Α-tubulin mRNA Research Articles

Lobaplatin-induced changes of F-actin and α-tubulin cytoskeleton in cholangiocarcinoma cell line RBE

Objective To investigate the F-actin and α- tubulin cytoskeleton changes in cholangiocarcinoma cell line RBE after treatment with Lobaplatin. Methods The RBE cells were cultured in vitro, followed by treatment with various concentrations of lobaplatin for 24 h. The changes of F-actin cytoskeleton were scanned by confocal laser scanning microscopy(CLSM)after staining with phalloidin-fluoresceine isothiocyanate(FITC)and DAPI. Immunohistochemistry and real-time polymerase chain reaction(Real-time PCR)were used to observe α-tubulin cytoskeleton changes and α-tubulin mRNA transcript levels respectively. Results CLSM and immunohistochemistry showed that both the F-actin and the α- tubulin cytoskeleton of RBE cells had disorganization and rearrangement after the treatment with lobaplatin. The staining of α - tubulin was getting deep(P<0.05),and the Real- time PCR determinedα- tubulin mRNA/β- actin ratio in 5.0 mg/L and 25.0 mg/L lobaplatin- treated groups was 2.08±0.13 and 1.91±0.46 respectively (P<0.05). Conclusion The changes of microfilaments and microtubules in RBE cells play a positive role in the apoptosis induced by lobaplatin. Key words: Cholangiocarcinoma; Lobaplatin; Cytoskeleton; F-actin; α-tubulin

PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells

BackgroundCombination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells.MethodsCell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively.ResultsWe found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner.ConclusionsThese results suggest that the synergy between PMA and apicularen A is involved by PKCα activation and microtubule disruption, and that may inform the development of novel approaches to treat cancer.

Alpha tubulin genes from Leishmania braziliensis:genomic organization, gene structure and insights on their expression

BackgroundAlpha tubulin is a fundamental component of the cytoskeleton which is responsible for cell shape and is involved in cell division, ciliary and flagellar motility and intracellular transport. Alpha tubulin gene expression varies according to the morphological changes suffered by Leishmania in its life cycle. However, the objective of studying the mechanisms responsible for the differential expression has resulted to be a difficult task due to the complex genome organization of tubulin genes and to the non-conventional mechanisms of gene regulation operating in Leishmania.ResultsWe started this work by analyzing the genomic organization of α-tubulin genes in the Leishmania braziliensis genome database. The genomic organization of L. braziliensis α-tubulin genes differs from that existing in the L. major and L. infantum genomes. Two loci containing α-tubulin genes were found in the chromosomes 13 and 29, even though the existence of sequence gaps does not allow knowing the exact number of genes at each locus. Southern blot assays showed that α-tubulin locus at chromosome 13 contains at least 8 gene copies, which are tandemly organized with a 2.08-kb repetition unit; the locus at chromosome 29 seems to contain a sole α-tubulin gene. In addition, it was found that L. braziliensis α-tubulin locus at chromosome 13 contains two types of α-tubulin genes differing in their 3′ UTR, each one presumably containing different regulatory motifs. It was also determined that the mRNA expression levels of these genes are controlled by post-transcriptional mechanisms tightly linked to the growth temperature. Moreover, the decrease in the α-tubulin mRNA abundance observed when promastigotes were cultured at 35°C was accompanied by parasite morphology alterations, similar to that occurring during the promastigote to amastigote differentiation.ConclusionsInformation found in the genome databases indicates that α-tubulin genes have been reorganized in a drastic manner along Leishmania speciation. In the L. braziliensis genome database, two loci containing α-tubulin sequences were found, but only the locus at chromosome 13 contains the prototypic α-tubulin genes, which are repeated in a head-to-tail manner. Also, we determined that the levels of α-tubulin mRNAs are down-regulated drastically in response to heat shock by a post-transcriptional mechanism which is dependent upon active protein synthesis.

An Aurora Kinase Homologue Is Involved in Regulating Both Mitosis and Cytokinesis in Trypanosoma brucei

The chromosomal passenger protein aurora kinases have been implicated in regulating chromosome segregation and cell division. Three aurora kinase homologues were identified (TbAUK1, -2 and -3) in the Trypanosome Genomic Data Base, and their expressions in the procyclic form of Trypanosoma brucei were knocked down individually by using the RNA interference technique. Only a knockdown of TbAUK1 arrested the cells in G(2)/M phase with each cell showing an extended posterior end, two kinetoplasts, and an enlarged nucleus, apparently the result of an inhibited kinetoplast multiplication and a failed mitosis. There is no mitotic spindle structure in the TbAUK1-depleted cell. The two kinetoplasts moved apart from each other but stopped just before cytokinesis, suggesting that cytokinesis was blocked in its early phase. Overexpression of TbAUK1 in the cells resulted in little change in cell growth. By immunofluorescence, TbAUK1 was primarily localized to the nucleus in interphase and to the mitotic spindle during apparent metaphase and anaphase. Thus, differing from other eukaryotes, TbAUK1 has an apparent triple function in coupling mitosis and kinetoplast replication with cytokinesis in T. brucei. T. brucei polo-like kinase, previously identified as the initiator of cytokinesis without apparent involvement in mitosis in the trypanosome, was either depleted or overexpressed in the TbAUK1-deficient cells. A dominant TbAUK1-depleted phenotype was demonstrated in both cases, suggesting that TbAUK1 plays an essential role in cytokinesis that cannot be affected by changes in the level of T. brucei polo-like kinase. To our knowledge, this is the first time that the function of an aurora B-like kinase is a prerequisite for polo-like kinase action in initiating cytokinesis. TbAUK1 is also the first identified protein that couples both mitosis and kinetoplast replication with cytokinesis in the trypanosome.

A transcriptional role for C/EBP β in the neuronal response to axonal injury

The molecular mechanisms responsible for inducing gene expression following neuronal injury are not well understood. Here, we address this issue by focusing upon C/EBPβ, a transcription factor implicated in cellular injury and regeneration. We show that C/EBPβ mRNA is expressed in neurons throughout the mature brain and that levels of both C/EBPβ mRNA and phosphoprotein are increased in facial motor neurons following axonal injury. To determine the importance of these increases, we examined the regeneration-associated Tα1 α-tubulin gene which contains functional C/EBP binding sites in its promoter. In transgenic mice, expression of a minimal 176 nucleotide Tα1 α-tubulin promoter:nlacZ reporter gene was upregulated in injured facial motor neurons. This injury-induced transcriptional increase was inhibited in C/EBPβ −/− mice. A similar inhibition was observed in C/EBPβ −/− mice that carried a larger 1.1-kb promoter Tα1:nlacZ reporter construct. Moreover, in situ hybridization revealed that the injury-induced upregulation of the endogenous mouse α1 α-tubulin mRNA, and of a second regeneration-associated mRNA, GAP-43, was inhibited in C/EBPβ −/− mice. Thus, C/EBPβ is essential for the neuronal injury response, acting to transcriptionally activate regeneration-associated gene expression.

Axonal reinjury reveals the survival and re-expression of regeneration-associated genes in chronically axotomized adult mouse motoneurons

Recently, we reported that chronically axotomized rubrospinal neurons survive for up to 1 year in an atrophied state. This finding contrasted previous work suggesting the death of up to 50% of the neurons over time. In the adult mouse, the majority of facial motoneurons appear to be lost as a result of chronic nerve resection. Here, we sought to determine if chronically resected adult mouse facial motoneurons, like rubrospinal neurons, survive in an atrophied state. To test this hypothesis, we asked whether a second nerve injury, 10 weeks after an initial nerve resection, could stimulate a regenerative cell body response. After chronic resection (10 weeks), mouse facial motoneurons underwent atrophy resulting in a loss of countable neuronal cell bodies. In addition, the motoneurons failed to maintain their initial increase in expression of GAP-43 and α-tubulin mRNA. Reinjury of 10-week chronically resected facial motoneurons by the removal of the neuroma reversed the atrophy of the cell bodies and increased the percentage of identifiable cell bodies from 36% of contralateral to 79% in C57BL/6-C3H mice and from 28% of contralateral to 40% in Balb/c mice. Moreover, the reinjured motoneurons displayed an increase in GAP-43 and α-tubulin mRNA expression. The results of this study indicate that a second axon injury stimulates regenerative cell body responses in chronically resected mouse facial motoneurons and suggest previous studies using this model may have overestimated the number of dying motoneurons.

Axonal reinjury reveals the survival and re-expression of regeneration-associated genes in chronically axotomized adult mouse motoneurons

Recently, we reported that chronically axotomized rubrospinal neurons survive for up to 1 year in an atrophied state. This finding contrasted previous work suggesting the death of up to 50% of the neurons over time. In the adult mouse, the majority of facial motoneurons appear to be lost as a result of chronic nerve resection. Here, we sought to determine if chronically resected adult mouse facial motoneurons, like rubrospinal neurons, survive in an atrophied state. To test this hypothesis, we asked whether a second nerve injury, 10 weeks after an initial nerve resection, could stimulate a regenerative cell body response. After chronic resection (10 weeks), mouse facial motoneurons underwent atrophy resulting in a loss of countable neuronal cell bodies. In addition, the motoneurons failed to maintain their initial increase in expression of GAP-43 and α-tubulin mRNA. Reinjury of 10-week chronically resected facial motoneurons by the removal of the neuroma reversed the atrophy of the cell bodies and increased the percentage of identifiable cell bodies from 36% of contralateral to 79% in C57BL/6-C3H mice and from 28% of contralateral to 40% in Balb/c mice. Moreover, the reinjured motoneurons displayed an increase in GAP-43 and α-tubulin mRNA expression. The results of this study indicate that a second axon injury stimulates regenerative cell body responses in chronically resected mouse facial motoneurons and suggest previous studies using this model may have overestimated the number of dying motoneurons.

Conserved Wat1/Pop3 WD-repeat protein of fission yeast secures genome stability through microtubule integrity and may be involved in mRNA maturation.

Accurate chromosome segregation is dependent upon the integrity of mitotic spindles, which pull each pair of sister chromatids towards opposite poles. In this study, we have characterised fission yeast pop3-5235, a diploidising mutant that is impaired in genome stability. Pop3 is the same as Wat1, a conserved protein containing 7 WD repeats. Pop3/Wat1 has also been isolated from a two-hybrid screen as a binding partner to Prp2, the large subunit of the essential splicing factor U2AF. In wat1 mutants, the cellular amount of alpha-tubulin is decreased to very low levels, which results in compromised microtubules and spindles, consequently leading to unequal chromosome separation. Further analysis shows that, in spite of the binding between Wat1 and Prp2, Wat1 may not be involved directly in splicing reactions per se. Instead, we find that Wat1 is required for the maintenance of alpha-tubulin mRNA levels; moreover, transcript levels of genes other than the alpha-tubulin gene are also equally decreased in this mutant. Wild-type Wat1, but not the mutant protein, forms a large complex in the cell with several other proteins, suggesting that Wat1 functions as a structural linker in the complex. The results suggest that Wat1 plays a role in mRNA maturation as a coupling protein between splicing and synthesis and/or stabilisation.

Glial Cell Line-Derived Neurotrophic Factor (GDNF) Gene Delivery Protects Dopaminergic Terminals from Degeneration

Previously, we observed that injection of an adenoviral (Ad) vector expressing glial cell line-derived neurotrophic factor (GDNF) into the striatum, but not the substantia nigra (SN), prior to a partial 6-OHDA lesion protects dopaminergic (DA) neuronal function and prevents the development of behavioral impairment in the aged rat. This suggests that striatal injection of AdGDNF maintains nigrostriatal function either by protecting DA terminals or by stimulating axonal sprouting to the denervated striatum. To distinguish between these possible mechanisms, the present study examines the effect of GDNF gene delivery on molecular markers of DA terminals and neuronal sprouting in the aged (20 month) rat brain. AdGDNF or a control vector coding for β-galactosidase (AdLacZ) was injected unilaterally into either the striatum or the SN. One week later, rats received a unilateral intrastriatal injection of 6-OHDA on the side of vector injection. Two weeks postlesion, rats injected with AdGDNF into either the striatum or the SN exhibited a reduction in the area of striatal denervation and increased binding of the DA transporter ligand [125I]IPCIT in the lesioned striatum compared to control animals. Furthermore, injections of AdGDNF into the striatum, but not the SN, increased levels of tyrosine hydroxylase mRNA in lesioned DA neurons in the SN and prevented the development of amphetamine-induced rotational asymmetry. In contrast, the level of T1 α-tubulin mRNA, a marker of neuronal sprouting, was not increased in lesioned DA neurons in the SN following injection of AdGDNF either into the striatum or into the SN. These results suggest that GDNF gene delivery prior to a partial lesion ameliorates damage caused by 6-OHDA in aged rats by inhibiting the degeneration of DA terminals rather than by inducing sprouting of nigrostriatal axons.

Sugar-regulated control of α-tubulin in maize cell suspension culture

The data reported here shows that α-tubulin, the subunit of microtubulin in the cytoskeleton, is regulated by sugars. A 48-h sugar-depletion treatment in BMS maize cell suspension culture medium led to a marked reduction in the levels of α-tubulin mRNA and protein. This response was reversible; the addition of metabolizable sugars led to a rapid increase in the levels of the α-tubulin transcript. Levels of α-tubulin protein also increased, albeit gradually. Surprisingly, there was little or no effect of phosphate depletion and phosphate addition on α-tubulin expression. Furthermore, the data show that sugars modulate a co-regulation of α-tubulin and cell-wall invertase. We propose that microtubules might be a major component of a sugar-signaling pathway that is regulated by, among other factors, cellular carbon metabolism.

Two trans-acting rat-brain proteins, MARTA1 and MARTA2, interact specifically with the dendritic targeting element in MAP2 mRNAs

Different isoforms of the microtubule-associated protein 2 (MAP2) are somatodendritic components of neurons that seem to regulate the stability of the dendritic cytoskeleton. MAP2 localization into dendrites appears to be a complex multicausal mechanism that involves the specific recruitment of MAP2 mRNAs into dendritic compartments. Recently, we have functionally characterized a 640-nucleotide dendritic targeting element (DTE) in the 3′ untranslated region (3′ UTR) of MAP2 transcripts that mediates extrasomatic mRNA localization in primary neurons (Blichenberg et al., 1999). In analogy to molecular mechanisms regulating cytoplasmic RNA translocation in other cell systems, we propose that, in vivo, the cis-acting MAP2-DTE interacts with specific protein factors present in neurons. To identify putative trans-acting DTE-binding proteins, we performed in vitro ultraviolet crosslinking assays. Using this experimental system, two 90-kDa and 65-kDa MA P2- R NA t rans - a cting proteins, MARTA1 and MARTA2, were identified in rat-brain extracts. Both MARTAs bind with high affinity to the MAP2-DTE, but not to other investigated regions of MAP2 transcripts or the somatically restricted α-tubulin mRNA. Moreover, MARTA1 and MARTA2 do not bind significantly to other dendritically localized transcripts encoding vasopressin and arg3.1, nor to a dendritic trafficking element from the mRNA encoding the α-subunit of the Ca 2+/calmodulin-dependent protein kinase II. Binding of MARTA1 and MARTA2 to the MAP2-DTE occurs with an affinity in the nanomolar range. Whereas MARTA1 is clearly detectable in crude lysates, cytosolic and ribosomal salt-wash fractions, and in nuclear extracts, MARTA2 is preferentially found in the ribosomal salt-wash preparation. Neither MARTA is restricted to rat brain, and both are present in a number of other rat tissues. Thus, both proteins may be involved in a variety of nuclear and cytoplasmic events that regulate RNA metabolism in different cell types.

DsRNA-induced mRNA degradation

Expression of double stranded RNA (dsRNA) in Caenorhabditis elegans has been shown, under certain circumstances, to cause genetic interference. In plants, expression of RNA leads to phenotypic co-suppression. However, the use of dsRNA to manipulate microorganisms genetically is still in its infancy. Ngo and colleagues1xDouble stranded RNA induces mRNA degradation in Trypanosoma brucei. Ngo, H. et al. Proc. Natl. Acad. Sci. U. S. A. 1999; 95: 14687–14692Crossref | Scopus (504)See all References1 have now used gene-specific dsRNA, either expressed in vivo or directly electroporated into Trypanosoma brucei cells, as a means to manipulate this important protozoan parasite. Constructs encoding dsRNA of 5′ untranslated regions of the α-tubulin gene give rise to cells with altered morphology and multi-nuclei. These cells are unable to undergo cytokinesis and eventually die. The dsRNA specifically induces degradation of mature α-tubulin mRNA but the exact mechanism of degradation is unknown. Development of an inducible system that can be regulated will allow better control over expression and allow the analysis of phenotypes which might otherwise be lethal if dsRNA expression was constitutive. Furthermore, these studies open the possibility of using dsRNA as a tool for genetic manipulation of other microorganisms.

Double-stranded RNA induces mRNA degradation in Trypanosoma brucei.

Double-stranded RNA (dsRNA) recently has been shown to give rise to genetic interference in Caenorhabditis elegans and also is likely to be the basis for phenotypic cosuppression in plants in certain instances. While constructing a plasmid vector for transfection of trypanosome cells, we serendipitously discovered that in vivo expression of dsRNA of the alpha-tubulin mRNA 5' untranslated region (5' UTR) led to multinucleated cells with striking morphological alterations and a specific block of cytokinesis. Transfection of synthetic alpha-tubulin 5' UTR dsRNA, but not of either strand individually, caused the same phenotype. On dsRNA transfection, tubulin mRNA, but not the corresponding pre-mRNA, was rapidly and specifically degraded, leading to a deficit of alpha-tubulin synthesis. The transfected cells were no longer capable of carrying out cytokinesis and eventually died. Analysis of cytoskeletal structures from these trypanosomes revealed defects in the microtubules of the flagellar axoneme and of the flagellar attachment zone, a complex cortical structure that we propose is essential for establishing the path of the cleavage furrow at cytokinesis. Last, dsRNA-mediated mRNA degradation is not restricted to alpha-tubulin mRNA but can be applied to other cellular mRNAs, thus establishing a powerful tool to genetically manipulate these important protozoan parasites.

Cloning and sequencing of an α-tubulin cDNA from Artemia franciscana: evidence for translational regulation of α-tubulin synthesis

The brine shrimp, Artemia franciscana, exhibits a limited number of tubulin isotypes which change little during early postgastrula growth. In order to better understand the synthesis of α-tubulins during Artemia development, a cDNA termed αAT1 was cloned and sequenced. Alignment analyses revealed that the polypeptide encoded by αAT1 is similar to α-tubulins from other species. Hybridization of αAT1 to restriction-digested DNA on Southern blots produced a simple banding pattern, indicating that Artemia have a small number of α-tubulin genes. Probing of Northern blots demonstrated an abundant supply of α-tubulin mRNA in dormant cysts, emerging nauplii and instar I larvae. However, it was not until instar I larvae were produced that the amount of polysomal α-tubulin mRNA increased, suggesting that synthesis of the tubulin corresponding to αAT1 is translationally controlled. This work provides one of the few examples where tubulin synthesis is thought to be translationally regulated. Moreover, when considered in the light of previous analyses, the findings imply that cell differentiation in postgastrula Artemia and the diversification of microtubule function certain to accompany this process occur with little or no change in α-tubulin composition.

Impaired molecular regenerative responses in sensory neurones of diabetic rats: gene expression changes in dorsal root ganglia after sciatic nerve crush.

This study investigated changes in gene expression in lumbar dorsal root ganglia (DRG), contralateral and ipsilateral to a sciatic nerve crush in control and streptozotocin (STZ)-induced diabetic rats. After 10 weeks of diabetes, the left sciatic nerves of all rats were crushed at mid-thigh level, and the rats were maintained for a further 2 weeks. Northern blots, with internal standards, were made from L4 and L5 (pooled) DRG on each side to compare RNA hybrids from ganglia attached to crushed nerves with those attached to intact nerves. The expression of growth-associated proteins, GAP-43 and Talpha1 alpha-tubulin mRNA in DRG, was stimulated (all P < 0.05) by crush injury in control and diabetic rats. Steady-state expression of transcripts for neurofilament (NF) proteins (NF-L, NF-H) and the high-affinity NGF receptor, trkA was decreased by diabetes in the contralateral ganglia to the crush (all P < 0.05). Crush injury further decreased expression of these transcripts in both control and diabetic rats (all P < 0.05). This reduced expression of mRNA coding for both growth-associated proteins, and neurofilament proteins in ganglia of diabetic rats could participate in the reduced competence of the regenerative response to nerve crush.

Brain‐derived neurotrophic factor modulates GAP‐43 but not tα1 expression in injured retinal ganglion cells of adult rats

The administration of neurotrophins affects neuronal survival and growth, but less is known about their ability to modify the expression of growth associated genes following injury to CNS neurons. Here we characterize the effect of brain-derived neurotrophic factor (BDNF) on mRNA levels for Tα1 α-tubulin, and for GAP-43, two genes whose expression levels in retinal ganglion cells (RGC) tend to correlate with growth. We first determined that most adult rat RGCs can retrogradely transport BDNF by injecting 125I-BDNF into RGC target sites in vivo. We then used quantitative in situ hybridization to characterize the effect of axotomy, or axotomy and BDNF administration on mRNA levels for GAP-43 and Tα1. Axotomy alone resulted in a general decrease in Tα1 α-tubulin mRNA levels by 2 weeks, and elicited an increase in GAP-43 mRNA levels in an average of 30% of surviving RGCs. The intravitreal administration of a single dose of BDNF (5 μg) to axotomized RGCs on the day of injury did not affect Tα1 α-tubulin mRNA levels, but was followed by a moderate (approximately 80%), and short-lasting enhancement of GAP-43 mRNA levels in most RGCs during the first week after axotomy. No significant increase in GAP-43 mRNA levels was observed when BDNF was injected into the uninjured eye. We conclude that BDNF specifically enhances GAP-43 but not Tα1 mRNA levels in injured RGCs. Because BDNF is known to stimulate branch length of injured RGCs, we suggest that changes in the expression of GAP-43, but not Tα1 tubulin, correlate with branching of injured neurons as opposed to long distance regrowth. J. Neurosci. Res. 47:561–572, 1997. © 1997 Wiley-Liss, Inc.

Rice β-tubulins mRNA levels are modulated during flower development and in response to external stimuli

Rice β-tubulin cDNA clones have been recently isolated by different laboratories (Kang et al. (1994) Plant Mol. Biol. 26, 1975–1979; Breviario et al. (1995) Plant Physiol. 108, 823–824; Koga-Ban et al. (1995) DNA Res. 2, 21–26). Analysis of their deduced amino acid sequences shows the conservation of all those structural motifs typical of plant β-tubulins. Putative sequences for autoregulation and tubulin mRNA stability, for GTP binding and exchange as well as for herbicides and Ca 2+ binding are present. With the use of a generic or isotype specific β-tubulin (OS-TUB16) probe we assayed the level of transcriptional accumulation in different rice tissues, during flower development, cell elongation and in response to anoxia. We found enhanced levels of total or isotype-16 specific β-tubulin mRNA at early stages of flower development in spikelets at 8–10 days before anthesis. The lowest levels were observed in adult leaves. Total β-tubulin as well as isotype-16 specific transcript levels were strongly decreased (10-fold) in rice coleoptiles treated with 50 μM abscisic acid (ABA); a more modest decrease (2-fold) in the total level of rice α-tubulin mRNA was instead observed in these samples. We also show a strict correlation between growth under anoxia and β-tubulin transcriptional abundance. Rice roots, unable to grow in anoxia, showed very low amount of total β-tubulin mRNA whereas rice coleoptiles, capable of elongating under anaerobiosis, maintained high levels of total β-tubulin transcripts. However, with the use of a 3′ non-coding probe specific for β-tubulin isotype-16, we found that the amount of this transcript is always decreased by anoxia regardless the type of tissue analyzed.

Distribution and Expression of the α-Tubulin mRNA in the Hippocampus and the Temporal Cortex in Alzheimer's Diseases

The distribution of the messenger RNA for alpha-tubulin has been investigated by in situ hybridization in the human hippocampus and temporal cortex in normal subjects and in Alzheimer's disease. The alpha-tubulin mRNA was strongly expressed in neurons in the gyrus dentatus, in the Ammon's horn and in cortical layers of the temporal cortex. The same distribution was observed in Alzheimer's disease. An important reduction of the hybridization signal was apparent, however, in areas rich in neurofibrillary lesions, e.g. as in layer II of entorhinal cortex. Neurons containing neurofibrillary tangles exhibited a weaker hybridization signal than adjacent neurons devoid of neurofibrillary tangles. The immunoreactivity for alpha-tubulin was drastically reduced in tangles-bearing neurons. These results indicate that tubulin transcription is reduced in tangles-bearing neurons, a reduction which might play a role in the reported decrease in the number of microtubules in neurons containing neurofibrillary tangles.

Effects of dexamethasone on protein degradation and protease gene expression in rat L8 myotube cultures

We examined the effects of a synthetic glucocorticoid (dexamethasone; Dex) on proteolysis and on protease messenger RNA (mRNA) concentrations in rat L8 skeletal myotube cultures. Protein degradation was measured as release of radioactive trichloroacetic acid-soluble material from intracellular proteins pre-labelled with [ 3H]tyrosine. Dex (1 μM) stimulated protein degradation ( P < 0.01). This effect was entirely blocked by the glucocorticoid antagonist, RU38486 (mifepristone; P < 0.01). Hence, actions of Dex on muscle protein degradation are mediated via intracellular glucocorticoid receptors. Molecular mechanisms by which glucocorticoids stimulate protein degradation in skeletal muscle are not known. Here, we investigated the regulation of protease (cathepsin B, cathepsin D, proteasome C2 subunit and m-calpain) mRNA concentrations by Dex in cultured L8 muscle cells. Cathepsin B mRNA concentration was enhanced 3.3-fold by Dex. This effect was blocked by RU38486. RU38486 alone did not affect cathepsin B mRNA concentration or mRNAs of other proteases. Concentrations of cathepsin D and m-calpain mRNAs were also increased by Dex. These effects were also abolished by RU38486. Proteasome C2 mRNA was unaffected by Dex and Dex reduced α-tubulin mRNA. Thus, glucocorticoids specifically regulate the concentrations of mRNAs encoding some proteases in muscle cells. The regulation of protease mRNA concentration is mediated via interaction between Dex with glucocorticoid receptors and is independent of the actions of Dex on mRNA encoding house-keeping proteins. These changes may underlie glucocorticoid-dependent control of proteolysis in muscle.

Tubulin and actin mRNAs in the young-adult and the aged rat brain: effects of repeated administration with bifemelane hydrochloride

In an attempt to identify the age-dependent changes in the potential synthesis of cytoskeletal proteins, we investigated changes in messenger RNA (mRNA) of α-tubulin and β-actin in the young-adult and the aged rat brain using Northern blot analysis. α-Tubulin mRNA levels in the frontal cortex and hippocampus, and β-actin mRNA levels in the hippocampus were significantly decreased in the aged rat brain. Age-dependent decreases in these mRNAs may be related to the neuronal dysfunction associated with aging, in addition to the reduction of several kinds of receptors previously reported. Repeated administration of bifemelane hydrochloride (4-(2-benzylphenoxy)-N-methylbutylamine hydrochloride) for 14 days increased the levels of β-actin mRNA in the frontal cortex and the striatum of both young-adult and aged rats, although the effect of bifemelane treatment was smaller and not significant in the aged group. These results suggest that bifemelane treatment may enhance the synthesis of cytoskeletal protein and promote neural plasticity by inducing neurite growth or synapse formation.