Β-tubulin Isotypes Research Articles

The tubulin code at a glance.

Microtubules are key cytoskeletal elements of all eukaryotic cells and are assembled of evolutionarily conserved α-tubulin-β-tubulin heterodimers. Despite their uniform structure, microtubules fulfill a large diversity of functions. A regulatory mechanism to control the specialization of the microtubule cytoskeleton is the 'tubulin code', which is generated by (i) expression of different α- and β-tubulin isotypes, and by (ii) post-translational modifications of tubulin. In this Cell Science at a Glance article and the accompanying poster, we provide a comprehensive overview of the molecular components of the tubulin code, and discuss the mechanisms by which these components contribute to the generation of functionally specialized microtubules.

Elucidation of the anticancer potential and tubulin isotype-specific interactions of β-sitosterol

Beta-sitosterol (β-SITO), a phytosterol present in many edible vegetables, has been reported to possess antineoplastic properties and cancer treatment potential. We have shown previously that it binds at a unique site (the ‘SITO-site’) compared to the colchicine binding site at the interface of α- and β-tubulin. In this study, we investigated the anticancer efficacy of β-SITO against invasive breast carcinoma using MCF-7 cells. Since ‘isotypes’ of β-tubulin show tissue-specific expression and many are associated with cancer drug resistance, using computer-assisted docking and atomistic molecular dynamic simulations, we also examined its binding interactions to all known isotypes of β-tubulin in αβ-tubulin dimer. β-SITO inhibited MCF-7 cell viability by up to 50%, compared to vehicle-treated control cells. Indicating its antimetastatic potential, the phytosterol strongly inhibited cell migration. Immunofluorescence imaging of β-SITO-treated MCF-7 cells exhibited disruption of the microtubules and chromosome organization. Far-UV circular dichroism spectra indicated loss of helical stability in tubulin when bound to β-SITO. Docking and MD simulation studies, combined with MM-PBSA and MM-GBSA calculations revealed that β-SITO preferentially binds with specific β-tubulin isotypes (βII and βIII) in the αβ-tubulin dimer. Both these β-tubulin isotypes have been implicated in drug resistance against tubulin-targeted chemotherapeutics. Our data show the tubulin-targeted anticancer potential of β-SITO, and its potential clinical utility against βII and βIII isotype-overexpressing neoplasms.

Genotypic and phenotypic evaluation for benzimidazole resistance or susceptibility in Haemonchus contortus isolates.

Haemonchus contortus isolates were evaluated for benzimidazole (BZ) resistance or susceptibility by allele-specific PCR based on β-tubulin isotype 1 gene polymorphisms at the F167Y, E198A, and F200Y sites. Two isolates, one presumed susceptible from wild pronghorn antelope (PH) and one known to be resistant from goats (VM), were also assayed phenotypically for BZ resistance or susceptibility in the larval development assay (Drenchrite®). The BZ EC50 was 0.198μM (intermediate between susceptible and weak resistant) for PH with critical well 5 (intermediate between susceptible and weak resistant) and 1.456μM (intermediate weak resistant and resistant) for VM with critical well 8.5 (resistant). Genotypically, DNA extracted from pooled VM L3 larvae in the Drenchrite® wells with the highest BZ concentration was homozygous susceptible (SS) at the F167Y and E198A sites and homozygous resistant (RR) at the F200Y site by PCR, and sequence analysis bore this out. PH L3 larvae DNA from a control well (no BZ) was SS at all three sites by PCR, confirmed by sequence analysis. All single adult worm samples (N=21) from PH, VM, Egypt goat (EG), and a Texas llama were SS at F167Y and E198A by PCR; however, only 3 PH worms and 1 EG worm were SS at F200Y. Three additional PH worms were RS and upon cloning two clones were identified as resistant by sequencing and two as susceptible. Clones from single adult worms VM, llama, and EG samples that were RR by PCR at F200Y were sequence verified as resistant. In this study, F200Y was the most frequently found genotypic marker for BZ resistance or susceptibility in the different Haemonchus isolates.

Novel mutations involving βI-, βIIA-, or βIVB-tubulin isotypes with functional resemblance to βIII-tubulin in breast cancer.

Tubulin is the target for very widely used anti-tumor drugs, including Vinca alkaloids, taxanes, and epothilones, which are an important component of chemotherapy in breast cancer and other malignancies. Paclitaxel and other tubulin-targeting drugs bind to the β subunit of tubulin, which is a heterodimer of α and β subunits. β-Tubulin exists in the form of multiple isotypes, which are differentially expressed in normal and neoplastic cells and differ in their ability to bind to drugs. Among them, the βIII isotype is overexpressed in many aggressive and metastatic cancers and may serve as a prognostic marker in certain types of cancer. The underpinning mechanisms accounting for the overexpression of this isotype in cancer cells are unclear. To better understand the role of β-tubulin isotypes in cancer, we analyzed over 1000 clones from 90 breast cancer patients, sequencing their β-tubulin isotypes, in search of novel mutations. We have elucidated two putative emerging molecular subgroups of invasive breast cancer, each of which involve mutations in the βI-, βIIA-, or βIVB isotypes of tubulin that increase their structural, and possibly functional, resemblance to the βIII isotype. A unifying feature of the first of the two subgroups is the mutation of the highly reactive C239 residue of βI- or βIVB-tubulin to L239, R239, Y239, or P239, culminating in probable conversion of these isotypes from ROS-sensitive to ROS-resistant species. In the second subgroup, βI, βIIA, and βIVB have up to seven mutations to the corresponding residues in βIII-tubulin. Given that βIII-tubulin has emerged as a pro-survival factor, overexpression of this isotype may confer survival advantages to certain cancer cell types. In this mini-review, we bring attention to a novel mechanism by which cancer cells may undergo adaptive mutational changes involving alternate β-tubulin isotypes to make them acquire some of the pro-survival properties of βIII-tubulin. These "hybrid" tubulins, combining the sequences and/or properties of two wild-type tubulins (βIII and either βI, βIIA, or βIVB), are novel isotypes expressed solely in cancer cells and may contribute to the molecular understanding and stratification of invasive breast cancer and provide novel molecular targets for rational drug development.

Isothermal Diagnostic Assays for Monitoring Single Nucleotide Polymorphisms in Necator americanus Associated with Benzimidazole Drug Resistance.

BackgroundSoil-transmitted helminths (STHs) are the most prevalent intestinal helminths of humans, and a major cause of morbidity in tropical and subtropical countries. The benzimidazole (BZ) drugs albendazole (ABZ) and mebendazole (MBZ) are used for treatment of human STH infections and this use is increasing dramatically with massive drug donations. Frequent and prolonged use of these drugs could lead to the emergence of anthelmintic resistance as has occurred in nematodes of livestock. Previous molecular assays for putative resistance mutations have been based mainly on PCR amplification and sequencing. However, these techniques are complicated and time consuming and not suitable for resource-constrained situations. A simple, rapid and sensitive genotyping method is required to monitor for possible developing resistance to BZ drugs.MethodsTo address this problem, single nucleotide polymorphism (SNP) detection assays were developed based on the Smart amplification method (SmartAmp2) to target codons 167, 198, and 200 in the β-tubulin isotype 1 gene for the hookworm Necator americanus.FindingsDiagnostic assays were developed and applied to analyze hookworm samples by both SmartAmp2 and conventional sequencing methods and the results showed high concordance. Additionally, fecal samples spiked with N. americanus larvae were assessed and the results showed that the Aac polymerase used has high tolerance to inhibitors in fecal samples.ConclusionThe N. americanus SmartAmp2 SNP detection assay is a new genotyping tool that is rapid, sensitive, highly specific and efficient with the potential to be used as a field tool for monitoring SNPs associated with BZ resistance. However, further validation on large numbers of field samples is required.

High levels of benzimidazole resistance and β-tubulin isotype 1 SNP F167Y in Haemonchus contortus populations from Ceará State, Brazil

Haemonchus contortus is the most prevalent parasitic nematode in tropical areas, and anthelmintic resistance is a global problem. Our objective was to characterize benzimidazole (BZ) resistance in gastrointestinal nematode populations in Ceará State, Brazil, using the egg hatch test (EHT) and in H. contortus populations using quantitative real-time polymerase chain reaction (qPCR). Twenty locations were surveyed, and fecal samples were collected from a minimum of 40 animals from each farm and pooled. Five thousand L3 from each farm were used to infect single animals at Embrapa (Brazilian Agricultural Research Company) to provide a source of eggs for both phenotypical and molecular tests. The mean EHT was 2.46μg/ml (±0.58μg/ml), and BZ resistance was detected at all surveyed locations. The mean resistant allelic frequencies at positions F200Y and F167Y were 34.16% (±12.13%) and 58.31% (±18.89%), respectively. The resistant allelic frequencies at F167Y were higher than those at F200Y in most studied locations. We also investigated the possibility that specific BZ utilization may influence resistant allelic frequencies. We selected three nematode populations based on the resistant SNP prevalence at F200Y and F167Y as follows: higher frequency at SNP F200Y, higher frequency at SNP F167Y and similar frequencies at both positions. Anthelmintic treatments included two BZs (oxfendazole and albendazole) and ivermectin. Three animals per population per treatment were infected with 5000 L3, and nematode eggs were collected for molecular test before and after anthelmintic treatments. The results showed preferential selection of SNP F167Y in response to oxfendazole, an increase in resistant SNP frequencies in general in response to albendazole and little change in relation to pre-treatment situations in response to ivermectin. Our results confirm that BZ resistance is common. The resistant allele at SNP F167Y in H. contortus prevails in Ceará State, and we provide evidence that this result may be due to the utilization of oxfendazole in recent years.

Mutations in TUBB8 cause a multiplicity of phenotypes in human oocytes and early embryos

BackgroundTUBB8 is a primate-specific β-tubulin isotype whose expression is confined to oocytes and the early embryo. We previously found that mutations in TUBB8 caused oocyte maturation arrest. The objective was...

2-(m-Azidobenzoyl)taxol binds differentially to distinct β-tubulin isotypes

There are seven β-tubulin isotypes present in distinct quantities in mammalian cells of different origin. Altered expression of β-tubulin isotypes has been reported in cancer cell lines resistant to microtubule stabilizing agents (MSAs) and in human tumors resistant to Taxol. To study the relative binding affinities of MSAs, tubulin from different sources, with distinct β-tubulin isotype content, were specifically photolabeled with a tritium-labeled Taxol analog, 2-(m-azidobenzoyl)taxol, alone or in the presence of MSAs. The inhibitory effects elicited by these MSAs on photolabeling were distinct for β-tubulin from different sources. To determine the exact amount of drug that binds to different β-tubulin isotypes, bovine brain tubulin was photolabeled and the isotypes resolved by high-resolution isoelectrofocusing. All bands were analyzed by mass spectrometry following cyanogen bromide digestion, and the identity and relative quantity of each β-tubulin isotype determined. It was found that compared with other β-tubulin isotypes, βIII-tubulin bound the least amount of 2-(m-azidobenzoyl)taxol. Analysis of the sequences of β-tubulin near the Taxol binding site indicated that, in addition to the M-loop that is known to be involved in drug binding, the leucine cluster region of βIII-tubulin contains a unique residue, alanine, at 218, compared with other isotypes that contain threonine. Molecular dynamic simulations indicated that the frequency of Taxol-accommodating conformations decreased dramatically in the T218A variant, compared with other β-tubulins. Our results indicate that the difference in residue 218 in βIII-tubulin may be responsible for inhibition of drug binding to this isotype, which could influence downstream cellular events.

Analisis Sekuen Gen Tubulin-β Isotipe 1 Cacing Haemonchus contortus Isolat Resisten terhadap Benzimidazole pada Domba di Indonesia

<p>Benzimidazole (BZ)<br />resistance to gastrointestinal nematodes in small ruminants<br />(sheep and goat) has become a significant problem worldwide.<br />Evidences of anthelmintic resistance to albendazole in<br />Indonesia has been reported from some government owned<br />farms in West Java, Central Java, and Yogyakarta. Previous<br />study on the sheep parasite H. contortus had shown that the<br />BZ resistance was related to selection for individuals in a<br />population possesing a spesific β-tubulin isotype 1 gene. The<br />study is aimed to determine mutation on coding region of<br />central part of β-tubulin isotype 1 gene of H. contortus resistant<br />strain from Indonesia. Seven H. contortus worms were<br />isolated from four BZ resistant sheep from two government<br />farms (SPTD Trijaya, Kuningan, West Java, and UPTD<br />Pelayanan Kesehatan Hewan, Bantul, Yogyakarta), and from<br />a BZ susceptible sheep from Cicurug, Sukabumi, West Java.<br />DNA was extracted individually from female H. contortus<br />worms. A fragment of 520 bp β-tubulin isotype 1 gene exon<br />3, 4, 5 was amplified using the PCR technique and then<br />sequenced. The results showed that a single mutation<br />occurred in codon 200 (from phenilalanine to tyrosine) had<br />caused benzimidazole resistance in H. contortus from SPTD<br />Trijaya, Kuningan, West Java. Mutation in β-tubulin isotype 1<br />gene of H. contortus from UPTD Pelayanan Kesehatan<br />Hewan, Yogyakarta, occurred in codon 198 (from glutamate<br />to glycine), codon 201 (from cystein to stop codon), and<br />codon 202 (from isoleucyne to stop codon).</p>

Abstract 2932: Impaired taxane binding to MT pore sites mediates intrinsic drug resistance in diffuse gastric cancer

Abstract Successful clinical application of taxanes (microtubule-stabilizers) is limited due to intrinsic or acquired drug resistance. Thus, it is critical to unveil the molecular mechanisms of taxane resistance to significantly improve clinical outcomes. Retrospective analysis of the TAX-325 gastric cancer (GC) trial revealed that the addition of docetaxel (DTX) to cisplatin/fluorouracil increased progression-free plus overall survival, in intestinal (INT) but not in diffuse (DIF) GC subtypes. Our preclinical data confirmed that DIF GC cell lines are intrinsically resistant to taxanes. The incidence of DTX resistance in DIF GC cell lines was 2.5 times higher than INT GC cell lines. Drug efflux, tubulin posttranslational modification and differential β-tubulin isotype expression were ruled out as potential mechanisms of intrinsic taxane resistance. Thus, a new molecular mechanism must underlie the intrinsic taxane resistance in DIF GC. To quantify the kinetics of taxol binding to cellular MTs, we treated both DIF and INT groups of GC lines with the fluorescein-conjugated paclitaxel analog, Flutax-2. The Flutax-2 staining intensity of cellular MTs was assessed by live-cell confocal microscopy at different time points. Following a 3h incubation, there was less than 20% decrease in Flutax-2 intensity in the sensitive cell lines, compared to 59∼89% decrease in the resistant cell lines. These data suggested different binding kinetics between sensitive and resistant cells. We added 1μM Flutax-2 to sensitive/resistant cells’ native cytoskeletons for 0∼60 seconds to determine the association rate (kon) of Flutax-2 binding to MTs. The kon of Flutax-2 in the sensitive cell lines was significantly higher in sensitive (5.8×104M−1s−1) versus resistant cells (0.3∼2.6×104M−1s−1). Next, we measured the dissociation rates (koff) by competing the pre-bound Flutax-2 with DTX (0∼600 seconds). Although 20∼40% faster koff was observed in resistant cells, it appeared that the association rate of Flutax-2 was the dominant factor of differential taxane binding to MTs in GC cells. The binding mode of taxanes to MTs involves 1) binding to MT pores and 2) internalization to the high-affinity binding site at the MT lumen. To differentiate between the two, we used hexaflutax, which binds exclusively to the MT pores. We observed hexaflutax decorating radial MT arrays in sensitive but not in the resistant cells. As tubulin mutations around the high-affinity taxane binding site or tubulin posttranslational modifications/differential expression of β-tubulin isotype is not the case in DIF GC cell lines, our data suggest that modifications of the MT pore conformation or occlusion of the pore site is responsible for the intrinsic taxane resistance in DIF GC. Determining the origin of the defect at the pores will help design better MT-stabilizing drugs to overcome chemo-resistance, the major obstacle hindering overall survival of patients. Citation Format: Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Chao Zhang, Isabel Barasoain, J. Fernando Díaz, Doron Betel, Manish A. Shah, Paraskevi Giannakakou. Impaired taxane binding to MT pore sites mediates intrinsic drug resistance in diffuse gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2932.

Abstract 1229: The next-generation taxanes, SB-T-1214 and SB-CST-10202, exhibit distinct inhibitory effects on photolabeling of β-tubulin from different eukaryotic sources

Abstract Next-generation taxanes are known to be active against taxane-resistant cell lines that overexpress P-glycoprotein (P-gp) and/or βIII-tubulin, both of which are associated with drug resistance. In this study, we examined the effect of two potent next-generation taxanes, SB-T-1214 and SB-CST-10202 (C-seco-taxane), on growth of the ovarian cancer cell line Hey and its drug-resistant daughter cell lines that overexpress different levels of P-gp and express increased levels of βIII-tubulin. We found that SB-T-1214 was ∼6-9-fold more potent than SB-CST-10202 against these drug resistant cells. Since the taxanes bind to both tubulin and P-gp, the relative binding affinity of the taxanes to these two cellular targets may influence the effectiveness of the taxanes in resistant cells. To determine the effects of these two taxanes on binding of Taxol® to P-gp and tubulin, a tritium-labeled Taxol® analog, 2-(m-azidobenzoyl)taxol (2-m-AzTax), was used. We have previously demonstrated that 2-m-AzTax photolabels a peptide (amino acids 217-231) in β-tubulin. To study the relative binding affinities of SB-T-1214 and SB-CST-10202, tubulins from bovine brain (BBT) and chicken erythrocytes (CET) were specifically photolabeled with [3H]2-m-AzTax, in the presence and absence of either Taxol®, Taxotere®, SB-T-1214 or SB-CST-10202. β-tubulin isotype content from BBT and CET is different, the former contains βI-, βII-, βIII- and βIV-tubulins, but the latter has only βVI. Taxol® had a minimal inhibitory effect (∼3%) on BBT, but a strong inhibitory effect (99%) on CET. Taxotere® appeared to have a stronger inhibitory effect than Taxol® on photolabeling (31% and 99% for BBT and CET, respectively). The inhibitory effects elicited by the two next-generation taxanes on photolabeling were distinct for β-tubulins from these two sources. SB-T-1214 and SB-CST-10202 inhibited BBT photolabeling by 38% and 32%, respectively. Interestingly, these two drugs caused a 41% and only 6% inhibition, respectively, on photolabeling of CET that does not contain βIII-tubulin. We are currently analyzing the effect of SB-T-1214 on binding of [3H]2-m-AzTax to different tubulin isotypes resolved by isoelectrofocusing. To study photolabeling of P-gp, plasma membranes from a multidrug resistant (MDR) cell line SKVLB1 that expresses very high levels of P-gp were prepared. SB-T-1214 and SB-CST-10202 inhibited photolabeling of P-gp markedly (> 80% inhibition), suggesting that these two taxanes are good substrates for P-gp. They also caused an increase in steady state [3H]2-m-AzTax accumulation in the MDR cell line SKVLB1. Since drug binding to tubulin and P-gp is the primary event that occurs in the cell following drug administration, our data on taxane binding affinity to the two targets will help in understanding the effectiveness of the next-generation taxanes in taxane-resistance. Citation Format: Chia-Ping H. Yang, Changwei Wang, Iwao Ojima, Susan Band Horwitz. The next-generation taxanes, SB-T-1214 and SB-CST-10202, exhibit distinct inhibitory effects on photolabeling of β-tubulin from different eukaryotic sources. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1229.

F200Y polymorphism of the β-tubulin isotype 1 gene in Haemonchus contortus and sheep flock management practices related to anthelmintic resistance in eastern Amazon

The objective of the present study was to determine the frequency of the F200Y polymorphism in the β-tubulin isotype 1 gene of Haemonchus contortus from various sheep flocks in eastern Amazon, and to identify management practices that may favor the emergence of resistance to anthelmintic drugs in the same area. In total, 305 specimens of H. contortus were collected from sheep at 12 farms located in the state of Pará. An allele-specific PCR was performed to detect the F200Y polymorphism, and questionnaires were used to obtain information about the farms and flocks. All genotypes were detected as follows: 31% of the parasites were RR, 37% of the parasites were SR, and 32% were SS. The completed questionnaires revealed that all farms employed semi-intensive farming systems, performed suppressive anthelmintic treatment, and based their choice of drug on cost and availability rather than on any knowledge regarding drugs that remained effective on their property. It can thus be concluded that the SNP in codon 200 of the β-tubulin isotype 1 gene is present in the H. contortus populations from eastern Amazon, and that a series of management practices that favor the emergence of anthelmintic resistance are employed on these farms.

Clinical observation of gene expression-guided chemoradiation therapy for nonsurgical esophageal squamous cell carcinoma patients: a retrospective analysis of 36 cases.

ObjectiveTo make an informed choice of chemotherapy drugs according to the oncogene mRNA expression and to explore whether it could increase the survival rate of patients.Patients and methodsThe study retrospectively analyzed 36 cases of nonsurgical esophageal squamous cell carcinoma patients treated at the Center for Oncology of Shandong Provincial Hospital from December 1, 2010, to November 1, 2013. Intensity-modulated radiation therapy was used for the treatment with a conventional radiotherapy dose of 60–66 Gy. Chemotherapy started 1–5 weeks after radiation therapy. The selection of the chemotherapy drug was based on the mRNA expression levels of excision repair cross-complementation 1, thymidylate synthetase, ribonucleotide reductase M1, and β-tubulin isotype III. The objective response rate, progression-free survival, and overall survival were observed.ResultsThe reason for poor prognosis of patients with high expression of excision repair cross-complementation 1 was unknown. No correlation was observed between patient survival and expression of thymidylate synthetase, ribonucleotide reductase M1, and β-tubulin isotype III. Complete response, partial response, stable disease, and progressive disease were observed in 25, five, three, and three patients, respectively. The objective response rate was 83.3%. The 1-year, 2-year, and 3-year progression-free survival rates were 79.8%, 58.9%, and 54.4%, respectively. The 1-year, 2-year, and 3-year overall survival rates were 83.3%, 68.1%, and 58.4%, respectively.ConclusionSelecting the chemotherapy drug according to the oncogene expression, combined with radiation therapy, could increase the 3-year survival rate in nonsurgical esophageal squamous cell carcinoma patients. Such conclusion needs to be further confirmed using a larger sample size.

Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends

The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. Here, we purify and characterize tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.

Characterizing the Epothilone Binding Site on β-Tubulin by Photoaffinity Labeling: Identification of β-Tubulin Peptides TARGSQQY and TSRGSQQY as Targets of an Epothilone Photoprobe for Polymerized Tubulin.

Photoaffinity labeling with an epothilone A photoprobe led to the identification of the β-tubulin peptides TARGSQQY and TSRGSQQY as targets of the photoprobe for polymerized tubulin. These peptides represent residues 274-281 in different β-tubulin isotypes. Placing the carbene producing 21-diazo/triazolo moiety of the photoprobe in the vicinity of the TARGSQQY peptide in a homology model of TBB3 predicted a binding pose and conformation of the photoprobe that are very similar to the ones reported for 1) the high resolution cocrystal structure of epothilone A with an α,β-tubulin complex and for 2) a saturation transfer difference NMR and transferred NOESY NMR study of dimeric and polymerized tubulin. Our findings thus provide additional support for these models as physiologically the most relevant among several modes of binding that have been proposed for epothilone A in the taxane pocket of β-tubulin.

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

Marginal vitamin C (vitC) deficiency affects 5% to 10% of adults including subpopulations such as pregnant women and newborns. Animal studies link vitC deficiency to deleterious effects on the developing brain, but exactly how the brain adapts to vitC deficiency and the mechanisms behind 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) subjected to either a sufficient (918mg vitC/kg feed) or deficient (100mg vitC/kg feed) maternal dietary regimen were assessed with regards to hippocampal volume and β-tubulin isotype III staining intensity at 2 gestational time points (45 and 56). We found a distinct differential regional growth pattern of the hippocampus with a clear effect of gestational age, whereas vitC status did not affect either investigated parameters. Within hippocampal subdivisions, the overall expansion of the hippocampus from gestational day 45 to 56 was found to reside in the dentate gyrus. In conclusion, the present study found that hippocampal volume and β-tubulin isotype III intensity in the prenatal guinea pig were influenced by gestational day but not by maternal vitC intake.

Mutations in TUBB8 and Human Oocyte Meiotic Arrest

BackgroundHuman reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown.MethodsWe sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse-transcriptase–polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes.ResultsWe identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes.ConclusionsTUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility. (Funded by the National Basic Research Program of China and others.)

Differential expression of β-tubulin isotypes in different life stages of Parascaris spp after exposure to thiabendazole

Anthelmintic resistance (AR) to macrocyclic lactones (ML) has been described in Parascaris of horses world-wide. In contrast, benzimidazoles (BZ) are still effective, although reduced efficacy to this drug class was recently reported. The mode of action of BZ is binding to β-tubulin, which prevents polymerisation of microtubules. In this study, β-tubulin gene expression of isotypes 1 and 2 was investigated at seven time points (0, 6, 24, 72, 96 and 120h) during embryogenesis and in adult worms. In addition, an in ovo larval developmental test was developed to study β-tubulin gene expression of both isotypes in parasacaris eggs after exposure to different concentrations of thiabendazole (TBZ) for five days at 25°C. A strong pattern of differential expression of β-tubulin and isotype 1 was observed in all stages, while isotype 2 expression was mainly found at an early phase of the embryogenesis. For isotype 1, a 5-fold increase was observed during the first 48h, but gene expression gradually decreased after 72, 96 and 120h. Isotype 2 was only expressed during the first 24h, followed by a 130-fold decrease at (time points) 72, 96 and 120h. The in ovo larval developmental test, in which we exposed initially unembryonated eggs to increased concentrations of TBZ, did affect isotype 1 gene expression but not isotype 2. This assumes that each isotype has specific functions in different life stages. This is in agreement with the ‘multi-tubulin’ hypothesis, which states that different tubulin isotypes are required for specialised microtubule functions. Isotype 1 is the most likely drug target for BZs, as isotype 2 was only expressed at very low levels later in development. Increasing concentrations of TBZ altered β-tubulin isotype 1 gene expression after exposure of the eggs for five days, but this was not seen for isotype 2.

Benzimidazole resistance in the ovine Haemonchus contortus from southern Poland - coproscopical and molecular findings.

Anthelmintic resistance within nematodes has become a very common issue, however, the data about its occurrence in the gastrointestinal nematodes of sheep in Poland are very limited. This study was carried out in order to evaluate the presence of benzimidazole resistance in highly pathogenic Haemonchus contortus by means of parasitological and molecular techniques. The research represents the first Polish attempt to confirm the presence of a mutation at codon 200 of the β-tubulin isotype 1 gene in H. contortus by PCR-RFLP. The occurrence of this mutation indicates the risk of ineffective benzimidazole treatment, nowadays commonly used for parasite control in Poland. The resistant parasites were detected by means of FECRT in a sheep flock (30 individuals) from southern Poland. To confirm the resistance on the molecular level, primers designed according to the sequences available in Genebank were used to detect the mutation. Unfortunately, as the PCR product was shorter than required (403 bp), further analyses are needed. The obtained results may indicate the high variability within the parasite population. Hence, it is essential to adapt the reaction conditions to our geographic strain of the nematode - and further analyses are required. Haemonchus contortus, benzimidazole resistance, PCR, Poland.

Mutations in Either TUBB or MAPRE2 Cause Circumferential Skin Creases Kunze Type

Circumferential skin creases Kunze type (CSC-KT) is a specific congenital entity with an unknown genetic cause. The disease phenotype comprises characteristic circumferential skin creases accompanied by intellectual disability, a cleft palate, short stature, and dysmorphic features. Here, we report that mutations in either MAPRE2 or TUBB underlie the genetic origin of this syndrome. MAPRE2 encodes a member of the microtubule end-binding family of proteins that bind to the guanosine triphosphate cap at growing microtubule plus ends, and TUBB encodes a β-tubulin isotype that is expressed abundantly in the developing brain. Functional analyses of the TUBB mutants show multiple defects in the chaperone-dependent tubulin heterodimer folding and assembly pathway that leads to a compromised yield of native heterodimers. The TUBB mutations also have an impact on microtubule dynamics. For MAPRE2, we show that the mutations result in enhanced MAPRE2 binding to microtubules, implying an increased dwell time at microtubule plus ends. Further, in vivo analysis of MAPRE2 mutations in a zebrafish model of craniofacial development shows that the variants most likely perturb the patterning of branchial arches, either through excessive activity (under a recessive paradigm) or through haploinsufficiency (dominant de novo paradigm). Taken together, our data add CSC-KT to the growing list of tubulinopathies and highlight how multiple inheritance paradigms can affect dosage-sensitive biological systems so as to result in the same clinical defect.