Accumulation Of Alpha Research Articles

Ectopic T cell receptor expression causes B cell immunodeficiency in transgenic mice.

Mice expressing transgenic T cell receptors (TCR) are used to explore important questions in immunity. However, transgene expression may have unexpected effects. We previously reported a B cell immunodeficiency, comprising decreased B cell numbers and diminished antibody responses, in mice that express a transgenic TCR specific for nicotinic acetylcholine receptor; the mice were generated using cassette vectors designed specifically for transgenic TCR expression [see Kouskoff et al. J. Immunol. Methods 1995. 180: 273-280]. We now show data suggesting that this defect is due to the expression and accumulation of TCR alpha and beta chains inside B cells and induction of an endoplasmic reticulum stress response, causing apoptosis at the pre B-I and later B cell stage. Thus, inappropriate transgene expression can profoundly affect B cells, leading to a previously undescribed mechanism of immunodeficiency.

Effects of extracts from Bangladeshi medicinal plants on in vitro proliferation of human breast cancer cell lines and expression of estrogen receptor α gene

In this study we determined the activity of extracts from Bangladeshi medicinal plants (Emblica officinalis, Aegle marmelos, Vernonia anthelmintica, Oroxylum indicum, Argemone mexicana) on human breast tumor cell lines. Extracts from E. officinalis and O. indicum displayed anti-proliferative activity on MCF7 and MDA-MB-231 breast cancer cell lines, while extracts from A. mexicana were active on MCF7 cells, exhibiting on the contrary low antiproliferative effects on MDA-MB-231 cells. Extracts from A. marmelos and V. anthelmintica were antiproliferative on both cell lines, but at higher concentrations. The accumulation of estrogen receptor alpha (ERalpha) mRNA, a marker of neoplastic status, was analysed by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The data obtained demonstrated that only extracts from E. officinalis induce an increase of ERalpha mRNA in MCF7 cells. When MDA-MB-231 cell line was employed, extracts from E. officinalis, V. anthelmintica and A. mexicana were found to be inducers of the increase of ERalpha mRNA accumulation. Since activation of ERalpha gene expression could have clinical impact, our results suggest a possible use of extracts from medicinal plants to identify compounds of possible interest in the treatment of breast cancer.

Inhibition of Receptor-mediated Endocytosis Demonstrates Generation of Amyloid β-Protein at the Cell Surface

Sequential cleavages of the amyloid beta-protein precursor (APP) by the beta- and gamma-secretases generate the amyloid beta-protein (A beta), which plays a central role in Alzheimer's disease. Previous work provided evidence for involvement of both the secretory and endocytic pathways in A beta generation. Here, we used HeLa cells stably expressing a tetracycline-regulated dominant-negative dynamin I (dyn K44A), which selectively inhibits receptor-mediated endocytosis, and analyzed the effects on the processing of endogenous APP. Upon induction of dyn K44A, levels of mature APP rose at the cell surface, consistent with retention of APP on the plasma membrane. The alpha-secretase cleavage products of APP were increased by dyn K44A, in that alpha-APPs in medium and the C83 C-terminal stub in the membrane both rose. The beta-secretase cleavage of APP, C99, also increased modestly. The use of specific gamma-secretase inhibitors to study the accumulation of alpha- and beta-cleavage products independent of their processing by gamma-secretase confirmed that retention of APP on the plasma membrane results in increased processing by both alpha- and beta-secretases. Unexpectedly, endogenous A beta secretion was significantly increased by dyn K44A, as detected by three distinct methods: metabolic labeling, immunoprecipitation/Western blotting, and enzyme-linked immunosorbent assay. Levels of p3 (generated by sequential alpha- and gamma-cleavage) also rose. We conclude that endogenous A beta can be produced directly at the plasma membrane and that alterations in the degree of APP endocytosis may help regulate its production. Our findings are consistent with a role for the gamma-secretase complex in the processing of numerous single-transmembrane receptors at the cell surface.

Mechanisms governing the accumulation of estrogen receptor alpha in MCF-7 breast cancer cells treated with hydroxytamoxifen and related antiestrogens

This study aimed at a better understanding of estrogen receptor α (ER) up regulation induced by partial estrogen antagonists. Effect of treatment with hydroxytamoxifen (OH-Tam) on ER level in MCF-7 cells was investigated by an approach combining ER measurement (enzyme immunoassay) and morphological demonstration (immunofluorescence). Furthermore, the influence of drug exposure on the rates of ER synthesis and degradation was assessed by determining [ 35 S ]methionine incorporated into the receptor in different experimental conditions (measurement of synthesis or pulse-chase experiments). ER up regulation was already induced by a 1-h pulse treatment with OH-Tam, thus a continuous exposure was not required. This process appeared reversible (i.e. ER accumulation due to OH-Tam rapidly vanished upon subsequent exposure to 17β-estradiol (E 2) or the pure antiestrogen RU 58668). While OH-Tam did not affect the rate of [ 35 S ]methionine incorporation into ER, it clearly caused an impairment of ER degradation (pulse-chase experiments) indicating that up regulation results from a stabilization of the receptor associated with the maintenance of its synthesis. Various tamoxifen derivatives, as well as a few related partial antiestrogens, were compared on the basis of binding ability and propensity to induce ER up regulation. A close relationship was found between both properties. Structure-activity analysis revealed that the capacity of these compounds to induce ER up regulation is associated with characteristics of their aminoalkyle side-chain, similar to those required for antiestrogenicity.

Understanding Emotion Regulation in Borderline Personality Disorder: Contributions of Neuroimaging

Understanding Emotion Regulation in Borderline Personality Disorder: Contributions of Neuroimaging

Amifostine induces anaerobic metabolism and hypoxia-inducible factor 1α

The cytoprotective mechanism of amifostine (WR-2721) implies free radical scavenging and DNA repair activities. We investigated additional cytoprotective pathways involving intracellular hypoxia and the activation of the hypoxia-inducible factor (HIF) pathway, a key transcription factor regulating glycolysis, angiogenesis and apoptosis, which is also linked with radioresistance. The glucose and oxygen levels in the peripheral blood of patients receiving 1000 mg amifostine were determined at various time-points in order to investigate the metabolic changes induced by amifostine. MDA468 breast tumor cell lines were incubated with a high amifostine concentration (10 m M) to overcome the natural resistance of cancer cells to influx of the non-hydrolyzed WR-2721, and the HIF1 alpha protein levels were determined by Western blot analysis. In vivo experiments with Wistar rats were performed in order to assess immunohistochemically changes in the intracellular accumulation of HIF1 alpha induced by amifostine (200 mg/kg). By 30 min following amifostine administration, the hemoglobin oxygen saturation and pO(2) levels had increased in the peripheral blood while glucose levels had reduced, providing evidence that normal tissue metabolism switches to glycolytic pathways. Incubation of cell lines with amifostine resulted in HIF1 alpha induction. In Wistar rats administration of amifostine resulted in increased HIF1 alpha accumulation in normal tissues. Since it is doubtful whether dephosphorylation of amifostine to the active metabolite WR-1065 occurs within tumoral tissues (an acidic environment that lacks vascular alkaline phosphatase activity), intracellular hypoxia and upregulation of HIF1 alpha represents an additional, normal tissue-specific, amifostine cytoprotective pathway.

Accumulation of airborne hexachlorocyclohexanes and DDT in pine needles.

The accumulation of alpha- and gamma-hexachlorocyclohexanes (HCHs) and p,p'-DDT in Scots pine needles was measured simultaneously with the concentrations in the surrounding air. All three compounds accumulate during the entire life span of the needles. For p,p'-DDT the general accumulation is overlaid by a concentration peak during winter, probably related to particle deposition. For the HCHs the accumulation is overlaid by a seasonal pattern with rapid accumulation during spring and summer months and constant concentrations during winter months. The concentrations of alpha-HCH in the air were more or less constant during the whole sampling period, whereas gamma-HCH shows an air concentration peak in spring and early summer. Air concentrations of p,p'-DDT were for the most part below the detection limit. There is little evidence of revolatilization from the needles, with the exception of a small decline of gamma-HCH concentration in summer after the peak air concentrations. The high accumulation rate of the HCHs during the warm season correlates with high levels of volatile organic compounds (VOCs) in the needles. We suggest that the variation of VOC content in the needles cause seasonal variation of the air-plant partitioning coefficient. An additional complicating factor is that the dry weight of the needles has to be corrected for the yearly variation of starch content. The results presented here question models of uptake of airborne contaminants by plants that assume rapid equilibria between the air and plants.

Nitric oxide, oxidative stress, and apoptosis

Life demands intra- and intercellular communication in and between cells to respond and adapt to changes in the environment. Among signaling molecules, reactive oxygen (ROS) and nitrogen (RNS) species gained attention in facilitating intracellular communication and causing cell demise during pathology. Complexity was added with the notion that ROS and RNS signals overlap and/or produce synergistic, as well as antagonistic, effects. This is exemplified by using oxidized lipoproteins (oxLDL), or NO donors, in provoking the stabilization of two well recognized transcription factors, such as tumor suppressor p53 and hypoxia-inducible factor-1 alpha (HIF-1 alpha). Radical (i.e., superoxide) (O2-) formation in response to oxLDL is associated with p53, as well as HIF-1 alpha accumulation in human macrophages, a process that is antagonized by NO. On the other side, NO-elicited HIF-1 alpha stabilization is modulated by O2-. Thus, ROS- and RNS-signaling is important in understanding cell physiology and pathology, with the notion that marginal changes in the flux rates of either NO or O2- may shift vital signals used for communication into areas of pathology in close association with human diseases.

A novel type of lycopene epsilon-cyclase in the marine cyanobacterium Prochlorococcus marinus MED4.

Chlorophyll- b-possessing cyanobacteria of the genus Prochlorococcus share the presence of high amounts of alpha- and beta-carotenoids with green algae and higher plants. The branch point in carotenoid biosynthesis is the cyclization of lycopene, for which in higher plants two distinct enzymes are required, epsilon- and beta-lycopene cyclase. All cyanobacteria studied so far possess a single beta-cyclase. Here, two different Prochlorococcus sp. MED4 genes were functionally identified by heterologous gene complementation in Escherichia coli to encode lycopene cyclases. Whereas one is both functionally and in sequence highly similar to the beta-cyclase of Synechococcus sp. strain PCC 7942 and other cyanobacteria, the other showed several intriguing features. It acts as a bifunctional enzyme catalyzing the formation of epsilon- as well as of beta-ionone end groups. Expression of this cyclase in E. coli resulted in the simultaneous accumulation of alpha- beta-, delta-, and epsilon-carotene. Such an activity is in contrast to all lycopene epsilon-cyclases known so far, including those of the higher plants. Thus, for the first time among prokaryotes, two individual enzymes were identified in one organism that are responsible for the formation of cyclic carotenoids with either beta- or epsilon-end groups. These two genes are suggested to be designated as crtL-b and crtL-e. The results indicate that both enzymes might have originated from duplication of a single gene. Consequently, we suggest that multiple gene duplications followed by functional diversification resulted several times, and in independent lineages, in the appearance of enzymes for the biosynthesis of cyclic carotenoids.

Targeting of platelet integrin alphaIIbbeta3 determines systemic reaction and bleeding in murine thrombocytopenia regulated by activating and inhibitory FcgammaR.

Previous work on cellular destruction induced by several clinically relevant anti-platelet IgG antibodies suggested antigen-specific mechanisms in the development of immune thrombocytopenia in mice. mAb directed against mouse platelet GPIbalpha and integrin alpha(IIb)beta(3) were highly pathogenic, and mediated their effects via different Fc-dependent (alpha(IIb)beta(3)) and Fc-independent (GPIbalpha) pathways, indicating that clearance of IgG-bound platelets is only one event in the pathogenesis of murine thrombocytopenia. Here, we demonstrate that in addition to thrombocytopenia, targeting of platelet integrin alpha(IIb)beta(3) results in acute systemic reaction and bleeding that is regulated by activating IgG Fc receptors (FcgammaR) and the inhibitory FcgammaRII. As shown by electron microscopy, anti-alpha(IIb)beta(3) IgG mediated initial loss of alpha(IIb)beta(3) integrin from platelet surfaces followed by rapid accumulation of alpha(IIb)beta(3) antibody-containing immune complex (IC)-like structures in spleen and liver in vivo. In FcRgamma chain deficiency, mice resisted bleeding, but not platelet destruction, while genetic ablation of FcgammaRII resulted in uncontrolled systemic reaction and severe hemorrhage leading to enhanced mortality. Together, these results provide evidence that IC formation and engagement of FcgammaR on effector cells determines the alpha(IIb)beta(3)-specific part of the platelet pathology of the systemic reaction and bleeding in murine thrombocytopenia.

Le corps de Lewy, marqueur abusif de la maladie de Parkinson ?

The Lewy body, an eosinophilic inclusion around 10 microns in diameter, is localised in the neuronal perikaryon. Its dense core is surrounded by a clear halo, which is lacking in the so-called "cortical Lewy bodies". Numerous proteins have been identified in Lewy bodies, among which the three neurofilament isoforms, ubiquitin and proteasome subunits. More recently, alpha-synuclein--a pre-synaptic protein--has been found to be the essential constituent of the Lewy body. Alpha-synuclein antibody has greatly increased the sensitivity of the neuropathological examination: it has emphasized the frequency of "Lewy neurites" (accumulation of alpha--synuclein in neuronal processes) and has shown the importance of extra-nigral pathology. Lewy bodies and neurites are indeed to be found in many areas of the central and peripheral nervous system: stellate ganglia, cardiac and enteric plexus, pigmented nuclei of the brainstem, basal nucleus of Meynert, amygdala, limbic nuclei of the thalamus, parahippocampal and cingulate gyri, insula and isocortex. Lewy body diseases include at least three clinical syndromes: 1) idiopathic Parkinson disease in which the brainstem bears the brunt of the pathology 2) Parkinson disease dementia in which Lewy lesions are found in the brainstem and are also abundant in the isocortex. A large number of senile plaques is frequently associated. 3) In dementia with Lewy bodies, the same lesions are observed but the cognitive deficit occurs first or shortly (less than one year) after the motor symptoms.

Adenosine A2A or A3 receptors are required for inhibition of inflammation by methotrexate and its analog MX-68.

Low-dose weekly methotrexate therapy remains a mainstay in the treatment of inflammatory arthritis. Results of previous studies demonstrated that adenosine, acting at one or more of its receptors, mediates the antiinflammatory effects of methotrexate in animal models of both acute and chronic inflammation. We therefore sought to establish which receptor(s) is involved in the modulation of acute inflammation by methotrexate and its nonpolyglutamated analog MX-68 (N-[[4-[(2,4-diaminopteridin-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]-carbonyl]-L-homoglutamic acid). We studied the effects of low-dose methotrexate (0.75 mg/kg intraperitoneally [IP] every week for 5 weeks), MX-68 (2 mg/kg IP 2 days and 1 hour before induction of inflammation), dexamethasone (1.5 mg/kg IP 1 hour before induction of inflammation), or vehicle control on acute inflammation in an air-pouch model in A(2A) and A(3) receptor knockout mice. Low-dose weekly methotrexate treatment increased the adenosine concentration in the exudates of all mice studied and reduced leukocyte and tumor necrosis factor alpha accumulation in the exudates of wild-type mice, but not in those of A(2A) or A(3) receptor knockout mice. Dexamethasone, an agent that suppresses inflammation by a different mechanism, was equally effective at suppressing leukocyte accumulation in A(2A) knockout, A(3) knockout, and wild-type mice, indicating that the lack of response was specific for methotrexate and MX-68. These findings confirm that adenosine, acting at A(2A) and A(3) receptors, is a potent regulator of inflammation. Moreover, these results provide strong evidence that adenosine, acting at either or both of these receptors, mediates the antiinflammatory effects of methotrexate and its analog MX-68.

Importin alpha can migrate into the nucleus in an importin beta- and Ran-independent manner.

A classical nuclear localization signal (NLS)-containing protein is transported into the nucleus via the formation of a NLS-substrate/importin alpha/beta complex. In this study, we found that importin alpha migrated into the nucleus without the addition of importin beta, Ran or any other soluble factors in an in vitro transport assay. A mutant importin alpha lacking the importin beta-binding domain efficiently entered the nucleus. Competition experiments showed that this import pathway for importin alpha is distinct from that of importin beta. These results indicate that importin alpha alone can enter the nucleus via a novel pathway in an importin beta- and Ran-independent manner. Furthermore, this process is evolutionarily conserved as similar results were obtained in Saccharomyces cerevisiae. Moreover, the import rate of importin alpha differed among individual nuclei of permeabilized cells, as demonstrated by time-lapse experiments. This heterogeneous nuclear accumulation of importin alpha was affected by the addition of ATP, but not ATPgammaS. These results suggest that the nuclear import machinery for importin alpha at individual nuclear pore complexes may be regulated by reaction(s) that require ATP hydrolysis.

Chronic Ethanol Increases Lipopolysaccharide-stimulated Egr-1 Expression in RAW 264.7 Macrophages: CONTRIBUTION TO ENHANCED TUMOR NECROSIS FACTOR α PRODUCTION

Increased production of tumor necrosis factor alpha (TNFalpha) is associated with the development of alcoholic liver disease. Culture of RAW264.7 macrophages with 25 mm ethanol for 48 h increased lipopolysaccharide (LPS)-stimulated accumulation of tumor necrosis factor alpha (TNFalpha) peptide and mRNA by 2-fold. We investigated whether chronic ethanol-induced increases in the DNA binding and/or promoter activity of the key transcription factors regulating LPS-stimulated TNFalpha promoter activity contribute to increased TNFalpha expression. Binding of Egr-1 to the TNFalpha promoter was increased by 2.5-fold after ethanol exposure, whereas NFkappaB binding was decreased to 30% of control. AP-1 binding was not affected. Changes in binding activity were paralleled by an increased contribution of the Egr-1 binding site and a decreased contribution of the NFkappaB site to LPS-stimulated TNFalpha promoter activity. Overexpression of dominant negative Egr-1 prevented the ethanol-induced increase in LPS-stimulated TNFalpha mRNA accumulation. Chronic ethanol exposure enhanced LPS-stimulated Egr-1 promoter-driven CAT expression and transcription of Egr-1. Induction of Egr-1 is dependent on ERK1/2 activation in other systems. Therefore, we investigated whether the ERK1/2 pathway mediated the chronic ethanol-induced increases in Egr-1 and TNFalpha. Increased Egr-1 promoter activity and TNFalpha mRNA accumulation after chronic ethanol were both prevented by overexpression of dominant negative ERK1/2. LPS-stimulated ERK1/2 phosphorylation was increased 2-fold in cells cultured with ethanol compared with controls. These results demonstrate that enhanced LPS-dependent activation of Egr-1 contributes to increased TNFalpha production after chronic ethanol exposure.

Role of integrin alpha(v)beta3 in the early phase of liver metastasis: PET and IVM analyses.

To clarify the function of integrin alpha(v)beta3 in the early stage of liver metastasis, we investigated the interactions of metastatic cells with their target organ under the actual blood flow by using positron emission tomography (PET). The cells used were CHO-K1 cells and their transfectants bearing human integrin alpha(v)beta3 cDNA (alpha(v)beta3-CHO-K1 cells). The liver accumulation of alpha(v)beta3-CHO-K1 cells was significantly higher than that of CHO-K1 cells after injection via the portal vein, whereas no significant difference was observed in the lung accumulation after tail vein injection, suggesting a specific interaction of alpha(v)beta3-CHO-K1 cells with the hepatic sinusoids. Furthermore, to clarify the precise location of each cell in the liver, i.e., to determine whether individual cells were intravascularly localized or had extravasated, we performed intravital fluorescence microscopy (IVM) on the liver by using stable transfectants bearing the green fluorescent protein (GFP) gene, namely, GFP-CHO-K1 and GFP-alpha(v)beta3-CHO-K1 cells. Both types of cells remained in the hepatic blood vessels 1 h after injection via the portal vein. On the other hand, expression of integrin alpha(v)beta3 promoted the cells to reach the extravascular region after 24 h. These results suggest the possibility that the specific accumulation of alpha(v)beta3-CHO-K1 cells in the liver is followed by migration of the cells into the extravascular region. Interestingly, the adhesion of the two types of cells to hepatic sinusoidal endothelial cells in vitro did not correspond to in vivo accumulation of these cells. Therefore, integrin alpha(v)beta3 may function to promote extravasation of integrin alpha(v)beta3-expressing tumor cells in liver through a process possibly mediated by vitronectin produced by this organ.

Post-translational modifications of cardiac tubulin during chronic heart failure in the rat.

Cytoskeletal reorganization has been shown to participate in cellular remodeling and in the alterations of mechanical function of isolated cardiomyocytes during pressure overload hypertrophy. Post-translational modifications of tubulin towards stabilization of microtubules have also been described in animal models of compensatory hypertrophy, but the status of the microtubules network in end stage heart failure is not clearly established. Using a rat model of congestive heart failure (CHF) induced by aortic banding, we studied the expression of alpha- and beta-tubulin, as well as their post-translational modification and distribution in the soluble and polymerized fraction by immunoblotting. We found an accumulation of alpha- and beta-tubulin protein content specifically in the soluble fraction with no change in the polymerized fraction. Amongst the several variants of alpha-tubulin examined, only detyrosinated Glu-tubulin and deglutamylated delta2-tubulin levels were selectively increased during heart failure. Glu-tubulin accumulated in the polymerized fraction while delta2-tubulin levels were increased in the soluble fraction in CHF hearts. These results show that a profound remodeling of the microtubule network occurs in heart failure. This remodeling suggests an increase in the stability of the microtubule network which is discussed in terms of possible functional consequences.

Regulation of the hypoxia-inducible factor-1 alpha. ARNT is not necessary for hypoxic induction of HIF-1 alpha in the nucleus.

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of mammalian oxygen homeostasis. HIF-1 consists of two subunits, HIF-1 alpha and the aryl hydrocarbon receptor nuclear translocator (ARNT). Whereas hypoxia prevents ubiquitination and proteasomal degradation of HIF-1 alpha, ARNT expression is thought to be oxygen-independent. We and others previously showed that ARNT is indispensable for HIF-1 DNA-binding and transactivation function. To examine the requirement of ARNT for accumulation and nuclear translocation of HIF-1 alpha in hypoxia, we used ARNT-mutant mouse hepatoma and ARNT-deficient embryonic stem cells. As shown by immunofluorescence, HIF-1 alpha accumulation in the nucleus of hypoxic cells did not require ARNT, demonstrating that nuclear translocation is intrinsic to HIF-1 alpha. During biochemical separation, both HIF-1 alpha and ARNT tend to leak from the nuclei in the absence of the corresponding subunit, suggesting that heterodimerization is required for stable association within the nuclear compartment. Nuclear stabilization of the heterodimer might also explain the hypoxically increased total cellular ARNT levels observed in some of the cell lines examined.

Carboxyl-terminal Fragments of Alzheimer β-Amyloid Precursor Protein Accumulate in Restricted and Unpredicted Intracellular Compartments in Presenilin 1-deficient Cells

Absence of functional presenilin 1 (PS1) protein leads to loss of gamma-secretase cleavage of the amyloid precursor protein (betaAPP), resulting in a dramatic reduction in amyloid beta peptide (Abeta) production and accumulation of alpha- or beta-secretase-cleaved COOH-terminal fragments of betaAPP (alpha- or beta-CTFs). The major COOH-terminal fragment (CTF) in brain was identified as betaAPP-CTF-(11-98), which is consistent with the observation that cultured neurons generate primarily Abeta-(11-40). In PS1(-/-) murine neurons and fibroblasts expressing the loss-of-function PS1(D385A) mutant, CTFs accumulated in the endoplasmic reticulum, Golgi, and lysosomes, but not late endosomes. There were some subtle differences in the subcellular distribution of CTFs in PS1(-/-) neurons as compared with PS1(D385A) mutant fibroblasts. However, there was no obvious redistribution of full-length betaAPP or of markers of other organelles in either mutant. Blockade of endoplasmic reticulum-to-Golgi trafficking indicated that in PS1(-/-) neurons (as in normal cells) trafficking of betaAPP to the Golgi compartment is necessary before alpha- and beta-secretase cleavages occur. Thus, although we cannot exclude a specific role for PS1 in trafficking of CTFs, these data argue against a major role in general protein trafficking. These results are more compatible with a role for PS1 either as the actual gamma-secretase catalytic activity or in other functions indirectly related to gamma-secretase catalysis (e.g. an activator of gamma-secretase, a substrate adaptor for gamma-secretase, or delivery of gamma-secretase to betaAPP-containing compartments).

Meiotic mutants of <i>Medicago</i> <i>sativa</i> show altered levels of α- and β-tubulin

We have analysed the level of accumulation of alpha- and beta-tubulin polypeptides in flowers collected from different meiotic mutants of alfalfa (Medicago sativa L.). The H33 mutant previously identified as a producer of male and female gametes with the somatic chromosome number (2n gametes) as a result of defective spindle orientation or, more rarely, abnormal cytokinesis, showed a higher level of alpha- and beta-tubulin compared to control diploid plants and approximately the same level as control tetraploid plants. A higher level of tubulin was likewise observed in diploid plants displaying abnormalities in spindle orientation and cytokinesis, which had gone through 3-4 cycles of phenotypic recurrent selection to increase 2n gamete production. A similar analysis was performed on another class of Medicago meiotic mutants characterized by production of 4n pollen (jumbo pollen, due to the absence of cytokinesis at the end of meiosis) and 2n eggs. Again, the level of alpha- and beta-tubulin was found to be higher in the mutants than in diploid controls. We conclude that meiotic defects, such as abnormal spindle orientation or cytokinesis leading to the formation of 2n gametes, determine an increased level of tubulin, the main constituent of plant microtubules (MTs).

Cell-cycle modulation of CK2 activity in tobacco BY-2 cells.

Protein kinase CK2 is an ubiquitous Ser/Thr kinase essential for cell growth. We have used the highly synchronizable tobacco BY-2 cell line to investigate whether CK2 activity and expression are regulated in a cell cycle phase-dependent manner in higher plants. Specific cDNA probes for tobacco CK2alpha and beta subunits, respectively, and polyclonal antibodies recognising alpha and beta subunits separately, were obtained to determine mRNA and protein levels of both subunits. Our results show that CK2 activity oscillates throughout the cell cycle, peaking at G1/S and M phases, due to a post-translational regulation of the tetrameric enzyme. Additional levels of control of CK2 expression operate in relation to the proliferative state of the cells, including differential accumulation of alpha and beta transcripts and post-transcriptional regulation of protein levels (beta subunit). Moreover, in vivo inhibition of CK2 activity corroborates the requirement of the functional CK2 to progress through the cell division cycle, and suggests that CK2 might play an important role at the G2/M checkpoint.