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Biomedical subjects

M Costa

Publications and source records attributed to M Costa.

At least 487 records · Page 27Linked to original sources
PubMed1983

Inhibition of LPS toxicity for macrophages by metallothionein-inducing agents.

Parenteral administration of adrenal corticosteroids or particular transition metal salts are known to protect mice from the lethal effects of bacterial lipopolysaccharides (LPS). To determine if both groups of substances act through similar biologic mechanisms, their capacity to protect macrophages from the direct toxic effects of LPS was examined in vitro. When added simultaneously with LPS at culture initiation, 10 to 100 microM cortisone increased the viability of normal peritoneal macrophages as determined by trypan blue exclusion. Prednisolone and corticosterone protected LPS-treated macrophages at even lower concentrations (0.1 to 1 microM); estradiol and testosterone failed to alter cell viability at any concentration tested. Protection was dependent on de novo synthesis because inclusion of 20 nM actinomycin C1 or 1 microM cycloheximide with 10 microM corticosterone during a 4-hr pretreatment period blocked induction of the protective effect. Murine macrophages were also protected by micromolar concentrations of zinc, cadmium, mercury, and manganese, but not by calcium or lead. As was obtained with corticosteroids, heavy metal-induced protection depended on de novo RNA and protein synthesis. Because all substances that protected against LPS are known inducers of metallothionein in somatic cells, peritoneal macrophages were assayed for the presence of this unique, cytoplasmic protein. Within 2 to 8 hr, 10 microM cadmium caused three to fivefold increases in the incorporation of 35S-cysteine and in the binding of 203Hg into the TCA-soluble fraction of cell lysates that was excluded on centrifugally accelerated Sephadex G-10 columns. These results suggest macrophages may be protected from LPS-mediated cytotoxicity through synthesis of a sulfhydryl-rich, metal-binding protein. Although its mechanism of action remains unknown, it is proposed that metallothionein may function homeostatically by altering intracellular concentrations of zinc or may play a regulatory role by facilitating transfer of heavy metals among metal-requiring apoproteins.

Animals↗
PubMed1983

Correlations of DNA strand breaks and their repair with cell survival following acute exposure to mercury(II) and X-rays.

Alkaline elution analysis demonstrates that both HgCl2 and X-rays result in a rapid induction of DNA single-strand breaks at acutely cytotoxic doses (HgCl2, 25-100 microM for 60 min; X-rays, 150-600 rads) in cultured Chinese hamster ovary cells. Cytotoxicity, as measured by cell-plating efficiency, correlates linearly with the level of DNA breakage induced by both agents (HgCl2, r = 0.97; X-rays, r = 0.99), although a substantial difference in axis intercepts of the two linear regression lines indicates that a higher level of DNA damage was required by X-rays as compared with HgCl2 to produce an equivalent level of cell killing. DNA damage induced by X-rays was rapidly repaired such that within 1 hr following treatment the elution rate of DNA from treated cells resembled that obtained in untreated cultures. In contrast, DNA damage after Hg2+ insult was not repaired, and further damage was evident following a similar 1-hr recovery period. Addition of noncytotoxic, non-DNA-damaging concentrations of HgCl2 (10 microM) to cells 15-45 min following treatment with X-rays greatly inhibited the repair of the DNA strand breaks. Thus, although both HgCl2 and X-rays induce rapid and striking single-strand breaks in the DNA, persistence of Hg2+ in the cell can inhibit the repair of these breaks. The inhibition of DNA repair by HgCl2 may explain why this agent is not severely mutagenic or carcinogenic despite its ability to induce an X-ray-like DNA damage and why a lower level of mercury-induced DNA damage, compared with that induced by X-rays, was required to produce an equivalent level of cell death.

Animals↗
PubMed1983

Influence of surface charge and dissolution on the selective phagocytosis of potentially carcinogenic particulate metal compounds.

Substantial evidence has accumulated which documents the active endocytosis by cells of particulate nickel compounds having potent carcinogenic and transforming capacity; compounds less potent in these respects exhibit a reduced tendency to be phagocytized by cultured fibroblasts. The surface charges (zeta potentials) of a number of particulate nickel compounds were measured in an attempt to identify the determinants of their variable degrees of cellular uptake. The carcinogenic particulates, crystalline NiS, Ni3S2, and NiO, exhibit strongly negative zeta potentials in distilled water and enter cells readily, while noncarcinogenic amorphous NiS, which is phagocytized to a lesser degree, is slightly positive in surface charge under similar conditions. The greater dissolution rate of amorphous NiS in comparison to crystalline NiS may contribute to its reduced uptake by cells by causing substantial alteration of the particle surface and/or by the generation of particle dissolution products at its site of cellular interaction which inhibit particle uptake. Addition of ionic nickel was found to be inhibitory toward the phagocytosis process in general, although the potency of ionic nickel in inhibiting particle uptake is not sufficiently high to attribute the selectivity of uptake of nickel-containing particulates solely to this inhibitory effect. Freshly suspended amorphous NiS particles were phagocytized more than particles aged in either H2O or culture medium for 1 to 7 days. This reduced tendency of the aged amorphous NiS particles to be phagocytized remained following removal of potential inhibitory dissolution products. Binding of amorphous NiS to DEAE paper, which represented an alternate method to determine the surface charge, was decreased by aging in H2O or culture medium, suggesting that a loss of negative surface charge during this aging process may have been associated with decreased uptake. These findings lend support to the hypothesis that surface charge may play a role in the phagocytosis of potentially carcinogenic nickel sulfide particles.

Animals↗
PubMed1983

Growth inhibition and metallothionein induction in cadmium-resistant cells by essential and non-essential metals.

Essential and non-essential metal ions were compared on the basis of their growth-inhibitory potency and their mediation of metallothionein induction in a Chinese hamster ovary cell line resistant to cadmium. Cadmium-resistant cells were found to be 20-fold and 6-fold more resistant than wild-type Chinese hamster ovary cells to the non-essential metals CdCl2 and HgCl2, respectively. In contrast, cadmium-resistant cells showed 2-fold or less resistance to growth inhibition due to the metals with known or possible biological essentiality, ZnCl2, CuSO4, CoCl2, and NiCl2. Resistance to either cadmium or mercury was not due to decreased uptake as measured isotopically or by X-ray fluorescence. At concentrations near the threshold of growth inhibition, CdCl2 and ZnCl2 induced metallothionein 8- to 10-fold above background levels in cadmium-resistant cells within 8-10 hr. A 2- to 3-fold induction of this protein was produced in resistant cells by levels of HgCl2, CuSO4, and CoCl2 near the threshold of growth inhibition whereas NiCl2 produced no measurable elevations of metallothionein at concentrations below, near, and above those that inhibit cell growth. Induction of metallothionein was measured by a modified 203Hg binding assay and by [35S]cysteine incorporation. No measurable induction of metallothionein was evident in wild-type cells with any metal treatment using a reasonable quantity of cells consistent with our assay. These results in cadmium-resistant cells demonstrate selective induction of metallothionein by various metals and suggest that induction of this protein alone is not solely responsible for differences in the growth-inhibitory potential of these elements.

Animals↗
PubMed1982

Soluble protein kinase fractions from DEAE-cellulose chromatography. A comparison between brain and heart from the rat.

The cytosol fraction from rat midbrain was chromatographed on DEAE-cellulose with a linear NaCl gradient (0-0.3 M). Two peaks of protein kinase activity were obtained when assayed with either histone or casein. A similar elution profile of the kinase activity was obtained from rat heart. The first peaks from midbrain and heart were compared in terms of their dependency upon cAMP and sensitivity to the endogenous protein kinase inhibitor. Neither of the two substances had an effect on the activity of the brain kinase. Furthermore, the dissociability of the midbrain and heart enzymes in the presence of cAMP or histone was compared by DEAE-cellulose chromatography. The heart enzyme was dissociated into a catalytic subunit characteristic of a cAMP-dependent protein kinase, whereas the brain kinase was totally unaffected by the cAMP or histone. The results of these tests indicate that although the elution profiles from DEAE-cellulose are similar between midbrain and heart, the first peak from brain contains a protein kinase that appears to be cAMP independent.

Animals↗
PubMed1982

Baroreceptor reflexes in conscious guinea-pigs are unaffected by depletion of cardiovascular substance P nerves.

Adult guinea-pigs were injected with doses of capsaicin increasing from 10 mg/kg to 400 mg/kg over 6-days. The guinea-pigs were supplied with indwelling arterial cannulae 14 days later and the next day cardiovascular function was assessed in the conscious animals. Resting blood pressures and heart rates were recorded and then baroreceptor reflex function was evaluated by recording the changes in heart period which were evoked by blood pressure responses to injections of phenylephrine and glyceryl trinitrate. Substance P was measured by radioimmunoassay and its localization examined by immunohistochemistry. The capsaicin treatment caused a substantial reduction in substance P levels in vascular and cardiac nerves. However, there were no changes in resting heart rate, blood pressure or baroreceptor sensitivity. It is concluded that the widespread substance P nerves associated with the heart and blood vessels are not necessary for baroreceptor reflex function. Their role or roles remain unknown.

Animals↗
PubMed1982

Reduction of beta-thiopyruvic acid by lactate dehydrogenase: a kinetic study.

In this paper a steady-state kinetic study on the system lactate dehydrogenase-beta-thiopyruvate-beta-thiolactate is presented and the possible mechanistic and physiological implications are discussed. At pH 7.4 the equilibrium between beta-thiopyruvate and beta-thiolactate, in the presence of NADH and lactate dehydrogenase is largely shifted towards the formation of beta-thiolactate as in the case of pyruvate and lactate. This can can be relevant in connection with the mixed disulfide between cysteine and beta-thiolactate that is observed to be present in the mammalian body fluids. The catalytic mechanism is of the bi-bi compulsory type, and rapid equilibrium conditions for the binding of the first substrate (NADH) are shown to apply. A complex inhibition pattern of inhibitions by both substrates, however, prevents simple suggestions about the nature of the dead-end species involved.

Binding, Competitive↗
PubMed1982

Improved immunohistochemical visualization of central serotonin nerves after loading with 5,7-dihydroxytryptamine.

5,7-Dihydroxytryptamine (5,7-DHT), which reacts with a monoclonal antibody raised against 5-hydroxytryptamine (5-HT), has been used to increase the sensitivity of the immunohistochemical detection by this antibody of central 5-HT nerves in the rat brain. Rats pretreated with tricyclic antidepressants were given a single intraventricular injection of 5,7-DHT and were sacrificed 30 min-3 h later. This treatment resulted in accumulation of 5,7-DHT by central 5-HT nerves and selectively enhanced the immunofluorescence reaction of these nerves, particularly the terminals and non-terminal fibres.

5,7-Dihydroxytryptamine↗
PubMed1982

Substance P in nerve tissue in the gut.

Substance P is present in intrinsic nerves in the intestine and in some extrinsic sensory nerves. It can be released from these nerves to act on the muscle layers and on other enteric neurons. Immunohistochemical studies show that the enteric substance P neurons are short and branch profusely to supply all intestinal layers. Pharmacological studies indicate that enteric substance P neurons receive inputs from enteric cholinergic interneurons. This suggests that these neurons are part of multineuronal nerve pathways in the intestine. The substance P neurons are not essential for the enteric reflexes which subserve peristalsis but they appear to modulate these reflexes.

Acetylcholine↗
PubMed1982

Characterization of substance P-like immunoreactivity in peripheral sensory nerves and enteric nerves by high pressure liquid chromatography and radioimmunoassay.

Material exhibiting immunoreactivity for substance P in enteric nerves, obtained from the myenteric plexus of the guinea pig small intestine, and in the peripheral ends of sensory nerves of the ureter, atrium and superior mesenteric artery, was characterized by separation by high pressure liquid chromatography, and quantified by radioimmunoassay of fractions collected from the chromatograph. Capsaicin, which depletes substance P-like immunoreactivity from sensory, but not from other substance P-containing nerves, reduced the content of substance P-like immunoreactivity in ureter, atrium and superior mesenteric artery by more than 99.5%, whereas the reduction in immunoreactive material in the myenteric plexus was less than 10%. Separation of extracts of myenteric plexus, ureter and atrium on a reversed-phase column gave major peaks corresponding to authentic substance P and minor peaks that coeluted with oxidized substance P. If the extracts were oxidized with hydrogen peroxide before chromatography, all the immunoreactivity was found in the peak corresponding to oxidized substance P. In the superior mesenteric artery extracts, in addition to the components corresponding to substance P and its oxidized derivative, there was a small intermediate peak that has yet to be identified. Physalaemin, which has been suggested to be present in mammalian nerves, was not detectable in any of the extracts. It is concluded that both enteric nerves and the peripheral processes of sensory nerves which show immunoreactivity for substance P in this species contain the authentic peptide.

Animals↗
PubMed1982

Surface reduction of amorphous NiS particles potentiates their phagocytosis and subsequent induction of morphological transformation in Syrian hamster embryo cells.

The incidence of morphological transformation following exposure of Syrian hamster embryo (SHE) cells to crystalline alphaNiS particles was considerably greater than that following a similar exposure to amorphous NiS particles. These differences in potency were attributable to the selective phagocytosis of crystalline alphaNiS particles into cells, since untreated amorphous NiS particles were not readily taken up. Chemical reduction of the amorphous NiS particles' surface with LiAlH4 resulted in an increase both in their phagocytic uptake by Chinese hamster ovary (CHO) cells and in their ability to induce transformation in SHE cells. The phagocytosis and transforming activity of crystalline alphaNiS particles was also enhanced by LiAlH4 reduction. These results are consistent with previous observations showing that untreated crystalline NiS particles have a negative surface charge while amorphous NiS particles possess positively charged surfaces. These findings support the general hypothesis that the transforming activity of particulate metal compounds is proportional to their phagocytic uptake. Specifically, these observations show that the entry of metal sulfide particles into cells is related to their surface properties and, in particular, to the degree of negative charge on the surface microenvironment.

Animals↗