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Biomedical subjects

M Costa

Publications and source records attributed to M Costa.

At least 307 records · Page 17Linked to original sources
PubMed1992

A conserved region in human and Chinese hamster X chromosomes can induce cellular senescence of nickel-transformed Chinese hamster cell lines.

Cellular senescence is the genetically programmed cessation of cellular proliferation. We have recently mapped a putative senescence gene(s) on the X chromosome of Chinese hamster embryo (CHE) cells. In the present study, we have utilized microcell-mediated chromosome transfer (microcell fusion) to test whether: (i) the human X chromosome exhibits similar genetic potential to induce senescence and (ii) the deletion or inactivation of the X-linked senescence gene(s) in CHE cells is associated with nickel-induced immortalization. A normal CHE or human X chromosome was first introduced into mouse-cell hybrids, then transferred by microcell fusion into a nickel-transformed, immortal male CHE cell line (Ni-2/TGR) with an X deletion (Xq1). Microcell fusion of the normal CHE X chromosome into tumorigenic Ni-2/TGR cells yielded senescence of all X recipient clones. The normal human X chromosome induced dominant senescence of tumorigenic Ni-2/TGR cells in only 17% of the resulting microcell hybrids (14/81). Karyotypic analyses of 13 non-senescing human X chromosome-derived microcell hybrid clones revealed that none of these clones retained the complete X. A normal CHE X chromosome induced senescence of 75% of hybrids obtained with another immortal and tumorigenic nickel-transformed male CHE cell line (Ni-6/TGR), which exhibited no visible deletion of the X chromosome, while the normal human X chromosome, only induced senescence in 19% of these hybrids. Transfer of the normal CHE or human X chromosome into spontaneously transformed and tumorigenic cell lines, CHO/TGR or V79/TGR, had little or no effect on their growth. These data suggest that both human and CHE cells possess similar X-linked genetic activities that regulate the process of cellular senescence, and that in Chinese hamster cells nickel-induced immortalization but not that of CHO or V79 cells is associated with inactivation of an X-linked senescence gene.

Animals↗
PubMed1992

A simple, sensitive assay to detect DNA-protein crosslinks in intact cells and in vivo.

Addition of potassium chloride to sodium dodecyl sulfate (SDS) resulted in the formation of an insoluble precipitate that was easily recovered by low-speed centrifugation. Since SDS tightly binds to proteins but not DNA, all proteins and detergent-resistant DNA--protein complexes were also effectively co-precipitated in the presence of potassium--SDS leaving free DNA in the supernatant. The amount of SDS-precipitable DNA represented a measure of DNA--protein crosslinks. We have adapted this method for determining DNA--protein crosslinks formed in cells following their exposure in culture or in vivo to crosslinking agents such as chromate, cis-Pt(II) diammine dichloride and formaldehyde. The critical parameters for application of the K--SDS assay to cells were rigorously reproducible shearing of chromosomal DNA and effective washing steps. We have found that freeze-thawing SDS lysed cells followed by vortexing and repeated resuspensions of the precipitate by pipeting resulted in a low background and high reproducibility of the assay. The method detected in a dose-dependent manner DNA--protein crosslinks induced in CHO cells by chromate, cis-platinum and formaldehyde, with sensitivity similar to the alkaline elution procedure. The K--SDS assay was also successfully utilized to determine DNA--protein crosslinks in rat and mouse white blood cells exposed in vivo to chromate. Its sensitivity and simplicity in sample handling and DNA--protein complex isolation potential allows wide application of the assay to measure formation of DNA--protein crosslinks. The ease of recovery of DNA--protein complexes allows for a more thorough investigation of this lesion.

Animals↗
PubMed1992

Distribution of peptide-containing nerve fibers in the gastric musculature of patients undergoing surgery for gastroesophageal reflux.

The distribution of nerve fibers and cell bodies reactive for the peptides enkephalin, neuropeptide Y, substance P, and vasoactive intestinal peptide were studied in biopsies of external muscle taken from the gastric body and antrum of 17 patients undergoing surgery for gastroesophageal reflux, and in regions of healthy stomach resected as part of gastric cancer operations. The results were correlated with preoperative measurements of liquid and solid emptying from the stomach in the patients with gastro-esophageal reflux. In three cases, no delay was detected in either liquid or solid emptying, and the distribution of peptide immunoreactive fibers in the external muscle was similar to that of healthy muscle. In 14 cases, the emptying of either liquids or solids or both was delayed, and in eight of these clearcut changes in the distribution of peptide-immunoreactive fibers occurred. In six cases, the number of enkephalin-immunoreactive fibers was fewer than normal in the biopsy from the gastric body (in one of these samples the number of substance P-immunoreactive fibers was also reduced). In another cae, the number of substance P-immunoreactive fibers in the antrum was reduced, and in another the number of vasoactive intestinal peptide and neuropeptide Y-immunoreactive fibers in the antral biopsy was reduced. It is concluded that in patients with gastroesophageal reflux who have delayed gastric emptying, a proportion demonstrate abnormalities of the peptide-immunoreactive fibers that innervate the gastric external muscle.

Enkephalins↗
PubMed1992

Modulation of peristalsis in the guinea-pig isolated small intestine by exogenous and endogenous opioids.

1. A recording method was developed to measure physiological parameters of the preparatory and emptying phases of peristalsis in vitro. This method enabled measurement of: the compliance of the intestinal wall during the preparatory phase (a reflection of the resistance of the wall to distension); longitudinal muscle contraction during the preparatory phase; the threshold volume required to trigger the emptying phase; the maximal ejection pressure and the average power generated during the emptying phase, which reflects the rate at which the intestine performs work. Modulation of these parameters by exogenous and endogenous opioids acting at mu, kappa and delta opioid receptors was investigated. 2. The compliance of the intestinal wall during the preparatory phase was reduced by the mu opioid receptor agonist, [D-Ala2, N-methyl-Phe4, Gly5-ol] enkephalin (DAMGO) but not by the kappa agonist, dynorphin, or the delta agonist, [D-penicillamine2, D-penicillamine5] enkephalin (DPDPE). Reflex contraction of the longitudinal muscle during the preparatory phase was inhibited by DAMGO, dynorphin and DPDPE. The threshold volume required to trigger the emptying phase of peristalsis was increased by DAMGO, dynorphin and DPDPE. 3. The maximal ejection pressure generated during the emptying phase was reduced by dynorphin and DPDPE, but not by DAMGO. The average power generated by the intestine when emptying was not altered by any of the agonists. 4. Electrically stimulated contractions of longitudinal muscle in strips of longitudinal muscle-myenteric plexus were not inhibited by DPDPE. Similarly, DPDPE did not significantly inhibit electrically induced contraction of circular muscle in strips of circular muscle-myenteric plexus.5. Each of the agonist effects on peristaltic parameters was antagonized by the appropriate antagonist:D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) (mu), norbinaltorphimine (nor-BNI) (kappa), naltrindole(delta).6. It is concluded that mu and kappa agonists act primarily on excitatory circular and longitudinal muscle motor neurones. The delta agonist probably acts on enteric neurones presynaptic to excitatory circular and longitudinal muscle motor neurones.7. Antagonists for mu, delta and kappa receptors did not affect any parameters of peristalsis when the intestine emptied against a low resistance. However, when emptying against a high outflow resistance, the average power generated by the intestine was increased by the kappa antagonist, nor-BNI, but not by CTOP or naltrindole.8. It is concluded that endogenous opioids appear to have little role in peristalsis when the intestine is working against a low outflow resistance. However endogenous opioids, acting primarily at kappa receptors,provide a braking mechanism by inhibiting the emptying phase of peristalsis in conditions in which the intestine empties against a higher resistance.

Algorithms↗
PubMed1992

[Intermittent prophylaxis in febrile convulsions with oral diazepam].

Intermittent prophylaxis with oral diazepam is presented as an optional treatment for febrile seizures. This proposition is justified by the severe side effects of the currently used chronic anticonvulsant drug therapy in febrile seizures (phenobarbital and valproate). Nineteen patients aged between 3 months and 5 years were treated. They had either simple or complex febrile seizures. Sixteen patients had at least one prognostic factor for recurrence of febrile seizures: first febrile seizure before 15 months of age, positive family history for epilepsy or febrile seizures, occurrence of a complex febrile seizure or abnormal neurological examination. Three patients had none (cases 8, 12 and 13). We recommended 2.5mg b.i.d. for children younger than 12 months, 5mg b.i.d. for children older than 12 months and younger than 3 years, and 7.5 b.i.d. for children older than 3 years. The results showed that only one patient had febrile convulsions while taking adequate diazepam dosage. Transient side effects occurred in 36.8% of the cases.

Administration, Oral↗
PubMed1992

[Resistance to antibiotics of vibrio cholerae strains isolated in Angola].

Among 87 strains of Vibrio choleare (78 Ogawa serotype and 9 Inaba serotype strains) isolated in Angola in 1987-1990, 86% exhibited multiple resistance to antimicrobials. Eighty-four to 86% of strains were resistant to ampicillin with beta-lactamase production (MIC greater than or equal to 512 mg/l), streptomycin (MIC greater than or equal to 64 mg/l), spectinomycin (MIC greater than or equal to 1,024 mg/l), and trimethoprime-sulfisoxazole (MIC greater than 1,024 mg/l). Seventy-four per cent of strains were resistant to kanamycin (MIC = 512 mg/l), 26% to chloramphenicol (MIC = 32 mg/l), 10% to tetracycline (MIC = 16 mg/l), and 10% to gentamycin (MIC greater than or equal to 32 mg/l). Transfer to E. coli K12 was associated with a substantial increase in expression of resistance to tetracycline and chloramphenicol (CAT type I), with MICs in the 128-512 mg/l range. Transfer rates to E. coli K12 of plasmids for the various resistance phenotypes were 10(-6)/10(-8). The size of the isolated plasmids was 100 Md in diameter and belonged to the incompatibility group inc 6-C.

Angola↗
PubMed1991

Sulfur-containing cyclic ketimines and imino acids. A novel family of endogenous products in the search for a role.

Aminoethylcysteine, lanthionine, cystathionine and cystine are mono-deaminated either by L-amino-acid oxidase or by a transaminase exhibiting the properties described for glutamine transaminase. The deaminated products cyclize producing the respective ketimines. Authentic samples of each ketimine were prepared by reacting the appropriate aminothiol compound with bromopyruvate, except cystine ketimine which required the interaction of thiopyruvate with cystine sulfoxide. Reduction of the first three mentioned ketimines with NaBH4 yields the respective derivatives with the saturated rings of thiomorpholine and hexahydrothiazepine. The same reduction is carried out enzymically by a reductase extracted from mammalian tissues. Properties of the members of this family of compounds are described. Gas chromatography followed by mass spectrometry permits the identification of most of these products. HPLC is very useful for the determination of the ketimines by taking advantage of specific absorbance at 380 nm obtained by prior derivatization with phenylisothiocyanate. Adaptation of these and other analytical procedures to biological samples disclosed the presence of most of these compounds in bovine brain and in human urine. By using [35S]lanthionine ketimine as a representative member of the ketimine group, the specific, high-affinity, saturable and reversible binding to bovine brain membranes has been demonstrated. The binding is removed by aminoethylcysteine ketimine and by cystathionine ketimine indicating the occurrence in bovine brain of a common binding site for ketimines. The reduced ketimines are totally ineffective in competing with [35S]lanthionine ketimine. Alltogether these findings are highly indicative for the existence in mammals of a novel class of endogenous sulfur-containing cyclic products provided with a possible neurochemical function to be investigated further.

Amino Acids, Sulfur↗
PubMed1991

Identification of myenteric neurons which project to the mucosa of the guinea-pig small intestine.

Myenteric neurons which innervate the mucosa of the guinea-pig ileum were characterized by combining retrograde transport of DiI in vitro with immunohistochemistry. Of DiI-labelled myenteric neurons, 43% were immunoreactive for calbindin and substance P, 25% were immunoreactive for calbindin alone, and 18% were immunoreactive for substance P alone. These 3 classes of neurons had Dogiel Type II morphology and are probably sensory neurons. Two classes of probable secretomotor neurons were characterized by immunoreactivity for neuropeptide Y (4%) and vasoactive intestinal peptide (2%). These 5 classes of myenteric neurons represent over 90% of the retrogradely labelled myenteric neurons that project to the mucosa.

Animals↗
PubMed1991

Differential DNA-protein crosslinking in lymphocytes and liver following chronic drinking water exposure of rats to potassium chromate.

Carcinogenic chromium (VI) compounds are persistent environmental contaminants with potential for human exposure through drinking water. One lesion associated with chromium (VI) exposure is the formation of DNA-protein crosslinks (DPC). In an attempt to develop markers of chromium exposure, the formation of DPC in lymphocytes was investigated. Fisher 344 rats were exposed to K2CrO4 in their drinking water for 3 and 6 weeks at concentrations of 100 and 200 ppm chromium. No DPC could be detected in isolated splenic lymphocytes using the alkaline elution technique or by using a polyclonal antibody to chromate-induced DPC. However, increased complexing of proteins with DNA was demonstrated in liver following 3 weeks of exposure at both 100 and 200 ppm chromium. Intraperitoneal administration of potassium chromate did not induce detectable DPC in lymphocytes; however, an increased association of proteins with isolated DNA was detected in the liver. DPC were also induced in isolated splenic lymphocytes following a 2-hr exposure in vitro to 100 microM K2CrO4 in a salts-glucose medium. Although chromium was detected in blood, liver, and kidney, blood levels were comparatively much lower. A comparison of chromium levels required to induce DPC in lymphocytes in vitro and the amount absorbed orally suggests that the white blood cell chromium levels following oral exposure may be too low to induce measurable DNA-protein crosslinks in lymphocytes.

Animals↗
PubMed1991

Distribution of chromium within cells of the blood.

Although a number of investigators have examined the uptake of chromium in red blood cells (RBC) or whole blood, little is known about chromium uptake in white blood cells (WBC). Radiolabeled chromium (51Cr) was used to determine chromium uptake and distribution. Isolated RBC and enriched WBC populations were exposed in vitro to potassium chromate (Cr+6) and uptake was determined over a 2-hr time period. Exposure of either rat or human blood cells to 50 microM K2CrO4 for 2 hr resulted in greater accumulation of chromium within WBC than RBC. Uptake by rat WBC was significantly greater than that of human; whereas, uptake by human RBC was greater than that of the rat. Exposure of human whole blood to 50 microM K2CrO4, prior to isolation of WBC, also resulted in an increased uptake of chromium by WBC. Fisher 344 rats were exposed either orally or intravenously to a single dose of K2CrO4 and the distribution of chromium within blood cells was determined 1 hr, 24 hr, or 7 days following exposure. Regardless of the route or time following exposure, WBC chromium levels were consistently greater than those of RBC. However, the absolute levels of chromium did change with time. A comparison of chromium distribution 24 hr following a single oral exposure (1 ppm Cr+6) to the distribution 7 days following exposure demonstrated a reduction in chromium levels for RBC (10-fold) and for WBC (approximately 2.5-fold). In contrast, intravenous administration of chromate resulted in no significant decrease in RBC chromium levels when compared 1 hr, 24 hr, and 7 days following exposure. Although no difference in WBC chromium content was observed at 1 and 24 hr after exposure, an approximate 1.7-fold decrease in chromium content was detected at Day 7 for WBC. Intravenous administration of chromic chloride (Cr+3) resulted in a low level of chromium associated with RBC following 1 hr, and chromium was undetected in the WBC. These data demonstrate that WBC accumulate hexavalent chromium following both in vitro and in vivo exposure. In addition, white blood cells accumulate chromium to a greater extent than red blood cells. Since WBC accumulate chromium, their use as a target for the development of biomarkers of chromium exposure may be warranted.

Animals↗
PubMed1991

Senescence of nickel-transformed cells by an X chromosome: possible epigenetic control.

Transfer of a normal Chinese hamster X chromosome (carried in a mouse A9 donor cell line) to a nickel-transformed Chinese hamster cell line with an Xq chromosome deletion resulted in senescense of these previously immortal cells. At early passages of the A9/CX donor cells, the hamster X chromosome was highly active, inducing senescence in 100% of the colonies obtained after its transfer into the nickel-transformed cells. However, senescence was reduced to 50% when Chinese hamster X chromosomes were transferred from later passage A9 cells. Full senescing activity of the intact hamster X chromosome was restored by treatment of the donor mouse cells with 5-azacytidine, which induced demethylation of DNA. These results suggest that a senescence gene or genes, which may be located on the Chinese hamster X chromosome, can be regulated by DNA methylation, and that escape from senescence and possibly loss of tumor suppressor gene activity can occur by epigenetic mechanisms.

Animals↗
PubMed1991

Bone marrow transplant in a case of mucopolysaccharidosis I Scheie phenotype: skin ultrastructure before and after transplantation.

An 11-year-old girl with mucopolysaccharidosis I Scheie phenotype (MPS I-S) received a bone marrow transplant (BMT) from her heterozygous HLA-identical LMC-non-reactive mother. Multidisciplinary studies were carried out and results evaluated 21 months after transplantation. Herein we report the ultrastructural findings pre- and post-BMT in skin. Multidisciplinary studies are commonly used to evaluate the benefits of metabolic correction following BMT in some MPS and other inherited metabolic disorders, and changes in morphology have been described in liver and few other tissues. In this case, we elected skin, since connective tissue is universally involved in MPS and is safely and easily obtainable. Comparison of skin biopsy specimens taken before and after BMT showed a considerable change in dermal fibroblast morphology, with marked reduction in cell size and the number and size of abnormal lysosomes, thus indicating the clearance of storage. Our results demonstrate that dermal cells respond to enzyme replacement therapy in MPS I-S, with the clearance of glycosaminoglycan lysosomal accumulation in connective tissue fibroblasts, which had near-normal morphology 21 months after BMT. Therefore, the practice of skin biopsy after BMT in MPS and other metabolic disorders in which dermal cells are involved should be encouraged.

Bone Marrow Transplantation↗
PubMed1991

Neurofilament protein triplet immunoreactivity in the dorsal root ganglia of the guinea-pig.

Immunoreactivity for the neurofilament protein triplet was investigated in neurons of the dorsal root ganglia of the guinea-pig by using a battery of antibodies. In unfixed tissue, nearly all neurons in these ganglia demonstrated some degree of neurofilament protein triplet immunoreactivity. Large neurons generally displayed intense immunoreactivity, whereas most small to medium-sized neurons showed faint to moderate immunoreactivity. Double-labelling immunofluorescence demonstrated that most antibodies to the individual subunits of the neurofilament protein triplet had the same distribution and intensity of labelling in sensory neurons. Increasing durations of tissue fixation in aldehyde solutions selectively diminished neurofilament protein triplet immunoreactivity in small to medium-sized neurons. Double-labelling with neurofilament protein triplet antibodies in combination with antibodies to other neuronal markers, such as neuron-specific enolase, substance P and tyrosine hydroxylase, showed that tissue processing conditions affect the degree of co-localization of immunoreactivity to the neurofilament protein triplet and to these other neuronal markers. These results indicate that, with a judicious manipulation of the duration of tissue fixation, neurofilament protein triplet immunoreactivity can be used in combination with other neuronal markers to distinguish groups of neurons according to their size and chemical coding.

Animals↗
PubMed1991

Calretinin immunoreactivity in cholinergic motor neurones, interneurones and vasomotor neurones in the guinea-pig small intestine.

Immunoreactivity for calretinin, a calcium-binding protein, was studied in neurones in the guinea-pig small intestine. 26 +/- 1% of myenteric neurones and 12 +/- 3% of submucous neurones were immunoreactive for calretinin. All calretinin-immunoreactive neurones were also immunoreactive for choline acetyltransferase and hence are likely to be cholinergic. In the myenteric plexus, two subtypes of Dogiel type-I calretinin-immunoreactive neurones could be distinguished from their projections and neurochemical coding. Some calretinin-immunoreactive myenteric neurones had short projections to the tertiary plexus, and hence are likely to be cholinergic motor neurones to the longitudinal muscle. Some of these cells were also immunoreactive for substance P. The remaining myenteric neurones, immunoreactive for calretinin, enkephalin, neurofilament protein triplet and substance P, are likely to be orad-projecting, cholinergic interneurones. Calretinin immunoreactivity was also found in cholinergic neurones in the submucosa, which project to the submucosal vasculature and mucosal glands, and which are likely to mediate vasodilation. Thus, calretinin immunoreactivity in the guinea-pig small intestine is confined to three functional classes of cholinergic neurones. It is possible, for the first time, to distinguish these classes of cells from other enteric neurones.

Animals↗
PubMed1991

Changes in protein phosphorylation in wild-type and nickel-resistant cells and their involvement in morphological elongation.

Treatment of wild-type Balb/c-3T3 cells with NiCl2 or N6,2-O-dibutyl-adenosine 3',5'-monophosphate (Bt2-cAMP) resulted in a high degree and frequency of cellular elongation. Nickel-resistant Balb/c-3T3 cells (B200) treated with Bt2-cAMP elongated at the same exposure concentration as wild-type cells. In contrast, treatment of the nickel-resistant cells with both non-cytotoxic and cytotoxic doses of NiCl2 failed to induce elongation. Nickel-resistant cells had two-thirds of the total protein-phosphorylation activity of wild-type cells. Both cAMP and NiCl2 enhanced phosphorylation of specific proteins in intact wild-type cells as detected by 32p autoradiography of these proteins separated on two-dimensional gels. A nickel-dependent phosphorylation of specific proteins is seen following NiCl2 treatment of wild-type cells but was not observed in B200 cells. In contrast, the pattern of Bt2-cAMP-stimulated protein phosphorylation was quite similar in both wild-type and nickel-resistant cells. Although it is unclear at present how nickel ions affect the cellular protein-phosphorylation system, these results suggested that targets controlling cellular elongation are sensitive to nickel, are altered in nickel-resistant cells and appear to involve protein phosphorylation. Further characterization of these targets may help in understanding the mechanisms of nickel carcinogenesis.

3T3 Cells↗
PubMed1991

Role of repeated exposure to morphine in determining its affective properties: place and taste conditioning studies in rats.

Male Sprague Dawley rats were injected daily with saline (morphine naive rats) or 20 mg/kg morphine (morphine experienced rats), starting at least 12 days before training. Subsequent place and taste conditioning indicated that 2.5 mg/kg morphine caused a significant increase in the amount of time spent on the least preferred side by morphine experienced but not by morphine naive rats; furthermore, saccharin consumption was markedly decreased and slightly increased by 10-20 mg/kg morphine in naive and experienced rats, respectively. It was concluded that morphine experience enhances the reinforcing efficacy of morphine and broadens the conditions under which the drug is reinforcing; thus it possibly increases morphine abuse potential.

Animals↗
PubMed1991

Differences in the effect of vitamin E on nickel sulfide or nickel chloride-induced chromosomal aberrations in mammalian cells.

Vitamin E (alpha-tocopherol succinate) pretreatment significantly inhibited formation of some but not all of the chromosomal aberrations induced by crystalline NiS particles but it did not affect the chromosome damage induced by NiCl2. Crystalline NiS particles are phagocytized by cells in contrast to water-soluble NiCl2 which enters the cells by a different mechanism. These and other previous studies suggest that the phagocytosis of crystalline NiS particles produces genetic damage as a consequence of oxygen radicals resulting from the irritant particle effect and also from the high intracellular levels of nickel ions dissolving from the endocytized particles.

Animals↗