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    120726154001-pcr-knockin-mouse-targatt-knockin-rat-pcr-premix-knockout-mouse-miceIan Dworkin, a zoologist at Michigan State University, was part of the team that first demonstrated why everywhere - from Moose rhinoceros beetles - and other decorative ma-attracting structures are sensitive to changes in diet. As reported in the current issue of the journal Science, a key component of this growth is insulin, Dworkin said.
    "Sure elk antlers, peacock tail feathers and beetle horns are very different, but it seems to have similar mechanisms such large structures," he said. "A reduction in insulin levels significantly reduces the size of the ornamental structures."
    Sexual selection has roots in Darwin's research. Later research showed that the so-called principle of "handicap", which marked a man loaded with such baggage carrying awkward. Dworkin team believes, however, that part of the image insulin, males are actually striking off. In contrast, insulin dependence of these big horns provides a way for men to show how good they are.
    "It's a sign that these men are thriving, some pretty robust and certainly worthy companion," said Dworkin, who led the research at the lighthouse, MSU's National Science Foundation Center for the Study of Evolution in Action.
    Dworkin and the team discovered that whenever such exaggerated traits evolve, but repeatedly, but independently of each other, seems to use insulin dependence. This suggests that the properties are more likely to have evolved as honest indicators of quality rather than disadvantages.
    "While there is work to be done, our results provide an important way to connect and genetic mechanism with the latest evolution exaggerated trait reason," said Dworkin.


    Cytokinins are plant hormones that play critical roles in growth and development. In Arabidopsis, the transcriptional response to cytokinin is regulated by action of type-B Arabidopsis response regulators (ARRs). Although central elements in the cytokinin signal transduction pathway have been identified, mechanisms controlling output remain to be elucidated. Here we demonstrate that a family of F-box proteins, called the KISS ME DEADLY (KMD) family, targets type-B ARR proteins for degradation. KMD proteins form an S-PHASE KINASE-ASSOCIATED PROTEIN1 (SKP1)/Cullin/F-box protein (SCF) E3 ubiquitin ligase complex and directly interact with type-B ARR proteins. Loss-of-function KMD mutants stabilize type-B ARRs and exhibit an enhanced cytokinin response. In contrast, plants with elevated KMD expression destabilize type-B ARR proteins leading to cytokinin insensitivity. Our results support a model in which an SCFKMD complex negatively regulates cytokinin responses by controlling levels of a key family of transcription factors.

    Cytokinins are mitogenic plant hormones that control multiple aspects of growth and development. Cytokinins were initially discovered and named based on their ability to promote cell division of plant cells grown in culture (1). Subsequent research defined a critical role for cytokinins in regulating cell division at stem cell niches throughout the life cycle of the plant (2, 3). Cytokinins also stimulate chloroplast development, modulate shoot and root development, delay leaf senescence, and regulate abiotic and biotic stress responses (4–6).

    Cytokinin signals are transmitted through a multistep histidine-to-aspartate phosphorelay system, evolutionarily related to the two-component signaling systems of prokaryotes (4, 7). In Arabidopsis, cytokinins are perceived by the three receptors ARABIDOPSIS HISTIDINE KINASE 2 (AHK2), AHK3, and AHK4, which upon perception of the cytokinin signal autophosphorylate on a conserved His residue (8, 9). The regulatory phosphoryl group is passed from receptor to a histidine-containing phosphotransfer (AHP) protein, and from there to a type-B Arabidopsis response regulator (ARR). Phosphorylation of the Arabidopsis type-B ARR proteins modulates their ability to control gene expression as the key transcription factors in the primary response pathway (10, 11). Among the transcriptional targets of type-B ARRs are a second class of response regulators, type-A ARRs, which act as negative regulators of the signal transduction pathway (12). This model of cytokinin signal transduction has been established largely based on studies in Arabidopsis, but similar two-component signaling elements have been identified in other plant species such as monocot rice and the moss Physcomitrella patens, supporting a common pathway for the transmission of the cytokinin signal in land plants (13, 14). Although central elements in the cytokinin signal transduction pathway have been identified, mechanisms controlling output remain to be elucidated.

    Control of protein stability through ubiquitin-mediated proteolysis has emerged as a central theme in plant growth and development over the past decade (15). E3 ubiquitin ligases have been identified that target key signaling elements for degradation in many plant hormone signaling pathways, including those for auxin, ethylene, gibberellin, and jasmonic acid (16). Identification of such a regulatory component for cytokinin signal transduction has proved elusive, although several studies suggest that the ubiquitin-proteasome system plays a role in the regulation of cytokinin signaling (17–20). Most significantly, the stability of the type-B response regulator ARR2 decreases in the presence of cytokinin, and a more stable mutant version of ARR2 enhances cytokinin sensitivity in various developmental processes (17). In this study, we identify a family of F-box proteins, designated the KISS ME DEADLY (KMD) family, that physically interact with and target type-B ARR proteins for degradation. Genetic analysis demonstrates that the KMD family members function as negative regulators of the cytokinin-signaling pathway. Thus, cytokinin joins the other classical plant hormones in having a key transcriptional regulator controlled by S-PHASE KINASE-ASSOCIATED PROTEIN1 (SKP1)/Cullin/F-box (SCF)/proteasome-mediated degradation.


    Cytokinin-Regulated KMD Genes Encode F-Box Proteins as Part of an SCF Complex.

    F-box proteins, which function as the specificity components within SCF-E3 ligases, are key regulators for several plant hormone-signaling pathways (15, 16). By examining microarray data (21, 22), we identified a cytokinin-regulated gene (accession no. At1g80440) encoding an F-box protein of unknown function. The protein encoded by At1g80440 contains an amino-terminal F-box motif followed by a Kelch-repeat domain that could mediate protein–protein interactions (23). We designated At1g80440 as KMD1 and its homologs At1g15670, At2g44130, and At3g59940 as KMD2, KMD3, and KMD4, respectively (Fig. S1 A and B). Expression of the three family members KMD1, -3, and -4 exhibited a twofold reduction in transcript levels within 1 h of cytokinin treatment (Fig. 1A). The effect of cytokinin on KMD gene expression was reduced in type-B ARR double mutant arr1, arr12 (24) (Fig. 1A), indicating the necessity of these type-B ARRs for mediating the transcriptional effect of cytokinin on KMD expression. Based on normalized microarray data from the Arabidopsis electronic fluorescent pictograph (eFP) browser (25, 26), KMD family members are broadly expressed, with expression at the shoot apical meristem (especially KMD1 and KMD2) and at the root tip (especially KMD2 and KMD3), tissues where cytokinin regulates cell division (2–4).

    cytokinin kmd

      Fig. 1.

      Cytokinin-regulated KMD genes encode F-box proteins interacting with components of the SCF complex. (A) Fourteen-day-old wild-type (Col) and arr1,arr12 mutant seedlings were treated with 10 µM benzyladenine (BA) or a DMSO vehicle control for 1 h, and expression of KMD1, KMD2, KMD3, and KMD4 analyzed by qRT-PCR in shoots excised from the treated seedlings. Error bars are ±SD. (B) Alignment of F-box motifs from Arabidopsis F-box proteins. Identities and similarities are highlighted by black and gray, respectively. (C) BiFC analysis of KMD1-ASK1 and KMD2-ASK1 interaction. The F-box domain (KMD1F-box or KMD2F-box) is sufficient for interaction with ASK1 in mesophyll protoplast cells. (D) Coimmunoprecipitation of KMD1-HA and Cullin1-HA with ASK1-GFP based on anti-GFP immunoprecipitation from transfected protoplasts. The immunoblot was probed with anti-HA and anti-GFP antibodies. GFP served as a negative control for the immunoprecipitation.


      KMD proteins contain a conserved amino-terminal F-box motif (Fig. 1B and Fig. S1B). To determine if KMDs function as canonical F-box proteins, we examined their interactions with known components of the Arabidopsis SCF complex. All four of the full-length KMD proteins interacted with ASK1, an Arabidopsis Skp1 protein of the SCF complex, in a bimolecular fluorescence complementation (BiFC) assay in protoplast cells (Fig. 1C and Fig. S1 C and D). The F-box domains of KMD1 and KMD2 were sufficient for interaction with ASK1 (Fig. 1C). The BiFC fluorescent signal was detected in both the nucleus and cytoplasm (Fig. 1C and Fig. S1D), consistent with the subcellular localization of KMD proteins (Fig. S1E), implying that KMD proteins may function as F-box proteins in the cytoplasm as well as in the nucleus. We performed coimmunoprecipitation analysis to confirm interactions of KMD1 within an SCF complex (Fig. 1D). Both hemagglutinin (HA)-tagged KMD1 and Cullin1 coimmunoprecipitated with green-fluorescence-protein (GFP)-tagged ASK1, consistent with the KMD proteins functioning within a SCF complex in plant cells.

      KMD Family Members Function as Negative Regulators of Cytokinin Responses.

      To investigate the functional role of KMDs in cytokinin signaling, we isolated transfer DNA (T-DNA) insertion mutations in the KMD1, KMD2, and KMD4 genes, and constructed single, double, and triple mutants. No full-length transcripts were detected for the mutant alleles based on RT-PCR analysis, suggesting, based on the position of the T-DNA inserts, that these represent null alleles (Fig. S2 A and B). We also generated independent antisense transgenic lines using a KMD4 antisense construct in the kmd1,2 background (kmd1,2,α4), which resulted in reduced transcript levels of both KMD3 and KMD4 (Fig. S2B). To examine the contributions of KMD genes to cytokinin responsiveness, the kmd mutants were examined for their inhibition of hypocotyl elongation in response to exogenous cytokinin. Single and double kmd mutants were comparable to the wild type, whereas the kmd1-1; kmd2-1; kmd4-1 (kmd1,2,4) and kmd1,2,α4 mutants showed enhanced sensitivity to cytokinin, indicating genetic redundancy among these genes (Fig. 2A and Fig. S2C). The higher order kmd mutants also exhibited increased sensitivity to cytokinin in a root growth response assay as well as in the ability of cytokinin to induce greening and shoot formation in tissue-cultured explants, the effects in tissue culture being most pronounced in the kmd1,2,α4–2 line (Fig. 2 AC and Fig. S2C). The effect of cytokinin on root development is in part due to a negative effect on root meristem size (4, 27). Consistent with this, both kmd1,2,4 and kmd1,2,α4 lines had fewer cells in their root meristems (Fig. 2D), suggesting that the absence of endogenous KMD proteins mimics the effect of cytokinin by reducing the size of the root meristem.

      kmd family

        Fig. 2.

        Members of the KMD family negatively regulate cytokinin signaling. (A and B) Effect of cytokinin on hypocotyl (A) and root (B) growth of Col, kmd1,2,4, kmd1,2,α4 antisense lines, and KMD-overexpression lines (35S::KMD1-3, 35S::KMD1-11, 35S::KMD2-7, and 35S::KMD2-13). In A, etiolated seedlings were grown for 4 d on the indicated t-zeatin concentrations. Error bars are ±SE (not shown if smaller than symbol; n ≥ 20). In B, seedlings were grown on vertical plates supplemented with t-zeatin under constant light. Increase in root length from day 4 through day 7 was measured. Error bars are ±SE (not shown if smaller than symbol; n ≥ 18). (C) Effect of a cytokinin on callus formation and shoot initiation. Calli were prepared from the hypocotyl tissues of the indicated lines, then incubated on shoot induction medium containing indole-3-butyric acid (0.2 mg/L) and the indicated concentrations of t-zeatin. (D) Reduced expression of the KMD family results in a decrease in root meristem size. Roots of 7-d-old seedlings of Col, kmd1,2,4, and kmd1,2,α4 antisense lines were stained with propidium iodide and visualized by confocal fluorescence microscopy, and cortex meristematic cell numbers measured. White arrows indicate the transition zone where cells leave the meristem and enter the elongation-differentiation zone. (Scale bar, 100 μm.) Asterisks indicate significant differences (P < 0.0001). Error bars are ±SE (n ≥ 20). (E and F) RNA levels of ARR7 and ARR15 were analyzed by qRT-PCR in Col, kmd1,2,4, and kmd1,2,α4 antisense lines treated with 0, 10, or 100 nM t-zeatin for 1 h (E); or in Col, 35S::KMD1-11, and 35S::KMD2-13 lines treated with 10 μM BA or a DMSO vehicle control for 1 h, the fold change in response being indicated for the lines (F).


        We confirmed that the KMD family functions as a negative regulator for cytokinin signaling by examining transgenic lines overexpressing KMD1 and KMD2 under the constitutive cauliflower mosaic virus (CaMV) 35S promoter (Fig. S2D). These overexpression lines exhibited a substantially reduced cytokinin response in several physiological assays, including hypocotyl and root growth in response to cytokinin as well as in shoot initiation assays (Fig. 2 AC). Consistent with their reduced sensitivity to cytokinin in the root growth assay (4, 27), the overexpression lines exhibited larger root meristems (Fig. S2E). Significantly, the 35S::KMD1 and -2 lines responded normally to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and the synthetic auxin naphthalene-1-acetic acid (NAA) (Fig. S2 F and G), indicating a specificity toward cytokinin for their effect on signaling. We also expressed KMD1 and KMD2 using the cassava vein mosaic virus (CsVMV) promoter, which yielded higher expression levels than that mediated by the CaMV 35S promoter (Fig. S2D). Seedlings of the CsVMV::KMD lines were stunted and exhibited premature termination of primary root growth (Fig. S3A). The shoot apical meristem (SAM) of the CsVMV::KMD lines exhibited a wild-type structure and organization, but was substantially smaller than that found in wild-type seedlings (Fig. S3B). In addition, the meristems of the prematurely aborted primary roots were substantially smaller in the CsVMV::KMD lines compared with wild type (Fig. S3 C and D). These meristematic changes resulting from CsVMV promoter-driven overexpression of KMD1 and KMD2 are similar to those previously reported for severe mutations in the primary cytokinin signaling pathway (11, 28, 29).

        To determine if the increased cytokinin sensitivity of higher order kmd mutants is associated with enhanced cytokinin signal transduction, we examined expression of the cytokinin primary response genes ARR7 and ARR15 by qRT-PCR (Fig. 2E). In wild-type plants, ARR7 and ARR15 were induced two to fourfold by 1-h treatment with 100 nM t-zeatin. Both the kmd1,2,4 and kmd1,2,α4 lines exhibited a greater amplitude in cytokinin-induced ARR7 and ARR15 expression compared with wild type (1.5- to twofold stronger induction; Fig. 2E), a similar change in responsiveness to what has been found with mutations in other negative regulators of the pathway (12). The molecular response to cytokinin was also altered in the 35S::KMD1 and -2 lines. The basal RNA levels of ARR7 and ARR15 were decreased compared with that in the wild type (Fig. 2F), indicating a reduced response to endogenous cytokinin in the 35S::KMD lines. Moreover, the degree of induction of the ARR genes by exogenous cytokinin was also reduced by KMD overexpression (Fig. 2F). This molecular phenotype is similar to what has been observed with mutations in other positive regulators of the cytokinin signaling pathway (11, 28, 29). Overall, these physiological and molecular results reveal that the KMD family members function as negative regulators of the cytokinin signaling pathway.

        KMD Proteins Physically Interact with Type-B ARR Proteins.

        The similarity of KMD mutant phenotypes (Fig. 2) to those involving type-B ARR mutations (Fig. S3) (11, 24, 30, 31), suggested that these F-box proteins might target type-B ARR proteins for degradation. To test for interaction between KMD and type-B ARR proteins, yeast two-hybrid assays were performed (Fig. 3A and Fig. S4 A and B). The KMD proteins interacted with ARR1 and ARR12, and to a lesser extent ARR2 and ARR10, of the subfamily-1 type-B ARRs. The KMD proteins also interacted with ARR20 of type-B ARR subfamily-3. Significantly, the KMD proteins did not interact with representative members of type-A ARRs (ARR4 and ARR7), indicating that interaction with the KMD proteins is not a general characteristic of the plant response regulators. We focused our subsequent interaction analysis on ARR1 and ARR12 because genetic studies have demonstrated that these two type-B ARRs are major contributors to multiple cytokinin responses (11, 24, 30). All four KMD proteins interacted with both ARR1 and ARR12 in a BiFC assay (Fig. 3B). Strong fluorescence was detected in the nuclei of cells, consistent with the subcellular localization of type-B ARRs (32, 33). In addition, HA-tagged ARR1 or ARR12 proteins coimmunoprecipitated with a GFP-tagged KMD1 protein from extracts of transfected protoplasts (Fig. 3C). ARR2 also interacted in vivo with KMD1 based on BiFC analysis (Fig. S4C), even though this interaction was weaker based on the yeast-two hybrid analysis (Fig. 3A and Fig. S4A), supporting the significance of the detectable yeast-two hybrid interactions. We did not find any effect of cytokinin on the interaction of KMDs with ARRs based on yeast-two hybrid and BiFC analyses. These results indicate that multiple type-B ARRs serve as direct targets for the KMD F-box proteins. In a BiFC assay, we found that KMD1 interacted more strongly with the C-terminal domains of ARR1 and ARR12 [ARR1 (153–669) and ARR12 (133–596)] than with their receiver domains [ARR1 (1–154) and ARR12 (1–134)] (Fig. S4D).

        kmd proteins

          Fig. 3.

          KMD proteins physically interact with type-B ARR proteins. (A) Yeast two-hybrid assay between KMD proteins (KMD1 or KMD2) and type-B ARR proteins. Pairs of indicated bait and prey vectors were transformed into yeast cells. The growth of a blue yeast colony on selective medium containing X-gal indicates a positive interaction. (B) Interaction between KMD proteins and ARR1 or ARR12 in BiFC assay. The indicated constructs were cotransfected into the Arabidopsis mesophyll protoplasts. (C) Coimmunoprecipitation of type-B ARR proteins (ARR1 or ARR12) and KMD1. Protoplast cells were cotransfected with ARR1-HA or ARR12-HA along with either KMD1-GFP or a GFP control. The KMD1-GFP or GFP proteins were immunoprecipitated with an anti-GFP antibody, and an immunoblot was probed with anti-HA and anti-GFP antibodies.


          KMD1 and KMD2 Target Type-B ARR Proteins for Degradation.

          Protein levels of ARR1 and ARR12 increase in the presence of the proteasome inhibitor MG132 (Fig. 4A and Fig. S5A), consistent with their degradation being controlled by the ubiquitin/proteasome system. In addition, treatment with the protein biosynthesis inhibitor cycloheximide revealed that both ARR1 and ARR12 are unstable proteins. Interestingly, their turnover was not affected by exogenous cytokinin (Fig. S5A), suggesting that the lifespan of type-B ARR may contribute to its ability to propagate the cytokinin signal. To determine if type-B ARRs are targeted by KMDs for degradation in planta, we crossed transgenic lines expressing 35S::ARR1-HA, 35S::ARR2-HA, or 35S::ARR12-Myc with wild-type or 35S::KMD-GFP plants, and analyzed the abundance of type-B ARR proteins in the F1 seedlings. Elevated expression of KMD1 and KMD2 resulted in a decrease in ARR1, ARR2, and ARR12 protein levels (Fig. 4 B and C and Fig. S5B). Furthermore, analysis of the turnover kinetics for ARR1 indicates that elevated levels of KMD1 result in an enhanced turnover rate for ARR1 (Fig. S5C). MG132 retarded degradation of ARR1 in the KMD1 overexpression line (Fig. S5D), consistent with degradation being mediated by the ubiquitin/proteasome pathway. Based on the overexpression analysis, KMD1 was more effective than KMD2 in targeting type-B ARRs for degradation. Consistent with a role for KMD proteins in controlling type-B ARR levels, we also found that the ARR1 protein is more abundant in the kmd1,2,α4 lines (Fig. 4D and Fig. S5E), providing a mechanistic basis for why these lines display enhanced cytokinin sensitivity (31). Because no changes in ARR1 and ARR12 transcript levels were observed in these lines (Fig. 4 BD), our results are consistent with the KMDs mediating the posttranslational degradation of type-B ARRs. To investigate whether canonical phosphoregulation of type-B ARRs is necessary for their SCFKMD-mediated degradation in planta, we generated ARR1Asp89Asn (ARR1D89N) and ARR12D69N, in which the phosphotransfer to ARR1 and ARR12 is predicted to be abolished due to mutation of the conserved Asp in the receiver domain (34, 35). The levels of ARR1D89N and ARR12D69N decreased in the presence of overexpressed KMD1 (Fig. 4E), suggesting that a cytokinin-induced phosphorelay to this conserved residue is not required for the SCFKMD-mediated degradation of ARR1 and ARR12.

          kmd1 kmd2

            Fig. 4.

            KMD1 and KMD2 target type-B ARR proteins for degradation. (A) Proteasome-dependent degradation of ARR1 and ARR12. Mesophyll protoplasts from 35S::ARR1-HA or 35S::ARR12-Myc lines were incubated for 3 h in the presence or absence of the proteasome inhibitor MG132 and protein extracts analyzed by immunoblotting. α-Tubulin served as a loading control. (B and C) Overexpression of KMD1 and KMD2 results in reduced protein levels of ARR1 (B) and ARR12 (C). Protein levels of the tagged proteins were determined by immunoblot analysis using anti-HA and anti-Myc antibodies, with α-tubulin as the loading control. Transcript levels for transgenes were detected using HA- or Myc-tag–specific primers by RT-PCR, with β-tubulin as the loading control. (D) Decreased expression of the KMD family results in increased protein levels of ARR1. Immunoblot detection of ARR1-HA in 10-d-old wild-type and kmd1,2,α4 seedlings, with α-tubulin as the loading control is shown. Each lane represents a protein sample from independent seedlings. (E) Degradation of ARR1 and ARR12 is not dependent on the conserved phosphorylation target Asp. The predicted phosphorylation target residues on ARR1 and ARR12 were mutated to Asn (ARR1D89N; ARR12D69N), and stability was examined in the presence or absence of KMD1 overexpression.




            We demonstrate a role for F-box proteins in regulating the cytokinin signal transduction pathway, identifying type-B ARR transcription factors as targets of the “orphan” KMD F-box proteins. A model integrating SCFKMD into the cytokinin signaling pathway is shown in Fig. S6A. SCFKMD interacts with and targets multiple type-B ARRs for degradation, notably the subfamily-1 members that mediate the majority of the cytokinin transcriptional response in Arabidopsis (11, 30, 35), changes in type-B ARR protein levels serving to regulate the cytokinin responsiveness of the plant. Consistent with this model are our data demonstrating that (i) loss-of-function mutations in the KMD family result in an enhanced sensitivity of plants to cytokinin, (ii) overexpression of KMD family members results in a decreased sensitivity of plants to cytokinin, (iii) KMDs physically interact with type-B ARRs, and (iv) the loss- and gain-of-function kmd mutant phenotypes correlate with changes in type-B ARR protein levels. Type-B ARRs are predicted to be direct targets of the KMD family based on the specificity of F-box proteins for their ubiquitination targets, our interaction data, and the proteasome-dependent turnover of type-B ARRs. The cytokinin signaling pathway is conserved in plants (13), and homologs to KMDs exist in the monocot rice (Fig. S1A) as well as the mosses P. patens (XP_001756872) and Selagenella moellendorffii (XP_002983138), supporting the broad conservation of this regulatory mechanism for controlling the cytokinin transcriptional output among the land plants.

            Our results, coupled with prior analyses of type-A and type-B ARR stability, point to a central role of the ubiquitin-proteasome pathway in controlling the protein levels of two families of plant response regulators. A subset of type-A response regulators, which act as negative regulators of the cytokinin signaling pathway, are stabilized in response to cytokinin (20). In addition, proteasome-dependent degradation of ARR2, a type-B ARR, is enhanced by cytokinin-induced phosphorylation of the conserved Asp residue in the receiver domain (17). The two-component signaling system is evolutionarily ancient (36), and our study points to how plants, like bacteria, use degradation of response regulators as a pivotal mechanism for transcriptional regulation (37, 38). Plants, unlike bacteria, accomplish this by using the eukaryotic-specific ubiquitin-proteasome system.

            Several additional studies implicate the ubiquitin-proteasome in the control of cytokinin signaling. Mutants of REGULATORY PARTICLE NON-ATPASE12 (RPN12), which is a subunit of proteasome regulatory structures, exhibit reduced cytokinin sensitivity, indicating that the 26S proteasome is required to degrade one or more repressors of cytokinin action in plants (19). Candidates for such a repressor include type-A ARRs as well as KMD proteins identified here. More recently, AUXIN UP-REGULATED F-BOX PROTEIN 1 (AUF1) was identified in the search for components of the ubiquitin-proteasome pathway that affect cytokinin signaling (18). No effects of AUF1 mutants were found on type-B ARR protein levels, suggesting that the role of AUF1 in cytokinin signaling is indirect, potentially via cross-talk through the auxin signaling pathway. Significantly, we found that the effects of KMD loss- and gain-of-function mutations are specific for cytokinin, mutant seedlings responding normally to auxin, based on several physiological assays, demonstrating the specificity of SCFKMD toward the cytokinin signaling pathway.

            Activity of response regulators such as type-B ARRs is typically regulated by phosphorylation of a conserved Asp residue within the receiver domain, phosphorylation thought to induce conformational changes that activate type-B ARRs (31, 39, 40). However, based on our analysis, SCFKMD appears to regulate stability of type-B ARRs in a phospho-Asp-independent manner. This is consistent with known characteristics for type-B ARR turnover which, with the exception of ARR2 (17), is not affected by cytokinin treatment. An ability of SCFKMD to mediate degradation of both inactive and activated forms of type-B ARRs could serve two purposes based on well-established models for signal transduction (Fig. S6B) (41). First, SCFKMD could regulate the abundance of type-B ARRs, thereby determining the threshold level for a cytokinin response, a mechanism that may facilitate cross-talk with other signaling pathways; for example, expression of KMD1, -3, and -4 exhibits circadian dependence in response to light and temperature (25, 26, 42), and could thus regulate the interaction between cytokinin and the circadian clock (43). Second, SCFKMD could remove activated type-B ARRs, thereby preventing continued transcriptional activation by cytokinin. In its simplest form, this suggests a “timer” model for transcriptional regulation, the stability and lifespan of the active type-B ARR modulating its signal output. Differences in the efficacy of the KMD family members in targeting type-B ARRs for degradation may allow for fine tuning of cytokinin responses; differences in efficacy have also been observed in the EIN3 BINDING F-BOX1 (EBF1) F-box family that target the ETHYLENE-INSENSITIVE3 (EIN3) transcription factor family to control the ethylene response, EBF2 being more effective than EBF1 based on overexpression analysis (44, 45). Although we do not observe an effect of cytokinin on KMD-mediated degradation of type-B ARRs, KMD interaction with the C-terminal portion of type-B ARRs suggests the DNA-binding/activation domain of the transcription factor may play a role in its degradation. In animals and bacteria, transcriptional activation domains often overlap with the degrons that target the transcription factor for proteolysis, such that activation is closely linked to degradation (41).

            With the identification of a role for SCFKMD, cytokinin joins the other classical phytohormones auxin, jasmonate, ethylene, and gibberellin in having key transcriptional regulators controlled by SCF/proteasome-mediated degradation (16, 46, 47). The role of degradation in the cytokinin signaling pathway, however, differs from that in these other hormonal signaling pathways. Auxin, jasmonate, and gibberellin all target transcriptional repressors for degradation. Ethylene, like cytokinin, employs a transcriptional activator (EIN3), but EIN3 is ubiquitinated and degraded in the absence of ethylene, stabilization of EIN3 in the presence of ethylene leading to activation of ethylene-response genes. In contrast, type-B ARRs functioning in cytokinin signaling are present in the absence of cytokinin but undergo continuous proteolysis. Although the mechanisms used for transcriptional control vary among these five phytohormones, constants are a role for SCF/proteasome-mediated degradation and a targeting of key elements involved in transcription as a means of modulating signal output.

            Materials and Methods

            Plant Materials and Growth Conditions.

            Arabidopsis thaliana Columbia (Col-0) ecotype was the parent strain for mutants and transgenic lines. The kmd1-1 (SALK_008497) and kmd4-1 (SALK_080249) T-DNA insertion lines were obtained from the Salk T-DNA insertion collections (48), and kmd2-1 (GABI-KAT 079A01) plants were obtained from the GABI-KAT T-DNA insertion collection. The genotype of each line was confirmed by PCR-based methodology. For detailed description on the growth conditions, see SI Materials and Methods.

            Cytokinin Growth and Molecular Response Analyses.

            Hypocotyl and root growth analysis was performed as described previously (11) and hypocotyl and root lengths were measured using ImageJ software (version 1.32; National Institutes of Health). Root meristem size was determined as described (49). The apical meristem was visualized by bright field microscopy, with tissue sectioning, fixation, and staining performed as described (50). Total RNA isolation, cDNA production, and quantitative RT-PCR (qRT-PCR) were performed as described (11). Primers used for qRT-PCR are listed in Table S1. Transcript abundances were calculated using the comparative CT method, with β-TUBULIN3 (β-TUB3) (At5g62700) as the normalized control. See SI Materials and Methods for detailed procedures.

            Tissue Regeneration Assay.

            Hypocotyls from 4-d-old dark-grown seedlings were excised and transferred to 1× Murashige and Skoog (MS) plate medium containing 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/L kinetin. After 4 d, hypocotyl segments were transferred to medium containing 0.2 mg/L indole-3-butyric acid (IBA) and t-zeatin ranging from 0 to 10 mg/L (24), and callus was examined after 3 wk.

            Transient Expression in Arabidopsis Protoplasts and in Vivo Coimmunoprecipitation Assay.

            Arabidopsis mesophyll protoplasts were isolated from mature leaves of the wild-type plants and transfected with various constructs as described (51). For BiFC assay, cDNA fragments were fused to plant expression vector containing either amino- or carboxyl-terminal fragments of the YFP (YFPN and YFPC) (52). Transfected protoplasts were examined with an Axioplan2 fluorescent microscope (Carl Zeiss). Coimmunoprecipitation (Co-IP) was performed using agarose-conjugated anti-GFP antibody (GFP-Trap; Chromotek) following the manufacturer’s protocol with slight modifications. To examine the role of the proteasome in ARR stability, protoplasts from ARR1-HA and ARR12-Myc overexpression lines were prepared and incubated for 3 h in the presence of 10 μM MG132 or 0.1% DMSO as a vehicle control, and protein levels were determined by immunoblot analysis. SI Materials and Methods has detailed information on BiFC and co-IP experiments.

            Wednesday, 17 July 2013 16:38

            Self-cleaning screens even killed E. coli


            A survey by the magazine Which? Conducted in 2010, the surface of a cell phone contains 18 times more harmful bacteria than a button in a public toilet cistern. For this reason, the company Corning introduces coating displays antimicrobial properties which kills virtually emptied microorganisms on it.

            CEO Jeff Evarsan says that innovative coverage will be effective against drug resistant bacteria and viruses. Originally designed for use in biomedical institutions, creators see huge potential and applied to the standard personal phones.

            Evarsan demonstrate some of the properties of the coating in public places such as fluorescently labeled bacteria Escherichia coli usually of glass and a specially crafted their antibacterial glass. While on common glass bacteria live undisturbed and full potential to infect someone on these patented by Corning coverage are completely destroyed in less than two hours.

            Escherichia coli is a Gram-negative rod-shaped bacterium that is a major cause of food poisoning and severe forms of gastrointestinal disorders. Study of the American Health Organization in 2012 concluded that one in six mobile phones is seriously contaminated with a large number of pathogens, mainly E. coli.
            The company informed that the first phones with their innovative hygienic coverage will reach the market by 2015

            mouseMedical research aimed at animal testing of therapies for the treatment of brain diseases are often skewed and show positive results, which are confirmed in humans - this according to a poll released Tuesday. The results of the study conducted by John Ioannidis and colleagues at Stanford University, explains why a large number of seemingly effective treatments for animals are not effective in humans.

            The authors have examined 160 publications 1411 developments involving more than 4000 test animals, associated with the treatment of Parkinson's disease, Alzheimer's disease or multiple sclerosis. Only eight of the developments show noticeable results, but only two of them allow to achieve satisfactory results in the last administration when people try therapy, say researchers from Stanford. The rest of the studies were skewed by a number of problems such as no good conduct, very little amount of attempts or publishing only studies with "positive results."

            Literature on studies of neurological diseases is probably very biased, according to a study published in the journal PLoS Biology. "Biased results from animal experiments may lead to ineffective or even harmful to human clinical trials or loss of money for research," the authors add. According to them, a possible explanation for this failure is in the biological and physiological differences between animals and humans, and these errors can be explained by abnormalities in research or publications for them.

            Researchers prefer to publish their research in prestigious scientific journals, but they tend to emphasize only studies with positive results, which may also explain biased results.

            White blood cells are reign as the heroes of the immune system. When the infection, the cells produced in the bone marrow, the race through the blood to combat pathogens. But new research shows that different bodies may also play a role in immune defense, which is essentially your own hero. In a study of a rare and deadly brain infection, researchers at Rockefeller University have discovered that the brain cells of healthy people can produce their own immune system molecules, demonstrating the "intrinsic immunity" that is crucial to stop the infection.

            Interfering with interferon Herpes simplex virus-infected neurons

            Interfering with interferon. Herpes simplex virus-infected neurons (above)
            are from patients with a genetic defect that impairs their brain’s ability to make
            interferon, an important immune system protein, and leaves their brain
            cells unable to fight off the infection. Healthy people, in turn,
            have an intrinsic immune response to the virus. (Credit: Image courtesy of Rockefeller University)

            Shen-Ying Zhang, clinical researcher at St. Giles Laboratory of Human Genetics of Infectious Diseases, has been studying children with herpes simplex encephalitis, a potentially deadly brain infection herpes virus, HSV-1, which may be important to brain damage. Scientists already knew from previous work that children with encephalitis have a genetic defect that affects the functions of the immune system receptor - Toll-like receptor 3 (TLR3) - in the brain. For this study we wanted to see how a defect in TLR3 prevent the brain's ability to fight herpes infection.
            When TLR3 recognizes pathogen that causes an immune response that causes the release of proteins known as interferons, and ringing "interfere" with the replication of the pathogen. Most commonly associated with white blood cells that are present within the body, but in this case researchers examines the existence of the receptor in neurons and other brain cells.
            "One interesting thing for these patients is that they have none of the other symptoms, the most frequent herpes. They had an infection of the skin or mouth only in their brains. Therefore hypothesis that TLR3 responses should be specifically responsible for the maintenance of herpes virus infects the brain and is not necessary in other parts of the body, "says Zhang.
            The laboratory, directed by Jean-Laurent Casanova, together with scientists from Harvard Medical School and the Institute of Memorial Sloan-Kettering Cancer to create induced pluripotent stem cells. Made from the patient's own tissue, the stem cells are converted into cells of the central nervous system, which leads to patient genetic defects. Zhang cells exposed to HSV-1 and synthetic double-stranded RNA, which mimics the product of the virus that stimulates Toll-like receptor activity. By measuring the levels of interferon, Zhang revealed that TLR3 response of patients was in fact defective, cells were making these important immune system proteins, preventing them from fighting infection.
            Zhang also exposed to the blood cells of patients with the virus and it was found that TLR3 defect is no problem there, as in the brain - interferons were released in another manner.
            Because Toll-like receptors in neurons was vital to prevent infection, encephalitis, researchers concluded that brain cells use as an internal mechanism to fight infection, rather than relying on the white blood cells. When the function deteriorates, patients who can not be improved.
            "This is proof of intrinsic immunity, recently discovered function of the immune system," says Zhang. "It is very likely that other organs also have their own specific tools to fight infection."
            Researchers are putting together a pilot study to test interferon-based therapy in patients with encephalitis, believing that will help speed recovery and increase survival when used in combination with antiviral drugs. They have also examined whether the brain shows intrinsic immunity to other viral infections.

            Tuesday, 16 July 2013 12:02

            Virus against tumor - who will win?

            myxoma-virus-rabbit-knockin-knock-out-mouse-ratWith the combination of myxoma virus and medications that suppress the immune system, it is possible to beat Glioblastoma multiforme - the most common and most lethal form of brain tumor, known to medicine.

            According to Dr. Peter Forsythe from the Center for Cancer Research "Moffitt" this therapy is effective even in the stage of the disease that many physicians would accept for the terminal. Another big plus of successful research is that the new treatment removes one of the most serious obstacles exist to date in the treatment of brain cancer: resistance to temozolomide.

            The new therapy is seen as a "target" for the oncolytic virus invades and destroys only the cancer cells, while chemotherapy affect any tissue of the patient. Myxoma virus that causes virulent diseases in rabbits will be used together with the immunosuppressant rapamycin.

            The precise mechanism by which rapamycin affects the infectious process in tumor cells is not yet well understood, but it is certainly effective - application in laboratory tests contribute to the destruction of more than 89% of the tumor cells by the virus.

            Therapy is now a candidate for clinical trials in humans, but the members of the research group carried out the study were confident of success. Florida State University, Texas, Calgary and the Center for Cancer Research of Ottawa devoted his most brilliant oncologists who are on track to achieve a revolution in the treatment of brain cancer.

            Buy Myxoma products from Gentaur

            Monday, 15 July 2013 09:36

            Component of soya kills cancer cells

            soya-gentaur-antibodies-molecular-productsResearch at the University of Illinois achieved in the laboratory incredible success: reducing tumor formations in colorectal cancer by 94%! Colon cancer is among the most common, aggressive and lethal forms of cancer.

            Impressive result was achieved with peptide lunasin found in soya and any other forms of therapy. Professor Elvira Mejia December revealed that peptide initially used in combination with physiotherapy drug oxaliplatin, which led to a sixfold reduction of metastatic tumors in the liver tissue. Researchers found that lunasina able to penetrate into cancer cells to induce their death and is associated with at least one type of cancer cell receptors, ready to metastasize. This means that the compound is able to recognize not only cancer cells but also to prevent their expansion from other tissues.

            On lunasina transcribed almost all beneficial effects of soya, the most studied is its ability to lower cholesterol. It affects the liver enzymes that are responsible for the regulation of the levels of "good" HDL, and "bad" LDL cholesterol levels.
            Currently, the research group developed a precise dosage of the medicament to be taken as the normal soybean meal. Their aim is to avoid unnecessary complication through therapy and hospital stays, and to reach the desired concentration of the compound in the blood of patients only by strict diet.

            The study was conducted for only 28 days, but their results were impressive. Professor considering encourage producers of fresh milk and yoghurt to include low concentrations of lunasin in their products, thereby preventing cancer before they occur. Market already has flour, enriched with soy peptide, because for years known its anti-inflammatory and cholesterol lowering properties.
            Research professor in December Mejia are published in the online Journal Portal cancer treatments in 2013 and enjoy a great deal of attention from around the scientific world.

            Researchers from Thomas Jefferson University's Kimmel Cancer Center have discovered that decorin, a naturally occurring protein that circulates in the blood, acts as a potent inhibitor of tumor growth modulating the tumor microenvironment.

            The study, published June 24 online in the Proceedings of the National Academy of Sciences, suggests it may be possible to harness the power of this naturally occurring anticancer agent as a way to treat cancer, including metastases.

            In several different publications it has been described the ability of decorin to affect a number of biological processes including inflammatory responses, wound healing, and angiogenesis.

            In this new article, the study's senior investigator, Renato Iozzo, M.D., Ph.D., has labeled decorin a "soluble tumor repressor" -- the first to be found that specifically targets new blood vessels, which are pushed to grow by the cancer, and forces the vessel cells to "eat" their internal components. This reduces their potential to feed the cancer overall causing an inhibition of tumor progression.

            "The tumor suppressors we all know are genes inside tumors that a cancer deletes or silences in order to continue growing. I call decorin a tumor repressor because its anti-tumor activity comes from the body, outside the cancer," says Dr. Iozzo, Professor of Pathology & Cell Biology, Biochemistry & Molecular Biology at Kimmel Cancer Center.

            "Decorin is a soluble compound that we found has a powerful, natural protective effect against cancer -- an exciting finding that we believe will open up a new avenue for both basic research and clinical application," Dr. Iozzo says. "Acting from the outside of the cells, decorin is able to modify the behavior of the cancer cells and of the normal cells in order to slow down the progression of the tumor. For this reason, decorin acts as a guardian of the matrix, the complicated structure built around the cells in our body."

            Absence of decorin promotes tumor growth

            Decorin has long been known to be involved in human development. It is so named because deposits of decorin "decorate" collagen fibrils after the human body forms.

            A second pool of decorin has been found circulating in blood after production by connective tissue throughout the body. This connective tissue is part of the extracellular matrix, which provides both structural support and biological regulation of tissue cells.

            But no one has understood the biological function of this second pool of decorin, according to Dr. Iozzo.

            The research team, including the two co-first authors, Simone Buraschi, Ph.D., and Thomas Neill, a graduate student, who work in the laboratory of Dr. Iozzo, decoded the function of soluble decorin. They found that addition of exogenous decorin to the tumor microenvironment induces autophagy, a mechanism by which cells discard unnecessary or damaged intracellular structures. "This process regulates a lot of cellular activities," says Dr. Iozzo.

            The researchers specifically found that decorin evoked autophagy in both microvascular and macrovascular endothelial cells -- cells that line the interior surface of blood vessels.

            "This matters because autophagy can exert a potential oncosupressive function by acting to discard critical cell components that would otherwise be involved in promotion of tumor growth through angiogenesis, the production of new blood vessels that can provide nutrition to the tumor," Dr. Iozzo says. "In contrast, absence of decorin permits tumor growth."

            Therefore, the presence of decorin in the surroundings of the tumor is essential to control tumorigenesis and formation of new blood vessels, he says. Moreover, Dr. Iozzo's laboratory has characterized for the first time Peg3, a known tumor-suppressor gene, as a master player in the autophagy process induced by decorin. "This discovery is important as it opens up to the study of new unexplored genes and signaling pathways in the field of autophagy," he says. 

            "Circulating decorin represents a fundamental cellular process that acts to combat tumor angiogenesis," Dr. Iozzo says. "Treatment based on systemic delivery of decorin may represent a genuine advance in our ongoing war against cancer."

            The study was funded by the National Institutes of Health grants R01 CA39481, R01 CA47282, and R01 CA120975.

            Collaborating researchers from LifeCell Corporation, in Branchburg, New Jersey, and Goethe University in Frankfurt, Germany, also contributed to the study.

            Scientists at Karolinska Institutet in Sweden, in collaboration with colleagues in Germany and the Netherlands, have identified a previously unknown group of nerve cells in the brain. The nerve cells regulate cardiovascular functions such as heart rhythm and blood pressure. It is hoped that the discovery, which is published in the Journal of Clinical Investigation, will be significant in the long term in the treatment of cardiovascular diseases in humans.

            The scientists have managed to identify in mice a previously totally unknown group of nerve cells in the brain. These nerve cells, also known as 'neurons', develop in the brain with the aid of thyroid hormone, which is produced in the thyroid gland. Patients in whom the function of the thyroid gland is disturbed and who therefore produce too much or too little thyroid hormone, thus risk developing problems with these nerve cells. This in turn has an effect on the function of the heart, leading to cardiovascular disease.

            It is well-known that patients with untreated hyperthyroidism (too high a production of thyroid hormone) or hypothyroidism (too low a production of thyroid hormone) often develop heart problems. It has previously been believed that this was solely a result of the hormone affecting the heart directly. The new study, however, shows that thyroid hormone also affects the heart indirectly, through the newly discovered neurons.

            "This discovery opens the possibility of a completely new way of combating cardiovascular disease," says Jens Mittag, group leader at the Department of Cell and Molecular Biology at Karolinska Institutet. "If we learn how to control these neurons, we will be able to treat certain cardiovascular problems like hypertension through the brain. This is, however, still far in the future. A more immediate conclusion is that it is of utmost importance to identify and treat pregnant women with hypothyroidism, since their low level of thyroid hormone may harm the production of these neurons in the fetus, and this may in the long run cause cardiovascular disorders in the offspring."

            The study has been financed with grants from the European Molecular Biology Organisation, Deutsche Forschungsgemeinschaft, the Fredrik and Ingrid Thuring Foundation, Karolinska Institutet Foundation, the American Thyroid Association, the Swedish Research Council, the Swedish Cancer Society, the Söderberg Foundations, the Swedish Heart-Lung Foundation, the Netherlands Organization for Health Research and Development, and the Ludgardine Bouwman Foundation.

            bottles-gentaur-antibodiesTwo patients have been taken off their HIV drugs after bone-marrow transplants seemed to clear the virus from their bodies, doctors report.

            One of the patients has spent nearly four months without taking medication with no sign of the virus returning.

            The team at Brigham and Women's Hospital, in the US, caution that it is far too soon to talk about a cure as the virus could return at any point. The findings were presented at the International Aids Society Conference.

            It is difficult to get rid of an HIV infection because it hides inside human DNA, forming untouchable "reservoirs" in body. Anti-retroviral drugs keep the virus in check within the bloodstream - but when the drugs stop, the virus comes back.

            The two men, who have not been identified, had lived with HIV for about 30 years. They both developed a cancer, lymphoma, which required a bone-marrow transplant.

            Bone marrow is where new blood cells are made and it is thought to be a major reservoir for HIV. After the transplant, there was no detectable HIV in the blood for two years in one patient and four in the other.

            It is far too early to call this a cure for HIV. And even if it was a cure, it wouldn't be a very good one.

            It is very expensive and often leads to "graft-v-host" disease. There is a 15-20% mortality rate within the first few years after the transplant.

            This occurs when new immune cells produced by the graft treat the rest of the body as foreign and attack it.

            The two patients in this study have replaced their regimen of anti-retroviral drugs, with those to suppress the immune system.

            The procedure was carried out in these patients only because they had cancer that needed to be treated. The real value of this research for the majority of people with HIV will come from a deeper understanding of the virus and HIV reservoirs. The pair came off their anti-retroviral drugs earlier this year.

            One has gone 15 weeks, and the other seven, since stopping treatment, and no signs of the virus have been detected so far.

            Dr Timothy Henrich told the BBC the results were exciting. But he added: "We have not demonstrated cure, we're going to need longer follow-up.

            "What we can say is if the virus does stay away for a year or even two years after we stopped the treatment, that the chances of the virus rebounding are going to be extremely low. "It's much too early at this point to use the C-word [cure]."

            It is thought that the transplanted bone marrow was initially protected from infection by the course of anti-retrovirals. Meanwhile the transplant also attacked the remaining bone marrow, which was harbouring the virus.

            However Dr Henrich cautioned that the virus could be still be hiding inside brain tissue or the gastrointestinal track.

            "If the virus does return, it would suggest that these other sites are an important reservoir of infectious virus and new approaches to measuring the reservoir at relevant sites will be needed to guide the development of HIV curative strategies," he said.

            The two US cases both received bone marrow from normal donors. There was also a report of an HIV cure in a baby born in Mississippi, US. She was treated with anti-retroviral drugs at birth so it is thought the virus was cleared from the body before reservoirs were established.

            Dr Michael Brady, the medical director of the Terrence Higgins Trust, said: "It is too early to know whether HIV has been eradicated from these men's bodies or whether it might return.

            Doctors say it is far too soon to talk about a cure for HIV, as James Gallagher reports "However, the case suggests that what happened to Timothy Brown, the Berlin Patient was perhaps not a one-off.

            "A bone marrow transplant is a complex and expensive procedure, which comes with significant risks. "For most people with HIV, it would be more dangerous to undergo a transplant than to continue managing the virus with daily medication.

            "So while this is by no means a workable cure, it does give researchers another signpost in the direction of one."

            The head of the Foundation for AIDS Research, Kevin Frost, said: "These findings clearly provide important new information that might well alter the current thinking about HIV and gene therapy.

            "While stem-cell transplantation is not a viable option for people with HIV on a broad scale because of its costs and complexity, these new cases could lead us to new approaches to treating, and ultimately even eradicating, HIV."