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Biomedical subjects

M Costa

Publications and source records attributed to M Costa.

At least 235 records · Page 13Linked to original sources
PubMed1995

Heterochromatic proteins specifically enhance nickel-induced 8-oxo-dG formation.

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) was measured as an indicator of nickel-induced oxidative base damage in the presence of H2O2. Heterochromatic proteins isolated from Chinese hamster liver cells enhanced the formation of 8-oxo-dG induced by NiCl2 and H2O2 in vitro, whereas euchromatic proteins inhibited this reaction. The inhibitory effect of euchromatic proteins on dG oxidation may be due to the oxygen radical scavenging effects of low molecular weight protein-rich fractions. Gel electrophoresis confirmed that histone H1 was present at a higher concentration in heterochromatin than in euchromatin. It is believed that the presence of nickel-protein complexes in cells is crucial for the formation of reactive oxygen species (ROS). We found that Ni2+ binds to histone H1 and core histones as determined by 63Ni autoradiography of proteins on nitrocellulose membranes. In vitro studies showed that commercially purified histone H1, and to a considerably lesser extent core histones, enhanced the NiCl2 and H2O2 catalyzed formation of 8-oxo-dG in a reaction containing free dG base. Since histone H1 is a lysine- and alanine-rich protein, the levels of 8-oxo-dG induced by NiCl2 and H2O2 were studied in the presence of these amino acids and found to be enhanced by them. These results suggest that nickel may specifically produce oxidative DNA damage in heterochromatin because of the nature of its binding to histone H1 and core histones. This selective oxidation of genetically inactive heterochromatin may explain why nickel compounds which generate oxygen radicals and oxidize DNA bases are inactive in most gene mutation assays.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1995

The receptor tyrosine kinase ARK mediates cell aggregation by homophilic binding.

The ARK (AXL, UFO) receptor is a member of a new family of receptor tyrosine kinases whose extracellular domain contains a combination of fibronectin type III and immunoglobulin motifs similar to those found in many cell adhesion molecules. ARK mRNA is expressed at high levels in the mouse brain, prevalently in the hippocampus and cerebellum, and this pattern of expression resembles that of adhesion molecules that are capable of promoting cell aggregation through homophilic or heterophilic binding. We report here the ability of the murine ARK receptor to mediate homophilic binding. Expression of the ARK protein in Drosophila S2 cells induces formation of cell aggregates consisting of ARK-expressing cells, and aggregation leads to receptor activation, with an increase in receptor phosphorylation. Homophilic binding does not require ARK tyrosine kinase activity, since S2 cells expressing a receptor in which the intracellular domain was deleted were able to undergo aggregation as well as cells expressing the wild-type ARK receptor. Similar results were obtained with NIH 3T3 and CHO cells expressing high levels of ARK, although in this case ARK expression appeared to be accompanied by constitutive activation. The purified recombinant extracellular domain of ARK can induce homotypic aggregation of coated fluorescent beads (Covaspheres), and this protein can also function as a substrate for adhesion by S2 and NIH 3T3 cells expressing ARK. These results suggest that ARK represents a new cell adhesion molecule that through its homophilic interaction may regulate cellular functions during cell recognition.

3T3 Cells↗
PubMed1995

Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: a new model for epigenetic carcinogens.

A transgenic gpt+ Chinese hamster cell line (G12) was found to be susceptible to carcinogenic nickel-induced inactivation of gpt expression without mutagenesis or deletion of the transgene. Many nickel-induced 6-thioguanine-resistant variants spontaneously reverted to actively express gpt, as indicated by both reversion assays and direct enzyme measurements. Since reversion was enhanced in many of the nickel-induced variant cell lines following 24-h treatment with the demethylating agent 5-azacytidine, the involvement of DNA methylation in silencing gpt expression was suspected. This was confirmed by demonstrations of increased DNA methylation, as well as by evidence indicating condensed chromatin and heterochromatinization of the gpt integration site in 6-thioguanine-resistant cells. Upon reversion to active gpt expression, DNA methylation and condensation are lost. We propose that DNA condensation and methylation result in heterochromatinization of the gpt sequence with subsequent inheritance of the now silenced gene. This mechanism is supported by direct evidence showing that acute nickel treatment of cultured cells, and of isolated nuclei in vitro, can indeed facilitate gpt sequence-specific chromatin condensation. Epigenetic mechanisms have been implicated in the actions of some nonmutagenic carcinogens, and DNA methylation changes are now known to be important in carcinogenesis. This paper further supports the emerging theory that nickel is a human carcinogen that can alter gene expression by enhanced DNA methylation and compaction, rather than by mutagenic mechanisms.

Animals↗
PubMed1995

Measurement of pleural fluid cholesterol and lactate dehydrogenase. A simple and accurate set of indicators for separating exudates from transudates.

OBJECTIVES: To evaluate the usefulness of diverse combinations of pleural cholesterol concentration, pleural or serum protein, and lactate dehydrogenase (LDH) levels for the differentiation of pleural exudates and transudates. DESIGN: Prospective laboratory study of pleural effusions. SETTING: Medical school hospital. PATIENTS: One hundred eighty consecutive internal medicine ward patients in whom the etiologic diagnosis of their pleural effusion was confirmed. MEASUREMENTS: Cholesterol concentration in pleural fluid and protein and LDH both in pleural fluid and blood serum. RESULTS: According to their etiology, 49 (27.2%) of the effusions were transudates and 131 (72.7%) were exudates. Using a cutoff point of 45 mg for pleural cholesterol and values for protein and LDH of Light et al, the best diagnostic power corresponded to the combination of pleural cholesterol and LDH: cholesterol level over 45 mg/dL and/or LDH over 200 IU/L identified exudates with a sensitivity of 99% and a specificity of 98%. All the other combinations showed inferior values and the criteria of Light et al reached 98 and 82%, respectively. CONCLUSIONS: The measurement of pleural cholesterol and LDH permits the separation of pleural exudates from transudates with an accuracy similar to the original report of Light et al, with the advantage of requiring only two laboratory determinations and no simultaneous blood sample.

Cholesterol↗
PubMed1995

Growth hormone response to growth hormone-releasing hormone (GHRH), insulin, clonidine and arginine after GHRH pretreatment in obese children: evidence of somatostatin increase?

To clarify the possible neuroendocrine mechanisms underlying the impairment in growth hormone (GH) secretion present in obesity, the GH response to GH-releasing hormone (GHRH, N = 6), insulin hypoglycemia (N = 6), clonidine (N = 7) and arginine (N = 8) after GHRH pretreatment (1 microgram/kg iv 2 h before the tests) was evaluated in 27 obese peripubertal children and in a group of normal-weight short-normal children (N = 26). Growth hormone-releasing hormone pretreatment and all further stimuli elicited a statistically significant GH response in both obese and short-normal children; in the latter group arginine did not induce a significant GH response. No differences were found among the GH responses after the second stimuli in obese children, while in short-normal children the arginine peak and area values were lower than after GHRH and clonidine. Comparison between the two groups showed similar baseline but higher stimulated GH levels in normal-weight children after all tests except arginine, after which no difference was present. In conclusion, the neuroregulation of GH release seems to be similar qualitatively in normal-weight and obese youngsters; the different behavior observed after arginine, which is supposed to act through somatostatin inhibition, might be due to a chronic increase in somatostatinergic tone responsible for the lower stimulated GH levels in obesity.

Adolescent↗
PubMed1995

Subcutaneous Nocardia asteroides abscess in a bone marrow transplant recipient.

We describe a case of cutaneous Nocardia asteroides infection in a 13-year-old Venezuelan boy with aplastic anemia, following allogeneic BMT. He was receiving immunosuppressive therapy with corticosteroids for GVHD and trimethoprim/sulfamethoxazole (TMP/SMX) for prophylaxis against Pneumocystis carinii pneumonia (PCP). He was not neutropenic and gave no history of cutaneous inoculation or trauma. He developed an abscess on the plantar surface of his right big toe from which Nocardia asteroides was isolated. He was successfully treated with a combination of TMP/SMX and minocycline. Despite prolonged immunosuppressive therapy for GVHD, the infection responded and did not recur. TMP/SMX prophylaxis for PCP in BMT recipients with GVHD does not always prevent nocardial infection but may prevent or delay the development of dissemination.

Abscess↗
PubMed1995

Long aboral projections of Dogiel type II, AH neurons within the myenteric plexus of the guinea pig small intestine.

Enteric AH neurons, with multipolar Dogiel type II morphology, project around the circumference of the intestine to myenteric ganglia, the submucosa and mucosa. Using retrograde labeling in vitro, intracellular recording, dye filling and immunohistochemistry, the projections of these neurons along the intestine were studied. When the retrograde tracer, Dil, was applied to the myenteric plexus, labeled nerve cell bodies were located up to 111 mm orally but only 13 mm aborally, demonstrating a marked difference in the lengths of projections up and down the small intestine. Of labeled nerve cell bodies located 2-110 mm orally, 43% had Dogiel type II morphology and of these, 70% were immunoreactive for calbindin, a calcium binding protein exclusive to Dogiel type II neurons. Intracellular filling with neurobiotin revealed several long circumferentially directed nerve fibers and short, filamentous dendrites; thus these were "dendritic" Dogiel type II neurons. This class accounts for approximately 3-4% of all myenteric neurons, and about 10% of all Dogiel type II neurons. Intracellular recordings revealed AH cell characteristics, with long afterhyperpolarizations following their action potentials, pronounced slow excitatory synaptic inputs and a lack of fast excitatory synaptic inputs. Antidromic action potentials could be evoked from the Dil application site in some cells, confirming their aboral projection. This is the first account of a major aboral projection of AH/Dogiel type II neurons and suggests an important role in aborally directed reflexes in the intestine.

Action Potentials↗
PubMed1995

Automatic assessment of scintmammographic images using a novelty filter.

99mTc-sestamibi scintmammograms provide a powerful non-invasive means for detecting breast cancer at early stages. This paper describes an automatic method for detecting breast tumors in such mammograms. The proposed method not only detects tumors but also classifies non-tumor images as "normal" or "diffuse increased uptake" mammograms. The detection method makes use of Kohonen's "novelty filter". In this technique an orthogonal vector basis is created from a normal set of images. Test images presented to the detection method are described as a linear combination of the images in the vector basis. Assuming that the image basis is representative of normal patterns, then it can be expected that there should be no major differences between a normal test image and its corresponding linear combination image. However, if the test image presents an abnormal pattern, then it is expected that the "abnormalities" will show as the difference between the original test image and the image built from the vector basis. In other words, the existing abnormality cannot be explained by the set of normal images and comes up as a "novelty." An important part of the proposed method are the steps taken for standardizing images before they can be used as part of the vector basis. Standardization is the keystone to the success of the proposed method, as the novelty filter is very sensitive to changes in shape and alignment.

Breast↗
PubMed1994

Altered oxidative stress responses in nickel-resistant mammalian cells.

BALB 3T3 cells exposed to NiCl2 acquired resistance to concentrations as high as 200 microM and retain resistance for many generations in the absence of nickel. This resistance was not due to alterations in uptake or to metallothionein overexpression. The nickel-resistant B200 cell line was found to also exhibit cross-resistance to hydrogen peroxide and menadione. These nickel-resistant cells had 1.8 times higher basal levels of glutathione compared to wild-type cells. Studies with the glutathione synthesis inhibitor buthionine sulfoximine showed that while glutathione turnover was more rapid in the nickel-resistant cells, its depletion following NiCl2 treatment of the parental BALB 3T3 cell line was greater than in the nickel-resistant B200 cells. The reduced level of binding of NFkB and AP-1 transcription factors to their DNA consensus sequences in B200 cells compared to wild-type cells, and their more reactive response following treatment of resistant cells with H2O2 or buthionine sulfoximine, strengthens the hypothesis that nickel resistance is closely allied to oxidative stress responses.

3T3 Cells↗
PubMed1994

The role of ascending excitatory and descending inhibitory pathways in peristalsis in the isolated guinea-pig small intestine.

1. The effects of experimental manipulations to alter the activation of ascending excitatory and descending inhibitory reflexes on peristalsis were studied in isolated segments of guinea-pig small intestine. 2. The normal site of initiation of the peristaltic contraction, the oral end, was always shifted to a point just anal to a crush which interrupted enteric neuronal pathways. 3. Shortening the functional length of the intestine by a series of crushes, thus effectively reducing the length of enteric neuronal reflex pathways, led to a progressive increase in the threshold distension for triggering the peristaltic contraction. 4. A sleeve placed around the intestine to prevent it from being distended by fluid led to a shift in the site of initiation of the peristaltic contraction to a point just anal to the sleeve. Furthermore, sleeves placed in the anal half of the intestine were able to stop propagation of the contraction. 5. The effect of these manipulations on peristalsis suggests that ascending excitatory and descending inhibitory enteric pathways, activated by distension, determine the pattern of peristaltic activity. The peristaltic contraction is initiated at the oral end as a result of the summation of ascending excitatory inputs and the relative absence of inputs from descending inhibitory pathways at this point. The magnitude of the distension for triggering this contraction is determined by a balance between ascending excitatory and descending inhibitory inputs to the circular muscle. Propagation of the circular muscle contraction requires the activation of ascending excitatory pathways at each point along the intestine and the sequential inactivation of the descending inhibitory reflex pathways anal to the contraction. The propagation of the circular muscle contraction stops when there is no longer a sufficient distension stimulus ahead.

Animals↗
PubMed1994

Pitfalls of genetic counselling in brachydactyly type C.

A familial case of brachydactyly type C is presented in which hand radiographs of the proposita's parents was considered normal. The metacarpophalangeal profile pattern of the mother's hands demonstrated minimal manifestations typical of brachydactyly type C.

Adult↗
PubMed1994

All calbindin-immunoreactive myenteric neurons project to the mucosa of the guinea-pig small intestine.

The projections of Dogiel type II myenteric neurons to the mucosa of the guinea-pig ileum were quantified by combining retrograde transport of DiI, in vitro, with immunohistochemistry. After DiI application to the mucosa over an area of 1.5 x 10 mm2, virtually all (> 97%) calbindin-immunoreactive Dogiel type II neurons in the myenteric plexus underneath the mucosal DiI application site were labelled, indicating that essentially all of these neurons project to the mucosa. From cell counts, on average 5 calbindin-immunoreactive neurons project to each villus, and each calbindin-immunoreactive neuron supplies on average 10 villi. Since Dogiel type II neurons that were not immunoreactive for calbindin (19% of all labelled nerve cells) also projected to the mucosa, it is likely that all Dogiel type II neurons, which are putative sensory neurons of the gut, project to the mucosa.

Animals↗
PubMed1994

High-performance liquid chromatographic measurement of the novel anti-HIV agent 7,8-dihydrocostatolide (NSC 661123).

An HPLC assay was used to determine levels of a promising anti-HIV agent 7,8-dihydrocostatolide (DC; NSC 661123) in human and murine plasma. The structurally related compound costatolide (C) was found to be a suitable internal standard. Drug was extracted from human or murine plasma using a solid-phase C18 cartridge. The compound was eluted from an ODS analytical HPLC column using an acetonitrile-water mobile phase. Drug was quantified over the assay range of 19.5 to 625 ng/ml with excellent within- and between-day reproducibility. Data resulting from the use of the assay method for determination of dihydrocostatolide pharmacokinetics in mice are presented. This is the first report of a validated HPLC assay for determining DC levels in human and mouse plasma.

Animals↗
PubMed1994

The role of enteric inhibitory motoneurons in peristalsis in the isolated guinea-pig small intestine.

1. Peristalsis is a co-ordinated motor behaviour in which an anally propagated contraction of the circular muscle propels intraluminal contents. The role of excitatory motoneurons in peristalsis is well established; however the role of enteric inhibitory motoneurons is unknown. 2. A combination of a nitric oxide synthase inhibitor and apamin, which blocks relaxation of the circular muscle of guinea-pig small intestine mediated by enteric inhibitory motoneurons, was used to investigate the role of inhibitory motoneurons in peristalsis in isolated segments of guinea-pig small intestine. 3. N omega-nitro-L-arginine methyl ester (L-NAME, 400 microM) and N omega-nitro-L-arginine (L-NOArg, 100 microM) significantly reduced the threshold volume required to trigger emptying of the intestine. This effect was reversed by L-arginine (4 mM) and L-arginine alone increased the threshold volume for initiation of peristalsis. Sodium nitroprusside (0.1-10 microM), which generates nitric oxide, also increased the threshold volume. L-NAME, L-NOArg, L-arginine and sodium nitroprusside did not alter the maximal intraluminal pressure generated during emptying. Contraction of the longitudinal muscle during the initial phase of fluid infusion was significantly increased by L-NAME and L-NOArg and reduced by sodium nitroprusside (1 nM to 10 microM). 4. Apamin (0.5 microM) did not significantly alter the threshold volume necessary to initiate peristalsis or contraction of the longitudinal muscle. However, the maximal pressure generated when the intestine was emptying was significantly increased. Furthermore, short segments of circular muscle contracted apparently randomly, before peristaltic emptying was triggered. 5. A combination of L-NAME and apamin completely disrupted peristalsis. Contractions of the circular muscle did not always start at the oral end. Stationary contractions as well as contractions propagating orally and anally were observed. 6. It is concluded that enteric inhibitory motoneurons are crucial for peristalsis to occur. They are important in setting the threshold at which peristaltic emptying is triggered, via nitric oxide. They are essential for the propagation of the circular muscle contraction, via an apamin-sensitive mechanism of transmission. Contraction of the longitudinal muscle during peristalsis is partly inhibited by a nitric oxide-mediated mechanism.

Animals↗
PubMed1994

Molecular mechanisms of nickel carcinogenesis.

Nickel treatment of intact cultured cells oxidized dichlorofluorescin to a fluorescent product indicating that nickel elevated the level of oxidants in cells. Nickel also caused an increase in crosslinking of amino acids to DNA and these complexes did not appear to involve the direct participation of Ni2+. Histidine, cysteine and tyrosine were prominent among the amino acids crosslinked to DNA. Nickel selectively damaged heterochromatin and this resulted in deletions of heterochromatic regions during nickel carcinogenesis. Thrombospondin was one of the genes expressed in normal cells that was not expressed in nickel-transformed cells. Other aspects of the molecular mechanism of nickel carcinogenesis are discussed.

Amino Acids↗
PubMed1994

Nickel-induced transformation of human cells causes loss of the phosphorylation of the retinoblastoma protein.

The retinoblastoma (Rb) protein (pRb) has been studied in various crystalline NiS-transformed cell clones derived from the human osteoblast cell line, HOS TE-85. The parental HOS cells were not able to proliferate in soft agar medium, but they acquired this property following treatment with crystalline NiS. The pRb was found only in the hypophosphorylated form in 8 of 9 nickel-transformed clones examined, whereas in the parental cells the pRb appeared in both phosphorylated and unphosphorylated forms. Neither Rb gene expression nor its phosphorylation was affected by acute nickel treatments of HOS cells. The nickel-transformed HOS clones expressed the major regulators of Rb phosphorylation, cyclin E and cdk-2, at levels similar to those of the parental cells. In coimmunoprecipitation assays with cell lysates from the transformed clones that exhibited the hypophosphorylated form of pRb, the Rb protein failed to form a complex with simian virus 40 large T-antigen, indicating a lack of functional activity. When a plasmid containing the normal Rb gene was transfected into these nickel-transformed cells, it restored the Rb phosphorylation pattern observed in parental cells and the cells acquired a normal phenotype (i.e., they were no longer able to grow in soft agar). This suggested that a mutation was induced in nickel-transformed cells that affected the ability of the Rb protein to be phosphorylated and function normally, and this mutation allowed the human nickel-transformed cells to acquire anchorage-independent growth.

Antigen-Antibody Reactions↗
PubMed1994

A putative cell signal encoded by the folded gastrulation gene coordinates cell shape changes during Drosophila gastrulation.

The folded gastrulation (fog) gene is required during Drosophila gastrulation for two morphogenetic movements, formation of the ventral furrow and invagination of the posterior midgut primordium. fog coordinates cell shape changes during these invaginations by inducing apical constriction of cells in spatially and temporally defined manners. fog is expressed in the invagination primordia in a pattern that precisely precedes the pattern of constrictions. Overexpression of fog in the dorsoanterior region of the embryo induces ectopic constrictions, indicating localization of fog transcripts may define domains of cell shape changes. fog encodes a novel protein with a putative signal sequence but no potential transmembrane domains. We suggest fog functions as a secreted signal that activates the G protein alpha subunit encoded by concertina in neighboring cells. Our analyses indicate that cell-cell communication ensures the rapid, orderly progression of constriction initiations from the middle of invagination primordia out toward the margins.

Amino Acid Sequence↗