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Sample records for arabidopsis tubulin mutants1woa

  1. Increase in expression level of alpha-tubulin gene in Arabidopsis seedlings under hypergravity conditions.

    Science.gov (United States)

    Saito, Yuka; Soga, Kouichi; Wakabayashi, Kazuyuki; Hoson, Takayuki

    2003-10-01

    Under hypergravity conditions, elongation growth of plant shoots is suppressed. The analysis of the changes in gene expression by hypergravity treatment in Arabidopsis hypocotyls by the differential display method showed that a gene encoding alpha-tubulin, which is a component of microtubules, was up-regulated by hypergravity. In Arabidopsis six genes encoding alpha-tubulin (TUA1-TUA6) have been identified. In the present study, we examined the dose-response and the time course relations of the changes in the expression of all six alpha-tubulin genes in Arabidopsis hypocotyls grown under hypergravity conditions. The expression levels of all six alpha-tubulin genes, TUA1-TUA6, were increased by increasing gravity, although the extent was variable among genes. The increase in expression of all alpha-tubulin genes was detected within a few hours, when the seedlings grown at 1 g were transferred to 300 g condition. These results suggest that Arabidopsis hypocotyls regulate the expression level of six alpha-tubulin genes promptly in response to gravity stimuli. The increase in the amount of microtubules due to the activation of tubulin gene expression may be involved in the regulation by gravity signal of shoot growth.

  2. Gravity-Induced Modifications to Development in Hypocotyls of Arabidopsis Tubulin Mutants1[W][OA

    Science.gov (United States)

    Matsumoto, Shouhei; Kumasaki, Saori; Soga, Kouichi; Wakabayashi, Kazuyuki; Hashimoto, Takashi; Hoson, Takayuki

    2010-01-01

    We investigated the roles of cortical microtubules in gravity-induced modifications to the development of stem organs by analyzing morphology and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis (Arabidopsis thaliana) tubulin mutants, tua3(D205N), tua4(S178Δ), and tua6(A281T), cultivated under 1g and hypergravity (300g) conditions. Hypocotyls of tubulin mutants were shorter and thicker than the wild type even at 1g, and hypergravity further suppressed elongation and stimulated expansion. The degree of such changes was clearly smaller in tubulin mutants, in particular in tua6. Hypocotyls of tubulin mutants also showed either left-handed or right-handed helical growth at 1g, and the degree of twisting phenotype was intensified under hypergravity conditions, especially in tua6. Hypergravity induced reorientation of cortical microtubules from transverse to longitudinal directions in epidermal cells of wild-type hypocotyls. In tubulin mutants, especially in tua6, the percentage of cells with longitudinal microtubules was high even at 1g, and it was further increased by hypergravity. The twisting phenotype was most obvious at cells 10 to 12 from the top, where reorientation of cortical microtubules from transverse to longitudinal directions occurred. Moreover, the left-handed helical growth mutants (tua3 and tua4) had right-handed microtubule arrays, whereas the right-handed mutant (tua6) had left-handed arrays. There was a close correlation between the alignment angle of epidermal cell files and the alignment of cortical microtubules. Gadolinium ions, blockers of mechanosensitive ion channels (mechanoreceptors), suppressed the twisting phenotype in tubulin mutants under both 1g and 300g conditions. Microtubule arrays in tubulin mutants were oriented more transversely by gadolinium treatment, irrespective of gravity conditions. These results support the hypothesis that cortical microtubules play an essential role in maintenance of normal growth

  3. Gravity-induced modifications to development in hypocotyls of Arabidopsis tubulin mutants.

    Science.gov (United States)

    Matsumoto, Shouhei; Kumasaki, Saori; Soga, Kouichi; Wakabayashi, Kazuyuki; Hashimoto, Takashi; Hoson, Takayuki

    2010-02-01

    We investigated the roles of cortical microtubules in gravity-induced modifications to the development of stem organs by analyzing morphology and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis (Arabidopsis thaliana) tubulin mutants, tua3(D205N), tua4(S178Delta), and tua6(A281T), cultivated under 1g and hypergravity (300g) conditions. Hypocotyls of tubulin mutants were shorter and thicker than the wild type even at 1g, and hypergravity further suppressed elongation and stimulated expansion. The degree of such changes was clearly smaller in tubulin mutants, in particular in tua6. Hypocotyls of tubulin mutants also showed either left-handed or right-handed helical growth at 1g, and the degree of twisting phenotype was intensified under hypergravity conditions, especially in tua6. Hypergravity induced reorientation of cortical microtubules from transverse to longitudinal directions in epidermal cells of wild-type hypocotyls. In tubulin mutants, especially in tua6, the percentage of cells with longitudinal microtubules was high even at 1g, and it was further increased by hypergravity. The twisting phenotype was most obvious at cells 10 to 12 from the top, where reorientation of cortical microtubules from transverse to longitudinal directions occurred. Moreover, the left-handed helical growth mutants (tua3 and tua4) had right-handed microtubule arrays, whereas the right-handed mutant (tua6) had left-handed arrays. There was a close correlation between the alignment angle of epidermal cell files and the alignment of cortical microtubules. Gadolinium ions, blockers of mechanosensitive ion channels (mechanoreceptors), suppressed the twisting phenotype in tubulin mutants under both 1g and 300 g conditions. Microtubule arrays in tubulin mutants were oriented more transversely by gadolinium treatment, irrespective of gravity conditions. These results support the hypothesis that cortical microtubules play an essential role in maintenance of normal

  4. Growth stimulation in inflorescences of an Arabidopsis tubulin mutant under microgravity conditions in space.

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    Hoson, T; Soga, K; Wakabayashi, K; Hashimoto, T; Karahara, I; Yano, S; Tanigaki, F; Shimazu, T; Kasahara, H; Masuda, D; Kamisaka, S

    2014-01-01

    Cortical microtubules are involved in plant resistance to hypergravity, but their roles in resistance to 1 g gravity are still uncertain. To clarify this point, we cultivated an Arabidopsis α-tubulin 6 mutant (tua6) in the Cell Biology Experiment Facility on the Kibo Module of the International Space Station, and analyzed growth and cell wall mechanical properties of inflorescences. Growth of inflorescence stems was stimulated under microgravity conditions, as compared with ground and on-orbit 1 g conditions. The stems were 10-45% longer and their growth rate 15-55% higher under microgravity conditions than those under both 1 g conditions. The degree of growth stimulation tended to be higher in the tua6 mutant than the wild-type Columbia. Under microgravity conditions, the cell wall extensibility in elongating regions of inflorescences was significantly higher than the controls, suggesting that growth stimulation was caused by cell wall modifications. No clear differences were detected in any growth or cell wall property between ground and on-orbit 1 g controls. These results support the hypothesis that cortical microtubules generally play an important role in plant resistance to the gravitational force.

  5. Arabidopsis GCP3-interacting protein 1/MOZART 1 is an integral component of the γ-tubulin-containing microtubule nucleating complex.

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    Nakamura, Masayoshi; Yagi, Noriyoshi; Kato, Takehide; Fujita, Satoshi; Kawashima, Noriyuki; Ehrhardt, David W; Hashimoto, Takashi

    2012-07-01

    Microtubules in eukaryotic cells are nucleated from ring-shaped complexes that contain γ-tubulin and a family of homologous γ-tubulin complex proteins (GCPs), but the subunit composition of the complexes can vary among fungi, animals and plants. Arabidopsis GCP3-interacting protein 1 (GIP1), a small protein with no homology to the GCP family, interacts with GCP3 in vitro, and is a plant homolog of vertebrate mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1), a recently identified component of the γ-tubulin complex in human cell lines. In this study, we characterized two closely related Arabidopsis GIP1s: GIP1a and GIP1b. Single mutants of gip1a and gip1b were indistinguishable from wild-type plants, but their double mutant was embryonic lethal, and showed impaired development of male gametophytes. Functional fusions of GIP1a with green fluorescent protein (GFP) were used to purify GIP1a-containing complexes from Arabidopsis plants, which contained all the subunits (except NEDD1) previously identified in the Arabidopsis γ-tubulin complexes. GIP1a and GIP1b interacted specifically with Arabidopsis GCP3 in yeast. GFP-GIP1a labeled mitotic microtubule arrays in a pattern largely consistent with, but partly distinct from, the localization of the γ-tubulin complex containing GCP2 or GCP3 in planta. In interphase cortical arrays, the labeled complexes were preferentially recruited to existing microtubules, from which new microtubules were efficiently nucleated. However, in contrast to complexes labeled with tagged GCP2 or GCP3, their recruitment to cortical areas with no microtubules was rarely observed. These results indicate that GIP1/MOZART1 is an integral component of a subset of the Arabidopsis γ-tubulin complexes.

  6. A Single Amino-Acid Substitution at Lysine 40 of an Arabidopsis thaliana α-tubulin Causes Extensive Cell Proliferation and Expansion Defects

    Institute of Scientific and Technical Information of China (English)

    Xue Xiong; Deyang Xu; Zhongnan Yang; HaiHuang; Xiaofeng Cui

    2013-01-01

    Microtubules are highly dynamic cytoskeletal polymers of α/β-tubulin heterodimers that undergo multiple post-translational modifications essential for various cellular functions in eukaryotes.The lysine 40 (K40) is largely conserved in α-tubulins in many eukaryote species,and the post-translational modification by acetylation at K40 is critical for neuronal development in vertebrates.However,the biological function of K40 of α-tubulins in plants remains unexplored.In this study,we show in Arabidopsis thaliana that constitutive expression of mutated forms of α-tubulin6 (TUA6) at K40 (TUA6κ40A or TUA6κ40Q),in which K40 is replaced by alanine or glutamine,result in severely reduced plant size.Phenotypic characterization of the 35S:TUA6κ40A transgenic plants revealed that both cell proliferation and cell expansion were affected.Cytological and biochemical analyses showed that the accumulation ofα-and β-tubulin proteins was significantly reduced in the transgenic plants,and the cortical microtubule arrays were severely disrupted,indicating that K40 of the plant α-tubulin is critical in maintaining microtubule stability.We also constructed 35S:TUA6κ40R transgenic plants in which K40 of the engineered TUA6 protein is replaced by an arginine,and found that the 35S:TUA6K40R plants were phenotypically indistinguishable from the wild-type.Since lysine and arginine are similar in biochemical nature but arginine cannot be acetylated,these results suggest a structural importance for K40 of α-tubulins in cell division and expansion.

  7. Cell edges accumulate gamma tubulin complex components and nucleate microtubules following cytokinesis in Arabidopsis thaliana.

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    Chris Ambrose

    Full Text Available Microtubules emanate from distinct organizing centers in fungal and animal cells. In plant cells, by contrast, microtubules initiate from dispersed sites in the cell cortex, where they then self-organize into parallel arrays. Previous ultrastructural evidence suggested that cell edges participate in microtubule nucleation but so far there has been no direct evidence for this. Here we use live imaging to show that components of the gamma tubulin nucleation complex (GCP2 and GCP3 localize at distinct sites along the outer periclinal edge of newly formed crosswalls, and that microtubules grow predominantly away from these edges. These data confirm a role for cell edges in microtubule nucleation, and suggest that an asymmetric distribution of microtubule nucleation factors contributes to cortical microtubule organization in plants, in a manner more similar to other kingdoms than previously thought.

  8. Arabidopsis CDS blastp result: AK069395 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069395 J023011N07 At1g71440.1 tubulin folding cofactor E / Pfifferling (PFI) almo...st identical to tubulin folding cofactor E (Pfifferling; PFI) GI:20514267 from [Arabidopsis thaliana]; identical to cDNA tubulin folding cofactor E, GI:20514266 7e-41 ...

  9. Arabidopsis CDS blastp result: AK102150 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK102150 J033086D17 At3g10220.1 tubulin folding cofactor B identical to tubulin folding... cofactor B GI:20514259 from [Arabidopsis thaliana]; identical to cDNA tubulin folding cofactor B GI:20514258 6e-91 ...

  10. Tubulin tyrosine ligase and stathmin compete for tubulin binding in vitro

    OpenAIRE

    Szyk, Agnieszka; Piszczek, Grzegorz; Roll-Mecak, Antonina

    2013-01-01

    Tubulin partition between soluble and polymeric forms is tightly regulated in cells. Stathmin and tubulin tyrosine ligase (TTL)a each form stable complexes with tubulin and inhibit tubulin polymerization. Here we explore the mutual relationship between these proteins in vitro and demonstrate that full-length stathmin and TTL compete for binding to tubulin and fail to make a stable tubulin:stathmin:TTL triple complex in solution. Moreover, stathmin depresses TTL tubulin tyrosination activity i...

  11. Tubulin tyrosine ligase and stathmin compete for tubulin binding in vitro.

    Science.gov (United States)

    Szyk, Agnieszka; Piszczek, Grzegorz; Roll-Mecak, Antonina

    2013-07-24

    Tubulin partition between soluble and polymeric forms is tightly regulated in cells. Stathmin and tubulin tyrosine ligase (TTL) each form stable complexes with tubulin and inhibit tubulin polymerization. Here we explore the mutual relationship between these proteins in vitro and demonstrate that full-length stathmin and TTL compete for binding to tubulin and fail to make a stable tubulin:stathmin:TTL triple complex in solution. Moreover, stathmin depresses TTL tubulin tyrosination activity in vitro. These results suggest either that TTL and stathmin have a partially overlapping footprint on the tubulin dimer or that stathmin induces a tubulin conformation incompatible with stable TTL binding.

  12. Microtubules, Tubulins and Associated Proteins.

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    Raxworthy, Michael J.

    1988-01-01

    Reviews much of what is known about microtubules, which are biopolymers consisting predominantly of subunits of the globular protein, tubulin. Describes the functions of microtubules, their structure and assembly, microtube associated proteins, and microtubule-disrupting agents. (TW)

  13. Purification of tubulin from porcine brain.

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    Gell, Christopher; Friel, Claire T; Borgonovo, Barbara; Drechsel, David N; Hyman, Anthony A; Howard, Jonathon

    2011-01-01

    Microtubules, polymers of the heterodimeric protein αβ-tubulin, give shape to cells and are the tracks for vesicle transport and chromosome segregation. In vitro assays to study microtubule functions and their regulation by microtubule-associated proteins require the availability of purified αβ-tubulin. In this chapter, we describe the process of purification of heterodimeric αβ-tubulin from porcine brain.

  14. Heterogeneity of the alpha subunit of tubulin and the variability of tubulin within a single organism.

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    Bibring, T; Baxandall, J; Denslow, S; Walker, B

    1976-05-01

    When tubulins obtained from particular microtubules of the sea urchin (ciliary doublet A tubules, flagellar doublet microtubules, and mitotic microtubules) are analyzed by electrophoresis in a polyacrylamide gel system containing sodium dodecyl sulfate and urea, heterogeneity of the alpha subunit, and differences between the tubulins are revealed. The alpha subunit of tubulin from mitotic apparatus and from A microtubules of ciliary doublets is resolved into two bands, while the alpha subunit of flagellar doublet tubulin gives a single band. The mitotic and ciliary tubulins differ in the mobilities of their two alpha species, or in the relative amounts present, or both. The existence of differences between the tubulins has been confirmed by a preliminary analysis of their cyanogen bromide peptides.

  15. Tubulin assembly is disordered in a hypogeomagnetic field.

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    Wang, Dong Liang; Wang, Xing Sheng; Xiao, Rong; Liu, Ying; He, Rong Qiao

    2008-11-14

    Although the effect of a magnetic field on functions of many proteins has been reported, tubulin assembly in a hypogeomagnetic field (HGMF) has not yet been characterized. Here, we show disorder in tubulin self-assembly in an HGMF. Absorbance at 350 nm, commonly used to monitor tubulin self-assembly, was altered in the HGMF, providing evidence for the effects of HGMF on tubulin. Measurements of intrinsic fluorescence (335 nm) also revealed a disordered change in tubulin conformation during assembly in the HGMF. Under the same conditions, microtubule-like filaments were not observed by electron microscopy, with the exception of amorphous oligomers. Incubation of tubulin with tau in the natural geomagnetic field (GMF) yielded microtubule-like filaments, while only amorphous oligomers were observed following the incubation in the HGMF. This distinction suggests that tubulin assembly depends upon the GMF, and that elimination of the GMF induces disorder in tubulin organization.

  16. Rationalization of paclitaxel insensitivity of yeast β-tubulin and human βIII-tubulin isotype using principal component analysis

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    Das Lalita

    2012-08-01

    Full Text Available Abstract Background The chemotherapeutic agent paclitaxel arrests cell division by binding to the hetero-dimeric protein tubulin. Subtle differences in tubulin sequences, across eukaryotes and among β-tubulin isotypes, can have profound impact on paclitaxel-tubulin binding. To capture the experimentally observed paclitaxel-resistance of human βIII tubulin isotype and yeast β-tubulin, within a common theoretical framework, we have performed structural principal component analyses of β-tubulin sequences across eukaryotes. Results The paclitaxel-resistance of human βIII tubulin isotype and yeast β-tubulin uniquely mapped on to the lowest two principal components, defining the paclitaxel-binding site residues of β-tubulin. The molecular mechanisms behind paclitaxel-resistance, mediated through key residues, were identified from structural consequences of characteristic mutations that confer paclitaxel-resistance. Specifically, Ala277 in βIII isotype was shown to be crucial for paclitaxel-resistance. Conclusions The present analysis captures the origin of two apparently unrelated events, paclitaxel-insensitivity of yeast tubulin and human βIII tubulin isotype, through two common collective sequence vectors.

  17. Molecular insight of isotypes specific β-tubulin interaction of tubulin heterodimer with noscapinoids

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    Santoshi, Seneha; Naik, Pradeep K.

    2014-07-01

    Noscapine and its derivatives bind stoichiometrically to tubulin, alter its dynamic instability and thus effectively inhibit the cellular proliferation of a wide variety of cancer cells including many drug-resistant variants. The tubulin molecule is composed of α- and β-tubulin, which exist as various isotypes whose distribution and drug-binding properties are significantly different. Although the noscapinoids bind to a site overlapping with colchicine, their interaction is more biased towards β-tubulin. In fact, their precise interaction and binding affinity with specific isotypes of β-tubulin in the αβ-heterodimer has never been addressed. In this study, the binding affinity of a panel of noscapinoids with each type of tubulin was investigated computationally. We found that the binding score of a specific noscapinoid with each type of tubulin isotype is different. Specifically, amino-noscapine has the highest binding score of -6.4, -7.2, -7.4 and -7.3 kcal/mol with αβI, αβII, αβIII and αβIV isotypes, respectively. Similarly 10 showed higher binding affinity of -6.8 kcal/mol with αβV, whereas 8 had the highest binding affinity of -7.2, -7.1 and -7.2 kcal/mol, respectively with αβVI, αβVII and αβVIII isotypes. More importantly, both amino-noscapine and its clinical derivative, bromo-noscapine have the highest binding affinity of -46.2 and -38.1 kcal/mol against αβIII (overexpression of αβIII has been associated with resistance to a wide range of chemotherapeutic drugs for several human malignancies) as measured using MM-PBSA. Knowledge of the isotype specificity of the noscapinoids may allow for development of novel therapeutic agents based on this class of drugs.

  18. Tubulin tyrosine ligase like 12 links to prostate cancer through tubulin posttranslational modification and chromosome ploidy.

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    Wasylyk, Christine; Zambrano, Alberto; Zhao, Chunhua; Brants, Jan; Abecassis, Joseph; Schalken, Jack A; Rogatsch, Hermann; Schaefer, Georg; Pycha, Armin; Klocker, Helmut; Wasylyk, Bohdan

    2010-12-01

    Prostate cancer is a common cause of death, and an important goal is to establish the pathways and functions of causative genes. We isolated RNAs that are differentially expressed in macrodissected prostate cancer samples. This study focused on 1 identified gene, TTLL12, which was predicted to modify tubulins, an established target for tumor therapy. TTLL12 is the most poorly characterized member of a recently discovered 14-member family of proteins that catalyze posttranslational modification of tubulins. We show that human TTLL12 is expressed in the proliferating layer of benign prostate. Expression increases during cancer progression to metastasis. It is highly expressed in many metastatic prostate cancer cell lines. It partially colocalizes with vimentin intermediate filaments and cellular structures containing tubulin, including midbodies, centrosomes, intercellular bridges and the mitotic spindle. Downregulation of TTLL12 affects several posttranslational modifications of tubulin (detyrosination and subsequent deglutamylation and polyglutamylation). Overexpression alters chromosomal ploidy. These results raise the possibility that TTLL12 could contribute to tumorigenesis through effects on the cytoskeleton, tubulin modification and chromosome number stability. This study contributes a step toward developing more selective agents targeting microtubules, an already successful target for tumor therapy.

  19. Tubulin nucleotide status controls Sas-4-dependent pericentriolar material recruitment.

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    Gopalakrishnan, Jayachandran; Chim, Yiu-Cheung Frederick; Ha, Andrew; Basiri, Marcus L; Lerit, Dorothy A; Rusan, Nasser M; Avidor-Reiss, Tomer

    2012-08-01

    Regulated centrosome biogenesis is required for accurate cell division and for maintaining genome integrity. Centrosomes consist of a centriole pair surrounded by a protein network known as pericentriolar material (PCM). PCM assembly is a tightly regulated, critical step that determines the size and capability of centrosomes. Here, we report a role for tubulin in regulating PCM recruitment through the conserved centrosomal protein Sas-4. Tubulin directly binds to Sas-4; together they are components of cytoplasmic complexes of centrosomal proteins. A Sas-4 mutant, which cannot bind tubulin, enhances centrosomal protein complex formation and has abnormally large centrosomes with excessive activity. These results suggest that tubulin negatively regulates PCM recruitment. Whereas tubulin-GTP prevents Sas-4 from forming protein complexes, tubulin-GDP promotes it. Thus, the regulation of PCM recruitment by tubulin depends on its GTP/GDP-bound state. These results identify a role for tubulin in regulating PCM recruitment independent of its well-known role as a building block of microtubules. On the basis of its guanine-bound state, tubulin can act as a molecular switch in PCM recruitment.

  20. The Diamagnetic Susceptibility of the Tubulin Dimer

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    Wim Bras

    2014-01-01

    Full Text Available An approximate value of the diamagnetic anisotropy of the tubulin dimer, Δχdimer, has been determined assuming axial symmetry and that only the α-helices and β-sheets contribute to the anisotropy. Two approaches have been utilized: (a using the value for the Δχα for an α-helical peptide bond given by Pauling (1979 and (b using the previously determined anisotropy of fibrinogen as a calibration standard. The Δχdimer≈4×10-27 JT−2 obtained from these measurements are similar to within 20%. Although Cotton-Mouton measurements alone cannot be used to estimate Δχ directly, the value we measured, CMdimer=1.41±0.03×10-8 T−2cm2mg−1, is consistent with the above estimate for Δχdimer. The method utilized for the determination of the tubulin dimer diamagnetic susceptibility is applicable to other proteins and macromolecular assemblies as well.

  1. Peloruside, laulimalide, and noscapine interactions with beta-tubulin.

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    Gajewski, Melissa M; Alisaraie, Laleh; Tuszynski, Jack A

    2012-11-01

    This article reviews the recent findings regarding the binding sites, binding modes and binding affinities of three novel antimitotic drugs peloruside, laulimalide and noscapine with respect to tubulin as the target of their action. These natural compounds are shown to bind to β-tubulin and stabilize microtubules for the cases of peloruside A and laulimalide, and prolong the time spent in pause for noscapine. Particular attention is focused on β-tubulin isotypes as targets for new cancer chemotherapy agents and the amino acid differences in the binding site for these compounds between isotypes. We propose a new strategy for antimitotic drug design that exploits differential distributions of tubulin isotypes between normal and cancer cells and corresponding differential affinities between various drug molecules and tubulin isotypes.

  2. A Unifying Hypothesis for the Conformational Change of Tubulin

    CERN Document Server

    Fygenson, D K

    2001-01-01

    Microtubule dynamic instability arises from the hydrolysis of GTP bound to the beta-monomer of the tubulin dimer. The conformational change induced by hydrolysis is unknown, but microtubules disassemble into protofilaments of GDP-bound tubulin that curve away from the microtubule axis. This paper presents the unfolding of a portion of the tubulin molecule into the microtubule interior as a plausible, unifying explanation for diverse structural and kinetic features of microtubules. This is the first specific structural hypothesis for the hydrolysis induced conformational change of tubulin that simultaneously explains weakening of lateral bonds, bending about longitudinal bonds, changes in protofilament supertwist associated with GTP hydrolysis, structural features of GDP-tubulin double rings, faster disassembly at higher temperatures and slower disassembly in the presence of glycerol and deuterium oxide. The hypothesis suggests further theoretical investigations and direct experimental tests.

  3. alpha-Tubulin of Histriculus cavicola (Ciliophora; Hypotrichea).

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    Pérez-Romero, P; Villalobo, E; Díaz-Ramos, C; Calvo, P; Santos-Rosa, F; Torres, A

    1997-03-01

    An alpha-tubulin gene fragment amplified by PCR from the hypotrichous ciliate Histriculus cavicola has been sequenced. This fragment, 1,182 bp long, contains an in-frame "stop" codon (UAA), which in other hypotrichous species codes for a glutamine residue. The comparison of the alpha-tubulin genes from several ciliates classes have revealed amino acid positions which could serve to distinguish these taxonomic groups.

  4. New benzimidazole-2-urea derivates as tubulin inhibitors.

    Science.gov (United States)

    Wang, Wenna; Kong, Dexin; Cheng, Huimin; Tan, Li; Zhang, Zhang; Zhuang, Xiaoxi; Long, Huoyou; Zhou, Yang; Xu, Yong; Yang, Xiaohong; Ding, Ke

    2014-09-01

    Emerging drug resistance and other drawbacks limit tubulin inhibitors' therapeutic applications and developing novel tubulin inhibitors still attracts intensive efforts. We describe the discovery and structure-activity relationship study of a series of benzimidazole-2-urea derivatives as novel β tubulin inhibitors. The representative compound 6o potently suppressed the proliferation of a panel of human cancer cells (NCI-H460, Colo205, K562, A431, HepG2, Hela, MDA-MB-435S) with IC50 values of 0.040, 0.050, 0.006, 0.026, 1.774, 0.452 and 0.052 μM, respectively. Compound 6o obviously inhibited NCI-H460 spindles formation and induced cell cycle arrest at G2/M phase at 0.10 μM. Computational study suggested that 6o interacts with β tubulin in a novel binding mode. Our results suggested that benzimidazole-2-urea derivatives might be promising tubulin inhibitors with novel binding mode for further development.

  5. Combretastatin A-4 and Derivatives: Potential Fungicides Targeting Fungal Tubulin.

    Science.gov (United States)

    Ma, Zhong-lin; Yan, Xiao-jing; Zhao, Lei; Zhou, Jiu-jiu; Pang, Wan; Kai, Zhen-peng; Wu, Fan-hong

    2016-02-01

    Combretastatin A-4, first isolated from the African willow tree Combretum caffrum, is a tubulin polymerization inhibitor in medicine. It was first postulated as a potential fungicide targeting fungal tubulin for plant disease control in this study. Combretastatin A-4 and its derivatives were synthesized and tested against Rhizoctonia solani and Pyricularia oryzae. Several compounds have EC50 values similar to or better than that of isoprothiolane, which is widely used for rice disease control. Structure-activity relationship study indicated the the cis configuration and hydroxyl group in combretastatin A-4 are crucial to the antifungal effect. Molecular modeling indicated the binding sites of combretastatin A-4 and carbendazim on fungal tubulin are totally different. The bioactivity of combretastatin A-4 and its derivatives against carbendazim-resistant strains was demonstrated in this study. The results provide a new approach for fungicide discovery and fungicide resistance management.

  6. Acetylated tubulin is essential for touch sensation in mice.

    Science.gov (United States)

    Morley, Shane J; Qi, Yanmei; Iovino, Loredana; Andolfi, Laura; Guo, Da; Kalebic, Nereo; Castaldi, Laura; Tischer, Christian; Portulano, Carla; Bolasco, Giulia; Shirlekar, Kalyanee; Fusco, Claudia M; Asaro, Antonino; Fermani, Federica; Sundukova, Mayya; Matti, Ulf; Reymond, Luc; De Ninno, Adele; Businaro, Luca; Johnsson, Kai; Lazzarino, Marco; Ries, Jonas; Schwab, Yannick; Hu, Jing; Heppenstall, Paul A

    2016-12-13

    At its most fundamental level, touch sensation requires the translation of mechanical energy into mechanosensitive ion channel opening, thereby generating electro-chemical signals. Our understanding of this process, especially how the cytoskeleton influences it, remains unknown. Here we demonstrate that mice lacking the α-tubulin acetyltransferase Atat1 in sensory neurons display profound deficits in their ability to detect mechanical stimuli. We show that all cutaneous afferent subtypes, including nociceptors have strongly reduced mechanosensitivity upon Atat1 deletion, and that consequently, mice are largely insensitive to mechanical touch and pain. We establish that this broad loss of mechanosensitivity is dependent upon the acetyltransferase activity of Atat1, which when absent leads to a decrease in cellular elasticity. By mimicking α-tubulin acetylation genetically, we show both cellular rigidity and mechanosensitivity can be restored in Atat1 deficient sensory neurons. Hence, our results indicate that by influencing cellular stiffness, α-tubulin acetylation sets the force required for touch.

  7. In vitro assembly of plant tubulin in the absence of microtubule-stabilizing reagents

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The assembly of microtubules is essential for physiological functions of microtubules. Addition of microtubule-stabilizing reagents or microtubule "seeds" is usually necessary for plant tubulin assembly in vitro, which hinders the investigation of plant microtubule dynamics. In the present note, highly purified plant tubulins have been obtained from lily pollen, a non-microtubule-stabilizing reagent or microtubule "seed" system for plant tubulin assembly has been established and the analysis of plant tubulin assembly performed. Experiment results showed that purified tubulin polymerized in vitro, and a typical microtubule structure was observed with electron microscopy. The kinetics curve of tubulin assembly exhibited typical "parabola". The presence of taxol significantly altered the character of plant tubulin assembly, including that abnormal microtubules were assembled and the critical concentration for plant tubulin assembly was decreased exceedingly from 3 mg/mL in the absence of taxol to 0.043 mg/mL in the presence of taxol.

  8. Taxol differentially modulates the dynamics of microtubules assembled from unfractionated and purified beta-tubulin isotypes.

    Science.gov (United States)

    Derry, W B; Wilson, L; Khan, I A; Luduena, R F; Jordan, M A

    1997-03-25

    Substoichiometric binding of taxol to tubulin in microtubules potently suppresses microtubule dynamics, which appears to be the most sensitive antiproliferative mechanism of taxol. To determine whether the beta-tubulin isotype composition of a microtubule can modulate sensitivity to taxol, we measured the effects of substoichiometric ratios of taxol bound to tubulin in microtubules on the dynamics of microtubules composed of purified alphabeta(II)-, alphabeta(III)-, or alphabeta(IV)-tubulin isotypes and compared the results with the effects of taxol on microtubules assembled from unfractionated tubulin. Substoichiometric ratios of bound taxol in microtubules assembled from purified beta-tubulin isotypes or unfractionated tubulin potently suppressed the shortening rates and the lengths shortened per shortening event. Correlation of the suppression of the shortening rate with the stoichiometry of bound taxol revealed that microtubules composed of purified alphabeta(II)-, alphabeta(III)-, and alphabeta(IV)-tubulin were, respectively, 1.6-, 7.4-, and 7.2-fold less sensitive to the effects of bound taxol than microtubules assembled from unfractionated tubulin. These results indicate that taxol differentially modulates microtubule dynamics depending upon the beta-tubulin isotype composition. The results are consistent with recent studies correlating taxol resistance in tumor cells with increased levels of beta(III0- and beta(IV)-tubulin expression and suggest that altered cellular expression of beta-tubulin isotypes can be an important mechanism by which tumor cells develop resistance to taxol.

  9. DSC Study on Brain Tubulin and the Effect of Cisplatin

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The thermal property of the polymerization of brain tubulin was studied by a high-sensitivity differential scanning calorimeter. The phenomenon that heat flows increased and decreased consistently and obviously was observed. This phenomenon was called heat flow oscillation. It was probably correlated to the dynamic instability of microtubules. The effect of cisplatin on it was reported, too.

  10. Anticancer Activity of Chamaejasmine: Effect on Tubulin Protein

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    Yingkun Nie

    2011-07-01

    Full Text Available In this work, the anticancer activity of chamaejasmine was studied by evaluating its in vitro cytotoxicity against several human cancer cell lines (MCF-7, A549, SGC-7901, HCT-8, HO-4980, Hela, HepG2, PC-3, LNCap, Vero and MDCK using the MTT assay. Results indicated chamaejasmine showed more notable anticancer activity than taxol against PC-3 cells, with IC50 values of 2.28 and 3.98 µM, respectively. Furthermore, Western blot analysis showed that chamaejasmine was able to increase the expression of β-tubulin, but not α-tubulin. In silico simulations indicated that chamaejasmine specifically interacts with the active site which is located at the top of β-tubulin, thanks to the presence of strong hydrophobic effects between the core templates and the hydrophobic surface of the TB active site. The binding energy (Einter was calculated to be −164.77 kcal·mol−1. Results presented here suggest that chamaejasmine possesses anti-cancer properties relating to β-tubulin depolymerization inhibition, and therefore is a potential source of anticancer leads for the pharmaceutical industry.

  11. Tubulin acetylation promoting potency and absorption efficacy of deacetylase inhibitors

    Science.gov (United States)

    Mangas-Sanjuan, V; Oláh, J; Gonzalez-Alvarez, I; Lehotzky, A; Tőkési, N; Bermejo, M; Ovádi, J

    2015-01-01

    Background and Purpose Histone deacetylase 6 (HDAC6) and silent information regulator 2 (SIRT2) control the dynamics of the microtubule network via their deacetylase activities. Tubulin polymerization promoting protein (TPPP/p25) enhances microtubule acetylation by its direct binding to HDAC6. Our objective was to characterize the multiple interactions of the deacetylases and to establish the inhibitory potency and the pharmacokinetic features of the deacetylase inhibitors, trichostatin A (TSA) and AGK2. Experimental Approach The interactions of deacetylases with tubulin and TPPP/p25 were quantified by elisa using human recombinant proteins. The effect of inhibitors on the tubulin acetylation was established in HeLa cells transfected with pTPPP and CG-4 cells expressing TPPP/p25 endogenously by celisa (elisa on cells), Western blot and immunofluorescence microscopy. The pharmacokinetic features of the inhibitors were evaluated by in situ kinetic modelling of their intestinal transport in rats. Key Results Deacetylases interact with both tubulin and TPPP/p25, notwithstanding piggy-back binding of HDAC6 or SIRT2 to the TPPP/p25-associated tubulin was established. Much higher inhibitory potency for TSA than for AGK2 was detected in both HeLa and CG-4 cells. Pioneer pharmacokinetic studies revealed passive diffusion and diffusion coupled with secretion for TSA and AGK2 respectively. Both inhibitors exhibited greater permeability than some other well-established drugs. Conclusions and Implications TPPP/p25-directed deacetylase inhibition provides mechanisms for the fine control of the dynamics and stability of the microtubule network. Deacetylase inhibitors with chemical structures similar to TSA and AGK2 appear to be excellent candidates for oral drug absorption. PMID:25257800

  12. Expression of alpha tubulin during the dimorphic transition of Paracoccidioides brasiliensis.

    Science.gov (United States)

    Silva, W P; Soares, R B; Jesuino, R S; Izacc, S M; Felipe, M S; Soares, C M

    2001-10-01

    In this study we analyzed the expression of (alpha-tubulin during the dimorphic transition of the human-pathogenic fungus Paracoccidioides brasiliensis. The alpha-tubulin from P. brasiliensis was recognized by a commercially available anti-tubulin antibody and was developmentally regulated during the dimorphic form transition. We detected at least two alpha-tubulin isoforms in the mycelial state and only one isoform in the yeast forms. This finding suggests specific roles for the alpha-tubulin isoforms in P. brasiliensis's yeast and mycelial forms.

  13. Increased expression of class III β-tubulin in castration-resistant human prostate cancer

    OpenAIRE

    Terry, S; Ploussard, G.; Allory, Y; Nicolaiew, N; Boissière-Michot, F; Maillé, P; Kheuang, L; Coppolani, E; Ali, A.; Bibeau, F; Culine, S; Buttyan, R; de la Taille, A; Vacherot, F

    2009-01-01

    Background: Class III β-tubulin (βIII-tubulin) is expressed in tissues of neuronal lineage and also in several human malignancies, including non-small-cell lung carcinoma, breast and ovarian cancer. Overexpression of βIII-tubulin in these tumours is associated with an unfavourable outcome and resistance to taxane-based therapies. At present, βIII-tubulin expression remains largely uncharacterised in prostate cancer. Methods: In this report, we evaluated the expression of βIII-tubulin in 138 d...

  14. A Direct Interaction between Leucine-rich Repeat Kinase 2 and Specific β-Tubulin Isoforms Regulates Tubulin Acetylation*

    Science.gov (United States)

    Law, Bernard M. H.; Spain, Victoria A.; Leinster, Veronica H. L.; Chia, Ruth; Beilina, Alexandra; Cho, Hyun J.; Taymans, Jean-Marc; Urban, Mary K.; Sancho, Rosa M.; Ramírez, Marian Blanca; Biskup, Saskia; Baekelandt, Veerle; Cai, Huaibin; Cookson, Mark R.; Berwick, Daniel C.; Harvey, Kirsten

    2014-01-01

    Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease. PMID:24275654

  15. Bacterial actin and tubulin homologs in cell growth and division.

    Science.gov (United States)

    Busiek, Kimberly K; Margolin, William

    2015-03-16

    In contrast to the elaborate cytoskeletal machines harbored by eukaryotic cells, such as mitotic spindles, cytoskeletal structures detectable by typical negative stain electron microscopy are generally absent from bacterial cells. As a result, for decades it was thought that bacteria lacked cytoskeletal machines. Revolutions in genomics and fluorescence microscopy have confirmed the existence not only of smaller-scale cytoskeletal structures in bacteria, but also of widespread functional homologs of eukaryotic cytoskeletal proteins. The presence of actin, tubulin, and intermediate filament homologs in these relatively simple cells suggests that primitive cytoskeletons first arose in bacteria. In bacteria such as Escherichia coli, homologs of tubulin and actin directly interact with each other and are crucial for coordinating cell growth and division. The function and direct interactions between these proteins will be the focus of this review.

  16. The solution structure of the N-terminal domain of human tubulin binding cofactor C reveals a platform for tubulin interaction.

    Directory of Open Access Journals (Sweden)

    Ma Flor Garcia-Mayoral

    Full Text Available Human Tubulin Binding Cofactor C (TBCC is a post-chaperonin involved in the folding and assembly of α- and β-tubulin monomers leading to the release of productive tubulin heterodimers ready to polymerize into microtubules. In this process it collaborates with other cofactors (TBC's A, B, D, and E and forms a supercomplex with TBCD, β-tubulin, TBCE and α-tubulin. Here, we demonstrate that TBCC depletion results in multipolar spindles and mitotic failure. Accordingly, TBCC is found at the centrosome and is implicated in bipolar spindle formation. We also determine by NMR the structure of the N-terminal domain of TBCC. The TBCC N-terminal domain adopts a spectrin-like fold topology composed of a left-handed 3-stranded α-helix bundle. Remarkably, the 30-residue N-terminal segment of the TBCC N-terminal domain is flexible and disordered in solution. This unstructured region is involved in the interaction with tubulin. Our data lead us to propose a testable model for TBCC N-terminal domain/tubulin recognition in which the highly charged N-terminus as well as residues from the three helices and the loops interact with the acidic hypervariable regions of tubulin monomers.

  17. The Solution Structure of the N-Terminal Domain of Human Tubulin Binding Cofactor C Reveals a Platform for Tubulin Interaction

    Science.gov (United States)

    Garcia-Mayoral, Mª Flor; Castaño, Raquel; Fanarraga, Monica L.; Zabala, Juan Carlos; Rico, Manuel; Bruix, Marta

    2011-01-01

    Human Tubulin Binding Cofactor C (TBCC) is a post-chaperonin involved in the folding and assembly of α- and β-tubulin monomers leading to the release of productive tubulin heterodimers ready to polymerize into microtubules. In this process it collaborates with other cofactors (TBC's A, B, D, and E) and forms a supercomplex with TBCD, β-tubulin, TBCE and α-tubulin. Here, we demonstrate that TBCC depletion results in multipolar spindles and mitotic failure. Accordingly, TBCC is found at the centrosome and is implicated in bipolar spindle formation. We also determine by NMR the structure of the N-terminal domain of TBCC. The TBCC N-terminal domain adopts a spectrin-like fold topology composed of a left-handed 3-stranded α-helix bundle. Remarkably, the 30-residue N-terminal segment of the TBCC N-terminal domain is flexible and disordered in solution. This unstructured region is involved in the interaction with tubulin. Our data lead us to propose a testable model for TBCC N-terminal domain/tubulin recognition in which the highly charged N-terminus as well as residues from the three helices and the loops interact with the acidic hypervariable regions of tubulin monomers. PMID:22028797

  18. Interaction of an Overexpressed gamma-Tubulin with Microtubules In Vivo and In Vitro.

    Science.gov (United States)

    Kofron, M; Nadezdina, E; Vassilev, A; Matuliene, J; Essner, R; Kato, J; Kuriyama, R

    1998-08-01

    gamma-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for gamma-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus gamma-tubulin. When CHO cells were transiently transfected with the gamma-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such gamma-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that gamma-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas gamma-tubulin purified from insect Sf9 cells (), interaction between gamma-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with gamma-tubulin monomers in vitro were associated with more gold particles conjugated with gamma-tubulin than in controls where no exogenous gamma-tubulin was added. However, binding of gamma-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although gamma-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (; ), which might be ascribed to the difference in the level of protein expression in transfected cells.

  19. Stathmin and interfacial microtubule inhibitors recognize a naturally curved conformation of tubulin dimers.

    Science.gov (United States)

    Barbier, Pascale; Dorléans, Audrey; Devred, Francois; Sanz, Laura; Allegro, Diane; Alfonso, Carlos; Knossow, Marcel; Peyrot, Vincent; Andreu, Jose M

    2010-10-08

    Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathmin-like domain of the RB3 protein (T(2)RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulin-stathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T(2)RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T(2)RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T(2)RB3, T(2)Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.

  20. Pironetin reacts covalently with cysteine-316 of α-tubulin to destabilize microtubule

    Science.gov (United States)

    Yang, Jianhong; Wang, Yuxi; Wang, Taijing; Jiang, Jian; Botting, Catherine H.; Liu, Huanting; Chen, Qiang; Yang, Jinliang; Naismith, James H.; Zhu, Xiaofeng; Chen, Lijuan

    2016-06-01

    Molecules that alter the normal dynamics of microtubule assembly and disassembly include many anticancer drugs in clinical use. So far all such therapeutics target β-tubulin, and structural biology has explained the basis of their action and permitted design of new drugs. However, by shifting the profile of β-tubulin isoforms, cancer cells become resistant to treatment. Compounds that bind to α-tubulin are less well characterized and unexploited. The natural product pironetin is known to bind to α-tubulin and is a potent inhibitor of microtubule polymerization. Previous reports had identified that pironetin reacts with lysine-352 residue however analogues designed on this model had much lower potency, which was difficult to explain, hindering further development. We report crystallographic and mass spectrometric data that reveal that pironetin forms a covalent bond to cysteine-316 in α-tubulin via a Michael addition reaction. These data provide a basis for the rational design of α-tubulin targeting chemotherapeutics.

  1. Tubulin dynamics during the cytoplasmic cohesiveness cycle in artificially activated sea urchin eggs.

    Science.gov (United States)

    Coffe, G; Foucault, G; Raymond, M N; Pudles, J

    1983-12-01

    Sedimentation studies and [3H]colchicine-binding assays have demonstrated a relationship between the cytoplasmic cohesiveness cycles and the changes in tubulin organization in Paracentrotus lividus eggs activated by 2.5 mM procaine. The same amount of tubulin (20-25% of the total egg tubulin) is involved in these cyclic process and appears to undergo polymerization and depolymerization cycles. Electron microscopy studies reveal that the microtubules formed during these cytoplasmic cohesiveness cycles are under a particulate form which is sedimentable at low speed. Activation experiments carried out in the presence of cytochalasin B (CB) show that the increase in the cytoplasmic cohesiveness is highly reduced while tubulin polymerization and depolymerization cycles and pronuclear centration are not affected. Although tubulin or actin polymerization can be independently triggered in procaine-activated eggs, the increase in cytoplasmic cohesiveness requires the polymerization of both proteins. However, the cytoplasmic cohesiveness cycles appear to be regulated by tubulin polymerization and depolymerization cycles.

  2. Evaluation of alpha-tubulin as an antigenic and molecular probe to detect Giardia lamblia.

    Science.gov (United States)

    Kim, Juri; Shin, Myeong Heon; Song, Kyoung-Ju; Park, Soon-Jung

    2009-09-01

    The alpha/beta-tubulin heterodimer is the basic subunit of microtubules in eukaryotes. Polyclonal antibodies specific to recombinant alpha-tubulin of Giardia lamblia were made, and found effective as a probe to specifically detect G. lamblia by immunofluorescence assays. Nucleotide sequences of alpha-tubulin genes were compared between G. lamblia WB and GS strains, prototypes of assemblage A and assemblage B, respectively. A set of primers was designed and used to amplify a portion of the alpha-tubulin gene from G. lamblia. PCR-RFLP analysis of this alpha-tubulin PCR product successfully differentiated G. lamblia into 2 distinct groups, assemblages A and B. The results indicate that alpha-tubulin can be used as a molecular probe to detect G. lamblia.

  3. In Vitro Trypanocidal Activity of Antibodies to Bacterially Ex­pressed Trypanosoma brucei Tubulin

    Directory of Open Access Journals (Sweden)

    GW Lubega

    2012-09-01

    Full Text Available Background: There are only four drugs for treating African trypanosomiasis, a devastating disease in sub-Saharan Africa. With slow discovery of better drugs, vaccination is viewed as the best method of control. We previously showed that antibodies to native Trypanosoma brucei brucei tubulin inhibit the growth of trypanosomes in culture. Here, we aimed to determine the effect of antibodies to bacte­rially expressed trypanosome tubulin on T. brucei brucei growth. Methods: T. brucei brucei alpha and beta tubulin genes were individually expressed in Escherichia coli under the tryptophan promoter. Monoclonal tubulin antibodies reacted specifically with the ex­pressed tubulins with no cross-reaction with the opposite tubulin. Rabbits were immunized with 450μg each of the concentrated recombinant tubulin, and production of antibodies assessed by ELISA and Western blotting. The effect of polyclonal antibodies on trypanosome growth was deter­mined by culturing bloodstream T. brucei brucei in up to 25% of antisera. Results: Low antisera dilutions (25% from the immunized rabbits inhibited trypanosome growth. The most cytotoxic antisera were from one rabbit immunized with a mixture of both alpha and beta tubulins. However, the result was not reproduced in other rabbits and there was no apparent effect on growth at higher antisera dilutions. Conclusion: Antibodies to bacterially expressed trypanosome tubulin are not effective at killing cul­tured bloodstream trypanosomes.

  4. MEC-17 deficiency leads to reduced α-tubulin acetylation and impaired migration of cortical neurons.

    Science.gov (United States)

    Li, Lei; Wei, Dan; Wang, Qiong; Pan, Jing; Liu, Rong; Zhang, Xu; Bao, Lan

    2012-09-12

    Neuronal migration is a fundamental process during the development of the cerebral cortex and is regulated by cytoskeletal components. Microtubule dynamics can be modulated by posttranslational modifications to tubulin subunits. Acetylation of α-tubulin at lysine 40 is important in regulating microtubule properties, and this process is controlled by acetyltransferase and deacetylase. MEC-17 is a newly discovered α-tubulin acetyltransferase that has been found to play a major role in the acetylation of α-tubulin in different species in vivo. However, the physiological function of MEC-17 during neural development is largely unknown. Here, we report that MEC-17 is critical for the migration of cortical neurons in the rat. MEC-17 was strongly expressed in the cerebral cortex during development. MEC-17 deficiency caused migratory defects in the cortical projection neurons and interneurons, and perturbed the transition of projection neurons from the multipolar stage to the unipolar/bipolar stage in the intermediate zone of the cortex. Furthermore, knockdown of α-tubulin deacetylase HDAC6 or overexpression of tubulin(K40Q) to mimic acetylated α-tubulin could reduce the migratory and morphological defects caused by MEC-17 deficiency in cortical projection neurons. Thus, MEC-17, which regulates the acetylation of α-tubulin, appears to control the migration and morphological transition of cortical neurons. This finding reveals the importance of MEC-17 and α-tubulin acetylation in cortical development.

  5. HDAC6 deacetylates alpha tubulin in sperm and modulates sperm motility in Holtzman rat.

    Science.gov (United States)

    Parab, Sweta; Shetty, Omshree; Gaonkar, Reshma; Balasinor, Nafisa; Khole, Vrinda; Parte, Priyanka

    2015-02-01

    Histone deacetylase 6 (HDAC6) is an alpha (α)-tubulin deacetylase and its over-expression has been demonstrated to promote chemotactic cell movement. Motility in sperm is driven by the flagella, the cytoskeletal structure comprising the microtubules, which are heterodimers of α- and β-tubulins. We have hypothesized that HDAC6, by virtue of being an α-tubulin deacetylase, might modulate sperm motility. However, the presence of HDAC6 on sperm has hitherto not been reported. In this study, we have demonstrated, for the first time, the presence of HDAC6 transcript and protein in the testicular and caudal sperm of rat. We have observed a significantly overlapping expression of HDAC6 with acetyl α-tubulin (Ac α-tubulin) in the mid-piece and principal piece of sperm flagella, and the co-precipitation of α-tubulin and Ac α-tubulin together with HDAC6 and vice versa in sperm lysates. This indicates that HDAC6 interacts with α-tubulin. The HDAC6 activity of sperm, sperm motility and status of Ac α-tubulin investigated in the presence of HDAC inhibitors Trichostatin A, Tubastatin A and sodium butyrate demonstrate that HDAC6 in sperm is catalytically active and that inhibitors of HDAC6 increase acetylation and restrict sperm motility. Thus, we show that (1) active HDAC6 enzyme is present in sperm, (2) HDAC6 in sperm is able to deacetylate α-tubulin, (3) inhibition of HDAC6 results in increased Ac α-tubulin expression and (4) HDAC6 inhibition affects sperm motility. This evidence suggests that HDAC6 is involved in modulating sperm movement.

  6. Dimethyl Sulfoxide Is Feasible for Plant Tubulin Assembly In vitro: A Comprehensive Analysis

    Institute of Scientific and Technical Information of China (English)

    Chun-Hua XU; Shan-Jin HUANG; Ming YUAN

    2005-01-01

    It is much more difficult for tubulin from plant sources to polymerize in vitro than tubulin from animal sources. Taxol, a most widely used reagent in microtubule studies, enhances plant microtubule assembly, but hinders microtubule dynamics. Dimethyl sulfoxide (DMSO), a widely used reagent in animal microtubule studies, is a good candidate for the investigation of plant microtubule assembly in vitro.However, proper investigation is lacking about the effects of DMSO on plant microtubule assembly in vitro.In the present study, DMSO was used to establish optimal conditions for the polymerization of plant tubulin. Tubulin, purified from lily pollen, polymerizes into microtubules at a critical concentration of 1.2mg/mL in the presence of 10% DMSO. The polymers appear to have a normal microtubule structure, as revealed by electron microscopy. In the presence of 10% DMSO, microtubule polymerization decreases when the pH of the medium is increased from 6.5 to 7.4. Both the polymerization rate and the mass of the polymers increase as temperature increases from 25 to 40 ℃. Tubulin polymerizes and depolymerizes along with cycling of temperature, from 37 to 4 ℃, or following the addition to or the removal of Ca2+ from the medium. When incubated with nuclei isolated from tobacco BY-2 suspension cells, tubulin assembles onto the nuclear surface in the presence of 10% DMSO. Labeling lily pollen tubulin with 5- (and 6-)carboxytetramethyl-rhodamine succinimidyl ester (NHS-rhodamine) was performed successfully in the presence of 10% DMSO. Labeled tubulin assembles into a radial structure on the surface of BY-2 nuclei. The polymerization of lily pollen tubulin is also enhanced by microtubule-associated proteins from animal sources in the presence of 10% DMSO. All the experimental results indicate that plant tubulin functions normally in the presence of DMSO. Therefore, DMSO is an appropriate reagent for plant tubulin polymerization and investigation of plant microtubules in

  7. JWA protein binds to α-tubulin in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Hairong; LI Aiqun; LI Aiping; ZHOU Jianwei

    2004-01-01

    Our previous study elucidated that JWA protein was a newly identified microtubule-associated protein (MAP), which combined to and co-localized with β-tubulin.In the present study, we designed a series of experiments to explore if any interactions between JWA protein and α-tubulin existed and how JWA protein would functionally link to α-tubulin, especially in cell mitosis. Results of coimmunoprecipitation, gene transfection and immunofluorescence microscopy from PC12 and HEK293 cells provided strong evidence for a linkage between JWA protein and α-tubulin. Our data showed that JWA protein bound to α-tubulin stably no matter whether α-tubulin was polymerized or not. In addition, by using antisense oligonucleotides, cell cycle blocking agents and hypothermia disposal techniques,we also found the interaction between JWA protein and α-tubulin. The further analysis using flow cytometry and confocal microscopy showed that both proteins co-existed in PC12 cells and were independent on the cell cycle. In conclusion, JWA protein is a newly identified microtubuleassociated protein, binds to α-tubulin, and probably plays an important role in regulation of microtubular stability.

  8. Arabidopsis hybrid speciation processes.

    Science.gov (United States)

    Schmickl, Roswitha; Koch, Marcus A

    2011-08-23

    The genus Arabidopsis provides a unique opportunity to study fundamental biological questions in plant sciences using the diploid model species Arabidopsis thaliana and Arabidopsis lyrata. However, only a few studies have focused on introgression and hybrid speciation in Arabidopsis, although polyploidy is a common phenomenon within this genus. More recently, there is growing evidence of significant gene flow between the various Arabidopsis species. So far, we know Arabidopsis suecica and Arabidopsis kamchatica as fully stabilized allopolyploid species. Both species evolved during Pleistocene glaciation and deglaciation cycles in Fennoscandinavia and the amphi-Beringian region, respectively. These hybrid studies were conducted either on a phylogeographic scale or reconstructed experimentally in the laboratory. In our study we focus at a regional and population level. Our research area is located in the foothills of the eastern Austrian Alps, where two Arabidopsis species, Arabidopsis arenosa and A. lyrata ssp. petraea, are sympatrically distributed. Our hypothesis of genetic introgression, migration, and adaptation to the changing environment during the Pleistocene has been confirmed: We observed significant, mainly unidirectional gene flow between the two species, which has given rise to the tetraploid A. lyrata. This cytotype was able to escape from the narrow ecological niche occupied by diploid A. lyrata ssp. petraea on limestone outcrops by migrating northward into siliceous areas, leaving behind a trail of genetic differentiation.

  9. Comparative modelling of human β tubulin isotypes and implications for drug binding

    Science.gov (United States)

    Torin Huzil, J.; Ludueña, Richard F.; Tuszynski, Jack

    2006-02-01

    The protein tubulin is a target for several anti-mitotic drugs, which affect microtubule dynamics, ultimately leading to cell cycle arrest and apoptosis. Many of these drugs, including the taxanes and Vinca alkaloids, are currently used clinically in the treatment of several types of cancer. Another tubulin binding drug, colchicine, although too toxic to be used as a chemotherapeutic agent, is commonly used for the treatment of gout. The main disadvantage that all of these drugs share is that they bind tubulin indiscriminately, leading to the death of both cancerous and healthy cells. However, the broad cellular distribution of several tubulin isotypes provides a platform upon which to construct novel chemotherapeutic drugs that could differentiate between different cell types, reducing the undesirable side effects associated with current chemotherapeutic treatments. Here, we report an analysis of ten human β tubulin isotypes and discuss differences within each of the previously characterized paclitaxel, colchicine and vinblastine binding sites.

  10. Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

    Science.gov (United States)

    Rezaei Darestani, Reza; Winter, Philip; Kitova, Elena N.; Tuszynski, Jack A.; Klassen, John S.

    2016-05-01

    Tubulin, which is the building block of microtubules, plays an important role in cell division. This critical role makes tubulin an attractive target for the development of chemotherapeutic drugs to treat cancer. Currently, there is no general binding assay for tubulin-drug interactions. The present work describes the application of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to investigate the binding of colchicinoid drugs to αβ-tubulin dimers extracted from porcine brain. Proof-of-concept experiments using positive (ligands with known affinities) and negative (non-binders) controls were performed to establish the reliability of the assay. The assay was then used to screen a library of seven colchicinoid analogues to test their binding to tubulin and to rank their affinities.

  11. Misato Controls Mitotic Microtubule Generation by Stabilizing the TCP-1 Tubulin Chaperone Complex [corrected].

    Science.gov (United States)

    Palumbo, Valeria; Pellacani, Claudia; Heesom, Kate J; Rogala, Kacper B; Deane, Charlotte M; Mottier-Pavie, Violaine; Gatti, Maurizio; Bonaccorsi, Silvia; Wakefield, James G

    2015-06-29

    Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies, but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs.

  12. Stathmin slows down guanosine diphosphate dissociation from tubulin in a phosphorylation-controlled fashion.

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    Amayed, P; Carlier, M F; Pantaloni, D

    2000-10-10

    Stathmin is an important protein that interacts with tubulin and regulates microtubule dynamics in a phosphorylation-controlled fashion. Here we show that the dissociation of guanosine 5'-diphosphate (GDP) from beta-tubulin is slowed 20-fold in the (tubulin)(2)-stathmin ternary complex (T(2)S). The kinetics of GDP or guanosine 5'-triphosphate (GTP) dissociation from tubulin have been monitored by the change in tryptophan fluorescence of tubulin upon exchanging 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-diphosphate (S6-GDP) for tubulin-bound guanine nucleotide. At molar ratios of stathmin to tubulin lower than 0.5, biphasic kinetics were observed, indicating that the dynamics of the complex is extremely slow, consistent with its high stability. The method was used to characterize the effects of phosphorylation of stathmin on its interaction with tubulin. The serine-to-glutamate substitution of all four phosphorylatable serines of stathmin (4E-stathmin) weakens the stability of the T(2)S complex by about 2 orders of magnitude. The phosphorylation of serines 16 and 63 in stathmin has a more severe effect and weakens the stability of T(2)S 10(4)-fold. The rate of GDP dissociation is lowered only 7-fold and 4-fold in the complexes of tubulin with 4E-stathmin and diphosphostathmin, respectively. Sedimentation velocity studies support the conclusions of nucleotide exchange data and show that the T(2)S complexes formed between tubulin and 4E-stathmin or diphosphostathmin are less compact than the highly stable T(2)S complex. The correlation between the effect of phosphorylation of stathmin on the stability of T(2)S complex measured in vitro and on the function of stathmin in vivo is discussed.

  13. Detection of beta-tubulin in the cytoplasm of the interphasic Entamoeba histolytica trophozoites.

    Science.gov (United States)

    Gómez-Conde, Eduardo; Vargas-Mejía, Miguel Ángel; Díaz-Orea, María Alicia; Hernández-Rivas, Rosaura; Cárdenas-Perea, María Elena; Guerrero-González, Tayde; González-Barrios, Juan Antonio; Montiel-Jarquín, Álvaro José

    2016-08-01

    It is known that the microtubules (MT) of Entamoeba histolytica trophozoites form an intranuclear mitotic spindle. However, electron microscopy studies and the employment of anti-beta-tubulin (β-tubulin) antibodies have not exhibited these cytoskeletal structures in the cytoplasm of these parasites. The purpose of this work was to detect β-tubulin in the cytoplasm of interphasic E. histolytica trophozoites. Activated or non-activated HMI-IMSS-strain E. histolytica trophozoites were used and cultured for 72 h at 37 °C in TYI-S-33 medium, and then these were incubated with the anti-β-tubulin antibody of E. histolytica. The anti-β-tubulin antibody reacted with the intranuclear mitotic spindle of E. histolytica-activated trophozoites as control. In contrast, in non-activated interphasic parasites, anti-β-tubulin antibody reacted with diverse puntiform structures in the cytoplasm and with ring-shaped structures localized in the cytoplasm, cellular membrane and endocytic stomas. In this work, for the first time, the presence of β-tubulin is shown in the cytoplasm of E. histolytica trophozoites.

  14. High-affinity accumulation of a maytansinoid in cells via weak tubulin interaction.

    Science.gov (United States)

    Goldmacher, Victor S; Audette, Charlene A; Guan, Yinghua; Sidhom, Eriene-Heidi; Shah, Jagesh V; Whiteman, Kathleen R; Kovtun, Yelena V

    2015-01-01

    The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4-6 × 10(7) copies). Efflux of 3[H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of 3 [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death.

  15. The interplay between tubulins and P450 cytochromes during Plasmodium berghei invasion of Anopheles gambiae midgut.

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    Rute C Félix

    Full Text Available BACKGROUND: Plasmodium infection increases the oxidative stress inside the mosquito, leading to a significant alteration on transcription of Anopheles gambiae detoxification genes. Among these detoxification genes several P450 cytochromes and tubulins were differently expressed, suggesting their involvement in the mosquito's response to parasite invasion. P450 cytochromes are usually involved in the metabolism and detoxification of several compounds, but are also regulated by several pathogens, including malaria parasite. Tubulins are extremely important as components of the cytoskeleton, which rearrangement functions as a response to malaria parasite invasion. METHODOLOGY/PRINCIPAL FINDINGS: Gene silencing methods were used to uncover the effects of cytochrome P450 reductase, tubulinA and tubulinB silencing on the A. gambiae response to Plasmodium berghei invasion. The role of tubulins in counter infection processes was also investigated by inhibiting their effect. Colchicine, vinblastine and paclitaxel, three different tubulin inhibitors were injected into A. gambiae mosquitoes. Twenty-four hours post injection these mosquitoes were infected with P. berghei through a blood meal from infected CD1 mice. Cytochrome P450 gene expression was measured using RT-qPCR to detect differences in cytochrome expression between silenced, inhibited and control mosquitoes. Results showed that cytochrome P450 reductase silencing, as well as tubulin (A and B silencing and inhibition affected the efficiency of Plasmodium infection. Silencing and inhibition also affected the expression levels of cytochromes P450. CONCLUSIONS: Our results suggest the existence of a relationship between tubulins and P450 cytochromes during A. gambiae immune response to P. berghei invasion. One of the P450 cytochromes in this study, CYP6Z2, stands out as the potential link in this association. Further work is needed to fully understand the role of tubulin genes in the response to

  16. Tubulin posttranslational modifications induced by cadmium in the sponge Clathrina clathrus

    Energy Technology Data Exchange (ETDEWEB)

    Ledda, F.D., E-mail: f.ledda@hotmail.it [Dipartimento di Scienze della Terra, dell’Ambiente e della Vita (DISTAV), Università di Genova, Corso Europa 26, I-16132 Genova (Italy); Dipartimento di Scienze della Natura e del Territorio (DIPNET), Università di Sassari, Via Muroni 25, I-07100 Sassari (Italy); Ramoino, P. [Dipartimento di Scienze della Terra, dell’Ambiente e della Vita (DISTAV), Università di Genova, Corso Europa 26, I-16132 Genova (Italy); Ravera, S. [Dipartimento di Farmacia (DIFAR), Viale Cembrano 4, I-16147 Genova (Italy); Perino, E. [Dipartimento di Scienze della Terra, dell’Ambiente e della Vita (DISTAV), Università di Genova, Corso Europa 26, I-16132 Genova (Italy); Bianchini, P. [Istituto Italiano di Tecnologia (IIT), Dipartimento di Nanofisica, Via Morego 30, I-16163 Genova (Italy); Diaspro, A. [Istituto Italiano di Tecnologia (IIT), Dipartimento di Nanofisica, Via Morego 30, I-16163 Genova (Italy); Dipartimento di Fisica (DIFI), Università di Genova, Via Dodecaneso 33, I-16146 Genova (Italy); Gallus, L.; Pronzato, R. [Dipartimento di Scienze della Terra, dell’Ambiente e della Vita (DISTAV), Università di Genova, Corso Europa 26, I-16132 Genova (Italy); Manconi, R. [Dipartimento di Scienze della Natura e del Territorio (DIPNET), Università di Sassari, Via Muroni 25, I-07100 Sassari (Italy)

    2013-09-15

    Highlights: •The effect of Cd{sup 2+} on Clathrina clathrus microtubule network was studied. •Cd{sup 2+} exposure increases acetylated and detyrosinated α-tubulin levels. •Microtubules enriched in acetylated/detyrosinated α-tubulin were resistant to cold. •Clathrina clathrus exposed to Cd{sup 2+} showed cytoplasmic microtubules with an enhanced stability. -- Abstract: As sessile filter feeders, sponges are exposed to environmental stress due to pollutants of both anthropogenic and natural origins and are able to accumulate harmful substances. Thus, sponges are considered a good tool for the biomonitoring of coastal areas. In this study, we used biochemical and immunocytochemical analyses to provide new data on the cadmium-related changes in sponge cells. In particular, we analyzed the effects of different concentrations of cadmium on the microtubule network in the calcisponge Clathrina clathrus. Quantitative densitometry of the immunoblots showed that, while the levels of α- and β-tubulin remained relatively constant in C. clathrus when exposed to 1 and 5 μM CdCl{sub 2}, there were progressive shifts in the levels of some tubulin isoforms. Exposure for 24 h to sublethal concentrations of cadmium reduced the level of tyrosinated α-tubulin and enhanced the levels of acetylated and detyrosinated α-tubulin relative to the levels in controls. Confocal microscopy analysis of immunolabeled tissue sections showed that the inhibitory effect of cadmium was associated with a decrease in the labeling of the cells with a monoclonal antibody that recognizes tyrosinated α-tubulin. By contrast, the reactivity with a monoclonal antibody that recognizes acetylated α-tubulin and with a polyclonal antibody specific for detyrosinated α-tubulin was enhanced at the same time points. Because the acetylation and detyrosination of α-tubulin occur on stable microtubules, the marked enhancement of α-tubulin acetylation and detyrosination in Cd{sup 2+}-treated cells

  17. Disruption of Cortical Microtubules by Overexpression of Green Fluorescent Protein-Tagged α-Tubulin 6 Causes a Marked Reduction in Cell Wall Synthesis

    Institute of Scientific and Technical Information of China (English)

    David H. Burk; Ruiqin Zhong; W. Herbert Morrison Ⅲ; Zheng-Hua Ye

    2006-01-01

    It has been known that the transverse orientation of cortical microtubules (MTs) along the elongation axis is essential for normal cell morphogenesis, but whether cortical MTs are essential for normal cell wall synthesis is still not clear. In the present study, we have investigated whether cortical MTs affect cell wall synthesis by direct alteration of the cortical MT organization in Arabidopsis thaliana. Disruption of the cortical MT organization by expression of an excess amount of green fluorescent protein-tagged α-tubulin 6 (GFP-TUA6)in transgenic Arabidopsis plants was found to cause a marked reduction in cell wall thickness and a decrease in the cell wall sugars glucose and xylose. Concomitantly, the stem strength of the GFP-TUA6overexpressors was markedly reduced compared with the wild type. In addition, expression of excess GFPTUA6 results in an alteration in cell morphogenesis and a severe effect on plant growth and development.Together, these results suggest that the proper organization of cortical MTs is essential for the normal synthesis of plant cell walls.

  18. Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin.

    Science.gov (United States)

    Vemu, Annapurna; Atherton, Joseph; Spector, Jeffrey O; Szyk, Agnieszka; Moores, Carolyn A; Roll-Mecak, Antonina

    2016-06-17

    Microtubules are polymers that cycle stochastically between polymerization and depolymerization, i.e. they exhibit "dynamic instability." This behavior is crucial for cell division, motility, and differentiation. Although studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure, and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogeneous isotypically pure engineered tubulin. Here, we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/βIII is a purely neuronal tubulin isoform. The 4.2-Å structure of post-translationally unmodified human α1A/βIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but it is distinguished by subtle differences at polymerization interfaces, which are hot spots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogeneous microtubules undergo dynamic instability, but they polymerize slower and have fewer catastrophes. Interestingly, we find that epitaxial growth of α1A/βIII microtubules from heterogeneous brain seeds is inefficient but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogeneous α1A/βIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well defined tubulin species and is a first and necessary step toward uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics.

  19. Role of delta-tubulin and the C-tubule in assembly of Paramecium basal bodies

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    Beisson Janine

    2001-03-01

    Full Text Available Abstract Background A breakthrough in the understanding of centriole assembly was provided by the characterization of the UNI3 gene in Chlamydomonas. Deletion of this gene, found to encode a novel member of the tubulin superfamily, delta-tubulin, results in the loss of the C-tubule, in the nine microtubule triplets which are the hallmark of centrioles and basal bodies. Delta-tubulin homologs have been identified in the genomes of mammals and protozoa, but their phylogenetic relationships are unclear and their function is not yet known. Results Using the method of gene-specific silencing, we have inactivated the Paramecium delta-tubulin gene, which was recently identified. This inactivation leads to loss of the C-tubule in all basal bodies, without any effect on ciliogenesis. This deficiency does not directly affect basal body duplication, but perturbs the cortical cytoskeleton, progressively leading to mislocalization and loss of basal bodies and to altered cell size and shape. Furthermore, additional loss of B- and even A-tubules at one or more triplet sites are observed: around these incomplete cylinders, the remaining doublets are nevertheless positioned according to the native ninefold symmetry. Conclusions The fact that in two distinct phyla, delta-tubulin plays a similar role provides a new basis for interpreting phylogenetic relationships among delta-tubulins. The role of delta-tubulin in C-tubule assembly reveals that tubulins contribute subtle specificities at microtubule nucleation sites. Our observations also demonstrate the existence of a prepattern for the ninefold symmetry of the organelle which is maintained even if less than 9 triplets develop.

  20. Anion-induced increases in the affinity of colcemid binding to tubulin.

    Science.gov (United States)

    Ray, K; Bhattacharyya, B; Biswas, B B

    1984-08-01

    Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37 degrees C the association rate constant for colcemid binding is 1.88 X 10(6) M-1 h-1, about 10 times higher than that for colchicine; this is reflected in the activation energies for binding which are 51.4 kJ/mol for colcemid and 84.8 kJ/mol for colchicine. Scatchard analysis indicates two binding sites on tubulin having different affinities for colcemid. The high-affinity site (Ka = 0.7 X 10(5) M-1 at 37 degrees C) is sensitive to temperature and binds both colchicine and colcemid and hence they are mutually competitive inhibitors. The low-affinity site (Kb = 1.2 X 10(4) M-1) is rather insensitive to temperature and binds only colcemid. Like colchicine, 0.6 mol of colcemid are bound/mol of tubulin dimer (at the high-affinity site) and the reaction is entropy driven (163 J K-1 mol-1). Similar to colchicine, colcemid binding to tubulin is stimulated by certain anions (viz. sulfate and tartrate) but by a different mechanism. Colcemid binding affinity at the lower-affinity site of tubulin is increased in the presence of ammonium sulfate. Interestingly, the lower-affinity site on tubulin for colcemid, even when converted to higher affinity in presence of ammonium sulfate, is not recognized by colchicine. We conclude that tubulin possesses two binding sites, one of which specifically recognized the groups present on the B-ring of colchicine molecule and is effected by the ammonium sulfate, whereas the higher-affinity site, which could accommodate both colchicine and colcemid, possibly recognized the A and C ring of colchicine.

  1. Polyamine sharing between tubulin dimers favours microtubule nucleation and elongation via facilitated diffusion.

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    Alain Mechulam

    2009-01-01

    Full Text Available We suggest for the first time that the action of multivalent cations on microtubule dynamics can result from facilitated diffusion of GTP-tubulin to the microtubule ends. Facilitated diffusion can promote microtubule assembly, because, upon encountering a growing nucleus or the microtubule wall, random GTP-tubulin sliding on their surfaces will increase the probability of association to the target sites (nucleation sites or MT ends. This is an original explanation for understanding the apparent discrepancy between the high rate of microtubule elongation and the low rate of tubulin association at the microtubule ends in the viscous cytoplasm. The mechanism of facilitated diffusion requires an attraction force between two tubulins, which can result from the sharing of multivalent counterions. Natural polyamines (putrescine, spermidine, and spermine are present in all living cells and are potent agents to trigger tubulin self-attraction. By using an analytical model, we analyze the implication of facilitated diffusion mediated by polyamines on nucleation and elongation of microtubules. In vitro experiments using pure tubulin indicate that the promotion of microtubule assembly by polyamines is typical of facilitated diffusion. The results presented here show that polyamines can be of particular importance for the regulation of the microtubule network in vivo and provide the basis for further investigations into the effects of facilitated diffusion on cytoskeleton dynamics.

  2. The impact of beta-elemene on beta-tubulin of human hepatoma hepg2 cells

    Institute of Scientific and Technical Information of China (English)

    Yuqiu Mao; Liying Ban; Jielin Zhang; Li Hou; Xiaonan Cui

    2014-01-01

    Objective:The aim of this study was to investigate the impact of beta-elemene injection on the growth and beta-tubulin of human hepatocarcinoma HepG2 cells. Methods:cellproliferation was assessed by MTT assay. cellcycle distribution was detected by flow cytometry (FCM). The mRNA expression of beta-tubulin was measured by RT-PCR. West-ern blot analysis was used to determine protein expression of beta-tubulin and the polymerization of beta-tubulin. Results:Beta-elemene injection inhibited HepG2 cells proliferation in a dose-and time-dependent manner;FCM analysis indicated beta-elemene injection induced cellcycle arrested at S phase. RT-PCR and western-blot analysis showed that beta-elemene injection down-regulated beta-tubulin expression at both mRNA and protein levels, presenting a dose-dependent manner. Moreover, beta-elemene injection reduced the polymerization of microtubules in a dose-dependent manner. Conclusion:Beta-elemene injection can inhibit the proliferation of hepatoma HepG2 cells, the mechanism might be partly related to the down-regulation of beta-tubulin and inhibition of microtubular polymerization.

  3. Microtubule-associated proteins and tubulin interaction by isothermal titration calorimetry.

    Science.gov (United States)

    Tsvetkov, P O; Barbier, P; Breuzard, G; Peyrot, V; Devred, F

    2013-01-01

    Microtubules play an important role in a number of vital cell processes such as cell division, intracellular transport, and cell architecture. The highly dynamic structure of microtubules is tightly regulated by a number of stabilizing and destabilizing microtubule-associated proteins (MAPs), such as tau and stathmin. Because of their importance, tubulin-MAPs interactions have been extensively studied using various methods that provide researchers with complementary but sometimes contradictory thermodynamic data. Isothermal titration calorimetry (ITC) is the only direct thermodynamic method that enables a full thermodynamic characterization (stoichiometry, enthalpy, entropy of binding, and association constant) of the interaction after a single titration experiment. This method has been recently applied to study tubulin-MAPs interactions in order to bring new insights into molecular mechanisms of tubulin regulation. In this chapter, we review the technical specificity of this method and then focus on the use of ITC in the investigation of tubulin-MAPs binding. We describe technical issues which could arise during planning and carrying out the ITC experiments, in particular with fragile proteins such as tubulin. Using examples of stathmin and tau, we demonstrate how ITC can be used to gain major insights into tubulin-MAP interaction.

  4. Pironetin reacts covalently with cysteine-316 of α-tubulin to destabilize microtubule

    Science.gov (United States)

    Yang, Jianhong; Wang, Yuxi; Wang, Taijing; Jiang, Jian; Botting, Catherine H.; Liu, Huanting; Chen, Qiang; Yang, Jinliang; Naismith, James H.; Zhu, Xiaofeng; Chen, Lijuan

    2016-01-01

    Molecules that alter the normal dynamics of microtubule assembly and disassembly include many anticancer drugs in clinical use. So far all such therapeutics target β-tubulin, and structural biology has explained the basis of their action and permitted design of new drugs. However, by shifting the profile of β-tubulin isoforms, cancer cells become resistant to treatment. Compounds that bind to α-tubulin are less well characterized and unexploited. The natural product pironetin is known to bind to α-tubulin and is a potent inhibitor of microtubule polymerization. Previous reports had identified that pironetin reacts with lysine-352 residue however analogues designed on this model had much lower potency, which was difficult to explain, hindering further development. We report crystallographic and mass spectrometric data that reveal that pironetin forms a covalent bond to cysteine-316 in α-tubulin via a Michael addition reaction. These data provide a basis for the rational design of α-tubulin targeting chemotherapeutics. PMID:27357539

  5. Trypanosoma cruzi tubulin eliminated in the urine of the infected host.

    Science.gov (United States)

    Bertot, G M; Corral, R S; Fresno, M; Rodríguez, C; Katzin, A M; Grinstein, S

    1998-06-01

    In previous studies we have identified and characterized an 80-kDa Trypanosoma cruzi urinary antigen (UAg) eliminated during acute infection. Polyclonal antibodies raised against this antigen revealed by western blotting and immunoprecipitation analyses showed the existence of another antigenic component of 50-55 kDa in the UAg preparation. The antiserum was also used for screening of a T. cruzi expression library. Sequencing of inserts from selected cDNA clones showed high homology with the 3' end of the T.cruzi beta-tubulin gene sequence encoding for the C-terminus of the protein. The presence of T. cruzi tubulin in the UAg was confirmed by immunoprecipitation of a 50-55-kDa protein from 125I-labeled UAg with monoclonal antibodies (MAbs) to human alpha/beta-tubulin. Interestingly, MAbs recognized radiolabeled T. cruzi tubulin eliminated in the urine of infected mice 24 hr postinoculation of [35S]methionine-labeled viable trypomastigotes. Tubulin found in the urine proved to be of T. cruzi origin because this protein could not be identified in urinary specimens from uninfected animals or mice acutely infected with Leishmania infantum or Toxoplasma gondii. We conclude that tubulin is one of the parasite antigens eliminated in the urine of T. cruzi-infected hosts. This finding may be used to develop a noninvasive procedure for early diagnosis of Chagas' disease.

  6. Efficient simulations of tubulin-driven axonal growth.

    Science.gov (United States)

    Diehl, Stefan; Henningsson, Erik; Heyden, Anders

    2016-08-01

    This work concerns efficient and reliable numerical simulations of the dynamic behaviour of a moving-boundary model for tubulin-driven axonal growth. The model is nonlinear and consists of a coupled set of a partial differential equation (PDE) and two ordinary differential equations. The PDE is defined on a computational domain with a moving boundary, which is part of the solution. Numerical simulations based on standard explicit time-stepping methods are too time consuming due to the small time steps required for numerical stability. On the other hand standard implicit schemes are too complex due to the nonlinear equations that needs to be solved in each step. Instead, we propose to use the Peaceman-Rachford splitting scheme combined with temporal and spatial scalings of the model. Simulations based on this scheme have shown to be efficient, accurate, and reliable which makes it possible to evaluate the model, e.g. its dependency on biological and physical model parameters. These evaluations show among other things that the initial axon growth is very fast, that the active transport is the dominant reason over diffusion for the growth velocity, and that the polymerization rate in the growth cone does not affect the final axon length.

  7. Resolving bundled microtubules using anti-tubulin nanobodies.

    Science.gov (United States)

    Mikhaylova, Marina; Cloin, Bas M C; Finan, Kieran; van den Berg, Robert; Teeuw, Jalmar; Kijanka, Marta M; Sokolowski, Mikolaj; Katrukha, Eugene A; Maidorn, Manuel; Opazo, Felipe; Moutel, Sandrine; Vantard, Marylin; Perez, Frank; van Bergen en Henegouwen, Paul M P; Hoogenraad, Casper C; Ewers, Helge; Kapitein, Lukas C

    2015-08-11

    Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20-40 nm and will thereby blend neighbouring microtubules into one structure. Here we develop single-chain antibody fragments (nanobodies) against tubulin to achieve super-resolution imaging of microtubules with a decreased apparent diameter. To test the resolving power of these novel probes, we generate microtubule bundles with a known spacing of 50-70 nm and successfully resolve individual microtubules. Individual bundled microtubules can also be resolved in different mammalian cells, including hippocampal neurons, allowing novel insights into fundamental mechanisms of microtubule organization in cell- and neurobiology.

  8. Tubulin evolution in insects: gene duplication and subfunctionalization provide specialized isoforms in a functionally constrained gene family

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    Gadagkar Sudhindra R

    2010-04-01

    Full Text Available Abstract Background The completion of 19 insect genome sequencing projects spanning six insect orders provides the opportunity to investigate the evolution of important gene families, here tubulins. Tubulins are a family of eukaryotic structural genes that form microtubules, fundamental components of the cytoskeleton that mediate cell division, shape, motility, and intracellular trafficking. Previous in vivo studies in Drosophila find a stringent relationship between tubulin structure and function; small, biochemically similar changes in the major alpha 1 or testis-specific beta 2 tubulin protein render each unable to generate a motile spermtail axoneme. This has evolutionary implications, not a single non-synonymous substitution is found in beta 2 among 17 species of Drosophila and Hirtodrosophila flies spanning 60 Myr of evolution. This raises an important question, How do tubulins evolve while maintaining their function? To answer, we use molecular evolutionary analyses to characterize the evolution of insect tubulins. Results Sixty-six alpha tubulins and eighty-six beta tubulin gene copies were retrieved and subjected to molecular evolutionary analyses. Four ancient clades of alpha and beta tubulins are found in insects, a major isoform clade (alpha 1, beta 1 and three minor, tissue-specific clades (alpha 2-4, beta 2-4. Based on a Homarus americanus (lobster outgroup, these were generated through gene duplication events on major beta and alpha tubulin ancestors, followed by subfunctionalization in expression domain. Strong purifying selection acts on all tubulins, yet maximum pairwise amino acid distances between tubulin paralogs are large (0.464 substitutions/site beta tubulins, 0.707 alpha tubulins. Conversely orthologs, with the exception of reproductive tissue isoforms, show little sequence variation except in the last 15 carboxy terminus tail (CTT residues, which serve as sites for post-translational modifications (PTMs and interactions

  9. Tubulin binding, protein-bound conformation in solution, and antimitotic cellular profiling of noscapine and its derivatives.

    Science.gov (United States)

    Bennani, Youssef L; Gu, Wenxin; Canales, Angeles; Díaz, Fernando J; Eustace, Brenda K; Hoover, Russell R; Jiménez-Barbero, Jesus; Nezami, Azin; Wang, Tiansheng

    2012-03-08

    Noscapine and its 7-hydroxy and 7-amino derivatives were characterized for their binding to tubulin. A solution NMR structure of these compounds bound to tubulin shows that noscapine and its 7-aniline derivative do not compete for the same binding site nor does its small molecule crystal structure match its tubulin-bound conformation. These compounds were also tested for their antiproliferative effects on a panel hepatocellular carcinoma cell lines.

  10. Tubulin binds to the cytoplasmic loop of TRESK background K⁺ channel in vitro.

    Directory of Open Access Journals (Sweden)

    Péter Enyedi

    Full Text Available The cytoplasmic loop between the second and third transmembrane segments is pivotal in the regulation of TRESK (TWIK-related spinal cord K+ channel, K2P18.1, KCNK18. Calcineurin binds to this region and activates the channel by dephosphorylation in response to the calcium signal. Phosphorylation-dependent anchorage of 14-3-3 adaptor protein also modulates TRESK at this location. In the present study, we identified molecular interacting partners of the intracellular loop. By an affinity chromatography approach using the cytoplasmic loop as bait, we have verified the specific association of calcineurin and 14-3-3 to the channel. In addition to these known interacting proteins, we observed substantial binding of tubulin to the intracellular loop. Successive truncation of the polypeptide and pull-down experiments from mouse brain cytosol narrowed down the region sufficient for the binding of tubulin to a 16 amino acid sequence: LVLGRLSYSIISNLDE. The first six residues of this sequence are similar to the previously reported tubulin-binding region of P2X2 purinergic receptor. The tubulin-binding site of TRESK is located close to the protein kinase A (PKA-dependent 14-3-3-docking motif of the channel. We provide experimental evidence suggesting that 14-3-3 competes with tubulin for the binding to the cytoplasmic loop of TRESK. It is intriguing that the 16 amino acid tubulin-binding sequence includes the serines, which were previously shown to be phosphorylated by microtubule-affinity regulating kinases (MARK kinases and contribute to channel inhibition. Although tubulin binds to TRESK in vitro, it remains to be established whether the two proteins also interact in the living cell.

  11. Evolutionary relationships among "jakobid" flagellates as indicated by alpha- and beta-tubulin phylogenies.

    Science.gov (United States)

    Edgcomb, V P; Roger, A J; Simpson, A G; Kysela, D T; Sogin, M L

    2001-04-01

    Jakobids are free-living, heterotrophic flagellates that might represent early-diverging mitochondrial protists. They share ultrastructural similarities with eukaryotes that occupy basal positions in molecular phylogenies, and their mitochondrial genome architecture is eubacterial-like, suggesting a close affinity with the ancestral alpha-proteobacterial symbiont that gave rise to mitochondria and hydrogenosomes. To elucidate relationships among jakobids and other early-diverging eukaryotic lineages, we characterized alpha- and beta-tubulin genes from four jakobids: Jakoba libera, Jakoba incarcerata, Reclinomonas americana (the "core jakobids"), and Malawimonas jakobiformis. These are the first reports of nuclear genes from these organisms. Phylogenies based on alpha-, beta-, and combined alpha- plus beta-tubulin protein data sets do not support the monophyly of the jakobids. While beta-tubulin and combined alpha- plus beta-tubulin phylogenies showed a sister group relationship between J. libera and R. americana, the two other jakobids, M. jakobiformis and J. incarcerata, had unclear affinities. In all three analyses, J. libera, R. americana, and M. jakobiformis emerged from within a well-supported large "plant-protist" clade that included plants, green algae, cryptophytes, stramenopiles, alveolates, Euglenozoa, Heterolobosea, and several other protist groups, but not animals, fungi, microsporidia, parabasalids, or diplomonads. A preferred branching order within the plant-protist clade was not identified, but there was a tendency for the J. libera-R. americana lineage to group with a clade made up of the heteroloboseid amoeboflagellates and euglenozoan protists. Jakoba incarcerata branched within the plant-protist clade in the beta- and the combined alpha- plus beta-tubulin phylogenies. In alpha- tubulin trees, J. incarcerata occupied an unresolved position, weakly grouping with the animal/fungal/microsporidian group or with amitochondriate parabasalid and

  12. Fodrin in centrosomes: implication of a role of fodrin in the transport of gamma-tubulin complex in brain.

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    Sasidharan Shashikala

    Full Text Available Gamma-tubulin is the major protein involved in the nucleation of microtubules from centrosomes in eukaryotic cells. It is present in both cytoplasm and centrosome. However, before centrosome maturation prior to mitosis, gamma-tubulin concentration increases dramatically in the centrosome, the mechanism of which is not known. Earlier it was reported that cytoplasmic gamma-tubulin complex isolated from goat brain contains non-erythroid spectrin/fodrin. The major role of erythroid spectrin is to help in the membrane organisation and integrity. However, fodrin or non-erythroid spectrin has a distinct pattern of localisation in brain cells and evidently some special functions over its erythroid counterpart. In this study, we show that fodrin and γ-tubulin are present together in both the cytoplasm and centrosomes in all brain cells except differentiated neurons and astrocytes. Immunoprecipitation studies in purified centrosomes from brain tissue and brain cell lines confirm that fodrin and γ-tubulin interact with each other in centrosomes. Fodrin dissociates from centrosome just after the onset of mitosis, when the concentration of γ-tubulin attains a maximum at centrosomes. Further it is observed that the interaction between fodrin and γ-tubulin in the centrosome is dependent on actin as depolymerisation of microfilaments stops fodrin localization. Image analysis revealed that γ-tubulin concentration also decreased drastically in the centrosome under this condition. This indicates towards a role of fodrin as a regulatory transporter of γ-tubulin to the centrosomes for normal progression of mitosis.

  13. [Regulation pattern of the FRUITFULL (FUL) gene of Arabidopsis thaliana].

    Science.gov (United States)

    Chu, Tingting; Xie, Hua; Xu, Yong; Ma, Rongcai

    2010-11-01

    FRUITFULL (FUL) is an MADS box gene that functions early in controlling flowering time, meristem identity and cauline leaf morphology and later in carpel and fruit development in Arabidopsis thaliana. In order to clarify the regulation of FUL expression the upstream regulatory region, -2148 bp - +96 bp and the first intron of the FUL gene were cloned, and vectors with a series of deletion of FUL promoter, and the ones fused with the first intron were constructed. Vectors harboring the fusion of cis-acting elements with the constitutive promoters of TUBULIN and ACTIN were also constructed. Beta-Glucuronidase activity assays of the transgenic Arabidopsis plants showed that two cis-elements were involved in the repression of FUL expression, with one of the two being probably the binding site of the transcriptional factor AP1. And the two CArG boxes played a important role in FUL initiation particularly. Furthermore, the first intron of FUL was shown to participate in the development of carpel and stamen as an enhancer.

  14. The ADNP derived peptide, NAP modulates the tubulin pool: implication for neurotrophic and neuroprotective activities.

    Directory of Open Access Journals (Sweden)

    Saar Oz

    Full Text Available Microtubules (MTs, key cytoskeletal elements in living cells, are critical for axonal transport, synaptic transmission, and maintenance of neuronal morphology. NAP (NAPVSIPQ is a neuroprotective peptide derived from the essential activity-dependent neuroprotective protein (ADNP. In Alzheimer's disease models, NAP protects against tauopathy and cognitive decline. Here, we show that NAP treatment significantly affected the alpha tubulin tyrosination cycle in the neuronal differentiation model, rat pheochromocytoma (PC12 and in rat cortical astrocytes. The effect on tubulin tyrosination/detyrosination was coupled to increased MT network area (measured in PC12 cells, which is directly related to neurite outgrowth. Tubulin beta3, a marker for neurite outgrowth/neuronal differentiation significantly increased after NAP treatment. In rat cortical neurons, NAP doubled the area of dynamic MT invasion (Tyr-tubulin into the neuronal growth cone periphery. NAP was previously shown to protect against zinc-induced MT/neurite destruction and neuronal death, here, in PC12 cells, NAP treatment reversed zinc-decreased tau-tubulin-MT interaction and protected against death. NAP effects on the MT pool, coupled with increased tau engagement on compromised MTs imply an important role in neuronal plasticity, protecting against free tau accumulation leading to tauopathy. With tauopathy representing a major pathological hallmark in Alzheimer's disease and related disorders, the current findings provide a mechanistic basis for further development. NAP (davunetide is in phase 2/3 clinical trial in progressive supranuclear palsy, a disease presenting MT deficiency and tau pathology.

  15. Regulation of tubulin expression by micro-RNAs: implications for drug resistance.

    Science.gov (United States)

    Lobert, Sharon; Graichen, Mary E

    2013-01-01

    In this chapter, we provide an overview of methods for studying micro-RNA regulation of tubulin isotypes. In clinical studies, β-tubulin isotypes were found to be biomarkers for tumor formation. In addition, because changes in the levels of specific β-tubulin isotypes alter the stability of microtubules in mitotic spindles in vitro, it has been hypothesized that changes in microtubule protein levels could contribute to chemotherapy resistance. Over the past 15 years, micro-RNAs have been shown to target mRNAs in signaling pathways involved in tumor suppression, as well as tumorigenesis. Investigating micro-RNA regulation of tubulin isotypes will shed light on the mechanisms underlying the processes that implicate tubulin isotypes as biomarkers for aggressive tumors or chemotherapy resistance. The methods discussed in this chapter include the use of micro-RNA superarrays, next-generation sequencing, real-time PCR experiments, upregulation of micro-RNAs, and immunoprecipitation of RNA-induced silencing complex. We will show examples of data collected using these methods and how these data contribute to understanding paclitaxel resistance.

  16. Roles for two partially redundant alpha-tubulins during mitosis in early Caenorhabditis elegans embryos.

    Science.gov (United States)

    Phillips, Jennifer B; Lyczak, Rebecca; Ellis, Gregory C; Bowerman, Bruce

    2004-06-01

    The Caenorhabditis elegans genome encodes multiple isotypes of alpha-tubulin and beta-tubulin. Roles for a number of these tubulins in neuronal development have been described, but less is known about the isoforms that function during early embryonic development. Microtubules are required for multiple events after fertilization produces a one-cell zygote in C. elegans, including pronuclear migration, mitotic spindle assembly and function, and proper spindle positioning. Here we describe a conditional and dominant mis-sense mutation in the C. elegans alpha-tubulin gene tba-1 that disrupts pronuclear migration and positioning of the first mitotic spindle, and results in a highly penetrant embryonic lethality, at the restrictive temperature of 26 degrees C. Our analysis of the dominant tba-1 (or346ts) allele suggests that TBA-1 assembles into microtubules in early embryonic cells. However, we also show that reduction of tba-1 function using RNA interference results in defects much less severe than those caused by the dominant or346ts mutation, due to partial redundancy of TBA-1 and another alpha-tubulin called TBA-2. Reducing the function of both TBA-1 and TBA-2 results in severe defects in microtubule-dependent processes. We conclude that microtubules in the early C. elegans embryo are composed of both TBA-1 and TBA-2, and that the dominant tba-1(or346ts) mutation disrupts MT assembly or stability. Cell Motil.

  17. Interaction of benzimidazole anthelmintics with Haemonchus contortus tubulin: binding affinity and anthelmintic efficacy.

    Science.gov (United States)

    Lubega, G W; Prichard, R K

    1991-08-01

    The ability of various benzimidazoles (BZs) to bind tubulin under different conditions was assessed by determining their IC50 values (the concentration of unlabeled drug required to inhibit 50% of the labeled drug binding), Ka (the apparent equilibrium association constant) and Bmax (the maximum binding at infinite [BZ] = [drug-receptor]). The ability of unlabeled benzimidazoles--fenbendazole, mebendazole (MBZ), oxibendazole (OBZ), albendazole (ABZ), rycobendazole (albendazole sulfoxide, ABZSO), albendazole sulfone, oxfendazole (OFZ), and thiabendazole--to bind tubulin was determined from their ability to inhibit the binding of [3H]MBZ or [3H]OBZ to tubulin in supernatants derived from unembryonated eggs or adult worms of Haemonchus contortus. The binding constants (IC50, Ka, and Bmax) correlated with the known anthelmintic potency (recommended therapeutic doses) of the BZ compounds except for OFZ and ABZSO whose Ka values were lower than could be expected from anthelmintic potency. The binding of [3H]ABZ or [3H]OFZ to tubulin in supernatants derived from BZ-susceptible and BZ-resistant H. contortus was compared. [3H]ABZ demonstrated saturable high-affinity binding but [3H]OFZ bound with low affinity. The high-affinity binding of [3H]ABZ was reduced for the R strain. Tubulin bound BZ drugs at 4 degrees C with lower apparent Ka than at 37 degrees C.

  18. Novel Suicide Ligands of Tubulin Arrest Cancer Cells in S-Phase

    Directory of Open Access Journals (Sweden)

    Ashley Davis

    1999-12-01

    Full Text Available It is presently accepted that the mechanism of action for all anti-tumor tubulin ligands involves the perturbation of microtubule dynamics during the G2/M phase of cell division and subsequent entry into apoptosis 1]. In this report, we challenge the established dogma by describing a unique mechanism of action caused by a novel series of tubulin ligands, halogenated derivatives of acetamido benzoyl ethyl ester. We have developed a suicide ligand for tubulin, which covalently attaches to the target and shows potent cancericidal activity in tissue culture assays and in animal tumor models. These compounds target early S-phase at the G1/S transition rather than the G2/M phase and mitotic arrest. Bcl-2 phosphorylation, a marker of mitotic microtubule inhibition by other tubulin ligands was dramatically altered, phosphorylation was rapid and biphasic rather than a slow linear event. The halogenated ethyl ester series of derivatives thus constitute a unique set of tubulin ligands which induce a novel mechanism of apoptosis.

  19. Molecular characterization of beta-tubulin from Phakopsora pachyrhizi, the causal agent of Asian soybean rust

    Directory of Open Access Journals (Sweden)

    Talles Eduardo Ferreira Maciel

    2010-01-01

    Full Text Available β-tubulins are structural components of microtubules and the targets of benzimidazole fungicides used to control many diseases of agricultural importance. Intron polymorphisms in the intron-rich genes of these proteins have been used in phylogeographic investigations of phytopathogenic fungi. In this work, we sequenced 2764 nucleotides of the β-tubulin gene (Pp tubB in samples of Phakopsora pachyrhizi collected from seven soybean fields in Brazil. Pp tubB contained an open reading frame of 1341 nucleotides, including nine exons and eight introns. Exon length varied from 14 to 880 nucleotides, whereas intron length varied from 76 to 102 nucleotides. The presence of only four polymorphic sites limited the usefulness of Pp tubB for phylogeographic studies in P. pachyrhizi. The gene structures of Pp tubB and orthologous β-tubulin genes of Melampsora lini and Uromyces viciae-fabae were highly conserved. The amino acid substitutions in β-tubulin proteins associated with the onset of benzimidazole resistance in model organisms, especially at His6, Glu198 and Phe200, were absent from the predicted sequence of the P. pachyrhizi β-tubulin protein.

  20. Binding of dihydroxynaphthyl aryl ketones to tubulin colchicine site inhibits microtubule assembly.

    Science.gov (United States)

    Gutierrez, Eunices; Benites, Julio; Valderrama, Jaime A; Calderon, Pedro Buc; Verrax, Julien; Nova, Esteban; Villanelo, Felipe; Maturana, Daniel; Escobar, Cristian; Lagos, Rosalba; Monasterio, Octavio

    2015-10-23

    Dihydroxynaphthyl aryl ketones 1-5 have been evaluated for their abilities to inhibit microtubule assembly and the binding to tubulin. Compounds 3, 4 and 5 displayed competitive inhibition against colchicine binding, and docking analysis showed that they bind to the tubulin colchicine-binding pocket inducing sheets instead of microtubules. Remarkable differences in biological activity observed among the assayed compounds seem to be related to the structure and position of the aryl substituent bonded to the carbonyl group. Compounds 2, 3 and 4, which contain a heterocyclic ring, presented higher affinity for tubulin compared to the carbocyclic analogue 5. Compound 4 showed the best affinity of the series, with an IC50 value of 2.1 μM for microtubule polymerization inhibition and a tubulin dissociation constant of 1.0 ± 0.2 μM, as determined by thermophoresis. Compound 4 was more efficacious in disrupting microtubule assembly in vitro than compound 5 although it contains the trimethoxyphenyl ring present in colchicine. Hydrogen bonds with Asn101 of α-tubulin seem to be responsible for the higher affinity of compound 4 respects to the others.

  1. In vitro study on the alterations of brain tubulin structure and assembly affected by magnetite nanoparticles.

    Science.gov (United States)

    Dadras, Ali; Riazi, Gholam Hossein; Afrasiabi, Ali; Naghshineh, Ali; Ghalandari, Behafarid; Mokhtari, Farzad

    2013-03-01

    In recent decades, considerable efforts have been made to understand the mechanism of memory, cognition, and relevant neurodegenerative diseases in the human brain. Several studies have shown the importance of microtubule proteins in the memory mechanism and memory dysfunction. Microtubules possess dynamicity, which is essential for functions of neuronal networks. Microtubule-associated proteins, i.e., tau, play vital roles in microtubule stability. On the other hand, the ferromagnetic mineral magnetite (Fe(3)O(4)) has been detected in the normal human brain, and elevated levels of magnetite are also observed in the brains of Alzheimer's disease patients. Therefore, we propose that a relationship between microtubule organization in axons and brain magnetite nanoparticles is possible. In this study we found alterations of microtubule polymerization in the presence of increasing concentrations of magnetite through transmission electron microscopy images and a turbidimetry method. Structural changes of microtubule and tau protein, as an essential microtubule-associated protein for tubulin assembly, were detected via circular dichroism spectroscopy, intrinsic fluorescence, and 8-anilino-1-naphthalenesulfonic acid fluorometry. We predicted three possible binding sites on tau protein and one possible binding site on tubulin dimer for magnetite nanoparticles. Magnetite also causes the morphology of PC12 cells to change abnormally and cell viability to decrease. Finally, we suggest that magnetite changes microtubule dynamics and polymerization through two paths: (1) changing the secondary and tertiary structure of tubulin and (2) binding to either tubulin dimer or tau protein and preventing tau-tubulin interaction.

  2. Significance of β-tubulin Expression in Breast Premalignant Lesions and Carcinomas

    Institute of Scientific and Technical Information of China (English)

    Yuxia Gao; Yun Niu; Xiumin Ding; Yong Yu

    2008-01-01

    OBJECTIVE To explore the expression of β-tubulin in premalignant lesions and carcinomas of the breast, and to observe the relationship of its expression with breast cancer pathological features.METHODS The expression of β-tubulin was detected immunohistochemically in 50 specimens of premalignant lesions of the breast (ADH and Peri-PM with ADH), 50 specimens of breast in situ ductal carcinomas (DCIS), and 50 specimens of invasive ductal carcinomas (IDC). Thirty specimens of normal breast tissues served as a control group.RESULTS Immunohistochemical analysis showed that: the differences among the 4 groups (normal breast tissues, breast premalignant lesions, DCIS and IDC, P < 0.05) were significant,and there were also statistically significant differences between any 2 groups (P < 0.05) except for the β-tubulin positive expression comparing DCIS versus IDC (P > 0.05). In addition, β-tubulin was expressed at a higher level in Peri-PM with ADH compared to ADH (P < 0.05). Following the degree of breast epithelial hyperplasia involved, and its development into carcinoma, the β-tubulin positive expression displayed an elevating tendency.We also found a significant positive relationship of β-tubulin expression with lymph node metastasis (P < 0.05), but no significant correlation with histological grading and nuclear grade.CONCLUSION Centrosome defects may be an early event in the development of breast cancer and they can also promote tumor progression. Studies of aberrations of centrosomal proteins provide a new way to explore the mechanism of breast tumorigenesis.

  3. Reference: 517 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available d isolated aleurone layers of Arabidopsis (Arabidopsis thaliana) were used in experiments designed to iden...tify components of the Arabidopsis seed that contribute to seed dormancy and to lea

  4. Tubulin binding cofactor C (TBCC suppresses tumor growth and enhances chemosensitivity in human breast cancer cells

    Directory of Open Access Journals (Sweden)

    Laurier Jean-Fabien

    2010-04-01

    Full Text Available Abstract Background Microtubules are considered major therapeutic targets in patients with breast cancer. In spite of their essential role in biological functions including cell motility, cell division and intracellular transport, microtubules have not yet been considered as critical actors influencing tumor cell aggressivity. To evaluate the impact of microtubule mass and dynamics on the phenotype and sensitivity of breast cancer cells, we have targeted tubulin binding cofactor C (TBCC, a crucial protein for the proper folding of α and β tubulins into polymerization-competent tubulin heterodimers. Methods We developed variants of human breast cancer cells with increased content of TBCC. Analysis of proliferation, cell cycle distribution and mitotic durations were assayed to investigate the influence of TBCC on the cell phenotype. In vivo growth of tumors was monitored in mice xenografted with breast cancer cells. The microtubule dynamics and the different fractions of tubulins were studied by time-lapse microscopy and lysate fractionation, respectively. In vitro sensitivity to antimicrotubule agents was studied by flow cytometry. In vivo chemosensitivity was assayed by treatment of mice implanted with tumor cells. Results TBCC overexpression influenced tubulin fraction distribution, with higher content of nonpolymerizable tubulins and lower content of polymerizable dimers and microtubules. Microtubule dynamicity was reduced in cells overexpressing TBCC. Cell cycle distribution was altered in cells containing larger amounts of TBCC with higher percentage of cells in G2-M phase and lower percentage in S-phase, along with slower passage into mitosis. While increased content of TBCC had little effect on cell proliferation in vitro, we observed a significant delay in tumor growth with respect to controls when TBCC overexpressing cells were implanted as xenografts in vivo. TBCC overexpressing variants displayed enhanced sensitivity to

  5. Probing FtsZ and tubulin with C8-substituted GTP analogs reveals differences in their nucleotide binding sites

    NARCIS (Netherlands)

    Läppchen, T.; Pinas, V.A.; Hartog, A.F.; Koomen, G.J.; Schaffner-Barbero, C.; Andreu, J.M.; Trambaiolo, D.; Löwe, J.; Juhem, A.; Popov, A.V.; den Blaauwen, T.

    2008-01-01

    The cytoskeletal proteins, FtsZ and tubulin, play a pivotal role in prokaryotic cell division and eukaryotic chromosome segregation, respectively. Selective inhibitors of the GTP-dependent polymerization of FtsZ could constitute a new class of antibiotics, while several inhibitors of tubulin are wid

  6. Effect of visual experience on tubulin synthesis during a critical period of visual cortex development in the hooded rat.

    Science.gov (United States)

    Cronly-Dillon, J; Perry, G W

    1979-08-01

    1. In some species, restriction of visual experience in early life may affect normal functional development of visual cortical cells. The purpose of the present study was to determine if visual deprivation during post-natal development in the hooded rat also affects the production in brain cells of certain molecular components such as tubulin, that are needed for growth and maintenance of synapses and neurites. 2. Norwegian black hooded rats were reared under a variety of conditions of visual deprivation. At various stages of development the animals were killed and the rate of synthesis of tubulin in visual and motor cortex determined. Tritiated colchicine was used to assay tubulin and L-[14C]leucine injected into the brain ventricles 2 hr before death was used to measure rate of tubulin synthesis. 3. In rats reared in normal light there is a marked elevation in visual cortex tubulin synthesis that spans the period from eye-opening (13 days) until approximately 35 days. This elevation in tubulin synthesis is absent in animals reared in darkness from birth or deprived of pattern vision by eyelid suture. Also the effect of visual deprivation on tubulin synthesis was specifically confined to visual cortex and was not found for the motor cortex. Similarly, the incorporation of L-[14C]leucine into total protein in visual cortex was unaffected by dark rearing. Hence the stimulation of tubulin synthesis by visual experience in rat visual cortex is not attributable to a general non-specific stimulation of protein synthesis. 4. Rats that were dark-reared from birth and then exposed to a lighted environment for 24 hr during a certain critical period that extends from eye-opening (13 days) until approximately 35 days, displayed a significant increase in visual cortex tubulin rats that were brought into the light later than 35 days showed no significant increase in tubulin synthesis when compared with their continuously dark-rearer controls. 5. It is suggested that the number

  7. (-)-Rhazinilam and the diphenylpyridazinone NSC 613241: Two compounds inducing the formation of morphologically similar tubulin spirals but binding apparently to two distinct sites on tubulin.

    Science.gov (United States)

    Bai, Ruoli; Hamel, Ernest

    2016-08-15

    The most potent microtubule assembly inhibitor of newer diphenylpyridazinone derivatives examined was NSC 613241. Because NSC 613241 and (-)-rhazinilam also induce the formation of similar 2-filament spirals, these aberrant reactions were compared. Spiral formation with both compounds was enhanced by GTP and inhibited by GDP and by 15 other inhibitors of microtubule assembly. Similarly, microtubule assembly induced by paclitaxel or laulimalide is enhanced by GTP and inhibited by GDP and assembly inhibitors, but neither [(3)H]NSC 613241 nor [(3)H](-)-rhazinilam bound to microtubules or inhibited the binding of [(3)H]paclitaxel or [(3)H]peloruside A to microtubules. Differences in the pitch of aberrant polymers were found: NSC 613241-induced and (-)-rhazinilam-induced spirals had average repeats of 85 and 79-80 nm, respectively. We found no binding of [(3)H]NSC 613241 or [(3)H](-)-rhazinilam to αβ-tubulin dimer, but both compounds were incorporated into the polymers they induced in substoichiometric reactions, with as little as 0.1-0.2 mol compound/mol of tubulin, and no cross-inhibition by NSC 613241 or (-)-rhazinilam into spirals occurred. Under reaction conditions where neither compound induced spiral formation, both compounds together synergistically induced substantial spiral formation. We conclude that (-)-rhazinilam and NSC 613241 bind to different sites on tubulin that differ from binding sites for other antitubulin agents.

  8. Oblongifolin C inhibits metastasis by up-regulating keratin 18 and tubulins.

    Science.gov (United States)

    Wang, Xiaoyu; Lao, Yuanzhi; Xu, Naihan; Xi, Zhichao; Wu, Man; Wang, Hua; Li, Xiyi; Tan, Hongsheng; Sun, Menghong; Xu, Hongxi

    2015-05-14

    Tumor metastasis is the main cause of cancer-related patient death. In this study, we performed a wound healing migration screen to search for a metastatic inhibitor within our library of natural compounds. We found that oblongifolin C (OC), a natural compound extracted from Garcinia yunnanensis Hu, is an effective inhibitor of metastasis in human esophageal squamous carcinoma Eca109 cells. The transwell migration and matrigel invasion assay results also showed that OC inhibits the migration of Eca109 cells and HepG2 cells. OC can increase the expression of tubulin, indicating that OC inhibits metastasis via tubulin aggregation. In addition, the Western blotting, real-time PCR, and immunostaining results indicated that OC increases the expression of keratin18. Furthermore, the knockdown of keratin 18 by small interfering RNAs inhibited the expression of tubulin and increased the metastasis of cancer cells, suggesting that keratin 18 is the upstream signal of tubulin and plays a vital role in metastasis. A subsequent study in a tail vein injection metastasis model showed that OC can significantly inhibit pulmonary metastasis, as revealed by immunohistochemistry staining. Taken together, our results suggest that OC inhibits metastasis through the induction of the expression of keratin 18 and may be useful in cancer therapy.

  9. Stereoselective synthesis of 3,3-diarylacrylonitriles as tubulin polymerization inhibitors.

    Science.gov (United States)

    Fang, Zhenglai; Song, Yunlong; Sarkar, Taradas; Hamel, Ernest; Fogler, William E; Agoston, Gregory E; Fanwick, Phillip E; Cushman, Mark

    2008-06-06

    A series of 3,3-diarylacrylonitriles were synthesized stereoselectively as tubulin polymerization inhibitors for potential use in cancer chemotherapy. This synthetic route features stannylcupration and palladium-catalyzed Stille cross-coupling chemistry, allowing both E and Z isomers of 3,3-diarylacrylonitriles to be prepared in a very short sequence of reactions.

  10. Imidazoles and benzimidazoles as tubulin-modulators for anti-cancer therapy.

    Science.gov (United States)

    Torres, Fernando C; García-Rubiño, M Eugenia; Lozano-López, César; Kawano, Daniel F; Eifler-Lima, Vera L; von Poser, Gilsane L; Campos, Joaquín M

    2015-01-01

    Imidazoles and benzimidazoles are privileged heterocyclic bioactive compounds used with success in the clinical practice of innumerous diseases. Although there are many advancements in cancer therapy, microtubules remain as one of the few macromolecular targets validated for planning active anti-cancer compounds, and the design of drugs that modulate microtubule dynamics in unknown sites of tubulin is one of the goals of the medicinal chemistry. The discussion of the role of new and commercially available imidazole and benzimidazole derivatives as tubulin modulators is scattered throughout scientific literature, and indicates that these compounds have a tubulin modulation mechanism different from that of tubulin modulators clinically available, such as paclitaxel, docetaxel, vincristine and vinblastine. In fact, recent literature indicates that these derivatives inhibit microtubule formation binding to the colchicine site, present good pharmacokinetic properties and are capable of overcoming multidrug resistance in many cell lines. The understanding of the mechanisms involved in the imidazoles/benzimidazoles modulation of microtubule dynamics is very important to develop new strategies to overcome the resistance to anti-cancer drugs and to discover new biomarkers and targets for cancer chemotherapy.

  11. Tubulin assembly, taxoid site binding, and cellular effects of the microtubule-stabilizing agent dictyostatin.

    Science.gov (United States)

    Madiraju, Charitha; Edler, Michael C; Hamel, Ernest; Raccor, Brianne S; Balachandran, Raghavan; Zhu, Guangyu; Giuliano, Kenneth A; Vogt, Andreas; Shin, Youseung; Fournier, Jean-Hugues; Fukui, Yoshikazu; Brückner, Arndt M; Curran, Dennis P; Day, Billy W

    2005-11-15

    (-)-Dictyostatin is a sponge-derived, 22-member macrolactone natural product shown to cause cells to accumulate in the G2/M phase of the cell cycle, with changes in intracellular microtubules analogous to those observed with paclitaxel treatment. Dictyostatin also induces assembly of purified tubulin more rapidly than does paclitaxel, and nearly as vigorously as does dictyostatin's close structural congener, (+)-discodermolide (Isbrucker et al. (2003), Biochem. Pharmacol. 65, 75-82). We used synthetic (-)-dictyostatin to study its biochemical and cytological activities in greater detail. The antiproliferative activity of dictyostatin did not differ greatly from that of paclitaxel or discodermolide. Like discodermolide, dictyostatin retained antiproliferative activity against human ovarian carcinoma cells resistant to paclitaxel due to beta-tubulin mutations and caused conversion of cellular soluble tubulin pools to microtubules. Detailed comparison of the abilities of dictyostatin and discodermolide to induce tubulin assembly demonstrated that the compounds had similar potencies. Dictyostatin inhibited the binding of radiolabeled discodermolide to microtubules more potently than any other compound examined, and dictyostatin and discodermolide had equivalent activity as inhibitors of the binding of both radiolabeled epothilone B and paclitaxel to microtubules. These results are consistent with the idea that the macrocyclic structure of dictyostatin represents the template for the bioactive conformation of discodermolide.

  12. HDAC6 regulates mutant SOD1 aggregation through two SMIR motifs and tubulin acetylation.

    Science.gov (United States)

    Gal, Jozsef; Chen, Jing; Barnett, Kelly R; Yang, Liuqing; Brumley, Erin; Zhu, Haining

    2013-05-24

    Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS.

  13. HDAC6 Regulates Mutant SOD1 Aggregation through Two SMIR Motifs and Tubulin Acetylation*

    Science.gov (United States)

    Gal, Jozsef; Chen, Jing; Barnett, Kelly R.; Yang, Liuqing; Brumley, Erin; Zhu, Haining

    2013-01-01

    Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS. PMID:23580651

  14. Dietary flavonoid fisetin binds to β-tubulin and disrupts microtubule dynamics in prostate cancer cells

    OpenAIRE

    Mukhtar, Eiman; Adhami, Vaqar Mustafa; Sechi, Mario; Mukhtar, Hasan

    2015-01-01

    Microtubule targeting based therapies have revolutionized cancer treatment; however, resistance and side effects remain a major limitation. Therefore, novel strategies that can overcome these limitations are urgently needed. We made a novel discovery that fisetin, a hydroxyflavone, is a microtubule stabilizing agent. Fisetin binds to tubulin and stabilizes microtubules with binding characteristics far superior than paclitaxel. Surface plasmon resonance and computational docking studies sugges...

  15. Tubulin dipole moment, dielectric constant and quantum behavior: computer simulations, experimental results and suggestions

    CERN Document Server

    Mershin, A; Schüssler, H A; Nanopoulos, Dimitri V; Mershin, Andreas; Kolomenski, Alexandre A.; Schuessler, Hans A.; Nanopoulos, Dimitri V.

    2004-01-01

    We used computer simulation to calculate the electric dipole moments of the alpha and beta tubulin monomers and dimer and found those to be |palpha|=552D, |pbeta|=1193D and |palpha-beta|=1740D respectively. Independent surface plasmon resonance (SPR) and refractometry measurements of the high-frequency dielectric constant and polarizability strongly corroborated our previous SPR-derived results giving delta-n/delta-c ~1.800x10^-3 ml/mg. The refractive index of tubulin was measured to be n_tub ~2.90 and the high frequency tubulin dielectric constant kappa_tub ~8.41 while the high-frequency polarizability was found to be alpha_tub ~ 2.1x10^-33 C m^2/V. Methods for the experimental determination of the low-frequency p are explored as well as ways to test the often conjectured quantum coherence and entanglement properties of tubulin. Biobits, bioqubits and other applications to bioelectronics are discussed.

  16. Defective tubulin organization and proplatelet formation in murine megakaryocytes lacking Rac1 and Cdc42

    DEFF Research Database (Denmark)

    Pleines, Irina; Dütting, Sebastian; Cherpokova, Deya;

    2013-01-01

    normally in vivo but displayed highly abnormal morphology and uncontrolled fragmentation. Consistently, a lack of Rac1/Cdc42 virtually abrogated proplatelet formation in vitro. Strikingly, this phenotype was associated with severely defective tubulin organization, whereas actin assembly and structure were...

  17. Orally Active and Selective Tubulin Inhibitors as Anti-Trypanosome Agents.

    Directory of Open Access Journals (Sweden)

    Vishal Nanavaty

    Full Text Available There is an urgent need to develop a safe, effective, orally active, and inexpensive therapy for African trypanosomiasis due to the drawbacks of current drugs. Selective tubulin inhibitors have the potential to be promising drug candidates for the treatment of this disease, which is based on the tubulin protein structural difference between mammalian and trypanosome cells. We propose to identify novel tubulin inhibitors from a compound library developed based on the lead compounds that selectively target trypanosomiasis.We used Trypanosoma brucei brucei as the parasite model, and human normal kidney cells and mouse microphage cells as the host model. Growth rates of both trypanosomes and mammalian cells were determined as a means to screen compounds that selectively inhibit the proliferation of parasites. Furthermore, we examined the cell cycle profile of the parasite and compared tubulin polymerization dynamics before and after the treatment using identified compounds. Last, in vivo anti-parasite activities of these compounds were determined in T. brucei-infected mice.Three compounds were selected that are 100 fold more effective against the growth of T. brucei cells than mammalian cells. These compounds caused cell cycle progression defects in T. brucei cells. Western analyses indicated that these compounds decreased tubulin polymerization in T. brucei cells. The in vivo investigation revealed that these compounds, when admitted orally, inhibited T. brucei cell proliferation in mouse blood. However, they were not potent enough to clear up the infection completely.These compounds are promising lead compounds as orally active agents for drug development of anti-trypanosome agents. A more detail structure activity relationship (SAR was summarized that will be used to guide future lead optimization to improve the selectivity and potency of the current compounds.

  18. Ansamitocin P3 depolymerizes microtubules and induces apoptosis by binding to tubulin at the vinblastine site.

    Science.gov (United States)

    Venghateri, Jubina B; Gupta, Tilak Kumar; Verma, Paul J; Kunwar, Ambarish; Panda, Dulal

    2013-01-01

    Maytansinoid conjugates are currently under different phases of clinical trials and have been showing promising activity for various types of cancers. In this study, we have elucidated the mechanism of action of ansamitocin P3, a structural analogue of maytansine for its anticancer activity. Ansamitocin P3 potently inhibited the proliferation of MCF-7, HeLa, EMT-6/AR1 and MDA-MB-231 cells in culture with a half-maximal inhibitory concentration of 20±3, 50±0.5, 140±17, and 150±1.1 pM, respectively. Ansamitocin P3 strongly depolymerized both interphase and mitotic microtubules and perturbed chromosome segregation at its proliferation inhibitory concentration range. Treatment of ansamitocin P3 activated spindle checkpoint surveillance proteins, Mad2 and BubR1 and blocked the cells in mitotic phase of the cell cycle. Subsequently, cells underwent apoptosis via p53 mediated apoptotic pathway. Further, ansamitocin P3 was found to bind to purified tubulin in vitro with a dissociation constant (Kd) of 1.3±0.7 µM. The binding of ansamitocin P3 induced conformational changes in tubulin. A docking analysis suggested that ansamitocin P3 may bind partially to vinblastine binding site on tubulin in two different positions. The analysis indicated that the binding of ansamitocin P3 to tubulin is stabilized by hydrogen bonds. In addition, weak interactions such as halogen-oxygen interactions may also contribute to the binding of ansamitocin P3 to tubulin. The study provided a significant insight in understanding the antiproliferative mechanism of action of ansamitocin P3.

  19. VDAC–Tubulin, an Anti-Warburg Pro-Oxidant Switch

    Science.gov (United States)

    Maldonado, Eduardo N.

    2017-01-01

    Aerobic enhanced glycolysis characterizes the Warburg phenotype. In cancer cells, suppression of mitochondrial metabolism contributes to maintain a low ATP/ADP ratio that favors glycolysis. We propose that the voltage-dependent anion channel (VDAC) located in the mitochondrial outer membrane is a metabolic link between glycolysis and oxidative phosphorylation in the Warburg phenotype. Most metabolites including respiratory substrates, ADP, and Pi enter mitochondria only through VDAC. Oxidation of respiratory substrates in the Krebs cycle generates NADH that enters the electron transport chain (ETC) to generate a proton motive force utilized to generate ATP and to maintain mitochondrial membrane potential (ΔΨ). The ETC is also the major source of mitochondrial reactive oxygen species (ROS) formation. Dimeric α-β tubulin decreases conductance of VDAC inserted in lipid bilayers, and high free tubulin in cancer cells by closing VDAC, limits the ingress of respiratory substrates and ATP decreasing mitochondrial ΔΨ. VDAC opening regulated by free tubulin operates as a “master key” that “seal–unseal” mitochondria to modulate mitochondrial metabolism, ROS formation, and the intracellular flow of energy. Erastin, a small molecule that binds to VDAC and kills cancer cells, and erastin-like compounds antagonize the inhibitory effect of tubulin on VDAC. Blockage of the VDAC–tubulin switch increases mitochondrial metabolism leading to decreased glycolysis and oxidative stress that promotes mitochondrial dysfunction, bioenergetic failure, and cell death. In summary, VDAC opening-dependent cell death follows a “metabolic double-hit model” characterized by oxidative stress and reversion of the pro-proliferative Warburg phenotype. PMID:28168164

  20. Ansamitocin P3 depolymerizes microtubules and induces apoptosis by binding to tubulin at the vinblastine site.

    Directory of Open Access Journals (Sweden)

    Jubina B Venghateri

    Full Text Available Maytansinoid conjugates are currently under different phases of clinical trials and have been showing promising activity for various types of cancers. In this study, we have elucidated the mechanism of action of ansamitocin P3, a structural analogue of maytansine for its anticancer activity. Ansamitocin P3 potently inhibited the proliferation of MCF-7, HeLa, EMT-6/AR1 and MDA-MB-231 cells in culture with a half-maximal inhibitory concentration of 20±3, 50±0.5, 140±17, and 150±1.1 pM, respectively. Ansamitocin P3 strongly depolymerized both interphase and mitotic microtubules and perturbed chromosome segregation at its proliferation inhibitory concentration range. Treatment of ansamitocin P3 activated spindle checkpoint surveillance proteins, Mad2 and BubR1 and blocked the cells in mitotic phase of the cell cycle. Subsequently, cells underwent apoptosis via p53 mediated apoptotic pathway. Further, ansamitocin P3 was found to bind to purified tubulin in vitro with a dissociation constant (Kd of 1.3±0.7 µM. The binding of ansamitocin P3 induced conformational changes in tubulin. A docking analysis suggested that ansamitocin P3 may bind partially to vinblastine binding site on tubulin in two different positions. The analysis indicated that the binding of ansamitocin P3 to tubulin is stabilized by hydrogen bonds. In addition, weak interactions such as halogen-oxygen interactions may also contribute to the binding of ansamitocin P3 to tubulin. The study provided a significant insight in understanding the antiproliferative mechanism of action of ansamitocin P3.

  1. Molecular and biomolecular-based nanomaterials: Tubulin and taxol as molecular constituents

    Science.gov (United States)

    Castro Carmona, Javier Servando

    The new field of protein-based nano-technology takes advantage of the complex interactions between proteins to form unique structures with properties that cannot be achieved with traditional components. Microtubules (MTs), self assembled proteinaceous hollow filaments, offer promise in the development of MT-based nano-systems. The compelling need for the controlled assembly of 3D MT arrays is the fundamental motivation for the first part of this research. We report on the morphology of MTs grown in a crowded environment in the form of high viscosity fluids containing agarose and a novel process that enables the assembly of MTs supported by gel-based 3D scaffolds. Our research on MTs and their interaction with other molecules lead us to discover extraordinary spherulitic structures that changed the course of the project. The novel subject situate us into a complicated dilemma that question the nature of MT asters reported in experiments carried out in cells. The second part of this research is focused in the crystallization of Taxol, a MT stabilizing molecule used as anti-cancer drug. It was confirmed via fluorescent and differential interference contrast microscopy that Taxol crystals can be decorated with fluorescent proteins and fluorochromes without perturbing their morphology. We used theoretical calculations to further investigate Taxol-fluorescent agent interactions. Furthermore, the crystallization of Taxol was studied in pure water, aqueous solutions containing tubulin proteins and tubulin-containing agarose gels. We demonstrated that tubulin is able to heterogeneously nucleate Taxol spherulites. To explain the formation of tubulin-Taxol nuclei a new, secondary Taxol-binding site within the tubulin heterodimer is suggested. Results presented in this work are important for in vivo and in vitro microtubule studies due to the possibility of mistaking these Taxol spherulites for microtubule asters. Thus, we are confirming the need for careful interpretation of

  2. Anion-induced increases in the rate of colchicine binding to tubulin.

    Science.gov (United States)

    Bhattacharyya, B; Wolff, J

    1976-06-01

    The rate of binding of colchicine to tubulin to tubulin is enhanced by certain anions. Among the inorganic anions tested, only sulfate was effective. The organic anions include mostly dicarboxylic acids, among which tartrate was the most effective. This effect occurs onlt at low concentrations of colchicine (less than 0.6 X 10(-5) M). The rate increase dor sulfate and L-(+)-tartrate is ca. 2.5-fold at 1.0 mM and plateaus at a limiting value of ca. 4-fold at 100mM. The overall dissociation rate of the colchicine from the complex, which includes both the true rate of dissociation and the rate of irreversible denaturation of tubulin, is not influenced by 1.0 mM tartrate. The affinity constants for colchicine determined from the rate constants are 8.7 X 10(6) and 2.1 X 10(7) M-1 in the absence and the presence of 1.0 mM L-(+)-tartrate. The limiting value is 3.2 X 10(7) M-1. The affinity constant calculated from steady-state measurements is 3.2 X 10(6) M-1 with or without anions. The binding of other ligands like podophyllotoxin, vinblastine, and 1 -anilino-8-naphthalenesulfonate to tubulin is not affected by tartrate. No major conformational changes resulting from anion treatment could be detected by circular dichroism or intrinsic fluorescence. However, the ability of tubulin to polymerize is inhibited by L-(+)-tartrate at concentrations that increase the rate of colchicine binding. We conclude that anions must have a local effect at or near the binding site which enhances the binding rate of colchicine and which may be related to inhibition of polymerization.

  3. Antitumor Activity of IMC-038525, a Novel Oral Tubulin Polymerization Inhibitor.

    Science.gov (United States)

    Tuma, Maria Carolina; Malikzay, Asra; Ouyang, Xiaohu; Surguladze, David; Fleming, James; Mitelman, Stan; Camara, Margarita; Finnerty, Bridget; Doody, Jacqueline; Chekler, Eugene L P; Kussie, Paul; Tonra, James R

    2010-10-01

    Microtubules are a well-validated target for anticancer therapy. Molecules that bind tubulin affect dynamic instability of microtubules causing mitotic arrest of proliferating cells, leading to cell death and tumor growth inhibition. Natural antitubulin agents such as taxanes and Vinca alkaloids have been successful in the treatment of cancer; however, several limitations have encouraged the development of synthetic small molecule inhibitors of tubulin function. We have previously reported the discovery of two novel chemical series of tubulin polymerization inhibitors, triazoles (Ouyang et al. Synthesis and structure-activity relationships of 1,2,4-triazoles as a novel class of potent tubulin polymerization inhibitors. Bioorg Med Chem Lett. 2005; 15:5154-5159) and oxadiazole derivatives (Ouyang et al. Oxadiazole derivatives as a novel class of antimitotic agents: synthesis, inhibition of tubulin polymerization, and activity in tumor cell lines. Bioorg Med Chem Lett. 2006; 16:1191-1196). Here, we report on the anticancer effects of a lead oxadiazole derivative in vitro and in vivo. In vitro, IMC-038525 caused mitotic arrest at nanomolar concentrations in epidermoid carcinoma and breast tumor cells, including multidrug-resistant cells. In vivo, IMC-038525 had a desirable pharmacokinetic profile with sustained plasma levels after oral dosing. IMC-038525 reduced subcutaneous xenograft tumor growth with significantly greater efficacy than the taxane paclitaxel. At efficacious doses, IMC-038525 did not cause substantial myelosuppression or peripheral neurotoxicity, as evaluated by neutrophil counts and changes in myelination of the sciatic nerve, respectively. These data indicate that IMC-038525 is a promising candidate for further development as a chemotherapeutic agent.

  4. GTP binding to the. beta. -subunit of tubulin is greatly reduced in Alzheimers disease

    Energy Technology Data Exchange (ETDEWEB)

    Khatoon, S.; Slevin, J.T.; Haley, B.E.

    1987-05-01

    A decrease occurs (80-100%) in the (/sup 32/P)8N/sub 3/GTP photoinsertion into a cytosolic protein (55K M/sub r/) of Alzheimer's (AD) brain, tentatively identified as the ..beta..-subunit of tubulin (co-migration with purified tubulin, concentration dependence of interaction with GTP, ATP and their 8-azido photoprobes, and similar effects of Ca/sup 2 +/ and EDTA on photoinsertion). This agrees with prior observations of (/sup 32/P)8N/sub 3/GTP interactions with brain tubulin and a recent report on faulty microtubular assembly in AD brain. The decrease in (/sup 32/P)8N/sub 3/GTP photoinsertion into the 55K M/sub r/ protein of AD brain was in contrast with other photolabeled proteins, which remained at equal levels in AD and age-matched normal brain tissues. The 55K and 45K M/sub r/ were the two major (/sup 32/P)8N/sub 3/GTP photoinsertion species in non-AD brain. Of 5 AD brains, the photoinsertion of (/sup 32/P)8N/sub 3/GTP into the 55K M/sub r/ region was low or absent in 4 (55K/45K=0.1); one was 75% below normals (55K/45K=0.24). Total protein migrating at 55K M/sub r/ was similar in AD and controls. AD brain tubulin, while present, has its exchangeable GTP binding site on ..beta..-tubulin blocked/modified such that (/sup 32/P)8N/sub 3/GTP cannot interact normally with this site.

  5. Study on Molecular Recognition between Euphorbia Factor L713283 and β-Tubulin via Molecular Simulation Methods

    Directory of Open Access Journals (Sweden)

    Shan Chang

    2015-01-01

    Full Text Available Euphorbia factor L713283 is a new lathyrane diterpene isolated from Euphorbia lathyris and shows strong anticancer activity. By using molecular similarity analysis, β-tubulin was identified as one of the possible targets of L713283. We further investigated the binding modes of L713283 with β-tubulin using molecular docking and molecular dynamics (MD simulation methods. The results indicated that the binding site between β-tubulin and L713283 was composed of the four regions, that is, residues Phe20~Glu27, Leu225~Thr232, Phe270~Gly277, and Ile356~Met363. MM/GBSA method was used to calculate the binding free energy and determine the key residues for the association of L713283 with β-tubulin. It was found that nonpolar interactions made the major contributions for the binding. In addition, we compared the binding pocket and motion modes of L713283-free and L713283-bound β-tubulin systems. It is proposed that L713283 may bind to β-tubulin and favor the formation of αβ-tubulin dimmer. This work provides possible explanation for molecular mechanism of the anticancer agent L713283, and the strategy used here could benefit the investigation of possible target profile for those bioactive agents with unknown mechanisms.

  6. Fluorescence resonance energy transfer and molecular modeling studies on 4',6-diamidino-2-phenylindole (DAPI) complexes with tubulin.

    Science.gov (United States)

    Arbildua, José J; Brunet, Juan E; Jameson, David M; López, Maribel; Nova, Esteban; Lagos, Rosalba; Monasterio, Octavio

    2006-03-01

    The goal of this work was to determine the binding properties and location of 4',6-diamidino-2-phenylindole (DAPI) complexed with tubulin. Using fluorescence anisotropy, a dissociation constant of 5.2+/-0.4 microM for the DAPI-tubulin complex was determined, slightly lower than that for the tubulin S complex. The influence of the C-terminal region on the binding of DAPI to tubulin was also characterized. Using FRET experiments, and assuming a kappa2 value of 2/3, distances between Co2+ bound to its high-affinity binding site and the DAPI-binding site and 2',3'-O-(trinitrophenyl)guanosine 5'-triphosphate bound to the exchangeable nucleotide and the DAPI-binding site were found to be 20+/-2 A and 43+/-2 A, respectively. To locate potential DAPI-binding sites on tubulin, a molecular modeling study was carried out using the tubulin crystal structure and energy minimization calculations. The results from the FRET measurements were used to limit the possible location of DAPI in the tubulin structure. Several candidate binding sites were found and these are discussed in the context of the various properties of bound DAPI.

  7. Fluorescence resonance energy transfer and molecular modeling studies on 4′,6-diamidino-2-phenylindole (DAPI) complexes with tubulin

    Science.gov (United States)

    Arbildua, José J.; Brunet, Juan E.; Jameson, David M.; López, Maribel; Nova, Esteban; Lagos, Rosalba; Monasterio, Octavio

    2006-01-01

    The goal of this work was to determine the binding properties and location of 4′,6-diamidino-2-phenylindole (DAPI) complexed with tubulin. Using fluorescence anisotropy, a dissociation constant of 5.2 ± 0.4 μM for the DAPI–tubulin complex was determined, slightly lower than that for the tubulin S complex. The influence of the C-terminal region on the binding of DAPI to tubulin was also characterized. Using FRET experiments, and assuming a κ2 value of 2/3, distances between Co2+ bound to its high-affinity binding site and the DAPI-binding site and 2′,3′-O-(trinitrophenyl)guanosine 5′-triphosphate bound to the exchangeable nucleotide and the DAPI-binding site were found to be 20 ± 2 Å and 43 ± 2 Å, respectively. To locate potential DAPI-binding sites on tubulin, a molecular modeling study was carried out using the tubulin crystal structure and energy minimization calculations. The results from the FRET measurements were used to limit the possible location of DAPI in the tubulin structure. Several candidate binding sites were found and these are discussed in the context of the various properties of bound DAPI. PMID:16452620

  8. Establishing a High-content Analysis Method for Tubulin Polymerization to Evaluate Both the Stabilizing and Destabilizing Activities of Compounds.

    Science.gov (United States)

    Sum, Chi Shing; Nickischer, Debra; Lei, Ming; Weston, Andrea; Zhang, Litao; Schweizer, Liang

    2014-01-01

    Microtubules are important components of the cellular cytoskeleton that play roles in various cellular processes such as vesicular transport and spindle formation during mitosis. They are formed by an ordered organization of α-tubulin and β-tubulin hetero-polymers. Altering microtubule polymerization has been known to be the mechanism of action for a number of therapeutically important drugs including taxanes and epothilones. Traditional cell-based assays for tubulin-interacting compounds rely on their indirect effects on cell cycle and/or cell proliferation. Direct monitoring of compound effects on microtubules is required to dissect detailed mechanisms of action in a cellular setting. Here we report a high-content assay platform to monitor tubulin polymerization status by directly measuring the acute effects of drug candidates on the cellular tubulin network with the capability to dissect the mechanisms of action. This high-content analysis distinguishes in a quantitative manner between compounds that act as tubulin stabilizers versus those that are tubulin destabilizers. In addition, using a multiplex approach, we expanded this analysis to simultaneously monitor physiological cellular responses and associated cellular phenotypes.

  9. Design, Synthesis and Biological Evaluation of 1,4-Disubstituted-3,4-dihydroisoquinoline Compounds as New Tubulin Polymerization Inhibitors

    Directory of Open Access Journals (Sweden)

    Ling Zhang

    2015-05-01

    Full Text Available A series of 1,4-disubstituted-3,4-dihydroisoquinoline derivatives designed as tubulin polymerization inhibitors were synthesized. Their cytotoxic activities against the CEM leukemia cell line were evaluated. Most of them displayed moderate cytotoxic activities, and compounds 21 and 32 showed good activities with IC50 of 4.10 and 0.64 μM, respectively. The most potent compound 32 was further confirmed to be able to inhibit tubulin polymerization, and its hypothetical binding mode with tubulin was obtained by molecular docking.

  10. Hypergravity-induced changes in gene expression in Arabidopsis hypocotyls.

    Science.gov (United States)

    Yoshioka, R; Soga, K; Wakabayashi, K; Takeba, G; Hoson, T

    2003-01-01

    Under hypergravity conditions, the cell wall of stem organs becomes mechanically rigid and elongation growth is suppressed, which can be recognized as the mechanism for plants to resist gravitational force. The changes in gene expression by hypergravity treatment were analyzed in Arabidopsis hypocotyls by the differential display method, for identifying genes involved in hypergravity-induced growth suppression. Sixty-two cDNA clones were expressed differentially between the control and 300 g conditions: the expression levels of 39 clones increased, whereas those of 23 clones decreased under hypergravity conditions. Sequence analysis and database searching revealed that 12 clones, 9 up-regulated and 3 down-regulated, have homology to known proteins. The expression of these genes was further analyzed using RT-PCR. Finally, six genes were confirmed to be up-regulated by hypergravity. One of such genes encoded 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor of terpenoids such as membrane sterols and several types of hormones. The expression of HMGR gene increased within several hours after hypergravity treatment. Also, compactin, an inhibitor of HMGR, prevented hypergravity-induced growth suppression, suggesting that HMGR is involved in suppression of Arabidopsis hypocotyl growth by hypergravity. In addition, hypergravity increased the expression levels of genes encoding CCR1 and ERD15, which were shown to take part in the signaling pathway of environmental stimuli such as temperature and water, and those of the alpha-tubulin gene. These genes may be involved in a series of cellular events leading to growth suppression of stem organs under hypergravity conditions.

  11. Proteomics indicates modulation of tubulin polymerization by L-menthol inhibiting human epithelial colorectal adenocarcinoma cell proliferation.

    Science.gov (United States)

    Faridi, Uzma; Sisodia, Brijesh S; Shukla, Ashutosh K; Shukla, Rakesh K; Darokar, Mahendra P; Dwivedi, Upendra N; Shasany, Ajit K

    2011-05-01

    Menthol is a naturally occurring cyclic monoterpene used in oral hygiene products, confectionary, pharmaceuticals, cosmetics, pesticides, and as a flavoring agent. In the present study, we analyzed the differentially expressing proteome in L-menthol-treated Caco-2 cell line as it was found to inhibit cell proliferation. Interestingly, free tubulin proteins were observed to be limited after menthol treatment. Semiquantitative RT-PCR with α-tubulin primers showed no change in the level of RNA expression in menthol-treated cell line. However, tubulin polymerization assay with menthol indicated a trend similar to taxol in promoting microtubule assembly. Further, physical counting of apoptotic nuclei and active caspase-3 assays confirmed onset of apoptosis though the rate was slower as compared with that of taxol treatment. This study is the first report of a monoterpene L-menthol modulating tubulin polymerization and apoptosis to inhibit cancer cell proliferation.

  12. In vitro porcine brain tubulin assembly inhibition by water extract from a Chinese medicinal herb, Tripterygium hypoglaucum Hutch

    Institute of Scientific and Technical Information of China (English)

    Zi-Qin Liang; Neng Cao; Zhong-Kui Song; Xu Wang

    2006-01-01

    AIM: To investigate the effect of Tripterygium hyp-oglaucum Hutch (THH) on the assembly and disassembly process of tubulin and its possible mode of action.METHODS: In vitro porcine brain tubulin assembly assay was employed to analyze the inhibitory effects of THH at different concentrations (0.05 μg/L, 0.07 μg/L, 0.09μg/L). Colchicine (0.0025 mmol/L, 0.0050 mmol/L, 0.0075mmol/L) was used as a positive control.RESULTS: THH could significantly inhibit the assembly of isolated porcine brain tubulin at all tested concentrations.CONCLUSION: THH is capable of inducing aneuploidy in mammals via tubulin polymerization inhibition pathway and may pose a genetic risk to human beings.

  13. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...... expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant....

  14. Down-regulated βIII-tubulin Expression Can Reverse Paclitaxel Resistance in A549/Taxol Cells Lines

    Directory of Open Access Journals (Sweden)

    Yinling ZHUO

    2014-08-01

    Full Text Available Background and objective Chemotherapy drug resistance is the primary causes of death in patients with pulmonary carcinoma which make tumor recurrence or metastasis. β-tubulin is the main cell targets of anti-microtubule drug. Increased expression of βIII-tubulin has been implicated in non-small cell lung cancer (NSCLC cell lines. To explore the relationship among the expression level of βIII-tubulin and the sensitivity of A549/Taxolcell lines to Taxol and cell cycles and cell apoptosis by RNA interference-mediated inhibition of βIII-tubulin in A549/Taxol cells. Methods Three pairs of siRNA targetd βIII-tubulin were designed and prepared, which were transfected into A549/Taxol cells using LipofectamineTM 2000. We detected the expression of βIII-tubulin mRNA using Real-time fluorescence qRT-PCR. Tedhen we selected the most efficient siRNA by the expression of βIII-tubulin mRNA in transfected group. βIII-tubulin protein level were mesured by Western blot. The taxol sensitivity in transfected group were evaluated by MTT assay. And the cell apoptosis and cell cycles were determined by flow cytometry. Results βIII-tubulin mRNA levels in A549/Taxol cells were significantly decreased in transfected grop by Real-time qRT-PCR than control groups. And βIII-tubulin siRNA-1 sequence showed the highest transfection efficiency, which was (87.73±4.87% (P<0.01; Western blot results showed that the expressional level of BIII tublin protein was significantly down-reulated in the transfectant cells than thant in the control cells. By MTT assay, we showed that the inhibition ratio of Taxol to A549/Taxol cells transfeced was higher than that of control group (51.77±4.60% (P<0.01. The early apoptosis rate of A549/Taxol cells in transfected group were significantly higher than that of control group (P<0.01; G2-M content in taxol group obviously increased than untreated samples by the cell cycle (P<0.05. Conclusion βIII-tubulin down-regulated significantly

  15. On the Nature and Shape of Tubulin Trails: Implications on Microtubule Self-Organization

    CERN Document Server

    Glade, Nicolas

    2012-01-01

    Microtubules, major elements of the cell skeleton are, most of the time, well organized in vivo, but they can also show self-organizing behaviors in time and/or space in purified solutions in vitro. Theoretical studies and models based on the concepts of collective dynamics in complex systems, reaction-diffusion processes and emergent phenomena were proposed to explain some of these behaviors. In the particular case of microtubule spatial self-organization, it has been advanced that microtubules could behave like ants, self-organizing by 'talking to each other' by way of hypothetic (because never observed) concentrated chemical trails of tubulin that are expected to be released by their disassembling ends. Deterministic models based on this idea yielded indeed like-looking spatio-temporal self-organizing behaviors. Nevertheless the question remains of whether microscopic tubulin trails produced by individual or bundles of several microtubules are intense enough to allow microtubule self-organization at a macr...

  16. Interaction of hepatitis C virus F protein with prefoldin 2 perturbs tubulin cytoskeleton organization.

    Science.gov (United States)

    Tsao, Mei-Ling; Chao, Chung-Hao; Yeh, Chau-Ting

    2006-09-15

    By use of the yeast two-hybrid system, hepatitis C virus (HCV) F protein was found to interact with a cellular protein named prefoldin 2. The interaction was confirmed by confocal immunofluorescence microscopy as well as coimmunoprecipitation experiments. Prefoldin 2 is a subunit of a hexameric molecular chaperone complex, named prefoldin, which delivers nascent actin and tubulin proteins to the eukaryotic cytosolic chaperonin for facilitated folding. Functional prefoldin spontaneously assembles from its six subunits (prefoldin 1-6). In the yeast three-hybrid system, it was found that expression of HCV F protein impeded the interaction between prefoldin 1 and 2. By performing immunofluorescence experiment and non-denaturing gel electrophoresis, it was shown that expression of HCV F protein resulted in aberrant organization of tubulin cytoskeleton. Since HCV replication requires intact microtubule and actin polymerization, HCV F protein may serve as a modulator to prevent high level of HCV replication and thus contributes to viral persistence in chronic HCV infection.

  17. Imaging of self-assembled tubulin polymorphs used as metallization templates

    Science.gov (United States)

    Habicht, W.; Behrens, S.; Böhm, Kj; Dinjus, E.

    2007-03-01

    Tubulin, a protein isolated from eukaryotic cells, is able to self-assemble in vitro under well-defined chemical conditions into highly ordered polymorphic suprastructures such as tubules, rings or sheets. Here we report about the imaging and the morphological appearance of such tubulin assemblies utilized as templates for the deposition of metal nanoparticles or continuous metallization. The structural imaging and analyzing tools like field emission scanning electron microscopy (FESEM) with respect to different electron detectors (Inlens-SE, BSE, TE) are addressed. An EDX-unit is applied for the verification of the deposited metals. Atomic force microscopy (AFM) in tapping-mode (TM) is adopted for 3D-rendering and morphological measurements (height). Tip-surface interactions, the influence of fixation and cantilever-types are considered. Transmission electron microscopy (TEM) is applied to visualize deposited nanoparticles.

  18. Light-microscopic observations of individual microtubules reconstituted from brain tubulin.

    Science.gov (United States)

    Kuriyama, R; Miki-Noumura, T

    1975-12-01

    The course of polymerization of individual brain microtubules could be observed with a light microscope employing dark-field illumination. Statistical analysis of the increase in microtubule length during the polymerization was in accordance with the time course of viscosity change of the tubulin solution. After a plateau level in viscosity was attained, there was no significant change in histograms showing length distribution. These observations were confirmed with fixed and stained microtubules, using a phase-contrast microscope. Observations with dark-field illumination revealed that reconstituted microtubules depolymerized and disappeared immediately upon exposure to buffer containing CaCl2 or sulphydryl reagents such as p-chloromercuriphenyl sulphonic acid (PCMPS) and p-chloromercuribenzoic acid (PCMB). They were also cold-labile. The growth of heterogeneous microtubules which were assembled by mixing purified tubulin dimers with ciliary outer fibres could also be followed with these optical systems.

  19. Design and synthesis of pyrazole-oxindole conjugates targeting tubulin polymerization as new anticancer agents.

    Science.gov (United States)

    Kamal, Ahmed; Shaik, Anver Basha; Jain, Nishant; Kishor, Chandan; Nagabhushana, Ananthamurthy; Supriya, Bhukya; Bharath Kumar, G; Chourasiya, Sumit S; Suresh, Yerramsetty; Mishra, Rakesh K; Addlagatta, Anthony

    2015-03-06

    A series of twenty one compounds with pyrazole and oxindole conjugates were synthesized by Knoevenagel condensation and investigated for their antiproliferative activity on different human cancer cell lines. The conjugates are comprised of a four ring scaffold; the structural isomers 12b and 12c possess chloro-substitution in the D ring. Among the congeners 12b, 12c, and 12d manifested significant cytotoxicity and inhibited tubulin assembly. Treatments with 12b, 12c and 12d resulted in accumulation of cells in G2/M phase, disruption of microtubule network, and increase in cyclin B1 protein. Zebrafish screening revealed that 12b, and 12d caused developmental defects. Docking analysis demonstrated that the congeners occupy the colchicine binding pocket of tubulin.

  20. Molecular modeling and competition binding study of Br-noscapine and colchicine provides insight into noscapinoid-tubulin binding site

    OpenAIRE

    Naik, Pradeep K.; Santoshi, Seneha; Rai, Ankit; Joshi, Harish C.

    2011-01-01

    We have previously discovered the tubulin-binding anti-cancer properties of noscapine and its derivatives (noscapinoids). Here, we present three lines of evidence that noscapinoids bind at or near the well studied colchicine binding site of tubulin: 1) In silico molecular docking studies of Br-noscapine and noscapine yield highest docking score with the well characterised colchicine-binding site from the co-crystal structure; 2) the molecular mechanics-generalized Born/surface area (MM-GB/SA)...

  1. Prognostic Value of Beta-Tubulin-3 and c-Myc in Muscle Invasive Urothelial Carcinoma of the Bladder

    Science.gov (United States)

    Massari, Francesco; Bria, Emilio; Ciccarese, Chiara; Munari, Enrico; Modena, Alessandra; Zambonin, Valentina; Sperduti, Isabella; Artibani, Walter; Cheng, Liang; Martignoni, Guido; Tortora, Giampaolo; Brunelli, Matteo

    2015-01-01

    Background To date, putative prognostic biomarkers have shown limited utility from the clinical perspective for bladder urothelial carcinoma. Herein, the expression of beta-tubulin-3 and c-Myc was evaluated to determine their prognostic potential. Methods In formalin fixed-paraffin embedded blocks, immunohistochemical expression of c-Myc and beta-tubulin-3 was evaluated. H score ranging from 0 to 300 was obtained by multiplying the percentage of positive cells by intensity (0–3); c-Myc and beta-tubulin-3 expression was defined: 0: negative, 1: weakly positive, 2: strongly positive. Results beta-tubulin-3 and c-Myc immunoexpression was available for 46 cases. At the univariate analysis, node-involvement, beta-tubulin-3 and c-Myc overexpression discriminate shorter DFS (HR 2.19, p = 0.043; HR 3.10, p = 0.24 and HR 3.05, p = 0.011, respectively); 2-yrs DFS log-rank analysis according to low versus high level of immunoexpression were statistically significant; beta-tubulin-3, 53% low vs 12.7% high (p = value 0.02) and c-Myc 28 low vs 8 high (p-value 0.007). Patients displaying negative beta-tubulin-3/c-Myc had statistically significant better 2-yrs DFS than those with mixed expression or double positivity (54.5% versus 18.7% versus 0%, log-rank p = 0.006). Conclusions c-Myc and beta-tubulin-3 show improvement for prognostic risk stratification in patients with muscle invasive bladder urothelial carcinoma. These molecular pathways may also be candidate to improve predictiveness to targeted therapies. PMID:26046361

  2. Prognostic Value of Beta-Tubulin-3 and c-Myc in Muscle Invasive Urothelial Carcinoma of the Bladder.

    Directory of Open Access Journals (Sweden)

    Francesco Massari

    Full Text Available To date, putative prognostic biomarkers have shown limited utility from the clinical perspective for bladder urothelial carcinoma. Herein, the expression of beta-tubulin-3 and c-Myc was evaluated to determine their prognostic potential.In formalin fixed-paraffin embedded blocks, immunohistochemical expression of c-Myc and beta-tubulin-3 was evaluated. H score ranging from 0 to 300 was obtained by multiplying the percentage of positive cells by intensity (0-3; c-Myc and beta-tubulin-3 expression was defined: 0: negative, 1: weakly positive, 2: strongly positive.beta-tubulin-3 and c-Myc immunoexpression was available for 46 cases. At the univariate analysis, node-involvement, beta-tubulin-3 and c-Myc overexpression discriminate shorter DFS (HR 2.19, p = 0.043; HR 3.10, p = 0.24 and HR 3.05, p = 0.011, respectively; 2-yrs DFS log-rank analysis according to low versus high level of immunoexpression were statistically significant; beta-tubulin-3, 53% low vs 12.7% high (p = value 0.02 and c-Myc 28 low vs 8 high (p-value 0.007. Patients displaying negative beta-tubulin-3/c-Myc had statistically significant better 2-yrs DFS than those with mixed expression or double positivity (54.5% versus 18.7% versus 0%, log-rank p = 0.006.c-Myc and beta-tubulin-3 show improvement for prognostic risk stratification in patients with muscle invasive bladder urothelial carcinoma. These molecular pathways may also be candidate to improve predictiveness to targeted therapies.

  3. Phylogenetic Characterization of β-Tubulins and Development of Pyrosequencing Assays for Benzimidazole Resistance in Cattle Nematodes

    Science.gov (United States)

    Demeler, Janina; Krüger, Nina; Krücken, Jürgen; von der Heyden, Vera C.; Ramünke, Sabrina; Küttler, Ursula; Miltsch, Sandra; López Cepeda, Michael; Knox, Malcolm; Vercruysse, Jozef; Geldhof, Peter; Harder, Achim; von Samson-Himmelstjerna, Georg

    2013-01-01

    Control of helminth infections is a major task in livestock production to prevent health constraints and economic losses. However, resistance to established anthelmintic substances already impedes effective anthelmintic treatment in many regions worldwide. Thus, there is an obvious need for sensitive and reliable methods to assess the resistance status of at least the most important nematode populations. Several single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene of various nematodes correlate with resistance to benzimidazoles (BZ), a major anthelmintic class. Here we describe the full-length β-tubulin isotype 1 and 2 and α-tubulin coding sequences of the cattle nematode Ostertagia ostertagi. Additionally, the Cooperia oncophora α-tubulin coding sequence was identified. Phylogenetic maximum-likelihood analysis revealed that both isotype 1 and 2 are orthologs to the Caenorhabditis elegans ben-1 gene which is also associated with BZ resistance upon mutation. In contrast, a Trichuris trichiura cDNA, postulated to be β-tubulin isotype 1 involved in BZ resistance in this human parasite, turned out to be closely related to C. elegans β-tubulins tbb-4 and mec-7 and would therefore represent the first non-ben-1-like β-tubulin to be under selection through treatment with BZs. A pyrosequencing assay was established to detect BZ resistance associated SNPs in β-tubulin isotype 1 codons 167, 198 and 200 of C. oncophora and O. ostertagi. PCR-fragments representing either of the two alleles were combined in defined ratios to evaluate the pyrosequencing assay. The correlation between the given and the measured allele frequencies of the respective SNPs was very high. Subsequently laboratory isolates and field populations with known resistance status were analyzed. With the exception of codon 167 in Cooperia, increases of resistance associated alleles were detected for all codons in at least one of the phenotypically resistant population. Pyrosequencing

  4. A photoaffinity analogue of discodermolide specifically labels a peptide in beta-tubulin.

    Science.gov (United States)

    Xia, Shujun; Kenesky, Craig S; Rucker, Paul V; Smith, Amos B; Orr, George A; Horwitz, Susan Band

    2006-10-01

    Discodermolide is a potentially important antitumor agent that stabilizes microtubules and blocks cells at the G2/M phase of the cell cycle in a manner similar to that of Taxol. Discodermolide also has unique properties that distinguish it from Taxol. In the present study, photoaffinity-labeled discodermolide analogues are used to investigate their binding site in tubulin. Three photoaffinity-labeled discodermolide analogues were synthesized, all of which promoted microtubule polymerization in the absence of GTP. The analogue, C19-[4-(4-(3)H-benzoyl-phenyl)-carbamate]-discodermolide (C19-[3H]BPC-discodermolide), was selected for photolabeling studies because it had the highest extent of photoincorporation, approximately 1%, of the three radiolabeled discodermolide analogues explored. Although compared to discodermolide, C19-BPC-discodermolide revealed no hypernucleation effect in the in vitro microtubule polymerization assay, it was more cytotoxic than discodermolide, and, like discodermolide, demonstrated synergism with Taxol. These results suggest that the hypernucleation effect of discodermolide is not involved in its cytotoxic activity. Similar to discodermolide, C19-BPC-discodermolide can effectively displace [3H]Taxol from microtubules, but Taxol cannot effectively displace C19-[3H]BPC-discodermolide binding. Discodermolide can effectively displace C19-[3H]BPC-discodermolide binding. Formic acid hydrolysis, immunoprecipitation experiments, and subtilisin digestion indicate that C19-BPC-discodermolide labels amino acid residues 305-433 in beta-tubulin. Further digestion with Asp-N and Arg-C enzymes suggested that C19-BPC-discodermolide binds to amino acid residues, 355-359, in beta-tubulin, which is in close proximity to the Taxol binding site. Molecular modeling guided by the above evidence led to a putative binding model for C19-BPC-discodermolide in tubulin.

  5. Synthesis of phenstatin/isocombretastatin-chalcone conjugates as potent tubulin polymerization inhibitors and mitochondrial apoptotic inducers.

    Science.gov (United States)

    Kamal, Ahmed; Kumar, G Bharath; Vishnuvardhan, M V P S; Shaik, Anver Basha; Reddy, Vangala Santhosh; Mahesh, Rasala; Sayeeda, Ibrahim Bin; Kapure, Jeevak Sopanrao

    2015-04-07

    A series of phenstatin/isocombretastatin–chalcones were synthesized and screened for their cytotoxic activity against various human cancer cell lines. Some representative compounds exhibited significant antiproliferative activity against a panel of sixty human cancer cell lines of the NCI, with GI50 values in the range of 0.11 to 19.0 μM. Three compounds (3b, 3c and 3e) showed a broad spectrum of antiproliferative efficacy on most of the cell lines in the sub-micromolar range. In addition, all the synthesized compounds (3a–l and 4a–l) displayed moderate to excellent cytotoxicity against breast cancer cells such as MCF-7 and MDA-MB-231 with IC50 values in the range of 0.5 to 19.9 μM. Moreover, the tubulin polymerization assay and immunofluorescence analysis results suggest that some of these compounds like 3c and 3e exhibited significant inhibitory effect on the tubulin assembly with an IC50 value of 0.8 μM and 0.6 μM respectively. A competitive binding assay suggested that these compounds bind at the colchicine-binding site of tubulin. A cell cycle assay revealed that these compounds arrest at the G2/M phase and lead to apoptotic cell death. Furthermore, this was confirmed by Hoechst 33258 staining, activation of caspase 9, DNA fragmentation, Annexin V-FITC and mitochondrial membrane depolarization. Molecular docking studies indicated that compounds like 3e occupy the colchicine binding site of tubulin.

  6. Stathmin Regulates Centrosomal Nucleation of Microtubules and Tubulin Dimer/Polymer Partitioning

    OpenAIRE

    Ringhoff, Danielle N.; Cassimeris, Lynne

    2009-01-01

    Stathmin is a microtubule-destabilizing protein ubiquitously expressed in vertebrates and highly expressed in many cancers. In several cell types, stathmin regulates the partitioning of tubulin between unassembled and polymer forms, but the mechanism responsible for partitioning has not been determined. We examined stathmin function in two cell systems: mouse embryonic fibroblasts (MEFs) isolated from embryos +/+, +/−, and −/− for the stathmin gene and porcine kidney epithelial (LLCPK) cells ...

  7. The tubulin-bound conformation of paclitaxel: T-taxol vs "PTX-NY".

    Science.gov (United States)

    Yang, Yutao; Alcaraz, Ana A; Snyder, James P

    2009-03-27

    Nearly 35 years after its discovery and 11 years after FDA approval of paclitaxel (PTX) as a breakthrough anticancer drug, the 3-D structure of the agent bound to its beta-tubulin target was proposed to be T-Taxol. The latter bioactive form has recently been challenged by the Ojima group with a structure, "PTX-NY" ("REDOR Taxol"), in which the C-13 side chain is proposed to adopt a different conformation and an alternative hydrogen-bonding pattern in the tubulin binding site. Previously, the two conformers were compared to show that only T-Taxol fits the PTX-derived electron crystallographic density. That work has been extended by molecular mechanics and quantum chemical methods to reveal that the PTX-NY conformation is relatively less stable, on average, by 10-11 kcal/mol. In agreement with NMR studies, an 11 ns molecular dynamics treatment for PTX in an explicit water pool locates T-Taxol along the trajectory, but not PTX-NY. Docking of various PTX conformers into the electron crystallographic binding site of tubulin demonstrates that PTX-NY cannot be accommodated unless the pocket is reorganized in violation of the experimental constraints. Finally, analysis of the structures of T-Taxol and PTX-NY for their capacity to predict the existence of superpotent PTX analogues discloses that only the former forecasts such analogues, as now established by the T-Taxol-inspired synthesis of bridged taxanes. In sum, all empirical criteria support T-Taxol as the bound conformation of PTX on beta-tubulin in microtubules.

  8. Phosphorylated LIM kinases colocalize with gamma-tubulin in centrosomes during early stages of mitosis.

    Science.gov (United States)

    Chakrabarti, Ratna; Jones, Jennifer L; Oelschlager, Denise K; Tapia, Tenekua; Tousson, Albert; Grizzle, William E

    2007-12-01

    LIM kinases (LIMK1 and LIMK2) are LIM domain containing serine/threonine kinases that modulate reorganization of actin cytoskeleton through inactivating phosphorylation of cofilin. The Rho family of small GTPases regulates the catalytic activity of LIMK1 and LIMK2 through activating phosphorylation by ROCK or by p21 kinase. Recent studies have suggested that LIMK1 could play a role in modulation of cellular growth by alteration of the cell cycle in breast and prostate tumor cells; however, the direct mitogenic effects of LIMK1 in these tumor cells is yet to be elucidated. Via immunofluorescence, in this study, we show that phosphorylated LIM kinases (pLIMK1/2) are colocalized with gamma-tubulin in the centrosomes during the early mitotic phases of human breast and prostate cancer cells (MDA-MB-231 and DU145); apparent colocalization begins in the centrosomes in prophase. As shown by both bright field (MDA-MB-231) and fluorescent immunohistochemistry (MDA-MB-231 and DU145), pLIMK1/2 does not localize to centrosomes during interphase. By bright field immunohistochemistry, the largest area of the centrosome that is stained with pLIMK1/2 occurs at anaphase. In early telophase, reduced staining of pLIMK1/2 at the spindle poles and concomitant accumulation of pLIMK1/2 at the cleavage furrow begins to occur. In late telophase, loss of staining of pLIMK1/2 and of colocalization with gamma-tubulin occurs at the poles and pLIMK1/2 became further concentrated at the junction between the two daughter cells. Co-immunoprecipitation studies indicated that gamma-tubulin associates with phosphorylated LIMK1 and LIMK2 but not with dephosphorylated LIMK1 or LIMK2. The results suggest that activated LIMK1/2 may associate with gamma-tubulin and play a role in mitotic spindle assembly.

  9. Review: tubulin function, action of antitubulin drugs, and new drug development.

    Science.gov (United States)

    Pellegrini, Federico; Budman, Daniel R

    2005-01-01

    Anticancer agents that interfere with microtubulin function are in widespread use in man and have a broad spectrum of activity against both hematological malignancies and solid tumors. The mechanisms of actions of these agents have been better defined during the past decade, indicating that there are distinct binding sites for these agents and that they interfere with microtubulin dynamics (growth and shortening of tubules) at low concentrations and only evoke microtubulin aggregation or dissociation at high concentrations. Tubulin has been recently described in the nucleus of cells and in mitochondria. Downstream events from tubulin binding are believed to be critical events for the generation of apoptosis in the malignant cell. The effects of vinca alkaloids and taxanes are distinct, suggesting that the interference with the tubulin cap by high-affinity binding of effective agents is not the only mechanism of cytotoxic effect, and the low-affinity binding of drug, which distorts microtubulin function, may also be important. The epothilones share some of the binding characteristics of the taxanes and are in clinical trials because of cytoxic activity in taxane resistant cells. Tubulin has additional target sites for anticancer drugs including interference with the binding and function of microtubule associated proteins and interference with motor proteins which are essential for the transport of substances within the cell. Because many of these microtubule associated proteins have an ATP binding site, both computer-aided design and combinatorial chemistry techniques can be used to make agents to interfere with their function analogous to imatinib mesylate (Gleevec). Agents that interfere with the motor protein kinesin are entering clinical trials.

  10. Molecular modelling and competition binding study of Br-noscapine and colchicine provide insight into noscapinoid-tubulin binding site.

    Science.gov (United States)

    Naik, Pradeep K; Santoshi, Seneha; Rai, Ankit; Joshi, Harish C

    2011-06-01

    We have previously discovered the tubulin-binding anti-cancer properties of noscapine and its derivatives (noscapinoids). Here, we present three lines of evidence that noscapinoids bind at or near the well studied colchicine binding site of tubulin: (1) in silico molecular docking studies of Br-noscapine and noscapine yield highest docking score with the well characterised colchicine-binding site from the co-crystal structure; (2) the molecular mechanics-generalized Born/surface area (MM-GB/SA) scoring results ΔΔG(bind-cald) for both noscapine and Br-noscapine (3.915 and 3.025 kcal/mol) are in reasonably good agreement with our experimentally determined binding affinity (ΔΔG(bind-Expt) of 3.570 and 2.988 kcal/mol, derived from K(d) values); and (3) Br-noscapine competes with colchicine binding to tubulin. The simplest interpretation of these collective data is that Br-noscapine binds tubulin at a site overlapping with, or very close to colchicine-binding site of tubulin. Although we cannot rule out a formal possibility that Br-noscapine might bind to a site distinct and distant from the colchicine-binding site that might negatively influence the colchicine binding to tubulin.

  11. The tubulin repertoire of Caenorhabditis elegans sensory neurons and its context‑dependent role in process outgrowth

    Science.gov (United States)

    Lockhead, Dean; Schwarz, Erich M.; O’Hagan, Robert; Bellotti, Sebastian; Krieg, Michael; Barr, Maureen M.; Dunn, Alexander R.; Sternberg, Paul W.; Goodman, Miriam B.

    2016-01-01

    Microtubules contribute to many cellular processes, including transport, signaling, and chromosome separation during cell division. They comprise αβ‑tubulin heterodimers arranged into linear protofilaments and assembled into tubes. Eukaryotes express multiple tubulin isoforms, and there has been a longstanding debate as to whether the isoforms are redundant or perform specialized roles as part of a tubulin code. Here we use the well‑characterized touch receptor neurons (TRNs) of Caenorhabditis elegans to investigate this question through genetic dissection of process outgrowth both in vivo and in vitro. With single‑cell RNA-seq, we compare transcription profiles for TRNs with those of two other sensory neurons and present evidence that each sensory neuron expresses a distinct palette of tubulin genes. In the TRNs, we analyze process outgrowth and show that four tubulins (tba‑1, tba‑2, tbb‑1, and tbb‑2) function partially or fully redundantly, whereas two others (mec‑7 and mec‑12) perform specialized, context‑dependent roles. Our findings support a model in which sensory neurons express overlapping subsets of tubulin genes whose functional redundancy varies among cell types and in vivo and in vitro contexts. PMID:27654945

  12. Anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize

    Science.gov (United States)

    Adams, Stephen R.; Yang, Howard C.; Savariar, Elamprakash N.; Aguilera, Joe; Crisp, Jessica L.; Jones, Karra A.; Whitney, Michael A.; Lippman, Scott M.; Cohen, Ezra E. W.; Tsien, Roger Y.; Advani, Sunil J.

    2016-01-01

    Tumour resistance to radiotherapy remains a barrier to improving cancer patient outcomes. To overcome radioresistance, certain drugs have been found to sensitize cells to ionizing radiation (IR). In theory, more potent radiosensitizing drugs should increase tumour kill and improve patient outcomes. In practice, clinical utility of potent radiosensitizing drugs is curtailed by off-target side effects. Here we report potent anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize to tumours based on surface receptor expression. While two classes of potent anti-tubulins, auristatins and maytansinoids, indiscriminately radiosensitize tumour cells, conjugating these potent anti-tubulins to anti-ErbB antibodies restrict their radiosensitizing capacity. Of translational significance, we report that a clinically used maytansinoid ADC, ado-trastuzumab emtansine (T-DM1), with IR prolongs tumour control in target expressing HER2+ tumours but not target negative tumours. In contrast to ErbB signal inhibition, our findings establish an alternative therapeutic paradigm for ErbB-based radiosensitization using antibodies to restrict radiosensitizer delivery. PMID:27698471

  13. Dietary flavonoid fisetin binds to β-tubulin and disrupts microtubule dynamics in prostate cancer cells.

    Science.gov (United States)

    Mukhtar, Eiman; Adhami, Vaqar Mustafa; Sechi, Mario; Mukhtar, Hasan

    2015-10-28

    Microtubule targeting based therapies have revolutionized cancer treatment; however, resistance and side effects remain a major limitation. Therefore, novel strategies that can overcome these limitations are urgently needed. We made a novel discovery that fisetin, a hydroxyflavone, is a microtubule stabilizing agent. Fisetin binds to tubulin and stabilizes microtubules with binding characteristics far superior than paclitaxel. Surface plasmon resonance and computational docking studies suggested that fisetin binds to β-tubulin with superior affinity compared to paclitaxel. Fisetin treatment of human prostate cancer cells resulted in robust up-regulation of microtubule associated proteins (MAP)-2 and -4. In addition, fisetin treated cells were enriched in α-tubulin acetylation, an indication of stabilization of microtubules. Fisetin significantly inhibited PCa cell proliferation, migration, and invasion. Nudc, a protein associated with microtubule motor dynein/dynactin complex that regulates microtubule dynamics, was inhibited with fisetin treatment. Further, fisetin treatment of a P-glycoprotein overexpressing multidrug-resistant cancer cell line NCI/ADR-RES inhibited the viability and colony formation. Our results offer in vitro proof-of-concept for fisetin as a microtubule targeting agent. We suggest that fisetin could be developed as an adjuvant for treatment of prostate and other cancer types.

  14. A second tubulin binding site on the kinesin-13 motor head domain is important during mitosis.

    Directory of Open Access Journals (Sweden)

    Dong Zhang

    Full Text Available Kinesin-13s are microtubule (MT depolymerases different from most other kinesins that move along MTs. Like other kinesins, they have a motor or head domain (HD containing a tubulin and an ATP binding site. Interestingly, kinesin-13s have an additional binding site (Kin-Tub-2 on the opposite side of the HD that contains several family conserved positively charged residues. The role of this site in kinesin-13 function is not clear. To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A. We show that the Kin-Tub-2 site enhances tubulin cross-linking and MT bundling properties of KLP10A in-vitro. Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells. The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.

  15. Prefoldins 3 and 5 Play an Essential Role in Arabidopsis Tolerance to Salt Stress

    Institute of Scientific and Technical Information of China (English)

    Miguel A.Rodriguez-Milla; Julio Salinas

    2009-01-01

    During the last years,our understanding of the mechanisms that control plant response to salt stress has been steadily progressing.Pharmacological studies have allowed the suggestion that the cytoskeleton may be involved in reg-ulating such a response.Nevertheless,genetic evidence establishing that the cytoskeleton has a role in plant tolerance to salt stress has not been reported yet.Here,we have characterized Arabidopsis T-DNA mutants for genes encoding proteins orthologous to prefoldin (PFD) subunits 3 and 5 from yeast and mammals.In these organisms,PFD subunits,also known as Genes Involved in Microtubule biogenesis (GIM),form a heterohexameric PFD complex implicated in tubulin and actin folding.We show that,indeed,PFD3 and PFD5 can substitute for the loss of their yeast orthologs,as they are able to complement yeast gim2△ and gim5△ mutants,respectively.Our results indicate thatpfd3 and pfd5 mutants have reduced levels of α- and β-tubulin compared to the wild-type plants when growing under both control and salt-stress conditions.In addition,pfd3 and pfd5 mutants display alterations in their developmental patterns and microtubule organization,and,more importantly,are hypersensitive to high concentrations of NaCI but not of LiCI or mannitol.These results demonstrate that the cytoskeleton plays an essential role in plant tolerance to salt stress.

  16. Rational design of biaryl pharmacophore inserted noscapine derivatives as potent tubulin binding anticancer agents

    Science.gov (United States)

    Santoshi, Seneha; Manchukonda, Naresh Kumar; Suri, Charu; Sharma, Manya; Sridhar, Balasubramanian; Joseph, Silja; Lopus, Manu; Kantevari, Srinivas; Baitharu, Iswar; Naik, Pradeep Kumar

    2015-03-01

    We have strategically designed a series of noscapine derivatives by inserting biaryl pharmacophore (a major structural constituent of many of the microtubule-targeting natural anticancer compounds) onto the scaffold structure of noscapine. Molecular interaction of these derivatives with α,β-tubulin heterodimer was investigated by molecular docking, molecular dynamics simulation, and binding free energy calculation. The predictive binding affinity indicates that the newly designed noscapinoids bind to tubulin with a greater affinity. The predictive binding free energy (ΔGbind, pred) of these derivatives (ranging from -5.568 to -5.970 kcal/mol) based on linear interaction energy (LIE) method with a surface generalized Born (SGB) continuum solvation model showed improved binding affinity with tubulin compared to the lead compound, natural α-noscapine (-5.505 kcal/mol). Guided by the computational findings, these new biaryl type α-noscapine congeners were synthesized from 9-bromo-α-noscapine using optimized Suzuki reaction conditions for further experimental evaluation. The derivatives showed improved inhibition of the proliferation of human breast cancer cells (MCF-7), human cervical cancer cells (HeLa) and human lung adenocarcinoma cells (A549), compared to natural noscapine. The cell cycle analysis in MCF-7 further revealed that these compounds alter the cell cycle profile and cause mitotic arrest at G2/M phase more strongly than noscapine. Tubulin binding assay revealed higher binding affinity to tubulin, as suggested by dissociation constant (Kd) of 126 ± 5.0 µM for 5a, 107 ± 5.0 µM for 5c, 70 ± 4.0 µM for 5d, and 68 ± 6.0 µM for 5e compared to noscapine (Kd of 152 ± 1.0 µM). In fact, the experimentally determined value of ΔGbind, expt (calculated from the Kd value) are consistent with the predicted value of ΔGbind, pred calculated based on LIE-SGB. Based on these results, one of the derivative 5e of this series was used for further toxicological

  17. Reference: 774 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available an essential gene, the disruption of which causes embryonic lethality. Plants carrying a hypomorphic smg7 mu...e progression from anaphase to telophase in the second meiotic division in Arabidopsis. Arabidopsis SMG7 is

  18. Reference: 398 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available plays attenuated chloroplast movements under intermediate and high light intensitie...hese movements. In this work, we describe plastid movement impaired 2 (pmi2), a mutant in Arabidopsis (Arabidopsis thaliana) that dis

  19. Reference: 173 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available mical approaches to elucidate the action mechanisms of sirtinol in Arabidopsis. A...tic and chemical analyses of the action mechanisms of sirtinol in Arabidopsis. 8 3129-34 15710899 2005 Feb P

  20. Reference: 718 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available displayed a moderate but significant decrease in germination in the presence of D...NA damage. This report links Ubc13-Uev with functions in DNA damage response in Arabidopsis. Arabidopsis UEV

  1. Arabidopsis CDS blastp result: AK068856 [KOME

    Lifescience Database Archive (English)

    Full Text Available eme oxygenase (HY1) [Arabidopsis thaliana] GI:4877362, heme oxygenase 1 [Arabidopsis thaliana] GI:4530591 GB:AF132475; annotation upd...ated per Seth J. Davis at University of Wisconsin-Madison 3e-90 ...

  2. Arabidopsis CDS blastp result: AK104955 [KOME

    Lifescience Database Archive (English)

    Full Text Available B:AF132475; annotation updated per Seth J. Davis at University of Wisconsin-Madison 3e-90 ... ...heme oxygenase (HY1) [Arabidopsis thaliana] GI:4877362, heme oxygenase 1 [Arabidopsis thaliana] GI:4530591 G

  3. Reference: 110 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available on process. Our study shows that an Arabidopsis SNM protein, although structurally closer to the SNM1/PSO2 members, shares some prope...rties with ARTEMIS but also has novel characteristics. Arabidopsis plants defective

  4. A mutant beta-tubulin confers resistance to the action of benzimidazole-carbamate microtubule inhibitors both in vivo and in vitro.

    Science.gov (United States)

    Foster, K E; Burland, T G; Gull, K

    1987-03-16

    The mutant BEN210 of Physarum polycephalum is highly resistant to a number of benzimidazole carbamate agents, including methylbenzimidazole-2-yl-carbamate and parbendazole. The resistance is conferred by the benD210 mutation in a structural gene for beta-tubulin. This mutant allele encodes a beta-tubulin with novel electrophoretic mobility. We have used this strain to determine whether the mutant beta-tubulin is used in microtubules and whether this usage permits microtubule polymerisation in the presence of drugs both in vivo and in vitro. In vitro assembly studies of tubulin purified from the mutant strain have shown that microtubules are formed both in the absence of drugs and in all drug concentrations tested (up to 50 microM parbendazole). In contrast, the assembly of microtubules from wild-type tubulin in vitro is totally inhibited by 2-5 microM parbendazole. Thus the resistance of BEN210 to parbendazole observed in vivo has been reproduced in vitro using tubulin purified from the mutant strain. Electrophoretic analysis of the microtubules formed in vitro has shown that both the wild-type and the mutant beta-tubulin are incorporated into the microtubules and that the proportion of mutant to wild-type beta-tubulin appears to remain constant with increasing drug concentration. This is the first demonstration of a single mutation in a tubulin structural gene causing an altered function of the gene product in vitro.

  5. Acyl-biotinyl exchange chemistry and mass spectrometry-based analysis of palmitoylation sites of in vitro palmitoylated rat brain tubulin.

    Science.gov (United States)

    Zhao, Zhiqiang; Hou, Junjie; Xie, Zhensheng; Deng, Jianwei; Wang, Xiaoming; Chen, Danfang; Yang, Fuquan; Gong, Weimin

    2010-11-01

    Research has shown that the palmitoyl group of α-tubulin mediates the hydrophobic interaction between microtubules and intracellular membranes and that palmitoylated tubulin plays a role in signal transduction. There are 20 cysteine residues per α/β tubulin heterodimer. C376 of α-tubulin was reported to be predominantly palmitoylated and C20, C213 and C305 of α-tubulin were palmitoylated at lower levels. The previous method used for the analysis of the palmitoylation sites on α-tubulin was based on ³H-labeling, enzymolysis, purification and sequencing. This approach, although efficient, is laborious. Mass spectrometry (MS), especially tandem MS, has been shown to be a successful method for identification of various post-translational modifications of proteins. We report here a convenient MS-based method to comprehensively analyze the palmitoylation sites of the α/β tubulin heterodimer. Acyl-biotinyl exchange chemistry and streptavidin agarose affinity purification were applied to enrich palmitoylated peptides from tubulin. After nano-LC-MS/MS analysis, database searching and manual analysis of the spectra revealed that 11 cysteine residues of the α/β tubulin heterodimer were palmitoylated.

  6. Efficient gusA Transient Expression in Porphyra yezoensis Protoplasts Mediated by Endogenous Beta-tubulin Flanking Sequences

    Institute of Scientific and Technical Information of China (English)

    GONG Qianhong; YU Wengong; DAI Jixun; LIU Hongquan; XU Rifu; GUAN Huashi; PAN Kehou

    2007-01-01

    Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are tmknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were constructed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5'- and 3'-flanking regions (Tub5'and Tub3') up- and down-stream of β-glucuronidase (GUS) gene (gusA), respectively,into pA, a derivative of pCAT(R)3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3 '. These constructs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5'-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when compared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35 S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

  7. CXI-benzo-84 reversibly binds to tubulin at colchicine site and induces apoptosis in cancer cells.

    Science.gov (United States)

    Rai, Ankit; Gupta, Tilak Kumar; Kini, Sudarshan; Kunwar, Ambarish; Surolia, Avadhesha; Panda, Dulal

    2013-08-01

    Here, we have discovered CXI-benzo-84 as a potential anticancer agent from a library of benzimidazole derivatives using cell based screening strategy. CXI-benzo-84 inhibited cell cycle progression in metaphase stage of mitosis and accumulated spindle assembly checkpoint proteins Mad2 and BubR1 on kinetochores, which subsequently activated apoptotic cell death in cancer cells. CXI-benzo-84 depolymerized both interphase and mitotic microtubules, perturbed EB1 binding to microtubules and inhibited the assembly and GTPase activity of tubulin in vitro. CXI-benzo-84 bound to tubulin at a single binding site with a dissociation constant of 1.2±0.2μM. Competition experiments and molecular docking suggested that CXI-benzo-84 binds to tubulin at the colchicine-site. Further, computational analysis provided a significant insight on the binding site of CXI-benzo-84 on tubulin. In addition to its potential use in cancer chemotherapy, CXI-benzo-84 may also be useful to screen colchicine-site agents and to understand the colchicine binding site on tubulin.

  8. Voltage-dependent anion channels modulate mitochondrial metabolism in cancer cells: regulation by free tubulin and erastin.

    Science.gov (United States)

    Maldonado, Eduardo N; Sheldon, Kely L; DeHart, David N; Patnaik, Jyoti; Manevich, Yefim; Townsend, Danyelle M; Bezrukov, Sergey M; Rostovtseva, Tatiana K; Lemasters, John J

    2013-04-26

    Respiratory substrates and adenine nucleotides cross the mitochondrial outer membrane through the voltage-dependent anion channel (VDAC), comprising three isoforms--VDAC1, 2, and 3. We characterized the role of individual isoforms in mitochondrial metabolism by HepG2 human hepatoma cells using siRNA. With VDAC3 to the greatest extent, all VDAC isoforms contributed to the maintenance of mitochondrial membrane potential, but only VDAC3 knockdown decreased ATP, ADP, NAD(P)H, and mitochondrial redox state. Cells expressing predominantly VDAC3 were least sensitive to depolarization induced by increased free tubulin. In planar lipid bilayers, free tubulin inhibited VDAC1 and VDAC2 but not VDAC3. Erastin, a compound that interacts with VDAC, blocked and reversed mitochondrial depolarization after microtubule destabilizers in intact cells and antagonized tubulin-induced VDAC blockage in planar bilayers. In conclusion, free tubulin inhibits VDAC1/2 and limits mitochondrial metabolism in HepG2 cells, contributing to the Warburg phenomenon. Reversal of tubulin-VDAC interaction by erastin antagonizes Warburg metabolism and restores oxidative mitochondrial metabolism.

  9. Distribution of tubulin, kinesin, and dynein in light- and dark-adapted octopus retinas.

    Science.gov (United States)

    Martinez, J M; Elfarissi, H; De Velasco, B; Ochoa, G H; Miller, A M; Clark, Y M; Matsumoto, B; Robles, L J

    2000-01-01

    Cephalopod retinas exhibit several responses to light and dark adaptation, including rhabdom size changes, photopigment movements, and pigment granule migration. Light- and dark-directed rearrangements of microfilament and microtubule cytoskeletal transport pathways could drive these changes. Recently, we localized actin-binding proteins in light-/dark-adapted octopus rhabdoms and suggested that actin cytoskeletal rearrangements bring about the formation and degradation of rhabdomere microvilli subsets. To determine if the microtubule cytoskeleton and associated motor proteins control the other light/dark changes, we used immunoblotting and immunocytochemical procedures to map the distribution of tubulin, kinesin, and dynein in dorsal and ventral halves of light- and dark-adapted octopus retinas. Immunoblots detected alpha- and beta-tubulin, dynein intermediate chain, and kinesin heavy chain in extracts of whole retinas. Epifluorescence and confocal microscopy showed that the tubulin proteins were distributed throughout the retina with more immunoreactivity in retinas exposed to light. Kinesin localization was heavy in the pigment layer of light- and dark-adapted ventral retinas but was less prominent in the dorsal region. Dynein distribution also varied in dorsal and ventral retinas with more immunoreactivity in light- and dark-adapted ventral retinas and confocal microscopy emphasized the granular nature of this labeling. We suggest that light may regulate the distribution of microtubule cytoskeletal proteins in the octopus retina and that position, dorsal versus ventral, also influences the distribution of motor proteins. The microtubule cytoskeleton is most likely involved in pigment granule migration in the light and dark and with the movement of transport vesicles from the photoreceptor inner segments to the rhabdoms.

  10. Conservation of tubulin-binding sequences in TRPV1 throughout evolution.

    Directory of Open Access Journals (Sweden)

    Puspendu Sardar

    Full Text Available BACKGROUND: Transient Receptor Potential Vanilloid sub type 1 (TRPV1, commonly known as capsaicin receptor can detect multiple stimuli ranging from noxious compounds, low pH, temperature as well as electromagnetic wave at different ranges. In addition, this receptor is involved in multiple physiological and sensory processes. Therefore, functions of TRPV1 have direct influences on adaptation and further evolution also. Availability of various eukaryotic genomic sequences in public domain facilitates us in studying the molecular evolution of TRPV1 protein and the respective conservation of certain domains, motifs and interacting regions that are functionally important. METHODOLOGY AND PRINCIPAL FINDINGS: Using statistical and bioinformatics tools, our analysis reveals that TRPV1 has evolved about ∼420 million years ago (MYA. Our analysis reveals that specific regions, domains and motifs of TRPV1 has gone through different selection pressure and thus have different levels of conservation. We found that among all, TRP box is the most conserved and thus have functional significance. Our results also indicate that the tubulin binding sequences (TBS have evolutionary significance as these stretch sequences are more conserved than many other essential regions of TRPV1. The overall distribution of positively charged residues within the TBS motifs is conserved throughout evolution. In silico analysis reveals that the TBS-1 and TBS-2 of TRPV1 can form helical structures and may play important role in TRPV1 function. CONCLUSIONS AND SIGNIFICANCE: Our analysis identifies the regions of TRPV1, which are important for structure-function relationship. This analysis indicates that tubulin binding sequence-1 (TBS-1 near the TRP-box forms a potential helix and the tubulin interactions with TRPV1 via TBS-1 have evolutionary significance. This interaction may be required for the proper channel function and regulation and may also have significance in the context

  11. Rapid Genotyping of β-tubulin Polymorphisms in Trichuris trichiura and Ascaris lumbricoides

    Science.gov (United States)

    Rashwan, Nour; Scott, Marilyn; Prichard, Roger

    2017-01-01

    Background The benzimidazole (BZ) anthelmintics, albendazole (ABZ) and mebendazole (MBZ) are the most common drugs used for treatment of soil-transmitted helminths (STHs). Their intensive use increases the possibility that BZ resistance may develop. In veterinary nematodes, BZ resistance is caused by a single nucleotide polymorphism (SNP) in the β-tubulin isotype 1 gene at codon position 200, 167 or 198, and these SNPs have also been correlated with poor response of human Trichuris trichiura to BZ treatment. It is important to be able to investigate the presence of resistance-associated SNPs in STHs before resistance becomes clinically established. Methods The objective of this study was to develop new genotyping assays to screen for the presence of β-tubulin SNPs in T. trichiura and Ascaris lumbricoides. Rapid, simple and accurate genotyping assays were developed based on the SmartAmp2 method. Primer sets were optimized and selected to distinguish the SNP-variant genotypes. After initial optimization on control plasmids, the feasibility of the assay was assessed in field samples from Haiti and Panama. Finally, spiked fecal samples were assessed to determine the tolerance of Aac polymerase to fecal inhibitors. Findings Rapid SNP genotyping assays were developed to target β-tubulin polymorphisms in T. trichiura and A. lumbricoides. The assays showed high sensitivity and specificity in field samples and also demonstrated high tolerance to PCR inhibitors in fecal samples. Conclusion These assays proved to be robust and efficient with the potential to be used as field tools for monitoring SNPs that could be associated with BZ resistance. However, further work is needed to validate the assays on large numbers of field samples before and after treatment. PMID:28081124

  12. Capzb2 interacts with beta-tubulin to regulate growth cone morphology and neurite outgrowth.

    Directory of Open Access Journals (Sweden)

    David A Davis

    2009-10-01

    Full Text Available Capping protein (CP is a heterodimer that regulates actin assembly by binding to the barbed end of F-actin. In cultured nonneuronal cells, each CP subunit plays a critical role in the organization and dynamics of lamellipodia and filopodia. Mutations in either alpha or beta CP subunit result in retinal degeneration in Drosophila. However, the function of CP subunits in mammalian neurons remains unclear. Here, we investigate the role of the beta CP subunit expressed in the brain, Capzb2, in growth cone morphology and neurite outgrowth. We found that silencing Capzb2 in hippocampal neurons resulted in short neurites and misshapen growth cones in which microtubules overgrew into the periphery and completely overlapped with F-actin. In searching for the mechanisms underlying these cytoskeletal abnormalities, we identified beta-tubulin as a novel binding partner of Capzb2 and demonstrated that Capzb2 decreases the rate and the extent of tubulin polymerization in vitro. We mapped the region of Capzb2 that was required for the subunit to interact with beta-tubulin and inhibit microtubule polymerization. A mutant Capzb2 lacking this region was able to bind F-actin and form a CP heterodimer with alpha2-subunit. However, this mutant was unable to rescue the growth cone and neurite outgrowth phenotypes caused by Capzb2 knockdown. Together, these data suggest that Capzb2 plays an important role in growth cone formation and neurite outgrowth and that the underlying mechanism may involve direct interaction between Capzb2 and microtubules.

  13. A Cell-Based Pharmacokinetics Assay for Evaluating Tubulin-Binding Drugs

    Science.gov (United States)

    Wang, Yuwei; Liu, Jihua; Zhang, Jun; Wang, Liping; Chan, Jonathon; Wang, Hai; Jin, Yi; Yu, Lei; Grainger, David W.; Ying, Wenbin

    2014-01-01

    Increasing evidence reveals that traditional pharmacokinetics parameters based on plasma drug concentrations are insufficient to reliably demonstrate accurate pharmacological effects of drugs in target organs or cells in vivo. This underscores the increasing need to improve the types and qualities of cellular pharmacokinetic information for drug preclinical screening and clinical efficacy assessments. Here we report a whole cell-based method to assess drugs that disturb microtubule dynamics to better understand different formulation-mediated intracellular drug release profiles. As proof of concept for this approach, we compared the well-known taxane class of anti-microtubule drugs based on paclitaxel (PTX), including clinically familiar albumin nanoparticle-based Abraxane™, and a polymer nanoparticle-based degradable paclitaxel carrier, poly(L-glutamic acid)-paclitaxel conjugate (PGA-PTX, also known as CT-2103) versus control PTX. This in vitro cell-based evaluation of PTX efficacy includes determining the cellular kinetics of tubulin polymerization, relative populations of cells under G2 mitotic arrest, cell proliferation and total cell viability. For these taxane tubulin-binding compounds, the kinetics of cell microtubule stabilization directly correlate with G2 arrest and cell proliferation, reflecting the kinetics and amounts of intracellular PTX release. Each individual cell-based dose-response experiment correlates with published, key therapeutic parameters and taken together, provide a comprehensive understanding of drug intracellular pharmacokinetics at both cellular and molecular levels. This whole cell-based evaluating method is convenient, quantitative and cost-effective for evaluating new formulations designed to optimize cellular pharmacokinetics for drugs perturbing tubulin polymerization as well as assisting in explaining drug mechanisms of action at cellular levels. PMID:24688312

  14. A cell-based pharmacokinetics assay for evaluating tubulin-binding drugs.

    Science.gov (United States)

    Wang, Yuwei; Liu, Jihua; Zhang, Jun; Wang, Liping; Chan, Jonathon; Wang, Hai; Jin, Yi; Yu, Lei; Grainger, David W; Ying, Wenbin

    2014-01-01

    Increasing evidence reveals that traditional pharmacokinetics parameters based on plasma drug concentrations are insufficient to reliably demonstrate accurate pharmacological effects of drugs in target organs or cells in vivo. This underscores the increasing need to improve the types and qualities of cellular pharmacokinetic information for drug preclinical screening and clinical efficacy assessments. Here we report a whole cell-based method to assess drugs that disturb microtubule dynamics to better understand different formulation-mediated intracellular drug release profiles. As proof of concept for this approach, we compared the well-known taxane class of anti-microtubule drugs based on paclitaxel (PTX), including clinically familiar albumin nanoparticle-based Abraxane™, and a polymer nanoparticle-based degradable paclitaxel carrier, poly(L-glutamic acid)-paclitaxel conjugate (PGA-PTX, also known as CT-2103) versus control PTX. This in vitro cell-based evaluation of PTX efficacy includes determining the cellular kinetics of tubulin polymerization, relative populations of cells under G2 mitotic arrest, cell proliferation and total cell viability. For these taxane tubulin-binding compounds, the kinetics of cell microtubule stabilization directly correlate with G2 arrest and cell proliferation, reflecting the kinetics and amounts of intracellular PTX release. Each individual cell-based dose-response experiment correlates with published, key therapeutic parameters and taken together, provide a comprehensive understanding of drug intracellular pharmacokinetics at both cellular and molecular levels. This whole cell-based evaluating method is convenient, quantitative and cost-effective for evaluating new formulations designed to optimize cellular pharmacokinetics for drugs perturbing tubulin polymerization as well as assisting in explaining drug mechanisms of action at cellular levels.

  15. Novel hybrid nocodazole analogues as tubulin polymerization inhibitors and their antiproliferative activity

    Science.gov (United States)

    Kale, Sangram S.; Jedhe, Ganesh S.; Meshram, Sachin N.; Santra, Manas K.; Hamel, Ernest; Sanjayan, Gangadhar J.

    2015-01-01

    We describe the design, synthesis and SAR profiling of a series of novel combretastatin–nocodazole conjugates as potential anticancer agents. The thiophene ring in the nocodazole moiety was replaced by a substituted phenyl ring from the combretastatin moiety to design novel hybrid analogues. The hydroxyl group at the ortho position in compounds 2, 3 and 4 was used as the conformationally locking tool by anticipated six-membered hydrogen bonding. The bioactivity profiles of all compounds as tubulin polymerization inhibitors and as antiproliferative agents against the A-549 human lung cancer cell line were investigated Compounds 1 and 4 showed μM IC50 values in both assays. PMID:25817588

  16. Divalent cation and ionic strength effects on Vinca alkaloid-induced tubulin self-association.

    OpenAIRE

    Lobert, S; Boyd, C A; Correia, J J

    1997-01-01

    We present here a systematic study of ionic strength and divalent cation effects on Vinca alkaloid-induced tubulin spiral formation. We used sedimentation velocity experiments and quantitative fitting of weight-average sedimentation coefficients versus free drug concentrations to obtain thermodynamic parameters under various solution conditions. The addition of 50-150 mM NaCl to our standard buffer (10 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM Mg, 50 microM GDP or GTP, pH 6.9) enhan...

  17. High LET Radiation Amplifies Centrosome Overduplication Through a Pathway of γ-Tubulin Monoubiquitination

    Energy Technology Data Exchange (ETDEWEB)

    Shimada, Mikio [Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Kyoto (Japan); Hirayama, Ryoichi [Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba (Japan); Komatsu, Kenshi, E-mail: komatsu@house.rbc.kyoto-u.ac.jp [Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Kyoto (Japan)

    2013-06-01

    Purpose: Radiation induces centrosome overduplication, leading to mitotic catastrophe and tumorigenesis. Because mitotic catastrophe is one of the major tumor cell killing factors in high linear energy transfer (LET) radiation therapy and long-term survivors from such treatment have a potential risk of secondary tumors, we investigated LET dependence of radiation-induced centrosome overduplication and the underlying mechanism. Methods and Materials: Carbon and iron ion beams (13-200 keV/μm) and γ-rays (0.5 keV/μm) were used as radiation sources. To count centrosomes after IR exposure, human U2OS and mouse NIH3T3 cells were immunostained with antibodies of γ-tubulin and centrin 2. Similarly, Nbs1-, Brca1-, Ku70-, and DNA-PKcs-deficient mouse cells and their counterpart wild-type cells were used for measurement of centrosome overduplication. Results: The number of excess centrosome-containing cells at interphase and the resulting multipolar spindle at mitosis were amplified with increased LET, reaching a maximum level of 100 keV/μm, followed by sharp decrease in frequency. Interestingly, Ku70 and DNA-PKcs deficiencies marginally affected the induction of centrosome overduplication, whereas the cell killings were significantly enhanced. This was in contrast to observation that high LET radiation significantly enhanced frequencies of centrosome overduplication in Nbs1- and Brca1-deficient cells. Because NBS1/BRCA1 is implicated in monoubiquitination of γ-tubulin, we subsequently tested whether it is affected by high LET radiation. As a result, monoubiquitination of γ-tubulin was abolished in 48 to 72 hours after exposure to high LET radiation, although γ-ray exposure slightly decreased it 48 hours postirradiation and was restored to a normal level at 72 hours. Conclusions: High LET radiation significantly reduces NBS1/BRCA1-mediated monoubiquitination of γ-tubulin and amplifies centrosome overduplication with a peak at 100 keV/μm. In contrast, Ku70 and DNA

  18. Insecticidal heterolignans--tubuline polymerization inhibitors with activity against chewing pests.

    Science.gov (United States)

    Frackenpohl, Jens; Adelt, Isabelle; Antonicek, Horst; Arnold, Christian; Behrmann, Patricia; Blaha, Nicole; Böhmer, Jutta; Gutbrod, Oliver; Hanke, Roman; Hohmann, Sabine; van Houtdreve, Marc; Lösel, Peter; Malsam, Olga; Melchers, Martin; Neufert, Valentina; Peschel, Elisabeth; Reckmann, Udo; Schenke, Thomas; Thiesen, Hans-Peter; Velten, Robert; Vogelsang, Kathrin; Weiss, Hans-Christoph

    2009-06-15

    Starting from natural product podophyllotoxin 1 substituted heterolignans were identified with promising insecticidal in vivo activity. The impact of substitution in each segment of the core structure was investigated in a detailed SAR study, and variation of substituents in both aromatic moieties afforded derivatives 5 and 43 with broad insecticidal activity against lepidopteran and coleopteran species. In vitro measurements supported by modeling studies indicate that heterolignans 3-134 act as tubuline polymerization inhibitors interacting with the colchicine-binding site. Insect specific structure-activity effects were observed showing that the insecticidal SAR described herein differs from reported cytotoxicity studies.

  19. New imidazoquinoxaline derivatives: Synthesis, biological evaluation on melanoma, effect on tubulin polymerization and structure-activity relationships.

    Science.gov (United States)

    Zghaib, Zahraa; Guichou, Jean-François; Vappiani, Johanna; Bec, Nicole; Hadj-Kaddour, Kamel; Vincent, Laure-Anaïs; Paniagua-Gayraud, Stéphanie; Larroque, Christian; Moarbess, Georges; Cuq, Pierre; Kassab, Issam; Deleuze-Masquéfa, Carine; Diab-Assaf, Mona; Bonnet, Pierre-Antoine

    2016-06-01

    Microtubules are considered as important targets of anticancer therapy. EAPB0503 and its structural imidazo[1,2-a]quinoxaline derivatives are major microtubule-interfering agents with potent anticancer activity. In this study, the synthesis of several new derivatives of EAPB0503 is described, and the anticancer efficacy of 13 novel derivatives on A375 human melanoma cell line is reported. All new compounds show significant antiproliferative activity with IC50 in the range of 0.077-122μM against human melanoma cell line (A375). Direct inhibition of tubulin polymerization assay in vitro is also assessed. Results show that compounds 6b, 6e, 6g, and EAPB0503 highly inhibit tubulin polymerization with percentages of inhibition of 99%, 98%, 90%, and 84% respectively. Structure-activity relationship studies within the series are also discussed in line with molecular docking studies into the colchicine-binding site of tubulin.

  20. In vitro and in vivo evaluation of tubulin inhibitors with non-small cell lung cancer pre-clinical models

    Directory of Open Access Journals (Sweden)

    Lama R

    2015-05-01

    Full Text Available Synthetic small molecule tubulin inhibitors have many advantages as novel anti-cancer agents compared to the current tubulin inhibitors generated from natural products. Our previous studies led to the design and synthesis of a series of novel tubulin inhibitors. Some of these compounds also inhibited heat shock protein 27 (Hsp27, and showed promising in vitro anti-cancer activities in several breast cancer cell lines at sub nano-molar concentrations. However, whether these compounds could suppress tumor growth in animals was not investigated yet. In the current study, to identify the best drug candidates, therapeutic efficacy of the representative compounds from previous analyses was evaluated using non-small cell lung cancer preclinical models. These agents dose-dependently inhibited the growth of lung cancer cells in both monolayer cultures and three-dimensional multicellular spheroids. Several compounds also showed promising tumor growth suppressive activity in nude mice xenograft model

  1. Probing FtsZ and tubulin with C8-substituted GTP analogs reveals differences in their nucleotide binding sites.

    Science.gov (United States)

    Läppchen, Tilman; Pinas, Victorine A; Hartog, Aloysius F; Koomen, Gerrit-Jan; Schaffner-Barbero, Claudia; Andreu, José Manuel; Trambaiolo, Daniel; Löwe, Jan; Juhem, Aurélie; Popov, Andrei V; den Blaauwen, Tanneke

    2008-02-01

    The cytoskeletal proteins, FtsZ and tubulin, play a pivotal role in prokaryotic cell division and eukaryotic chromosome segregation, respectively. Selective inhibitors of the GTP-dependent polymerization of FtsZ could constitute a new class of antibiotics, while several inhibitors of tubulin are widely used in antiproliferative therapy. In this work, we set out to identify selective inhibitors of FtsZ based on the structure of its natural ligand, GTP. We found that GTP analogs with small hydrophobic substituents at C8 of the nucleobase efficiently inhibit FtsZ polymerization, whereas they have an opposite effect on the polymerization of tubulin. The inhibitory activity of the GTP analogs on FtsZ polymerization allowed us to crystallize FtsZ in complex with C8-morpholino-GTP, revealing the binding mode of a GTP derivative containing a nonmodified triphosphate chain.

  2. Arabidopsis thaliana peroxidase N

    DEFF Research Database (Denmark)

    Mirza, Osman Asghar; Henriksen, A; Ostergaard, L

    2000-01-01

    The structure of the neutral peroxidase from Arabidopsis thaliana (ATP N) has been determined to a resolution of 1.9 A and a free R value of 20.5%. ATP N has the expected characteristic fold of the class III peroxidases, with a C(alpha) r.m.s.d. of 0.82 A when compared with horseradish peroxidase C...... (HRP C). HRP C is 54% identical to ATP N in sequence. When the structures of four class III plant peroxidases are superimposed, the regions with structural differences are non-randomly distributed; all are located in one half of the molecule. The architecture of the haem pocket of ATP N is very similar...... to that of HRP C, in agreement with the low small-molecule substrate specificity of all class III peroxidases. The structure of ATP N suggests that the pH dependence of the substrate turnover will differ from that of HRP C owing to differences in polarity of the residues in the substrate-access channel. Since...

  3. Low dose tubulin-binding drugs rescue peroxisome trafficking deficit in patient-derived stem cells in Hereditary Spastic Paraplegia

    Directory of Open Access Journals (Sweden)

    Yongjun Fan

    2014-05-01

    Full Text Available Hereditary Spastic Paraplegia (HSP is a genetically heterogeneous group of disorders, diagnosed by progressive gait disturbances with muscle weakness and spasticity, for which there are no treatments targeted at the underlying pathophysiology. Mutations in spastin are a common cause of HSP. Spastin is a microtubule-severing protein whose mutation in mouse causes defective axonal transport. In human patient-derived olfactory neurosphere-derived (ONS cells, spastin mutations lead to lower levels of acetylated α-tubulin, a marker of stabilised microtubules, and to slower speed of peroxisome trafficking. Here we screened multiple concentrations of four tubulin-binding drugs for their ability to rescue levels of acetylated α-tubulin in patient-derived ONS cells. Drug doses that restored acetylated α-tubulin to levels in control-derived ONS cells were then selected for their ability to rescue peroxisome trafficking deficits. Automated microscopic screening identified very low doses of the four drugs (0.5 nM taxol, 0.5 nM vinblastine, 2 nM epothilone D, 10 µM noscapine that rescued acetylated α-tubulin in patient-derived ONS cells. These same doses rescued peroxisome trafficking deficits, restoring peroxisome speeds to untreated control cell levels. These results demonstrate a novel approach for drug screening based on high throughput automated microscopy for acetylated α-tubulin followed by functional validation of microtubule-based peroxisome transport. From a clinical perspective, all the drugs tested are used clinically, but at much higher doses. Importantly, epothilone D and noscapine can enter the central nervous system, making them potential candidates for future clinical trials.

  4. Chromosomal proteins of Arabidopsis thaliana.

    Science.gov (United States)

    Moehs, C P; McElwain, E F; Spiker, S

    1988-07-01

    In plants with large genomes, each of the classes of the histones (H1, H2A, H2B, H3 and H4) are not unique polypeptides, but rather families of closely related proteins that are called histone variants. The small genome and preponderance of single-copy DNA in Arabidopsis thaliana has led us to ask if this plant has such families of histone variants. We have thus isolated histones from Arabidopsis and analyzed them on four polyacrylamide gel electrophoretic systems: an SDS system; an acetic acid-urea system; a Triton transverse gradient system; and a two-dimensional system combining SDS and Triton-acetic acid-urea systems. This approach has allowed us to identify all four of the nucleosomal core histones in Arabidopsis and to establish the existence of a set of H2A and H2B variants. Arabidopsis has at least four H2A variants and three H2B variants of distinct molecular weights as assessed by electrophoretic mobility on SDS-polyacrylamide gels. Thus, Arabidopsis displays a diversity in these histones similar to the diversity displayed by plants with larger genomes such as wheat.The high mobility group (HMG) non-histone chromatin proteins have attracted considerable attention because of the evidence implicating them as structural proteins of transcriptionally active chromatin. We have isolated a group of non-histone chromatin proteins from Arabidopsis that meet the operational criteria to be classed as HMG proteins and that cross-react with antisera to HMG proteins of wheat.

  5. Exploring the Contribution of Collective Motions to the Dynamics of Forced-Unfolding in Tubulin

    Science.gov (United States)

    Joshi, Harshad; Momin, Farhana; Haines, Kelly E.; Dima, Ruxandra I.

    2010-01-01

    Abstract Decomposition of the intrinsic dynamics of proteins into collective motions among distant regions of the protein structure provides a physically appealing approach that couples the dynamics of the system with its functional role. The cellular functions of microtubules (an essential component of the cytoskeleton in all eukaryotic cells) depend on their dynamic instability, which is altered by various factors among which applied forces are central. To shed light on the coupling between forces and the dynamic instability of microtubules, we focus on the investigation of the response of the microtubule subunits (tubulin) to applied forces. We address this point by adapting an approach designed to survey correlations for the equilibrium dynamics of proteins to the case of correlations for proteins forced-dynamics. The resulting collective motions in tubulin have a number of functional implications, such as the identification of long-range couplings with a role in blocking the dynamic instability of microtubules. A fundamental implication of our study for the life of a cell is that, to increase the likelihood of unraveling of large cytoskeletal filaments under physiological forces, molecular motors must use a combination of pulling and torsion rather than just pulling. PMID:20159162

  6. βIII-Tubulin Regulates Breast Cancer Metastases to the Brain.

    Science.gov (United States)

    Kanojia, Deepak; Morshed, Ramin A; Zhang, Lingjiao; Miska, Jason M; Qiao, Jian; Kim, Julius W; Pytel, Peter; Balyasnikova, Irina V; Lesniak, Maciej S; Ahmed, Atique U

    2015-05-01

    Brain metastases occur in about 10% to 30% of breast cancer patients, which culminates in a poor prognosis. It is, therefore, critical to understand the molecular mechanisms underlying brain metastatic processes to identify relevant targets. We hypothesized that breast cancer cells must express brain-associated markers that would enable their invasion and survival in the brain microenvironment. We assessed a panel of brain-predominant markers and found an elevation of several neuronal markers (βIII-tubulin, Nestin, and AchE) in brain metastatic breast cancer cells. Among these neuronal predominant markers, in silico analysis revealed overexpression of βIII-tubulin (TUBB3) in breast cancer brain metastases (BCBM) and its expression was significantly associated with distant metastases. TUBB3 knockdown studies were conducted in breast cancer models (MDA-Br, GLIM2, and MDA-MB-468), which revealed significant reduction in their invasive capabilities. MDA-Br cells with suppressed TUBB3 also demonstrated loss of key signaling molecules such as β3 integrin, pFAK, and pSrc in vitro. Furthermore, TUBB3 knockdown in a brain metastatic breast cancer cell line compromised its metastatic ability in vivo, and significantly improved survival in a brain metastasis model. These results implicate a critical role of TUBB3 in conferring brain metastatic potential to breast cancer cells.

  7. Phosphorylation of the yeast γ-tubulin Tub4 regulates microtubule function

    DEFF Research Database (Denmark)

    Lin, Tien-chen; Gombos, Linda; Neuner, Annett;

    2011-01-01

    The yeast ¿-tubulin Tub4 is assembled with Spc97 and Spc98 into the small Tub4 complex. The Tub4 complex binds via the receptor proteins Spc72 and Spc110 to the spindle pole body (SPB), the functional equivalent of the mammalian centrosome, where the Tub4 complex organizes cytoplasmic and nuclear...... microtubules. Little is known about the regulation of the Tub4 complex. Here, we isolated the Tub4 complex with the bound receptors from yeast cells. Analysis of the purified Tub4 complex by mass spectrometry identified more than 50 phosphorylation sites in Spc72, Spc97, Spc98, Spc110 and Tub4. To examine...... the functional relevance of the phosphorylation sites, phospho-mimicking and non-phosphorylatable mutations in Tub4, Spc97 and Spc98 were analyzed. Three phosphorylation sites in Tub4 were found to be critical for Tub4 stability and microtubule organization. One of the sites is highly conserved in ¿-tubulins...

  8. Specific expression of a β-tubulin gene (GhTub1) in developing cotton fibers

    Institute of Scientific and Technical Information of China (English)

    LI; Yuanli; (李园莉); SUN; Jie; (孙杰); LI; Chunhong; (李春红); ZHU; Yongqing; (朱勇清); XIA; Guixian; (夏桂先)

    2003-01-01

    A cDNA library was constructed using poly (A)+ RNA isolated from -1-15 DPA fibers of upland cotton (Gossypium hirsutum). The cDNA encoding a β-tubulin isoform (designated as GhTub1) was identified through EST search. Northern blot analysis using 3′-UTR of the cDNA as a gene-specific probe was performed to investigate the expression levels of GhTub1 in various organs and in the developing fibers. The results showed that GhTub1 gene was specifically expressed in cotton fiber cells. During fiber development, GhTub1 transcripts accumulated highlyat the stage of cell rapid elongation with the highest expression appearing at the time when fiber expansion reaches the peak rate. To probe the in vivo function of GhTub1, its cDNA was cloned in the yeast expression vector pREP1 and transformed into the fission yeast Schizosaccharomyces pombe. Overexpression of GhTub1 in yeast cells caused severe changes in the cell morphology. These results suggest that GhTub1 may play a role in the polar elongation of cotton fibers. To our knowledge, this is the first report on the fiber-specific transcript accumulation of a cotton β-tubulin gene.

  9. Motor neuron synapse and axon defects in a C. elegans alpha-tubulin mutant.

    Directory of Open Access Journals (Sweden)

    Renee Baran

    Full Text Available Regulation of microtubule dynamics underlies many fundamental cellular mechanisms including cell division, cell motility, and transport. In neurons, microtubules play key roles in cell migration, axon outgrowth, control of axon and synapse growth, and the regulated transport of vesicles and structural components of synapses. Loss of synapse and axon integrity and disruption of axon transport characterize many neurodegenerative diseases. Recently, mutations that specifically alter the assembly or stability of microtubules have been found to directly cause neurodevelopmental defects or neurodegeneration in vertebrates. We report here the characterization of a missense mutation in the C-terminal domain of C. elegans alpha-tubulin, tba-1(ju89, that disrupts motor neuron synapse and axon development. Mutant ju89 animals exhibit reduction in the number and size of neuromuscular synapses, altered locomotion, and defects in axon extension. Although null mutations of tba-1 show a nearly wild-type pattern, similar axon outgrowth defects were observed in animals lacking the beta-tubulin TBB-2. Genetic analysis reveals that tba-1(ju89 affects synapse development independent of its role in axon outgrowth. tba-1(ju89 is an altered function allele that most likely perturbs interactions between TBA-1 and specific microtubule-associated proteins that control microtubule dynamics and transport of components needed for synapse and axon growth.

  10. Divalent cation and ionic strength effects on Vinca alkaloid-induced tubulin self-association.

    Science.gov (United States)

    Lobert, S; Boyd, C A; Correia, J J

    1997-01-01

    We present here a systematic study of ionic strength and divalent cation effects on Vinca alkaloid-induced tubulin spiral formation. We used sedimentation velocity experiments and quantitative fitting of weight-average sedimentation coefficients versus free drug concentrations to obtain thermodynamic parameters under various solution conditions. The addition of 50-150 mM NaCl to our standard buffer (10 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM Mg, 50 microM GDP or GTP, pH 6.9) enhances overall vinblastine- or vincristine-induced tubulin self-association. As demonstrated in previous studies, GDP enhances overall self-association more than GTP, although in the presence of salt, GDP enhancement is reduced. For example, in 150 mM NaCl, GDP enhancement is 0.24 kcal/mol for vinblastine and 0.36 kcal/mol for vincristine versus an average enhancement of 0.87 (+/- 0.34) kcal/mol for the same drugs in the absence of salt. Wyman linkage analysis of experiments with vinblastine or vincristine over a range of NaCl concentrations showed a twofold increase in the change in NaCl bound to drug-induced spirals in the presence of GTP compared to GDP. These data indicate that GDP enhancement of Vinca alkaloid-induced tubulin self-association is due in part to electrostatic inhibition in the GTP state. In the absence of NaCl, we found that vinblastine and 1 mM Mn2+ or Ca2+ causes immediate condensation of tubulin. The predominant aggregates observed by electron microscopy are large sheets. This effect was not found with 1 mM Mg2+. At 100 microM cation concentrations (Mn2+, Mg2+, or Ca2+), GDP enhances vinblastine-induced spiral formation by 0.55 (+/- 0.26) kcal/mol. This effect is found only in K2, the association of liganded heterodimers at the ends of growing spirals. There is no GDP enhancement of K1, the binding of drug to heterodimer, although K1 is dependent upon the divalent cation concentration. NaCl diminishes tubulin condensation, probably by inhibiting lateral

  11. Class III β-tubulin overexpression within the tumor microenvironment is a prognostic biomarker for poor overall survival in ovarian cancer patients treated with neoadjuvant carboplatin/paclitaxel.

    Science.gov (United States)

    Roque, Dana M; Buza, Natalia; Glasgow, Michelle; Bellone, Stefania; Bortolomai, Ileana; Gasparrini, Sara; Cocco, Emiliano; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Rutherford, Thomas J; Schwartz, Peter E; Santin, Alessandro D

    2014-01-01

    Critics have suggested that neoadjuvant chemotherapy (NACT) followed by interval debulking may select for resistant clones or cancer stem cells when compared to primary cytoreduction. β-tubulins are chemotherapeutic targets of taxanes and epothilones. Class III β-tubulin overexpression has been linked to chemoresistance and hypoxia. Herein, we describe changes in class III β-tubulin in patients with advanced ovarian carcinoma in response to NACT, in relationship to clinical outcome, and between patients who underwent NACT versus primary debulking; we characterize in vitro chemosensitivity to paclitaxel/patupilone of cell lines established from this patient population, and class III β-tubulin expression following repeated exposure to paclitaxel. Using immunohistochemistry, we observed among 22 paired specimens obtained before/after NACT decreased expression of class III β-tubulin following therapy within stroma (p=0.07), but not tumor (p=0.63). Poor median overall survival was predicted by high levels of class III β-tubulin in both tumor (HR 3.66 [1.11,12.05], p=0.03) and stroma (HR 4.53 [1.28,16.1], p=0.02). Class III β-tubulin expression by quantitative-real-time-polymerase-chain-reaction was higher among patients who received NACT (n=12) compared to primary cytoreduction (n=14) (mean±SD fold-change: 491.2±115.9 vs. 224.1±55.66, p=0.037). In vitro subculture with paclitaxel resulted in class III β-tubulin upregulation, however, cell lines that overexpressed class III β-tubulin remained sensitive to patupilone. Overexpression of class III β-tubulin in patients dispositioned to NACT may thus identify an intrinsically aggressive phenotype, and predict poor overall survival and paclitaxel resistance. Decreases in stromal expression may represent normalization of the tumor microenvironment following therapy. Epothilones warrant study for patients who have received neoadjuvant carboplatin and paclitaxel.

  12. Oxalone and lactone moieties of podophyllotoxin exhibit properties of both the B and C rings of colchicine in its binding with tubulin.

    Science.gov (United States)

    Gupta, Suvroma; Das, Lalita; Datta, Ajit B; Poddar, Asim; Janik, Mark E; Bhattacharyya, Bhabatarak

    2006-05-23

    Thermodynamics of podophyllotoxin binding to tubulin and its multiple points of attachment with tubulin has been studied in detail using isothermal titration calorimetry. The calorimetric enthalpy of the association of podophyllotoxin with tubulin is negative and occurs with a negative heat capacity change (DeltaC(p) = -2.47 kJ mol(-)(1) K(-)(1)). The binding is unique with a simultaneous participation of both hydrophobic and hydrogen-bonding forces with unfavorable negative entropic contribution at higher temperature, favored with an enthalpy-entropy compensation. Interestingly, the binding of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone (AC, a colchicine analogue without the B ring) with tubulin is enthalpy-favored. However, the podophyllotoxin-tubulin association depending upon the temperature of the reaction has a favorable entropic and enthalpic component, which resembles both B- and C-ring properties of colchicine. On the basis of the crystal structure of the podophyllotoxin-tubulin complex, distance calculations have indicated a possible interaction between threonine 179 of alpha-tubulin and the hydroxy group on the D ring of podophyllotoxin. To confirm the involvement of the oxalone moiety as well as the lactone ring of podophyllotoxin in tubulin binding, analogues of podophyllotoxin are synthesized with methoxy substitution at the 4' position of ring D along with its isomer and another analogue epimerized at ring E. From these results, involvement of oxalone as well as the lactone ring of the drug in a specific orientation inclusive of ring A is indicated for podophyllotoxin-tubulin binding. Therefore, podophyllotoxin, like colchicine, behaves as a bifunctional ligand having properties of both the B and C rings of colchicine by making more than one point of attachment with the protein tubulin.

  13. Exploiting Natural Variation in Arabidopsis

    NARCIS (Netherlands)

    Molenaar, J.A.; Keurentjes, J.J.B.

    2014-01-01

    Natural variation for many traits is present within the species Arabidopsis thaliana . This chapter describes the use of natural variation to elucidate genes underlying the regulation of quantitative traits. It deals with the development and use of mapping populations, the detection and handling of

  14. Exploiting natural variation in Arabidopsis

    NARCIS (Netherlands)

    J.A. Molenaar; J.J.B. Keurentjes

    2014-01-01

    Natural variation for many traits is present within the species Arabidopsis thaliana. This chapter describes the use of natural variation to elucidate genes underlying the regulation of quantitative traits. It deals with the development and use of mapping populations, the detection and handling of g

  15. The salty tale of Arabidopsis.

    Science.gov (United States)

    Sanders, D

    2000-06-29

    High concentrations of sodium chloride are toxic to most plant species. New insights into the mechanisms by which plants tolerate salt have emerged from the identification of genes in Arabidopsis thaliana that play a critical part in physiological resistance to salt.

  16. Rapid and dynamic subcellular reorganization following mechanical stimulation of Arabidopsis epidermal cells mimics responses to fungal and oomycete attack

    Directory of Open Access Journals (Sweden)

    Takemoto Daigo

    2008-06-01

    Full Text Available Abstract Background Plant cells respond to the presence of potential fungal or oomycete pathogens by mounting a basal defence response that involves aggregation of cytoplasm, reorganization of cytoskeletal, endomembrane and other cell components and development of cell wall appositions beneath the infection site. This response is induced by non-adapted, avirulent and virulent pathogens alike, and in the majority of cases achieves penetration resistance against the microorganism on the plant surface. To explore the nature of signals that trigger this subcellular response and to determine the timing of its induction, we have monitored the reorganization of GFP-tagged actin, microtubules, endoplasmic reticulum (ER and peroxisomes in Arabidopsis plants – after touching the epidermal surface with a microneedle. Results Within 3 to 5 minutes of touching the surface of Arabidopsis cotyledon epidermal cells with fine glass or tungsten needles, actin microfilaments, ER and peroxisomes began to accumulate beneath the point of contact with the needle. Formation of a dense patch of actin was followed by focusing of actin cables on the site of contact. Touching the cell surface induced localized depolymerization of microtubules to form a microtubule-depleted zone surrounding a dense patch of GFP-tubulin beneath the needle tip. The concentration of actin, GFP-tubulin, ER and peroxisomes remained focused on the contact site as the needle moved across the cell surface and quickly dispersed when the needle was removed. Conclusion Our results show that plant cells can detect the gentle pressure of a microneedle on the epidermal cell surface and respond by reorganizing subcellular components in a manner similar to that induced during attack by potential fungal or oomycete pathogens. The results of our study indicate that during plant-pathogen interactions, the basal defence response may be induced by the plant's perception of the physical force exerted by the

  17. γ-tubulin is differentially expressed in mitotic and non-mitotic cardiomyocytes in the regenerating zebrafish heart

    Directory of Open Access Journals (Sweden)

    Pauline Sallin

    2015-06-01

    Full Text Available This data article contains complementary figures related to the research article entitled, “ A dual epimorphic and compensatory mode of heart regeneration” ([10], http://dx.doi.org/10.1016/j.ydbio.2014.12.002, which presents a spatial and temporal characterization of cardiomyocyte proliferation and dedifferentiation after cryoinjury-induced myocardial infarction. This study demonstrated that mitotic divisions occur in cardiac cells at distinct differentiation status, namely in dedifferentiated cells at the injury border as well as in mature cardiac cells within the remaining intact myocardium. One of the important aspects supporting our conclusions is a characterization of proteins that are upregulated during mitosis in the regenerating hearts. The data presented here reveal a dynamic change in the expression level and in the subcellular distribution of γ-tubulin between mitotic and non-mitotic cardiac cells. We report that in the non-mitotic cells, γ-tubulin expression is restricted to the centrosome. By contrast, during the mitosis, γ-tubulin strongly expands its localization within the spindle apparatus that interacts with the condensed chromosomes. We demonstrated that the differential distribution of γ-tubulin in non-mitotic and mitotic cells requires adjusted image processing for the appropriate visualization of both expression patterns in the same histological specimens.

  18. Class III β-tubulin in advanced NSCLC of adenocarcinoma subtype predicts superior outcome in a randomized trial

    DEFF Research Database (Denmark)

    Vilmar, Adam Christian; Santoni-Rugiu, Eric; Sørensen, Jens Benn

    2011-01-01

    Platinum-based doublets are the cornerstone of treatment in advanced non-small-cell lung cancer (NSCLC) and often include vinorelbine or taxanes. A predictive biomarker is greatly needed to select chemotherapy-sensitive patients for these microtubule-interfering agents. Class III ß-tubulin (TUBB3...

  19. GTP analogue inhibits polymerization and GTPase activity of the bacterial protein FtsZ without affecting its eukaryotic homologue tubulin.

    Science.gov (United States)

    Läppchen, Tilman; Hartog, Aloysius F; Pinas, Victorine A; Koomen, Gerrit-Jan; den Blaauwen, Tanneke

    2005-05-31

    The prokaryotic tubulin homologue FtsZ plays a key role in bacterial cell division. Selective inhibitors of the GTP-dependent polymerization of FtsZ are expected to result in a new class of antibacterial agents. One of the challenges is to identify compounds which do not affect the function of tubulin and various other GTPases in eukaryotic cells. We have designed a novel inhibitor of FtsZ polymerization based on the structure of the natural substrate GTP. The inhibitory activity of 8-bromoguanosine 5'-triphosphate (BrGTP) was characterized by a coupled assay, which allows simultaneous detection of the extent of polymerization (via light scattering) and GTPase activity (via release of inorganic phosphate). We found that BrGTP acts as a competitive inhibitor of both FtsZ polymerization and GTPase activity with a Ki for GTPase activity of 31.8 +/- 4.1 microM. The observation that BrGTP seems not to inhibit tubulin assembly suggests a structural difference of the GTP-binding pockets of FtsZ and tubulin.

  20. GTP analogue inhibits polymerization and GTPase activity of the bacterial protein FtsZ without affecting its eukaryotic homologue tubulin.

    NARCIS (Netherlands)

    Läppchen, T.; Hartog, A.F.; Pinas, V.; Koomen, G.J.; den Blaauwen, T.

    2005-01-01

    The prokaryotic tubulin homologue FtsZ plays a key role in bacterial cell division. Selective inhibitors of the GTP-dependent polymerization of FtsZ are expected to result in a new class of antibacterial agents. One of the challenges is to identify compounds which do not affect the function of tubul

  1. Tubulin bond energies and microtubule biomechanics determined from nanoindentation in silico

    CERN Document Server

    Kononova, Olga; Theisen, Kelly E; Marx, Kenneth A; Dima, Ruxandra I; Ataullakhanov, Fazly I; Grishchuk, Ekaterina L; Barsegov, Valeri

    2015-01-01

    Microtubules, the primary components of the chromosome segregation machinery, are stabilized by longitudinal and lateral non-covalent bonds between the tubulin subunits. However, the thermodynamics of these bonds and the microtubule physico-chemical properties are poorly understood. Here, we explore the biomechanics of microtubule polymers using multiscale computational modeling and nanoindentations in silico of a contiguous microtubule fragment. A close match between the simulated and experimental force-deformation spectra enabled us to correlate the microtubule biomechanics with dynamic structural transitions at the nanoscale. Our mechanical testing revealed that the compressed MT behaves as a system of rigid elements interconnected through a network of lateral and longitudinal elastic bonds. The initial regime of continuous elastic deformation of the microtubule is followed by the transition regime, during which the microtubule lattice undergoes discrete structural changes, which include first the reversib...

  2. ESTs library from embryonic stages reveals tubulin and reflectin diversity in Sepia officinalis (Mollusca — Cephalopoda).

    Science.gov (United States)

    Bassaglia, Yann; Bekel, Thomas; Da Silva, Corinne; Poulain, Julie; Andouche, Aude; Navet, Sandra; Bonnaud, Laure

    2012-05-01

    New molecular resources regarding the so-called “non-standard models” in biology extend the present knowledge and are essential for molecular evolution and diversity studies (especially during the development) and evolutionary inferences about these zoological groups, or more practically for their fruitful management. Sepia officinalis, an economically important cephalopod species, is emerging as a new lophotrochozoan developmental model. We developed a large set of expressed sequence tags (ESTs) from embryonic stages of S. officinalis, yielding 19,780 non-redundant sequences (NRS). Around 75% of these sequences have no homologs in existing available databases. This set is the first developmental ESTs library in cephalopods. By exploring these NRS for tubulin, a generic protein family, and reflectin, a cephalopod specific protein family,we point out for both families a striking molecular diversity in S. officinalis.

  3. [1,2]Oxazolo[5,4-e]isoindoles as promising tubulin polymerization inhibitors.

    Science.gov (United States)

    Spanò, Virginia; Pennati, Marzia; Parrino, Barbara; Carbone, Anna; Montalbano, Alessandra; Lopergolo, Alessia; Zuco, Valentina; Cominetti, Denis; Diana, Patrizia; Cirrincione, Girolamo; Zaffaroni, Nadia; Barraja, Paola

    2016-11-29

    A series of [1,2]Oxazolo [5,4-e]isoindoles has been synthesized through a versatile and high yielding sequence. All the new structures showed in the (1)HNMR spectra, the typical signal in the 8.34-8.47 ppm attributable to the H-3 of the [1,2]oxazole moiety. Among all derivatives, methoxy benzyl substituents at positions 3 and 4 or/and 5 were very effective in reducing the growth of different tumor cell lines, including diffuse malignant peritoneal mesothelioma (DMPM), an uncommon and rapidly malignancy poorly responsive to available therapeutic options. The most active compound 6j was found to impair tubulin polymerization, cause cell cycle arrest at G2/M phase and induce apoptosis in DMPM cells, making it as a new lead for the discovery of new potent antimitotic drugs.

  4. PAX3 inhibits β-Tubulin-III expression and neuronal differentiation of neural stem cell.

    Science.gov (United States)

    Cao, Sixian; Du, Jinfeng; Lv, Yan; Lin, Hengrong; Mao, Zuming; Xu, Man; Liu, Mei; Liu, Yan

    2017-02-20

    PAX3 functions at the nodal point in neural stem cell maintenance and differentiation. Using bioinformatics methods, we identified PAX3 as a potential regulator of β-Tubulin-III (TUBB3) gene transcription, and the results indicated that PAX3 might be involved in neural stem cell (NSC) differentiation by orchestrating the expression of cytoskeletal proteins. In the present study, we reported that PAX3 could inhibit the differentiation of NSCs and the expression of TUBB3. Further, using luciferase and electrophoretic mobility shift assays, we demonstrated that PAX3 could bind to the promoter region of TUBB3 and inhibit TUBB3 transcription. Finally, we confirmed that PAX3 could bind to the promoter region of endogenous TUBB3 in the native chromatin of NSCs. These findings indicated that PAX3 is a pivotal factor targeting various molecules during differentiation of NSCs in vitro.

  5. Tubulin and actin interplay at the T cell and Antigen-presenting cell interface

    Directory of Open Access Journals (Sweden)

    Noa B Martín-Cófreces

    2011-07-01

    Full Text Available T cells reorganize their actin and tubulin-based cytoskeletons to provide a physical basis to the immune synapse. However, growing evidence shows that their roles on T cell activation are more dynamic than merely serving as tracks or scaffold for different molecules. The cross-talk between both skeletons may be important for the formation and movement of the lamella at the IS by increasing the adhesion of the T cell to the APC, thus favoring the transport of components towards the plasma membrane and in turn regulating the T-APC intercellular communication. Microtubules and F-actin appear to be essential for the transport of the different signaling microclusters along the membrane, therefore facilitating the propagation of the signal. Finally, they can also be important for regulating the endocytosis, recycling and degradation of the TCR signaling machinery, thus helping both to sustain the activated state and to switch it off.

  6. In silico inspired design and synthesis of a novel tubulin-binding anti-cancer drug: folate conjugated noscapine (Targetin)

    Science.gov (United States)

    Naik, Pradeep K.; Lopus, Manu; Aneja, Ritu; Vangapandu, Surya N.; Joshi, Harish C.

    2012-02-01

    Our screen for tubulin-binding small molecules that do not depolymerize bulk cellular microtubules, but based upon structural features of well known microtubule-depolymerizing colchicine and podophyllotoxin, revealed tubulin binding anti-cancer property of noscapine (Ye et al. in Proc Natl Acad Sci USA 95:2280-2286, 1998). Guided by molecular modelling calculations and structure-activity relationships we conjugated at C9 of noscapine, a folate group—a ligand for cellular folate receptor alpha (FRα). FRα is over-expressed on some solid tumours such as ovarian epithelial cancers. Molecular docking experiments predicted that a folate conjugated noscapine (Targetin) accommodated well inside the binding cavity (docking score -11.295 kcal/mol) at the interface between α- and β-tubulin. The bulky folate moiety of Targetin is extended toward lumen of microtubules. The binding free energy (Δ G bind) computed based on molecular mechanics energy minimization was -221.01 kcal/mol that revealed favourable interaction of Targetin with the receptor. Chemical synthesis, tubulin-binding experiments, and anti-cancer activity in vitro corroborate fully well with the molecular modelling experiments. Targetin binds tubulin with a dissociation constant ( K d value) of 149 ± 3.0 μM and decreases the transition frequencies between growth and shortening phases of microtubule assembly dynamics at concentrations that do not alter the total polymer mass. Cancer cells in general were more sensitive to Targetin compared with the founding compound noscapine (IC50 in the range of 15-40 μM). Quite strikingly, ovarian cancer cells (SKOV3 and A2780), known to overexpress FRα, were much more sensitive to targetin (IC50 in the range of 0.3-1.5 μM).

  7. In silico inspired design and synthesis of a novel tubulin-binding anti-cancer drug: folate conjugated noscapine (Targetin).

    Science.gov (United States)

    Naik, Pradeep K; Lopus, Manu; Aneja, Ritu; Vangapandu, Surya N; Joshi, Harish C

    2012-02-01

    Our screen for tubulin-binding small molecules that do not depolymerize bulk cellular microtubules, but based upon structural features of well known microtubule-depolymerizing colchicine and podophyllotoxin, revealed tubulin binding anti-cancer property of noscapine (Ye et al. in Proc Natl Acad Sci USA 95:2280-2286, 1998). Guided by molecular modelling calculations and structure-activity relationships we conjugated at C9 of noscapine, a folate group-a ligand for cellular folate receptor alpha (FRα). FRα is over-expressed on some solid tumours such as ovarian epithelial cancers. Molecular docking experiments predicted that a folate conjugated noscapine (Targetin) accommodated well inside the binding cavity (docking score -11.295 kcal/mol) at the interface between α- and β-tubulin. The bulky folate moiety of Targetin is extended toward lumen of microtubules. The binding free energy (ΔG (bind)) computed based on molecular mechanics energy minimization was -221.01 kcal/mol that revealed favourable interaction of Targetin with the receptor. Chemical synthesis, tubulin-binding experiments, and anti-cancer activity in vitro corroborate fully well with the molecular modelling experiments. Targetin binds tubulin with a dissociation constant (K (d) value) of 149 ± 3.0 μM and decreases the transition frequencies between growth and shortening phases of microtubule assembly dynamics at concentrations that do not alter the total polymer mass. Cancer cells in general were more sensitive to Targetin compared with the founding compound noscapine (IC(50) in the range of 15-40 μM). Quite strikingly, ovarian cancer cells (SKOV3 and A2780), known to overexpress FRα, were much more sensitive to targetin (IC(50) in the range of 0.3-1.5 μM).

  8. Intracellular ascorbate tightens the endothelial permeability barrier through Epac1 and the tubulin cytoskeleton.

    Science.gov (United States)

    Parker, William H; Rhea, Elizabeth Meredith; Qu, Zhi-Chao; Hecker, Morgan R; May, James M

    2016-10-01

    Vitamin C, or ascorbic acid, both tightens the endothelial permeability barrier in basal cells and also prevents barrier leak induced by inflammatory agents. Barrier tightening by ascorbate in basal endothelial cells requires nitric oxide derived from activation of nitric oxide synthase. Although ascorbate did not affect cyclic AMP levels in our previous study, there remains a question of whether it might activate downstream cyclic AMP-dependent pathways. In this work, we found in both primary and immortalized cultured endothelial cells that ascorbate tightened the endothelial permeability barrier by ∼30%. In human umbilical vein endothelial cells, this occurred at what are likely physiologic intracellular ascorbate concentrations. In so doing, ascorbate decreased measures of oxidative stress and also flattened the cells to increase cell-to-cell contact. Inhibition of downstream cyclic AMP-dependent proteins via protein kinase A did not prevent ascorbate from tightening the endothelial permeability barrier, whereas inhibition of Epac1 did block the ascorbate effect. Although Epac1 was required, its mediator Rap1 was not activated. Furthermore, ascorbate acutely stabilized microtubules during depolymerization induced by colchicine and nocodazole. Over several days in culture, ascorbate also increased the amount of stable acetylated α-tubulin. Microtubule stabilization was further suggested by the finding that ascorbate increased the amount of Epac1 bound to α-tubulin. These results suggest that physiologic ascorbate concentrations tighten the endothelial permeability barrier in unstimulated cells by stabilizing microtubules in a manner downstream of cyclic AMP that might be due both to increasing nitric oxide availability and to scavenging of reactive oxygen or nitrogen species.

  9. Structural complexity of filaments formed from the actin and tubulin folds

    Science.gov (United States)

    Jiang, Shimin; Ghoshdastider, Umesh; Narita, Akihiro; Popp, David

    2016-01-01

    ABSTRACT From yeast to man, an evolutionary distance of 1.3 billion years, the F-actin filament structure has been conserved largely in line with the 94% sequence identity. The situation is entirely different in bacteria. In comparison to eukaryotic actins, the bacterial actin-like proteins (ALPs) show medium to low levels of sequence identity. This is extreme in the case of the ParM family of proteins, which often display less than 20% identity. ParMs are plasmid segregation proteins that form the polymerizing motors that propel pairs of plasmids to the extremities of a cell prior to cell division, ensuring faithful inheritance of the plasmid. Recently, exotic ParM filament structures have been elucidated that show ParM filament geometries are not limited to the standard polar pair of strands typified by actin. Four-stranded non-polar ParM filaments existing as open or closed nanotubules are found in Clostridium tetani and Bacillus thuringiensis, respectively. These diverse architectures indicate that the actin fold is capable of forming a large variety of filament morphologies, and that the conception of the “actin” filament has been heavily influenced by its conservation in eukaryotes. Here, we review the history of the structure determination of the eukaryotic actin filament to give a sense of context for the discovery of the new ParM filament structures. We describe the novel ParM geometries and predict that even more complex actin-like filaments may exist in bacteria. Finally, we compare the architectures of filaments arising from the actin and tubulin folds and conclude that the basic units possess similar properties that can each form a range of structures. Thus, the use of the actin fold in microfilaments and the tubulin fold for microtubules likely arose from a wider range of filament possibilities, but became entrenched as those architectures in early eukaryotes. PMID:28042378

  10. Expression of recombinant alpha and beta tubulins from the yew Taxus cuspidata and analysis of the microtubule assembly in the presence of taxol.

    Science.gov (United States)

    Kudo, Yuma; Abe, Akihiro; Ito, Kumiko; Cho, Yuko; Yotsu-Yamashita, Mari; Konoki, Keiichi

    2014-01-01

    Taxol was originally isolated from the yew Taxus brevifolia. Because taxol inhibits the depolymerization of microtubules, the presence of a self-resistance mechanism in Taxus spp. was hypothesized. The cloning of the cDNA for alpha and beta tubulins from Taxus cuspidata and those from the human embryonic kidney cell line HEK293T revealed that the (26)Asp, (359)Arg, and (361)Leu residues in the human beta tubulin, which are important for taxol binding, were replaced with Glu, Trp, and Met in the beta tubulin of T. cuspidata, respectively. The microtubule assembly of the recombinant alpha and beta tubulins was monitored turbidimetrically, and the results clearly demonstrated that the microtubule from T. cuspidata is less sensitive to taxol than that from HEK293T cells. The Taxus microtubule composed of the wild-type alpha tubulin and the beta tubulin with the E26D mutation restored the sensitivity to taxol. We thus postulated that the mutation identified in the beta tubulin of T. cuspidata plays a role in the self-resistance of this species against taxol.

  11. Water-stress-induced inhibition of α-tubulin gene expression during growth, and its implications for reproductive success in rice.

    Science.gov (United States)

    Sheoran, Inder S; Koonjul, Priyum; Attieh, Jihad; Saini, Hargurdeep S

    2014-07-01

    A drought-suppressed cDNA (RiP-3), encoding a putative α-tubulin protein was isolated from rice panicle at pollen-mother-cell meiosis stage. Analysis of its deduced amino acid sequence showed all the typical structural motifs for plant α-tubulins. The expression of α-tubulin transcripts was observed in all the reproductive organs of rice panicle, and in 5- or 15-day old seedlings, but not in mature leaves. Expression levels were positively correlated with the regions and periods of high growth, and the transcript level declined in parallel with drought-induced reduction in growth rates in all tissues examined. Immunoblot analysis of proteins separated by SDS-PAGE with anti-α-tubulin monoclonal antibody showed that the level of protein paralleled the changes in the transcript abundance in these organs. In situ immunolocalization of the α-tubulin protein in sections of the basal part of 5-day old seedlings showed that the highest levels of the protein were associated with the fastest growing leaf whorls, and the protein level declined upon a brief episode of water stress. Given the known critical role of tubulin in cell division and elongation, the results indicate that the expression of α-tubulin gene may be part of the events that suppress panicle elongation during water deficit, which is in turn a suspected cause of male reproductive failure and yield reduction in rice.

  12. Dependency of microtubule-associated proteins (MAPs) for tubulin stability and assembly; use of estramustine phosphate in the study of microtubules.

    Science.gov (United States)

    Fridén, B; Wallin, M

    1991-07-10

    Microtubule-associated proteins (MAPs) were separated from tubulin with several different methods. The ability of the isolated MAPs to reinduce assembly of phosphocellulose purified tubulin differed markedly between the different methods. MAPs isolated by addition of 0.35 M NaCl to taxol-stabilized microtubules stimulated tubulin assembly most effectively, while addition of 0.6 M NaCl produced MAPs with a substantially lower ability to stimulate tubulin assembly. The second best preparation was achieved with phosphocellulose chromatographic separation of MAPs with 0.6 M NaCl elution. The addition of estramustine phosphate to microtubules reconstituted of MAPs prepared by 0.35 M NaCl or phosphocellulose chromatography, induced less disassembly than for microtubules assembled from unseparated proteins, and was almost without effect on microtubules reconstituted from MAPs prepared by taxol and 0.6 M NaCl. Estramustine phosphate binds to the tubulin binding part of the MAPs, and the results do therefore indicate that the MAPs are altered by the separation methods. Since the MAPs are regarded as highly stable molecules, one probable alteration could be aggregation of the MAPs, as also indicated by the results. The purified tubulin itself seemed not to be affected by the phosphocellulose purification, since the microtubule proteins were unchanged by the low buffer strenght used during the cromatography. However, the assembly competence after a prolonged incubation of the microtubule proteins at 4 degrees C was dependent on intact bindings between the tubulin and MAPs.

  13. Reference: 710 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available n factor family in Arabidopsis (Arabidopsis thaliana). Treatment with abscisic acid (ABA) induced AtMYB44 tr...anscript accumulation within 30 min. The gene was also activated under various abiotic stre...sses, such as dehydration, low temperature, and salinity. In transgenic Arabidopsis carrying an At...MYB44 promoter-driven beta-glucuronidase (GUS) construct, strong GUS activity was observed in the vasculature... and leaf epidermal guard cells. Transgenic Arabidopsis overexpressing AtMYB44 is more

  14. Rearrangements of microtubule cytoskeleton in stomatal closure of Arabidopsis induced by nitric oxide

    Institute of Scientific and Technical Information of China (English)

    ZHANG YongMei; WU ZhongYi; WANG XueChen; YU Rong

    2008-01-01

    NO (nitric oxide), known as a key signal molecule in plant, plays important roles in regulation of stomatal movement. In this study, microtubule dynamics and its possible mechanism in the NO signal pathway were investigated. The results were as follows: (ⅰ) In vivo stomatal aperture assays revealed that both vinblastine (microtubule-disrupting drug) and SNP (exogenous NO donor) prevented stomatal opening in the light, and vinblastine even could enhance the inhibitory effect of SNP, whereas taxol (a microtubule-stabilizing agent) was able to reduce this effect; (ⅱ) microtubules in the opening Arabi-dopsis guard cells expressing GFP:α-tubulin-6 (AtGFP:α-tubulin-6) were organized in parallel, straight and dense bundles, radiating from the ventral side to the dorsal side, and most of them were localized perpendicularly to the ventral wall; (ⅲ) under the same environmental conditions, treated with SNP for 30 min, the radial arrays of microtubules in guard cells began to break down, twisted partially and be-came oblique or exhibited a random pattern; (ⅳ) furthermore, the involvement of cytosolic Ca2+ in this event was tested. Stomatal aperture assays revealed that BAPTA-AM (a chelator of Ca2+) greatly sup-pressed the effect of NO on stomatal closure; however, it did not show the same function on stomatal closure induced by vinblastine. When BAPTA-AM was added to the SNP-pretreated solution, the SNP-induced disordered microtubulue cytoskeleton in guard cells underwent rearrangement in a time-dependent manner. After 30 min of treatment with BAPTA-AM, the cortical microtubules resumed the original radial distribution, almost the same as the control. All this indicates that NO may promote rearrangement of microtubule cytoskeleton via elevation of [Ca2+]cyt (free Ca2+ concentration in the cy-toplasm), finally leading to stomatal closure.

  15. Cell type- and isotype-specific expression and regulation of β-tubulins in primary olfactory ensheathing cells and Schwann cells in vitro.

    Science.gov (United States)

    Omar, Mohamed; Hansmann, Florian; Kreutzer, Robert; Kreutzer, Mihaela; Brandes, Gudrun; Wewetzer, Konstantin

    2013-05-01

    Olfactory ensheathing cells (OECs) and Schwann cells (SCs) are closely-related cell types with regeneration-promoting properties. Comparative gene expression analysis is particularly relevant since it may explain cell type-specific effects and guide the use of each cell type into special clinical applications. In the present study, we focused on β-tubulin isotype expression in primary adult canine glia as a translational large animal model. β-tubulins so far have been studied mainly in non-neuronal tumors and implied in tumorigenic growth. We show here that primary OECs and SCs expressed βII-V isotype mRNA. Interestingly, βIII-tubulin mRNA and protein expression was high in OECs and low in SCs, while fibroblast growth factor-2 (FGF-2) induced its down-regulation in both cell types to the same extent. This was in contrast to βV-tubulin mRNA which was similarly expressed in both cell types and unaltered by FGF-2. Immunocytochemical analysis revealed that OEC cultures contained a higher percentage of βIII-tubulin-positive cells compared to SC cultures. Addition of FGF-2 reduced the number of βIII-tubulin-positive cells in both cultures and significantly increased the percentage of cells with a multipolar morphology. Taken together, we demonstrate cell type-specific expression (βIII) and isotype-specific regulation (βIII, βV) of β-tubulin isotypes in OECs and SCs. While differential expression of βIII-tubulin in primary glial cell types with identical proliferative behaviour argues for novel functions unrelated to tumorigenic growth, strong βIII-tubulin expression in OECs may help to explain the specific properties of this glial cell type.

  16. G protein betagamma subunits interact with alphabeta- and gamma-tubulin and play a role in microtubule assembly in PC12 cells.

    Science.gov (United States)

    Montoya, Valentina; Gutierrez, Christina; Najera, Omar; Leony, Denisse; Varela-Ramirez, Armando; Popova, Juliana; Rasenick, Mark M; Das, Siddhartha; Roychowdhury, Sukla

    2007-12-01

    The betagamma subunit of G proteins (Gbetagamma) is known to transfer signals from cell surface receptors to intracellular effector molecules. Recent results suggest that Gbetagamma also interacts with microtubules and is involved in the regulation of the mitotic spindle. In the current study, the anti-microtubular drug nocodazole was employed to investigate the mechanism by which Gbetagamma interacts with tubulin and its possible implications in microtubule assembly in cultured PC12 cells. Nocodazole-induced depolymerization of microtubules drastically inhibited the interaction between Gbetagamma and tubulin. Gbetagamma was preferentially bound to microtubules and treatment with nocodazole suggested that the dissociation of Gbetagamma from microtubules is an early step in the depolymerization process. When microtubules were allowed to recover after removal of nocodazole, the tubulin-Gbetagamma interaction was restored. Unlike Gbetagamma, however, the interaction between tubulin and the alpha subunit of the Gs protein (Gsalpha) was not inhibited by nocodazole, indicating that the inhibition of tubulin-Gbetagamma interactions during microtubule depolymerization is selective. We found that Gbetagamma also interacts with gamma-tubulin, colocalizes with gamma-tubulin in centrosomes, and co-sediments in centrosomal fractions. The interaction between Gbetagamma and gamma-tubulin was unaffected by nocodazole, suggesting that the Gbetagamma-gamma-tubulin interaction is not dependent on assembled microtubules. Taken together, our results suggest that Gbetagamma may play an important and definitive role in microtubule assembly and/or stability. We propose that betagamma-microtubule interaction is an important step for G protein-mediated cell activation. These results may also provide new insights into the mechanism of action of anti-microtubule drugs.

  17. Design, synthesis and biological evaluation of a simplified fluorescently labeled discodermolide as a molecular probe to study the binding of discodermolide to tubulin.

    Science.gov (United States)

    Qi, Jun; Blanden, Adam R; Bane, Susan; Kingston, David G I

    2011-09-01

    The design, synthesis, and biological evaluation of a simplified fluorescently labeled discodermolide analogue possessing a dimethylaminobenzoyl fluorophore has been achieved. Stereoselective Suzuki coupling and Horner-Wadsworth-Emmons reaction comprised the key tactics for its construction. The analogue exhibited qualitatively similar activity to paclitaxel in a tubulin assembly assay, and it can thus be used as a fluorescent molecular probe to explore the local environment of the discodermolide binding site on tubulin. The results of fluorescence measurements on the tubulin-bound analogue are reported.

  18. Integrating docking and molecular dynamics approaches for a series of proline-based 2,5-diketopiperazines as novel αβ-tubulin inhibitors.

    Science.gov (United States)

    Fani, Najmeh; Bordbar, Abdol-Khalegh; Ghayeb, Yousef; Sepehri, Saghi

    2015-01-01

    In this work, docking tools were utilized in order to study the binding properties of more than five hundred of proline-based 2,5-diketopiperazine in the binding site of αβ-tubulin. Results revealed that 20 compounds among them showed lower binding energies in comparison with Tryprostatin-A, a well known tubulin inhibitor and therefore could be potential inhibitors of tubulin. However, the precise evaluation of binding poses represents the similar binding modes for all of these compounds and Tryprostatin-A. Finally, the best docked complex was subjected to a 25 ns molecular dynamics simulation to further validate the proposed binding mode of this compound.

  19. An International Bioinformatics Infrastructure to Underpin the Arabidopsis Community

    Science.gov (United States)

    The future bioinformatics needs of the Arabidopsis community as well as those of other scientific communities that depend on Arabidopsis resources were discussed at a pair of recent meetings held by the Multinational Arabidopsis Steering Committee (MASC) and the North American Arabidopsis Steering C...

  20. Arabidopsis CDS blastp result: AK240652 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240652 J023098G11 At5g63090.2 68418.m07919 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 1e-13 ...

  1. Arabidopsis CDS blastp result: AK241761 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241761 J065205C18 At5g63090.1 68418.m07918 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 5e-32 ...

  2. Arabidopsis CDS blastp result: AK240652 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240652 J023098G11 At5g63090.1 68418.m07918 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 1e-13 ...

  3. Arabidopsis CDS blastp result: AK240652 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240652 J023098G11 At5g63090.4 68418.m07921 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 1e-13 ...

  4. Arabidopsis CDS blastp result: AK241761 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241761 J065205C18 At5g63090.3 68418.m07920 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 5e-32 ...

  5. Arabidopsis CDS blastp result: AK241761 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241761 J065205C18 At5g63090.2 68418.m07919 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 5e-32 ...

  6. Arabidopsis CDS blastp result: AK241761 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241761 J065205C18 At5g63090.4 68418.m07921 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 5e-32 ...

  7. Arabidopsis CDS blastp result: AK240652 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240652 J023098G11 At5g63090.3 68418.m07920 LOB domain protein / lateral organ boundaries... protein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 1e-13 ...

  8. Arabidopsis CDS blastp result: AK105527 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105527 001-127-G05 At5g63090.4 LOB domain protein / lateral organ boundaries prot...ein (LOB) identical to LOBa [Arabidopsis thaliana] GI:17484100, SP|Q9FML4 LATERAL ORGAN BOUNDARIES protein {Arabidopsis thaliana} 3e-52 ...

  9. Using "Arabidopsis" Genetic Sequences to Teach Bioinformatics

    Science.gov (United States)

    Zhang, Xiaorong

    2009-01-01

    This article describes a new approach to teaching bioinformatics using "Arabidopsis" genetic sequences. Several open-ended and inquiry-based laboratory exercises have been designed to help students grasp key concepts and gain practical skills in bioinformatics, using "Arabidopsis" leucine-rich repeat receptor-like kinase (LRR…

  10. Arabidopsis CDS blastp result: AK240730 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240730 J043030K09 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 2e-11 ...

  11. Arabidopsis CDS blastp result: AK288052 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288052 J075151I09 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 6e-14 ...

  12. Arabidopsis CDS blastp result: AK240911 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240911 J065037E05 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 4e-22 ...

  13. Arabidopsis CDS blastp result: AK241119 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241119 J065094C22 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 2e-13 ...

  14. Arabidopsis CDS blastp result: AK243149 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243149 J100032I21 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 7e-12 ...

  15. Arabidopsis CDS blastp result: AK241581 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241581 J065181K09 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 4e-15 ...

  16. Arabidopsis CDS blastp result: AK287479 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287479 J043023O14 At2g32440.1 68415.m03963 ent-kaurenoic acid hydroxylase, putati...ve / cytochrome P450, putative identical to ent-kaurenoic acid hydroxylase / cytochrome P450 CYP88A (GI:1302...1856) [Arabidopsis thaliana]; similar to ent-kaurenoic acid hydroxylase [Arabidopsis thaliana] GI:13021853 1e-17 ...

  17. Reference: 631 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ggest that atRZ-1a has a negative impact on seed germination and seedling growth of Arabidopsis under salt o...binding protein, atRZ-1a, has a negative impact on seed germination and seedling growth of Arabidopsis thali

  18. The microtubule-stabilizing agent discodermolide competitively inhibits the binding of paclitaxel (Taxol) to tubulin polymers, enhances tubulin nucleation reactions more potently than paclitaxel, and inhibits the growth of paclitaxel-resistant cells.

    Science.gov (United States)

    Kowalski, R J; Giannakakou, P; Gunasekera, S P; Longley, R E; Day, B W; Hamel, E

    1997-10-01

    The lactone-bearing polyhydroxylated alkatetraene (+)-discodermolide, which was isolated from the sponge Discodermia dissoluta, induces the polymerization of purified tubulin with and without microtubule-associated proteins or GTP, and the polymers formed are stable to cold and calcium. These effects are similar to those of paclitaxel (Taxol), but discodermolide is more potent. We confirmed that these properties represent hypernucleation phenomena; we obtained lower tubulin critical concentrations and shorter polymers with discodermolide than paclitaxel under a variety of reaction conditions. Furthermore, we demonstrated that discodermolide is a competitive inhibitor with [3H]paclitaxel in binding to tubulin polymer, with an apparent Ki value of 0.4 microM. Multidrug-resistant human colon and ovarian carcinoma cells overexpressing P-glycoprotein, which are 900- and 2800-fold resistant to paclitaxel, respectively, relative to the parental lines, retained significant sensitivity to discodermolide (25- and 89-fold more resistant relative to the parental lines). Ovarian carcinoma cells that are 20-30-fold more resistant to paclitaxel than the parental line on the basis of expression of altered beta-tubulin polypeptides retained nearly complete sensitivity to discodermolide. The effects of discodermolide on the reorganization of the microtubules of Potorous tridactylis kidney epithelial cells were examined at different times. Intracellular microtubules were reorganized into bundles in interphase cells much more rapidly after discodermolide treatment compared with paclitaxel treatment. A variety of spindle aberrations were observed after treatment with both drugs. The proportions of the different types of aberration were different for the two drugs and changed with the length of drug treatment.

  19. Physiological characterization and genetic modifiers of aberrant root thigmomorphogenesis in mutants of Arabidopsis thaliana MILDEW LOCUS O genes.

    Science.gov (United States)

    Bidzinski, Przemyslaw; Noir, Sandra; Shahi, Shermineh; Reinstädler, Anja; Gratkowska, Dominika Marta; Panstruga, Ralph

    2014-12-01

    Root architecture and growth patterns are plant features that are still poorly understood. When grown under in vitro conditions, seedlings with mutations in Arabidopsis thaliana genes MLO4 or MLO11 exhibit aberrant root growth patterns upon contact with hard surfaces, exemplified as tight root spirals. We used a set of physiological assays and genetic tools to characterize this thigmomorphogenic defect in detail. We observed that the mlo4/mlo11-associated root curling phenotype is not recapitulated in a set of mutants with altered root growth patterns or architecture. We further found that mlo4/mlo11-conditioned root curling is not dependent upon light and endogenous flavonoids, but is pH-sensitive and affected by exogenous calcium levels. Based upon the latter two characteristics, mlo4-associated root coiling appears to be mechanistically different from the natural strong root curvature of the Arabidopsis ecotype Landsberg erecta. Gravistimulation reversibly overrides the aberrant thigmomorphogenesis of mlo4 seedlings. Mutants with dominant negative defects in α-tubulin modulate the extent and directionality of mlo4/mlo11-conditioned root coils, whereas mutants defective in polar auxin transport (axr4, aux1) or gravitropism (pgm1) completely suppress the mlo4 root curling phenotype. Our data implicate a joint contribution of calcium signalling, pH regulation, microtubular function, polar auxin transport and gravitropism in root thigmomorphogenesis.

  20. A canonical FtsZ protein in Verrucomicrobium spinosum, a member of the Bacterial phylum Verrucomicrobia that also includes tubulin-producing Prosthecobacter species

    Directory of Open Access Journals (Sweden)

    Staley James T

    2007-03-01

    Full Text Available Abstract Background The origin and evolution of the homologous GTP-binding cytoskeletal proteins FtsZ typical of Bacteria and tubulin characteristic of eukaryotes is a major question in molecular evolutionary biology. Both FtsZ and tubulin are central to key cell biology processes – bacterial septation and cell division in the case of FtsZ and in the case of tubulins the function of microtubules necessary for mitosis and other key cytoskeleton-dependent processes in eukaryotes. The origin of tubulin in particular is of significance to models for eukaryote origins. Most members of domain Bacteria possess FtsZ, but bacteria in genus Prosthecobacter of the phylum Verrucomicrobia form a key exception, possessing tubulin homologs BtubA and BtubB. It is therefore of interest to know whether other members of phylum Verrucomicrobia possess FtsZ or tubulin as their FtsZ-tubulin gene family representative. Results Verrucomicrobium spinosum, a member of Phylum Verrucomicrobia of domain Bacteria, has been found to possess a gene for a protein homologous to the cytoskeletal protein FtsZ. The deduced amino acid sequence has sequence signatures and predicted secondary structure characteristic for FtsZ rather than tubulin, but phylogenetic trees and sequence analysis indicate that it is divergent from all other known FtsZ sequences in members of domain Bacteria. The FtsZ gene of V. spinosum is located within a dcw gene cluster exhibiting gene order conservation known to contribute to the divisome in other Bacteria and comparable to these clusters in other Bacteria, suggesting a similar functional role. Conclusion Verrucomicrobium spinosum has been found to possess a gene for a protein homologous to the cytoskeletal protein FtsZ. The results suggest the functional as well as structural homology of the V. spinosum FtsZ to the FtsZs of other Bacteria implying its involvement in cell septum formation during division. Thus, both bacteria-like FtsZ and eukaryote

  1. Use of substitute Nonidet P-40 nonionic detergents in intracellular tubulin polymerization assays for screening of microtubule targeting agents.

    Science.gov (United States)

    Sinha, Saptarshi; Field, Jessica J; Miller, John H

    2017-01-10

    Shell Chemical Company Nonidet P-40 has been used for decades in many biochemical assays as a nonionic, nondenaturing detergent; however, Shell no longer produces this product. Four commercially available substitutes were investigated and their activities titrated in an intracellular tubulin polymerization assay. Although claimed by the supply companies to be identical to the Shell Nonidet P-40, all four substitutes were about 10-fold more potent and needed to be diluted accordingly. As microtubule targeting drugs are a major class of anticancer agent, and many researchers use the intracellular tubulin polymerization assay, this information is important to help troubleshoot assay development with the new substitutes. As the Shell Nonidet P-40 has been used in many biochemical buffers, these results will be of general interest to the biochemical, cell, and molecular research community.

  2. Quantum chemistry calculation-aided structural optimization of combretastatin A-4-like tubulin polymerization inhibitors: improved stability and biological activity.

    Science.gov (United States)

    Jiang, Junhang; Zheng, Canhui; Zhu, Kongkai; Liu, Jia; Sun, Nannan; Wang, Chongqing; Jiang, Hualiang; Zhu, Ju; Luo, Cheng; Zhou, Youjun

    2015-03-12

    A potent combretastatin A-4 (CA-4) like tubulin polymerization inhibitor 22b was found with strong antitumor activity previously. However, it easily undergoes cis-trans isomerization under natural light, and the resulting decrease in activity limits its further applications. In this study, we used quantum chemistry calculations to explore the molecular basis of its instability. Aided by the calculations, two rounds of structural optimization of 22b were conducted. Accelerated quantitative light stability testing confirmed that the stability of these designed compounds was significantly improved as predicted. Among them, compounds 1 and 3b displayed more potent inhibitory activity on tumor cell growth than 22b. In addition, the potent in vivo antitumor activity of compound 1 was confirmed. Quantum chemistry calculations were used in the optimization of stilbene-like molecules, providing new insight into stilbenoid optimization and important implications for the future development of novel CA-4-like tubulin polymerization inhibitors.

  3. Rational design, synthesis and biological evaluations of amino-noscapine: a high affinity tubulin-binding noscapinoid

    Science.gov (United States)

    Naik, Pradeep K.; Chatterji, Biswa Prasun; Vangapandu, Surya N.; Aneja, Ritu; Chandra, Ramesh; Kanteveri, Srinivas; Joshi, Harish C.

    2011-05-01

    Noscapine and its derivatives are important microtubule-interfering agents shown to have potent anti-tumor activity. The binding free energies (Δ G bind) of noscapinoids computed using linear interaction energy (LIE) method with a surface generalized Born (SGB) continuum solvation model were in agreement with the experimental Δ G bind with average root mean square error of 0.082 kcal/mol. This LIE-SGB model guided us in designing a novel derivative of noscapine, amino-noscapine [(S)-3-((R)-9-amino-4-methoxy-6-methyl-5,6,7,8-tetrahydro [1, 3] dioxolo[4,5- g]isoquinolin-5-yl)-6,7-dimethoxy isobenzo-furan-1(3 H)-one] that has higher tubulin binding activity (predicted Δ G bind = -6.438 kcal/mol and experimental Δ G bind = -6.628 kcal/mol) than noscapine, but does not significantly change the total extent of the tubulin subunit/polymer ratio. The modes of interaction of amino-noscapine with the binding pocket of tubulin involved three hydrogen bonds and are distinct compared to noscapine which involved only one hydrogen bond. Also the patterns of non-bonded interactions are albeit different between both the lignads. The `blind docking' approach (docking of ligand with different binding sites of a protein and their evaluations) as well as the reasonable accuracy of calculating Δ G bind using LIE-SGB model constitutes the first evidence that this class of compounds binds to tubulin at a site overlapping with colchicine-binding site or close to it. Our results revealed that amino-noscapine has better anti-tumor activity than noscapine.

  4. Maternal vitamin C deficiency does not reduce hippocampal volume and beta-tubulin III intensity in prenatal Guinea pigs

    DEFF Research Database (Denmark)

    Hansen, Stine Normann; Schjoldager, Janne Gram; Paidi, Maya Devi

    2016-01-01

    the observed deficits remain largely unknown. We hypothesized that vitC deficiency in utero may lead to a decreased neuronal maturation and increased cellular death giving rise to alterations of the hippocampal morphology in a guinea pig model. Brains from prenatal guinea pig pups (n = 9-10 in each group...... study found that hippocampal volume and beta-tubulin isotype III intensity in the prenatal guinea pig were influenced by gestational day but not by maternal vitC intake...

  5. Class III β-tubulin in advanced NSCLC of adenocarcinoma subtype predicts superior outcome in a randomized trial

    DEFF Research Database (Denmark)

    Vilmar, Adam Christian; Santoni-Rugiu, Eric; Sørensen, Jens Benn

    2011-01-01

    Platinum-based doublets are the cornerstone of treatment in advanced non-small-cell lung cancer (NSCLC) and often include vinorelbine or taxanes. A predictive biomarker is greatly needed to select chemotherapy-sensitive patients for these microtubule-interfering agents. Class III β-tubulin (TUBB3......) has been shown of value in NSCLC, but evidence is not uniform. Accordingly, we explored the predictive role of TUBB3 in advanced NSCLC....

  6. Analysis of the Strength of Interfacial Hydrogen Bonds between Tubulin Dimers Using Quantum Theory of Atoms in Molecules

    OpenAIRE

    Ayoub, Ahmed T.; Craddock, Travis J.A.; Klobukowski, Mariusz; Tuszynski, Jack

    2014-01-01

    Microtubules are key structural elements that, among numerous biological functions, maintain the cytoskeleton of the cell and have a major role in cell division, which makes them important cancer chemotherapy targets. Understanding the energy balance that brings tubulin dimers, the building blocks of microtubules, together to form a microtubule is especially important for revealing the mechanism of their dynamic instability. Several studies have been conducted to estimate various contribution...

  7. Photoinduced Partial Unfolding of Tubulin Bound to Meso-tetrakis(sulfonatophenyl) Porphyrin Leads to Inhibition of Microtubule Formation In Vitro

    Science.gov (United States)

    2013-07-30

    hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and...completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information...morphogen- esis, mitosis and meiosis [15, 16]. Because tubulin het- erodimers and MTs are vital for cellular processes, they have become a prime target in

  8. Increase in α-tubulin modifications in the neuronal processes of hippocampal neurons in both kainic acid-induced epileptic seizure and Alzheimer’s disease

    Science.gov (United States)

    Vu, Hang Thi; Akatsu, Hiroyasu; Hashizume, Yoshio; Setou, Mitsutoshi; Ikegami, Koji

    2017-01-01

    Neurodegeneration includes acute changes and slow-developing alterations, both of which partly involve common cellular machinery. During neurodegeneration, neuronal processes are impaired along with dysregulated post-translational modifications (PTMs) of cytoskeletal proteins. In neuronal processes, tubulin undergoes unique PTMs including a branched form of modification called glutamylation and loss of the C-terminal tyrosine residue and the penultimate glutamic acid residue forming Δ2-tubulin. Here, we investigated the state of two PTMs, glutamylation and Δ2 form, in both acute and slow-developing neurodegenerations, using a newly generated monoclonal antibody, DTE41, which had 2-fold higher affinity to glutamylated Δ2-tubulin, than to unmodified Δ2-tubulin. DTE41 recognised glutamylated Δ2-tubulin preferentially in immunostaining than in enzyme-linked immunosorbent assay and immunoblotting. In normal mouse brain, DTE41 stained molecular layer of the cerebellum as well as synapse-rich regions in pyramidal neurons of the cerebral cortex. In kainic acid-induced epileptic seizure, DTE41-labelled signals were increased in the hippocampal CA3 region, especially in the stratum lucidum. In the hippocampi of post-mortem patients with Alzheimer’s disease, intensities of DTE41 staining were increased in mossy fibres in the CA3 region as well as in apical dendrites of the pyramidal neurons. Our findings indicate that glutamylation on Δ2-tubulin is increased in both acute and slow-developing neurodegeneration. PMID:28067280

  9. Direct measurement of tubulin and bulk message distributions on polysomes of growing, starved and deciliated Tetrahymena using RNA gel blots of sucrose gradients containing acrylamide.

    Science.gov (United States)

    Calzone, F J; Callahan, R; Gorovsky, M A

    1988-10-25

    A method was developed using sucrose gradients containing acrylamide which greatly simplifies the measurement of the polysomal distribution of messages. After centrifugation, the acrylamide was polymerized, forming a "polysome gel". RNA gel blots of polysome gels were used to determine the polysomal distributions of alpha-tubulin and total polyadenylated mRNA in growing, starved (nongrowing) and starved-deciliated Tetrahymena and the number of messages loaded onto polysomes was calculated. These measurements indicated that the translational efficiencies of alpha-tubulin mRNA and total polyadenylated mRNA are largely unaffected when the rates of tubulin and total protein synthesis vary dramatically. Thus, differential regulation of alpha-tubulin mRNA translation initiation does not contribute to the greater than 100-fold induction of tubulin synthesis observed during cilia regeneration and in growing cells. The major translation-level process regulating tubulin synthesis in Tetrahymena appears to be a change in message loading mediated by a non-specific message recruitment or unmasking factor.

  10. Luminal localization of α-tubulin K40 acetylation by cryo-EM analysis of fab-labeled microtubules.

    Directory of Open Access Journals (Sweden)

    Virupakshi Soppina

    Full Text Available The αβ-tubulin subunits of microtubules can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs that provide functional specialization to subsets of cellular microtubules. Acetylation of α-tubulin residue Lysine-40 (K40 has been correlated with increased microtubule stability, intracellular transport, and ciliary assembly, yet a mechanistic understanding of how acetylation influences these events is lacking. Using the anti-acetylated tubulin antibody 6-11B-1 and electron cryo-microscopy, we demonstrate that the K40 acetylation site is located inside the microtubule lumen and thus cannot directly influence events on the microtubule surface, including kinesin-1 binding. Surprisingly, the monoclonal 6-11B-1 antibody recognizes both acetylated and deacetylated microtubules. These results suggest that acetylation induces structural changes in the K40-containing loop that could have important functional consequences on microtubule stability, bending, and subunit interactions. This work has important implications for acetylation and deacetylation reaction mechanisms as well as for interpreting experiments based on 6-11B-1 labeling.

  11. In vitro anti-tubulin effects of mebendazole and fenbendazole on canine glioma cells.

    Science.gov (United States)

    Lai, S R; Castello, S A; Robinson, A C; Koehler, J W

    2017-01-12

    Benzimidazole anthelmintics have reported anti-neoplastic effects both in vitro and in vivo. The purpose of this study was to evaluate the in vitro chemosensitivity of three canine glioma cell lines to mebendazole and fenbendazole. The mean inhibitory concentration (IC50 ) (±SD) obtained from performing the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay after treating J3T, G06-A, and SDT-3G cells for 72 h with mebendazole were 0.030 ± 0.003, 0.080 ± 0.015 and 0.030 ± 0.006 μM respectively, while those for fenbendazole were 0.550  ± 0.015, 1.530 ± 0.159 and 0.690 ± 0.095 μM; treatment of primary canine fibroblasts for 72 h at IC50 showed no significant effect. Immunofluorescence studies showed disruption of tubulin after treatment. Mebendazole and fenbendazole are cytotoxic in canine glioma cell lines in vitro and may be good candidates for treatment of canine gliomas. Further in vivo studies are required.

  12. Beta4 tubulin identifies a primitive cell source for oligodendrocytes in the mammalian brain.

    Science.gov (United States)

    Wu, Chuanshen; Chang, Ansi; Smith, Maria C; Won, Roy; Yin, Xinghua; Staugaitis, Susan M; Agamanolis, Dimitri; Kidd, Grahame J; Miller, Robert H; Trapp, Bruce D

    2009-06-17

    We have identified a novel population of cells in the subventricular zone (SVZ) of the mammalian brain that expresses beta4 tubulin (betaT4) and has properties of primitive neuroectodermal cells. betaT4 cells are scattered throughout the SVZ of the lateral ventricles in adult human brain and are significantly increased in the SVZs bordering demyelinated white matter in multiple sclerosis brains. In human fetal brain, betaT4 cell densities peak during the latter stages of gliogenesis, which occurs in the SVZ of the lateral ventricles. betaT4 cells represent 95% of cells in neurospheres treated with the anti-mitotic agent Ara C. betaT4 cells produce oligodendrocytes, neurons, and astrocytes in vitro. We compared the myelinating potential of betaT4-positive cells with A2B5-positive oligodendrocyte progenitor cells after transplantation (25,000 cells) into postnatal day 3 (P3) myelin-deficient rat brains. At P20, the progeny of betaT4 cells myelinated up to 4 mm of the external capsule, which significantly exceeded that of transplanted A2B5-positive progenitor cells. Such extensive and rapid mature CNS cell generation by a relatively small number of transplanted cells provides in vivo support for the therapeutic potential of betaT4 cells. We propose that betaT4 cells are an endogenous cell source that can be recruited to promote neural repair in the adult telencephalon.

  13. Jasmonate Signal Pathway in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Yi Shan; Zhi-Long Wang; Daoxin Xie

    2007-01-01

    Jasmonates (JAs), which include jasmonic acid and its cyclopentane derivatives are synthesized from the octadecanoid pathway and widely distributed throughout the plant kingdom. JAs modulate the expression of numerous genes and mediate responses to stress, wounding, insect attack, pathogen infection, and UV damage. They also affect a variety of processes in many plant developmental processes. The JA signal pathway involves two important events: the biosynthesis of JA and the transduction of JA signal. Several important Arabidopsis mutants in jasmonate signal pathway were described in this review.

  14. Reference: 572 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available et al. 2007 May. Plant J. 50(3):439-51. Although glycine-rich RNA-binding protein 2 (GRP2) has been implicated in plant re...sponses to environmental stresses, the function and importance of GRP2 in stress responses are largely unknown. Here...haliana under high-salinity, cold or osmotic stress. GRP2 affects seed germination of Arabidopsis plants under salt stre...ss, but does not influence seed germination and seedling growth of Arabidopsis plants under osmotic stre...ss. GRP2 accelerates seed germination and seedling growth in Arabidopsis plants under cold stre

  15. Reference: 446 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available rk E et al. 2006 Nov. Plant Physiol. 142(3):1004-13. Arabidopsis (Arabidopsis thaliana) QUARTET (QRT) genes are require...d for pollen separation during normal floral development. In qrt mutants, the four products of microsporogenesis re...main fused and pollen grains are released as tetrads. In Arabid...opsis, tetrad analysis in qrt mutants has been used to map all five centromeres, easily distinguish sporophy...tic from gametophytic mutations, and accurately assess crossover interference. Using a combination of forward and re

  16. Tau Tubulin Kinase TTBK2 Sensitivity of Glutamate Receptor GluK2

    Directory of Open Access Journals (Sweden)

    Kristina Nieding

    2016-09-01

    Full Text Available Background/Aims: Inherited, autosomal dominant spinocerebellar ataxia type 11 (SCA11 is caused by loss of function mutations of TTBK2 (tau tubulin kinase 2. Mutations observed in patients with SCA11 include truncated TTBK2(450. The present study explored the possibility that TTBK2 influences the function of the glutamate receptor GluK2. Methods: GluK2 was expressed in Xenopus oocytes without and with additional expression of wild type TTBK2, the truncated mutant TTBK2(450, or the kinase dead mutants TTBK2(KD and TTBK2(450/KD. GluK2 current was determined by dual electrode voltage clamp and GluK2 protein abundance in the cell membrane utilizing confocal microscopy. Results: Glutamate exposure of GluK2 expressing oocytes generated a current, which was significantly lower in oocytes expressing GluK2 together with TTBK2 wt or TTBK2(KD than in oocytes expressing GluK2 alone or together with either TTBK2(450 or TTBK2(450/KD. According to confocal microscopy of EGFP-tagged GluK2, TTBK2 wt decreased the GluK2 protein abundance in the cell membrane. Overexpression of an inactive RAB5(N133I mutant but not RAB5wt could reverse the TTBK2 effect on GluK2 suggesting that RAB5 function is required for the effect. Conclusions: TTBK2 down-regulates GluK2 activity by decreasing the receptor protein abundance in the cell membrane via RAB5-dependent endocytosis, an effect that may protect against neuroexcitotoxicity.

  17. Hypocotyl expression and light downregulation of the soybean tubulin gene, tubB1.

    Science.gov (United States)

    Tonoike, H; Han, I S; Jongewaard, I; Doyle, M; Guiltinan, M; Fosket, D E

    1994-03-01

    The tubB1 beta-tubulin gene of Glycine max (previously named s beta 1) is highly expressed only in rapidly elongating regions of etiolated seedling hypocotyls and this expression is strongly downregulated when the seedlings are exposed to light. Primer extension demonstrated that the gene was transcribed in these tissues and contained two sites of transcriptional initiation. To determine the mechanism regulating tubB1 expression, a chimeric reporter gene was constructed by fusing 5' upstream regions of tubB1 to a promoterless beta-glucuronidase (GUS) gene and these constructs were introduced into protoplasts by electroporation. Strong transient expression of the reporter gene was obtained after electroporation of chimeric constructs containing 1 kb of tubB1 5' upstream sequence into tobacco protoplasts. Deletion of the distal most 300 bp from the 5' sequence of tubB1 enhanced expression, suggesting the possibility of a negative transcriptional regulator in this region. Additional deletions of the 5' sequence reduced expression substantially. Constructs containing a tubB1 3' terminus were expressed at much lower levels than those containing a nopaline synthase (NOS) 3' terminus. The tubB1-GUS chimeric gene also was introduced into tobacco by Agrobacterium-mediated Ti plasmid transformation and the organ-specific expression pattern of the chimeric gene was determined in seedlings of the transgenic plants. Hypocotyls exhibited strong GUS activity when the seedlings were germinated in darkness, but lacked the GUS enzyme when the seedlings were germinated in the light.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Arabidopsis CDS blastp result: AK065259 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK065259 J013002J18 At5g16910.1 cellulose synthase family protein similar to gi:2827143 cellulose... synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 0.0 ...

  19. Arabidopsis CDS blastp result: AK102134 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK102134 J033085F12 At5g16910.1 cellulose synthase family protein similar to gi:2827143 cellulose... synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 0.0 ...

  20. Arabidopsis CDS blastp result: AK066835 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066835 J013087I16 At5g16910.1 cellulose synthase family protein similar to gi:2827143 cellulose... synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 1e-171 ...

  1. Arabidopsis CDS blastp result: AK100523 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100523 J023100P04 At5g16910.1 cellulose synthase family protein similar to gi:2827143 cellulose... synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 0.0 ...

  2. Arabidopsis CDS blastp result: AK102695 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK102695 J033103F21 At5g16910.1 cellulose synthase family protein similar to gi:2827143 cellulose... synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 0.0 ...

  3. Reference: 488 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available Inactivation of ATAB2 strongly affects Arabidopsis development and thylakoid mem...n center subunits is decreased and the association of their mRNAs with polysomes is affected. ATAB2 is a chl

  4. Reference: 212 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available identified in pea (Pisum sativum) using biochemical approaches. The Arabidopsis (...C75-IV, which we studied using a range of molecular, genetic, and biochemical techniques. Expression of atTO

  5. Reference: 480 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available activity was analyzed. Compared to all other Suc transporters, AtSUC9 had an ult...abidopsis (Arabidopsis thaliana) L. Heynh., was expressed in Xenopus (Xenopus laevis) oocytes, and transport

  6. Reference: 507 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available een them. However, little is known about the mechanisms that regulate the two pathways and the metabolic cro...ss-talk. To identify such regulatory mechanisms, we isolated and characterized the Arabidopsis T-DNA inserti

  7. Reference: 278 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available functional ERA1 gene, which encodes the beta-subunit of protein farnesyltransferase (PFT), exhibit pleiotropic effects...gnaling and meristem development. Here, we report the effects of T-DNA insertion mutations in the Arabidopsi

  8. Reference: 185 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available organisms, we suggest that AtARP4 is likely to exert its effects on plant develop...nuclear actin-related protein AtARP4 in Arabidopsis has multiple effects on plant development, including ear

  9. Arabidopsis CDS blastp result: AK069960 [KOME

    Lifescience Database Archive (English)

    Full Text Available thyltransferase 1 / caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT1) identical to O-methyltrans...T1) (Flavonol 3- O-methyltransferase 1) (Caffeic acid/5-hydroxyferulic acid O- methyltransferase) {Arabidopsis thaliana} 5e-60 ...

  10. Arabidopsis CDS blastp result: AK064768 [KOME

    Lifescience Database Archive (English)

    Full Text Available thyltransferase 1 / caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT1) identical to O-methyltrans...T1) (Flavonol 3- O-methyltransferase 1) (Caffeic acid/5-hydroxyferulic acid O- methyltransferase) {Arabidopsis thaliana} 1e-112 ...

  11. Arabidopsis CDS blastp result: AK061551 [KOME

    Lifescience Database Archive (English)

    Full Text Available ethyltransferase 1 / caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT1) identical to O-methyltran...MT1) (Flavonol 3- O-methyltransferase 1) (Caffeic acid/5-hydroxyferulic acid O- methyltransferase) {Arabidopsis thaliana} 2e-67 ...

  12. Arabidopsis CDS blastp result: AK104764 [KOME

    Lifescience Database Archive (English)

    Full Text Available ethyltransferase 1 / caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT1) identical to O-methyltran...MT1) (Flavonol 3- O-methyltransferase 1) (Caffeic acid/5-hydroxyferulic acid O- methyltransferase) {Arabidopsis thaliana} 2e-67 ...

  13. Arabidopsis CDS blastp result: AK098998 [KOME

    Lifescience Database Archive (English)

    Full Text Available thyltransferase 1 / caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT1) identical to O-methyltrans...T1) (Flavonol 3- O-methyltransferase 1) (Caffeic acid/5-hydroxyferulic acid O- methyltransferase) {Arabidopsis thaliana} 8e-57 ...

  14. Arabidopsis CDS blastp result: AK061859 [KOME

    Lifescience Database Archive (English)

    Full Text Available ethyltransferase 1 / caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT1) identical to O-methyltran...MT1) (Flavonol 3- O-methyltransferase 1) (Caffeic acid/5-hydroxyferulic acid O- methyltransferase) {Arabidopsis thaliana} 1e-100 ...

  15. Arabidopsis CDS blastp result: AK103387 [KOME

    Lifescience Database Archive (English)

    Full Text Available ntical to SC35-like splicing factor SCL28, 28 kD [Arabidopsis thaliana] GI:9843655; contains Pfam profile PF00076: RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) 2e-34 ...

  16. Reference: 564 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 39-44 17360695 2007 Feb Proceedings of the National Academy of Sciences of the Un...tion in plants. Arabidopsis plasma membrane protein crucial for Ca2+ influx and touch sensing in roots. 9 36

  17. Reference: 796 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ceedings of the National Academy of Sciences of the United States of America DeBolt...required for normal microtubule dynamics and organization in Arabidopsis. 46 18064-9 19004800 2008 Nov Pro

  18. Reference: 67 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available A complete knockout of AGD2 renders embryos inviable. We suggest that AGD2 synthesizes an important amino a...no acid-derived molecule important for activating defense signaling. Divergent roles in Arabidopsis thaliana

  19. Reference: 420 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available are found in various compartments in plant cells. The cytosolic and chloroplast APXs appear to play important...d development, suggesting that APX3 may not be an important antioxidant enzyme in Arabidopsis, at least unde

  20. Reference: 771 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available RCADIAN TIMEKEEPER (XCT), an Arabidopsis thaliana gene important for light regula...l elongation in xct is hyposensitive to red light but hypersensitive to blue light. Finally, XCT is important

  1. Reference: 797 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available that the level of GMPase activity regulates Arabidopsis sensitivity to NH(4)(+). Further analysis showed that defective N-glycosylati...on of proteins, unfolded protein response, and cell death in the roots are likely i

  2. Arabidopsis CDS blastp result: AK241712 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241712 J065197H24 At4g37750.1 68417.m05344 ovule development protein aintegumenta... (ANT) identical to ovule development protein aintegumenta (ANT) (GI:1244708) ) [Arabidopsis thaliana] 6e-27 ...

  3. Arabidopsis CDS blastp result: AK242957 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242957 J090089I15 At4g37750.1 68417.m05344 ovule development protein aintegumenta... (ANT) identical to ovule development protein aintegumenta (ANT) (GI:1244708) ) [Arabidopsis thaliana] 1e-28 ...

  4. Arabidopsis CDS blastp result: AK287726 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287726 J065138E17 At4g37750.1 68417.m05344 ovule development protein aintegumenta... (ANT) identical to ovule development protein aintegumenta (ANT) (GI:1244708) ) [Arabidopsis thaliana] 1e-88 ...

  5. Arabidopsis CDS blastp result: AK242387 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242387 J080051E14 At4g37750.1 68417.m05344 ovule development protein aintegumenta... (ANT) identical to ovule development protein aintegumenta (ANT) (GI:1244708) ) [Arabidopsis thaliana] 2e-45 ...

  6. Arabidopsis CDS blastp result: AK106306 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK106306 002-101-C10 At4g37750.1 ovule development protein aintegumenta (ANT) ident...ical to ovule development protein aintegumenta (ANT) (GI:1244708) ) [Arabidopsis thaliana] 3e-89 ...

  7. Arabidopsis CDS blastp result: AK241272 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241272 J065132I19 At4g37750.1 68417.m05344 ovule development protein aintegumenta... (ANT) identical to ovule development protein aintegumenta (ANT) (GI:1244708) ) [Arabidopsis thaliana] 1e-88 ...

  8. Arabidopsis CDS blastp result: AK240892 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240892 J065030K10 At4g37750.1 68417.m05344 ovule development protein aintegumenta... (ANT) identical to ovule development protein aintegumenta (ANT) (GI:1244708) ) [Arabidopsis thaliana] 5e-88 ...

  9. Arabidopsis CDS blastp result: AK109848 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109848 002-148-F05 At4g37750.1 ovule development protein aintegumenta (ANT) ident...ical to ovule development protein aintegumenta (ANT) (GI:1244708) ) [Arabidopsis thaliana] 5e-73 ...

  10. Arabidopsis CDS blastp result: AK287673 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287673 J065121E18 At4g37750.1 68417.m05344 ovule development protein aintegumenta... (ANT) identical to ovule development protein aintegumenta (ANT) (GI:1244708) ) [Arabidopsis thaliana] 6e-17 ...

  11. Arabidopsis CDS blastp result: AK287621 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287621 J065066I09 At4g37750.1 68417.m05344 ovule development protein aintegumenta... (ANT) identical to ovule development protein aintegumenta (ANT) (GI:1244708) ) [Arabidopsis thaliana] 5e-85 ...

  12. Reference: 142 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available te S-glucosyltransferase, UGT74B1, to determine its role in the Arabidopsis glucosinolate pathway. Biochem...ical analyses demonstrate that recombinant UGT74B1 specifically glucosylates the th

  13. Reference: 522 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available tol phosphate (InsP) and phosphoinositide phosphate (PtdInsP) substrates. Arabidopsis thaliana has 15 genes encoding 5PTases. Biochem...ical analyses of a subgroup of 5PTase enzymes suggest th

  14. Reference: 459 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available plants. These results suggest an additive contribution of AMT1;1 and AMT1;3 to the overall ammonium uptake ...capacity in Arabidopsis roots under nitrogen-deficiency conditions. Additive contribution

  15. Arabidopsis CDS blastp result: AK288065 [KOME

    Lifescience Database Archive (English)

    Full Text Available al to sulfate tansporter Sultr1;3 [Arabidopsis thaliana] GI:10716805; contains Pfam profile PF00916: Sulfate... transporter family; contains Pfam profile PF01740: STAS domain; contains TIGRfam profile TIGR00815: sulfate permease 1e-145 ...

  16. Reference: 645 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available rter AtDUR3 in nitrogen nutrition in Arabidopsis. In transgenic lines expressing ... impaired growth on urea as a sole nitrogen source were used to investigate a role of the H+/urea co-transpo

  17. The fifth international conference on Arabidopsis research

    Energy Technology Data Exchange (ETDEWEB)

    Hangarter, R.; Scholl, R.; Davis, K.; Feldmann, K.

    1993-12-31

    This volume contains abstracts of oral and poster presentations made in conjunction with the Fifth International Conference on Arabidopsis Research held August 19--22, 1993 at the Ohio State University, Columbus, Ohio.

  18. Reference: 711 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available of the RLK signaling pathway, which also mediates adaptation to Na(+) stress. RLK pathway components, known... The Arabidopsis kinase-associated protein phosphatase regulates adaptation to Na+ stress. 2 612-22 18162596

  19. Reference: 734 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available umi et al. 2008 Apr. Development 135(7):1335-45. CAPRICE (CPC) encodes a small protein with an R3 MYB motif ...doreduplication. Arabidopsis CAPRICE-LIKE MYB 3 (CPL3) controls endoreduplication and flowering development

  20. Arabidopsis CDS blastp result: AK101526 [KOME

    Lifescience Database Archive (English)

    Full Text Available ucosaminyltransferase, putative similar to N-acetylglucosaminyltransferase I from Arabidopsis thaliana [gi:5139335]; contains AT-AC non-consensus splice sites at intron 13 1e-179 ...

  1. Reference: 733 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available role in this transition. Specifically, two autonomous factors in the Arabidopsis...tes FCA alternative polyadenylation and promotes flowering as a novel factor in the autonomous pathway. Firs

  2. Reference: 343 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available the characterization of a T-DNA insertion mutant of the Arabidopsis CAP-C gene. Analysis of the progeny of selfe...matin was observed between segregating mitotic chromosomes in pollen produced by selfed heterozygotes. Addit

  3. Arabidopsis CDS blastp result: AK241281 [KOME

    Lifescience Database Archive (English)

    Full Text Available 2 protein) [Arabidopsis thaliana]; a false single bp exon was added to circumvent a single basepair insertion in the genomic sequence, supported by cDNA/genome alignment. 3e-19 ...

  4. Arabidopsis CDS blastp result: AK241243 [KOME

    Lifescience Database Archive (English)

    Full Text Available 2 protein) [Arabidopsis thaliana]; a false single bp exon was added to circumvent a single basepair insertion in the genomic sequence, supported by cDNA/genome alignment. 6e-11 ...

  5. Arabidopsis CDS blastp result: AK243188 [KOME

    Lifescience Database Archive (English)

    Full Text Available 2 protein) [Arabidopsis thaliana]; a false single bp exon was added to circumvent a single basepair insertion in the genomic sequence, supported by cDNA/genome alignment. 8e-23 ...

  6. Arabidopsis CDS blastp result: AK242986 [KOME

    Lifescience Database Archive (English)

    Full Text Available 2 protein) [Arabidopsis thaliana]; a false single bp exon was added to circumvent a single basepair insertion in the genomic sequence, supported by cDNA/genome alignment. 1e-17 ...

  7. Reference: 30 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ponse to various biotic and abiotic stresses. However the physiological role of t...his pathway remains obscure. To elucidate its role in plants, we analyzed Arabidopsis T-DNA knockout mutants

  8. Arabidopsis CDS blastp result: AK062082 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK062082 001-044-F11 At3g59970.3 methylenetetrahydrofolate reductase 1 (MTHFR1) ide...ntical to methylenetetrahydrofolate reductase MTHFR1 [Arabidopsis thaliana] GI:5911425 4e-81 ...

  9. Reference: 783 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available sis ACBP6 was confirmed by analyses of transgenic Arabidopsis expressing autofluorescence-tagged ACBP6 and w... mRNA encoding phospholipase Ddelta. Lipid profiling analyses of rosettes from co

  10. Reference: 789 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ylakoid membranes. Microarray analysis of the chl27-t mutant showed repression of numerous nuclear genes involved in photosynthesis...d CHL27 proteins. Role of Arabidopsis CHL27 protein for photosynthesis, chloroplast development and gene exp

  11. Reference: 352 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available em II and has a specific function distinct from 2-Cys peroxiredoxin in protecting photosynthesis. Its absenc...f Arabidopsis thaliana is attached to the thylakoids and functions in context of photosynthesis

  12. Reference: 21 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ication of a number of mutant lines with altered Chl fluorescence characteristics. Analysis of photosynthesis...cation of mutants of Arabidopsis defective in acclimation of photosynthesis to th

  13. Reference: 413 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ollination and fertilization, and, in the absence of fertilization, flowers senesce. In the Arabidopsis thal...ARF8 acts as an inhibitor to stop further carpel development in the absence of fertilization and the generat

  14. Reference: 405 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available as previously thought. These mutants will prove to be valuable resources for understanding laccase functions in vivo. Mutant identifi...cation and characterization of the laccase gene family in Arabidopsis. 11 2563-9 16

  15. Reference: 263 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available idopsis leaves GLB1 expression and PII protein levels were not significantly affected by either the day/nigh...bolism. Physiological characterisation of Arabidopsis mutants affected in the expression of the putative reg

  16. Reference: 160 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available excessive accumulation of these toxic compounds impairs cell death containment and counteracts the effect...iveness of the plant defenses to restrict pathogen infection. Arabidopsis SHMT1, a

  17. Arabidopsis CDS blastp result: AK242550 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242550 J080319D10 At2g35630.1 68415.m04369 microtubule organization 1 protein (MO...R1) identical to microtubule organization 1 protein GI:14317953 from [Arabidopsis thaliana] 5e-44 ...

  18. Reference: 301 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available n phosphatidylinositol metabolism and is encoded by an At5PTase gene family in Arabidopsis thaliana. A previous study...ntracellular calcium levels. In this study, we provide evidence that At5PTase13 m

  19. Reference: 724 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available is required in the roots during early signaling steps of rhizobacteria-mediated ...ISR. MYB72 is required in early signaling steps of rhizobacteria-induced systemic resistance in Arabidopsis.

  20. Reference: 289 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available f flavonoids in Arabidopsis seed coat. 11 2966-80 16243908 2005 Nov The Plant cell Caboche Michel|Debeaujon Isabelle|Kerhoas Lucien|Lepiniec Loïc|Pourcel Lucille|Routaboul Jean-Marc

  1. Reference: 684 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available cellular proliferation and expansion at nanomolar concentrations. PSY1 is widely expressed in various Arabi...ulfated glycopeptide involved in cellular proliferation and expansion in Arabidopsis. 46 18333-8 17989228 20

  2. Reference: 147 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available the region-specific control of trichome development of Arabidopsis. 3 389-98 15604688 2004 May Plant molecular biology Hulskamp Mart...in|Kirik Victor|Schiefelbein John|Simon Marissa|Wester Katja

  3. Arabidopsis CDS blastp result: AK241043 [KOME

    Lifescience Database Archive (English)

    Full Text Available upted by a stop codon, creating non-consensus donor and acceptor splice sites. 2e-41 ... ...tical to SP|P92997 Germin-like protein subfamily 1 member 13 precursor {Arabidopsis thaliana}; exon 2 interr

  4. Arabidopsis CDS blastp result: AK243135 [KOME

    Lifescience Database Archive (English)

    Full Text Available upted by a stop codon, creating non-consensus donor and acceptor splice sites. 7e-43 ... ...tical to SP|P92997 Germin-like protein subfamily 1 member 13 precursor {Arabidopsis thaliana}; exon 2 interr

  5. Reference: 798 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available iption factors, control the delicately tuned reorientation and timing of cell div...EZ and SOMBRERO control the orientation of cell division plane in Arabidopsis root stem cells. 6 913-22 1908

  6. Arabidopsis CDS blastp result: AK071710 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK071710 J023110L07 At4g14030.1 selenium-binding protein, putative contains Pfam profile PF05694: 56kDa sele...nium binding protein (SBP56); identical to Putative selenium-binding protein (Swiss...-Prot:O23264) [Arabidopsis thaliana]; similar to selenium binding protein (GI:15485232) [Arabidopsis thalian...a]; identical to cDNA from partial mRNA for selenium binding protein (sbp gene) GI:15485231 1e-162 ...

  7. Reference: 221 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ell cycle. In addition, RAD51 is required for meiosis and its Arabidopsis (Arabidopsis thaliana) ortholog is important... cell cultures, the RAD51 paralog RAD51C is also important for mitotic homologous...ortant for recombination and DNA repair in the mitotic c...chromosome (homolog) pairing, synapsis, and recombination. The budding yeast (Saccharomyces cerevisiae) RAD51 gene is known to be imp

  8. Reference: 598 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available omoter is markedly reduced in the cdkc;2 and cyct1;5 mutants, indicating that the kinase complexes are important... flowering. These results establish Arabidopsis CDKC kinase complexes as important...T1;4 and CYCT1;5, play important roles in infection with Cauliflower mosaic virus...hat Arabidopsis thaliana CDK9-like proteins, CDKC;1 and CDKC;2, and their interacting cyclin T partners, CYC

  9. Synthesis and biological evaluation of cis-restricted triazole/tetrazole mimics of combretastatin-benzothiazole hybrids as tubulin polymerization inhibitors and apoptosis inducers.

    Science.gov (United States)

    Subba Rao, A V; Swapna, Konderu; Shaik, Siddiq Pasha; Lakshma Nayak, V; Srinivasa Reddy, T; Sunkari, Satish; Shaik, Thokhir Basha; Bagul, Chandrakant; Kamal, Ahmed

    2017-02-01

    A series of colchicine site binding tubulin inhibitors were synthesized by the modification of the combretastatin pharmacophore. The ring B was replaced by the pharmacologically relevant benzothiazole scaffolds, and the cis configuration of the olefinic bond was restricted by the incorporation of a triazole and tetrazole rings which is envisaged by the structural resemblance to a tubulin inhibitor like combretastatin (CA-4). These compounds were evaluated for their antiproliferative activity on selected cancer cell lines and an insight in the structure activity relationship was developed. The most potent compounds (9a and 9b) demonstrated an antiproliferative effect comparable to that of CA-4. Mitotic cell cycle arrest in G2/M phase revealed the disruption of microtubule dynamics that was confirmed by tubulin polymerization assays and immunocytochemistry studies at the cellular level. Western blot analysis revealed that these compounds accumulate more tubulin in the soluble fraction. The colchicine competitive binding assay and the molecular docking studies suggested that the binding of these mimics at the colchicine site of the tubulin is similar to that of CA-4. Moreover, the triggering of apoptotic cell death after mitotic arrest was investigated by studying their effect by Hoechst staining, Annexin-V-FITC assay, mitochondrial membrane potential, ROS generation and caspase-3 activation.

  10. The elongation of primary cilia via the acetylation of α-tubulin by the treatment with lithium chloride in human fibroblast KD cells.

    Science.gov (United States)

    Nakakura, Takashi; Asano-Hoshino, Anshin; Suzuki, Takeshi; Arisawa, Kenjiro; Tanaka, Hideyuki; Sekino, Yoshihisa; Kiuchi, Yoshiko; Kawai, Kazuhiro; Hagiwara, Haruo

    2015-03-01

    Primary cilium, an organelle found on nearly every cell in the human body, typically serves as the mechanical sensor of the cell. Lithium ion is known to promote the elongation of primary cilia in a variety of cell types, but it is unknown whether lithium is involved in the acetylation of α-tubulin which is essential for the assembly of primary cilia. In order to reveal the relationship between the elongation of primary cilia with lithium and the acetylation of α-tubulin, we first observed the formation and structure of primary cilia in KD cells, a cell line deriving fibroblasts in human labium. Subsequently, by immunohistochemical and western blot analysis we elucidated that the length of primary cilia and acetylation of α-tubulin are regulated by lithium chloride (LiCl) in the medium in a time- and concentration-dependent manner. We next performed the RT-PCR, RNAi-based experiments and biochemical study using an inhibitor of glycogen synthase kinase-3βGSK-3β). We found that LiCl mobilizes the α-tubulin N-acetyltransferase 1 (αTAT1) in the signaling pathway mediating GSK-3β and adenylate cyclase III. In conclusion, our results suggested that LiCl treatments activate αTAT1 by the inhibition of GSK-3β and promote the α-tubulin acetylation, and then elongate the primary cilia.

  11. Transient increase in the levels of γ-tubulin complex and katanin are responsible for reorientation by ethylene and hypergravity of cortical microtubules.

    Science.gov (United States)

    Soga, Kouichi; Yamaguchi, Aya; Kotake, Toshihisa; Wakabayashi, Kazuyuki; Hoson, Takayuki

    2010-11-01

    The body shape of a plant is primarily regulated by orientation of cortical microtubules. γ-Tubulin complex and katanin are required for the nucleation and the severing of microtubules, respectively. Here we discuss the role of γ-tubulin complex and katanin during reorientation of cortical microtubules. 1-Aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, modifies growth anisotropy of azuki bean epicotyls; it inhibits elongation growth and promotes lateral growth. The ACC-induced reorientation of cortical microtubules from transverse to longitudinal directions preceded the modification of growth anisotropy. The transcript level of γ-tubulin complex (VaTUG and VaGCP3) and katanin (VaKTN1) was increased transiently by ACC treatment. During reorientation of cortical microtubules by hypergravity, which also modifies growth anisotropy of shoots, the expression levels of both γ-tubulin complex and katanin genes were increased transiently. The increase in the number of the nucleated microtubule branch as well as the microtubule-severing activity via upregulation of γ-tubulin complex genes and katanin genes may be involved in the reorientation of cortical microtubules, and contribute to the regulation of the shape of plant body.

  12. Sequence variation in the Trichuris trichiura beta-tubulin locus: implications for the development of benzimidazole resistance.

    Science.gov (United States)

    Bennett, A B; Anderson, T J C; Barker, G C; Michael, E; Bundy, D A P

    2002-11-01

    Benzimidazole resistance has evolved in a variety of organisms and typically results from mutations in the beta-tubulin locus at specific amino acid sites. Despite widespread treatment of human intestinal nematodes with benzimidazole drugs, there have been no unambiguous reports of resistance. However, since beta-tubulin mutations conferring resistance are generally recessive, frequencies of resistance alleles less than 30% would be difficult to detect on the basis of drug treatment failures. Here we investigate sequence variation in a 1079 bp segment of the beta-tubulin locus in the human whipworm Trichuris trichiura from 72 individual nematodes from seven countries. We did not observe any alleles with amino acid mutations indicative of resistance, and of 40 point mutations there were only four non-synonymous mutations all of which were singletons. Estimated effective population sizes are an order of magnitude lower than those from another nematode species in which benzimidazole resistance has developed (Haemonchus contortus). Both the lower diversity and reduced population sizes suggest that benzimidazole resistance is likely to evolve less rapidly in Trichuris than in trichostrongyle parasites of livestock. We observed moderate levels of population subdivision (Phi(ST)=0.26) comparable with that previously observed in Ascaris lumbricoides, and identical alleles were frequently found in parasites from different continents, suggestive of recent admixture. A particularly interesting feature of the data is the high nucleotide diversities observed in nematodes from the Caribbean. This genetic complexity may be a direct result of extensive admixture and complex history of human populations in this region of the world. These data should encourage (but not make complacent) those involved in large-scale benzimidazole treatment of human intestinal nematodes.

  13. Postmortem proteomic analysis in human amygdala of drug addicts: possible impact of tubulin on drug-abusing behavior.

    Science.gov (United States)

    Zill, P; Vielsmeier, V; Büttner, A; Eisenmenger, W; Siedler, F; Scheffer, B; Möller, H-J; Bondy, B

    2011-03-01

    Besides the ventral tegmental area and the nucleus accumbens as the most investigated brain reward structures, several reports about the relation between volume and activity of the amygdala and drug-seeking behavior have emphasized the central role of the amygdala in the etiology of addiction. Considering its proposed important role and the limited number of human protein expression studies with amygdala in drug addiction, we performed a human postmortem proteomic analysis of amygdala tissue obtained from 8 opiate addicts and 7 control individuals. Results were validated by Western blot in an independent postmortem replication sample from 12 opiate addicts compared to 12 controls and 12 suicide victims, as a second "control sample". Applying 2D-electrophoresis and MALDI-TOF-MS analysis, we detected alterations of beta-tubulin expression and decreased levels of the heat-shock protein HSP60 in drug addicts. Western blot analysis in the additional sample demonstrated significantly increased alpha- and beta-tubulin concentrations in the amygdala of drug abusers versus controls (P = 0.021, 0.029) and to suicide victims (P = 0.006, 0.002). Our results suggest that cytoskeletal alterations in the amygdala determined by tubulin seem to be involved in the pathophysiology of drug addiction, probably via a relation to neurotransmission and cellular signaling. Moreover, the loss of neuroprotection against stressors by chaperons as HSP60 might also contribute to structural alteration in the brain of drug addicts. Although further studies have to confirm our results, this might be a possible pathway that may increase our understanding of drug addiction.

  14. Nano-ZnO leads to tubulin macrotube assembly and actin bundling, triggering cytoskeletal catastrophe and cell necrosis

    Science.gov (United States)

    García-Hevia, Lorena; Valiente, Rafael; Martín-Rodríguez, Rosa; Renero-Lecuna, Carlos; González, Jesús; Rodríguez-Fernández, Lidia; Aguado, Fernando; Villegas, Juan C.; Fanarraga, Mónica L.

    2016-05-01

    Zinc is a crucial element in biology that plays chief catalytic, structural and protein regulatory roles. Excess cytoplasmic zinc is toxic to cells so there are cell-entry and intracellular buffering mechanisms that control intracellular zinc availability. Tubulin and actin are two zinc-scavenging proteins that are essential components of the cellular cytoskeleton implicated in cell division, migration and cellular architecture maintenance. Here we demonstrate how exposure to different ZnO nanostructures, namely ZnO commercial nanoparticles and custom-made ZnO nanowires, produce acute cytotoxic effects in human keratinocytes (HaCat) and epithelial cells (HeLa) triggering a dose-dependent cell retraction and collapse. We show how engulfed ZnO nanoparticles dissolve intracellularly, triggering actin filament bundling and structural changes in microtubules, transforming these highly dynamic 25 nm diameter polymers into rigid macrotubes of tubulin, severely affecting cell proliferation and survival. Our results demonstrate that nano-ZnO causes acute cytoskeletal collapse that triggers necrosis, followed by a late reactive oxygen species (ROS)-dependent apoptotic process.Zinc is a crucial element in biology that plays chief catalytic, structural and protein regulatory roles. Excess cytoplasmic zinc is toxic to cells so there are cell-entry and intracellular buffering mechanisms that control intracellular zinc availability. Tubulin and actin are two zinc-scavenging proteins that are essential components of the cellular cytoskeleton implicated in cell division, migration and cellular architecture maintenance. Here we demonstrate how exposure to different ZnO nanostructures, namely ZnO commercial nanoparticles and custom-made ZnO nanowires, produce acute cytotoxic effects in human keratinocytes (HaCat) and epithelial cells (HeLa) triggering a dose-dependent cell retraction and collapse. We show how engulfed ZnO nanoparticles dissolve intracellularly, triggering actin

  15. Advances in Arabidopsis research in China from 2006 to 2007

    Institute of Scientific and Technical Information of China (English)

    LIANG Yan; ZUO JianRu; YANG WeiCai

    2007-01-01

    @@ Arabidopsis thaliana, a model plant species, has a number of advantages over other plant species as an experimental organism due to many of its genetic and genomic features. The Chinese Arabidopsis community has made significant contributions to plant biology research in recent years[1,2]. In 2006, studies of plant biology in China received more attention than ever before, especially those pertaining to Arabidopsis research. Here we briefly summarize recent advances in Arabidopsis research in China.

  16. Exploring natural product chemistry and biology with multicomponent reactions. 5. Discovery of a novel tubulin-targeting scaffold derived from the rigidin family of marine alkaloids.

    Science.gov (United States)

    Frolova, Liliya V; Magedov, Igor V; Romero, Anntherese E; Karki, Menuka; Otero, Isaiah; Hayden, Kathryn; Evdokimov, Nikolai M; Banuls, Laetitia Moreno Y; Rastogi, Shiva K; Smith, W Ross; Lu, Shi-Long; Kiss, Robert; Shuster, Charles B; Hamel, Ernest; Betancourt, Tania; Rogelj, Snezna; Kornienko, Alexander

    2013-09-12

    We developed synthetic chemistry to access the marine alkaloid rigidins and over 40 synthetic analogues based on the 7-deazaxanthine, 7-deazaadenine, 7-deazapurine, and 7-deazahypoxanthine skeletons. Analogues based on the 7-deazahypoxanthine skeleton exhibited nanomolar potencies against cell lines representing cancers with dismal prognoses, tumor metastases, and multidrug resistant cells. Studies aimed at elucidating the mode(s) of action of the 7-deazahypoxanthines in cancer cells revealed that they inhibited in vitro tubulin polymerization and disorganized microtubules in live HeLa cells. Experiments evaluating the effects of the 7-deazahypoxanthines on the binding of [(3)H]colchicine to tubulin identified the colchicine site on tubulin as the most likely target for these compounds in cancer cells. Because many microtubule-targeting compounds are successfully used to fight cancer in the clinic, we believe the new chemical class of antitubulin agents represented by the 7-deazahypoxanthine rigidin analogues have significant potential as new anticancer agents.

  17. Synthesis, biological evaluation, and molecular docking studies of novel 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives as potential tubulin polymerization inhibitors.

    Science.gov (United States)

    Wang, Yan-Ting; Qin, Ya-Juan; Yang, Na; Zhang, Ya-Liang; Liu, Chang-Hong; Zhu, Hai-Liang

    2015-06-24

    A series of 1-benzene acyl-2-(1-methylindol-3-yl)-benzimidazole derivatives were designed, synthesized and evaluated as potential tubulin polymerization inhibitors and for the cytotoxicity against anthropic cancer cell lines. Among the novel compounds, compound 11f was demonstrated the most potent tubulin polymerization inhibitory activity (IC50 = 1.5 μM) and antiproliferative activity against A549, HepG2 and MCF-7 (GI50 = 2.4, 3.8 and 5.1 μM, respectively), which was compared with the positive control colchicine and CA-4. We also evaluated that compound 11f could effectively induce apoptosis of A549 associated with G2/M phase cell cycle arrest. Docking simulation and 3D-QSAR model in these studies provided more information that could be applied to design new molecules with more potent tubulin inhibitory activity.

  18. SAS-4 is recruited to a dynamic structure in newly forming centrioles that is stabilized by the gamma-tubulin-mediated addition of centriolar microtubules.

    Science.gov (United States)

    Dammermann, Alexander; Maddox, Paul S; Desai, Arshad; Oegema, Karen

    2008-02-25

    Centrioles are surrounded by pericentriolar material (PCM), which is proposed to promote new centriole assembly by concentrating gamma-tubulin. Here, we quantitatively monitor new centriole assembly in living Caenorhabditis elegans embryos, focusing on the conserved components SAS-4 and SAS-6. We show that SAS-4 and SAS-6 are coordinately recruited to the site of new centriole assembly and reach their maximum levels during S phase. Centriolar SAS-6 is subsequently reduced by a mechanism intrinsic to the early assembly pathway that does not require progression into mitosis. Centriolar SAS-4 remains in dynamic equilibrium with the cytoplasmic pool until late prophase, when it is stably incorporated in a step that requires gamma-tubulin and microtubule assembly. These results indicate that gamma-tubulin in the PCM stabilizes the nascent daughter centriole by promoting microtubule addition to its outer wall. Such a mechanism may help restrict new centriole assembly to the vicinity of preexisting parent centrioles that recruit PCM.

  19. Biochemical studies of mouse brain tubulin: colchicine binding (DEAE-cellulose filter) assay and subunits (. cap alpha. and. beta. ) biosynthesis and degradation (in newborn brain)

    Energy Technology Data Exchange (ETDEWEB)

    Tse, Cek-Fyne

    1978-01-01

    A DEAE-cellulose filter assay, measuring (/sup 3/H)colchicine bound to colchicine binding protein (CBP) absorbed on filter discs, has been modified to include lM sucrose in the incubation medium for complexing colchicine to CBP in samples before applying the samples to filter discs (single point assay). Due to the much greater stability of colchicine binding capacity in the presence of lM sucrose, multiple time-point assays and least squares linear regression analysis were not necessary for accurate determination of CBP in hybrid mouse brain at different stages of development. The highest concentrations of CBP were observed in the 160,000g supernatant and pellet of newborn brain homogenate. Further studies of the modified filter assay documented that the assay has an overall counting efficiency of 27.3%, that DEAE-cellulose filters bind and retain all tubulin in the assay samples, and that one molecule of colchicine binds approximately one molecule of tubulin dimer. Therefore, millimoles of colchicine bound per milligram total protein can be used to calculate tubulin content. With this technique tubulin content of brain supernatant was found to be 11.9% for newborn, and 7.15% for 11 month old mice. Quantitative densitometry was also used to measure mouse brain supernatant actin content for these two stages. In vivo synthesis and degradation rates of tubulin ..cap alpha.. and ..beta.. subunits of two day mouse brain 100,000g supernatant were studied after intracerebral injection of (/sup 3/H)leucine. Quantitative changes of the ratio of tritium specific activities of tubulin ..cap alpha.. and ..beta.. subunits with time were determined. The pattern of change was biphasic. During the first phase the ratio decreased; during the second phase the ratio increased continuously. An interpretation consistent with all the data in this study is that the ..cap alpha.. subunit is synthesized at a more rapid rate than the ..beta.. subunit. (ERB)

  20. TGF-{beta}-stimulated aberrant expression of class III {beta}-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Eun Jee [Department of Ophthalmology, National Health Insurance Corporation Ilsan Hospital, Gyeonggi-do (Korea, Republic of); Chun, Ji Na; Jung, Sun-Ah [Konyang University Myunggok Medical Research Institute, Kim' s Eye Hospital, Konyang University College of Medicine, Seoul (Korea, Republic of); Cho, Jin Won [Department of Biology, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Lee, Joon H., E-mail: joonhlee@konyang.ac.kr [Konyang University Myunggok Medical Research Institute, Kim' s Eye Hospital, Konyang University College of Medicine, Seoul (Korea, Republic of)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer TGF-{beta} induces aberrant expression of {beta}III in RPE cells via the ERK pathway. Black-Right-Pointing-Pointer TGF-{beta} increases O-GlcNAc modification of {beta}III in RPE cells. Black-Right-Pointing-Pointer Mature RPE cells have the capacity to express a neuron-associated gene by TGF-{beta}. -- Abstract: The class III {beta}-tubulin isotype ({beta}{sub III}) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III {beta}-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-{beta} (TGF-{beta}) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-{beta} on the aberrant expression of class III {beta}-tubulin and the intracellular signaling pathway mediating these changes. TGF-{beta}-induced aberrant expression and O-linked-{beta}-N-acetylglucosamine (O-GlcNac) modification of class III {beta}-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-{beta} also stimulated phosphorylation of ERK. TGF-{beta}-induced aberrant expression of class III {beta}-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-{beta} stimulated aberrant expression of class III {beta}-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-{beta} stimulation and provide useful information

  1. Neoadjuvant Doxorubicin/Cyclophosphamide Followed by Ixabepilone or Paclitaxel in Early Stage Breast Cancer and Evaluation of βIII-Tubulin Expression as a Predictive Marker

    Science.gov (United States)

    Tseng, Ling-Ming; Chan, Stephen; Chacko, Raju T.; Campone, Mario; Manikhas, Alexy; Nag, Shona M.; Leichman, Cynthia G.; Dasappa, Lokanatha; Fasching, Peter A.; Hurtado de Mendoza, Fernando; Symmans, W. Fraser; Liu, David; Mukhopadhyay, Pralay; Horak, Christine; Xing, Guan; Pusztai, Lajos

    2013-01-01

    Background. This randomized phase II trial was designed to compare the rate of pathologic complete response (pCR) induced by neoadjuvant cyclophosphamide plus doxorubicin (AC) followed by ixabepilone or paclitaxel in women with early stage breast cancer (BC). Expression of βIII-tubulin as a predictive marker was also evaluated. Patients and Methods. Women with untreated, histologically confirmed primary invasive breast adenocarcinoma received four cycles of AC followed by 1:1 randomization to either ixabepilone 40 mg/m2 (3-hour infusion) every 3 weeks for four cycles (n = 148) or weekly paclitaxel 80 mg/m2 (1-hour infusion) for 12 weeks (n = 147). All patients underwent a core needle biopsy of the primary cancer for molecular marker analysis prior to chemotherapy. βIII-Tubulin expression was assessed using immunohistochemistry. Results. There was no significant difference in the rate of pCR in the ixabepilone treatment arm (24.3%; 90% confidence interval [CI], 18.6–30.8) and the paclitaxel treatment arm (25.2%; 90% CI, 19.4–31.7). βIII-Tubulin-positive patients obtained higher pCR rates compared with βIII-tubulin-negative patients in both treatment arms; however, βIII-tubulin expression was not significantly associated with a differential response to ixabepilone or paclitaxel. The safety profiles of both regimens were generally similar, although neutropenia occurred more frequently in the ixabepilone arm (grade 3/4: 41.3% vs. 8.4%). The most common nonhematologic toxicity was peripheral neuropathy. Conclusions. Neoadjuvant treatment of early stage BC with AC followed by ixabepilone every 3 weeks or weekly paclitaxel was well tolerated with no significant difference in efficacy. Higher response rates were observed among βIII-tubulin-positive patients. PMID:23853246

  2. Upregulation of Na+,Cl--Coupled Betaine/ γ-Amino-Butyric Acid Transporter BGT1 by Tau Tubulin Kinase 2

    Directory of Open Access Journals (Sweden)

    Ahmad Almilaji

    2013-08-01

    Full Text Available Background/Aims: The serine/threonine kinase Tau-tubulin-kinase 2 (TTBK2 is expressed in various tissues including kidney, liver and brain. Loss of function mutations of TTBK2 lead to autosomal dominant spinocerebellar ataxia type 11 (SCA11. Cell survival is fostered by cellular accumulation of organic osmolytes. Carriers accomplishing cellular accumulation of organic osmolytes include the Na+, Cl--coupled betaine/γ-amino-butyric acid transporter BGT1. The present study explored whether TTBK2 participates in the regulation of BGT1 activity. Methods: Electrogenic transport of GABA was determined in Xenopus oocytes expressing BGT1 with or without wild-type TTBK2, truncated TTBK2[1-450] or kinase inactive mutants TTBK2- KD and TTBK2[1-450]-KD. Results: Coexpression of wild-type TTBK2, but not of TTBK2[1-450], TTBK2-KD or TTBK2[1-450]-KD, increased electrogenic GABA transport. Wildtype TTBK2 increased the maximal transport rate without significantly modifying affinity of the carrier. Coexpression of wild-type TTBK2 significantly delayed the decline of transport following inhibition of carrier insertion with brefeldin A, indicating that wild-type TTBK2 increased carrier stability in the cell membrane. Conclusion: Tau-tubulin-kinase 2 TTBK2 is a powerful stimulator of the osmolyte and GABA transporter BGT1.

  3. Synthesis of 2-aryl-1,2,4-oxadiazolo-benzimidazoles: Tubulin polymerization inhibitors and apoptosis inducing agents.

    Science.gov (United States)

    Kamal, Ahmed; Reddy, T Srinivasa; Vishnuvardhan, M V P S; Nimbarte, Vijaykumar D; Subba Rao, A V; Srinivasulu, Vunnam; Shankaraiah, Nagula

    2015-08-01

    A new series of 2-aryl 1,2,4-oxadiazolo-benzimidazole conjugates have been synthesized and evaluated for their antiproliferative activity in the sixty cancer cell line panel of the National Cancer Institute (NCI). Compounds 5l (NSC: 761109/1) and 5x (NSC: 761814/1) exhibited remarkable cytotoxic activity against most of the cancer cell lines in the one dose assay and were further screened at five dose concentrations (0.01, 0.1, 1, 10 and 100 μM) which showed GI50 values in the range of 0.79-28.2 μM. Flow cytometric data of these compounds showed increased cells in G2/M phase, which is suggestive of G2/M cell cycle arrest. Further, compounds 5l and 5x showed inhibition of tubulin polymerization and disruption of the formation of microtubules. These compounds induce apoptosis by DNA fragmentation and chromatin condensation as well as by mitochondrial membrane depolarization. In addition, structure activity relationship studies within the series are also discussed. Molecular docking studies of compounds 5l and 5x into the colchicine-binding site of the tubulin, revealed the possible mode of interaction by these compounds.

  4. Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

    Directory of Open Access Journals (Sweden)

    CB Toaldo

    2001-01-01

    Full Text Available The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70 and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

  5. Genetic analysis of a novel tubulin mutation that redirects synaptic vesicle targeting and causes neurite degeneration in C. elegans.

    Directory of Open Access Journals (Sweden)

    Jiun-Min Hsu

    2014-11-01

    Full Text Available Neuronal cargos are differentially targeted to either axons or dendrites, and this polarized cargo targeting critically depends on the interaction between microtubules and molecular motors. From a forward mutagenesis screen, we identified a gain-of-function mutation in the C. elegans α-tubulin gene mec-12 that triggered synaptic vesicle mistargeting, neurite swelling and neurodegeneration in the touch receptor neurons. This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin. Synaptic vesicle mistargeting in the mutant neurons was suppressed by reducing dynein function, suggesting that aberrantly high dynein activity mistargeted synaptic vesicles. We demonstrated that dynein showed preference towards binding mutant microtubules over wild-type in microtubule sedimentation assay. By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal. This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy. Our work provides insights into how microtubule-dynein interaction instructs synaptic vesicle targeting and the importance of microtubule in the maintenance of neuronal structures against constant mechanical stress.

  6. Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes.

    Science.gov (United States)

    Waring, R B; May, G S; Morris, N R

    1989-06-30

    Plasmids have been constructed in which expression of a gene can be placed under the control of the inducible promoter of the alcA gene encoding alcohol dehydrogenase I in Aspergillus nidulans. Simplified shuttle vectors carrying pyr4 which complements pyrG89 mutations have also been constructed. These are based on pUC19 and retain alpha-peptide expression. The beta-tubulin genes, tubC and benA, have been placed under the control of alcA and their expression studied. Levels of expression can be assayed phenotypically because increased synthesis of beta-tubulin inhibits vegetative growth. Sensitivity of asexual spore formation to the anti-microtubule drug benomyl provides a means of detecting very low levels of expression of the chimeric genes. Glucose almost completely represses the chimeric genes. Induction is rapid and is maximal within an hour. When a strain carrying seven copies of an alcA::tubC gene fusion was grown under inducing conditions, 6.5% of total sulfate labelled protein consisted of tubC product. Cyclopentanone was the most potent inducer of the chimeric genes on solid media but it also partially inhibited growth. Chimeric alcA::tubC and alcA::benA genes were expressed to very similar levels despite the fact that tubC utilizes many rare codons.

  7. PCR-RFLP on β-tubulin gene for rapid identification of the most clinically important species of Aspergillus.

    Science.gov (United States)

    Nasri, Tuba; Hedayati, Mohammad Taghi; Abastabar, Mahdi; Pasqualotto, Alessandro C; Armaki, Mojtaba Taghizadeh; Hoseinnejad, Akbar; Nabili, Mojtaba

    2015-10-01

    Aspergillus species are important agents of life-threatening infections in immunosuppressed patients. Proper speciation in the Aspergilli has been justified based on varied fungal virulence, clinical presentations, and antifungal resistance. Accurate identification of Aspergillus species usually relies on fungal DNA sequencing but this requires expensive equipment that is not available in most clinical laboratories. We developed and validated a discriminative low-cost PCR-based test to discriminate Aspergillus isolates at the species level. The Beta tubulin gene of various reference strains of Aspergillus species was amplified using the universal fungal primers Bt2a and Bt2b. The PCR products were subjected to digestion with a single restriction enzyme AlwI. All Aspergillus isolates were subjected to DNA sequencing for final species characterization. The PCR-RFLP test generated unique patterns for six clinically important Aspergillus species, including Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, Aspergillus clavatus and Aspergillus nidulans. The one-enzyme PCR-RFLP on Beta tubulin gene designed in this study is a low-cost tool for the reliable and rapid differentiation of the clinically important Aspergillus species.

  8. Development a diagnostic pan-dermatophyte TaqMan probe real-time PCR assay based on beta tubulin gene.

    Science.gov (United States)

    Mirhendi, Hossein; Motamedi, Marjan; Makimura, Koichi; Satoh, Kazuo

    2016-08-01

    Early differentiation of dermatophytosis from other cutaneous mycoses is essential to avoid inaccurate therapy. DNA-based techniques including real-time PCR have increasingly been considered for detection of fungal elements in clinical specimens. In this study, after partial sequence analysis of beta tubulin (BT2) gene in 13 common and rare pathogenic dermatophyte species, a pan-dermatophyte primer and probe set was designed in a TaqMan probe-based PCR format. The sensitivity and specificity of the system was tested with 22 reference strains of dermatophytes, 234 positive clinical specimens, 32 DNA samples extracted from normal nails, several fungi other than dermatophytes and human DNAs. Analytical detection limit of the designed PCR on serially diluted DNAs of prepared recombinant plasmid indicated that only five molecules per sample are the minimum number for reliable detection by the assay. A total of 226 out of 234 (96.5%) DNAs extracted from clinical samples, but none of the 32 nail samples, from healthy volunteers were positive in PCR. The real-time PCR targeted beta tubulin gene established in this study could be a sensitive diagnostic tool which is significantly faster than the conventional culture method and should be useful in the clinical settings, in large-scale epidemiological studies and in clinical trials of antifungal therapy.

  9. Design and synthesis of pyrazole/isoxazole linked arylcinnamides as tubulin polymerization inhibitors and potential antiproliferative agents.

    Science.gov (United States)

    Kamal, Ahmed; Shaik, Anver Basha; Rao, Bala Bhaskara; Khan, Irfan; Bharath Kumar, G; Jain, Nishant

    2015-10-28

    As pyrazole and isoxazole based derivatives are well-known for displaying a considerable biological profile, an attempt has been made to unravel their cytotoxic potential. In this context, a number of pyrazole/isoxazole linked arylcinnamide conjugates (15a-o and 21a-n) have been synthesized by employing a straight forward route. The basic structure comprised three ring scaffolds (A, B and C): methoxyphenyl rings as A and C rings and a five membered heterocyclic ring (pyrazole or isoxazole) as the B-ring. To achieve clear understanding, these derivatives are categorized as pyrazole-phenylcinnamides (PP) and isoxazole-phenylcinnamides (IP). These compounds have been evaluated for their ability to inhibit the growth of various human cancer cell lines such as HeLa, DU-145, A549 and MDA-MB231 and most of them exhibit considerable cytotoxic effects. Some of them like 15a, 15b, 15e, 15i and 15l exhibit promising cytotoxicity in HeLa cells (IC50 = 0.4, 1.8, 1.2, 2.7 and 1.7 μM). Amongst them 15a, 15b and 15e were taken up for detailed biological studies, they were found to arrest the cells in the G2/M phase of the cell cycle. Moreover, they were investigated for their effect on the microtubular cytoskeletal system by using a tubulin polymerization assay, immunofluroscence and molecular docking studies; interestingly they demonstrate a significant inhibition of tubulin polymerization.

  10. Mining the active proteome of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Renier A. L. Van Der Hoorn

    2011-11-01

    Full Text Available Assigning functions to the >30.000 proteins encoded by the Arabidopsis genome is a challenging task of the Arabidopsis Functional Genomics Network. Although genome-wide technologies like proteomics and transcriptomics have generated a wealth of information that significantly accelerated gene annotation, protein activities are poorly predicted by transcript or protein levels as protein activities are post-translationally regulated. To directly display protein activities in Arabidopsis proteomes, we developed and applied Activity-based Protein Profiling (ABPP. ABPP is based on the use of small molecule probes that react with the catalytic residues of distinct protein classes in an activity-dependent manner. Labeled proteins are separated and detected from proteins gels and purified and identified by mass spectrometry. Using probes of six different chemotypes we have displayed of activities of 76 Arabidopsis proteins. These proteins represent over ten different protein classes that contain over 250 Arabidopsis proteins, including cysteine- serine- and metallo-proteases, lipases, acyltransferases, and the proteasome. We have developed methods for identification of in vivo labeled proteins using click-chemistry and for in vivo imaging with fluorescent probes. In vivo labeling has revealed novel protein activities and unexpected subcellular activities of the proteasome. Labeling of extracts displayed several differential activities e.g. of the proteasome during immune response and methylesterases during infection. These studies illustrate the power of ABPP to display the functional proteome and testify to a successful interdisciplinary collaboration involving chemical biology, organic chemistry and proteomics.

  11. Recent Progress in Arabidopsis Research in China: A Preface

    Institute of Scientific and Technical Information of China (English)

    Zhi-Hong Xu

    2006-01-01

    @@ In 2002, a workshop on Arabidopsis research in China was held in Shanghai, when a small group of Chinese plant scientists was working on this model species. Since then, we have witnessed the rapid growth of Arabidopsis research in China. This special issue of Journal of Integrative Plant Biology is dedicated exclusively to the Fourth Workshop on Arabidopsis Research in China, scheduled on November 30, 2005, in Beijing. In addition to reports collected in this special issue, the Chinese Arabidopsis community has been able to make significant contributions to many research fields. Here, I briefly summarize recent advances in Arabidopsis research in China.

  12. The arabidopsis cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara

    2016-05-11

    Background Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals. Methods An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling. Results A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment. Conclusions We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.

  13. Reference: 510 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ch stabilizes the water-oxidizing complex, is represented in Arabidopsis thaliana (Arabidopsis) by two isofo...rms. Two T-DNA insertion mutant lines deficient in either the PsbO1 or the PsbO2 protein were re...ally. Both PsbO proteins were able to support the oxygen evolution activity of PSII, although PsbO2 was less... efficient than PsbO1 under photoinhibitory conditions. Prolonged high light stress led to re...duced growth and fitness of the mutant lacking PsbO2 as compared with the wild type and the muta

  14. Reference: 600 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available n M et al. 2007 Jun. Plant J. 50(5):810-24. A novel abscisic acid (ABA)-deficient mutant, aba4, was identified in a scre...en for paclobutrazol-resistant germination. Compared with wild-type, the mutant showed reduced e...by map-based cloning, and found to be a unique gene in the Arabidopsis genome. The predicted protein has fou...r putative helical transmembrane domains and shows significant similarity to pred...icted proteins from tomato, rice and cyanobacteria. Constitutive expression of the ABA4 gene in Arabidopsis

  15. Reference: 59 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 59 http://metadb.riken.jp/db/SciNetS_ria224i/cria224u4ria224u14563930i Kaczorowski Kare...naling network in Arabidopsis, we used a sensitized genetic screen for deetiolation-defective seedlings. Two allelic mutants were... isolated that exhibited reduced sensitivity to both continuous red and far-re...d light, suggesting involvement in both phyA and phyB signaling. The molecular lesions res...ponsible for the phenotype were shown to be mutations in the Arabidopsis PSEUDO-RESPONSE REGULATOR7 (PRR7) g

  16. Reference: 640 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available er Alois et al. 2007 Jul. Plant Cell 19(7):2213-24. Wound signaling pathways in plants are mediated by mitog...en-activated protein kinases (MAPKs) and stress hormones, such as ethylene and jasmonates. In Arabidopsis th...ed investigations; however, the involvement of specific phosphatases in wound signaling is not known. Here, ...we show that AP2C1, an Arabidopsis Ser/Thr phosphatase of type 2C, is a novel stress signal regulator that inactivates the stress-re... significantly higher amounts of jasmonate upon wounding and are more resistant to phytophagous mites (Tetra

  17. Reference: 756 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available elle et al. 2008 Jun. Plant Physiol. 147(2):595-610. Treatment of Arabidopsis (Arabidopsis thaliana) alterna...tive oxidase1a (aox1a) mutant plants with moderate light under drought conditions resulted in a phenotypic difference compare...d with ecotype Columbia (Col-0), as evidenced by a 10-fold incre...ase in the accumulation of anthocyanins in leaves, alterations in photosynthetic efficiency, and increased superoxide radical and re...duced root growth at the early stages of seedling growth. Analysis of metabolite profiles re

  18. Reference: 457 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available n et al. 2006 Oct. Plant J. 48(2):238-48. The Arabidopsis BAP1 gene encodes a small protein with a C2-like domain. Here...er and is associated with membranes in vivo. We identify multiple roles of BAP1 in negatively re...gulating defense responses and cell death in Arabidopsis thaliana. The loss of BAP1 function ...confers an enhanced disease resistance to virulent bacterial and oomycete pathogens. The enhanced resistance... is mediated by salicylic acid, PAD4 and a disease resistance gene SNC1. BAP1 is

  19. Gibberellins control fruit patterning in Arabidopsis thaliana.

    Science.gov (United States)

    Arnaud, Nicolas; Girin, Thomas; Sorefan, Karim; Fuentes, Sara; Wood, Thomas A; Lawrenson, Tom; Sablowski, Robert; Østergaard, Lars

    2010-10-01

    The Arabidopsis basic helix-loop-helix (bHLH) proteins INDEHISCENT (IND) and ALCATRAZ (ALC) specify tissues required for fruit opening that have major roles in seed dispersal and plant domestication. Here, we show that synthesis of the phytohormone gibberellin is a direct and necessary target of IND, and that ALC interacts directly with DELLA repressors, which antagonize ALC function but are destabilized by gibberellin. Thus, the gibberellin/DELLA pathway has a key role in patterning the Arabidopsis fruit, and the interaction between DELLA and bHLH proteins, previously shown to connect gibberellin and light responses, is a versatile regulatory module also used in tissue patterning.

  20. Triazole linked mono carbonyl curcumin-isatin bifunctional hybrids as novel anti tubulin agents: Design, synthesis, biological evaluation and molecular modeling studies.

    Science.gov (United States)

    Sharma, Sahil; Gupta, Manish K; Saxena, Ajit K; Bedi, Preet Mohinder S

    2015-11-15

    Keeping in view the limitations associated with currently available anticancer drugs, molecular hybrids of mono carbonyl curcumin and isatin tethered by triazole ring have been synthesized and evaluated for in vitro cytotoxicity against THP-1, COLO-205, HCT-116, A549, HeLa, CAKI-I, PC-3, MiaPaca-2 human cancer cell lines. The results revealed that the compounds SA-1 to SA-9, SB-2, SB-3, SB-4, SB-7 and SC-2 showed a good range of IC50 values against THP-1, COLO-205, HCT-116 and PC-3 cell lines, while the other four cell lines among these were found to be almost resistant. Structure activity relationship revealed that the nature of Ring X and substitution at position R influences the activity. Methoxy substituted phenyl ring as Ring X and H as R were found to be the ideal structural features. The most potent compounds (SA-2, SA-3, SA-4, SA-7) were further tested for tubulin inhibition. Compound SA-2 was found to significantly inhibit the tubulin polymerization (IC50=1.2 μM against HCT-116). Compound SA-2, moreover, lead to the disruption of microtubules as confirmed by immunofluorescence technique. The significant cytotoxicity and tubulin inhibition by SA-2 was streamlined by molecular modeling studies where it was docked at the curcumin binding site of tubulin.

  1. The alpha-tubulin gene AmTuba1: a marker for rapid mycelial growth in the ectomycorrhizal basidiomycete Amanita muscaria.

    Science.gov (United States)

    Tarkka, Mika T; Schrey, Silvia; Nehls, Uwe

    2006-05-01

    The apical extension of hyphae is of central importance for extensive spread of fungal mycelium in forest soils and for effective ectomycorrhiza development. Since the tubulin cytoskeleton is known to be important for fungal tip growth, we have investigated the expression of an alpha-tubulin gene from the ectomycorrhizal basidiomycete Amanita muscaria (AmTuba1). The phylogenetic analysis of protein sequences revealed the existence of two subgroups of alpha-tubulins in homobasidiomycetes, clearly distinguishable by defined amino acids. AmTuba1 belongs to subgroup1. The AmTuba1 transcript level is related to mycelial growth rate. Growth induction of carbohydrate starved (non-growing) hyphae resulted in an enhanced AmTuba1 expression as soon as hyphal growth started, reaching a maximum at highest mycelial growth rate. Bacterium-induced hyphal elongation also leads to increased AmTuba1 transcript levels. In mature A. muscaria/P. abies ectomycorrhizas, where fungal hyphae are highly branched, and slowly growing, AmTuba1 expression were even lower than in carbohydrate-starved mycelium, indicating a further down-regulation of gene expression in symbiosis. In conclusion, our analyses show that the AmTuba1 gene can be used as a marker for active apical extension in fly agaric, and that alpha-tubulin proteins are promising tools for the classification of fungi.

  2. Synthesis, molecular docking and biological evaluation of 1-phenylsulphonyl-2-(1-methylindol-3-yl)-benzimidazole derivatives as novel potential tubulin assembling inhibitors.

    Science.gov (United States)

    Wang, Yan-Ting; Cai, Xun-Chao; Shi, Tian-Qi; Zhang, Ya-Liang; Wang, Zhong-Chang; Liu, Chang-Hong; Zhu, Hai-Liang

    2016-12-29

    A series of new 1-phenylsulphonyl-2-(1-methylindol-3-yl)-benzimidazole derivatives were designed, synthesized and evaluated as potential inhibitors of tubulin polymerization and anthropic cancer cell lines. Among them, compound 33 displayed the most potent tubulin polymerization inhibitory activity in vitro (IC50  = 1.41 μM) and strong antiproliferative activities against A549, Hela, HepG2 and MCF-7 cell lines in vitro with GI50 value of 1.6, 2.7, 2.9 and 4.3 μM, respectively, comparable with the positive control colchicine (GI50 value of 4.1, 7.2, 9.5 and 14.5 μM, respectively) and CA-4 (GI50 value of 2.2, 4.3, 6.4 and 11.4 μM, respectively). Simultaneously, we evaluated that compound 33 could effectively induce apoptosis of A549 associated with G2/M phase cell cycle arrest. Immunofluorescence microscopy also clearly indicated compound 33 a potent antimicrotubule agent. Docking simulation showed that compound 33 could bind tightly with the colchicine-binding site and act as a tubulin inhibitor. Three-dimensional-QSAR model was also built to provide more pharmacophore understanding that could be used to design new agents with more potent tubulin assembling inhibitory activity in the future.

  3. N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)colcemid, a probe for different classes of colchicine-binding site on tubulin.

    Science.gov (United States)

    Sengupta, S; Puri, K D; Surolia, A; Roy, S; Bhattacharyya, B

    1993-03-01

    The nature of binding of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-colcemid (NBD-colcemid), an environment-sensitive fluorescent analogue of colchicine, to tubulin was tested. This article reports the first fluorometric study where two types of binding site of a colchicine analogue on tubulin were detected. Binding of NBD-colcemid to one of these sites equilibrates slowly. NBD-colcemid competes with colchicine for this site. Binding of NBD-colcemid to this site also causes inhibition of tubulin self-assembly. In contrast, NBD-colcemid binding to the other site is characterised by rapid equilibration and lack of competition with colchicine. Nevertheless, binding to this site is highly specific for the colchicine nucleus, as alkyl-NBD analogues have no significant binding activity. Fast-reaction-kinetic studies gave 1.76 x 10(5) M-1 s-1 for the association and 0.79 s-1 for the dissociation rate constants for the binding of NBD-colcemid to the fast site of tubulin. The association rate constants for the two phases of the slow site are 444.4 M-1 s-1 and 11.67 M-1 s-1 [corrected], respectively. These two sites may be related to the two sites of colchicine reported earlier, with binding characteristics altered by the increased hydrophobic nature of NBD-colcemid.

  4. Fission yeast MOZART1/Mzt1 is an essential γ-tubulin complex component required for complex recruitment to the microtubule organizing center, but not its assembly.

    Science.gov (United States)

    Masuda, Hirohisa; Mori, Risa; Yukawa, Masashi; Toda, Takashi

    2013-09-01

    γ-Tubulin plays a universal role in microtubule nucleation from microtubule organizing centers (MTOCs) such as the animal centrosome and fungal spindle pole body (SPB). γ-Tubulin functions as a multiprotein complex called the γ-tubulin complex (γ-TuC), consisting of GCP1-6 (GCP1 is γ-tubulin). In fungi and flies, it has been shown that GCP1-3 are core components, as they are indispensable for γ-TuC complex assembly and cell division, whereas the other three GCPs are not. Recently a novel conserved component, MOZART1, was identified in humans and plants, but its precise functions remain to be determined. In this paper, we characterize the fission yeast homologue Mzt1, showing that it is essential for cell viability. Mzt1 is present in approximately equal stoichiometry with Alp4/GCP2 and localizes to all the MTOCs, including the SPB and interphase and equatorial MTOCs. Temperature-sensitive mzt1 mutants display varying degrees of compromised microtubule organization, exhibiting multiple defects during both interphase and mitosis. Mzt1 is required for γ-TuC recruitment, but not sufficient to localize to the SPB, which depends on γ-TuC integrity. Intriguingly, the core γ-TuC assembles in the absence of Mzt1. Mzt1 therefore plays a unique role within the γ-TuC components in attachment of this complex to the major MTOC site.

  5. TUBA1A mutations cause wide spectrum lissencephaly (smooth brain) and suggest that multiple neuronal migration pathways converge on alpha tubulins

    NARCIS (Netherlands)

    R.A. Kumar (Ravinesh); D.T. Pilz (Daniela); T.D. Babatz (Timothy); T.D. Cushion (Thomas); K. Harvey (Kirsten); M. Topf (Maya); L. Yates (Laura); S. Robb (Stephanie); G. Uyanik (Gökhan); G.M.S. Mancini (Grazia); M.I. Rees (Mark); R.J. Harvey (Robert); W.B. Dobyns (William)

    2010-01-01

    textabstracte previously showed that mutations in LIS1 and DCX account for ~85% of patients with the classic form of lissencephaly (LIS). Some rare forms of LIS are associated with a disproportionately small cerebellum, referred to as lissencephaly with cerebellar hypoplasia (LCH). Tubulin alpha1A (

  6. Thiabendazole resistance and mutations in the beta-tubulin gene of Penicillium expansum strains isolated from apples and pears with blue mold decay.

    Science.gov (United States)

    Cabañas, Romualdo; Castellá, Gemma; Abarca, M Lourdes; Bragulat, M Rosa; Cabañes, F Javier

    2009-08-01

    Penicillium expansum is the causal agent of blue mold rot, a postharvest decay of stored fruits. This fungus also produces the mycotoxins patulin and citrinin. Control of P. expansum still relies mainly on the use of fungicides such as thiabendazole. Since its introduction, resistant strains have been reported. The aim of this work was to investigate the thiabendazole resistance and mutations in the beta-tubulin gene of P. expansum strains isolated from apples and pears with blue mold decay from Spain. A total of 71 strains of P. expansum were scored for resistance to thiabendazole and the beta-tubulin gene was sequenced. Out of 71 strains, 37 were sensitive and 34 were resistant to thiabendazole. Regarding the beta-tubulin gene sequence, 10 different genetic types were determined, with a 99.7-100% similarity. When the amino acid sequence was deduced, five different amino acid sequences were found. All except one of the sensitive strains lacked mutations in the region sequenced. Of the 34 resistant strains, only eight had mutations that involved the residues 198 and 240. All the strains with mutations at position 198 always corresponded to resistant isolates. However, a high percentage of resistant strains had no mutations in the region of the beta-tubulin gene sequenced, and so other mechanisms may be involved in thiabendazole resistance.

  7. [Down-regulated βIII-tubulin expression can reverse paclitaxel resistance in A549/taxol cells lines].

    Science.gov (United States)

    Zhuo, Yinling; Guo, Qisen

    2014-08-20

    背景与目的 化疗耐药导致肿瘤很快复发和/或转移,是目前肺癌死亡的主要原因之一。β-tubulin是抗微管药物的主要细胞靶点。已有的研究证明:βIII-tubulin高表达与非小细胞肺癌(non-small cell lung cancer, NSCLC)耐药有关。利用RNA干扰技术沉默耐紫杉醇A549细胞(A549/Taxol)中βIII-tubulin基因表达,探讨靶基因下调后对化疗药物紫杉醇的敏感性的变化以及细胞周期和细胞凋亡情况。方法 构建靶向βIII-tubulin的siRNA,以脂质体为载体介导βIII-tubulin siRNA转染A549/Taxol细胞,利用qRT-PCR检测细胞内βIII-tubulin mRNA的变化情况,并筛选出最佳干扰序列;Western blot法检测A549/Taxol细胞内βIII-tubulin蛋白表达的变化;MTT法检测转染后细胞株对紫杉醇敏感性的变化;流式细胞仪检测细胞周期和细胞凋亡的变化。结果 实时荧光qRT-PCR法显示转染后细胞株靶基因水平较对照组降低,其中βIII-tubulin siRNA-1序列抑制率最高为(87.73±4.87)%(P<0.01);Western blot显示转染后靶蛋白水平较对照组明显降低;MTT法表明紫杉醇处理转染后细胞株的细胞抑制率较对照组明显增加(51.77±4.60)%(P<0.01);细胞凋亡显示βIII-tubulin siRNA+Taxol组细胞早期凋亡率较对照组明显增加(P<0.01),两者的差异有统计学意义;细胞周期检测结果显示紫杉醇处理组的G2/M期细胞百分率高于对照组,且转染后紫杉醇处理组的细胞晚期凋亡率较对照组增加。结论 βIII-tubulin表达下调明显提高A549/Taxol细胞株对Taxol的敏感性。

  8. Association between response to albendazole treatment and β-tubulin genotype frequencies in soil-transmitted helminths.

    Directory of Open Access Journals (Sweden)

    Aïssatou Diawara

    Full Text Available BACKGROUND: Albendazole (ABZ, a benzimidazole (BZ anthelmintic (AH, is commonly used for treatment of soil-transmitted helminths (STHs. Its regular use increases the possibility that BZ resistance may develop, which, in veterinary nematodes is caused by single nucleotide polymorphisms (SNPs in the β-tubulin gene at positions 200, 167 or 198. The relative importance of these SNPs varies among the different parasitic nematodes of animals studied to date, and it is currently unknown whether any of these are influencing BZ efficacy against STHs in humans. We assessed ABZ efficacy and SNP frequencies before and after treatment of Ascaris lumbricoides, Trichuris trichiura and hookworm infections. METHODS: Studies were performed in Haiti, Kenya, and Panama. Stool samples were examined prior to ABZ treatment and two weeks (Haiti, one week (Kenya and three weeks (Panama after treatment to determine egg reduction rate (ERR. Eggs were genotyped and frequencies of each SNP assessed. FINDINGS: In T. trichiura, polymorphism was detected at codon 200. Following treatment, there was a significant increase, from 3.1% to 55.3%, of homozygous resistance-type in Haiti, and from 51.3% to 67.8% in Kenya (ERRs were 49.7% and 10.1%, respectively. In A. lumbricoides, a SNP at position 167 was identified at high frequency, both before and after treatment, but ABZ efficacy remained high. In hookworms from Kenya we identified the resistance-associated SNP at position 200 at low frequency before and after treatment while ERR values indicated good drug efficacy. CONCLUSION: Albendazole was effective for A. lumbricoides and hookworms. However, ABZ exerts a selection pressure on the β-tubulin gene at position 200 in T. trichiura, possibly explaining only moderate ABZ efficacy against this parasite. In A. lumbricoides, the codon 167 polymorphism seemed not to affect drug efficacy whilst the polymorphism at codon 200 in hookworms was at such low frequency that conclusions

  9. Natural product Celastrol destabilizes tubulin heterodimer and facilitates mitotic cell death triggered by microtubule-targeting anti-cancer drugs.

    Directory of Open Access Journals (Sweden)

    Hakryul Jo

    Full Text Available BACKGROUND: Microtubule drugs are effective anti-cancer agents, primarily due to their ability to induce mitotic arrest and subsequent cell death. However, some cancer cells are intrinsically resistant or acquire a resistance. Lack of apoptosis following mitotic arrest is thought to contribute to drug resistance that limits the efficacy of the microtubule-targeting anti-cancer drugs. Genetic or pharmacological agents that selectively facilitate the apoptosis of mitotic arrested cells present opportunities to strengthen the therapeutic efficacy. METHODOLOGY AND PRINCIPAL FINDINGS: We report a natural product Celastrol targets tubulin and facilitates mitotic cell death caused by microtubule drugs. First, in a small molecule screening effort, we identify Celastrol as an inhibitor of neutrophil chemotaxis. Subsequent time-lapse imaging analyses reveal that inhibition of microtubule-mediated cellular processes, including cell migration and mitotic chromosome alignment, is the earliest events affected by Celastrol. Disorganization, not depolymerization, of mitotic spindles appears responsible for mitotic defects. Celastrol directly affects the biochemical properties of tubulin heterodimer in vitro and reduces its protein level in vivo. At the cellular level, Celastrol induces a synergistic apoptosis when combined with conventional microtubule-targeting drugs and manifests an efficacy toward Taxol-resistant cancer cells. Finally, by time-lapse imaging and tracking of microtubule drug-treated cells, we show that Celastrol preferentially induces apoptosis of mitotic arrested cells in a caspase-dependent manner. This selective effect is not due to inhibition of general cell survival pathways or mitotic kinases that have been shown to enhance microtubule drug-induced cell death. CONCLUSIONS AND SIGNIFICANCE: We provide evidence for new cellular pathways that, when perturbed, selectively induce the apoptosis of mitotic arrested cancer cells, identifying a

  10. Arabidopsis CDS blastp result: AK242890 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242890 J090079L19 At2g32540.1 68415.m03975 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 4e-47 ...

  11. Arabidopsis CDS blastp result: AK242585 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242585 J090010M20 At5g16910.1 68418.m01982 cellulose synthase family protein similar to gi:2827143 cellulo...se synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 1e-28 ...

  12. Arabidopsis CDS blastp result: AK242601 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242601 J090014G03 At4g23990.1 68417.m03448 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 2e-26 ...

  13. Arabidopsis CDS blastp result: AK242601 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242601 J090014G03 At2g32540.1 68415.m03975 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 2e-45 ...

  14. Arabidopsis CDS blastp result: AK242601 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242601 J090014G03 At5g16910.1 68418.m01982 cellulose synthase family protein similar to gi:2827143 cellulo...se synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 0.0 ...

  15. Arabidopsis CDS blastp result: AK242585 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242585 J090010M20 At1g32180.1 68414.m03958 cellulose synthase family protein similar to cellulose... synthase catalytic subunit gi:2827143 from [Arabidopsis thaliana], cellulose synthase-9 (gi:9622890) from Zea mays 1e-24 ...

  16. Arabidopsis CDS blastp result: AK242585 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242585 J090010M20 At5g16910.1 68418.m01982 cellulose synthase family protein similar to gi:2827143 cellulo...se synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 2e-65 ...

  17. Arabidopsis CDS blastp result: AK110534 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110534 002-168-A07 At5g16910.1 cellulose synthase family protein similar to gi:2827143 cellulose... synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 1e-114 ...

  18. Arabidopsis CDS blastp result: AK242890 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242890 J090079L19 At2g32530.1 68415.m03974 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 4e-50 ...

  19. Arabidopsis CDS blastp result: AK242601 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242601 J090014G03 At4g38190.1 68417.m05391 cellulose synthase family protein similar to cellulose... synthase catalytic subunit gi:2827143 from [Arabidopsis thaliana], cellulose synthase-5 (gi:9622882) from Zea mays 0.0 ...

  20. Arabidopsis CDS blastp result: AK242601 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242601 J090014G03 At4g23990.1 68417.m03448 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 5e-25 ...

  1. Arabidopsis CDS blastp result: AK242585 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242585 J090010M20 At2g32530.1 68415.m03974 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 8e-98 ...

  2. Arabidopsis CDS blastp result: AK061162 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061162 006-209-A01 At2g32540.1 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 3e-35 ...

  3. Arabidopsis CDS blastp result: AK242601 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242601 J090014G03 At1g32180.1 68414.m03958 cellulose synthase family protein similar to cellulose... synthase catalytic subunit gi:2827143 from [Arabidopsis thaliana], cellulose synthase-9 (gi:9622890) from Zea mays 0.0 ...

  4. Arabidopsis CDS blastp result: AK242585 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242585 J090010M20 At1g32180.1 68414.m03958 cellulose synthase family protein similar to cellulose... synthase catalytic subunit gi:2827143 from [Arabidopsis thaliana], cellulose synthase-9 (gi:9622890) from Zea mays 3e-66 ...

  5. Arabidopsis CDS blastp result: AK069071 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069071 J023010H01 At2g32540.1 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 1e-167 ...

  6. Arabidopsis CDS blastp result: AK121003 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121003 J023045B21 At2g32540.1 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 1e-167 ...

  7. Arabidopsis CDS blastp result: AK242890 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242890 J090079L19 At4g23990.1 68417.m03448 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 1e-45 ...

  8. Arabidopsis CDS blastp result: AK242585 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242585 J090010M20 At2g32540.1 68415.m03975 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 4e-98 ...

  9. Arabidopsis CDS blastp result: AK060286 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060286 001-006-C08 At2g32540.1 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 6e-78 ...

  10. Arabidopsis CDS blastp result: AK242890 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242890 J090079L19 At4g38190.1 68417.m05391 cellulose synthase family protein similar to cellulose... synthase catalytic subunit gi:2827143 from [Arabidopsis thaliana], cellulose synthase-5 (gi:9622882) from Zea mays 1e-125 ...

  11. Arabidopsis CDS blastp result: AK242601 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242601 J090014G03 At4g23990.1 68417.m03448 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 8e-25 ...

  12. Arabidopsis CDS blastp result: AK242601 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242601 J090014G03 At2g32540.1 68415.m03975 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 3e-31 ...

  13. Arabidopsis CDS blastp result: AK242890 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242890 J090079L19 At5g16910.1 68418.m01982 cellulose synthase family protein similar to gi:2827143 cellulo...se synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 1e-130 ...

  14. Arabidopsis CDS blastp result: AK105393 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105393 001-123-B04 At5g16910.1 cellulose synthase family protein similar to gi:2827143 cellulose... synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 0.0 ...

  15. Arabidopsis CDS blastp result: AK242601 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242601 J090014G03 At2g32530.1 68415.m03974 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 5e-48 ...

  16. Arabidopsis CDS blastp result: AK242601 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242601 J090014G03 At2g32530.1 68415.m03974 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 2e-29 ...

  17. Arabidopsis CDS blastp result: AK109812 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109812 002-147-H02 At5g16910.1 cellulose synthase family protein similar to gi:2827143 cellulose... synthase catalytic subunit, Arabidopsis thaliana, gi:9622886 cellulose synthase-7 from Zea mays 5e-90 ...

  18. Arabidopsis CDS blastp result: AK242585 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242585 J090010M20 At4g38190.1 68417.m05391 cellulose synthase family protein similar to cellulose... synthase catalytic subunit gi:2827143 from [Arabidopsis thaliana], cellulose synthase-5 (gi:9622882) from Zea mays 8e-63 ...

  19. Arabidopsis CDS blastp result: AK242890 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242890 J090079L19 At1g32180.1 68414.m03958 cellulose synthase family protein similar to cellulose... synthase catalytic subunit gi:2827143 from [Arabidopsis thaliana], cellulose synthase-9 (gi:9622890) from Zea mays 1e-126 ...

  20. Arabidopsis CDS blastp result: AK242585 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242585 J090010M20 At4g23990.1 68417.m03448 cellulose synthase family protein similar to cellulose... synthase catalytic subunit from Arabidopsis thaliana [gi:5230423], cellulose synthase-5 from Zea mays [gi:9622882] 1e-124 ...

  1. Arabidopsis CDS blastp result: AK242585 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242585 J090010M20 At4g38190.1 68417.m05391 cellulose synthase family protein similar to cellulose... synthase catalytic subunit gi:2827143 from [Arabidopsis thaliana], cellulose synthase-5 (gi:9622882) from Zea mays 4e-27 ...

  2. Reference: 415 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available study focuses on the seven other Arabidopsis CAD for which functions are not yet elucidated. Their expression patterns were determine...ession of CAD 1, B1, and G genes was determined using their promoters fused to the GUS reporter gene. CAD 1

  3. Arabidopsis CDS blastp result: AK243408 [KOME

    Lifescience Database Archive (English)

    Full Text Available subunit ClpX, putative similar to CLP protease regulatory subunit CLPX GI:2674203 from [Arabidopsis thaliana]; non-consensus... splice donor GC at exon 4; non-consensus splice donor AA at exon 7 1e-151 ...

  4. Arabidopsis CDS blastp result: AK242797 [KOME

    Lifescience Database Archive (English)

    Full Text Available subunit ClpX, putative similar to CLP protease regulatory subunit CLPX GI:2674203 from [Arabidopsis thaliana]; non-consensus... splice donor GC at exon 4; non-consensus splice donor AA at exon 7 2e-23 ...

  5. Arabidopsis CDS blastp result: AK243408 [KOME

    Lifescience Database Archive (English)

    Full Text Available subunit ClpX, putative similar to CLP protease regulatory subunit CLPX GI:2674203 from [Arabidopsis thaliana]; non-consensus... splice donor GC at exon 4; non-consensus splice donor AA at exon 7 2e-12 ...

  6. Reference: 767 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available Arabidopsis thaliana genome. Mutation analysis of 25 of the 27 member genes representing 13 of the 14 sub-families... of the UBP gene family revealed that single-gene mutants of three genes in two sub-families exhibit v

  7. Reference: 158 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available onika et al. 2005 Feb. Plant J. 41(3):386-99. Cullin proteins, which belong to multigenic families in all eu...ic search revealed the existence of at least 76 BTB-domain proteins in Arabidopsis belonging to 11 major families.... Yeast two-hybrid experiments indicate that representative members of certain families are able to phy

  8. Reference: 456 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available h other Spo11/topo VIA proteins, but their functional relationship during meiosis or other processes is not ...s. Thus, the three Arabidopsis Spo11 homologues appear to function in two discrete processes, i.e. AtSPO11-1

  9. Reference: 412 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available the tobacco arcA gene, mediates hormone responses and plays a regulatory role in multiple developmental processes...in RACK1A confer defects in multiple developmental processes including seed germination, leaf production, an...ltiple hormone responsiveness and developmental processes in Arabidopsis. 11 2697-708 16829549 2006 Journal

  10. Reference: 51 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available urce of acetyl-CoA formation in the plastids of plants and is composed of multiple copies of four different ...astidic E2 (dihydrolipoyl acetyltransferase) subunit, plE2, of the complex in Arabidopsis destroys the expre

  11. Reference: 567 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ith findings that noxy2 and mutants with defective 9-LOX activity showed increased numbers of lateral roots,...or of lateral root formation. Histochemical and molecular analyses revealed that 9-HOT activated events comm...in Arabidopsis regulate lateral root development and defense responses through a specific signaling cascade.

  12. Arabidopsis CDS blastp result: AK287911 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287911 J065213B08 At1g12110.1 68414.m01402 nitrate/chlorate transporter (NRT1.1) ...(CHL1) identical to nitrate/chlorate transporter SP:Q05085 from [Arabidopsis thaliana]; contains Pfam profile: PF00854 POT family 3e-85 ...

  13. Arabidopsis CDS blastp result: AK318551 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK318551 J075138M12 At1g12110.1 68414.m01402 nitrate/chlorate transporter (NRT1.1) ...(CHL1) identical to nitrate/chlorate transporter SP:Q05085 from [Arabidopsis thaliana]; contains Pfam profile: PF00854 POT family 4e-27 ...

  14. Arabidopsis CDS blastp result: AK241823 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241823 J065212G21 At1g12110.1 68414.m01402 nitrate/chlorate transporter (NRT1.1) ...(CHL1) identical to nitrate/chlorate transporter SP:Q05085 from [Arabidopsis thaliana]; contains Pfam profile: PF00854 POT family 1e-150 ...

  15. Arabidopsis CDS blastp result: AK243378 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243378 J100063A13 At1g12110.1 68414.m01402 nitrate/chlorate transporter (NRT1.1) ...(CHL1) identical to nitrate/chlorate transporter SP:Q05085 from [Arabidopsis thaliana]; contains Pfam profile: PF00854 POT family 5e-18 ...

  16. Arabidopsis CDS blastp result: AK288351 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288351 J090024C17 At1g12110.1 68414.m01402 nitrate/chlorate transporter (NRT1.1) ...(CHL1) identical to nitrate/chlorate transporter SP:Q05085 from [Arabidopsis thaliana]; contains Pfam profile: PF00854 POT family 2e-24 ...

  17. Arabidopsis CDS blastp result: AK242252 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242252 J075182G16 At1g12110.1 68414.m01402 nitrate/chlorate transporter (NRT1.1) ...(CHL1) identical to nitrate/chlorate transporter SP:Q05085 from [Arabidopsis thaliana]; contains Pfam profile: PF00854 POT family 6e-88 ...

  18. Arabidopsis CDS blastp result: AK073411 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK073411 J033041P20 At4g02060.1 prolifera protein (PRL) / DNA replication licensing... factor Mcm7 (MCM7) identical to DNA replication licensing factor Mcm7 SP|P43299 PROLIFERA protein {Arabidopsis thaliana}; contains Pfam profile PF00493: MCM2/3/5 family 0.0 ...

  19. Arabidopsis CDS blastp result: AK100867 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100867 J023124E13 At2g29640.1 josephin family protein contains Pfam domain PF02099: Jose...phin; similar to Josephin-like protein (Swiss-Prot:O82391) [Arabidopsis thaliana] 7e-59 ...

  20. Arabidopsis CDS blastp result: AK241402 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241402 J065159A02 At4g19070.1 68417.m02810 cadmium-responsive protein / cadmium i...nduced protein (AS8) identical to cadmium induced protein AS8 SP:P42735 from [Arabidopsis thaliana] 3e-11 ...

  1. Proteomics of Arabidopsis seed germination and priming

    NARCIS (Netherlands)

    Gallardo, K.; Job, C.; Groot, S.P.C.; Puype, M.; Demol, H.; Vandekerckhove, J.; Job, D.

    2003-01-01

    To better understand seed germination, a complex developmental process, we developed a proteome analysis of the model plant Arabidopsis for which complete genome sequence is now available. Among about 1,300 total seed proteins resolved in two-dimensional gels, changes in the abundance (up- and down-

  2. Arabidopsis CDS blastp result: AK241096 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241096 J065076O13 At3g10520.1 68416.m01262 non-symbiotic hemoglobin 2 (HB2) (GLB2...) identical to SP|O24521 Non-symbiotic hemoglobin 2 (Hb2) (ARAth GLB2) {Arabidopsis thaliana} 1e-40 ...

  3. Arabidopsis CDS blastp result: AK240885 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240885 J065029A17 At3g10520.1 68416.m01262 non-symbiotic hemoglobin 2 (HB2) (GLB2...) identical to SP|O24521 Non-symbiotic hemoglobin 2 (Hb2) (ARAth GLB2) {Arabidopsis thaliana} 6e-34 ...

  4. Arabidopsis CDS blastp result: AK241096 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241096 J065076O13 At2g16060.1 68415.m01841 non-symbiotic hemoglobin 1 (HB1) (GLB1...) identical to SP|O24520 Non-symbiotic hemoglobin 1 (Hb1) (ARAth GLB1) {Arabidopsis thaliana} 1e-59 ...

  5. Arabidopsis CDS blastp result: AK240885 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240885 J065029A17 At2g16060.1 68415.m01841 non-symbiotic hemoglobin 1 (HB1) (GLB1...) identical to SP|O24520 Non-symbiotic hemoglobin 1 (Hb1) (ARAth GLB1) {Arabidopsis thaliana} 3e-49 ...

  6. Protease gene families in Populus and Arabidopsis

    Directory of Open Access Journals (Sweden)

    Jansson Stefan

    2006-12-01

    Full Text Available Abstract Background Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution. Results We have performed a comparative analysis of protease genes in the two sequenced dicot genomes, Arabidopsis thaliana and Populus trichocarpa by using genes coding for proteases in the MEROPS database 1 for Arabidopsis to identify homologous sequences in Populus. A multigene-based phylogenetic analysis was performed. Most protease families were found to be larger in Populus than in Arabidopsis, reflecting recent genome duplication. Detailed studies on e.g. the DegP, Clp, FtsH, Lon, rhomboid and papain-Like protease families showed the pattern of gene family expansion and gene loss was complex. We finally show that different Populus tissues express unique suites of protease genes and that the mRNA levels of different classes of proteases change along a developmental gradient. Conclusion Recent gene family expansion and contractions have made the Arabidopsis and Populus complements of proteases different and this, together with expression patterns, gives indications about the roles of the individual gene products or groups of proteases.

  7. Arabidopsis CDS blastp result: AK241728 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241728 J065199H08 At1g50310.1 68414.m05640 monosaccharide transporter (STP9) iden...tical to monosaccharide transporter STP9 protein [Arabidopsis thaliana] GI:15487254; contains Pfam profile PF00083: major facilitator superfamily protein 3e-36 ...

  8. Arabidopsis CDS blastp result: AK240645 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK240645 J023003B03 At1g50310.1 68414.m05640 monosaccharide transporter (STP9) iden...tical to monosaccharide transporter STP9 protein [Arabidopsis thaliana] GI:15487254; contains Pfam profile PF00083: major facilitator superfamily protein 1e-17 ...

  9. Arabidopsis CDS blastp result: AK243302 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243302 J100054J17 At1g50310.1 68414.m05640 monosaccharide transporter (STP9) iden...tical to monosaccharide transporter STP9 protein [Arabidopsis thaliana] GI:15487254; contains Pfam profile PF00083: major facilitator superfamily protein 4e-82 ...

  10. Arabidopsis CDS blastp result: AK241015 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241015 J065054A13 At1g50310.1 68414.m05640 monosaccharide transporter (STP9) iden...tical to monosaccharide transporter STP9 protein [Arabidopsis thaliana] GI:15487254; contains Pfam profile PF00083: major facilitator superfamily protein 8e-37 ...

  11. Arabidopsis CDS blastp result: AK288091 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288091 J075184D14 At1g50310.1 68414.m05640 monosaccharide transporter (STP9) iden...tical to monosaccharide transporter STP9 protein [Arabidopsis thaliana] GI:15487254; contains Pfam profile PF00083: major facilitator superfamily protein 4e-29 ...

  12. Arabidopsis CDS blastp result: AK318617 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK318617 J100090H20 At1g19850.1 68414.m02490 transcription factor MONOPTEROS (MP) /... auxin-responsive protein (IAA24) / auxin response factor 5 (ARF5) identical to transcription factor MONOPTEROS (MP/IAA24/ARF5) SP:P93024 from [Arabidopsis thaliana] 2e-63 ...

  13. Arabidopsis CDS blastp result: AK103452 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103452 J033129I11 At1g19850.1 transcription factor MONOPTEROS (MP) / auxin-respon...sive protein (IAA24) / auxin response factor 5 (ARF5) identical to transcription factor MONOPTEROS (MP/IAA24/ARF5) SP:P93024 from [Arabidopsis thaliana] 1e-166 ...

  14. Arabidopsis CDS blastp result: AK243230 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243230 J100044L04 At1g19850.1 68414.m02490 transcription factor MONOPTEROS (MP) /... auxin-responsive protein (IAA24) / auxin response factor 5 (ARF5) identical to transcription factor MONOPTEROS (MP/IAA24/ARF5) SP:P93024 from [Arabidopsis thaliana] 2e-65 ...

  15. Reference: 346 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 346 http://metadb.riken.jp/db/SciNetS_ria224i/cria224u4ria224u16496096i Todd Christopher...midohydrolase activity from Arabidopsis thaliana. 5 1108-13 16496096 2006 Apr Planta Polacco Joe C|Todd Christopher D

  16. Arabidopsis CDS blastp result: AK242980 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242980 J090094F15 At3g58780.1 68416.m06551 agamous-like MADS box protein AGL1 / shatterproof... 1 (AGL1) (SHP1) identical to SP|P29381 Agamous-like MADS box protein AGL1 (Protein Shatterproof 1) {Arabidopsis thaliana} 2e-19 ...

  17. Arabidopsis CDS blastp result: AK241644 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241644 J065189M04 At3g58780.1 68416.m06551 agamous-like MADS box protein AGL1 / shatterproof... 1 (AGL1) (SHP1) identical to SP|P29381 Agamous-like MADS box protein AGL1 (Protein Shatterproof 1) {Arabidopsis thaliana} 3e-37 ...

  18. Arabidopsis CDS blastp result: AK241055 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241055 J065063N18 At3g58780.1 68416.m06551 agamous-like MADS box protein AGL1 / shatterproof... 1 (AGL1) (SHP1) identical to SP|P29381 Agamous-like MADS box protein AGL1 (Protein Shatterproof 1) {Arabidopsis thaliana} 1e-26 ...

  19. Arabidopsis CDS blastp result: AK242211 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242211 J075171C16 At3g58780.1 68416.m06551 agamous-like MADS box protein AGL1 / shatterproof... 1 (AGL1) (SHP1) identical to SP|P29381 Agamous-like MADS box protein AGL1 (Protein Shatterproof 1) {Arabidopsis thaliana} 5e-21 ...

  20. Arabidopsis CDS blastp result: AK243669 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243669 J100089N11 At3g58780.1 68416.m06551 agamous-like MADS box protein AGL1 / shatterproof... 1 (AGL1) (SHP1) identical to SP|P29381 Agamous-like MADS box protein AGL1 (Protein Shatterproof 1) {Arabidopsis thaliana} 6e-14 ...

  1. Arabidopsis CDS blastp result: AK100613 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100613 J023107M18 At4g10180.1 light-mediated development protein 1 / deetiolated1... (DET1) identical to Light-mediated development protein DET1 (Deetiolated1) (Swiss-Prot:P48732) [Arabidopsis thaliana] 0.0 ...

  2. Arabidopsis CDS blastp result: AK058683 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK058683 001-019-A06 At4g10180.1 light-mediated development protein 1 / deetiolated...1 (DET1) identical to Light-mediated development protein DET1 (Deetiolated1) (Swiss-Prot:P48732) [Arabidopsis thaliana] 0.0 ...

  3. Arabidopsis CDS blastp result: AK241645 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241645 J065189N07 At5g20000.1 68418.m02380 26S proteasome AAA-ATPase subunit, putative almost... identical to 26S proteasome AAA-ATPase subunit RPT6a GI:6652888 from [Arabidopsis thaliana]; almost

  4. Arabidopsis CDS blastp result: AK243043 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243043 J100008P08 At5g20000.1 68418.m02380 26S proteasome AAA-ATPase subunit, putative almost... identical to 26S proteasome AAA-ATPase subunit RPT6a GI:6652888 from [Arabidopsis thaliana]; almost

  5. Arabidopsis CDS blastp result: AK241277 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241277 J065134P20 At5g20000.1 68418.m02380 26S proteasome AAA-ATPase subunit, putative almost... identical to 26S proteasome AAA-ATPase subunit RPT6a GI:6652888 from [Arabidopsis thaliana]; almost

  6. Arabidopsis CDS blastp result: AK241074 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241074 J065068E03 At5g20000.1 68418.m02380 26S proteasome AAA-ATPase subunit, putative almost... identical to 26S proteasome AAA-ATPase subunit RPT6a GI:6652888 from [Arabidopsis thaliana]; almost

  7. Reference: 386 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 386 http://metadb.riken.jp/db/SciNetS_ria224i/cria224u4ria224u16698900i Hricová Andrea...d mesophyll cell proliferation in Arabidopsis. 3 942-56 16698900 2006 Jul Plant physiology Hricová Andrea|Micol José Luis|Quesada Victor

  8. Reference: 394 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 394 http://metadb.riken.jp/db/SciNetS_ria224i/cria224u4ria224u16766689i Rudella Andrea...and defects in chloroplast biogenesis in Arabidopsis. 7 1704-21 16766689 2006 Jul The Plant cell Alonso Jose M|Ecker Joseph R|Friso Giulia|Rudella Andrea|van Wijk Klaas J

  9. Arabidopsis CDS blastp result: AK243428 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243428 J100067L15 At5g14750.1 68418.m01731 myb family transcription factor (MYB66) / werewolf...iption factor (MYB66) mRNA, partial cds GI:3941491; identical to GP:9755743 myb transcription factor werewolf (WER)/ MYB66 {Arabidopsis thaliana} 8e-36 ...

  10. Arabidopsis CDS blastp result: AK288699 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288699 J090061C22 At5g14750.1 68418.m01731 myb family transcription factor (MYB66) / werewolf...iption factor (MYB66) mRNA, partial cds GI:3941491; identical to GP:9755743 myb transcription factor werewolf (WER)/ MYB66 {Arabidopsis thaliana} 8e-36 ...

  11. Arabidopsis CDS blastp result: AK243271 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243271 J100049K04 At5g14750.1 68418.m01731 myb family transcription factor (MYB66) / werewolf...iption factor (MYB66) mRNA, partial cds GI:3941491; identical to GP:9755743 myb transcription factor werewolf (WER)/ MYB66 {Arabidopsis thaliana} 4e-35 ...

  12. Arabidopsis CDS blastp result: AK241812 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241812 J065210K15 At5g14750.1 68418.m01731 myb family transcription factor (MYB66) / werewolf...iption factor (MYB66) mRNA, partial cds GI:3941491; identical to GP:9755743 myb transcription factor werewolf (WER)/ MYB66 {Arabidopsis thaliana} 1e-22 ...

  13. Arabidopsis CDS blastp result: AK241549 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241549 J065176M15 At5g14750.1 68418.m01731 myb family transcription factor (MYB66) / werewolf...iption factor (MYB66) mRNA, partial cds GI:3941491; identical to GP:9755743 myb transcription factor werewolf (WER)/ MYB66 {Arabidopsis thaliana} 3e-32 ...

  14. Arabidopsis CDS blastp result: AK241615 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241615 J065186D02 At5g14750.1 68418.m01731 myb family transcription factor (MYB66) / werewolf...iption factor (MYB66) mRNA, partial cds GI:3941491; identical to GP:9755743 myb transcription factor werewolf (WER)/ MYB66 {Arabidopsis thaliana} 8e-35 ...

  15. Arabidopsis CDS blastp result: AK288487 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288487 J090040H24 At5g14750.1 68418.m01731 myb family transcription factor (MYB66) / werewolf...iption factor (MYB66) mRNA, partial cds GI:3941491; identical to GP:9755743 myb transcription factor werewolf (WER)/ MYB66 {Arabidopsis thaliana} 5e-37 ...

  16. Arabidopsis CDS blastp result: AK287469 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287469 J043021L20 At5g14750.1 68418.m01731 myb family transcription factor (MYB66) / werewolf...iption factor (MYB66) mRNA, partial cds GI:3941491; identical to GP:9755743 myb transcription factor werewolf (WER)/ MYB66 {Arabidopsis thaliana} 2e-36 ...

  17. Arabidopsis CDS blastp result: AK241370 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241370 J065154C10 At5g14750.1 68418.m01731 myb family transcription factor (MYB66) / werewolf...iption factor (MYB66) mRNA, partial cds GI:3941491; identical to GP:9755743 myb transcription factor werewolf (WER)/ MYB66 {Arabidopsis thaliana} 2e-31 ...

  18. Arabidopsis CDS blastp result: AK288415 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288415 J090031E07 At5g14750.1 68418.m01731 myb family transcription factor (MYB66) / werewolf...iption factor (MYB66) mRNA, partial cds GI:3941491; identical to GP:9755743 myb transcription factor werewolf (WER)/ MYB66 {Arabidopsis thaliana} 3e-37 ...

  19. Arabidopsis CDS blastp result: AK287447 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287447 J043016O04 At2g46590.1 68415.m05811 Dof zinc finger protein DAG2 / Dof affecting germination... 2 (DAG2) identical to SP|Q9ZPY0 DOF zinc finger protein DAG2 (Dof affecting germination 2) {Arabidopsis thaliana} 2e-30 ...

  20. Arabidopsis CDS blastp result: AK241364 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241364 J065152E11 At2g46590.1 68415.m05811 Dof zinc finger protein DAG2 / Dof affecting germination... 2 (DAG2) identical to SP|Q9ZPY0 DOF zinc finger protein DAG2 (Dof affecting germination 2) {Arabidopsis thaliana} 2e-20 ...

  1. Arabidopsis CDS blastp result: AK242393 [KOME

    Lifescience Database Archive (English)

    Full Text Available ctor, putative / enhancer of shoot regeneration (ESR1) similar to gb|D38124 EREBP-3 from Nicotiana tabacum a...nd contains PF|00847 AP2 domain; identical to cDNA enhancer of shoot regeneration ESR1 GI:18028939, enhancer of shoot regeneration ESR1 [Arabidopsis thaliana] GI:18028940 3e-13 ...

  2. Arabidopsis CDS blastp result: AK241281 [KOME

    Lifescience Database Archive (English)

    Full Text Available ctor, putative / enhancer of shoot regeneration (ESR1) similar to gb|D38124 EREBP-3 from Nicotiana tabacum a...nd contains PF|00847 AP2 domain; identical to cDNA enhancer of shoot regeneration ESR1 GI:18028939, enhancer of shoot regeneration ESR1 [Arabidopsis thaliana] GI:18028940 1e-12 ...

  3. Arabidopsis CDS blastp result: AK241762 [KOME

    Lifescience Database Archive (English)

    Full Text Available ctor, putative / enhancer of shoot regeneration (ESR1) similar to gb|D38124 EREBP-3 from Nicotiana tabacum a...nd contains PF|00847 AP2 domain; identical to cDNA enhancer of shoot regeneration ESR1 GI:18028939, enhancer of shoot regeneration ESR1 [Arabidopsis thaliana] GI:18028940 9e-17 ...

  4. Arabidopsis CDS blastp result: AK242986 [KOME

    Lifescience Database Archive (English)

    Full Text Available ctor, putative / enhancer of shoot regeneration (ESR1) similar to gb|D38124 EREBP-3 from Nicotiana tabacum a...nd contains PF|00847 AP2 domain; identical to cDNA enhancer of shoot regeneration ESR1 GI:18028939, enhancer of shoot regeneration ESR1 [Arabidopsis thaliana] GI:18028940 1e-13 ...

  5. Arabidopsis CDS blastp result: AK287689 [KOME

    Lifescience Database Archive (English)

    Full Text Available avonol 3-O-methyltransferase 1 / caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT1) identical to ...1.1.76) (AtOMT1) (Flavonol 3- O-methyltransferase 1) (Caffeic acid/5-hydroxyferulic acid O- methyltransferase) {Arabidopsis thaliana} 5e-23 ...

  6. Arabidopsis CDS blastp result: AK240736 [KOME

    Lifescience Database Archive (English)

    Full Text Available avonol 3-O-methyltransferase 1 / caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT1) identical to ...1.1.76) (AtOMT1) (Flavonol 3- O-methyltransferase 1) (Caffeic acid/5-hydroxyferulic acid O- methyltransferase) {Arabidopsis thaliana} 1e-22 ...

  7. Arabidopsis CDS blastp result: AK241705 [KOME

    Lifescience Database Archive (English)

    Full Text Available avonol 3-O-methyltransferase 1 / caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT1) identical to ...1.1.76) (AtOMT1) (Flavonol 3- O-methyltransferase 1) (Caffeic acid/5-hydroxyferulic acid O- methyltransferase) {Arabidopsis thaliana} 1e-11 ...

  8. Arabidopsis CDS blastp result: AK287483 [KOME

    Lifescience Database Archive (English)

    Full Text Available avonol 3-O-methyltransferase 1 / caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT1) identical to ...1.1.76) (AtOMT1) (Flavonol 3- O-methyltransferase 1) (Caffeic acid/5-hydroxyferulic acid O- methyltransferase) {Arabidopsis thaliana} 1e-37 ...

  9. Arabidopsis CDS blastp result: AK107208 [KOME

    Lifescience Database Archive (English)

    Full Text Available Ala hydrolase, putative virtually identical to gr1-protein from [Arabidopsis thaliana] GI:3559811; similar t...AK107208 002-125-B11 At1g44350.1 IAA-amino acid hydrolase 6, putative (ILL6) / IAA-

  10. Arabidopsis CDS blastp result: AK062144 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK062144 001-045-G08 At5g54080.2 homogentisate 1,2-dioxygenase / homogentisicase/ho... (EC 1.13.11.5) (Homogentisicase) (Homogentisate oxygenase) (Homogentisic acid oxidase) {Arabidopsis thaliana}; contains Pfam profile PF04209: homogentisate 1,2-dioxygenase 1e-155 ...

  11. Arabidopsis CDS blastp result: AK061294 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061294 006-301-D01 At3g08900.1 reversibly glycosylated polypeptide-3 (RGP3) nearl...y identical to reversibly glycosylated polypeptide-3 [Arabidopsis thaliana] GI:11863238; contains non-consensus GA-donor splice site at intron 2 0.0 ...

  12. Reference: 119 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available of the Arabidopsis homolog of MSH4 (AtMSH4). We demonstrate that AtMSH4 expression can only be detected in floral tissues, consisten...chromosomes. A T-DNA insertional mutant (Atmsh4) exhibited normal vegetative growth but a severe reduction in fertility, consistent

  13. Reference: 428 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available on was delayed in the psb27 mutant, suggesting that Psb27 is required for efficient...icient repair of photodamaged photosystem II. 4-5 567-75...he involvement of this lumenal protein in the recovery process of PSII. A Psb27 homologue in Arabidopsis thaliana is required for eff

  14. Arabidopsis CDS blastp result: AK105724 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105724 001-201-G07 At1g07110.1 fructose-6-phosphate 2-kinase / fructose-2,6-bisph...osphatase (F2KP) identical to fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (F2KP) [Arabidopsis thaliana] GI:13096098 0.0 ...

  15. Arabidopsis CDS blastp result: AK072243 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072243 J023003N10 At1g07110.1 fructose-6-phosphate 2-kinase / fructose-2,6-bispho...sphatase (F2KP) identical to fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (F2KP) [Arabidopsis thaliana] GI:13096098 0.0 ...

  16. Arabidopsis CDS blastp result: AK243221 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243221 J100043L21 At5g15410.2 68418.m01803 cyclic nucleotide-regulated ion channel / cyclic... nucleotide-gated channel (CNGC2) identical to cyclic nucleotide-gated cation channel GI:3894399 from [Arabidopsis thaliana] 5e-40 ...

  17. Arabidopsis CDS blastp result: AK067626 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK067626 J013112I06 At5g15410.1 cyclic nucleotide-regulated ion channel / cyclic nu...cleotide-gated channel (CNGC2) identical to cyclic nucleotide-gated cation channel GI:3894399 from [Arabidopsis thaliana] 0.0 ...

  18. Arabidopsis CDS blastp result: AK243602 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243602 J100084P18 At5g15410.2 68418.m01803 cyclic nucleotide-regulated ion channel / cyclic... nucleotide-gated channel (CNGC2) identical to cyclic nucleotide-gated cation channel GI:3894399 from [Arabidopsis thaliana] 2e-98 ...

  19. Arabidopsis CDS blastp result: AK288592 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288592 J090051B06 At5g15410.2 68418.m01803 cyclic nucleotide-regulated ion channel / cyclic... nucleotide-gated channel (CNGC2) identical to cyclic nucleotide-gated cation channel GI:3894399 from [Arabidopsis thaliana] 1e-145 ...

  20. Arabidopsis CDS blastp result: AK060339 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060339 001-008-C12 At5g15410.2 cyclic nucleotide-regulated ion channel / cyclic n...ucleotide-gated channel (CNGC2) identical to cyclic nucleotide-gated cation channel GI:3894399 from [Arabidopsis thaliana] 1e-175 ...

  1. Regulatory Proteolysis in Arabidopsis-Pathogen Interactions

    OpenAIRE

    Miklós Pogány; Tamás Dankó; Evelin Kámán-Tóth; Ildikó Schwarczinger; Zoltán Bozsó

    2015-01-01

    Approximately two and a half percent of protein coding genes in Arabidopsis encode enzymes with known or putative proteolytic activity. Proteases possess not only common housekeeping functions by recycling nonfunctional proteins. By irreversibly cleaving other proteins, they regulate crucial developmental processes and control responses to environmental changes. Regulatory proteolysis is also indispensable in interactions between plants and their microbial pathogens. Proteolytic cleavage is s...

  2. Reference: 566 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available utations in the MKK3-MPK6 cascade, which indicates important roles in JA signaling. We provide a model expla...tress - into three different sets of responses in Arabidopsis. The mitogen-activated protein kinase cascade MKK3-MPK6 is an important

  3. Reference: 392 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available pment. The Arabidopsis SUPPRESSOR OF AUXIN RESISTANCE proteins are nucleoporins with an important role in ho...olyadenylated RNA within the nucleus, indicating that SAR1 and SAR3 are required for mRNA export. Our results demonstrate the importa...nt role of the plant NPC in hormone signaling and develo

  4. Reference: 438 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ity and drought tolerance in Arabidopsis thaliana. 18 6902-12 16943431 2006 Sep Molecular and cellular bio...logy Chen Zhizhong|Gong Zhizhong|Hong Xuhui|Jablonowski Daniel|Ren Xiaozhi|Schaffrath Raffael|Zhang Hairong|Zhou Xiaofeng|Zhu Jian-Kang

  5. Reference: 356 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 006 Mar Plant molecular biology Deng Xingwang|Dong Li|Wang Lei|Xue Yongbiao|Zhang Yansheng|Zhang Yu'e ...ein CEGENDUO negatively regulates auxin-mediated lateral root formation in Arabidopsis. 4 599-615 16525894 2

  6. Arabidopsis CDS blastp result: AK059353 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059353 001-026-D01 At1g01170.1 ozone-responsive stress-related protein, putative ...similar to stress-related ozone-induced protein AtOZI1 (GI:790583) [Arabidopsis thaliana]; contains 1 predicted transmembrane domain; 2e-29 ...

  7. Arabidopsis CDS blastp result: AK066771 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066771 J013083K07 At1g01170.1 ozone-responsive stress-related protein, putative s...imilar to stress-related ozone-induced protein AtOZI1 (GI:790583) [Arabidopsis thaliana]; contains 1 predicted transmembrane domain; 2e-29 ...

  8. Arabidopsis CDS blastp result: AK059160 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059160 001-023-D05 At1g01170.1 ozone-responsive stress-related protein, putative ...similar to stress-related ozone-induced protein AtOZI1 (GI:790583) [Arabidopsis thaliana]; contains 1 predicted transmembrane domain; 3e-28 ...

  9. Reference: 234 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 234 http://metadb.riken.jp/db/SciNetS_ria224i/cria224u4ria224u15980261i Stepanova ...ion of two root-specific ethylene-insensitive mutants in Arabidopsis. 8 2230-42 15980261 2005 Aug The Plant cell Alonso Jose M|Hamilton Alexandra A|Hoyt Joyce M|Stepanova Anna N

  10. Arabidopsis CDS blastp result: AK101721 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101721 J033061A20 At3g57040.1 two-component responsive regulator / response reactor... 4 (RR4) identical to responce reactor4 GI:3273202 from [Arabidopsis thaliana]; contains Pfam profile: PF00072 response regulator receiver domain 9e-49 ...

  11. Arabidopsis CDS blastp result: AK058585 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK058585 001-017-G01 At3g57040.1 two-component responsive regulator / response reactor... 4 (RR4) identical to responce reactor4 GI:3273202 from [Arabidopsis thaliana]; contains Pfam profile: PF00072 response regulator receiver domain 6e-55 ...

  12. Arabidopsis CDS blastp result: AK066153 [KOME

    Lifescience Database Archive (English)

    Full Text Available pC almost identical to ClpC GI:2921158 from [Arabidopsis thaliana]; contains Pfam profile PF02861: Clp amino... terminal domain; contains Pfam profile PF00004: ATPase, AAA family; contains Pfam profile PF02151: UvrB/uvrC motif 0.0 ...

  13. Arabidopsis CDS blastp result: AK287906 [KOME

    Lifescience Database Archive (English)

    Full Text Available subunit / ClpC almost identical to ClpC GI:2921158 from [Arabidopsis thaliana]; contains Pfam profile PF028...61: Clp amino terminal domain; contains Pfam profile PF00004: ATPase, AAA family; contains Pfam profile PF02151: UvrB/uvrC motif 0.0 ...

  14. Arabidopsis CDS blastp result: AK100126 [KOME

    Lifescience Database Archive (English)

    Full Text Available pC almost identical to ClpC GI:2921158 from [Arabidopsis thaliana]; contains Pfam profile PF02861: Clp amino... terminal domain; contains Pfam profile PF00004: ATPase, AAA family; contains Pfam profile PF02151: UvrB/uvrC motif 0.0 ...

  15. Arabidopsis CDS blastp result: AK058510 [KOME

    Lifescience Database Archive (English)

    Full Text Available lpC almost identical to ClpC GI:2921158 from [Arabidopsis thaliana]; contains Pfam profile PF02861: Clp amin...o terminal domain; contains Pfam profile PF00004: ATPase, AAA family; contains Pfam profile PF02151: UvrB/uvrC motif 0.0 ...

  16. Arabidopsis CDS blastp result: AK069552 [KOME

    Lifescience Database Archive (English)

    Full Text Available pC almost identical to ClpC GI:2921158 from [Arabidopsis thaliana]; contains Pfam profile PF02861: Clp amino... terminal domain; contains Pfam profile PF00004: ATPase, AAA family; contains Pfam profile PF02151: UvrB/uvrC motif 0.0 ...

  17. Arabidopsis CDS blastp result: AK288349 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288349 J090023P19 At2g46590.1 68415.m05811 Dof zinc finger protein DAG2 / Dof affect...ing germination 2 (DAG2) identical to SP|Q9ZPY0 DOF zinc finger protein DAG2 (Dof affecting germination 2) {Arabidopsis thaliana} 1e-23 ...

  18. Reference: 396 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ht to be encoded in Arabidopsis by the ATS1 locus. A number of genetic mutants deficient in this activity have been described. How...hosphatidylglycerol raised the question of whether an alternative pathway of phosphatidylglycerol assembly in the plastid exists. How

  19. Arabidopsis CDS blastp result: AK103126 [KOME

    Lifescience Database Archive (English)

    Full Text Available 0S proteasome beta subunit PBB1 (PBB1) GB:AAC32066 [Arabidopsis thaliana] (Genetics 149 (2), 677-692 (1998)); contains Pfam profile: PF00227 proteasome A-type and B-type; 1e-129 ...

  20. Reference: 750 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 750 http://metadb.riken.jp/db/SciNetS_ria224i/cria224u4ria224u18390594i Fulton Daniel...in Arabidopsis chloroplasts. 4 1040-58 18390594 2008 Apr The Plant cell Dorken Gary|Eicke Simona|Francisco Perigio|Fulton Daniel

  1. Reference: 161 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available sis have not been identified. We tested whether several Arabidopsis thaliana enzy...ith the fact that GH3.6 was active on each of these auxins. By contrast, GH3.6 and the other five enzymes tested

  2. Reference: 267 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available tien et al. 2005 Sep. Plant J. 43(6):824-36. The sucrose transporter gene AtSUC5 was studied as part of a programme aimed at identify...ing and studying the genes involved in seed maturation in Arabidopsis. Expression p

  3. Arabidopsis CDS blastp result: AK242807 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242807 J090060H17 At5g37500.1 68418.m04516 guard cell outward rectifying K+ chann...el (GORK) identical to guard cell outward rectifying K+ channel [Arabidopsis thaliana] gi|11414742|emb|CAC17

  4. Arabidopsis gene expression patterns during spaceflight

    Science.gov (United States)

    Paul, A.-L.; Ferl, R. J.

    The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments resulted in the differential expression of hundreds of genes. A 5 day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β -Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on two fronts. First, expression patterns visualized with the Adh/GUS transgene were used to address specifically the possibility that spaceflight induces a hypoxic stress response, and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. (Paul et al., Plant Physiol. 2001, 126:613). Second, genome-wide patterns of native gene expression were evaluated utilizing the Affymetrix ATH1 GeneChip? array of 8,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes identified with the arrays was further characterized with quantitative Real-Time RT PCR (ABI - TaqmanTM). Comparison of the patterns of expression for arrays of hybridized with RNA isolated from plants exposed to spaceflight compared to the control arrays revealed hundreds of genes that were differentially expressed in response to spaceflight, yet most genes that are hallmarks of hypoxic stress were unaffected. These results will be discussed in light of current models for plant responses to the spaceflight environment, and with regard to potential future flight opportunities.

  5. Arabidopsis CDS blastp result: AK110694 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110694 002-170-A08 At5g59560.2 sensitivity to red light reduced protein (SRR1) id...entical to sensitivity to red light reduced protein [Arabidopsis thaliana] GI:25527089; supporting cDNA gi|25527088|gb|AY127047.1| 1e-18 ...

  6. Arabidopsis CDS blastp result: AK243061 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243061 J100014C18 At5g24520.2 68418.m02892 transparent testa glabra 1 protein (TTG1) identical to transpar...ent testa glabra 1 (Ttg1) protein (GI:10177852) {Arabidopsis thaliana}; contains Pfam PF00400: WD domain, G-beta repeat (4 copies,1 weak); 1e-102 ...

  7. Arabidopsis CDS blastp result: AK288081 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288081 J075172F18 At5g24520.3 68418.m02893 transparent testa glabra 1 protein (TTG1) identical to transpar...ent testa glabra 1 (Ttg1) protein (GI:10177852) {Arabidopsis thaliana}; contains Pfam PF00400: WD domain, G-beta repeat (4 copies,1 weak); 4e-13 ...

  8. Arabidopsis CDS blastp result: AK287566 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK287566 J065027L04 At1g34790.1 68414.m04337 transparent testa 1 protein (TT1) / zi...nc finger (C2H2 type) protein TT1 identical to transparent testa 1 GI:18253279 from [Arabidopsis thaliana]; contains Pfam profile PF00096: Zinc finger, C2H2 type 2e-77 ...

  9. Arabidopsis CDS blastp result: AK288081 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288081 J075172F18 At5g24520.1 68418.m02891 transparent testa glabra 1 protein (TTG1) identical to transpar...ent testa glabra 1 (Ttg1) protein (GI:10177852) {Arabidopsis thaliana}; contains Pfam PF00400: WD domain, G-beta repeat (4 copies,1 weak); 4e-13 ...

  10. Arabidopsis CDS blastp result: AK289209 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK289209 J100058I16 At1g34790.1 68414.m04337 transparent testa 1 protein (TT1) / zi...nc finger (C2H2 type) protein TT1 identical to transparent testa 1 GI:18253279 from [Arabidopsis thaliana]; contains Pfam profile PF00096: Zinc finger, C2H2 type 1e-12 ...

  11. Arabidopsis CDS blastp result: AK243061 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243061 J100014C18 At5g24520.1 68418.m02891 transparent testa glabra 1 protein (TTG1) identical to transpar...ent testa glabra 1 (Ttg1) protein (GI:10177852) {Arabidopsis thaliana}; contains Pfam PF00400: WD domain, G-beta repeat (4 copies,1 weak); 1e-102 ...

  12. Arabidopsis CDS blastp result: AK243061 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243061 J100014C18 At5g24520.3 68418.m02893 transparent testa glabra 1 protein (TTG1) identical to transpar...ent testa glabra 1 (Ttg1) protein (GI:10177852) {Arabidopsis thaliana}; contains Pfam PF00400: WD domain, G-beta repeat (4 copies,1 weak); 1e-102 ...

  13. Arabidopsis CDS blastp result: AK243285 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243285 J100051N01 At1g34790.1 68414.m04337 transparent testa 1 protein (TT1) / zi...nc finger (C2H2 type) protein TT1 identical to transparent testa 1 GI:18253279 from [Arabidopsis thaliana]; contains Pfam profile PF00096: Zinc finger, C2H2 type 1e-24 ...

  14. Arabidopsis CDS blastp result: AK288081 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288081 J075172F18 At5g24520.2 68418.m02892 transparent testa glabra 1 protein (TTG1) identical to transpar...ent testa glabra 1 (Ttg1) protein (GI:10177852) {Arabidopsis thaliana}; contains Pfam PF00400: WD domain, G-beta repeat (4 copies,1 weak); 4e-13 ...

  15. Arabidopsis CDS blastp result: AK242346 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242346 J080012M07 At2g41100.1 68415.m05076 touch-responsive protein / calmodulin-related protein 3, touch...-induced (TCH3) identical to calmodulin-related protein 3, touch-induced SP:P25071 from [Arabidopsis thaliana] 8e-44 ...

  16. Arabidopsis CDS blastp result: AK243656 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243656 J100088L22 At2g41100.2 68415.m05077 touch-responsive protein / calmodulin-related protein 3, touch...-induced (TCH3) identical to calmodulin-related protein 3, touch-induced SP:P25071 from [Arabidopsis thaliana] 5e-20 ...

  17. Arabidopsis CDS blastp result: AK242346 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242346 J080012M07 At2g41100.1 68415.m05076 touch-responsive protein / calmodulin-related protein 3, touch...-induced (TCH3) identical to calmodulin-related protein 3, touch-induced SP:P25071 from [Arabidopsis thaliana] 4e-41 ...

  18. Arabidopsis CDS blastp result: AK242346 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242346 J080012M07 At2g41100.2 68415.m05077 touch-responsive protein / calmodulin-related protein 3, touch...-induced (TCH3) identical to calmodulin-related protein 3, touch-induced SP:P25071 from [Arabidopsis thaliana] 3e-26 ...

  19. Arabidopsis CDS blastp result: AK242346 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242346 J080012M07 At5g37770.1 68418.m04547 touch-responsive protein / calmodulin-related protein 2, touch...-induced (TCH2) identical to calmodulin-related protein 2,touch-induced SP:P25070 from [Arabidopsis thaliana] 2e-11 ...

  20. Arabidopsis CDS blastp result: AK242428 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242428 J080089P09 At2g41100.2 68415.m05077 touch-responsive protein / calmodulin-related protein 3, touch...-induced (TCH3) identical to calmodulin-related protein 3, touch-induced SP:P25071 from [Arabidopsis thaliana] 3e-16 ...