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Biomedical subjects

M Costa

Publications and source records attributed to M Costa.

At least 253 records · Page 14Linked to original sources
PubMed1994

Accommodation mediated by enteric inhibitory reflexes in the isolated guinea-pig small intestine.

1. The aim of the present study was to investigate whether the guinea-pig small intestine shows accommodation to infused fluid, similarly to other regions of the gastrointestinal tract. Tetrodotoxin, papaverine and transmitter antagonists were used to establish the existence of reflex pathways and the nature of the neurotransmitters involved. 2. Compliance, measured as the change in volume of infused fluid divided by the intraluminal pressure change, was reduced by tetrodotoxin (0.6 microM), indicating that there is an overall neurally mediated relaxation of the circular muscle in response to low rates of distension. Papaverine (10 microM) did not have any significant effect on compliance at the low rates of distension, suggesting that the circular muscle is fully relaxed. 3. At each rate of distension, 400 microM N omega-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) significantly decreased the compliance of the intestinal wall, indicating that the circular muscle was relaxed by a nitric oxide-mediated mechanism. Apamin (0.5 microM), which blocks a component of inhibitory transmission, did not have a significant effect. 4. In control preparations, the intestinal wall was less compliant when distended by fluid at a fast rate, compared with the lower rates of distension. This was not due to changes in passive components of the intestinal wall or a myogenic response to rapid stretch. 5. When the intestine was distended rapidly, 1 microM hyoscine and 100 microM hexamethonium increased intestinal compliance. However, they had no detectable effect on compliance with low rates of distension.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

MCF-10A cells infected with the int-2 oncogene induce angiogenesis in the chick chorioallantoic membrane and in the rat mesentery.

A growing body of evidence demonstrates the relevant role of the int-2 (FGF-3) oncogene in human carcinomas. To investigate its angiogenic activity, the human epithelial mammary cell line MCF-10A was infected with a retroviral expression vector carrying the int-2 oncogene. Infected cells were entrapped in an alginate pellet and placed on the chorioallantoic membrane of chick embryos. After 7 days, a dense capillary network was found to grow toward the pellet, whereas parental cells did not show any angiogenic activity. Conditioned medium from int-2-infected cells was injected i.p. twice daily into rats over a period of 10 days. The mesentery of treated rats showed numerous small blood vessels originating from larger vascular arcades and growing through the stromal layer of the mesentery. In control experiments, neither medium for cell culture nor conditioned medium from parental cells was found to induce angiogenesis. In conclusion, the stimulation of blood vessel growth by int-2-infected cells suggests that the production of the int-2 protein is associated with the acquisition of the angiogenic phenotype.

Allantois↗
PubMed1994

Protein oxidation and amino acid-DNA crosslinking by nickel compounds in intact cultured cells.

Oxidative damage induced by NiCl2 to protein has been investigated. We found that nickel induced a dose-dependent increase in the oxidation of bovine serum albumin (BSA) as detected by carbonyl formation in the presence of H2O2 in vitro, as well as producing carbonyl of proteins in intact cultured Chinese hamster ovary (CHO) cells. Other metals capable of producing oxidative damage to BSA in the presence of H2O2 included Cu, Co, Fe, and chromate. However, Cd2+, Hg2+, and Pb2+ were not active even in the presence of H2O2. As an indicator of nickel-induced genotoxic damage, the crosslinking of amino acids to DNA was also examined. Cysteine, histidine, and tyrosine were increased in their association with DNA based upon their persistent binding to DNA following washes with EDTA or SDS. The results suggest that DNA-protein or DNA-amino acids crosslinks induced by nickel may result from interaction of the nickel-oxidized carbonyl group of protein, peptides, and free amino acids.

Amino Acids↗
PubMed1994

Ventilatory responses to hypercapnia and hypoxia in elite breath-hold divers.

It was recently hypothesized that elite breath-hold divers may display blunted ventilatory responses to hypoxia and/or hypercapnia (Ferretti et al., J. Appl. Physiol. 70: 794-802, 1991). To test this hypothesis, the following measurements were made on three elite breath-hold divers (members of the same family), and on 9 healthy untrained control subjects (C): (1) Steady-state pulmonary ventilation (VE) at rest in the supine posture while breathing room air or normoxic CO2-enriched mixtures. (2) Breath-by-breath VE changes (delta VE), with respect to baseline conditions, after 4 breaths of 100% O2, under the following conditions: normoxia (PIO2 = 146 Torr) at rest (NR); normoxic exercise (60 watt on a bicycle ergometer) (NE); hypoxia (PIO2 = 77 Torr) at rest (HR); hypoxic exercise (HE). The results were as follows: (1) In hypercapnic experiments VE (normalized per unit of body surface area) was significantly lower in the divers than in C (4.32 +/- 0.04 [mean +/- SD]L.min-1.m-2 vs. 5.31 +/- 0.62 at FICO2 = 1.5%; 5.21 +/- 0.17 vs. 7.72 +/- 1.39 at FICO2 = 3%; 8.86 +/- 0.76 vs. 13.14 +/- 2.27 at FICO2 = 5%), as well as than in subjects described by previous authors as being characterized by 'low CO2 sensitivity'. (2) The 100% O2-breathing maneuvers did not induce significant delta VE both in NR and in HR, whereas peak delta VE were -6.73 +/- 1.38 L.min-1 (divers) vs. -5.24 +/- 3.10 (C) in NE, and -17.39 +/- 4.92 (divers) vs. -17.52 +/- 6.32 (C) in HE (no significant differences). It is concluded that the divers, compared to C, had a blunted ventilatory response to hypercapnia, but not to hypoxia. The former may represent an adaptive or genetically inherited phenomenon.

Adaptation, Physiological↗
PubMed1994

NADPH-diaphorase and other neuronal markers in nerves and ganglia supplying the guinea-pig vas deferens.

Enzyme histochemistry, in combination with immunohistochemistry was used to establish the neurochemistry of neurons in the vas deferens and pelvic ganglia of the guinea-pig. Nerve fibres characterised by reactivity for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase reactivity formed a dense network in the lamina propria and circular muscle layer of the vas deferens, but were very sparse in the longitudinal muscle layer of the vas deferens. NADPH-diaphorase reactivity was also present in nerve fibres forming a dense perivascular plexus in many of the arteries in the pelvic region and in some of the endothelial cells, especially near the origin of the capillaries. Nerves with vasoactive intestinal polypeptide (VIP)-immunoreactivity had a similar distribution to NADPH-diaphorase reactive nerves. Tyrosine hydroxylase (TH)-immunoreactive nerve fibres were found in both muscle layers of the vas deferens. There was no coexistence of VIP- and TH-immunoreactivities in nerve fibres in the vas deferens. In the anterior pelvic ganglia, the origin of the nerve fibres in the vas deferens, several classes of neurons could be identified by the presence or absence of the reactivity for NADPH-diaphorase and immunoreactivity for VIP and TH. Neurons containing both VIP and NADPH-diaphorase reactivity accounted for 40% of neurons in the ganglia. Neurons with VIP-immunoreactivity but not NADPH-diaphorase reactivity accounted for 6%. TH-immunoreactive neurons accounted for 22% of neurons in the anterior pelvic ganglia. Very rare cells (< 1%) contained both VIP- and TH-immunoreactivities. The remaining neurons, which were not labelled by any of these markers, comprised 31% of neurons in anterior pelvic ganglia. These results demonstrate the existence of NADPH-diaphorase reactivity in neurons containing VIP-immunoreactivity, thus suggest that nitric oxide may be a neurotransmitter in guinea-pig vas deferens, especially in the circular muscle layer, in the arteries, and in other pelvic organs innervated by pelvic ganglia.

Animals↗
PubMed1994

Characterization of alkaline phosphatase-reactive neurons in the guinea-pig small intestine.

Endogenous alkaline phosphatase activity has been localized histochemically on the surface of enteric neurons of the guinea-pig small intestine by both light and electron microscopy. The enzyme activity was associated with some myenteric neurons that had Dogiel type I morphology, and the histochemical reaction products typically formed a honeycomb-like structure on labelled cell bodies. No Dogiel type II neurons in the myenteric plexus or submucous neurons showed alkaline phosphatase reactivity. Nerve fibres reactive for alkaline phosphatase were present in the myenteric plexus and ran in bundles in the circular muscle and deep muscular plexus. In addition, reactive varicose axons supplied the submucous plexus and non-ganglionated plexus of the mucosa. The results of interruption of the enteric neuronal pathways demonstrated that alkaline phosphatase-reactive myenteric neurons project anally to other myenteric ganglia, to the circular muscle and to the submucous plexus. Sequential enzyme histochemistry showed that virtually all alkaline phosphatase-reactive neurons also contained nitric oxide synthase, revealed by NADPH-diaphorase reactivity. It was estimated that 14-18% of all myenteric neurons showed alkaline phosphatase reactivity. About one-third of nitric oxide synthase-containing myenteric neurons, however, did not contain alkaline phosphatase activity. At the ultrastructural level, alkaline phosphatase activity was associated specifically with the plasma membranes of nerve cell bodies, axons and dendrites of some myenteric neurons. Reactive nerve fibres made close appositions with non-reactive submucous neurons and, within myenteric ganglia, predominantly with other alkaline phosphatase-reactive neurons. In addition to its presence in neurons, alkaline phosphatase reactivity was also present in some endothelial cells in blood vessels in the submucosa and in capillary pericytes. It is concluded, on the basis of the projections and neurochemistry, that in the guinea-pig small intestine alkaline phosphatase activity is associated with nitric oxide synthase-containing neurons which include inhibitory motor neurons to the circular muscle, and anally-directed interneurons to other myenteric and submucous neurons.

Alkaline Phosphatase↗
PubMed1994

Crystalline Ni3S2 specifically enhances the formation of oxidants in the nuclei of CHO cells as detected by dichlorofluorescein.

Dichlorofluorescein (DCF) was used as a fluorescent probe to detect oxidants formed in cultured CHO cells during nickel treatment. Crystalline Ni3S2 specifically enhanced the formation of oxidants in the nuclei of these living cells, but Ni3S2 particles did not enhance DCF fluorescence as much when added in vitro to isolated nuclei. Our results add to the emerging concept that oxidants mediated by nickel compounds may play an important role in nickel-induced genotoxicity.

Animals↗
PubMed1994

Loss of thrombospondin transcriptional activity in nickel-transformed cells.

mRNA from normal Chinese hamster embryo (CHE) cells was transcribed to cDNA and subtracted with an excess of mRNA from Chinese hamster embryo cells transformed by nickel compounds. Here we report the recovery of a sequence found to be highly homologous to the mouse thrombospondin 1 gene that was obtained by this subtraction procedure. Since thrombospondin is antiangiogenic, cancer cells expressing high levels of thrombospondin cannot grow in vivo because capillaries will not proliferate to cells secreting thrombospondin. To examine expression of thrombospondin, normal CHE cells were stained with monoclonal antibodies to human thrombospondin. The protein was present abundantly in the cytoplasm of normal cells but at greatly reduced levels in Ni-transformed cells. Analysis of mRNA by Northern (RNA) blot revealed transcripts in normal cells but little thrombospondin mRNA in Ni-transformed cells. Loss of thrombospondin mRNA expression was related to Ni treatment rather than transformation, since Ni-resistant cells also exhibited fewer thrombospondin transcripts than did wild-type cells. Digestion of genomic DNA with various combinations of restriction enzymes revealed thrombospondin gene patterns that were identical in both cell types, suggesting that there were no major deletions or rearrangements of the gene in the nickel-transformed cells. The inactivation of the thrombospondin gene was further investigated by analyzing the promoter activity of this gene linked to a chloramphenicol acetyltransferase (CAT) reporter plasmid that was transfected into normal and Ni-transformed cells. The CAT activity in normal cells was significantly higher than in Ni-transformed cells, suggesting that the promoter region of thrombospondin was less efficiently transcribed in Ni-transformed cells. We studied the consequences of enhanced expression of the retinoblastoma (Rb) gene, a known tumor suppressor gene, on CAT transcription driven by the human thrombospondin promoter. Cotransfection of an expression vector containing the mouse Rb gene greatly enhanced the transcription from the thrombospondin promoter such that the expression was higher in normal cells than in transformed cells.

Amino Acid Sequence↗
PubMed1994

Application of reliability models to studies of biomarker validation.

We present a model of biomarker validation developed in our laboratory, the results of the validation study, and the impact of the estimation of the variance components on the design of future molecular epidemiologic studies. Four different biomarkers of exposure are illustrated: DNA-protein cross-link (DNA-PC), DNA-amino acid cross link (DNA-AA), metallothionein gene expression (MT), and autoantibodies to oxidized DNA bases (DNAox). The general scheme for the validation experiments involves n subjects measured on k occasions, with j replicate samples analyzed on each occasion. Multiple subjects, occasions, and replicates provide information on intersubject, intrasubject, and analytical measurement variability, respectively. The analysis of variance showed a significant effect of batch variability for DNA-PC and MT gene expression, whereas DNAox showed a significant between-subject variability. Among the amino acids tested, cysteine and methionine showed a significant contribution of both batch and between-subject variability, threonine showed between-subject variability only, and tyrosine showed between-batch and between-subject variability. The total variance estimated through the experiment was used to calculate the minimum sample size required for a future epidemiologic study including the same biomarkers used for the reliability study. Such validation studies can detect the various components of variability of a biomarker and indicate needed improvements of the assay, along with possible use in field studies.

Amino Acids↗
PubMed1994

Molecular mechanisms of nickel carcinogenesis.

Carcinogenic, water-insoluble Ni compounds are phagocytized by cells; and the particles undergo dissolution inside the cell, releasing Ni ions that interact with chromatin. Ni produces highly selective damage to heterochromatin. The longest contiguous region of heterochromatin in the Chinese hamster genome is found on the q arm of the X chromosome, and this region is selectively damaged by Ni. More than half of the male mice in which there were Ni-induced transformations of Chinese hamster cells exhibited complete deletion of the long arm of the X chromosome. The introduction of a normal X chromosome into these cells resulted in cellular senescence, suggesting that the Ni interacted with Chinese hamster genome to inactivate a senescence gene. Investigations were conducted into the mechanisms by which Ni produced damage to chromatin. Ni ions have a much higher affinity for proteins and amino acids than for DNA (by five to seven orders of magnitude). Therefore, Ni interacted with chromatin because of the protein present, not because of its reactivity for DNA. Studies have shown that Ni produced an increase in oxidative products in cells as indicated by oxidation of the fluorescent dye dichlorofluorescein; Ni has also been shown to produce oxidation of proteins in cells, as measured by carbonyl formation. Ni cross-linked certain amino acids and proteins to DNA. These covalent cross-links were not dissociated by EDTA and are inconsistent with direct Ni involvement, but they are consistent with Ni acting catalytically. Using subtractive hybridization, we have isolated a number of clones that are expressed in normal but not in Ni-transformed cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Complexing of amino acids to DNA by chromate in intact cells.

Using o-pthaldialdehyde (OPT) fluorescence, the amino acids associated with DNA were studied following exposure of intact Chinese hamster ovary cells to chromate. Rigorous extraction with EDTA, acid, or base was required to release the amino acids cross-linked to the DNA isolated from control or chromate-treated cells by standard procedures (i.e., proteinase K, phenol, etc.). Amino acids resisting extraction from DNA were not studied since analysis was limited to those that could be released by these procedures. There was a chromate dose-dependent increase in amino acids complexed with the DNA that could be released by EDTA, acid, and base, and these amino acids were separated by HPLC and identified. Substantial increases in cysteine, glutamine, glutamic acid, histidine, threonine, and tyrosine were found as a function of increasing concentrations of chromate. There was also a time-dependent increase in complexing of these amino acids to the DNA by chromate. The amino acids found complexed to DNA in intact cells by chromate were thought to originate from reactions of free amino acids or small peptides with the DNA rather than being proteolytic products derived from larger proteins that were cross-linked to the DNA. This was supported by a number of experiments: a) free amino acids or bovine serum albumin (BSA) were cross-linked by chromium to DNA in vitro and the DNA was isolated by standard procedures.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1994

The role of nickel and nickel-mediated reactive oxygen species in the mechanism of nickel carcinogenesis.

Increasing evidence demonstrates the reactive oxygen species (ROS) are implicated in metal carcinogenesis. Exposure of cultured Chinese hamster ovary (CHO) cells to several nickel compounds, i.e. NiS, Ni3S2, NiO (black and green), and NiCl2 has been shown to increase oxidation of 2',7-dichlorofluorescein to the fluorescent 2',7-dichlorofluorescein (DCF), suggesting that nickel compounds increased the concentration of oxidants in CHO cells. This fluorescence can be attenuated by addition of exogenous catalase to the extracellular media, indicating that H2O2 is one of the formed oxidants in this system. Fluorimetric measurements of chromogens following thiobarbituric acid reaction showed that nickel compounds also induce lipid peroxidation with a decreasing potency NiS, Ni3S2 > black NiO > green NiO > NiCl2. These results suggest that lipid hydroperoxides may also be produced through the action of nickel in intact cells. MgCl2, an antagonist of Ni-induced DNA strand breaks and cell transformation, has no effect on the formation of DCF fluorescence induced in CHO cells by nickel. The results suggest that nickel is an active inducer of ROS in intact mammalian cells and that the molecular mechanism of nickel carcinogenesis may involve multiple steps of nickel-mediated ROS.

Animals↗
PubMed1994

Transformation of human osteoblasts to anchorage-independent growth by insoluble nickel particles.

Nickel compounds are well established by epidemiologic studies as human carcinogens. Although the carcinogenicity of nickel compounds has been studied in experimental animals and in a variety of cultured mammalian cells, there are only sporadic reports of nickel-induced transformation of human cells. In attempts to study the mechanisms of nickel-induced carcinogenesis in human cells, an immortalized human osteoblastic cell line (HOS) that could not grow in soft agar or form tumors in athymic nude mouse was repeatedly treated with a water-soluble nickel compound (NiCl2) or a less water-soluble nickel compound crystalline (NiS). After three rounds of NiS treatment, there was an increase in anchorage-independent (AI) colony formation. This was not found in untreated or NiCl2-treated cells. Ten AI colonies obtained from NiS-treated cells were isolated. All of these clones showed changes in cell morphology, including the appearance of uniform polygon shape, growth in multilayers, and heavy staining with Giemsa. Most of these clones were retested for their ability to grow in soft agar and showed growth efficiencies of 5 to 50%. It has been shown by other investigators that aggregate growth is well correlated with tumorigenic potential in viral or chemical transformants of HOS cells. Four of seven tested NiS-transformed clones were able to form large aggregates compared to their untransformed counterparts, and continued to proliferate in aggregate form when they were plated on 0.9% agar. Current investigations focus on the molecular and genetic changes induced by nickel compounds in these human cells.

Carcinogens↗
PubMed1994

Development of an 125I-postlabeling assay as a simple, rapid, and sensitive index of DNA-protein cross-links.

A rapid, simple, and sensitive 125I-postlabeling technique has been developed to allow detection of DNA-protein cross-links induced by environmental contaminants and carcinogens. This method is based on specific incorporation of 125I into tyrosine residues associated with DNA. Cultured Chinese hamster ovary cells were exposed to various crosslinking agents, e.g., UV light, K2CrO4, or NiCl2. DNA was isolated by proteinase K/phenol/chloroform. The residual peptides cross-linked to DNA were radioiodinated with Na125I and chloramine T. After repeated precipitation with ethanol, the radioactivity was determined. The 125I method was compared with a 3[H]-tyrosine prelabeling method and found to be of similar sensitivity.

Animals↗
PubMed1994

Metal mutagenesis in transgenic Chinese hamster cell lines.

Metals are toxic agents for which genotoxic effects are often difficult to demonstrate. To study metal mutagenesis, we have used two stable hprt/gpt+ transgenic cell lines that were derived from Chinese hamster V79 cells. Both the G12 and G10 cell lines are known to be very sensitive to clastogens such as X-rays and bleomycin, with the mutagenic response of the integrated xanthine guanine phosphoribosyl transferase (gpt) gene in G10 usually exceeding that of the same gene in the transgenic G12 cells. In studies with carcinogenic insoluble nickel compounds, a high level of mutagenesis was found at the gpt locus of G12 cells but not at the endogenous hypoxanthine phosphoribosyl transferase (hprt) locus of V79 cells. We have since demonstrated the similar recovery of a high frequency of viable G12 mutants with other insoluble nickel salts including nickel oxides (black and green). The relative mutant yield for the insoluble nickel compounds (G12 > G10) is the opposite of that obtained with nonmetal clastogens (G10 > G12). In the G12 cells, nickel mutagenesis may be related to the integration of the gpt sequence into a heterochromatic region of the genome. For some of the insoluble nickel compounds, significant inhibition of both cytotoxicity and mutant yield resulted when the G12 cells were pretreated with vitamin E. In comparison with the nickel studies, the mutagenic responses to chromium compounds in these cell lines were not as dramatic. Mutagenesis of the gpt target could not be demonstrated with other metals such as mercury or vanadium.

Animals↗
PubMed1994

[Oculodentodigital dysplasia: report of 2 familial cases].

We describe a father and his child with bilateral syndactyly of fingers IV and V and with pinched nose, hypoplastic alae nasi and thin anteverted nares. The patients also showed a small nodule on the tongue tip. Both had no ocular or dental anomalies. The clinical features of our patients resemble those of the patients described by Brueton et al. The hypothesis that the oculodentodigital dysplasia may belong to a contiguous gene spectrum could be confirmed.

Abnormalities, Multiple↗
PubMed1993

A subpopulation of chicken primary sensory neurons defined by complete co-localization of peripherin-and ovalbumin-immunoreactivities.

In a previous study, we have demonstrated that an ovalbumin-like antigen is present within approximately one-half of all neurons of chicken spinal ganglia. The current study demonstrates this antigen co-localizes absolutely with neural intermediate filament protein (Peripherin) in small to medium-sized neurons of spinal ganglia. While the function of ovalbumin in neurons is unknown, its precise co-localization with Peripherin suggests a functional role restricted to neurons of a defined phenotype.

Animals↗