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L R Ferreira

Publications and source records attributed to L R Ferreira.

16 recordsLinked to original sources
PubMed2004

Anti-inflammatory activity of Arnica montana 6cH: preclinical study in animals.

The anti-inflammatory effect of Arnica montana 6cH was evaluated using acute and chronic inflammation models. In the acute, model, carrageenin-induced rat paw oedema, the group treated with Arnica montana 6cH showed 30% inhibition compared to control (P < 0.05). Treatment with Arnica 6cH, 30 min prior to carrageenin, did not produce any inhibition of the inflammatory process. In the chronic model, Nystatin-induced oedema, the group treated 3 days previously with Arnica montana 6cH had reduced inflammation 6 h after the inflammatory agent was applied (P < 0.05). When treatment was given 6 h after Nystatin treatment, there was no significant inhibitory effect. In a model based on histamine-induced increase of vascular permeability, pretreatment with Arnica montana 6cH blocked the action of histamine in increasing vascular permeability.

Acute Disease↗
PubMed2003

Expression of Sec61 alpha in F9 and P19 teratocarcinoma cells after retinoic acid treatment.

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (RE) membrane through a protein-conducting channel called the translocon. Sec61 alpha, a multispanning membrane translocon protein, has been implicated as essential for translocation of polypeptides chains into the cisterns of the ER. However, it is not known whether Sec61 alpha is ubiquitously expressed in collagen producing teratocarcinoma cells. Furthermore, the production, expression, and utilization of Sec61 alpha may depend on the cell differentiation stage. Stem cells from many cultured teratocarcinoma cell lines such as F9 and P19 cells are capable of differentiation in response to low retinoic acid concentrations. This differentiation of the tumorigenic stem cells results in tumorigenicity loss. For this study, mouse F9 and P19 teratocarcinoma cells were grown in culture medium treated with or without retinoic acid. Expression of Sec61 alpha was determined by reverse trancriptase polimerase chain reaction (RT-PCR). In untreated conditions, F9 cells expressed undetected Sec61 alpha amounts. It was also demonstrated that Sec61 alpha expression is stimulated in F9 cells after retinoic acid treatment for 72 hours. No changes were found in Sec61 alpha expression in P19 cells after retinoic acid treatment. These data indicate that the expression of Sec61 alpha is enhanced with retinoic acid induced differentiation of F9 teratocarcinoma cells.

Animals↗
PubMed2003

Sec61alpha synthesis is enhanced during translocation of nascent chains of collagen type IV in F9 teratocarcinoma cells after retinoic acid treatment.

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (ER) membrane through a protein-conducting channel called translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as being essential for translocation of polypeptide chains into the cisterns of the ER. Sec61alpha forms a protein complex with collagen and Hsp47, an ER-resident heat shock protein that binds specifically to collagen. However, it is not known whether Sec61alpha is ubiquitously produced in collagen-producing F9 teratocarcinoma cells or under heat shock treatment. Furthermore, the production and utilization of Sec61alpha may depend on the stage of cell differentiation. Cultured F9 teratocarcinoma cells are capable of differentiation in response to low concentrations of retinoic acid. This differentiation results in loss of tumorigenicity. Mouse F9 cells were grown in culture medium at 37 degrees C and 43 degrees C (heat shock treatment) treated or not with retinoic acid, and labeled in certain instances with 35S-methionine. Membrane-bound polysomes of procollagen IV were then isolated. Immunoprecipitation and Western blot analysis were performed using polyclonal antibodies against collagen IV, Hsp47 and Sec61alpha. Under retinoic acid-untreated conditions, F9 cells produced undetectable amounts of Sec61alpha. Sec61alpha, Hsp47 and type IV collagen levels were increased after retinoic acid treatment. Heat shock treatment did not alter Sec61alpha levels, suggesting that Sec61alpha production is probably not affected by heat shock. These data indicate that the enhanced production of Sec61alpha in retinoic acid-induced F9 teratocarcinoma cells parallels the increased synthesis of Hsp47 and collagen type IV.

Animals↗
PubMed2000

Action of ozonized water in preclinical inflammatory models.

Ozonized water increases oxygen local tension. The ozone by itself produces inflammatory reactions in lung epithelium. The action of ozonized water during the inflammatory process was investigated. In rat paw edema induced by carrageenin, the inflammatory process was stimulated by ozonized water (1.2 microg, p.o.) when applied 30 min prior to the induction. In the inflammatory process induced by Freund's Complete Adjuvant, the ozonized water did not interfere in the inflammatory reaction. Furthermore, in gastric ulcers models induced by stress, there was a significant reduction in the incidence of ulcers types I, II and III (P<0.05, Student's t -test).

Animals↗
PubMed1999

Expression of serum amyloid A3 mRNA by inflammatory macrophages exposed to membrane glycoconjugates from Trypanosoma cruzi.

Differential display reverse transcriptase-polymerase chain reaction (PCR) was used to identify genes expressed by murine macrophages exposed to glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi trypomastigotes. Among the different PCR product bands identified in the differential display gel, one showed high homology with the serum amyloid A3 protein (SAA3). Northern blot assays showed augmentation of SAA3 mRNA expression by inflammatory macrophages exposed to live trypomastigotes or parasite glycolipids, as compared to unstimulated macrophages. Our results also showed the expression of SAA3 mRNA, in liver and heart from animals in the acute phase of Chagas disease. It is important that expression of SAA3 mRNA was closely associated with tissue parasitism and presence of inflammatory cells. Together, our findings indicate the possible involvement of SAA3 protein on immunopathology of Chagas disease and establish a new infectious disease model to study the pathophysiological role of this acute-phase protein.

Animals↗
PubMed1999

Application of the differential display RT-PCR strategy for the identification of inflammation-related mouse genes.

The inflammatory response elicited by various stimuli such as microbial products or cytokines is determined by differences in the pattern of cellular gene expression. We have used the differential display RT-PCR (DDRT-PCR) strategy to identify mRNAs that are differentially expressed in various murine cell types stimulated with pro-inflammatory cytokines, microbial products or anti-inflammatory drugs. Mouse embryonic fibroblasts (MEFs) were treated with IFNs, TNF, or sodium salicylate. Also, peritoneal macrophages from C3H/Hej mice were stimulated with T. cruzi-derived GPI-mucin and/or IFN-gamma. After DDRT-PCR, various cDNA fragments that were differentially represented on the sequencing gel were recovered, cloned and sequenced. Here, we describe a summary of several experiments and show that, when 16 of a total of 28 recovered fragments were tested for differential expression, 5 (31%) were found to represent mRNAs whose steady-state levels are indeed modulated by the original stimuli. Some of the identified cDNAs encode for known proteins that were not previously associated with the inflammatory process triggered by the original stimuli. Other cDNA fragments (8 of 21 sequences, or 38%) showed no significant homology with known sequences and represent new mouse genes whose characterization might contribute to our understanding of inflammation. In conclusion, DDRT-PCR has proven to be a potent technology that will allow us to identify genes that are differentially expressed when cells are subjected to changes in culture conditions or isolated from different organs.

Animals↗
PubMed1999

Increase in expression of Hsp47 and collagen in hereditary gingival fibromatosis is modulated by stress and terminal procollagen N-propeptides.

HGF is a rare oral condition characterized by a slow, progressive enlargement of the gingiva, involving both the maxilla and mandible. HGF provides a model for the study of regulatory features of conditions characterized by connective tissue hyperplasia. In this study, the culture characteristics of gingival fibroblasts derived from patients of the same family with HGF (n = 4) were similar with regard to cell cycle analysis. Flow cytometric DNA content analysis revealed uniform DNA diploidy for fibroblasts cultured from NG and HGF. NG cells showed a low S-phase fraction (19.8%) and G2/M fraction (5.8%) and a relatively high G1 phase fraction (74%). In contrast, HGF cells from all members of the tested kindred, exhibited diploid cells with a higher S-phase (40.9%) and G2/M (10.1%) fraction and a relatively low G1 phase fraction (40.9%). Furthermore, we demonstrated that the expression and production of Hsp47 parallels the increased levels of collagen secretion observed in HGF. In addition, we show that Hsp47 and collagen are coordinately regulated following stress via a feedback mechanism mediated by N-terminal procollagen propeptides. Utilizing confocal microscopy and antibodies directed against GST-fusion proteins encompassing the pro alpha1(I) N-propeptide globular domain (NP1) (residues 23-108), it was apparent that this regulatory mechanism does not involve significant interaction with Hsp47's chaperoning of procollagen.

Cells, Cultured↗
PubMed1998

Interleukin-12 enhances in vivo parasiticidal effect of benznidazole during acute experimental infection with a naturally drug-resistant strain of Trypanosoma cruzi.

The roles of gamma interferon (IFN-gamma) and interleukin-12 (IL-12) in mediating and/or enhancing the in vivo trypanosomicidal activity of the nitroheterocyclic derivative benznidazole (Bz) were evaluated during early stages of experimental Chagas' disease. Our results show that treatment of Trypanosoma cruzi-infected mice with anti-cytokine monoclonal antibodies (MAbs) had no apparent effect when the optimal dose of Bz (100 mg/kg of body weight) was used. In contrast, treatment with anti-IL-12 or anti-IFN-gamma MAbs enhanced the parasitemia and accelerated the mortality of mice treated with a suboptimal dose of Bz (25 mg/kg). Simultaneous treatment with a suboptimal dose of Bz and recombinant IL-12 (rIL-12) enhanced the efficacy of drug treatment in terms of parasitemia and mouse survival. Interestingly, we found that drug-resistant T. cruzi strains were found to be poor inducers of IL-12 both in vitro and in vivo compared to strains of T. cruzi which are susceptible or partially resistant to Bz treatment. These results suggest that early activation of the cellular compartment of the immune system by IL-12 may favor in vivo Bz activity against T. cruzi. In order to test this hypothesis mice infected with the drug-resistant Colombiana strain of T. cruzi were treated with 100 mg of Bz per kg plus different concentrations of rIL-12. By using the results of PCR and serological and parasitological methods as the criteria of a cure, our results indicate that a higher percentage of mice treated with Bz combined with rIL-12 than mice treated with Bz alone are cured.

Acute Disease↗
PubMed1996

Hsp47 and other ER-resident molecular chaperones form heterocomplexes with each other and with collagen type IV chains.

Collagen type IV is a genetically distinct secretory protein that constitutes a major component of the basement membranes. Despite the differences between collagen types I and IV, it appears that collagen IV alpha-chain translation/translocation through the ER should follow similar pathways to those established for procollagen I. Using a collagen IV-producing mouse teratocarcinoma cell line, we sought to determine if evolving nascent chains of collagen IV are associated with Hsp47 and other ER-resident molecular chaperones. We show that Hsp47, Grp78 and Grp94 appear to form heteromeric complexes associated with each other and with nascent chains of collagen IV. In addition, we investigated whether ATP depletion, a condition known to promote stable association of Grp78 and Grp94 proteins with their chaperone substrates, extended this response to include a chaperone independent of ATP, Hsp47. These studies reveal that ATP depletion increased the levels of newly synthesized collagen IV associated with Hsp47, Grp78, Grp94 and with each other. These results indicate that Hsp47, Grp78 and Grp94 exist as oligomers and form heterocomplexes during the maturation of newly synthesized collagen IV chains.

Adenosine Triphosphate↗
PubMed1995

Hsp47 and cyclophilin B traverse the endoplasmic reticulum with procollagen into pre-Golgi intermediate vesicles. A role for Hsp47 and cyclophilin B in the export of procollagen from the endoplasmic reticulum.

Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated alpha 1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5'-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [35S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for concentration of procollagen for ER export.

Amino Acid Isomerases↗
PubMed1995

Quantitative image analysis of p53 protein accumulation in keratoacanthomas.

Keratoacanthomas are benign skin tumors that grow rapidly but eventually regress. They occur most commonly in sun-exposed skin and are histologically remarkably similar to squamous cancers. Since mutations of the p53 tumor suppressor gene are found frequently in cutaneous squamous cell carcinomas, we hypothesized that p53 mutations might contribute to the development of keratoacanthomas. To address this question, we did p53 immunohistochemistry with a polyclonal rabbit antiserum, CM-1, that binds both mutant and wild-type p53 proteins. Although wild-type p53 protein degrades rapidly and is generally undetected by immunohistochemistry, mutant p53 protein has a longer half-life and accumulates to detectable levels. We tested 26 formalin-fixed keratoacanthomas and 4 normal skin biopsies. Positive nuclear staining was detected in 20 of 26 (77%) of the keratoacanthomas and in none of the normal skin samples. Nuclear staining occurred in the outermost layer of the neoplasms and not in the keratin-filled central cores. Since nuclear p53 protein within a cutaneous squamous cell carcinoma usually correlates with missense mutation, these data suggest that p53 mutations contribute to the development of this benign neoplasm. The histologic similarity to squamous cell carcinoma and the accumulation of p53 protein suggest progression toward malignancy, but the invariable regression of these tumors suggests an arrest at some point in multistage carcinogenesis. If this model is correct, then genetic analysis of keratoacanthomas may provide clues to the later stages of squamous carcinogenesis including local invasion and metastasis.

Animals↗
PubMed1995

Audit of clinical information and diagnoses supplied to the pathologist following biopsy of oral squamous cell carcinomas.

Strict criteria for the biopsy of oral lesions suspected to be squamous cell carcinoma (SCCA) are difficult to find. Evaluation of the patient's history and clinical findings should help the clinician to accurately diagnose oral cancer. Our study attempts to compare and correlate cases that have the histopathologic diagnosis of oral SCCA with the data submitted by the clinician to the oral pathologist. We find the more information the clinician lists in the biopsy request form, the more likely the correct clinical diagnosis of oral SCCA is. We also show that when risk factors such as tobacco and alcohol use are mentioned by the clinician, the percentage of correct clinical diagnosis is increased.

Biopsy↗
PubMed1994

Association of Hsp47, Grp78, and Grp94 with procollagen supports the successive or coupled action of molecular chaperones.

Hsp47, Grp78, and Grp94 have been implicated with procollagen maturation events. In particular, Hsp47 has been shown to nascent procollagen alpha 1(I) chains in the course of synthesis and/or translocation into the endoplasmic reticulum (ER). Although, Hsp47 binding to gelatin and collagen has previously been suggested to be independent of ATP. Grp78 and Grp94 are known to dissociate from its substrates by an ATP-dependent release mechanism. The early association of Hsp47 with procollagen and its relatively late release suggested that other chaperones, Grp78 and Grp94, interact successively or concurrently with Hsp47. Herein, we examined how these events occur in cells metabolically stressed by depletion of ATP. In cells depleted of ATP, the release of Hsp47, Grp78, and Grp94 from maturing procollagen is delayed. Thus, in cells experiencing metabolic stress, newly synthesized procollagen unable to properly fold became stably bound to a complex of molecular chaperones. In that Hsp47, Grp78, and Grp94 could be recovered with nascent procollagen and as oligomers in ATP depleted cells suggests that these chaperones function in a series of coupled or successive reactions.

2,2'-Dipyridyl↗
PubMed1994

Rearrangement of intermediate filament network of BHK-21 cells infected with vaccinia virus.

Association between vaccinia virus (VV) structures and intermediate filaments in specific areas of the cytoplasm of infected cells (virus "factories") suggests that VV infection interferes with the cellular architecture by modifying the intermediate filament network. To analyse this question, we examined the array of intermediate filaments of BHK-21 cells infected with VV by laser scanning confocal microscopy using an anti-vimentin mouse monoclonal antibody. We observed a marked reorganization of intermediate filaments around the nucleus of infected cells. Bidimensional analysis of 32PO4-labeled intermediate filament proteins revealed that the acidic isoform of vimentin and two isoforms of desmin have increased phosphorylation levels in infected cells. Our results suggest that the reorganization of intermediate filaments observed during VV infection could be promoted by an increase in the phosphorylation level of the intermediate filament proteins, vimentin and desmin.

Animals↗
PubMed1991

Evaluation of different staining procedures for the quantification of fibroblasts cultured in 96-well plates.

Comparison of published methods for the quantification of adherent cell numbers by the measurement of absorbance of bound stain indicates a wide variation in their sensitivity. This study aimed at comparing the sensitivities of five different staining procedures (Coomassie brilliant blue G in perchloric acid, Coomassie brilliant blue G in phosphoric acid, methylene blue, crystal violet, and toluidine blue) applied to three separate types of cultured fibroblasts (3T3 cells, Vero cells, and human gingival fibroblasts) at concentrations from 0.125 x 10(4) to 10 x 10(4) per well in 96-well microplates. Absorbance values of Coomassie blue-stained cells were measured in situ. Those of the remaining cells were measured after solubilization of the dye with 1% sodium dodecyl sulfate. All absorbance values were measured using an Elisa reader at 620 or 570 nm for crystal violet. The relationship between cell number and absorbance over the entire cell concentration range was best fitted with quadratic regression analysis, in contrast with the linear relationship described elsewhere. The order of sensitivity of the staining procedures was the same for each cell type: Coomassie blue in perchloric acid less than Coomassie blue in phosphoric acid less than methylene blue less than crystal violet less than toluidine blue. With the latter two stains absorbance values began to plateau at approximately 8 x 10(4) cells per well. However, staining with Coomassie blue in perchloric acid and methylene blue resulted in an almost linear relationship between cell number and absorbance over the entire concentration range tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Count↗