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Biomedical subjects

D Job

Publications and source records attributed to D Job.

At least 19 recordsLinked to original sources
PubMed2004

Astrocytes and oligodendrocytes express different STOP protein isoforms.

Many cell types contain subpopulations of microtubules that resist depolymerizing conditions, such as exposure to cold or to the drug nocodazole. This stabilization is due mainly to polymer association with STOP proteins. In mouse, neurons express two major variants of these proteins, N-STOP and E-STOP (120 kDa and 79 kDa, respectively), whereas fibroblasts express F-STOP (42 kDa) and two minor variants of 48 and 89 kDa. N- and E-STOP induce microtubule resistance to both cold and nocodazole exposure, whereas F-STOP confers microtubule stability only to the cold. Here, we investigated the expression of STOP proteins in oligodendrocytes and astrocytes in culture. We found that STOP proteins were expressed in precursor cells, in immature and mature oligodendrocytes, and in astrocytes. We found that oligodendrocytes express a major STOP variant of 89 kDa, which we called O-STOP, and two minor variants of 42 and 48 kDa. The STOP variants expressed by oligodendrocytes induce microtubule resistance to the cold and to nocodazole. For astrocytes, we found the expression of two STOP variants of 42 and 48 kDa and a new STOP isoform of 60 kDa, which we called A-STOP. The STOP variants expressed by astrocytes induce microtubule resistance to the cold but not to nocodazole, as fibroblast variants. In conclusion, astrocytes and oligodendrocytes express different isoforms of STOP protein, which show different microtubule-stabilizing capacities.

Animals↗
PubMed2004

Suppression of nuclear oscillations in Saccharomyces cerevisiae expressing Glu tubulin.

In most eukaryotic cells, the C-terminal amino acid of alpha-tubulin is aromatic (Tyr in mammals and Phe in Saccharomyces cerevisiae) and is preceded by two glutamate residues. In mammals, the C-terminal Tyr of alpha-tubulin is subject to cyclic removal from the peptide chain by a carboxypeptidase and readdition to the chain by a tubulin-Tyr ligase. There is evidence that tubulin-Tyr ligase suppression and the resulting accumulation of detyrosinated (Glu) tubulin favor tumor growth, both in animal models and in human cancers. However, the molecular basis for this apparent stimulatory effect of Glu tubulin accumulation on tumor progression is unknown. Here we have developed S. cerevisiae strains expressing only Glu tubulin and used them as a model to assess the consequences of Glu tubulin accumulation in cells. We find that Glu tubulin strains show defects in nuclear oscillations. These defects are linked to a markedly decreased association of the yeast ortholog of CLIP170, Bik1p, with microtubule plus-ends. These results indicate that the accumulation of Glu tubulin in cells affects microtubule tip complexes that are important for microtubule interactions with the cell cortex.

Amino Acid Substitution↗
PubMed2004

[A role for microtubules in mental diseases?].

Microtubules are key cytoskeletal components in the cytoplasm of eukaryotic cells where they have pleiotropic and vital roles in functions such as cell division, trafficking or morphogenesis. Microtubules are especially abundant in neurons. Although microtubules are in many cells dynamic polymers, they exhibit an extreme state of stability in neurons. Previous work has indicated a central role of microtubule associated proteins called STOPs in neuronal microtubule stabilization. We have recently developed STOP null mice. These mice were devoid of stable brain microtubules but to our surprise had nevertheless an apparently normal brain anatomy. However the mice showed synaptic defects affecting different forms of long- and short-term synaptic plasticity. These synaptic defects were associated with severe behavioral defects that showed a remarkable sensitivity to long-term treatment with neuroleptics. We discuss the relationship of the phenotypes observed in STOP null mice with current models of schizophrenia in which the multiple, severe, and neuroleptic sensitive mental disorders caused by the disease are due to a "disease of the synapse".

Animals↗
PubMed2003

Structural disconnectivity in schizophrenia: a diffusion tensor magnetic resonance imaging study.

BACKGROUND: There is growing evidence that schizophrenia is a disorder of cortical connectivity. Specifically, frontotemporal and frontoparietal connections are thought to be functionally impaired. Diffusion tensor magnetic resonance imaging (DT-MRI) is a technique that has the potential to demonstrate structural disconnectivity in schizophrenia. AIMS: To investigate the structural integrity of frontotemporal and frontoparietal white matter tracts in schizophrenia. METHOD: Thirty patients with DSM-IV schizophrenia and thirty matched control subjects underwent DT-MRI and structural MRI. Fractional anisotropy - an index of the integrity of white matter tracts - was determined in the uncinate fasciculus, the anterior cingulum and the arcuate fasciculus and analysed using voxel-based morphometry. RESULTS: There was reduced fractional anisotropy in the left uncinate fasciculus and left arcuate fasciculus in patients with schizophrenia compared with controls. CONCLUSIONS: The findings of reduced white matter tract integrity in the left uncinate fasciculus and left arcuate fasciculus suggest that there is frontotemporal and frontoparietal structural disconnectivity in schizophrenia.

Anisotropy↗
PubMed2001

Tubulin detyrosination is a frequent occurrence in breast cancers of poor prognosis.

Tubulin, the dimeric subunit of microtubules, is a major cell protein that is centrally involved in cell division. Tubulin is subject to specific enzymatic posttranslational modifications including cyclic tyrosine removal and addition at the COOH terminus of the alpha-subunit. Tubulin is normally extensively tyrosinated in cycling cells. However, we have previously shown that detyrosinated tubulin accumulates in cancer cells during tumor progression in nude mice. Tubulin detyrosination, resulting from suppression of tubulin tyrosine ligase and the resulting unbalanced activity of tubulin-carboxypeptidase, apparently represents a strong selective advantage for cancer cells. We have now analyzed the occurrence and significance of tubulin detyrosination in human breast tumors. We studied a total of 134 breast cancer tumors from patients with or without known complications over a follow-up period of 31 +/- 10 months. The mean age of the patients at the time of diagnosis was 57 years. For each patient, detailed data concerning the histology and extension of the tumor were available. Tumor cells containing detyrosinated tubulin were visualized by immunohistochemical staining of paraffin-embedded tissue sections. Cancer cells with detyrosinated tubulin were observed in 53% of the tumors and were predominant in 19.4% of the tumors. Tubulin detyrosination correlated to a high degree of significance (P < 0.001) with a high Scarf-Bloom-Richardson (SBR) grade, a known marker of tumor aggressiveness. Among SBR grade 1 tumors, 3.8% were strongly positive for tubulin detyrosination compared with 65.4% of the SBR grade 3 tumors. The SBR component showing the strongest correlation with tubulin detyrosination was the mitotic score. In the entire patient population, neither the SBR grade nor the detyrosination index had significant prognostic value (P = 0.11, P = 0.27, respectively), whereas a combined index was significantly correlated with the clinical outcome (P = 0.02). A preliminary subgroup analysis indicated that tubulin detyrosination may define high- and low- risk groups in breast cancer tumors with an SBR grade of 2. Our study shows that tubulin detyrosination is a frequent occurrence in breast cancer, easy to detect, and linked to tumor aggressiveness.

Adult↗
PubMed2001

Identification of novel bifunctional calmodulin-binding and microtubule-stabilizing motifs in STOP proteins.

Although microtubules are intrinsically labile tubulin assemblies, many cell types contain stable polymers, resisting depolymerizing conditions such as exposure to the cold or the drug nocodazole. This microtubule stabilization is largely due to polymer association with STOP proteins. There are several STOP variants, some with capacity to induce microtubule resistance to both the cold and nocodazole, others with microtubule cold stabilizing activity only. These microtubule-stabilizing effects of STOP proteins are inhibited by calmodulin and we now demonstrate that they are determined by two distinct kinds of repeated modular sequences (Mn and Mc), both containing a calmodulin-binding peptide, but displaying different microtubule stabilizing activities. Mn modules induce microtubule resistance to both the cold and nocodazole when expressed in cells. Mc modules, which correspond to the STOP central repeats, have microtubule cold stabilizing activity only. Mouse neuronal STOPs, which induce both cold and drug resistance in cellular microtubules, contain three Mn modules and four Mc modules. Compared with neuronal STOPs, the non-neuronal F-STOP lacks multiple Mn modules and this corresponds with an inability to induce nocodazole resistance. STOP modules represent novel bifunctional calmodulin-binding and microtubule-stabilizing sequences that may be essential for the generation of the different patterns of microtubule stabilization observed in cells.

Amino Acid Motifs↗
PubMed2001

Microtubule dynamics.

Microtubules are fibrous elements in the cytoplasm of eukaryotic cells, where they perform a wide variety of functions. Microtubules are major organizers of the cell interior and are vitally involved in motility events such as chromosome migration during cell division. To fulfill their physiological function, microtubule arrays have to undergo dramatic changes in their spatial arrangement, and this depends to a large extent on the complex and special dynamic properties of the individual polymers. In this review we first describe the intrinsic dynamic properties of microtubules assembled in vitro from purified tubulin and examine the relationships between these properties and microtubule functions. Subsequent sections concern microtubule dynamics in vivo, their similarity and differences with microtubule dynamics in vitro, and the nature of the cellular regulators which act on microtubule assemblies in physiological conditions.

Animals↗
PubMed2001

Proteomic analysis of arabidopsis seed germination and priming.

To better understand seed germination, a complex developmental process, we developed a proteome analysis of the model plant Arabidopsis for which complete genome sequence is now available. Among about 1,300 total seed proteins resolved in two-dimensional gels, changes in the abundance (up- and down-regulation) of 74 proteins were observed during germination sensu stricto (i.e. prior to radicle emergence) and the radicle protrusion step. This approach was also used to analyze protein changes occurring during industrial seed pretreatments such as priming that accelerate seed germination and improve seedling uniformity. Several proteins were identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Some of them had previously been shown to play a role during germination and/or priming in several plant species, a finding that underlines the usefulness of using Arabidopsis as a model system for molecular analysis of seed quality. Furthermore, the present study, carried out at the protein level, validates previous results obtained at the level of gene expression (e.g. from quantitation of differentially expressed mRNAs or analyses of promoter/reporter constructs). Finally, this approach revealed new proteins associated with the different phases of seed germination and priming. Some of them are involved either in the imbibition process of the seeds (such as an actin isoform or a WD-40 repeat protein) or in the seed dehydration process (e.g. cytosolic glyceraldehyde-3-phosphate dehydrogenase). These facts highlight the power of proteomics to unravel specific features of complex developmental processes such as germination and to detect protein markers that can be used to characterize seed vigor of commercial seed lots and to develop and monitor priming treatments.

Arabidopsis↗
PubMed2001

Evidence for a role of the (alpha)-tubulin C terminus in the regulation of cyclin B synthesis in developing oocytes.

Microinjected mAb YL1/2, an (alpha)-tubulin antibody specific for the tyrosinated form of the protein, blocks the cell cycle in developing oocytes. Here, we have investigated the mechanism involved in the mAb effect. Both developing starfish and Xenopus oocytes were injected with two different (alpha)-tubulin C terminus antibodies. The injected antibodies blocked cell entry into mitosis through specific inhibition of cyclin B synthesis. The antibody effect was independent of the presence or absence of polymerized microtubules and was mimicked by injected synthetic peptides corresponding to the tyrosinated (alpha)-tubulin C terminus, whereas peptides lacking the terminal tyrosine were ineffective. These results indicate that tyrosinated (alpha)-tubulin, or another protein sharing the same C-terminal epitope, is involved in specific regulation of cyclin B synthesis in developing oocytes.

Amino Acid Sequence↗
PubMed2000

Inhibition of p-hydroxyphenylpyruvate dioxygenase by the diketonitrile of isoxaflutole: a case of half-site reactivity.

p-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the formation of homogentisate from p-hydroxyphenylpyruvate and molecular oxygen. In plants, this enzyme is the molecular target of new families of very active bleaching herbicides. In the study presented here, we report for the first time on the purification to homogeneity of a plant enzyme, as obtained from recombinant Escherichia coli cells expressing a cDNA encoding carrot HPPD. The purified enzyme allowed us to carry out a detailed characterization of the inhibitory properties of a diketonitile (DKN), the active inhibitor formed from the benzoylisoxazole herbicide isoxaflutole. Inhibition kinetic analyses confirmed that DKN exerts a slow and tight-binding inhibition of HPPD, competitive with respect to the p-hydroxyphenylpyruvate substrate. The stoichiometry of DKN binding to HPPD determined by kinetic analyses or by direct binding of [(14)C]DKN revealed a half-site reactivity of DKN.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed2000

A reassessment of the factors affecting microtubule assembly and disassembly in vitro.

Current models of microtubule assembly from pure tubulin involve a nucleation phase followed by microtubule elongation at a constant polymer number. Both the rate of microtubule nucleation and elongation are thought to be tightly influenced by the free GTP-tubulin concentration, in a law of mass action-dependent manner. However, these basic hypotheses have remained largely untested due to a lack of data reporting actual measurements of the microtubule length and number concentration during microtubule assembly.Here, we performed simultaneous measurements of the polymeric tubulin concentration, of the free GTP-tubulin concentration, and of the microtubule length and number concentration in both polymerizing and depolymerizing conditions. In agreement with previous work we find that the microtubule nucleation rate is strongly dependent on the initial GTP-tubulin concentration. But we find that microtubule nucleation persists during microtubule elongation. At any given initial tubulin-GTP concentration, the microtubule nucleation rate remains constant during polymer assembly, despite the wide variation in free GTP-tubulin concentration. We also find a remarkable constancy of the rate of microtubule elongation during assembly. Apparently, the rate of microtubule elongation is intrinsic to the polymers, insensitive to large variations of the free GTP-tubulin concentration. Finally we observe that when, following assembly, microtubules depolymerize below the free GTP-tubulin critical concentration, the rate-limiting factor for disassembly is the frequency of microtubule catastrophe. At all time-points during disassembly, the microtubule catastrophe frequency is independent of the free GTP-tubulin concentration but, as the microtubule nucleation rate, is strongly dependent on the initial free GTP-tubulin concentration. We conclude that the dynamics of both microtubule assembly and disassembly depend largely on factors other than the free GTP-tubulin concentration. We propose that intrinsic structural factors and endogenous regulators, whose concentration varies with the initial conditions, are also major determinants of these dynamics.

Animals↗
PubMed2000

Preliminary crystallographic study of a complex formed between the alpha/beta-tubulin heterodimer and the neuronal growth-associated protein SCG10.

Crystals of a complex formed between the alpha/beta-tubulin heterodimer and SCG10, a neuron-specific growth-associated protein, have been obtained by the hanging drop method. They belong to the space group P2(1)2(1)2(1), with unit cell parameters a = 56 A, b = 353 A, c = 466 A and four molecular complexes in the asymmetric unit. A complete X-ray diffraction data set to 6.1 A resolution has been collected using synchrotron radiation. This represents a challenging opportunity to study at a molecular level the structure-function relationships between a microtubule-destabilizing protein, SCG10, and tubulin.

Animals↗
PubMed2000

Mechanisms to account for maintenance of the soluble methionine pool in transgenic Arabidopsis plants expressing antisense cystathionine gamma-synthase cDNA.

To investigate the role of cystathionine gamma-synthase (CGS) in the regulation of methionine synthesis Arabidopsis plants were transformed with a full-length antisense CGS cDNA and transformants analysed. Plants that were heterozygous for the transgene showed a 20-fold reduction of CGS activity that was accompanied by severe growth retardation and morphological abnormalities, from germination to flowering. Application of exogenous methionine to the transgenic lines restored normal growth. Surprisingly, transformed Arabidopsis plants exhibited a modest decrease in methionine content (35% reduction of the wild-type level) but a seven-fold decrease in the soluble pool of S-methylmethionine (SMM), a compound that plays a major role in storage and transport of reduced sulphur and labile methyl moieties. Several mechanisms can account for the maintenance of the soluble pool of methionine. First, the observed 20-fold increase in O-phosphohomoserine, a substrate of CGS, could compensate for the depressed level of CGS polypeptide by increasing the net rate of catalysis supported by the remaining enzyme. Second, the transgenic plants exhibited a two-fold increased level of cystathionine beta-lyase, the second enzyme in the methionine biosynthetic pathway. This indicates that enzymes other than CGS are subjected to a regulatory control by methionine or one of its metabolites. In addition to these mechanisms affecting de novo methionine synthesis, the recruitment of SMM to produce methionine may account for the small change of methionine levels in transgenic lines.

Arabidopsis↗
PubMed2000

The third tubulin pool.

Tubulin normally undergoes a cycle of detyrosination/tyrosination on the carboxy terminus of its alpha-subunit and this results in subpopulations of tyrosinated tubulin and detyrosinated tubulin. Brain tubulin preparations also contain a third major tubulin subpopulation which is non-tyrosinatable. This review describes the purification and the structural characterization of non-tyrosinatable tubulin. This tubulin variant lacks a carboxyterminal glutamyl-tyrosine group on its alpha-subunit (delta2-tubulin). Delta2-tubulin is generated from detyrosinated tubulin through an irreversible reaction. Delta2-tubulin accumulates in neurons and in stable microtubule assemblies. It also accumulates in some tumor cells due to the frequent loss of tubulin tyrosine ligase in such cells. Delta2-tubulin may be a useful marker of malignancy in human tumors.

Biomarkers, Tumor↗
PubMed1999

Tubulin binding sites on gamma-tubulin: identification and molecular characterization.

gamma-Tubulin is essential to microtubule organization in eukaryotic cells. It is believed that gamma-tubulin interacts with tubulin to accomplish its cellular functions. However, such an interaction has been difficult to demonstrate and to characterize at the molecular level. gamma-Tubulin is a poorly soluble protein, not amenable to biochemical studies in a purified form as yet. Therefore basic questions concerning the existence and properties of tubulin binding sites on gamma-tubulin have been difficult to address. Here we have performed a systematic search for tubulin binding sites on gamma-tubulin using the SPOT peptide technique. We find a specific interaction of tubulin with six distinct domains on gamma-tubulin. These domains are clustered in the central part of the gamma-tubulin primary amino acid sequence. Synthetic peptides corresponding to the tubulin binding domains of gamma-tubulin bind with nanomolar K(d)s to tubulin dimers. These peptides do not interfere measurably with microtubule assembly in vitro and associate with microtubules along the polymer length. On the tertiary structure, the gamma-tubulin peptides cluster to surface regions on both sides of the molecule. Using SPOT analysis, we also find peptides interacting with gamma-tubulin in both the alpha- and beta-tubulin subunits. The tubulin peptides cluster to surface regions on both sides of the alpha- and beta- subunits. These data establish gamma-tubulin as a tubulin ligand with unique tubulin-binding properties and suggests that gamma-tubulin and tubulin dimers associate through lateral interactions.

Amino Acid Sequence↗
PubMed1999

STOP proteins.

Microtubules assembled from pure tubulin in vitro are labile, rapidly depolymerized upon exposure to the cold. In contrast, in a number of cell types, cytoplasmic microtubules are stable, resistant to prolonged cold exposure. During the past years, the molecular basis of this microtubule stabilization in cells has been elucidated. Cold stability is due to polymer association with different variants of a calmodulin-regulated protein, STOP protein. The dynamic and hence the physiological consequences of STOP association with microtubules vary in different tissues. In neurons, STOP seems almost permanently associated with microtubules. STOP is apparently a major determinant of microtubule turnover in such cells and is required for normal neuronal differentiation. In cycling cells, only minor amounts of STOP are associated with interphase microtubules and STOP does not measurably affects microtubule dynamics. However, STOP is associated with mitotic microtubules in the spindle. Recent results indicate that such an association could be vital for meiosis and for the long-term fidelity of the mitotic process.

Animals↗