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PubMed1994

Microtubular protein in its polymerized or nonpolymerized states differentially modulates in vitro and intracellular fluxes catalyzed by enzymes of carbon metabolism.

The fluxes through HK/G6PDH and PK/LDH coupled-enzymatic reactions were quantified in the presence of physiological concentrations (1-15 microM) of polymerized or non-polymerized microtubular protein (MTP) from rat brain and in a permeabilized yeast cell system. In vitro enzymatic fluxes were increased by either polymerized or nonpolymerized brain MTP mainly in the lower range of MTP concentration. At fixed MTP concentrations in the flux stimulatory range of HK/G6PDH (1 mg/ml MTP) or PK/LDH (0.4 mg/ml MTP), a hyperbolic and sigmoidal response to NADP and PEP, respectively, was detected. That dependence varied according to the polymeric status of MTP. The specificity of the phenomenon observed in vitro, was tested for the PK/LDH and HK/G6PDH enzymatic couples in the presence of neutral polymers such as glycogen (< or = 10 mg/ml), poly(ethylene glycol) (up to 10% w/w) or G-actin (< or = 1 mg/ml). In permeabilized Saccharomyces cerevisiae cells, the PK-catalyzed flux was sensitive to microtubule disruption by nocodazole (15 micrograms/ml). The HK/G6PDH system was not affected by nocodazole showing values of kinetic parameters close to those obtained in vitro in the presence of polymerized brain MTP. Indirect immunofluorescence with specific antibodies against tubulin allowed to confirm the microtubules disruption in the presence of nocodazole in permeabilized yeast cells under the same conditions in which enzymes were assayed intracellularly. The experimental evidence is in agreement with the observed phenomenon of increase in fluxes in the enzymatic reactions assayed to be specifically induced by MTP either in vitro or in situ. The results presented are discussed in terms of the assembly of large supramolecular structures as a supraregulatory mechanism of synchronization of systemic cellular processes such as metabolic fluxes.

Actins↗
PubMed2004

Differential up-regulation of cytosolic and membrane-bound heat shock protein 70 in tumor cells by anti-inflammatory drugs.

PURPOSE: Modulation of the heat shock protein (HSP) response affects sensitivity to therapeutic agents in cancer. Here, drugs with anti-inflammatory potential (cyclooxygenase 1/2 inhibitors) and peroxidase proliferator-activated receptor-gamma agonists were analyzed for their capacity to affect Hsp70 expression in human cancer cells with a divergent Hsp70 membrane expression pattern. EXPERIMENTAL DESIGN: In dose kinetics, the nonlethal concentration of acetyl-salicyl acid, celecoxib, rofecoxib, and the insulin-sensitizer pioglitazone was identified for the human adenocarcinoma cell line CX-. With the exception of CLX, which was diluted in DMSO, all reagents were dissolved in water. After treatment with the different compounds at nontoxic concentrations for 6 h, followed by a 1-h recovery period, the cytosolic Hsp70 levels were measured in CX-2 and CX- tumor cells by Western blot analysis. Fold increase was calculated in relation to the housekeeping protein tubulin. Membrane-bound Hsp70 was analyzed by flow cytometry using a FITC-labeled Hsp70-specific monoclonal antibody. Untreated cells and cells incubated with equivalent amounts of the diluting agents served as controls. The immunological function was tested in granzyme B apoptosis assays, standard (51)Cr release assays, and antibody blocking studies. RESULTS: Compared with aqua dest, the cytoplasmic amount of Hsp70 was equally enhanced in CX-2 and CX- cells by all compounds. An increase in membrane-bound Hsp70, detected selectively in CX- cells, corresponded to an enhanced sensitivity to granzyme B- and natural killer cell-mediated kill that was blockable by using a Hsp70-specific antibody. CONCLUSIONS: Although increase in cytosolic Hsp70 levels conferred resistance to further stress, membrane-bound Hsp70 rendered tumor cells more sensitive to the immunological attack mediated by granzyme B and natural killer cells. Our data provide a biological rational for combining anti-inflammatory drugs with immunotherapy in cancer therapy.

Anti-Inflammatory Agents↗
PubMed2000

Housekeeping genes commanded to commit suicide in CpG-cleavage commitment upstream of Bcl-2 inhibition in caspase-dependent and -independent pathways.

A CpG-specific commitment common to both caspase-dependent and -independent cell deaths implies critical gene activity from epigenetic modulation. Using a focused microarray (genechip) of 22 housekeeping genes, which have canonical CpG islands at 5'-promoter regions, here we show critical regulation of vital intermediary metabolism and cell structure that are common to both caspase-dependent fasL-mediated and caspase-independent etoposide-mediated cell deaths. Gene activity of at least twofold under or over control levels and common to both cell death pathways was considered to be significantly regulated in common. Seven genes controlling energy production in glycolysis, tricarboxylic acid cycle, and the respiratory electron transport chain were significantly downregulated in common. Energy depletion is lethal. Downregulated pyruvate dehydrogenase E1 gene, in addition, suggested primary metabolic acidification. Cell acidification is also lethal. Critical derangement of the cell structure was suggested by common downregulation of the basal histone gene H2A.X which is required for nucleosome assembly. Common upregulation of the alpha-tubulin gene suggested perturbation of vital microtubular dynamics. Gene-commanded cell suicide was suggested. We further show that a Bcl-2 overexpression of three- to fourfold above normal levels could not prevent the CpG-specific megabase DNA cleavages in the two cell death pathways, but abolished their low-molecular-weight 200-bp ladder cleavages. Together with incomplete suppression of the other apoptotic expressions, the Bcl-2 inhibition point appeared downstream from the CpG-cleavage commitment point.

Annexin A5↗
PubMed1981

Changes in axonally transported proteins during axon regeneration in toad retinal ganglion cells.

In an effort to understand the regulation of the transition of a mature neuron to the growth, or regenerating, state we have analyzed the composition of the axonally transported proteins in the retinal ganglion cells of the toad Bufo marinus after inducing axon regeneration by crushing the optic nerve. At increasing intervals after axotomy, we labeled the retinal ganglion cells with [35S]methionine and subsequently analyzed the labeled transported polypeptides in the crushed optic nerve by means of one- and two-dimensional electrophoretic techniques. The most significant conclusion from these experiments is that, while the transition from the mature to the regenerating state does not require a gross qualitative alteration in the composition of axonally transported proteins, the relative labeling of a small subset of rapidly transported proteins is altered dramatically (changes of more than 20-fold) and reproducibly (more than 30 animals) by axotomy. One of these growth-associated proteins (GAPs) was soluble in an aqueous buffer, while three were associated with a crude membrane fraction. The labeling of all three of the membrane-associated GAPs increased during the first 8 d after axotomy, and they continued to be labeled for at least 4 wk. The modulation of these proteins after axotomy is consistent with the possibility that they are involve in growth-specific functions and that the altered expression of a small number of genes is a crucial regulatory event in the transition of a mature neuron to a growth state. In addition to these selective changes in rapidly transported proteins, we observed the following more general metabolic correlates of the regeneration process: The total radioactive label associated with the most rapidly transported proteins (groups I and II) increased three to fourfold during the first 8 d after the nerve was crushed, while the total label associated with more slowly moving proteins (group IV) increased about 10-fold during this same period. Among these more slowly transported polypeptides, five were observed whose labeling increased much more than the average. Three of these five polypeptides resemble actin and alpha- and beta-tubulin in their electrophoretic properties.

Animals↗
PubMed2003

Involvement of protein kinase C in the adaptive changes of cholinergic neurons to sympathetic denervation in the guinea pig myenteric plexus.

Supersensitivity to muscarinic, kappa- and mu-opioid agents modulating cholinergic neurons in the guinea pig colon develops after chronic sympathetic denervation. A possible role for protein kinase C (PKC) in contributing to development of these sensitivity changes was investigated. The PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced acetylcholine (ACh) overflow in preparations obtained from normal animals. The facilitatory effect of PMA was significantly reduced after prolonged exposure to the phorbol ester and by the PKC inhibitors, chelerythrine and calphostin C. Subsensitivity to the facilitatory effect of PMA developed after chronic sympathetic denervation. In this experimental condition, immunoblot analysis revealed reduced levels of PKC in myenteric plexus synaptosomes. The facilitatory effect of the muscarininc antagonist, scopolamine, on ACh overflow was significantly reduced by the phospolipase C (PLC) inhibitor, U73122, chelerythrine and calphostin C, both in normal and denervated animals. However, in both experimental groups, PLC antagonists and PKC antagonists did not affect the inhibitory effect of the muscarinic agonist, oxotremorine-M on ACh overflow. The inhibitory effects of U69593 (kappa-opioid receptor agonist) and DAMGO (mu-opioid receptor agonist) on ACh overflow significantly increased in the presence of U73122, chelerythrine and calphostin C in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals. These results indicate that activation of PKC enhances ACh release in the myenteric plexus of the guinea pig colon. At this level, chronic sympathetic denervation entails a reduced efficiency of the enzyme. In addition, PKC is involved in the inhibitory modulation of ACh release mediated by muscarinic-, kappa- and mu-opioid receptors, although with different modalities. Muscarinic receptors inhibit PKC activity, whereas kappa- and mu-opioid receptors increase PKC activity. Both the inhibitory and the facilitatory effect on PKC involve modulation of PLC activity. The possibility that the change in PKC activity represents one of the biochemical mechanisms at the basis of development of sensitivity changes to opioid and muscarinic agents after chronic sympathetic denervation is discussed.

Acetylcholine↗
PubMed2004

Ascorbic acid responsive genes during neuronal differentiation of embryonic stem cells.

Ascorbic acid has been reported to enhance differentiation of embryonic stem (ES) cells into neurons, however, the specific functions of ascorbic acid have not been defined yet. To address this issue, gene expression profiling was performed using cDNA microarray. Ascorbic acid increased the expressions of genes involved in neurogenesis, maturation, and neurotransmission. Furthermore, statistical analysis using Fisher's exact test revealed ascorbic acid significantly modulated the genes involved in cell adhesion and development category. These results provide information on the role for ascorbic acid during neuronal differentiation of ES cells and might contribute to large-scale generation of neurons for future clinical treatment.

Animals↗
PubMed2003

Light control of nuclear gene mRNA abundance and translation in tobacco.

Photosynthetic signals modulate expression of nuclear genes at the levels of mRNA transcription, mRNA stability, and translation. In transgenic tobacco (Nicotiana tabacum), the pea (Pisum sativum) Ferredoxin 1 (Fed-1) mRNA dissociates from polyribosomes and becomes destabilized when photosynthesis is inhibited by photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We used polymerase chain reaction suppressive-subtractive hybridization to identify similarly regulated endogenous tobacco genes. This screen identified 14 nuclear-encoded tobacco mRNAs whose light-induced increase in abundance is suppressed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Sequence analysis of the cognate cDNAs revealed that nine of the mRNAs encode putative chloroplast-targeted proteins. We asked whether the abundance of these mRNAs was regulated transcriptionally or posttranscriptionally. Of the five mRNAs with sufficient abundance to detect using nuclear run-on assays, we observed transcriptional regulation of alpha-tubulin, thiazole biosynthetic enzyme, and pSKA10 (an unknown gene). Photosystem A subunit L and, to a lesser extent, alpha-tubulin and pSKA10 mRNAs, may also be stabilized in the light. In contrast, Rubisco small subunit mRNA abundance appears to be transcriptionally up-regulated but posttranscriptionally down-regulated in the light. To determine whether, like Fed-1 mRNA, the mRNAs identified in this screen were translationally responsive to light, we characterized the polyribosome association of these mRNAs in the light and after a 15-min dark treatment. A subset of the mRNAs showed dramatic dark-induced polyribosome dissociation, similar to Fed-1 mRNA, and all of the mRNAs showed at least slight polyribosome dissociation. Thus, both posttranscriptional and translational regulation appear to be important mechanisms regulating the expression of many nuclear-encoded mRNAs encoding proteins involved in photosynthesis.

Cell Nucleus↗
PubMed2003

The DCX-domain tandems of doublecortin and doublecortin-like kinase.

The doublecortin-like domains (DCX), which typically occur in tandem, are novel microtubule-binding modules. DCX tandems are found in doublecortin, a 360-residue protein expressed in migrating neurons; the doublecortin-like kinase (DCLK); the product of the RP1 gene that is responsible for a form of inherited blindness; and several other proteins. Mutations in the gene encoding doublecortin cause lissencephaly in males and the 'double-cortex syndrome' in females. We here report a solution structure of the N-terminal DCX domain of human doublecortin and a 1.5 A resolution crystal structure of the equivalent domain from human DCLK. Both show a stable, ubiquitin-like tertiary fold with distinct structural similarities to GTPase-binding domains. We also show that the C-terminal DCX domains of both proteins are only partially folded. In functional assays, the N-terminal DCX domain of doublecortin binds only to assembled microtubules, whereas the C-terminal domain binds to both microtubules and unpolymerized tubulin.

Amino Acid Sequence↗
PubMed1983

Nuclear-envelope nucleoside triphosphatase: stimulation by poly(A) (+)mRNA and modulation by microtubule protein.

Nuclear envelopes contain a nucleoside triphosphatase which is thought to be involved in the supply of energy for nucleo-cytoplasmic RNA transport. This enzyme is stimulated most efficiently by poly(A) and to a lesser extent by poly(G) and poly(dT). Half-maximal stimulation of the enzyme from rat liver nuclei, which was associated with the poly(A)-specific Endoribonuclease IV and was free from poly(A) polymerase and Endoribonuclease V activity, was determined to occur at a concentration of 1.1 X 10(6) poly(A) molecules/nuclear ghost. Double-reciprocal plot analysis revealed a 2.8-fold stimulation of the enzyme by poly(A). Poly(A) in the hybrid form had no influence on the activity of the nucleoside triphosphatase. Stimulation by oligo(A) required a minimal chain length of 18 nucleotide units. Naturally occurring RNA species enhanced the nucleoside triphosphatase activity, provided they contained a poly(A) segment. Using poly(A)(+)mRNA, half-maximal stimulation was determined to proceed at 0.5 X 10(6) molecules/nuclear ghost. Removal of the poly(A) segment from mRNA mRNA abolished the stimulatory effect on the enzyme. Microtubule protein was found to inhibit the nucleoside triphosphatase efficiently. At a concentration of 2.0 mg/ml, polymerized microtubule protein reduced the enzyme activity by 96%. Dimeric tubulin was less inhibitory, while actin was without any significant effect. From these findings it is suggested that a possible nucleoside triphosphatase-mediated transport of poly(A)(+) mRNA through nuclear envelope is controlled firstly, by the poly(A) segment of this RNA species and secondly, by cytoplasmic microtubules.

Animals↗
PubMed2001

Gene expression profiling of exposure to TZT-1027, a novel microtubule-interfering agent, in non-small cell lung cancer PC-14 cells and astrocytes.

Clinical use of TZT-1027, a microtubule-interfering agent that inhibits the polymerization of tubulin, is expected because of its potent effects on solid tumors. TZT-1027 is thought to act directly on cellular microtubules, and arrest cell mitosis, however, the molecular mechanisms of the microtubule damage by TZT-1027 have not been fully identified. To investigate the possible novel mechanisms of action of TZT-1027, we used the cDNA macroarray technique to examine its effect on the expression of hundreds of tightly transcriptionally controlled genes. We used two cell lines, one was human non-small cell lung carcinoma PC-14 cells as a model for cancer cells, and the other was human astrocytes as a model for normal neuronal cells, because the dose-limiting-factor of microtubule-interfering agents is mainly peripheral neurotoxicity. mRNAs prepared from the PC-14 and astrocyte cell lines treated with TZT-1027 were compared with 588 genes spotted onto the filter, and which gene groups TZT-1027 modulated between the two cell lines was investigated. TZT-1027 exposure modulated expression of a variety of genes including the genes encoding cell-cycle and growth regulators, receptors, angiogenesis and invasion regulators, rho family small GTPases and their regulators and growth factors and cytokines. However, the way of gene regulation by TZT-1027 exposure was different between PC-14 cells and astrocytes. Genes up-regulated in both PC-14 cells and astrocytes were those for RAR-epsilon, TNFR 1 and 2 and so on. Specifically altered genes in PC-14 cells, such as the genes coding for cytokeratin 8, XPG, fau and the genes regulated only in PC-14 cells may be involved in the antitumor activity of TZT-1027. On the other hand, growth factor receptor precursors was upregulated specifically in astrocytes by TZT-1027 and this gene regulation only in astrocytes may be candidates related with neurotoxicity.

Antineoplastic Agents↗
PubMed2002

Low intracellular zinc impairs the translocation of activated NF-kappa B to the nuclei in human neuroblastoma IMR-32 cells.

In the current work, we studied how variations in extracellular zinc concentrations modulate different steps involved in nuclear factor kappaB (NF-kappaB) activation in human neuroblastoma IMR-32 cells. Cells were incubated in media containing varying concentrations of zinc (1.5, 5, 15, and 50 microm). Within 3 h, the intracellular zinc content was lower in cells exposed to 1.5 and 5 microm, compared with the other groups. Low intracellular zinc concentrations were associated with the activation of NF-kappaB, based on high levels of IkappaBalpha phosphorylation, low IkappaBalpha concentrations, and high NF-kappaB binding activity in total cell fractions. However, the active dimer accumulated in the cytosol, as shown by a low ratio of nuclear/cytosolic NF-kappaB binding activity. This altered nuclear translocation was accompanied by a decreased transactivation of an endogenous NF-kappaB-driven gene (ikba) and of a reporter gene (pNF-kappaB-luc). In cells with low intracellular zinc concentrations, a low rate of in vitro tubulin polymerization was measured compared with the other groups. We conclude that low intracellular zinc concentrations induce tubulin depolymerization, which may be one signal for NF-kappaB activation. However, NF-kappaB nuclear translocation is impaired, which inhibits the transactivation of NF-kappaB-driven genes. This could affect cell survival, and be an important factor in certain zinc deficiency-associated pathologies.

Active Transport, Cell Nucleus↗
PubMed1996

Modulation of the hepatic alpha 1-adrenoceptor responsiveness by colchicine: dissociation of free cytosolic Ca(2+)-dependent and independent responses.

1. The cytoskeletal depolymerizing agent, colchicine, prevents the hepatic alpha 1-adrenoceptor-mediated stimulation of respiration, H+ and Ca2+ release to the effluent perfusate, intracellular alkalosis, and glycogenolysis. Unlike the other parameters, colchicine does not perturb the alpha 1-agonist-induced stimulation of gluconeogenesis or phosphorylase 'a' activation, and enhances the increase in portal pressure response. The lack of effect of colchicine on the hepatic alpha 2-adrenoceptor-mediated effects indicates that its actions are alpha 1-specific. 2. Colchicine enhances the acute alpha 1-adrenoceptor-mediated intracellular Ca2+ mobilization and prevents the activation of protein kinase C. This differential effect on the two branches of the alpha 1-adrenoceptor signalling pathway is a distinctive feature of the colchicine action. 3. The lack of effect of colchicine in altering the alpha 1-adrenoceptor ligand binding affinity suggests that it might interact with some receptor-coupled regulatory element(s). 4. The acuteness of the colchicine effect and the ability of its isomer beta-lumicolchicine to prevent all the alpha 1-adrenoceptor-mediated responses but the increase in vascular resistance, indicate that its action cannot be merely ascribed to its effects in depolymerizing tubulin. 5. Colchicine perturbs the hepatic responses to vasoactive peptides. It enhances the vasopressin-induced rise of cytosolic free Ca2+ in isolated hepatocytes and prevents the sustained decrease of Ca2+ in the effluent perfusate. It also inhibits the stimulation of glycogenolysis, without altering the stimulation of gluconeogenesis. 6. It is concluded that there are at least two major alpha 1-adrenoceptor signalling pathways. One is colchicine-sensitive, independent of variations in free cytosolic Ca2+, and protein kinase C-dependent; the other one is colchicine-insensitive, dependent on variations in free cytosolic Ca2+, and protein kinase C-independent.

Animals↗
PubMed1996

Modulation of vinblastine cytotoxicity by dilantin (phenytoin) or the protein phosphatase inhibitor okadaic acid involves the potentiation of anti-mitotic effects and induction of apoptosis in human tumour cells.

Cellular insensitivity to vinca alkaloids is suggested to be primarily due to drug efflux by P-glycoprotein (P-gp). The anti-epileptic phenytoin (DPH), which does not bind to P-gp, can selectively enhance vincristine (VCR) cytotoxicity in wild-type (WT) or multidrug-resistant (MDR) cells. We now demonstrate that the protein phosphatase inhibitor okadaic acid (OKA) can mimic the effect of DPH by selectively enhancing cytotoxicity of vinblastine (VBL), but not taxol and doxorubicin, in human leukaemia HL-60 cells. Both DPH and OKA potentiate the anti-mitotic effects of VBL by enhanced damage to the mitotic spindle, resulting in prolonged growth arrest. Also, unlike VBL alone, in human leukaemia or non-small-cell lung carcinoma cells treated with VBL plus DPH, recovery from damage to the mitotic spindle is compromised in drug-free medium and cell death by apoptosis in interphase ensues. Since protein phosphatases are involved with the regulation of metaphase to anaphase transit of cells during the mitotic cycle, enhanced VBL cytotoxicity in the presence of DPH or OKA may involve effects during metaphase on the mitotic spindle tubulin leading to growth arrest and apoptosis in interphase. These novel results suggest that DPH or OKA could be powerful tools to study cellular effects of vinca alkaloids and possibly for the development of novel therapeutic strategies.

Anticonvulsants↗
PubMed1995

Human macrophages contain a stretch-sensitive potassium channel that is activated by adherence and cytokines.

A variety of stimuli, including cytokines and adhesion to surfaces and matrix proteins, can regulate macrophage function, in part through changes in Ca(2+)- dependent second messengers. While fluctuation in intracellular Ca2+ is an important modulator of cellular activation, little attention has been paid to the roles of other ions whose cytoplasmic concentrations can be rapidly regulated by ion channels. To examine the role of ion channels in macrophage function, we undertook patch clamp studies of human culture-derived macrophages grown under serum-free conditions. The major ionic current in these cells was carried by an outwardly rectifying K+ channel, which had a single-channel conductance of 229 pS in symmetrical K(+)-rich solution and macroscopic whole-cell conductance of 9.8 nS. These channels opened infrequently in resting cells but were activated immediately by (i) adhesion of mobile cells onto a substrate, (ii) stretch applied to isolated membrane patches in Ca(2+)-free buffers, (iii) intracellular Ca2+ (EC50 of 0.4 microM), and (iv) the cytokine IL-2. Furthermore, barium and 4-aminopyridine, blockers of this channel, altered the organization and structure of the cytoskeletal proteins actin, tubulin and vimentin. These cytoskeletal changes were associated with reversible alteration to the morphology of the cells. Thus, we have identified an outwardly rectifying K+ channel that appeared to be involved in cytokine and adherence-mediated macrophage activation, and in the maintenance of cytoskeletal integrity and cell shape.

Cell Adhesion↗
PubMed2003

TAC-1 and ZYG-9 form a complex that promotes microtubule assembly in C. elegans embryos.

BACKGROUND: Modulation of microtubule dynamics is crucial for proper cell division. While a large body of work has made important contributions to our understanding of the mechanisms governing microtubule dynamics in vitro, much remains to be learned about how these mechanisms operate in vivo. RESULTS: We identified TAC-1 as the sole TACC (Transforming Acidic Coiled Coil) protein in C. elegans. TAC-1 consists essentially of a TACC domain, in contrast to the much larger members of this protein family in other species. We find that tac-1 is essential for pronuclear migration and spindle elongation in one-cell-stage C. elegans embryos. Using an in vivo FRAP-based assay, we establish that inactivation of tac-1 results in defective microtubule assembly. TAC-1 is present in the cytoplasm and is enriched at centrosomes in a cell cycle-dependent manner. Centrosomal localization is independent of microtubules but requires the activity of gamma-tubulin and the Aurora-A kinase AIR-1. By conducting FRAP analysis in embryos expressing GFP-TAC-1, we find that centrosomal TAC-1 exchanges rapidly with the cytoplasmic pool. Importantly, we establish that TAC-1 physically interacts with ZYG-9, a microtubule-associated protein (MAP) of the XMAP215 family, both in vitro and in vivo. Furthermore, we also uncover that TAC-1 and ZYG-9 stabilize each other in C. elegans embryos. CONCLUSIONS: Our findings identify TAC-1 as a core structural and functional member of the evolutionarily conserved TACC family of proteins and suggest that mutual stabilization between TACC and XMAP215 proteins is a key feature ensuring microtubule assembly in vivo.

Animals↗
PubMed2001

mDll1 and mDll3 expression in the developing mouse brain: role in the establishment of the early cortex.

The Delta/Notch signalling system is involved in several developmental processes. During fly neurogenesis, Delta expression defines the fate of neuronal precursors and inhibits neighboring Notch-expressing cells from acquiring a neural fate, a process known as lateral inhibition. In vertebrates, recent evidence demonstrates that Notch activation can positively determine cell fate and affect neuronal process extension. Nevertheless, Delta-like expression patterns during brain development are relatively unknown. Using a transgenic mouse, which expresses LacZ under the mDll1 promoter, we show by immunofluorescence that in the developing telencephalon mDll1 is expressed in undifferentiated cells in close contact with radial glial cells. Based on in situ hybridization data on mDll1 and mDll3 mRNA expression and on the immunohistochemical detection of beta-galactosidase in the Dll1-lacZ transgenic mouse, we suggest that mDll1 and mDll3 are involved in the establishment of the early cortical plate and that mDll1-expressing cells are in close contact with radial glial cells, thereby modulating the latter population, which is known to express Notch1. Furthermore, we suggest that the decrease in mDll1 mRNA found toward the end of gestation could be related, first, to the slowing of neurogenesis and, second, to the differentiation of the radial glial cell population into astrocytes.

Animals↗
PubMed1994

Focal ischemia causes an extensive induction of immediate early genes that are sensitive to MK-801.

BACKGROUND AND PURPOSE: There is strong evidence to implicate glutamate in the cerebral damage caused by ischemia. In this study we investigated the role of glutamate receptors in mediating effects of middle cerebral artery occlusion (MCAO) on immediate early gene expression in the rat by quantitation of mRNA levels. METHODS: The effect of MCAO on the induction of immediate early genes was studied in five regions, both ipsilateral and contralateral to the occlusion: the "core" ischemic area of the cortex in the central region of the middle cerebral artery territory, the surrounding area, frontal cortex, occipital cortex, and hippocampus. Levels of c-fos, c-jun, zif-268, and krox-20 mRNA were measured by Northern and slot blot analysis. RESULTS: A large induction of c-fos mRNA was obtained in all four cortical regions ipsilateral to the occlusion, with the greatest effect detected in the core area. Little effect was detected in the ipsilateral hippocampus and in all contralateral regions. Pretreatment with MK-801 (3 mg/kg) largely inhibited the induction of c-fos mRNA, indicating that the induction was mediated through an N-methyl-D-aspartate subtype of glutamate receptor. MCAO also produced a significant induction of c-jun and zif-268 mRNA in ipsilateral cortical regions. CONCLUSIONS: These results indicate that MCAO causes a profound modulation of the expression of multiple genes in an extensive area of cerebral cortex extending beyond the immediate area supplied by the middle cerebral artery. The marked effect of MK-801 indicates the potential importance of glutamate antagonists in restricting the widespread deleterious effects of glutamate.

Animals↗
PubMed2004

A model of organotypic rat spinal slice culture and biolistic transfection to elucidate factors that drive the preprotachykinin-A promoter.

The tachykinin substance P (SP) is a neuropeptide that is expressed in some nociceptive primary sensory afferents and in discrete populations of spinal cord neurons. Expression of spinal SP and the preprotachykinin-A (PPT-A) gene that encodes SP exhibits plasticity in response to conditions such as peripheral inflammation but the mechanisms that regulate expression are poorly understood. We have developed a spinal cord organotypic culture system that is suitable for the analysis of PPT-A gene promoter activity following biolistic transfection of recombinant DNA constructs. Spinal cord organotypic slices showed good viability over a 7-day culture period. Immunostaining for phenotypic markers such as NeuN and beta-III tubulin demonstrated preservation of neurons and their structure, although there was evidence of axotomy-induced down-regulation of NeuN in certain neuronal populations. Neurokinin-1 receptor (NK-1R) immunostaining in laminae I and III was similar to that seen in acute slices. Biolistic transfection was used to introduce DNA constructs into neurons of these organotypic cultures. Following transfection with a construct in which expression of enhanced green fluorescent protein (EGFP) is controlled by the PPT-A promoter, we showed that induction of neuronal activity by administration of a forskolin analogue/high K(+) (10 microM/10 mM) for 24 h resulted in a fourfold increase in the number of EGFP-positive cells. Similarly, a twofold increase was obtained after treatment with the NK-1R-specific agonist [Sar(9),Met (O(2))(11)]-substance P (10 microM). These data demonstrate the usefulness of this model to study physiological and pharmacological factors relevant to nociceptive processing that can modulate PPT-A promoter activity.

Animals↗