Biomedical subjects

A L Carvalho

Publications and source records attributed to A L Carvalho.

At least 19 recordsLinked to original sources

PubMed2006

Excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors involves the AP-1 transcription factor.

Cells preferentially expressing GluR4-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors are particularly sensitive to excitotoxicity mediated through non-N-methyl-D-aspartate receptors. However, the excitotoxic signalling pathways associated with GluR4-containing AMPA receptors are not known. In this work, we investigated the downstream signals coupled to excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors, using a HEK 293 cell line constitutively expressing the GluR4flip subunit of AMPA receptors (HEK-GluR4). Glutamate stimulation of GluR4-containing AMPA receptors decreased cell viability, in a calcium-dependent manner, when the receptor desensitisation was prevented with cyclothiazide. The excitotoxic stimulation mediated through GluR4-containing AMPA receptors increased activator protein-1 (AP-1) DNA-binding activity. Inhibition of the AP-1 activity by overexpression of a c-Jun dominant-negative form protected HEK-GluR4 cells against excitotoxic damage. Taken together, the results indicate that overactivation of Ca2+-permeable GluR4-containing AMPA receptors is coupled to a death pathway mediated, at least in part, by the AP-1 transcription factor.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2005

Prognostic and predictive value of the thymidylate synthase expression in patients with non-metastatic colorectal cancer.

AIMS: To assess the value of thymidylate synthase (TS) expression as a predictive factor in the efficacy of adjuvant chemotherapy in colorectal cancer, as well as its independent prognostic value for survival. METHODS: We studied 114 high risk colorectal carcinoma patients (high risk stage II and stage III), distributed in two treatment groups: surgery alone (61 patients) or surgery followed by 5-FU-based chemotherapy (53 patients). TS protein expression in the tumour tissue was assessed by immunohistochemistry. RESULTS: In the surgery alone subgroup, overall survival (OS) at 5 years were 77.5% for the patients with low TS expression, against 57.7% for the patients with high TS expression (p=0.006). Among patients with low TS, there was no difference in OS as a result of whether adjuvant chemotherapy was carried out or not (65.8 vs 77.5%, p=0.29). Among the patients with high TS, there was a significant gain in OS in favor of chemotherapy (87.8 vs 57.7%, p=0.04). Analyzing the complete sample, TS expression was not shown as an independent prognostic factor for survival in the multivariate analysis. CONCLUSIONS: The immunohistochemical TS expression may be used for selecting patients for better adjuvant chemotherapy protocols. In this sample, TS expression was not an independent prognostic factor for survival.

Aged↗

PubMed2004

Pelvic exenteration and sphincter preservation in the treatment of soft tissue sarcomas.

BACKGROUND: Pelvic sarcomas are rare and there are very few effective therapeutic alternatives. A complete resection is considered the main factor associated to a good prognosis, which justifies the employment of a pelvic exenteration (PE) in selected cases. METHODS: Between 1980 and 2000, 96 PE were performed, nine of which were for sarcomas. The clinical characteristics, surgical and anatomopathological aspects and the patients' evolution were described. RESULTS: The median follow-up time was 24 months (ranging from 1 to 57 months). In relation to the sphincters preservation, at least one sphincter was preserved in five patients. There were two post-operative deaths. In the last follow-up, six patients were alive without any evidence of the disease. CONCLUSION: Pelvic exenterations should be performed for the treatment of selected cases of locally advanced pelvic sarcomas. Sphincter preservation may be performed, provided that oncological resection principles are obeyed.

Adult↗

PubMed2004

Two African swine fever virus proteins derived from a common precursor exhibit different nucleocytoplasmic transport activities.

African swine fever virus (ASFV), a large icosahedral deoxyvirus, is the causative agent of an economically relevant hemorrhagic disease that affects domestic pigs. The major purpose of the present study was to investigate the nuclear transport activities of the ASFV p37 and p14 proteins, which result from the proteolytic processing of a common precursor. Experiments were performed by using yeast-based nucleocytoplasmic transport assays and by analysis of the subcellular localization of different green fluorescent and Myc fusion proteins in mammalian cells. The results obtained both in yeast and mammalian cells clearly demonstrated that ASFV p14 protein is imported into the nucleus but not exported to the cytoplasm. The ability of p37 protein to be exported from the nucleus to the cytoplasm of both yeast and mammalian cells was also demonstrated, and the results clearly indicate that p37 nuclear export is dependent on the interaction of the protein with the CRM-1 receptor. In addition, p37 was shown to exhibit nuclear import activity in mammalian cells. The p37 protein nuclear import and export abilities described here constitute the first report of a nucleocytoplasmic shuttling protein encoded by the ASFV genome. Overall, the overlapping results obtained for green fluorescent protein fusions and Myc-tagged proteins undoubtedly demonstrate that ASFV p37 and p14 proteins exhibit nucleocytoplasmic transport activities. These findings are significant for understanding the role these proteins play in the replication cycle of ASFV.

Active Transport, Cell Nucleus↗

PubMed2003

Sites of recurrence in oral and oropharyngeal cancers according to the treatment approach.

OBJECTIVE: The purpose of this study is to evaluate the rates and the sites of tumour recurrence in patients with oral and oropharyngeal carcinomas. DESIGN: This is a retrospective study of a series of cases treated in a single institution. PATIENTS AND METHODS: A series of 2067 patients with oral and oropharyngeal squamous carcinoma, treated from 1954 to 1998 were analysed. The treatment approach was: surgery, 624 cases (30.2%); radiotherapy alone, 729 cases (35.3%); radiotherapy and surgery, 552 cases (26.7%) and radiotherapy and chemotherapy, 162 cases (7.8%). MAIN OUTCOME MEASURES: Tumour recurrence was observed in 1079 patients (52.2%): 561 cases of local recurrences (27.1%); 168 neck recurrences (8.1%); 252 locoregional recurrences (12.2%); 59, distant metastasis (2.9%) and 39 (1.9%), combination of distant metastasis with local, neck or locoregional recurrence. RESULTS: The rates of recurrence varied significantly according to the treatment performed. Oral cavity cancer patients undergoing radiotherapy alone or in combination with chemotherapy presented the highest rates of neck recurrences (22.5 and 40.0%, respectively) for clinical stage (CS) I/II and of local (41.2 and 30.1%) and locoregional (21.7 and 31.1%) recurrences for CS III/IV; yet, for CS III/IV, surgery without neck dissection was associated with the highest rates of neck recurrences (20.7%), but no differences were observed in the rates of local or locoregional recurrences for CS I/II patients. For oropharynx cancer patients with CS I/II there was no difference in the rate of locoregional failures according to the treatment. However, patients with CS III/IV undergoing radiotherapy present a highest rate of local (42.3%) and locoregional (28.8%) failures. CONCLUSION: The results suggest that surgery should be the first option for initial clinical stage oral and oropharyngeal cancers. For advanced cases independently of the site of the tumour, surgery and postoperative radiotherapy should be the standard of care because it is associated with the lowest rates of locoregional recurrence.

Adolescent↗

PubMed2001

Differential postreceptor signaling events triggered by excitotoxic stimulation of different ionotropic glutamate receptors in retinal neurons.

The aim of this work was to investigate whether excitotoxicity induced by overstimulation of different ionotropic glutamate receptors could trigger different intracellular signaling cascades. Cultured chick neuronal retina cells, essentially amacrine-like, were particularly sensitive to the toxicity induced by non-NMDA glutamate receptor agonists. One hour stimulation with 100 microM kainate induced a reduction of cell viability of about 44%, as assessed by the MTT test 24 hr after stimulation. Kainate-induced toxicity was mediated through AMPA receptors. Glutamate (100 microM, 1 hr) reduced cell viability by 26%, essentially acting through N-methyl-D-aspartate receptors. Five hours after stimulation, neuronal retina cells had an apoptotic-like nuclear morphology. In retinal neurons, the excitotoxic stimulation, with either glutamate or kainate, induced a calcium-dependent enhancement of the DNA-binding activity of the activating protein-1 (AP-1) transcription factor, which was maximal 2 hr after stimulation. Glutamate induced a greater increase in the AP-1 DNA-binding activity than did kainate. Supershift assays using antibodies directed against different members of the Fos and Jun protein families showed that the AP-1 complex in retinal neurons includes proteins of the Fos family, namely, Fra-2, c-Jun, and Jun D. The DNA-binding activity of the nuclear factor-kappaB transcription factor was not significantly changed upon excitotoxic stimulation with any agonist. Stimulation of glutamate receptors with 100 microM kainate or 100 microM glutamate for 2 min was sufficient to induce the activation of the extracellular signal-regulated kinase (ERK). Inhibition of the ERK activation with the MEK inhibitors U 0126 and PD 98059 increased the toxicity induced by kainate but was without effect on the toxicity induced by glutamate. These results indicate that, although stimulation with both glutamate receptor agonists increased ERK phosphorylation, only kainate-induced ERK activation correlates with the activation of a survival signaling pathway. Our results suggest that, in chick embryo retinal neurons, the signaling pathways that mediate excitotoxic cell death and neuroprotection are stimulus specific.

Amacrine Cells↗

PubMed2001

Purification, crystallization and identification by X-ray analysis of a prostate kallikrein from horse seminal plasma.

The purification, crystallization and identification by X-ray diffraction analysis of a horse kallikrein is reported. The protein was purified from horse seminal plasma. Crystals belong to space group C2 and the structure was solved by the MIRAS method, with two heavy-atom derivatives of mercury and platinum. X-ray diffraction data to 1.42 A resolution were collected at the ESRF synchrotron-radiation source.

Amino Acid Sequence↗

PubMed2001

Influence of time delay and clinical upstaging in the prognosis of head and neck cancer.

The delayed time period between the first diagnosis of a head and neck cancer and its treatment can affect not only the clinical staging of the disease, but also its prognosis. The time period for clinical upstaging is studied in a retrospective cohort series. A retrospective case/control series was established to assess the prognosis of head and neck cancer patients due to a delay in treatment. In the first part of the study we included 69 patients who had confirmed clinical upstage before starting the oncologic treatment (cases). The survival of this series was compared to a historical control group of 138 patients matched by tumour site and stage, having received treatment within a short period after their first medical evaluation (controls). We observed that the natural evolution of a head and neck cancer was progressively faster, from an initial clinical stage to an advanced clinical stage until it becomes untreatable. It was observed that the delay in beginning oncologic treatment influenced the prognosis of the patients (P=0.030), especially in clinical stage IV patients (P=0.001) and oral cavity cancer patients (P=0.007).

Adult↗

PubMed2000

Ipsilateral neck cancer recurrences after elective supraomohyoid neck dissection.

DESIGN: Retrospective analysis of a case series. SETTING: Referral center, private or institutional practice, hospitalized care. OBJECTIVE: To analyze the level (site) of ipsilateral neck recurrences after supraomohyoid (SOH) dissection in patients with lip, oral, and oropharyngeal cancer treated in a single institution. INTERVENTION: Supraomohyoid neck dissection. PATIENTS AND METHODS: From 1979 to 1997, 154 patients with oral and oropharyngeal carcinoma and no palpable lymph nodes at the neck underwent ipsilateral elective SOH dissection. RESULTS: Tumor sites were the lip, 5 cases (3.3%); oral cavity, 128 cases (83.1%); and oropharynx, 21 cases (13.6%). Tumor stages were T1, 13 cases (8.4%); T2, 77 cases (50.0%); T3, 40 cases (27.0%); and T4, 22 cases (14.3%). There were 7 cases (4.5%) of ipsilateral neck recurrences. Three were beyond the limits of the SOH dissection, and 4 were inside these limits. There was no association of neck recurrences with the pathological status of the lymph nodes. Six of the 7 recurrences were in patients who underwent postoperative radiotherapy. CONCLUSIONS: The incidence of neck recurrence after selective neck dissection was 4.5%, and it occurred either inside (57.1%) or beyond (42.9%) the limits of the selective neck dissection.

Adult↗

PubMed2000

A comparison of radiotherapy or radiochemotherapy with symptomatic treatment alone in patients with advanced head and neck carcinomas.

The choice of palliative treatment and the prognostic factors in unresectable head and neck cancer cases continue to be controversial. In the present study we compared the survival rates of untreated stage IV head and neck cancer patients with cases managed prospectively at A.C. Camargo Hospital for Cancer with neoadjuvant chemotherapy, concomitant chemotherapy or radiotherapy alone. Previous results had shown that while the type of treatment did not influence survival rates (P = 0.706), tumor response to treatment (whether complete, partial or none) significantly influenced survival (P = 0.00002). In the present study we compared the survival rates in the groups with untreated patients (who remained untreated until death) with the same demographic and clinical characteristics of patients receiving treatment. We found that there was a significant difference between the survival rates of the untreated group and those of the treated groups that was independent of the type of treatment performed (P < 0.00001) or the tumor response to treatment (P < 0.0001).

Carcinoma, Squamous Cell↗

PubMed2000

Natural history of untreated head and neck cancer.

This paper describes the characteristics and natural history of the largest reported group of patients with untreated head and neck cancer. From 1953 to 1990, 808 untreated head and neck cancer patients were followed-up until their death. All patients were given supportive care, but specific oncological treatment was not pursued secondary to advanced tumour stage, poor performance status and/or patients' refusal of any treatment. The overall survival ranged from 1 day to 53.8 months (median 3.82 months). Performance status was the most significant predictor of survival (P < 0.001). A subgroup of 357 patients with good performance status and aged less than 70 years old had a 12.9% 1-year survival. Approximately 50% of untreated head and neck cancer patients will die within 4 months of their diagnosis. However, the remaining patients can survive up to 4 or more years, depending on their tumour location, extent, performance status and level of supportive care.

Adult↗

PubMed2000

Regulation of AMPA receptors by phosphorylation.

The AMPA receptors for glutamate are oligomeric structures that mediate fast excitatory responses in the central nervous system. Phosphorylation of AMPA receptors is an important mechanism for short-term modulation of their function, and is thought to play an important role in synaptic plasticity in different brain regions. Recent studies have shown that phosphorylation of AMPA receptors by cAMP-dependent protein kinase (PKA) and Ca2+- and calmodulin-dependent protein kinase II (CaMKII) potentiates their activity, but phosphorylation of the receptor subunits may also affect their interaction with intracellular proteins, and their expression at the plasma membrane. Phosphorylation of AMPA receptor subunits has also been investigated in relation to processes of synaptic plasticity. This review focuses on recent advances in understanding the molecular mechanisms of regulation of AMPA receptors, and their implications in synaptic plasticity.

Animals↗

PubMed1999

Characterization of phosphorylation sites on the glutamate receptor 4 subunit of the AMPA receptors.

Recent studies have suggested that protein phosphorylation of glutamate receptors may play an important role in synaptic transmission. Specifically, the phosphorylation of AMPA receptors has been implicated in cellular models of synaptic plasticity. The phosphorylation of the glutamate receptor 1 (GluR1) subunit of AMPA receptors by protein kinase A (PKA), protein kinase C (PKC), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been characterized extensively. Phosphorylation of this subunit occurs exclusively on the intracellular C-terminal domain. However, the GluR1 subunit C terminus shows low homology to the other AMPA receptor subunits. In this paper we characterized the phosphorylation of AMPA receptor subunit GluR4, using site-specific mutagenesis and biochemical techniques. We found that GluR4 is phosphorylated on serine 842 within the C-terminal domain in vitro and in vivo. Serine 842 is phosphorylated by PKA, PKC, and CaMKII in vitro and is phosphorylated in transfected cells by PKA. Two-dimensional phosphopeptide analysis indicates that serine 842 is the major phosphorylation site on GluR4. In addition, we identified threonine 830 as a potential PKC phosphorylation site. These results suggest that GluR4, which is the most rapidly desensitizing AMPA receptor subunit, may be modulated by phosphorylation.

Amino Acid Sequence↗

PubMed1999

Structural studies by X-ray diffraction on metal substituted desulforedoxin, a rubredoxin-type protein.

Desulforedoxin (Dx), isolated from the sulfate reducing bacterium Desulfovibrio gigas, is a small homodimeric (2 x 36 amino acids) protein. Each subunit contains a high-spin iron atom tetrahedrally bound to four cysteinyl sulfur atoms, a metal center similar to that found in rubredoxin (Rd) type proteins. The simplicity of the active center in Dx and the possibility of replacing the iron by other metals make this protein an attractive case for the crystallographic analysis of metal-substituted derivatives. This study extends the relevance of Dx to the bioinorganic chemistry field and is important to obtain model compounds that can mimic the four sulfur coordination of metals in biology. Metal replacement experiments were carried out by reconstituting the apoprotein with In3+, Ga3+, Cd2+, Hg2+, and Ni2+ salts. The In3+ and Ga3+ derivatives are isomorphous with the iron native protein; whereas Cd2+, Hg2+, and Ni2+ substituted Dx crystallized under different experimental conditions, yielding two additional crystal morphologies; their structures were determined by the molecular replacement method. A comparison of the three-dimensional structures for all metal derivatives shows that the overall secondary and tertiary structures are maintained, while some differences in metal coordination geometry occur, namely, bond lengths and angles of the metal with the sulfur ligands. These data are discussed in terms of the entatic state theory.

Amino Acid Sequence↗

PubMed1998

Glutamate in life and death of retinal amacrine cells.

1. Glutamate is the neurotransmitter released by bipolar cells at their synapses with amacrine cells. The amacrine cells express ionotropic (NMDA, AMPA and kainate) and metabotropic (mGluR1, mGluR2, mGluR4 and mGluR7) glutamate receptors and may take up glutamate from the synaptic cleft. 2. Activation of the ionotropic glutamate receptors increases the intracellular free calcium concentration ([Ca2+]i), owing to Ca2+ entry through the receptor-associated channels as well as through voltage-gated Ca2+ channels. The [Ca2+]i response to glutamate may be amplified by Ca2+-induced Ca2+ release from intracellular sources. 3. Activation of NMDA and non-NMDA glutamate receptors stimulates the release of GABA and acetylcholine from amacrine cells. GABA is released by a Ca2+-dependent mechanism and by reversal of the neurotransmitter transporter. 4. Excessive activation of glutamate receptors during ischemia leads to amacrine cell death. An increase in [Ca2+]i due to Ca2+ influx through NMDA and AMPA/kainate receptor channels is related to cell death in studies in vitro. In other studies, it was shown that nitric oxide may also take part in the process of cell damage during ischemia.

Animals↗

PubMed1998

Differential acetylcholine and GABA release from cultured chick retina cells.

In the present work we investigated the mechanisms controlling the release of acetylcholine (ACh) and of gamma-aminobutyric acid (GABA) from cultures of amacrine-like neurons, containing a subpopulation of cells which are simultaneously GABAergic and cholinergic. We found that 81.2 +/- 2.8% of the cells present in the culture were stained immunocytochemically with an antibody against choline acetyltransferase, and 38.5 +/- 4.8% of the cells were stained with an antibody against GABA. Most of the cells containing GABA (87.0 +/- 2.9%) were cholinergic. The release of acetylcholine and GABA was mostly Ca2+-dependent, although a significant release of [3H]GABA occurred by reversal of its transporter. Potassium evoked the Ca2+-dependent release of [3H]GABA and [3H]acetylcholine, with EC50 of 31.0 +/- 1.0 mm and 21.6 +/- 1.1 mm, respectively. The Ca2+-dependent release of [3H]acetylcholine was significantly inhibited by 1 micrometer tetrodotoxin and by low (30 nm) omega-conotoxin GVIA (omega-CgTx GVIA) concentrations, or by high (300 nm) nitrendipine (Nit) concentrations. On the contrary, the release of [14C]GABA was reduced by 30 nm nitrendipine, or by 500 nm omega-CgTx GVIA, but not by this toxin at 30 nm. The release of either transmitters was unaffected by 200 nm omega-Agatoxin IVA (omega-Aga IVA), a toxin that blocks P/Q-type voltage-sensitive Ca2+ channels (VSCC). The results show that Ca2+-influx through omega-CgTx GVIA-sensitive N-type VSCC and through Nit-sensitive L-type VSCC induce the release of ACh and GABA. However, the significant differences observed regarding the Ca2+ channels involved in the release of each neurotransmitter suggest that in amacrine-like neurons containing simultaneously GABA and acetylcholine the two neurotransmitters may be released in distinct regions of the cells, endowed with different populations of VSCC.

Acetylcholine↗

PubMed1998

Calcium influx through AMPA receptors and through calcium channels is regulated by protein kinase C in cultured retina amacrine-like cells.

The functional modulation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors by protein kinase C (PKC) was investigated in cultures enriched in retinal amacrine-like cells. The kainate-evoked [Ca2+]i increase is due to Ca2+ entry through open AMPA receptor channels, because it was blocked by the active isomer of a 2,3-benzodiazepine (LY 303070), an AMPA receptor antagonist. The AMPA receptor response to kainate was potentiated by phorbol 12-myristate 13-acetate, which specifically stimulates PKC, and it was decreased by bisindolylmaleimide I, a selective inhibitor of PKC, as well as by PKC down-regulation. The results indicate not only that the AMPA receptor activation has a PKC requirement, but also that PKC amplifies maximal receptor activation by 100 microM kainate. The effect of PKC activation or inhibition on voltage-gated Ca2+-channel activity was also investigated. Activation of PKC caused inhibition of Ca2+ channels, and the same effect was produced by inhibition of PKC, whereas the inactive analogue of the phorbol ester did not affect channel activity. Our results show an important role for PKC in regulating the function of both AMPA receptors and Ca2+ channels in cultured retina cells.

Animals↗

PubMed1997

Crystal structure of acidic seminal fluid protein (aSFP) at 1.9 A resolution: a bovine polypeptide of the spermadhesin family.

We report the three-dimensional crystal structure of acidic seminal fluid protein (aSFP), a 12.9 kDa polypeptide of the spermadhesin family isolated from bovine seminal plasma, solved by the multiple isomorphous replacement method and refined with data to 1.9 A resolution with a final R-factor of 17.3%. aSFP is built by a single CUB domain architecture, a 100 to 110 amino-acid-residue extracellular module found in 16 functionally diverse proteins. The structure of aSFP reveals that the CUB domain displays a beta-sandwich topology organised into two 5-stranded beta-sheets, each of which contain two parallel and four antiparallel strands. The structure of aSFP is almost identical to that of porcine spermadhesins PSP-I and PSP-II, which in turn show limited structural similarity with jellyroll topologies of certain virus capsid proteins. Essentially, topologically conserved residues in these proteins are those internal amino acids forming the hydrophobic core of the CUB and the jellyroll domains, suggesting their importance in maintaining the integrity of these protein folds. On the other hand, the structure of aSFP shows structural features that are unique to this protein and which may provide a structural ground for understanding the distinct biological properties of different members of the spermadhesin protein family.

Amino Acid Sequence↗