cells of F1 hybrids between the responder strain B10.STA12 and strains C57BL/6, C57BL/10, C57L, BALB/c, A, AKR, WB, DBA/1, and DBA/2 but fail to react with (C3H x B10.STA12) F1 and (CBA x B10.STA12) F1 cells. The B10.STA10 anti-B10.STA12 CTL react with B10.STA12, B10.P, and C3H.NB cells but fail to react to (B6 x B10.STA10) F1 target cells. The CTL reactivity in both combinations is Dp restricted. The B10.STA10 anti-B10.STA12 CTL exhibit, in addition, a cross-reactivity with B10.SAA48 cells that may be directed at one of the alloantigens controlled by the H-2 haplotype of this strain.     

IL-15-induced IL-10 increases the cytolytic activity of human natural killer cells

Interleukin 10 (IL-10) is a multifunctional cytokine that regulates diverse functions of immune cells. Natural killer (NK) cells express the IL-10 and IL-10 receptor, but little is known about the function of IL-10 on NK cell activation. In this study, we show the expression and role of IL-10 in human NK cells. Among the cytokines tested, IL-15 was the most potent inducer of IL-10, with a maximal peak expression at 5 h after treatment. Furthermore, IL-10 receptor was shown to be expressed in NK cells. IL-10 alone had a significant effect on NK cytotoxicity which additively increased NK cell cytotoxicity in the presence of IL-15. Neutralizing IL-10 with anti-IL-10 antibody suppressed the inductive effect of IL-10 on NK cell cytotoxicity; however, IL-10 had no effect on IFN-γ or TNF-α production or NK cell activatory receptor expression. STAT signals are implicated as a key mediator of IL-10/IL-15 cytotoxicity response. Thus, the effect of IL-10 on NK cells is particularly interesting with regard to the STAT3 signal that was enhanced by IL-10 or IL-15.     

Same structure, different function crystal structure of the Epstein-Barr virus IL-10 bound to the soluble IL-10R1 chain

Human IL-10 (hIL-10) is a cytokine that modulates diverse immune responses. The Epstein-Barr virus (EBV) genome contains an IL-10 homolog (vIL-10) that shares high sequence and structural similarity with hIL-10. Although vIL-10 suppresses inflammatory responses like hIL-10, it cannot activate many other immunostimulatory functions performed by the cellular cytokine. These functional differences have been correlated with the approximately 1000-fold lower affinity of vIL-10, compared to hIL-10, for the IL-10R1 receptor chain. To define the structural basis for these observations, crystal structures of vIL-10 and a vIL-10 point mutant were determined bound to the soluble IL-10R1 receptor fragment (sIL-10R1) at 2.8 and 2.7 A resolution, respectively. The structures reveal that subtle changes in the conformation and dynamics of the vIL-10 AB and CD loops and an orientation change of vIL-10 on sIL-10R1 are the main factors responsible for vIL-10's reduced affinity for sIL-10R1 and its distinct biological profile.     

Phosphorylation of grb10 regulates its interaction with 14-3-3

Grb10 is a member of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated signal transduction. Grb10 expression levels can influence Akt activity, and Grb10 may act as an adapter involved in the relocalization of Akt to the cell membrane. Here we identified 14-3-3 as a binding partner of Grb10 by employing a yeast two-hybrid screen. The 14-3-3.Grb10 interaction requires phosphorylation of Grb10, and only the phosphorylated form of Grb10 co-immunoprecipitates with endogenous 14-3-3. We could identify a putative phosphorylation site in Grb10, which is located in a classical 14-3-3 binding motif, RSVSEN. Mutation of this site in Grb10 diminished binding to 14-3-3. Thus, Grb10 exists in two different states of phosphorylation and complexes with 14-3-3 when phosphorylated on serine 428. We provide evidence that Akt directly binds Grb10 and is able to phosphorylate Grb10 in an in vitro kinase assay. Based on these findings, we propose a regulatory circuitry involving a phosphorylation-regulated complex formation of Grb10 with 14-3-3 and Akt.     

Characterization of the sequence specificity determinants required for processing and control of sex pheromone by the intramembrane protease Eep and the plasmid-encoded protein PrgY

Conjugative transfer of the Enterococcus faecalis plasmid pCF10 is induced by the peptide pheromone cCF10 when recipient-produced cCF10 is detected by donors. cCF10 is produced by proteolytic processing of the signal sequence of a chromosomally encoded lipoprotein (CcfA). In donors, endogenously produced cCF10 is carefully controlled to prevent constitutive expression of conjugation functions, an energetically wasteful process, except in vivo, where endogenous cCF10 induces a conjugation-linked virulence factor. Endogenous cCF10 is controlled by two plasmid-encoded products; a membrane protein PrgY reduces pheromone levels in donors, and a secreted inhibitor peptide iCF10 inhibits the residual endogenous pheromone that escapes PrgY control. In this study we genetically determined the amino acid specificity determinants within PrgY, cCF10, and the cCF10 precursor that are necessary for cCF10 processing and for PrgY-mediated control. We showed that amino acid residues 125 to 241 of PrgY are required for specific recognition of cCF10 and that PrgY recognizes determinants within the heptapeptide cCF10 sequence, supporting a direct interaction between PrgY and mature cCF10. In addition, we found that a regulated intramembrane proteolysis (RIP) family pheromone precursor-processing protein Eep recognizes amino acids N-terminal to cCF10 in the signal sequence of CcfA. These results support a model where Eep directly targets pheromone precursors for RIP and PrgY interacts directly with the mature cCF10 peptide during processing. Despite evidence that both PrgY and Eep associate with cCF10 in or near the membrane, results presented here indicate that these two proteins function independently.     

CXCL10/IP-10 in infectious diseases pathogenesis and potential therapeutic implications

C–X–C motif chemokine 10 (CXCL10) also known as interferon γ-induced protein 10 kDa (IP-10) or small-inducible cytokine B10 is a cytokine belonging to the CXC chemokine family. CXCL10 binds CXCR3 receptor to induce chemotaxis, apoptosis, cell growth and angiostasis. Alterations in CXCL10 expression levels have been associated with inflammatory diseases including infectious diseases, immune dysfunction and tumor development. CXCL10 is also recognized as a biomarker that predicts severity of various diseases. A review of the emerging role of CXCL10 in pathogenesis of infectious diseases revealed diverse roles of CXCL10 in disease initiation and progression. The potential utilization of CXCL10 as a therapeutic target for infectious diseases is discussed.     

Association of endosperm reduction with parental imprinting in maize

Among 38 reciprocal translocations between the maize B chromosome and the proximal region of the long arm of chromosome 10 were six interchanges associated with reduced endosperm development. These six have breakpoints that are the most proximal of the set and constitute a graded series with those broken nearer the centromere which have the most abnormal phenotypes. The group of six defines three major regions that produce the endosperm effects. The remaining 32 translocations reduce kernel size very slightly, suggesting the presence of a fourth region distal to all break-points.-The affected class of kernels lacks a paternally derived representative of that segment of 10L translocated to the B centromeric element (B(10) chromosome; 10 10 B(10)). An accompanying class of kernel in which the paternal B(10) chromosome is duplicated in the endosperm (10 10 10(B) B(10) B(10)) is normal. Kernels of the same endosperm constitution synthesized by introducing both 10 and B(10) maternally, however, are defective, resembling 10 10 10(B). Maternal B(10)'s are therefore unable to compensate for the absence of a paternal B(10). Clearly expression of the 10L genes involved supports normal endosperm growth only following pollen transmission.     

Quantitative analysis of the elemental composition and the mass of bacterial polyphosphate bodies using STEM EDX

The quantitative analysis of laboratory grown organisms (Plectonema boryanum and Staphylococcus aureus) revealed that a typical in vivo polyphosphate body (PPB) contains O (4.3 x 10(-8) microg), C (1.2 x 10(-8) microg), P (6.7 x 10(-9) microg), Mg (1.3 x 10(-9) microg), Ca (6.7 x 10(-10) microg), K (6.7 x 10(-10) microg), Fe (6.0 x 10(-10) microg), S (5.4 x 10(-10) microg) and Al (5.9 x 10(-10) microg). Quantitative X-ray analysis of samples from nature showed PPB contain O (1.63 x 10(-8) microg), C (4.75 x 10(-9) microg), P (2.50 x 10(-9) microg), Mg (5.0 x 10(-10) microg), Ca (2.50 x 10(-10) microg), K (2.50 x 10(-10) microg), Fe (2.25 x 10(-10) microg) and S (2.0 x 10(-10) microg). The mass of an average polyphosphate body was 6.7 x 10(-8) microg for P. boryanum, 2.5 x 10(-8) microg for S. aureus and for microbes from the natural environment 6.3 x 10(-8) microg. The results indicate that the PPB may have other unknown functions in addition to essential element storage, acting as a detoxification method by sequestering heavy metals and providing a homeostasis system in the cell.