Novel mutations in β-tubulin gene in Trichoderma harzianum mutants resistant to methyl benzimidazol-2-YL carbamate

Twelve low resistant (LR) mutants of Trichoderma harzianum with the capability of grow fast at 0.8 μg/mL methyl benzimidazol-2-yl carbamate (MBC) were obtained using UV mutagenesis. MR and HR mutants which could grow fast at 10 and 100 μg/mL MBC, respectively, were isolated by step-up selection protocols in which UV-treated mutants were induced and mycelial sector screening was made in plates with growth medium. Subsequently, β-tubulin genes of 14 mutants were cloned to describe the molecular lesion likely to be responsible for MBC resistance. Comparison of the β-tubulin sequences of the mutant and sensitive strains of T. harzianum revealed 2 new MBC-binding sites differed from those in other plant pathogens. A single mutation at amino acid 168, having Phe (TTC) instead of Ser (TCC), was demonstrated for the HR mutant; a double mutation in amino acid 13 resulting in the substitution of Gly (GGC) by Val (GTG) was observed in β-tubulin gene of MR mutant. On the other hand, no substitutions were identified in the β-tubulin gene and its 5’-flanking regions in 12 LR mutants of T. harzianum.     

The hematopoietic-specific β1-tubulin is naturally resistant to 2-Methoxyestradiol and protects patients from drug-induced myelosuppression

Taxanes and other microtubule-targeting drugs (MTDs) represent one of the most effective classes of cancer chemotherapeutics. However, ultimately their utility is limited due to drug-induced myelosuppression. Here we identify 2-Methoxyestradiol (2ME2) as the first MTD able to specifically target tumor cells while sparing the bone marrow from dose-limiting, life-threatening toxicities. Following drug selection with 2ME2, epithelial cancer cells acquired a tubulin mutation at Vβ236I that impaired the 2ME2-tubulin interaction and rendered cells resistant to 2ME2. We further show that the hematopoietic-specific Hβ1 tubulin isotype naturally encodes Iβ236 and is insensitive to 2ME2. Systemic administration of 2ME2 in C57BL6 mice revealed that there was no effect on bone marrow microtubules, in contrast to the taxane or Vinca alkaloid induced toxicities. Similar results were obtained upon drug treatment of human bone marrow and CD34-positive stem/progenitor cells. Herein, we describe the first isotype-targeted chemotherapeutic, setting a new paradigm for the entire class of MTDs, and providing a model that could allow the design of new tubulin inhibitors devoid of myelosuppression. The ability to design a drug with minimal side-effects would significantly augment the chances of clinical success by allowing the use of a truly therapeutic dose rather than the maximally tolerated.     

Functional analysis of OsTUB8, an anther-specific β-tubulin in rice

Microtubules play important roles in many cellular processes, such as cell division and cell elongation in plants. β-tubulins (TUB), which are one of the basic components of microtubules, are encoded by multigene family in eukaryotes and their nucleotide sequences are highly conserved in protein coding regions. OsTUB8 that was expressed in rice anthers was characterized with a multi-level approach. At the protein level, OsTUB8 was expressed mainly in anthers compared to callus, root, leaf sheath and leaf blade. In situ hybridization and GUS fusion analysis revealed that OsTUB8 was expressed in vascular bundles of anther filaments and in pollen. OsTUB8 expression was lower in the anthers of GA-deficient mutants, ‘Tanginbozu’ and ‘Akibrarewaisei’, compared to those of their respective wild types. Transgenic rice expressing OsTUB8 in an antisense orientation were suppressed in the amount of seed set upon maturity. Antisense-transgenic rice plants were 20–60% shorter compared to the vector-only control. These results suggest that OsTUB8 might be differentially expressed in rice anthers due to the action of GA, and involved in the processes of vegetative growth and seed set in rice.     

Design, overexpression, and purification of polymerization-blocked yeast αβ-tubulin mutants

Microtubule dynamics play essential roles in intracellular organization and cell division. They result from structural and biochemical properties of αβ-tubulin heterodimers and how these polymerizing subunits interact with themselves and with regulatory proteins. A broad understanding of the underlying mechanisms has been established, but fundamental questions remain unresolved. The lack of routine access to recombinant αβ-tubulin represents an obstacle to deeper insight into αβ-tubulin structure, biochemistry, and recognition. Indeed, the widespread reliance on animal brain αβ-tubulin means that very few in vitro studies have taken advantage of powerful and ordinarily routine techniques like site-directed mutagenesis. Here we report new methods for purifying wild-type or mutant yeast αβ-tubulin from inducibly overexpressing strains of Saccharomyces cerevisiae. Inducible overexpression is an improvement over existing approaches that rely on constitutive expression: it provides higher yields while also allowing otherwise lethal mutants to be purified. We also designed and purified polymerization-blocked αβ-tubulin mutants. These "blocked" forms of αβ-tubulin give a dominant lethal phenotype when expressed in cells; they cannot form microtubules in vitro and when present in mixtures inhibit the polymerization of wild-type αβ-tubulin. The effects of blocking mutations are very specific, because purified mutants exhibit normal hydrodynamic properties, bind GTP, and interact with a tubulin-binding domain. The ability to overexpress and purify wild-type αβ-tubulin, or mutants like the ones we report here, creates new opportunities for structural studies of αβ-tubulin and its complexes with regulatory proteins, and for biochemical and functional studies of microtubule dynamics and its regulation.     

Sorbose-resistant mutants ofNeurospora crassa do not have alteredβ(1–3)glucan synthase activity

A number of mutants ofNeurospora crassa (sor-1, sor-3, sor-4, sor-5, sor-6, sor-15, sor-T9, andpatch) were found to be resistant to the growth-inhibiting effect of sorbose.(1–3)-Glucan synthase activity from each strain was found to be as sensitive to sorbose as wild-type enzyme activity. Four of these strains (sor-1, sor-4, sor-5, sor-T9) had altered sorbose transport; the remaining strains tested had normal sorbose transport. All of these strains (except forsor-3) were found to metabolize sorbose to glucose (and other compounds). This may explain their sorbose resistance.     

Molecular characterization of four β-tubulin genes from dinitroaniline susceptible and resistant biotypes of Eleusine indica

Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, resistant biotypes of goosegrass (Eleusine indica) developed in previously susceptible wild-type populations. We have previously reported that -tubulin missense mutations correlate with dinitroaniline response phenotypes (Drp) (Plant Cell 10: 297–308, 1998). In order to ascertain associations of other tubulins with dinitroaniline resistance, four -tubulin cDNA classes (designated TUB1, TUB2, TUB3, and TUB4) were isolated from dinitroaniline-susceptible and -resistant biotypes. Sequence analysis of the four -tubulin cDNA classes identified no missense mutations. Identified nucleotide substitutions did not result in amino acid replacements. These results suggest that the molecular basis of dinitroaniline resistance in goosegrass differs from those of colchicine/dinitroaniline cross-resistant Chlamydomonas reinhardtii and benzimidazole-resistant fungi and yeast. Expression of the four -tubulins was highest in inflorescences. This is in contrast to -tubulin TUA1 that is expressed predominantly in roots. Collectively, these results imply that -tubulin genes are not associated with dinitroaniline resistance in goosegrass. Phylogenetic analysis of the four -tubulins, together with three -tubulins, suggests that the resistant biotype developed independently in multiple locations rather than spreading from one location.     

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.     

Engineering Chinese hamster ovary (CHO) cells to achieve an inverse growth – associated production of a foreign protein, β-galactosidase

Protein synthesis in mammalian cells can be observed in two strikingly different patterns: 1) production of monoclonal antibodies in hybridoma cultures is typically inverse growth associated and 2) production of most therapeutic glycoproteins in recombinant mammalian cell cultures is found to be growth associated. Production of monoclonal antibodies has been easily maximized by culturing hybridoma cells at very low growth rates in high cell density fed- batch or perfusion bioreactors. Applying the same bioreactor techniques to recombinant mammalian cell cultures results in drastically reduced production rates due to their growth associated production kinetics. Optimization of such growth associated production requires high cell growth conditions, such as in repeated batch cultures or chemostat cultures with attendant excess biomass synthesis. Our recent research has demonstrated that this growth associated production in recombinant Chinese hamster ovary (CHO) cells is related to the S (DNA synthesis)-phase specific production due to the SV40 early promoter commonly used for driving the foreign gene expression. Using the stably transfected CHO cell lines synthesizing an intracellular reporter protein under the control of SV40 early promoter, we have recently demonstrated in batch and continuous cultures that the product synthesis is growth associated. We have now replaced this S-phase specific promoter in new expression vectors with the adenovirus major late promoter which was found to be active primarily in the G1-phase and is expected to yield the desirable inverse growth associated production behavior. Our results in repeated batch cultures show that the protein synthesis kinetics in this resulting CHO cell line is indeed inverse growth associated. Results from continuous and high cell density perfusion culture experiments also indicate a strong inverse growth associated protein synthesis. The bioreactor optimization with this desirable inverse growth associated production behavior would be much simpler than bioreactor operation for cells with growth associated production. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mice with dysfunctional TGF-β signaling develop altered intestinal microbiome and colorectal cancer resistant to 5FU

Emerging data show a rise in colorectal cancer (CRC) incidence in young men and women that is often chemoresistant. One potential risk factor is an alteration in the microbiome. Here, we investigated the role of TGF-β signaling on the intestinal microbiome and the efficacy of chemotherapy for CRC induced by azoxymethane and dextran sodium sulfate in mice. We used two genotypes of TGF-β-signaling-deficient mice (Smad4^+/? and Smad4^+/?Sptbn1^+/?), which developed CRC with similar phenotypes and had similar alterations in the intestinal microbiome. Using these mice, we evaluated the intestinal microbiome and determined the effect of dysfunctional TGF-β signaling on the response to the chemotherapeutic agent 5-Fluoro-uracil (5FU) after induction of CRC. Using shotgun metagenomic sequencing, we determined gut microbiota composition in mice with CRC and found reduced amounts of beneficial species of Bacteroides and Parabacteroides in the mutants compared to the wild-type (WT) mice. Furthermore, the mutant mice with CRC were resistant to 5FU. Whereas the abundances of E. boltae, B.dorei, Lachnoclostridium sp., and Mordavella sp. were significantly reduced in mice with CRC, these species only recovered to basal amounts after 5FU treatment in WT mice, suggesting that the alterations in the intestinal microbiome resulting from compromised TGF-β signaling impaired the response to 5FU. These findings could have implications for inhibiting the TGF-β pathway in the treatment of CRC or other cancers.     

Selection and characterization of in vitro–induced mutants of Dendrobium ‘Earsakul’ resistant to black rot

In Vitro Cellular & Developmental Biology - Plant - The development of Dendrobium ‘Earsakul’ with improved resistance to black rot is essential for sustainable orchid production. In...     

Class III β-tubulin,a novel biomarker in the human melanocyte lineage

It is generally thought that class III β-tubulin expression is limited to cells of the neural lineage and is therefore often used to identify neurons amongst other cell types, both in vivo and in vitro. Melanocytes are derived from the neural crest and share both morphological features and functional characteristics with peripheral neurons. Here, we show that these similarities extend to class III β-tubulin (TUBB3) expression, and that human melanocytes express this protein both in vivo and in vitro. In addition, we studied the expression of class III β-tubulin in two murine melanogenic cell lines and show that expression of this protein starts as melanoblasts mature into melanocytes. Melanin bleaching experiments revealed close proximity between melanin and TUBB3 proteins. In vitro stimulation of primary human melanocytes by α-MSH indicated separate regulatory mechanisms for melanogenesis and to TUBB3 expression. Together, these observations imply that human melanocytes express TUBB3 and that this protein should be recognized as a wider marker for multiple neural crest-derived cells.     

A mutant β-D-glucoside transport system of Escherichia coli resistant to catabolite inhibition

A mutant strain of Escherichia coli in which β-glucoside transport is resistant to catabolite inhibition by methyl α-glucoside was characterized. The mutation was probably within the gene, bglC, coding for the β-glucoside enzyme II. The mutant organism is shown to transport the β-glucoside substrate, salicin, in preference to methyl α-glucoside or fructose. Salicin also caused inducer exclusion of lactose in the mutant strain.     

Diseased muscles that lack dystrophin or laminin-α2 have altered compositions and proliferation of mononuclear cell populations

Background  

Multiple types of mononucleate cells reside among the multinucleate myofibers in skeletal muscles and these mononucleate cells function in muscle maintenance and repair. How neuromuscular disease might affect different types of muscle mononucleate cells had not been determined. In this study, therefore, we examined how two neuromuscular diseases, dystrophin-deficiency and laminin-α2-deficiency, altered the proliferation and composition of different subsets of muscle-derived mononucleate cells.     

Specific β-tubulin isotypes can functionally enhance or diminish epothilone B sensitivity in non-small cell lung cancer cells

Epothilones are a new class of microtubule stabilizing agents with promising preclinical and clinical activity. Their cellular target is β-tubulin and factors influencing intrinsic sensitivity to epothilones are not well understood. In this study, the functional significance of specific β-tubulin isotypes in intrinsic sensitivity to epothilone B was investigated using siRNA gene knockdown against βII-, βIII- or βIVb-tubulins in two independent non-small cell lung cancer (NSCLC) cell lines, NCI-H460 and Calu-6. Drug-treated clonogenic assays showed that sensitivity to epothilone B was not altered following knockdown of βII-tubulin in both NSCLC cell lines. In contrast, knockdown of βIII-tubulin significantly increased sensitivity to epothilone B. Interestingly, βIVb-tubulin knockdowns were significantly less sensitive to epothilone B, compared to mock- and control siRNA cells. Cell cycle analysis of βIII-tubulin knockdown cells showed a higher percentage of cell death with epothilone B concentrations as low as 0.5 nM. In contrast, βIVb-tubulin knockdown cells displayed a decrease in epothilone B-induced G(2)-M cell cycle accumulation compared to control siRNA cells. Importantly, βIII-tubulin knockdowns displayed a significant dose-dependent increase in the percentage of apoptotic cells upon treatment with epothilone B, as detected using caspase 3/7 activity and Annexin-V staining. Higher concentrations of epothilone B were required to induce apoptosis in the βIVb-tubulin knockdowns compared to control siRNA, highlighting a potential mechanism underlying decreased sensitivity to this agent. This study demonstrates that specific β-tubulin isotypes can influence sensitivity to epothilone B and may influence differential sensitivity to this promising new agent.     

Shared or distinct responses between intermediate and satellite stream fish species in an altered Amazonian River?

Environmental and spatial variables can distinctly influence the occupancy frequency distributions in stream fish. From a metacommunity context, we tested the following hypothesis, intermediate species are governed by dispersal and niche-based processes; in contrast, satellite species are governed by niche-based processes. To test this, we separately analyzed three data sets, the entire metacommunity, the intermediate species and the satellite species, using a forward selection of explanatory variables, and a partial Redundancy Distance Analysis. The fish and 31 variables of 52 stream reaches of a Brazilian river basin in the Western Amazon were collected during the dry period of 2012. The results for all of the data set revealed two different patterns: on one side, satellite species revealed that niche and dispersal-based processes were the most important; on the other side, for intermediate species and for all of the species set, only dispersal-based processes were the most important. For the data set including all of the species and the intermediate species, the variance was explained mainly by landscape scale variables. By contrast, the variance within the satellite species set was explained by local scale variables. Management efforts for intermediate species should be taking at larger scale, but they are usually less critical for the maintenance of aquatic biodiversity; on the other hand, management efforts for satellite species should be taken at smaller scale and based on specific biological and ecological information for the focal species.     

Addition of lutein, lycopene, or β-carotene to LDL or serum in vitro: Effects on carotenoid distribution, LDL composition, and LDL oxidation

Carotenoids are dietary antioxidants transported with plasma lipoproteins, primarily low-density lipoprotein (LDL). In this study in vitro methods were used to increase the amounts of specific, individual carotenoids in LDL. By addition of carotenoid to isolated LDL or to serum, followed by (re)isolation of the lipoproteins, samples of LDL were enriched 4- to 150-fold with lutein, 2- to 15-fold with lycopene, or 3- to 25-fold with β-carotene. Enrichment with specific carotenoids was achieved without affecting the electrophoretic mobility of the lipoprotein, its cholesterol to protein ratio, or the levels of other cartenoids or -tocopherol. The distributions among lipoproteins of carotenoid added to serum were similar, but not identical, to the distributions of the endogenous carotenoids. In particular, for added lutein, a greater proportion was found in HDL, and for added β-carotene, more was found in very low-density lipoprotein (VLDL). We then studied the effect of enriching LDL with specific carotenoids on its susceptibility to oxidation by copper ions. Lutein, β-cryptoxanthin, lycopene, and β-carotene, the four major plasma carotenoids, and -tocopherol were destroyed before the formation of lipid peroxidation products. The rates of destruction of the individual carotenoids differed; lycopene was destroyed most rapidly and lutein most slowly. Upon oxidation of β-carotene-enriched LDL, the rates of destruction of β-carotene, lycopene, and lutein were slowed and the lag times before the initiation of lipid peroxidation increased from 19 to 65 min. Neither effect was observed in LDL enriched with lutein or lycopene. Thus, β-carotene was unique among the carotenoids studied in having a small, but significant effect on LDL oxidation in vitro.     

Do physicians have an ethical obligation to care for patients with AIDS?

This paper responds to the question: Do physicians have an ethical obligation to care for patients with acquired immunodeficiency syndrome (AIDS)? First, the social and political milieu in which this question arises is sampled. Here physicians as well as other members of the community are found declaring an unwillingness to be exposed to people with AIDS. Next, laws, regulations, ethical codes and principles, and the history of the practice of medicine are examined, and the literature as it pertains to these areas is reviewed. The obligation to care for patients with AIDS, however, cannot be located in an orientation to morality defined in rules and codes and an appeal to legalistic fairness. By turning to the orientation to morality that emerges naturally from connection and is defined in caring, the physicians'' ethical obligation to care for patients with AIDS is found. Through an exploration of the writings of modern medical ethicists, it is clear that the purpose of the practice of medicine is healing, which can only be accomplished in relationship to the patient. It is in relationship to patients that the physician has the opportunity for self-realization. In fact, the physician is physician in relationship to patients and only to the extent that he or she acts virtuously by being morally responsible for and to those patients. Not to do so diminishes the physician''s ethical ideal, a vision of the physician as good physician, which has consequences for the physician''s capacity to care and for the practice of medicine.     

What to do, or how to do it?

In this issue of Neuron, Ajemian et al. present a computational model of the activity of neurons in primary motor cortex (M1) during isometric movements in different postures. By modeling the output of M1 neurons in terms of their influence on muscles, they find each M1 neuron maps its output into a particular pattern of muscle actions.     

Peroxisomal Localization of Enzymes Related to Fatty Acid β-Oxidation in an n-Alkane-grown Yeast,Candida tropicalis

In Candida tropicalis cells grown on n-alkanes (C₁₀-C₁₃), the levels of the activities of the enzymes related to fatty acid β—oxidation—acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase—were found to be higher than those in cells grown on glucose, indicating that these enzymes were induced by alkanes. The enzymes were first confirmed to be localized only in peroxisomes, while none of these enzymes nor acyl-CoA dehydrogenase, which is known to participate in the initial step of mitochondrial β-oxidation in mammalian cells, were detected in yeast mitochondria under the conditions employed.

The significance of the peroxisomal β-oxidation system in the metabolism of alkanes by the yeast was also discussed.