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M Costa

Publications and source records attributed to M Costa.

At least 145 records · Page 8Linked to original sources
PubMed1999

In vivo localisation of the mitotic POLO kinase shows a highly dynamic association with the mitotic apparatus during early embryogenesis in Drosophila.

The gene polo encodes a highly conserved serine/threonine protein kinase that has been implicated in several functions during cell division. Polo-like kinases are important positive regulators of cell cycle progression and have also been implicated in the exit from mitosis through the activation of the anaphase-promoting complex. Several data indicate that Plks are required for centrosome function, bipolar spindle organisation and cytokinesis. The intracellular localisation of Plks reflects their multiple roles in cell division, however, in vivo studies that describe the distribution of this protein during different stages of mitosis have never been performed. In the present work, we report the in vivo distribution of a GFP-POLO fusion protein expressed in stable transformants and analysed during the early embryonic development of Drosophila melanogaster. The GFP-POLO protein can be detected in unfertilised oocytes associated with the centromeric region of chromosomes of the polar body and followed until the formation of mitotic domains in later development. Detailed analysis of the dynamic localisation of GFP-POLO during syncytial mitotic cycles shows the timing of localisation to the centrosomes, centromeres and midbody. The results also indicate that GFP-POLO is present in astral microtubules early in mitosis, accumulates around the nuclear envelope until nuclear envelop breakdown and at metaphase associates to spindle microtubules. These in vivo studies show a highly dynamic association of POLO with multiple compartments of the mitotic apparatus. Furthermore, the wide distribution of the GFP-POLO protein to all compartments of the mitotic apparatus provides a valuable tool for future studies on cell cycle during development.

Animals↗
PubMed1998

Transcriptional regulation of the tissue-type plasminogen-activator gene in human endothelial cells: identification of nuclear factors that recognise functional elements in the tissue-type plasminogen-activator gene promoter.

The gene encoding human tissue-type plasminogen activator (t-PA) is regulated in a cell-type-specific manner. Previous studies in non-endothelial cells have indicated that basal and phorbol ester mediated induction is controlled by a cAMP response element (CRE) referred to as the tPACRE, and an activating protein 2 (AP-2)-like site. The classification of the AP-2-like site was assigned on the basis of its sequence homology, but has been shown in some cell systems to be recognised by promoter-specific transcription factor-1 (Sp-1). Here, we have investigated the transcriptional regulation of the t-PA gene in endothelial cells and addressed the functional roles of the tPACRE and the Sp-1/AP-2-like sites. 5'-RACE experiments indicate that the t-PA gene uses two transcription initiation sites in these cells with the downstream site being preferred. Functional analyses of the t-PA promoter using reporter-gene constructs transfected into C11STH endothelial cells demonstrate that the first 410 bp of the t-PA promoter confers an increase in reporter-gene activity on treatment with 4beta-phorbol 12-myristate 13-acetate (PMA). Mutagenesis of either the tPACRE or the Sp-1/AP-2 site weakens both basal and inducible expression, while disruption of both sites renders the promoter completely unresponsive. Using supershift assays, we identify the predominant tPACRE-binding proteins in nuclear extracts prepared from both C11STH cells and primary umbilical vein endothelial cells (HUVECs) as activating transcription factor 2, CREB (cAMP-responsive-element-binding protein), CREM (cAMP response element modulator) and c-jun. Treatment of cells with PMA results in a selective recruitment of jun-D to the tPACRE, while Sp-1 was identified as the major transcription factor that recognises the AP-2-like site. Based on this data and previous reports, we have reassigned this as a Sp-1-binding site. Finally, the identification of specific endothelial-derived t-PACRE-binding proteins suggests an integral role for these factors in the regulation of t-PA gene expression in human endothelial cells.

Base Sequence↗
PubMed1998

Projections of submucous neurons to the myenteric plexus in the guinea pig small intestine.

The distribution of submucous neurons that project to the myenteric plexus of the guinea pig small intestine was established by retrograde transport of the carbocyanine dye 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) from myenteric ganglia in organ culture in combination with immunohistochemistry. Following the application of DiI to the serosal surface of a single myenteric ganglion, from 2 to 15 DiI-labelled nerve cell bodies were labelled in the submucous plexus up to 7.9 mm circumferentially, 4.5 mm orally, and 3.4 mm aborally to the DiI application site. No cells were labelled in preparations in which connections between myenteric and submucous plexuses had been severed prior to DiI application. Cells that were immunoreactive for vasoactive intestinal polypeptide (VIP) or for substance P (SP) accounted for about 75% and 11% of DiI-labelled cells, respectively. Neither neuropeptide Y- nor calretinin-immunoreactive submucous neurons were labelled by DiI, indicating that these classes of neurons do not project to the myenteric plexus. Retrograde tracing from the myenteric plexus with Neurobiotin revealed that labelled VIP-immunoreactive neurons had several short, filamentous processes and a single long axon that could be followed through the circular muscle to myenteric ganglia without branches to the mucosa. The previously described projection of submucous, SP-immunoreactive putative sensory neurons to the myenteric plexus was confirmed. However, this study has identified a considerably larger population of presumed interneurons that are immunoreactive for VIP that likely transmit information from the submucous plexus to the myenteric plexus and presumably coordinate activity between the two ganglionated plexuses.

Animals↗
PubMed1998

Effects of nickel on DNA methyltransferase activity and genomic DNA methylation levels.

Methylation of DNA plays an important role in organizing the genome into transcriptionally active and inactive zones. Nickel compounds cause chromatin condensation and DNA methylation in the transgenic gpt+ Chinese hamster cell line (G12). Here we show that nickel is an inhibitor of cytosine 5-methyltransferase activity in vivo and in vitro. In living cells, this inhibition is transient and following a recovery period after nickel treatment, Mtase activity slightly rebounds. Genomic DNA methylation levels are also somewhat decreased following nickel treatment, but with time, there is an elevation of total DNA methylation above basal levels and before any rebound of methyltransferase activity. These results suggest that nickel exposure can elevate total genomic DNA methylation levels even when DNA methyltransferase activity is depressed. These findings may explain the hypermethylation of senescence and tumor suppressor genes found during nickel carcinogenesis and support the model of a direct effect of Ni2+ on chromatin leading to de novo DNA methylation.

Aging↗
PubMed1998

Cap43, a novel gene specifically induced by Ni2+ compounds.

To better understand the molecular mechanism(s) involved in the essentiality, toxicity, and/or carcinogenicity of nickel compounds, a mRNA differential display technique was used to identify gene(s) that were specifically induced by these carcinogens. Differential expression of several genes was observed in human lung A549 cells exposed to nickel subsulfide. One gene, Cap43, which expressed a 3.0-kb mRNA encoding a Mr 43,000 protein, was found to be induced within 4-6 h by either Ni3S2 or NiCl2 in A549 cells and attained a level as high as 30-fold within 24-36 h of treatment. Twelve other tested metal compounds failed to induce Cap43 expression, leading to the conclusion that, with regard to metals, the induction of this gene was nickel-specific. Oxidative stress that is often caused by metals and heat shock did not induce Cap43 further, suggesting a specific nature in the signaling pathway involved in Cap43 induction. Activation of signaling pathways with vanadate did not induce Cap43 nor did trifluoperazine block its induction by nickel; however, okadaic acid, a serine/threonine phosphatase inhibitor, induced Cap43 to a greater extent than any nickel compound tested. Homocysteine did not induce Cap43 in a number of cell lines, with the exception of human endothelial cells. The Cap43 gene was found to be induced by nickel not only in all tested human and rodent cell lines in vitro but also in several rat organs after oral exposure to NiCl2. We have found that the primary signal for Cap43 induction was an elevation of free intracellular Ca2+ caused by Ni2+ exposure because Cap43 was induced by calcium ionophores and its induction was attenuated by bis-(O-aminophenyl)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)-ester, a chelator of intracellular Ca2+. We found that the Cap43 gene was evolutionarily conserved and similarly regulated in humans, mice, and rats. Recent studies have shown that Cap43 is expressed at lower levels in colon cancer. Further studies of Cap43 regulation by Ca2+ should enhance our understanding of the role of Cap43 in cell function and cancer pathogenesis.

Amino Acid Sequence↗
PubMed1998

Cr(III)-mediated crosslinks of glutathione or amino acids to the DNA phosphate backbone are mutagenic in human cells.

Carcinogenic Cr(VI) compounds were previously found to induce amino acid/glutathione-Cr(III)-DNA crosslinks with the site of adduction on the phosphate backbone. Utilizing the pSP189 shuttle vector plasmid we found that these ternary DNA adducts were mutagenic in human fibroblasts. The Cr(III)-glutathione adduct was the most potent in this assay, followed by Cr(III)-His and Cr(III)-Cys adducts. Binary Cr(III)-DNA complexes were only weakly mutagenic, inducing a significant response only at a 10 times higher number of adducts compared with Cr(III)-glutathione. Single base substitutions at the G:C base pairs were the predominant type of mutations for all Cr(III) adducts. Cr(III), Cr(III)-Cys and Cr(III)-His adducts induced G:C-->A:T transitions and G:C-->T:A transversions with almost equal frequency, whereas the Cr(III)-glutathione mutational spectrum was dominated by G:C-->T:A transversions. Adduct-induced mutations were targeted toward G:C base pairs with either A or G in the 3' position to the mutated G, while spontaneous mutations occurred mostly at G:C base pairs with a 3' A. No correlation was found between the sites of DNA adduction and positions of base substitution, as adducts were formed randomly on DNA with no base specificity. The observed mutagenicity of Cr(III)-induced phosphotriesters demonstrates the importance of a Cr(III)-dependent pathway in Cr(VI) carcinogenicity.

Adenine↗
PubMed1998

A putative catenin-cadherin system mediates morphogenesis of the Caenorhabditis elegans embryo.

During morphogenesis of the Caenorhabditis elegans embryo, hypodermal (or epidermal) cells migrate to enclose the embryo in an epithelium and, subsequently, change shape coordinately to elongate the body (Priess, J.R., and D.I. Hirsh. 1986. Dev. Biol. 117:156- 173; Williams-Masson, E.M., A.N. Malik, and J. Hardin. 1997. Development [Camb.]. 124:2889-2901). We have isolated mutants defective in morphogenesis that identify three genes required for both cell migration during body enclosure and cell shape change during body elongation. Analyses of hmp-1, hmp-2, and hmr-1 mutants suggest that products of these genes anchor contractile actin filament bundles at the adherens junctions between hypodermal cells and, thereby, transmit the force of bundle contraction into cell shape change. The protein products of all three genes localize to hypodermal adherens junctions in embryos. The sequences of the predicted HMP-1, HMP-2, and HMR-1 proteins are related to the cell adhesion proteins alpha-catenin, beta-catenin/Armadillo, and classical cadherin, respectively. This putative catenin-cadherin system is not essential for general cell adhesion in the C. elegans embryo, but rather mediates specific aspects of morphogenetic cell shape change and cytoskeletal organization.

Animals↗
PubMed1998

Endotoxin contamination may be responsible for the unexplained failure of human pancreatic islet transplantation.

Clinical transplantation of human islets has a disappointingly low rate of success. We report here the identification of a possible causative factor: endotoxin present in the collagenase preparations used to disperse the pancreatic tissue before islet purification and transplantation. Supporting evidence includes (1) detection of unexpectedly high levels of endotoxin in most collagenase solutions currently used to digest human pancreases; (2) demonstration that supernatants generated during islet separation are able to induce the inflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha) in macrophages; and (3) induction of IL-1, IL-6, and TNF-alpha in the islets during the separation procedure. Cytokine expression was assessed by reverse transcription-polymerase chain reaction and, for TNF-alpha, confirmed by enzyme-linked immunoabsorbent assay. It is proposed that endotoxin and locally induced cytokines carried over with the graft activate the endothelium and promote lymphomonocytic infiltration of grafted islets and surrounding liver tissue favoring primary nonfunction and early rejection. These results also have implications for the numerous experimental procedures that use collagenase, and they point to possible ways to improve islet preparation and transplantation protocols.

Adolescent↗
PubMed1998

Abnormalities of nerve fibers in the circular muscle of patients with slow transit constipation.

Abnormalities of the enteric nervous system are thought to explain the pathophysiology of motility disorders. Our aim was to determine if particular classes of enteric neurons are affected in slow transit constipation (STC). Specimens were taken from the terminal ileum and ascending, transverse and descending colon of patients undergoing subtotal colectomy for STC. Immunohistochemistry was performed using antisera to neuron-specific enolase, tachykinin, leu-enkephalin, choline acetyltransferase, vasoactive intestinal peptide, nitric oxide synthase, tyrosine hydroxylase and neuropeptide Y. The density of nerve fibres labelled with these antibodies in each layer was compared with age-matched controls. The density of nerve fibres with tachykinin and enkephalin immunoreactivity was reduced in the colonic circular muscle of the 15 patients with STC, whereas innervation of all other layers was normal. This reduction of tachykinin-immunoreactive nerve fibres also occurred in nine of the 12 specimens of terminal ileum examined. No difference was detected in the density or distribution of nerve fibres using the other antisera. Excitatory nerve fibres are present in the circular muscle in STC but they are deficient in tachykinins and enkephalin.

Adult↗
PubMed1998

The projections of 5-hydroxytryptamine-accumulating neurones in the myenteric plexus of the small intestine of the guinea-pig.

Retrograde tracing, combined with immunohistochemistry, was used to study the projections of 5-hydroxytryptamine (5-HT)-accumulating neurones within the ileum of the guinea-pig, with confocal microscopy being used to characterise further their morphology. Two classes of neurones in the myenteric plexus, capable of taking up 5-HT or analogues, were distinguished. One class had Dogiel type I morphology with lamellar dendrites, was located on the edge or in the middle of ganglia and lacked immunoreactivity for somatostatin (SOM). The other class had smooth ovoid cell bodies with multiple filamentous dendrites and a single axon and represented a subset of the SOM-immunoreactive interneurones in the myenteric plexus. Varicosities immunoreactive for 5-HT alone, 5-HT/SOM or SOM alone were present in the myenteric ganglia. Both classes of 5-HT-accumulating neurones had long aboral projections within the myenteric plexus (up to 100 mm long) and to the submucous plexus and probably function as descending interneurones.

Animals↗
PubMed1998

Neuronal pathways and transmission to the lower esophageal sphincter of the guinea Pig.

BACKGROUND & AIMS: The lower esophageal sphincter (LES) normally controls the opening and closing of the gastroesophageal junction to resist gastric reflux but allow swallowing. Neuronal pathways controlling the guinea pig LES were investigated anatomically and physiologically in isolated preparations. METHODS: Intracellular recording from the LES with focal electrical stimulation and retrograde and anterograde neuronal tracing were used. RESULTS: Electrical stimulation on the LES evoked inhibitory junction potentials (IJPs), which were reduced by 60% by 100 micromol/L N-nitro-L-arginine and subsequently blocked by 0.5 micromol/L apamin, unmasking excitatory junction potentials, which were abolished by 1 micromol/L hyoscine. Esophageal or vagal stimulation evoked IJPs, which were blocked by 100 micromol/L hexamethonium. Focal stimulation of the upper stomach evoked IJPs at 5-8 of 20 stimulation sites, which were abolished by cutting between the stimulation site and sphincter. Application of 1,1'-didodecyl-3,3,3', 3'-tetramethyl indocarbocyanine perchlorate (DiI) to the gastric sling muscle anterogradely labeled many motor axons in the sling muscle but few in the LES, confirming that the two muscles are separately innervated. DiI on the esophagus labeled nerve fibers, but not cell bodies, in the upper stomach. CONCLUSIONS: The inhibitory motor neurons of the LES receive inputs from the vagus nerve, esophagus, and upper stomach.

Animals↗
PubMed1998

Differential chemical probing of a group II self-splicing intron identifies bases involved in tertiary interactions and supports an alternative secondary structure model of domain V.

Dimethyl sulfate modification was used to probe for tertiary structural elements in the group II intron PI.LSU/2 from the mitochondrial pre-ribosomal RNA of the brown alga Pylaiella littoralis. Modification of the lariat form of the intron under conditions that allow both native folding and conformational homogeneity is found to be generally consistent with secondary and tertiary structural features identified previously for group II ribozymes. A comparison of chemical probing at temperatures just below and above the first melting transition illustrates the cooperative unfolding of tertiary structure and identifies novel candidates for tertiary interactions in addition to defining elements of secondary structure. Substitution of the GAAA terminal loop of domain V is shown to be compatible with retention of conformational homogeneity (despite the loss of an important tertiary interaction), but produces a concise methylation footprint in domain I at the site previously shown to harbor the receptor for that loop. The analysis also identified two nucleotide positions in domain V with novel secondary and potential tertiary structural roles. The proposed refinement of domain V secondary structure is supported by an expanded comparative analysis of group II sequences and bears increased resemblance to U2:U6 snRNA pairing in the spliceosome.

Alkylating Agents↗
PubMed1998

Hyperactivation of the folded gastrulation pathway induces specific cell shape changes.

During Drosophila gastrulation, mesodermal precursors are brought into the interior of the embryo by formation of the ventral furrow. The first steps of ventral furrow formation involve a flattening of the apical surface of the presumptive mesodermal cells and a constriction of their apical diameters. In embryos mutant for folded gastrulation (fog), these cell shape changes occur but the timing and synchrony of the constrictions are abnormal. A similar phenotype is seen in a maternal effect mutant, concertina (cta). fog encodes a putative secreted protein whereas cta encodes an (alpha)-subunit of a heterotrimeric G protein. We have proposed that localized expression of the fog signaling protein induces apical constriction by interacting with a receptor whose downstream cellular effects are mediated by the cta G(alpha)protein. In order to test this model, we have ectopically expressed fog at the blastoderm stage using an inducible promoter. In addition, we have examined the constitutive activation of cta protein by blocking GTP hydrolysis using both in vitro synthesized mutant alleles and cholera toxin treatment. Activation of the fog/cta pathway by any of these procedures results in ectopic cell shape changes in the gastrula. Uniform fog expression rescues the gastrulation defects of fog null embryos but not cta mutant embryos, arguing that cta functions downstream of fog expression. The normal location of the ventral furrow in embryos with uniformly expressed fog suggests the existence of a fog-independent pathway determining mesoderm-specific cell behaviors and invagination. Epistasis experiments indicate that this pathway requires snail but not twist expression.

Animals↗
PubMed1998

Utilization of DNA-protein cross-links as a biomarker of chromium exposure.

Human exposure to carcinogenic Cr(VI) compounds is found among workers in a large number of professional groups, and it can also occur through environmental pollution. A significant number of toxic waste sites contain Cr as a major contaminant. In this paper we summarize our efforts to apply measurements of DNA-protein cross-links (DPC) as test for biologically active doses of Cr(VI). DPC were found at elevated levels in lymphocytes in several human populations with low to medium Cr exposures. At high exposure to Cr(VI), exemplified by a group of Bulgarian chromeplaters, DPC plateaued and adducts' levels were similar to those found in environmentally exposed individuals. Lymphocytic DPC correlated strongly with Cr levels in erythrocytes that are indicative of Cr(VI) exposure. DPC in lymphocytes were not confounded by such variables as smoking, age, body weight, gender, or ethnicity. A new version of the cross-link assay offers improved sensitivity and requires a small amount of biologic material. Preliminary results indicate that the ability of DPC to reach detectable levels at low levels of Cr exposure could be related to a lack of repair of these lesions in lymphoid cells. Cr(III)-mediated cross-links of DNA with peptide glutathione or single amino acids were mutagenic in human cells, with a relationship of higher molecular weight of the peptide/amino acid correlating with a more potent mutagenic response. We speculate that bulky DPC could also have a significant promutagenic effect. The current methodology does not allow specific determination of Cr-induced DPC; however, demonstrated sensitivity of DPC measurements and the assay's large sample capacity may allow this assay to be used as the initial screening test for the occurrence of DNA damage in Cr(VI)-exposed populations.

Biomarkers↗
PubMed1998

Muscarinic drugs affect cholinesterase activity and development of eye structures during early chick development.

During neurogenesis, markers of the cholinergic system are present in the eye and visual cortex of vertebrates. In adult vertebrates, a role for these molecules, including muscarinic acetylcholine receptors (mAChRs), in eye growth non-accommodative regulation is also known. In order to understand the biological mechanisms triggered by the cholinergic system in these events, we analysed the effects of a cholinergic agonist (10(-4) M carbachol) and an antagonist (10(-4) M atropine) of the muscarinic receptors, on early chick development. To establish if the cholinergic system also plays a role in the regulation of early neurogenetic signals, the drug treatments were made at stage 5-6 HH, during the formation of the cephalic process. Specific effects on forehead, and in particular on eye development were found; carbachol treated embryos presented huge and well pigmented eyes, significantly different from controls. The eyes of atropine-exposed embryos presented anomalies with different phenotypes ranging from strongly affected features to normal-like appearance. Generally, the eyes were smaller as compared to the controls, with a number of anomalies, also in the normal-like phenotype, including retina and lens defects. In these structures, distribution of cholinesterase activities was checked by histochemical methods, and the amount of cells undergoing nuclear disgregation was revealed by DAPI staining. We propose that the drugs affected the known nervous and pre-nervous functions of the cholinergic markers, such as cell signalling during primary induction, and regulation of cell death by ACh receptors.

Acetylcholinesterase↗