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T. Schnieder - One of the best experts on this subject based on the ideXlab platform.
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Beta-Tubulin complementary DNA sequence variations observed between cyathostomins from benzimidazole-susceptible and -resistant populations.
The Journal of parasitology, 2004Co-Authors: Michaela Drogemuller, T. Schnieder, Gvon Samson-himmelstjernaAbstract:The molecular mechanism of benzimidazole (BZ) resistance in cyathostomins of horses is still unclear. Previous studies revealed that the TTC or TAC polymorphism in codon 200 of the Beta-Tubulin isotype 1 gene is not as strictly correlated with BZ resistance as in trichostrongyles in sheep. To identify further sites of polymorphism within the Beta-Tubulin gene related to BZ resistance, complete complementary DNAs (cDNAs) encoding Beta-Tubulin of adult worms of Cylicocyclus nassatus, Cyathostomum pateratum, Cyathostomum coronatum, Cyathostomum catinatum, Cylicostephanus longibursatus, and Cylicostephanus goldi of a BZ-resistant cyathostomin population were characterized using specific primers. The cDNA sequence of each species spans 1,429 bp, encoding a protein of 448 amino acids. The interspecific identities are 95.2–99.6% at the nucleotide and 98.7–100.0% at the peptide level. The comparison of the amino acid sequences of individuals isolated from the BZ-resistant cyathostomin population with those from i...
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allele specific pcr for the Beta Tubulin codon 200 ttc tac polymorphism using single adult and larval small strongyle cyathostominae stages
Journal of Parasitology, 2002Co-Authors: G Von Samsonhimmelstjerna, M. Pape, C Von Witzendorff, T. SchniederAbstract:It has been shown that benzimidazole (BZ) resistance in sheep gastrointestinal nematodes is linked with an increase in Beta-Tubulin codon 200 tyrosine-expressing alleles in the resistant parasite populations. Here, an allele-specific PCR has been developed for the discrimination of the TAC/TTC polymorphism in the Beta-Tubulin 200 codon of small strongyles. One reverse primer was used in 2 separate amplifications with 1 of 2 forward primers that differed only in their final 3′ nucleotide. The primers flank a facultative intron/exon. Therefore, the amplified fragments are either 251 or 308 bp in size, depending on the presence or absence of the intron in individual worms. Amplification of genomic DNA isolated from single adult small strongyles from a set of 7 species consistently generated allele-specific products. Three worms each of the following species were used: Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus elongatus, Cylicocyclus radiatus, Cyathostomum pateratum, Cyathostomum catinatum, an...
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Investigation of diversity and isotypes of the Beta-Tubulin cDNA in several small strongyle (Cyathostominae) species.
Journal of Parasitology, 2002Co-Authors: M. Pape, T. Schnieder, G. Von Samson-himmelstjernaAbstract:The diversity of the Beta-Tubulin cDNAs of the cyathostominae and the occurrence of further isotypes were examined in adult worms isolated from an anthelmintic-naive horse. cDNAs encoding Beta-Tubulin from Cyathostomum catinatum, Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus radiatus, Cylicocyclus elongatus, Cyathostomum coronatum, and Cyathostomum pateratum were characterized using specific primers developed from the cDNA sequence of Cc. nassatus. The cDNA sequences span 1,429 bp and show identities ranging from 95.6 to 100%. The deduced protein sequences span 448 amino acids and were 98–100% identical. The amino acid sequences of the 7 species varied within and between species at 10 positions. A 3′ Rapid Amplification of cDNA ends using a degenerate forward primer was carried out with cDNA from Cy. pateratum, Cy. coronatum, Cy. catinatum, and Cc. nassatus to investigate the occurrence of further Beta-Tubulin isotypes. The expected polymerase chain reaction (PCR) product of 400 bp, including ...
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Novel small strongyle (Cyathostominae) Beta-Tubulin sequences
Parasitology Research, 2001Co-Authors: G. Von Samson-himmelstjerna, A. Harder, M. Pape, T. SchniederAbstract:Several coding sequences of the benzimidazole (BZ) target Beta-Tubulin have been described for different parasitic nematodes. However, until recently no Tubulin sequences from Cyathostome species were available, despite the importance of BZ resistance in horses in the field. Here, we describe several full-length Beta-Tubulin coding sequences of two major small strongyle species, namely Cylicocyclus nassatus and Cyathostomum coronatum . In the latter sequence, the putative BZ resistant mutation in codon 200 leading to a Phe to Tyr exchange is present. High nucleotide sequence similarities (>95%) were found among the Tubulin sequences of the two different genera. This will be of advantage for the development of an allele-specific BZ resistance polymerase chain reaction (PCR) for multiple small strongyle species.
Maria Kavallaris - One of the best experts on this subject based on the ideXlab platform.
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Tubulin targeted drug action functional significance of class ii and class ivb Beta Tubulin in vinca alkaloid sensitivity
Cancer Research, 2008Co-Authors: Pei Pei Gan, Maria KavallarisAbstract:Aberrant expression of Beta-Tubulin isotypes is frequently described in tumor tissues and Tubulin-binding agent (TBA)-resistant cell lines. There is limited understanding of the role of specific Beta-Tubulin isotypes in cellular sensitivity to TBAs, and to gain insights into the functional role of BetaII- and BetaIVb-Tubulin, we examined these isotypes in lung cancer cell lines NCI-H460 (H460) and Calu-6. Drug-treated clonogenic assays revealed that small interfering RNA-mediated knockdown of either BetaII- or BetaIVb-Tubulin hypersensitized the lung cancer cell lines to Vinca alkaloids, with the effects more pronounced following BetaIVb-Tubulin knockdown. In contrast, there was no change in paclitaxel sensitivity following knockdown of either isotype. Cell cycle analysis revealed a greater propensity for the BetaII- and BetaIVb-Tubulin knockdown cells to undergo G2-M cell cycle block following 5 nmol/L vincristine treatment, with the BetaIVb knockdown cells being more sensitive than the BetaII-Tubulin knockdown cells compared with control. In contrast to BetaII-Tubulin knockdown, BetaIVb-Tubulin knockdown cells showed a significant increase in the sub-G1 population (cell death) following treatment with both 5 and 40 nmol/L of vincristine compared with controls. Importantly, BetaIVb-Tubulin knockdown in H460 cells caused a significant dose-dependent increase in Annexin V staining in response to vincristine but not paclitaxel. Therefore, increased sensitivity to induction of apoptosis is one mechanism underlying the Vinca alkaloid hypersensitivity. This study provides direct evidence that BetaII- or BetaIVb-Tubulins have functionally distinct roles and expression of these isotypes may serve as strong predictors of Vinca alkaloid response and resistance.
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neuronal associated microtubule proteins class iii Beta Tubulin and map2c in neuroblastoma role in resistance to microtubule targeted drugs
Molecular Cancer Therapeutics, 2004Co-Authors: Sima Don, Murray D Norris, Michelle Haber, Nicole M Verrills, Tracy Y E Liaw, Marjorie Liu, Maria KavallarisAbstract:Advanced stage neuroblastoma has a poor clinical outcome and microtubule-destabilizing agents, such as the Vinca alkaloids, are an important component in the treatment of this childhood cancer. Vinca alkaloids bind to Beta-Tubulin on the alpha/Beta-Tubulin heterodimer and disrupt microtubule dynamics, leading to cell death. To date, studies examining the contribution of microtubules and associated proteins to the efficacy of microtubule-destabilizing agents in neuroblastoma have been limited. In this study, BE2-C neuroblastoma cells previously selected for resistance to either vincristine (BE/VCR10) or colchicine (BE/CHCb0.2) were found to display significant decreases in neuronal-specific class III Beta-Tubulin. Interestingly, vincristine-selected cells exhibited increased levels of polymerized Tubulin that were not due to alpha-Tubulin and class I, II, or III Beta-Tubulin mutations. Expression levels of the microtubule-depolymerizing protein stathmin were significantly increased in BE/VCR10 cells. In contrast, levels of MAP2a and MAP2b were relatively unaltered. A marked decrease in the neuronal protein, MAP2c, was identified in the vincristine-selected cells and, to a lesser extent, in the colchicine-selected cells. This is the first report describing specific microtubule alterations in neuroblastoma cells resistant to Tubulin-targeted agents. The results indicate a need to identify the factors responsible for resistance to Tubulin-targeted agents in neuroblastoma so that improved and novel treatment strategies can be developed for this drug refractory disease.
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taxol resistant epithelial ovarian tumors are associated with altered expression of specific Beta Tubulin isotypes
Journal of Clinical Investigation, 1997Co-Authors: Maria Kavallaris, Dennis Yishin Kuo, Catherine Burkhart, Donna Lee Regl, Murray D Norris, Michelle Haber, Susan Band HorwitzAbstract:The treatment of advanced ovarian cancer with taxol is hindered by the development of drug resistance. The cellular target for taxol is the microtubule that is stabilized by the drug. Taxol preferentially binds to the Beta subunit of Tubulin of which there are six distinct isotypes in mammalian cells. We have used highly specific oligonucleotides and polymerase chain reaction to analyze expression of all six Beta-Tubulin genes. Human lung cancer cells (A549) were selected in 12 and 24 nM taxol resulting in cell lines that were 9- and 17-fold resistant, respectively. These cells displayed an altered ratio of classes I, II, III, and IVa Beta-Tubulin isotypes. Ovarian tumors, seven untreated primary and four taxol- resistant tumor-bearing ascites, displayed significant increases (P < 0.005) in classes I (3.6-fold), III (4.4-fold), and IVa (7.6-fold) isotypes in the taxol-resistant samples as compared with untreated primary ovarian tumors. The increased expression appears to be related to the resistance phenotype, as the basal levels of the class III and IVa isotypes in the untreated tumors were extremely low. This is the first report of altered expression of specific Beta-Tubulin genes in taxol-resistant ovarian tumors and we propose that the latter may play a role in clinical resistance to taxol.
M. Pape - One of the best experts on this subject based on the ideXlab platform.
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analysis of the Beta Tubulin codon 200 genotype distribution in a benzimidazole susceptible and resistant cyathostome population
Parasitology, 2003Co-Authors: M. Pape, Janez Posedi, Klaus Failing, Thomas Schnieder, G Von SamsonhimmelstjernaAbstract:To study the prevalence of the polymorphism in position 200 of the Beta-Tubulin gene in the mechanism of benzimidazole (BZ) resistance in cyathostomes of horses, an allele-specific PCR was used to detect the genotype of individuals of BZ-susceptible and BZ-resistant populations. The molecular analysis of 100 adults recovered from an anthelmintic-naive horse revealed 80% homozygous TTC/TTC individuals, 17% heterozygous TTC/TAC and 3% homozygous TAC/TAC. A naturally infected horse was treated with increasing fenbendazole (FBZ) dosages to select a BZ-resistant population of cyathostomes. The PCR based analysis of 3rd-stage larvae (L3) during the experiment revealed a decrease of the homozygous TTC/TTC genotype and an increase in heterozygous TTC/TAC and homozygous TAC/TAC individuals. After treatment 42·3% of the adults ( n =104) were homozygous TTC/TTC, 55·8% were heterozygous TTC/TAC and only 1·9% showed the homozygous genotype TAC/TAC. The results of the molecular analysis lead to the proposal that polymorphism within codon 200 is not the only reason for the development of BZ resistance in small strongyles.
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allele specific pcr for the Beta Tubulin codon 200 ttc tac polymorphism using single adult and larval small strongyle cyathostominae stages
Journal of Parasitology, 2002Co-Authors: G Von Samsonhimmelstjerna, M. Pape, C Von Witzendorff, T. SchniederAbstract:It has been shown that benzimidazole (BZ) resistance in sheep gastrointestinal nematodes is linked with an increase in Beta-Tubulin codon 200 tyrosine-expressing alleles in the resistant parasite populations. Here, an allele-specific PCR has been developed for the discrimination of the TAC/TTC polymorphism in the Beta-Tubulin 200 codon of small strongyles. One reverse primer was used in 2 separate amplifications with 1 of 2 forward primers that differed only in their final 3′ nucleotide. The primers flank a facultative intron/exon. Therefore, the amplified fragments are either 251 or 308 bp in size, depending on the presence or absence of the intron in individual worms. Amplification of genomic DNA isolated from single adult small strongyles from a set of 7 species consistently generated allele-specific products. Three worms each of the following species were used: Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus elongatus, Cylicocyclus radiatus, Cyathostomum pateratum, Cyathostomum catinatum, an...
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Investigation of diversity and isotypes of the Beta-Tubulin cDNA in several small strongyle (Cyathostominae) species.
Journal of Parasitology, 2002Co-Authors: M. Pape, T. Schnieder, G. Von Samson-himmelstjernaAbstract:The diversity of the Beta-Tubulin cDNAs of the cyathostominae and the occurrence of further isotypes were examined in adult worms isolated from an anthelmintic-naive horse. cDNAs encoding Beta-Tubulin from Cyathostomum catinatum, Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus radiatus, Cylicocyclus elongatus, Cyathostomum coronatum, and Cyathostomum pateratum were characterized using specific primers developed from the cDNA sequence of Cc. nassatus. The cDNA sequences span 1,429 bp and show identities ranging from 95.6 to 100%. The deduced protein sequences span 448 amino acids and were 98–100% identical. The amino acid sequences of the 7 species varied within and between species at 10 positions. A 3′ Rapid Amplification of cDNA ends using a degenerate forward primer was carried out with cDNA from Cy. pateratum, Cy. coronatum, Cy. catinatum, and Cc. nassatus to investigate the occurrence of further Beta-Tubulin isotypes. The expected polymerase chain reaction (PCR) product of 400 bp, including ...
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Novel small strongyle (Cyathostominae) Beta-Tubulin sequences
Parasitology Research, 2001Co-Authors: G. Von Samson-himmelstjerna, A. Harder, M. Pape, T. SchniederAbstract:Several coding sequences of the benzimidazole (BZ) target Beta-Tubulin have been described for different parasitic nematodes. However, until recently no Tubulin sequences from Cyathostome species were available, despite the importance of BZ resistance in horses in the field. Here, we describe several full-length Beta-Tubulin coding sequences of two major small strongyle species, namely Cylicocyclus nassatus and Cyathostomum coronatum . In the latter sequence, the putative BZ resistant mutation in codon 200 leading to a Phe to Tyr exchange is present. High nucleotide sequence similarities (>95%) were found among the Tubulin sequences of the two different genera. This will be of advantage for the development of an allele-specific BZ resistance polymerase chain reaction (PCR) for multiple small strongyle species.
Cristina Martin - One of the best experts on this subject based on the ideXlab platform.
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paclitaxel resistance in non small cell lung cancer associated with Beta Tubulin gene mutations
Journal of Clinical Oncology, 1999Co-Authors: Mariano Monzo, Rafael Rosell, Jose Javier Sanchez, Jin S Lee, Aurora Obrate, Jose Luis Gonzalezlarriba, V Alberola, Juan Carlos Lorenzo, Laura Nunez, Cristina MartinAbstract:PURPOSE: The mechanisms that cause chemoresistance in non–small-cell lung cancer (NSCLC) patients have yet to be clearly elucidated. Paclitaxel is a Tubulin-disrupting agent that binds preferentially to Beta-Tubulin. Tubulins are guanosine triphosphate (GTP)–binding proteins. Beta-Tubulin is a GTPase, whereas alpha-Tubulin has no enzyme activity. We reasoned that polymerase chain reaction (PCR) and DNA sequencing of the Beta-Tubulin gene could reveal more information regarding the connection between Beta-Tubulin mutations and primary paclitaxel resistance. PATIENTS AND METHODS: Constitutional genomic DNA and paired tumor DNA were isolated from 49 biopsies from 43 Spanish and six American stage IIIB and IV NSCLC patients who had been treated with a 3-hour, 210 mg/m2 paclitaxel infusion and a 24-hour, 200 mg/m2 infusion, respectively. Oligonucleotides specific to Beta-Tubulin were designed for PCR amplification and sequencing of GTP- and paclitaxel-binding Beta-Tubulin domains. RESULTS: Of 49 patients with ...
Anthony Frankfurter - One of the best experts on this subject based on the ideXlab platform.
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Beta Tubulin isotype classes ii and v expression patterns in nonsmall cell lung carcinomas
Cytoskeleton, 2008Co-Authors: Valeria Cucchiarelli, Laree Hiser, Hilda Smith, Anthony Frankfurter, Anthony J Spano, John J Correia, Sharon LobertAbstract:Previous studies suggest that Beta-Tubulin isotype protein levels could be useful as indicators of nonsmall cell lung cancer (NSCLC) aggressiveness. However, measurement of protein amounts in tissue samples by staining techniques is semiquantitative at best. Since technologies for measuring mRNA levels have become more efficient and quantitative, we wanted to determine whether Beta-Tubulin message levels may be useful as biomarkers. Quantitative real-time RT-PCR was used to measure the seven classes of Beta-Tubulin isotypes, stathmin and MAP4 mRNA levels in 64 NSCLC and 12 normal lung tissue samples. We found significantly higher fractions of Beta-Tubulin classes II and V mRNA compared to the other isotypes in all lung tumor samples (P < 0.05). In addition, the ratio of Beta-Tubulin classes II/V mRNA was significantly higher in NSCLCs compared to normal lung tissues (P < 0.001). The data suggest that the ratio of Beta-Tubulin classes II and V mRNA could be useful as a biomarker for NSCLC tumor differentiation and/or NSCLC aggressiveness. Furthermore, the ratio of MAP4 to stathmin mRNA was found to be higher in diseased lung tissues compared to normal lung tissues, suggesting this ratio might also be used as a clinically relevant biomarker for NSCLCs.
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characterization of posttranslational modifications in neuron specific class iii Beta Tubulin by mass spectrometry
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Janice E Alexander, Donald F Hunt, Michael K Lee, Jeffrey Shabanowitz, Hanspeter Michel, Sunetary C Berlin, Timothy L Macdonald, Richard J Sundberg, Lionel I Rebhun, Anthony FrankfurterAbstract:Abstract Class III Beta-Tubulin, isolated from adult bovine brain, is resolved into at least seven charge variants on isoelectric focusing gels. To identify the posttranslational modifications responsible for this heterogeneity, a mixture of brain Tubulins was treated with cyanogen bromide and the C-terminal fragments from the class III Beta-Tubulin isoforms were then isolated by binding them to the monoclonal antibody TuJ1. Combined use of tandem mass spectrometry and both subtractive and automated Edman degradation chemistry on the isolated peptides indicates that many of the isoforms differ by phosphorylation at Ser-444 plus attachment of one to six glutamic acid molecules to the side chain of the first glutamate residue, Glu-438, in the C-terminal sequence Tyr-Glu-Asp-Asp-Glu-Glu-Glu-Ser-glu-Ala-Gln-Gly-Pro-Lys.