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Biomedical subjects

C A Gonçalves

Publications and source records attributed to C A Gonçalves.

At least 19 recordsLinked to original sources
PubMed2006

Confounding factors in water EMG recordings: an approach to a definitive standard.

This work presents a study on the influence of the aqueous environment on the surface EMG (sEMG) signal recorded in bipolar montage from the abductor pollicis brevis muscle, when only the forearm is immersed in water. Ten men, 30.1+/-4.0 (mean +/- SD) years old, performed ten 2-s 40% MVC isometric contractions of the abductor pollicis brevis muscle in two controlled environments (air and water, at a temperature of 32 degrees C). They were always equipped with electrodes protected with a waterproof adhesive tape. No significant variations (paired Wilcoxon test) due to the environments were observed in the median frequency of the power spectrum (MDF) and in the root mean square (RMS) value of the sEMG signal. These results allow us to assess the methodological criteria to properly record sEMG signals in water and provide the basis to explain different findings obtained by other authors.

Adult↗
PubMed2005

Transitory gliosis in the CA3 hippocampal region in rats fed on a ketogenic diet.

The ketogenic diet (KD) is a high-fat, low-protein and low-carbohydrate diet included as medical practice against seizure disorders, particularly in children refractory to conventional anti-epileptic drug treatment. However, the molecular basis of its therapeutic effect remains unclear. Considering the growing evidence for the importance of glial cells for neuronal development, survival and plasticity, we investigated astrocyte protein markers from KD fed rats, in different regions of hippocampus, a brain structure commonly involved in seizure disorders. We found a transitory increment in GFAP in the CA3 hippocampal region, but not in the CA1 or dentate gyrus (DG). This change was not accompanied by changes in S100B content or glutamine synthetase activity. In order to evaluate possible hippocampal involvement we investigated spatial-cognitive behavior using the water-maze task. No changes were observed. This transitory gliosis in CA3 could be related to, or precede, other associated changes proposed to be involved in the attenuation of seizure disorders. These data reinforce the importance of hippocampal astrocytes as cell targets during KD feeding.

Animals↗
PubMed2003

Interictal serum S100B levels in chronic neurocysticercosis and idiopathic epilepsy.

OBJECTIVE: To assess whether serum S100B levels could reflect a glial response in patients with epilepsy secondary to neurocysticercosis (NCC) and with idiopathic epilepsy. SUBJECTS AND METHODS: Serum S100B levels were measured using an immunoluminometric assay in 20 patients with focal epilepsy related to chronic NCC (NCC group), and 19 patients with focal epilepsy (EPI group), matched by epidemiological and clinical data. Epileptic patients were compared with 20 healthy controls (CON group) matched by age and sex. RESULTS: No difference was observed in S100B levels among NCC, EPI and CON groups (P>0.39). Serum S100B levels were not affected by antiepileptic drugs, frequency and type of seizures. Preliminarily, significantly higher levels of S100B were observed in patients with bilateral electroencephalographic (EEG) findings than in patients with unilateral and normal EEG findings (P<0.05). CONCLUSION: Serum S100B is normal in patients with focal epilepsy related or not to chronic NCC.

Acute Disease↗
PubMed2002

Elevated serum S100B protein in drug-free bipolar patients during first manic episode: a pilot study.

S100B protein is a calcium-binding protein mostly derived from glial cells, which exerts trophic or toxic effects on neural cells depending on its concentration. Since serum S100B levels has been tested as a potential marker in neuropsychiatric disorders, and structural abnormalities on glial cells have been recently associated with bipolar disorder patients, we conducted this preliminary study to examine if S100B serum levels are altered during first manic episode. We quantitated S100B in serum of 40 subjects (20 unmedicated patients during manic episode and 20 healthy matched controls). The mean+/-S.D. values for S100B for bipolar subjects were 0.065+/-0.068 microg/l and 0.018+/-0.029 microg/l for healthy controls. Increased levels of S100B in bipolar mania was statistically significant (Wilcoxon signed ranks test, Z=-2.45, P=0.01). These preliminary findings suggest that mania may increase the levels of S100B in serum of bipolar disorder patients, which could be related to adaptative neural mechanisms in bipolar mania.

Adult↗
PubMed2001

Adherence of Giardia lamblia trophozoites to Int-407 human intestinal cells.

Attachment of Giardia lamblia trophozoites to enterocytes is essential for colonization of the small intestine and is considered a prerequisite for parasite-induced enterocyte dysfunction and clinical disease. In this work, coincubation of Giardia with Int-407 cells, was used as an in vitro model to study the role of cytoskeleton and surface lectins involved in the attachment of the parasite. This interaction was also studied by scanning and transmission electron microscopy. Adherence was dependent on temperature and was maximal at 37 degrees C. It was reduced by 2.5 mM colchicine (57%), mebendazole (10 microg/ml) (59%), 100 mM glucose (26%), 100 mM mannose (22%), 40 mM mannose-6-phosphate (18%), and concanavalin A (100 microg/ml) (21%). No significant modification was observed when Giardia was pretreated with cytochalasins B and D and with EDTA. Giardia attachment was also diminished by preincubating Int-407 cells with cytochalasin B and D (5 microg/ml) (16%) and by glutaraldehyde fixation of intestinal cells and of G. lamblia trophozoites (72 and 100%, respectively). Ultrastructural studies showed that Giardia attaches to the Int-407 monolayer predominantly by its ventral surface. Int-407 cells contact trophozoites with elongated microvilli, and both trophozoite imprints and interactions of Giardia flagella with intestinal cells were also observed. Transmission electron microscopy showed that Giardia lateral crest and ventrolateral flange were important structures in the adherence process. Our results suggest a combination of mechanical and hydrodynamic forces in trophozoite attachment; surface lectins also seem to mediate binding and may be involved in specific recognition of host cells.

Animals↗
PubMed2000

Immunocontent and secretion of S100B in astrocyte cultures from different brain regions in relation to morphology.

Primary astrocyte cultures prepared from neonatal hippocampus, cerebral cortex and cerebellum were morphologically distinct. Cells from hippocampus and cortex were almost entirely protoplasmic, whereas cerebellar astrocytes had many processes; in the absence of serum these differences were accentuated. We compared the immunocontent and secretion of the mitogenic protein S100B in these cultures. Immunocontent was 2.5 times higher in cerebellar astrocytes than in hippocampal or cortical astrocytes. Cells from all three regions secreted S100B under basal conditions, but the secretion rate was higher in cerebellar astrocytes. Secretion depended on protein synthesis and was increased by incubation with forskolin or lysophosphatidic acid in mechanisms which were additive. The stellate morphology induced by forskolin was reversed by lysophosphatidic acid in hippocampal but not in cerebellar cultures, suggesting that S100B secretion was not associated with a process-bearing phenotype of astrocytes.

Animals↗
PubMed2000

Decreased S100-beta protein in schizophrenia: preliminary evidence.

The S100 proteins are a family of calcium-binding proteins found in the central and peripheral nervous systems of vertebrates. S100beta, the most abundant member of this family in the CNS, mediates calcium signal transduction, and shows neurotrophic, gliotrophic and mitogenic actions that influence the development and maintenance of the nervous system. Another member of the S100 family (S100A10) was found to modulate phospholipid turnover by inhibiting the activity of enzyme phospholipase A2 (PLA2). We determined the concentration of S100beta protein in the plasma of 23 medicated schizophrenic patients and 23 healthy controls. S100beta protein accounts for 96% of the total S100 in the brain. Schizophrenic patients showed reduced S100beta concentrations (p=0.003), and this finding was not related to clinical variables or to intake of antipsychotic medication. Decreased S100beta could be related to the findings of increased PLA2 activity and to brain maldevelopment in schizophrenia. These results are discussed further with respect to the role of adenosine in S100beta release.

Adult↗
PubMed2000

Extracellular S100B protein modulates ERK in astrocyte cultures.

S100B is a calcium binding protein expressed and secreted by astrocytes. Extracellular S100B stimulates the proliferation of astroglial cells and the survival of neurons. Extracellular signal regulated kinases (ERK) are involved in the transduction of proliferating signals in astrocytes. Here we report that S100B significantly increases the activity of ERK in primary cultures of astrocytes, a result which may be related to previous observations of the effect of this protein on glial proliferation. We further confirm that conversion of S100B to its covalent dimer by oxidation of cysteine residues increases its extracellular activity. Although we cannot exclude S100B involvement in other mechanisms of signal transduction, these results suggest that ERK activity in astrocytes is modulated by extracellular S100B.

Animals↗
PubMed2000

GFAP phosphorylation studied in digitonin-permeabilized astrocytes: standardization of conditions.

Cycles of assembly/disassembly of the intermediate filaments of astrocytes are modulated by the phosphorylation of glial fibrillary acidic protein (GFAP). The sites on GFAP are localized at the N-terminal where they are phosphorylated by cAMP-dependent and Ca(2+)-dependent protein kinases. Phosphorylation of GFAP has been investigated in brain slices, astrocyte cultures, cytoskeletal fractions and purified systems. Here we describe a different approach to study GFAP phosphorylation. We show that permeabilization of astrocytes in culture with digitonin allows direct access to the systems phosphorylating GFAP. Conditions for the permeabilization were established with an assay based on the exclusion of Trypan blue. Incubation of permeabilized cells with cAMP and Ca(2+) increased the phosphorylation state of GFAP. Immunocytochemistry with anti-GFAP showed that permeabilized astrocytes retained their typical flat, fibroblast morphology and exhibited well preserved glial filaments. On incubation with cAMP the filaments apparently condensed to form long processes. The results suggest the approach of studying structural changes in glial filaments in parallel to protein phosphorylation, in the presence of specific modulators of protein kinases and phosphatases has considerable potential.

Adenosine Triphosphate↗
PubMed2000

Digitonin-permeabilization of astrocytes in culture monitored by trypan blue exclusion and loss of S100B by ELISA.

The present protocol details a procedure to permeabilize astrocytes in cultures with digitonin as well as to discuss some data about factors that interfere in permeabilization, particularly divalent cations and nucleotides. Two methods to assess astrocyte permeabilization are described: trypan blue exclusion and ELISA for S100B, a specific protein expressed by these cells. Digitonin-permeabilization of astrocytes has been used to investigate intracellular pools of Ca(2+), internal stores of metabolites, phosphoinositide hydrolysis, and recently we standardized a procedure to study protein phosphorylation (Brain Res. 853 (2000) 32-40). A short incubation time (10 min) with 30 microM digitonin permeabilized at least 75% of cells. A range of media with different ionic nature can be used in cell permeabilization without affecting significantly the extent of permeabilization, but calcium and ATP of the order of 10(-5) M induced a partial resealing which deserves to be considered in assays of permeabilized preparations of astrocytes.

Adenosine Triphosphate↗
PubMed2000

The use of permeabilized cells to assay protein phosphorylation and catecholamine release.

A number of approaches can be used to determine the protein kinases and protein phosphatases acting on particular phosphoproteins in vivo. Cell permeabilization represents one such approach. In this overview we discuss the different permeabilization procedures used in bovine adrenal chromaffin cells and in particular the use of digitonin. The effect of various factors on the extent of digitonin-permeabilization, protein phosphorylation and catecholamine release are also discussed. The factors include the permeabilization medium, the ions such as calcium, and the second messengers, such as cAMP, IP3, cADPR and calmodulin. The effect of specific peptide inhibitors of protein kinases on tyrosine hydroxylase phosphorylation is illustrated. Advantages and disadvantages of cell permeabilization procedures are discussed throughout the text.

Animals↗
PubMed1999

Simultaneous measurement of tyrosine hydroxylase activity and phosphorylation in bovine adrenal chromaffin cells.

A method for simultaneous measurement of tyrosine hydroxylase (TH) activation and phosphorylation in permeabilised and intact bovine adrenal chromaffin cells (BACCs) was established. Permeabilised cells were stimulated with cyclic AMP (1--10 microM) in the presence of [32P]ATP and L-[carboxyl-(14)C]tyrosine. Intact BACCs were preincubated with 32P(i) for 3 h and stimulated with forskolin (1--5 microM) in the presence of L-[carboxyl-(14)C]tyrosine. On stimulation each well was covered with a sealed 'chimney' fitted with a small plastic cup containing 300 microl of 1.0 M NaOH that trapped the 14CO(2) released. TH activity was determined by measuring 14C radioactivity. TH phosphorylation was measured in the same cells by separating the solubilized proteins on SDS PAGE followed by autoradiography and/or HPLC analysis. It was found that H89, a protein kinase A inhibitor, significantly blocked both TH phosphorylation and activation in response to cyclic AMP in permeabilised cells. However, in intact cells, H89 was effective only in respect to forskolin-stimulated TH activity and did not block the forskolin-stimulated TH phosphorylation of Ser-40. The reason(s) for this lack of correlation between TH activation and phosphorylation is presently not understood.

Adrenal Medulla↗
PubMed1998

Regulation of the phosphorylation of glial fibrillary acidic protein (GFAP) by glutamate and calcium ions in slices of immature rat spinal cord: comparison with immature hippocampus.

The effect of glutamate and lack of external Ca2+ on the phosphorylation of the astrocyte cell marker glial fibrillary acidic protein (GFAP) was studied in slices of hippocampus and thoracic spinal cord from immature (P12-16) rats. Confirming previous work with immature hippocampal slices (Wofchuk, S.T. and Rodnight, R., Neurochem. Int., 24 (1994) 517-523; Wofchuk, S.T. and Rodnight, R., Dev. Brain Res., 85 (1995) 181-186), glutamate strongly stimulated GFAP phosphorylation in media with Ca2+ and in media lacking Ca2+ a quantitatively similar stimulation of basal GFAP phosphorylation was observed. By contrast in slices of immature thoracic spinal cord, glutamate had no effect on GFAP phosphorylation and in media lacking Ca2+ phosphorylation of GFAP was inhibited. Since GFAP phosphorylation is Ca2+-dependent and is not stimulated by glutamate in slices of adult hippocampus, the present results suggest that astrocytic functions in the rat spinal cord mature more rapidly than in the hippocampus. The possibility that the difference in the control of GFAP phosphorylation in the two structures is related to differences in the control of GFAP dephosphorylation was investigated by incubating spinal cord slices with the calcineurin inhibitor FK506 in the presence of Ca2+. In contrast to results obtained with hippocampal slices FK506 had no effect on the phosphorylation state of GFAP in spinal cord slices.

Animals↗
PubMed1998

Circadian latency variability of sympathetic skin responses.

Sympathetic skin responses (SSRs) have been increasingly used as tests for autonomic function in the clinical setting. In spite of the known circadian rhythmicity of sympathetic function, however, normative studies have not addressed the possibility of circadian variability of SSR parameters. Ten normal volunteers (7 men, 3 women, aged 19 to 43) had SSR testing performed in the morning, at noon, and in the early evening, and response latencies and amplitudes were compared for the different day periods. Although amplitude values varied in a random fashion, regardless of the time of testing, there was a statistically significant variability in response latencies, which were, on the average, approximately 150 ms shorter in the morning trials, as compared to the early evening ones. This difference was statistically significant (ANOVA, p < 0.01). We propose that circadian variability of SSR latencies should be taken into account in normative studies of SSR parameters.

Adult↗
PubMed1998

The S100B protein inhibits phosphorylation of GFAP and vimentin in a cytoskeletal fraction from immature rat hippocampus.

The S100B protein belongs to a family of small Ca2+-binding proteins involved in several functions including cytoskeletal reorganization. The effect of S 100B on protein phosphorylation was investigated in a cytoskeletal fraction prepared from immature rat hippocampus. An inhibitory effect of 5 microM S100B on total protein phosphorylation, ranging from 25% to 40%, was observed in the presence of Ca2+ alone, Ca2+ plus calmodulin or Ca2+ plus cAMP. Analysis by two dimensional electrophoresis revealed a Ca2+/calmodulin-dependent and a Ca2+/cAMP-dependent inhibitory effect of S100B, ranging from 62% to 67% of control, on the phosphorylation of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The fact that S100B binds to the N-terminal domain of GFAP and that the two proteins are co-localized in astrocytes suggests a potential in vivo role for S100B in modulating the phosphorylation of intermediate filament proteins in glia.

Animals↗
PubMed1997

Calcium-dependent phosphorylation of glial fibrillary acidic protein (GFAP) in the rat hippocampus: a comparison of the kinase/phosphatase balance in immature and mature slices using tryptic phosphopeptide mapping.

In previous work we showed that phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in hippocampal slices from adult rats is dependent on external Ca2+, whereas in slices from immature rats aged 12-16 days postnatal 32P incorporation into GFAP is inhibited by external Ca2+. The nature of this late developmental change in Ca2+ sensitivity for GFAP phosphorylation was investigated in the present work by comparing in immature and adult animals phosphorylation of GFAP by endogenous protein kinase activity in cytoskeletal fractions and tryptic phosphopeptide maps prepared from cytoskeletal fractions labelled with [gamma-32P]ATP and from slices labelled with [32P]phosphate. Cytoskeletal fractions prepared from immature and adult hippocampus both contained endogenous protein kinase activity towards GFAP and other proteins stimulated by Ca2+/calmodulin and by cyclic AMP. The maps of GFAP isolated from the cytoskeletal fractions labelled in the presence of Ca2+/calmodulin were very similar and exhibited two major and several minor phosphopeptides. Comparison with maps derived from these fractions labelled in the presence of cyclic AMP showed that one of the major phosphopeptides was either directly or indirectly phosphorylated by Ca2+/calmodulin-stimulated kinase activity. Maps derived from GFAP isolated from adult slices labelled in the presence of Ca2+ and immature slices labelled in the absence of Ca2+ were qualitatively identical, with minor differences from the cytoskeletal maps. At both ages the slice maps displayed the phosphopeptide phosphorylated through the activity of a Ca2+/calmodulin kinase in the cytoskeletal fractions. By its migration properties this peptide appears to correspond to a sequence containing a site shown by other workers to be phosphorylated in vitro by CaM kinase II, suggesting that even in the absence of external Ca2+, kinase activity directly or indirectly dependent on Ca2+ was occurring in the immature slices. The near identity of the phosphorylation sites at the two ages suggest that the change in Ca2+ sensitivity of GFAP phosphorylation during development is not due to a change in the balance of kinase and phosphatase activities, but rather to a change in the mechanism(s) whereby Ca2+ controls the relative activity of these enzymes.

Animals↗