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M Costa

Publications and source records attributed to M Costa.

At least 451 records · Page 25Linked to original sources
PubMed1985

Light and electron microscopic immunocytochemistry of the same nerves from whole mount preparations.

A technique for performing correlated light and electron microscopic immunocytochemical studies on whole mount preparations has been developed using myenteric plexus from guinea pig small intestine as a model. With this method a structure containing a particular antigen can first be located by light microscopy and then examined with the electron microscope. Pieces of intestine pinned on balsa were incubated in oxygenated Krebs solution at 37 degrees C for 90-120 min and then fixed for 1 hr at room temperature in 4% formaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.4. The tissue was washed vigorously in several changes of 50% ethanol until the picric acid had been removed, stored overnight in phosphate buffer, and then exposed to 0.1% sodium cyanoborohydride in buffer for 30 min. Vasoactive intestinal peptide (VIP) was localized in separated layers containing myenteric plexus and longitudinal muscle using the peroxidase-antiperoxidase technique with imidazole intensification of the diaminobenzidine reaction product. At the light microscope level, tissue stained by this technique showed VIP-immunoreactive nerve cell bodies and processes throughout the thickness of the myenteric ganglia in numbers approximately equivalent to those seen in whole mounts processed by an established technique for the light microscopic demonstration of VIP, which does not involve exposure of tissue to glutaraldehyde. VIP-immunoreactive structures that were first identified at the light microscope level were subsequently examined at the electron microscope level. VIP-immunoreactive axon profiles were found to form synapses on both immunoreactive and nonimmunoreactive myenteric neurons. The fine structural appearance of the different cell types present in whole mount preparations prepared by this method was similar to that seen in conventionally fixed tissue, except that free and bound ribosomes were absent from the tissue processed for immunocytochemistry. The method described here is reliable and no more difficult than presently available methods for preembedding electron microscopic immunocytochemistry on sections. Its main advantage is that immunoreactive structures for ultrastructural study can be selected from the entire population of chemically identified nerves within a whole mount rather than from a smaller sample present within a section. This technique is applicable to other tissues that can be stained immunohistochemically in whole mounts. The fixation and penetration enhancement procedures can also be adapted for immunocytochemical studies on vibratome or frozen sections.

Animals↗
PubMed1985

Induction of chromosomal damage in Chinese hamster ovary cells by soluble and particulate nickel compounds: preferential fragmentation of the heterochromatic long arm of the X-chromosome by carcinogenic crystalline NiS particles.

Treatment of intact Chinese hamster ovary cells with crystalline NiS and NiCl2 resulted in the induction of chromosomal aberrations which included gaps, breaks, and exchanges. The incidence of these aberrations increased in a time- and concentration-dependent fashion. NiCl2 was more potent in inducing chromosomal aberrations in cells that were maintained with a salts/glucose medium during metal treatment than when cells were treated in culture growth medium. Chromosomal aberrations induced by NiCl2 occurred randomly among the autosomal arms; however, the heterochromatic centromeric regions of the chromosomes were preferentially damaged. In addition to inducing the same type of aberrations found with NiCl2, crystalline NiS particles also caused the selective fragmentation of the heterochromatic long arms of the X-chromosomes. This fragmentation was attributed to the difference in the mechanism of delivery of nickel ions from phagocytized crystalline NiS particles which aggregate around the nuclear membrane and release large amounts of nickel ions from a dissolving phagocytized particle. Previous studies have demonstrated that treatment of intact cells with crystalline NiS particles produces a considerably higher level of nickel in the nucleus compared with similar exposure to water-soluble NiCl2. Since heterochromatin is known to form the inside lining of the interface nucleus, nickel ions, as they are solubilized from a phagocytized particle and enter the nucleus, are likely to encounter heterochromatin before they interact with euchromatin. In contrast, nickel ions derived from NiCl2 do not preferentially accumulate in the cell, and those ions that enter the cell are taken up by a nonphagocytic mechanism. It is proposed that when cells are treated with high levels of NiCl2 in an attempt to achieve the cellular levels of nickel produced by NiS phagocytosis, this overloading results in cytotoxic responses rather than the preferential fragmentation of heterochromatin observed with particles. Since liposome-mediated delivery of NiCl2 also results in fragmentation of the long arm of the X-chromosome, the selective breakage of heterochromatin by NiS particles may be due solely to the mechanism of Ni2+ delivery in cells.

Animals↗
PubMed1985

The conversion of L-cystathionine into the cyclic ketimine form by heated rat liver extracts containing cystathionase and transaminase activities.

Rat liver homogenates heated for 10 min at 60 degrees C incubated with L-cystathionine yield cystathionine ketimine which was identified by its typical UV spectrum and by cochromatography with authentic samples on the amino acid analyzer. Alanine and alpha-amino butyric acid have been also detected among the final products. The reaction is due to heat stable gamma-cystathionase and transaminases present in the extracts. Cystathionase produces alpha-keto butyric acid and pyruvic acid which are then used for the transamination of the remaining cystathionine to yield the ketimine. This is the first report indicating the occurrence in a mammalian tissue of an enzymatic system using cystathionine for reactions differing from the traditional transulfuration to cysteine.

Animals↗
PubMed1985

New enzymatic changes of L-cystathionine catalyzed by bovine tissue extracts.

L-Cystathionine was used as substrate for enzyme systems prepared by heating bovine tissue extracts in the presence of pyruvate at 60 degrees C for 10 min. Analysis of the products indicated that the systems converted L-cystathionine into the cyclic ketimine form which was detected by its spectral properties and by chromatography on the amino acid analyzer. Alanine, alpha-aminobutyrate and cystine were also produced. Pyruvate and alpha-ketobutyrate enhance the production of the ketimine by liver, kidney and heart extracts, and are necessary for the brain extracts: alpha-Ketoglutarate is much less effective and its presence favors the production of homocystine by all the extracts. Homocystine was found in the brain incubates when any of the ketoacids assayed were added. The overall reaction is explained by the action of heat stable cystathionine gamma-lyase and beta-synthase which produce alpha-ketobutyrate and pyruvate used for the transamination of the remaining cystathionine to the monoketoacid. This last compound cyclizes spontaneously into the ketimine form thus avoiding the removal of the second amino group. This represents a new nontransulfurative path leading to the production of a seven membered etherocyclic product whose biochemical implications are yet unexplored.

Amino Acids↗
PubMed1985

Preferential DNA-protein cross-linking by NiCl2 in magnesium-insoluble regions of fractionated Chinese hamster ovary cell chromatin.

Intracellular nickel ions (Ni2+) have been shown to cause single-strand breaks in DNA, that were rapidly repaired, and DNA-protein cross-links, that persisted for at least 24 h following removal of extracellular ionic nickel. In this study, we have used the techniques of alkaline elution, chromatin fractionation, and sodium dodecyl sulfate:polyacrylamide gel electrophoresis to examine the DNA-protein cross-linking induced by NiCl2 in Chinese hamster ovary cells. Continuous treatment of logarithmically growing Chinese hamster ovary cells with 2.5 mM NiCl2 in complete medium resulted in DNA single-strand breaks within 1 h, followed by a time-dependent increase in the induction of DNA-protein cross-links at 2, 3, and 6 h. Since the entry of nickel into cells was maximal within 2 h of exposure, the time delay for the formation of DNA-protein cross-links was not limited by metal uptake. The nickel-induced DNA-protein cross-linking appeared to require active cell cycling, since single-strand breaks but no cross-linking could be detected in confluent cells treated with 1, 2.5, or 5 mM NiCl2 for 3 h. DNA-protein cross-linking induced by nickel occurred in late S phase of the cell cycle. High-molecular-weight nonhistone chromatin proteins and possibly histone H1 migrating at the Mr 30,000 range became cross-linked to DNA after treatment of cells with NiCl2. All nickel-cross-linked proteins were concentrated in the magnesium-insoluble regions of fractionated chromatin and were stable to urea, 2-mercaptoethanol, and Nonidet P-40. Some proteins (Mr 48,000, 52,000, 55,000, 70,000, and 95,000), the association of which with DNA was also stable to Sarkosyl, salt, and EDTA, were detectable in DNA rigorously fractionated from untreated cells. Nickel therefore appeared to cause the cross-linking of proteins that normally reside in close association with DNA. Alterations of the normal association of these proteins with DNA by nickel may be an early event in the nickel transformation process.

Animals↗
PubMed1985

Plasma exchange in progressive primary IgA nephropathy.

Five patients with progressive primary IgA nephropathy (PIgAGN) were treated by plasma-exchange (PE) combined with immunosuppressive drugs. Circulating IgA-containing immune complexes (IgAIC), detected by a specific conglutinin solid phase assay, were monitored. Two patients with acute nephritic syndrome and rapidly progressing course, crescent formations and high levels of IgAIC had substantial lasting clinical improvement after several PE, with a fall in IgAIC levels. Another rapidly progressive case with marked sclerotic changes and a longer history of nephritic syndrome, but with normal levels of IgAIC, did not show any clinical improvement after PE. Two patients with a PIgAGN diagnosed several years before and presenting slowly evolutive course had no substantial clinical benefit from PE treatment. IgAIC levels, very high before PE temporarily decreased, but returned to the previous values after the end of the treatment. We conclude that PE combined with immunosuppressive treatment may be of clinical benefit for cases with acute nephritic syndrome of recent onset who still have high levels of IgAIC, even when important crescent formations are present.

Adolescent↗
PubMed1984

Measurement and chromatographic characterization of vasoactive intestinal peptide from guinea-pig enteric nerves.

The material exhibiting immunoreactivity for vasoactive intestinal peptide in guinea-pig enteric nerves has been characterized by high-performance liquid chromatography in three modes: reversed-phase, cation-exchange and gel permeation. In each case a major portion of the material contained in acetic acid extracts of guinea-pig gut showed the same chromatographic properties as the synthetic porcine peptide of defined amino acid sequence. It is therefore concluded that this immunoreactive material is authentic vasoactive intestinal peptide. The study illustrates a number of the problems encountered in attempting to characterize, and measure reliably, peptides in tissue extracts.

Animals↗
PubMed1984

[Hemispheric asymmetries in cortical electrical activity during sleep. I].

The hypothesis of a predominance of the right hemisphere in stage REM as compared to NREM has been tested through a spectral analysis of the EEG recorded from left (T3) and right (T4) temporal sites in 5 young healthy right-handed male subjects. Variations in the asymmetry coefficient R - L/R + L in different sleep stages have been analyzed by one way ANOVAs and Sheffé's tests. The hypothesis of a progressive increase in left hemisphere activity throughout different REM cycles as one approaches final awakenings have been investigated by comparing variations in the asymmetry coefficient for epochs of REM and stage 2 NREM sampled in different phases of the REM cycle. EEG results do not support either the hypothesized stage dependent or cycle dependent variation in EEG activity during sleep. We question whether variations in EEG amplitude and synchronization can be used as indices of hemispheric asymmetries during sleep.

Cerebral Cortex↗
PubMed1984

Use of mammalian DNA repair-deficient mutants to assess the effects of toxic metal compounds on DNA.

Wild-type and repair-deficient cell lines ( EM9 ) of Chinese Hamster Ovary cells were utilized to assess cytotoxic responses towards metals that produce lesions in DNA. Alkaline elution studies indicated that both CaCrO4 and HgCl2 induced single-strand breaks in the DNA. CaCrO4 and HgCl2 treatments of intact Chinese hamster ovary cells also caused the induction of DNA cross links. The mutant cells, which are thought to have a defect in the repair polymerase enzyme and therefore exhibit greater sensitivity towards a variety of agents that produce lesions in the DNA such as X-rays and ultraviolet-light, also displayed a greater sensitivity, compared to wild-type cells, towards the cytotoxic response of HgCl2 and CaCrO4 . For example, the IC50 (concentration producing a 50% growth inhibition) following exposure for 6-hr to CaCrO4 or 1 hr to HgCl2 was 3.4-fold or 1.8- to 3.9-fold greater in wild-type cells compared to repair-deficient cells respectively. Mutant cells compared to wild-type cells were not more sensitive to growth inhibition by agents whose primary site of action was not at the DNA level (i.e. amphotericin B, trifluoroperazine and cycloheximide). The DNA crosslinks induced by exposure to 10 microM CaCrO4 for 6 hr were almost completely repaired in wild-type cells within 24 hr, whereas in similarly exposed mutant cells this lesion was initially more pronounced and was only partially repaired following a 24-hr recovery period in the absence of CaCrO4 . The repair of single-strand breaks induced by CaCrO4 was more rapid and similar in both wild-type and mutant cells. Since Hg(II) inhibits repair of single-strand breaks, we could not study repair of this lesion induced by this agent; however, at very low concentrations (1 microM) binding of 203Hg(II) to DNA was greater in the mutant cells compared to the wild-type cells. Following removal of 203Hg(II) from the media, mutant cells generally retained more 203Hg bound to DNA relative to the total 203Hg(II) present in the cell. These results demonstrate that an important toxic action of CaCrO4 and HgCl2 involves injury to DNA since the concentrations of these metals causing measurable DNA damage were consistent with their respective cytotoxic concentrations and DNA repair-deficient mutants displayed both enhanced cytotoxicity and decreased repair of metal-induced lesions.

Amphotericin B↗
PubMed1984

Somatostatin in human enteric nerves. Distribution and characterization.

Somatostatin-immunoreactive nerves and endocrine cells were localized by use of immunohistochemistry in human stomach, small and large intestine. The nature of the immunoreactivity in acid extracts of separated layers of intestine was determined with separation by high pressure liquid chromatography followed by detection with radioimmunoassay; authentic somatostatin-14 was found in the external musculature, which contains nerves, and in the submucosa and mucosa, which contain both nerve fibres and endocrine cells. The distribution of somatostatin nerves in the gastric antrum, duodenum, jejunum, ileum, ascending and sigmoid colon, and rectum is described. In the intestine many positive perikarya and fine varicose fibres were seen. Mucosal fibres formed a sub-epithelial plexus and a looser network in the lamina propria; this nerve supply was less dense in the large intestine. Submucous ganglia contained positive perikarya and terminals; many terminals formed pericellular baskets, mainly around non-reactive cells. A small number of nerve fibres were associated with submucosal blood vessels. The innervation of the circular and longitudinal muscle was sparse. Positive nerve terminals were seen in the myenteric plexus, although fewer than in the submucous ganglia; positive perikarya were scarce in myenteric ganglia. Somatostatin-immunoreactive nerves were found in the muscle layers and myenteric plexus of the gastric antrum, but were not detected in the antral mucosa and all layers of the gastric body. The distribution of human enteric somatostatin nerves is compared to that in small laboratory animals, and possible roles for these nerves are discussed.

Colon↗
PubMed1984

Choline acetyltransferase- and peptide immunoreactivity of submucous neurons in the small intestine of the guinea-pig.

The peptides cholecystokinin (CCK), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP), and the synthesizing enzyme for acetylcholine, choline acetyltransferase (ChAT) were localized immunohistochemically in nerve cell bodies of the submucous ganglia in the small intestine of the guinea-pig. VIP-like immunoreactivity was found in 45% of submucous neurons. ChAT immunoreactivity was observed in a separate group of nerve cells, which made up 54% of the total population. There were three subsets of neurons immunoreactive for ChAT: (1) ChAT neurons that also contained immunoreactivity for each of the peptides CCK, SOM and NPY, representing 29% of all submucous neurons; (2) ChAT neurons that also contained SP-like immunoreactivity, representing 11% of all submucous neurons, and (3) ChAT cells that did not contain any detectable amount of the peptides that were localized in this study.

Animals↗
PubMed1984

Distribution and projections of neurons with immunoreactivity for both gastrin-releasing peptide and bombesin in the guinea-pig small intestine.

Bombesin-like and gastrin-releasing peptide (GRP)-like immunoreactivities were localized in nerves of the guinea-pig small intestine and celiac ganglion with the use of antibodies raised against the synthetic peptides. The anti-bombesin serum (preincubated to avoid cross reactivity with substance P) and the anti-GRP serum revealed the same population of neurons. Preincubation of the anti-bombesin serum with bombesin abolished the immunoreactivity in nerves while absorption of the anti-GRP serum with either bombesin or the 14-27 C-terminal of GRP only reduced the immunoreactivity. The immunoreactivity was abolished by incubation with GRP 1-27. Immunoreactive nerves were found in the myenteric plexus, circular muscle, submucous plexus and in the celiac ganglion. Faintly reactive nerve cell bodies were found in the myenteric ganglia (3.2% of all neurons) but not in submucous ganglia. After all ascending and descending pathways in the myenteric plexus had been cut, reactive terminals disappeared in the myenteric plexus, circular muscle (including the deep muscular plexus) and the submucous plexus on the anal side. After the mesenteric nerves were cut no changes were observed in the intestinal wall but the reactive fibres in celiac ganglia disappeared. It is deduced that GRP/bombesin-immunoreactive nerve cell bodies in myenteric ganglia project from the myenteric plexus to other myenteric ganglia situated further anally (average length 12 mm), anally to the circular muscle (average length 9 mm), anally to submucous ganglia (average length 13 mm) and external to the intestine to the celiac ganglia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Characterization of DNA lesions produced by HgCl2 in cell culture systems.

HgCl2 is extremely cytotoxic to Chinese hamster ovary (CHO) cells in culture since a 1-h exposure to a 75- microM concentration of this compound reduced cell plating efficiency to 0 and cell growth was completely inhibited at 7.5 microM . The level of HgCl2 toxicity depended upon the culture incubation medium and has previously been shown to be inversely proportional to the extracellular concentration of metal chelating amino acids such as cysteine. Thus, HgCl2 toxicity in a minimal salts/glucose maintenance medium was about 10-fold greater than the toxicity in McCoy's culture medium. The HgCl2 toxicity in the latter medium was 3-fold greater than that in alpha-MEM which contains more of the metal chelating amino acids. When cells were exposed to HgCl2 there was a rapid and pronounced induction of single strand breaks in the DNA at time intervals and concentrations that paralleled the cellular toxicity. The DNA damage was shown to be true single strand breaks and not alkaline sensitive sites or double strand breaks by a variety of techniques. Consistent with the toxicity of HgCl2, the DNA damage under an equivalent exposure situation was more pronounced in the salts/glucose than in the McCoy's medium and more striking in the latter medium than in alpha-MEM. Most of the single strand breaks occurred within 1 h of exposure to the metal. We believe that the DNA damage caused by HgCl2 leads to cell death because the DNA single strand breaks are not readily repaired. DNA repair activity measured by CsCl density gradient techniques was elevated above the untreated levels at HgCl2 concentrations that produced little measurable binding of the metal to DNA or few single strand breaks assessed by the alkaline elution procedure. DNA repair activity decreased at HgCl2 concentrations that produced measurable DNA binding and single strand breaks. These irreversible interactions of HgCl2 with DNA may be responsible for its cytotoxic action in cells.

Animals↗
PubMed1984

Time course of effect of capsaicin on ultrastructure and histochemistry of substance P-immunoreactive nerves associated with the cardiovascular system of the guinea-pig.

Capsaicin, a neurotoxin which depletes substance P from primary afferent nerve fibres, was injected systematically into adult guinea pigs. The effects of capsaicin were studied by immunohistochemistry, electron microscopy and radioimmunoassay at times from 5 min to 1 year. Within 5 min after a single injection of capsaicin (50 mg/kg) substance P immunofluorescence appeared less intense and less homogeneous than normal (i.e. it appeared granular). Large nerve trunks remained evident, but there were fewer fine single nerve fibres. With increasing time there was a progressive decrease in the number of immunoreactive fibres; by 4 h there was a marked reduction in the number of fibres and by 24 h only an occasional fibre was evident. In animals sacrificed 2 or more hours after treatment large brightly fluorescent swellings were seen in many nerves. Depletion of substance P-immunoreactivity persisted for as long as 365 days after treatment. Electron microscopy revealed alterations in capsaicin-sensitive nerve fibres within 5 min after treatment. Many fibres appeared swollen and there was disruption of their internal morphology, e.g. loss of microtubules and filaments and presence of an amorphous flocculent material in the axons. With increasing time after treatment, electron-dense profiles, indicative of degenerating nerve fibres, were commonly seen associated with Schwann cells. These findings demonstrate that the effects of systemic administration of capsaicin to adult guinea pigs occur rapidly in capsaicin-sensitive nerve fibres. The long lasting depletion of substance P-containing fibres is due to their degeneration.

Animals↗
PubMed1984

Excitatory synaptic potentials due to activation of neurons with short projections in the myenteric plexus.

Intracellular microelectrodes have been used to examine the effects, on excitatory inputs to myenteric nerve cells, of lesions of intrinsic pathways in the myenteric plexus of the guinea-pig small intestine. The lesions consisted of circumferential cuts (myotomies) which severed the external musculature to the depth of the submucosa and thus interrupted pathways in the myenteric plexus. Sufficient time was allowed between creating the lesions and recording from the neurons for the endings of severed neurites to degenerate and this was confirmed histochemically by examining the distribution of varicose fibres with 5-hydroxytryptamine immunoreactivity in myenteric ganglia from which recordings were made. Two types of excitatory input, eliciting fast and slow excitatory post-synaptic potentials, respectively, were demonstrable in response to focal stimulation of nerves in the ganglia from which recordings were made. There were no differences in the proportions of neurons in which fast or slow excitatory synaptic potentials were evoked in unoperated preparations (controls), in islands 1.5-4 mm wide between myotomies, or within 1 mm on the oral or anal sides of myotomies. Possible differences in the amplitudes, durations at half amplitude, and threshold numbers of stimuli for initiation of slow excitatory synaptic potentials were analyzed. The only significant differences were found when data from control and oral areas were pooled and compared with combined data from island and anal areas (this assessed differences that could arise from severing nerve fibres running from oral to anal).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Somatostatin is present in a subpopulation of noradrenergic nerve fibres supplying the intestine.

Somatostatin and dopamine beta-hydroxylase have been localized in the coeliaco-mesenteric ganglia, in mesenteric nerves and in the wall of the guinea-pig small intestine. Nerve lesions were used to determine the sources of the nerves. Nerve cell bodies in the coeliaco-mesenteric ganglia with immunoreactivity for both somatostatin and dopamine beta-hydroxylase project to the intestine via the mesenteric nerves. Most of their terminals are in the submucous ganglia, where they make up the full complement of noradrenergic terminals, and in the mucosa where other noradrenergic terminals, not containing somatostatin immunoreactivity, are also present. The small number of noradrenergic fibres present in the tertiary component of the myenteric plexus and in the circular muscle all show immunoreactivity for somatostatin. The noradrenergic fibres supplying the mesenteric and intestinal blood vessels and those ramifying in the myenteric ganglia do not contain somatostatin. The numerous somatostatin-immunoreactive nerves in the enteric plexuses that do not contain dopamine beta-hydroxylase come from enteric nerve cell bodies. These results, considered in the context of other published work, indicate that post-ganglionic sympathetic noradrenergic neurons are chemically coded according to the target tissue they supply and suggest that neurons that were hitherto thought to be neurochemically equivalent, but which serve different functions, are in fact chemically distinct.

Adrenergic Fibers↗