Assembly and disassembly of brain tubulin is affected by high ammonia levels

Rats were fed a diet containing ammonium for up to 6 months. High ammonia levels were attained in brain. The amount of polymerized tubulin in microtubules increased, while the amount of free tubulin remained unchanged. Polymerization of tubulin from brain of ammonium fed rats (30 min, 37°C) was approximately 60% of control. Depolymerization of the microtubules was also affected and took approximately 3 times longer than in controls. These results indicate that both assembly and disassembly of tubulin in brain are impaired by high ammonia levels. Interestingly, the amount of microtubule-associated proteins was not affected.     

The phylogenetic distribution of tubulin:Tyrosine ligase

Summary The post-translational addition of tyrosine toa-tubulin, catalyzed by tubulin:tyrosine ligase, has been previously reported in mammals and birds. The present study demonstrated that significant ligase activity was present in representative organisms from several other major vertebrate classes (chondrichthyes through reptiles) and that both substrate and enzyme from all vertebrates investigated were compatible with mammalian ligase and tubulin in the tyrosination reaction. None of the invertebrate tissues examined showed incorporation of tyrosine, phenylalanine or dihydroxyphenylalanine intoa tubulin under conditions allowing significant incorporation of these compounds in vertebrate supernatant samples. The failure of invertebrate tubulin to incorporate tyrosine in vitro did not appear to be due to saturation of the carboxyl terminal position with tyrosine or the presence of a soluble inhibitor of ligase activity.Although tubulin amino acid composition has been highly conserved throughout evolution, a major evolutionary divergence is described based upon biochemical differences whereby invertebrate tubulin cannot be tyrosinated or posttranslationally modified with phenylalanine or dihydroxyphenylalanine under conditions suitable for the incorporation of these compounds by vertebratea tubulin.     

Colchicine-resistant assembly of tubulin in plant mitosis

Summary Tubulin distribution in c-mitoses (induced by 1 mM colchicine) has been studied by indirect immunofluorescence with monoclonal antibodies inAllium cepa L. meristems proliferating under steady state kinetics. Two hours after colchicine treatment was initiated tubulin is detected in approximately 25% of the cells as arrowheads on the kinetochores, as if these structures stabilize microtubules against disassembly. Total disassembly of microtubules occurs in 70% of the c-mitoses six hours after the initiation of the colchicine treatment, when restitution nuclei also start appearing. After 2 to 14 hours of colchicine treatment, tubulin is detected in about 30% of the c-mitoses, both in small kinetochores-like dots and in a strand which apparently connects sister kinetochores. Other larger microtubule-like structures, up to 20 m long, apparently unassociated with kinetochores, are assembled in the presence of cholchicine in c-mitoses after 10 hours. Such structures disappear when chromosomes decondense and the nuclear envelope reforms in the restitution nucleus; they do not seem to be related to interphase cortical microtubules which reappear in control telophase.     

Time and cell cycle dependent formation of heterogeneous tubulin arrays induced by colchicine in Triticum aestivum root meristem

We have investigated the appearance and reorganization of tubulin-containing arrays induced by colchicine in the root meristem of wheat Triticum aestivum, using immunostaining and electron microscopy. Colchicine caused depolymerization of microtubules and formation of tubulin cortical strands composed of filamentous material only in C-mitotic cells. After prolonged exposure to the drug, both interphase and C-mitotic cells acquired needle-type bundles, arranged as different crystalloids and/or macrotubules. The unmodified tyrosinated form of alpha-tubulin was detected within microtubules in control cells, but was not found within cortical strands. It was identified, however, within needle-type bundles. The modified acetylated form of alpha-tubulin, which was absent in control cells, was detected within needle-type bundles. Thus, cortical strands were transitory arrays, transformed into needle-type bundles during prolonged exposure to colchicine. Cortical strands appeared in a cell cycle-dependent manner, whereas needle-type bundles were cell cycle stable arrays. The diverse morphological organization, intracellular distribution and stability of tubulin-containing arrays may be associated with heterogeneity of alpha-tubulin isoforms. We assume that non-microtubular arrays substitute for microtubules in conditions where normal tubulin polymerization is inhibited.     

In vivo and in vitro effects of diisopropyl phosphorofluoridate (DFP) on the rate of hen brain tubulin polymerization

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in sensitive species. We have investigated the in vivo and in vitro effects of DFP on hen brain tubulin polymerization. Hens were treated with a single dose of DFP (1.7 mg/kg, sc.), and were sacrificed after 18–21 days. Tubulin from DFP-treated hen brains showed small but significant decrease (14.42%) in the rate of polymerization and 11.05% decrease in rise in O.D. at 340 nm in 30 min. DFP in vivo treatment also resulted in decreased concentration of tau and an enhanced concentration of two peptides (45 kDa, 35 kDa) in the brain supernatant. These peptides seemed to be the degradation products of MAP-2. The decrease in the rate of brain tubulin polymerization in treated hens is consistent with neurochemical alterations and the focal degeneration and aggregation of these filamentous structures in OPIDN.Abbreviations DFP Diisopropyl phosphorofluoridate - DMSO dimethyl sulfoxide - DTT dithiothreitol - EGTA ethyleneglycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid - EDTA ethylenediaminetetraacetic acid - 2, 5-DH 2, 5-hexanedione - DMHD 3, 4-dimethyl-2, 5-hexanedione - OPIDN organophosphorus ester-induced delayed neurotoxicity - PMSF phenylmethylsulfonyl fluoride - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - TOCP tri-o-cresyl phosphate     

Tubulin subunit sorting: Analysis of the mechanisms involved in the segregation of unique tubulin isotypes within microtubule copolymers

Summary Vertebrate cells contain biochemical and genetic isotypes of tubulin which are expressed in unique combinations in different tissues and cell types. To determine if mixtures of tubulin isotypes assemblein vitro to form different classes of microtubules, we analyzed the composition of microtubule copolymers assembled from mixtures of chicken brain and erythrocyte tubulin. During microtubule elongation brain tubulin assembled onto the ends of microtubules faster than erythrocyte tubulin, resulting in copolymers with continually changing ratios of isotypes along their lengths. Unlike examples of microtubule assembly where the rate of polymerization depends on the association rate constant (k⁺) and the subunit concentration, the rate and extent of sorting in copolymers appear to depend on the dissociation rate constant (k^–), which governs the rate at which subunits are released from tubulin oligomers and microtubules and thereby made available for reassembly into copolymers. The type of microtubule seed used to initiate elongation was also found to influence the composition of copolymers, indicating that polymerization favors association of subunits of the same isotype.     

Why do tubulin gene families lack diversity in flagellate/ciliate protists?

Summary Considerable amino acid sequence diversity is found among tubulin isotypes encoded by tubulin gene families in animal, higher plant, and fungal systems. In contrast, relatively little diversity is found among the isotypes produced by the gene families in a number of flagellate or ciliate protists. It is possible that proper assembly of the axoneme requires a homogeneous pool of tubulin subunits and that the axoneme thus provides a stringent selection against amino acid replacement substitutions among tubulin genes in these systems.     

General features of the recognition by tubulin of colchicine and related compounds

The kinetic mechanisms of the binding to tubulin of colchicine and eight different analogues have been studied to elucidate details of the recognition mechanism. All of the analogues follow a two step binding mechanism i.e. binding occurs via an initial step with low affinity, followed by an isomerisation of the initial complex leading to the final high affinity state. For several analogues the kinetic and thermodynamic data of both processes are compared here. For all the analogues the ΔG°₁ of initial binding at 25 °C varies between –13.3 and –28.8 kJ ⋅ mol^–1. For the second step ΔG°₂ varies between –2.4 and –27 kJ ⋅ mol^–1. These limited ranges of free energy change are, however, obtained by a great variety of enthalpy changes and compensatory entropy changes. Comparison of the data for the first and second steps indicates that structural alterations of the drugs always change the thermodynamic parameters of the two steps, and the changes in the first and the second steps are in opposite directions. The fact that this range of experimental behaviour can be incorporated into a general mechanism encourages the extension of these investigations to other colchicine analogues and related compounds with potential pharmaceutical applications. Received: 9 January 1998 / Revised version: 2 March 1998 / Accepted: 7 March 1998     

The interactions of tropolone with magnesium ions and tubulin

Tropolone, a simple analog of colchicine, interacts with Mg²⁺ with the formation of a 1:1 complex and an apparent equilibrium binding constant K_b of 1.4 · 10⁴ M^?1 in neutral aqueous solution at 25°C. The tropolone-Mg²⁺ complex, but not tropolone, is fluorescent. Since tubulin binds Mg²⁺ (Frigon, R.P. and Timasheff, S.N. (1975) Biochemistry 14, 4567–4573), previous reports of tropolone interaction with tubulin in Mg²⁺-containing buffers must be critically re-examined. Fluorescence and difference absorption spectroscopy experiments performed at essentially constant Mg²⁺ activity indicate that tubulin does bind tropolone, but the optical effects are too weak to use in quantitative studies.     

Identification of proteins binding the native tubulin dimer

Microtubules play an essential role in eukaryotic cells, where they perform a wide variety of functions. In this paper, we describe the characterization of proteins associated to tubulin dimer in its native form, using affinity chromatography and mass spectrometry. We used an immunoaffinity column with coupled-monoclonal antibody directed against the alpha-tubulin C-terminus. Tubulin was first loaded onto the column, then interphase and mitotic cell lysates were chromatographed. Tubulin-binding proteins were eluted using a peptide mimicking the alpha-tubulin C-terminus. Elution fractions were analyzed by SDS-PAGE, and a total of 14 proteins were identified with high confidence by mass spectrometry. These proteins could be grouped in four classes: known tubulin-binding proteins, one microtubule-associated protein, heat shock proteins, and proteins that were not shown previously to bind tubulin dimer or microtubules.     

Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

A growing body of evidence points to an association of prion protein (PrP) with microtubular cytoskeleton. Recently, direct binding of PrP to tubulin has also been found. In this work, using standard light scattering measurements, sedimentation experiments, and electron microscopy, we show for the first time the effect of a direct interaction between these proteins on tubulin polymerization. We demonstrate that full-length recombinant PrP induces a rapid increase in the turbidity of tubulin diluted below the critical concentration for microtubule assembly. This effect requires magnesium ions and is weakened by NaCl. Moreover, the PrP-induced light scattering structures of tubulin are cold-stable. In preparations of diluted tubulin incubated with PrP, electron microscopy revealed the presence of approximately 50 nm disc-shaped structures not reported so far. These unique tubulin oligomers may form large aggregates. The effect of PrP is more pronounced under the conditions promoting microtubule formation. In these tubulin samples, PrP induces formation of the above oligomers associated with short protofilaments and sheets of protofilaments into aggregates. Noticeably, this is accompanied by a significant reduction of the number and length of microtubules. Hence, we postulate that prion protein may act as an inhibitor of microtubule assembly by inducing formation of stable tubulin oligomers.     

An investigation of the plausibility of stochastic resonance in tubulin dimers

This paper considers the possibility of stochastic resonance (SR) in tubulin dimers. A formula for the signal-to-noise ratio (SNR) of tubulin as a function of temperature is derived. The effective potential experienced by a delocalized electron in such a dimer is postulated to be a symmetric bimodal well. Inter-well and intra-well motions are described by Kramers rate theory and the Langevin formalism respectively. The frequency-dependent expression for the SNR shows that the response of the electron-tubulin dimer system is enhanced by ambient dipolar oscillations in specific frequency regimes. This is a characteristic of SR. Biophysical implications of this property such as the relevance to 8.085 MHz microtubule resonance and the clocking mechanism are detailed.     

Kidney ischemia-reperfusion regulates expression and distribution of tubulin subunits, beta-actin and rho GTPases in proximal tubules

Ischemic injury is characterized by a loss of cell polarity and a release of proximal tubule epithelial cells resulting from cytoskeletal reorganization. This study used a reversible unilateral renal ischemia-reperfusion model to investigate the expression and distribution of cytoskeletal components and Rho GTPases at protein and mRNA levels in proximal tubule fractions. Ischemia strongly increased beta-actin and alpha-tubulin expressions that were predominantly found in nuclear fractions. Rho GTPases and caveolin-1 expression were upregulated by ischemia and were enriched mainly in Triton-soluble membranes. Rac1 expression was stimulated in the soluble fractions during reperfusion. Rho GTPases mRNA levels were similarly regulated by ischemia-reperfusion suggesting that changes in their expressions could occur at gene or mRNA levels. ERM protein expression and distribution were unaffected by ischemia-reperfusion. Together, these data show that renal ischemia-reperfusion induced expression and redistribution of actin and microtubule cytoskeleton components in addition to Rho GTPases in proximal tubules, suggesting that they participate in an adaptive response to cellular lesions.     

Sequence divergence of Entamoeba histolytica tubulin is responsible for its altered tertiary structure

Atypical microtubular structures of the protozoan parasite Entamoeba histolytica (Eh) have been attributed to amino acid sequence divergence of Eh tubulin. To investigate if this sequence divergence leads to significant differences in the tertiary structure of the Eh alphabeta-tubulin heterodimer, we have modeled alphabeta-tubulin heterodimer of Eh based on the crystal structure of mammalian tubulin. The predicted 3D homology model exhibits an overall resemblance with the known crystal structure of mammalian tubulin except for the 16 residue long carboxy terminal region of Eh beta-tubulin. We propose that this C-terminal region may provide steric hindrance in the polymerization of Eh alphabeta-tubulin for microtubule formation. Using docking studies, we have identified the binding sites for different microtubule specific drugs on Eh beta-tubulin. Our model provides a rational framework, both for understanding the contribution of Eh beta-tubulin C-terminal region to alphabeta-tubulin polymerization and design of new anti-protozoan drugs in order to control amoebiasis.     

One step flow-through adsorptive purification of tubulin from tissue homogenate

Tubulin, a potential target for anti-cancer drugs, has been purified in one step and obtained as flow-through fraction directly from an extract of a mammalian brain tissue by adsorption chromatography on H-CELBEADS, an indigenously developed rigid, superporous cross-linked cellulose based weakly hydrophobic adsorbent. The fibrous polymerized tubulin mass passed through the H-CELBEADS bed while the associated proteins were separated by adsorption. The final tubulin preparation was obtained free from other proteins as seen on SDS-PAGE. Purified tubulin was obtained in a yield of about 29 mg/100 g brain, and its bioactivity, evaluated through its ability to bind colchicine, was found to be preserved.     

Ingestion of Lactobacillus strain reduces anxiety and improves cognitive function in the hyperammonemia rat

Evidence suggests that the hyperammonemia(HA)-induced neuroinflammation and alterations in the serotonin(5-HT)system may contribute to cognitive decline and anxiety disorder during hepatic encephalopathy(HE).Probiotics that maintain immune system homeostasis and regulate the 5-HT system may be potential treatment for HA-mediated neurological disorders in HE.In this study,we tested the efficacy of probiotic Lactobacillus helveticus strain NS8 in preventing cognitive decline and anxiety-like behavior in HA rats.Chronic HA was induced by intraperitoneal injection of ammonium acetate for four weeks in male Sprague-Dawley rats.HA rats were then given Lactobacillus helveticus strain NS8(109 CFU mL?1)in drinking water as a daily supplementation.The Morris water maze task assessed cognitive function,and the elevated plus maze test evaluated anxiety-like behavior.Neuroinflammation was assessed by measuring the inflammatory markers:inducible nitric oxide synthase,prostaglandin E2,and interleukin-1βin the brain.5-HT system activity was evaluated by measuring 5-HT and its metabolite,5-HIAA,and the 5-HT precursor,tryptophan.Probiotic treatment of HA rats significantly reduced the level of inflammatory markers,decreased 5-HT metabolism,restored cognitive function and improved anxiety-like behavior.These results indicate that probiotic L.helveticus strain NS8 is beneficial for the treatment of cognitive decline and anxiety-like behavior in HA rats.     

Design,synthesis and biological studies of novel tubulin inhibitors

A series of compounds originally derived from the vascular endothelial growth factor receptor tyrosine kinase inhibitor, SU5416, were synthesized and evaluated. The most potent compound in this series, compound 3, which structurally resembles the potent anti-microtubule agent combretastatin A-4, inhibited tubulin polymerization and showed potent growth inhibitory activities on both prostate and breast cancer lines with IC₅₀ values in the low nanomolar range.     

The effect of hyperammonemia on myostatin and myogenic regulatory factor gene expression in broiler embryos

Myogenesis is facilitated by four myogenic regulatory factors and is significantly inhibited by myostatin. The objective of the current study was to examine embryonic gene regulation of myostatin/myogenic regulatory factors, and subsequent manipulations of protein synthesis, in broiler embryos under induced hyperammonemia. Broiler eggs were injected with ammonium acetate solution four times over 48 h beginning on either embryonic day (ED) 15 or 17. Serum ammonia concentration was significantly higher (P<0.05) in ammonium acetate injected embryos for both ED17 and ED19 collected samples when compared with sham-injected controls. Expression of mRNA, extracted from pectoralis major of experimental and control embryos, was measured using real-time quantitative PCR for myostatin, myogenic regulatory factors myogenic factor 5, myogenic determination factor 1, myogenin, myogenic regulatory factor 4 and paired box 7. A significantly lower (P<0.01) myostatin expression was accompanied by a higher serum ammonia concentration in both ED17 and ED19 collected samples. Myogenic factor 5 expression was higher (P<0.05) in ED17 collected samples administered ammonium acetate. In both ED17 and ED19 collected samples, myogenic regulatory factor 4 was lower (P⩽0.05) in ammonium acetate injected embryos. No significant difference was seen in myogenic determination factor 1, myogenin or paired box 7 expression between treatment groups for either age of sample collection. In addition, there was no significant difference in BrdU staining of histological samples taken from treated and control embryos. Myostatin protein levels were evaluated by Western blot analysis, and also showed lower myostatin expression (P<0.05). Overall, it appears possible to inhibit myostatin expression through hyperammonemia, which is expected to have a positive effect on embryonic myogenesis and postnatal muscle growth.