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C M Silva

Publications and source records attributed to C M Silva.

At least 37 records · Page 2Linked to original sources
PubMed1995

Red-shifted excitation mutants of the green fluorescent protein.

Using optimized combinatorial mutagenesis techniques and Digital Imaging Spectroscopy (DIS), we have isolated mutants of the cloned Aequorea victoria green fluorescent protein (GFP) that show red-shifted excitation spectra similar to that of Renilla reniformis GFP. Selective excitation of wild-type versus Red-Shifted GFP (RSGFP) enables spectral separation of these proteins. Six contiguous codons spanning the tyrosine chromophore region were randomized and sequence analysis of the mutants revealed a tyrosineglycine consensus. These mutants will enable the simultaneous analysis of two promoters or proteins per cell or organism. In consideration of the multitude of applications which are developing for GFP alone, we envisage that spectrally shifted fluorescent proteins will be of value to a diversity of research programs, including developmental and cell biology, drug-screening, and diagnostic assays.

Amino Acid Sequence↗
PubMed1994

Differential tyrosine phosphorylation of JAK1, JAK2, and STAT1 by growth hormone and interferon-gamma in IM-9 cells.

Both the growth hormone (GH) and interferon gamma (IFN gamma) receptors are members of the cytokine receptor family that activate tyrosine phosphorylation despite the lack of a tyrosine kinase domain. Recently, the Janus kinase (JAK) family of tyrosine kinases have been shown to play an integral role in intracellular signaling by the cytokine receptors. We demonstrate that, in the human IM-9 lymphocyte, both JAK1 and JAK2 are tyrosine-phosphorylated in response to IFN gamma, whereas only JAK2 is tyrosine-phosphorylated in response to GH. Furthermore, dimerization of the GH receptor appears to be necessary for GH stimulated tyrosine phosphorylation of JAK2. We provide two lines of evidence that the JAK2 kinases can be regulated independently by GH and IFN gamma in IM-9 cells: 1) desensitization of JAK2 to GH stimulation does not affect the IFN gamma stimulated tyrosine phosphorylation of JAK2; and 2) JAK2 tyrosine phosphorylation by GH and IFN gamma is additive to that seen with either hormone alone. Furthermore, we demonstrate that although IFN gamma activates the tyrosine phosphorylation of the p91 signal transducer and activator of transcription (STAT1) in IM-9 cells, GH does not. GH does activate the tyrosine phosphorylation of a 93-kDa protein that appears to be distinct from STAT1.

Cells, Cultured↗
PubMed1994

Regulation of the human glucocorticoid receptor by long-term and chronic treatment with glucocorticoid.

HeLa S3 cells that contain endogenous glucocorticoid receptors (GR) were treated with dexamethasone (DEX) for periods of time ranging from 24 h to 2 weeks or chronically over a 2-year period. Regulation of GR protein and mRNA were examined by affinity labeling, Western blotting, and Northern blotting. Relatively short-term treatment of cells with DEX for 24 or 48 h revealed more profound down-regulation of GR protein than of GR mRNA. However, by 2 weeks of DEX treatment, the levels of both receptor protein and mRNA were both maximally down-regulated. Cells that had been chronically DEX treated (for up to 2 years) had no measurable GR protein or mRNA. The down-regulation of receptor protein and RNA that occurred after 2 weeks of DEX treatment is completely reversible upon DEX removal, whereas reversibility did not occur with cells that had been chronically treated with DEX. Furthermore, transfection of a glucocorticoid responsive reporter plasmid into these chronically DEX-treated cells demonstrated that these cells were no longer responsive to steroid treatment. However, cotransfection of a plasmid encoding the human GR into these chronically DEX-treated cells resulted in restored production of GR and responsiveness to hormone, indicating that the defect in these cells occurs only at the receptor level.

Blotting, Northern↗
PubMed1994

Growth hormone and erythropoietin differentially activate DNA-binding proteins by tyrosine phosphorylation.

Binding of growth hormone (GH) and erythropoietin (EPO) to their respective receptors results in receptor clustering and activation of tyrosine kinases that initiate a cascade of events resulting not only in the rapid tyrosine phosphorylation of several proteins but also in the induction of early-response genes. In this report, we show that GH and EPO induce the tyrosine phosphorylation of cellular proteins with molecular masses of 93 kDa and of 91 and 84 kDa, respectively, and that these proteins form DNA-binding complexes which recognize an enhancer that has features in common with several rapidly induced genes such as c-fos. Assembly of the protein complexes required tyrosine phosphorylation, which occurred within minutes after addition of ligand. The activated complexes translocated from the cytoplasm to the nucleus. The protein activated by GH is antigenically similar to p91, a protein common to several transcription complexes that are activated by interferons and other cytokines. In contrast, the proteins activated by EPO are distinct from p91. These findings establish the outlines for a cytokine-induced intracellular signaling pathway, which begins with ligand-induced receptor clustering that activates one or more tyrosine kinases. These data are the first to demonstrate that GH- and EPO-activated tyrosine-phosphorylated proteins can specifically recognize a well-defined enhancer and therefore provide a mechanism for rapidly transducing signals from the membrane to the nucleus.

Base Sequence↗
PubMed1994

Growth hormone induces tyrosine phosphorylation but does not alter insulin-like growth factor-I gene expression in human IM9 lymphocytes.

GH induces hepatic IGF-I synthesis by increasing transcription of its gene. IGF-I is synthesized, however, in many other tissues where the effect of GH on its gene expression is less well characterized. IGF-I and GH are produced by human lymphocytes and may function as autocrine regulators of lymphoproliferation. We have therefore used the human IM9 lymphocyte cell line to (A) define the IGF-I gene transcripts expressed and (B) investigate the effect of GH on early (protein tyrosine phosphorylation) and late (changes in IGF-I mRNA levels) events in intracellular signal transduction. Multiple IGF-I mRNA species, ranging in size from 0.9 to 5.8 kb, were detected by Northern hybridization of poly(A)+ mRNA from IM9 cells. The human IGF-I gene contains at least six exons and alternative splicing produces a number of transcripts. Solution hybridization with exon-specific riboprobes and amplification by PCR using exon-specific primers revealed that multiple transcripts were expressed in IM9 cells, and that exon 2 was the dominant leader exon. Treatment of IM9 cells with 200 ng recombinant human (rh)GH/ml led to the specific tyrosine phosphorylation of three intracellular proteins (93, 120 and 134 kDa), which are involved in the initial signalling of the GH transduction pathway. However a solution hybridization assay using the IGF-IA specific riboprobe on IM9 cell RNA from similar experiments revealed that GH treatment did not change IGF-I gene expression. This study has demonstrated (A) that the IGF-I gene is expressed in human IM9 lymphocytes, (B) that in contrast to other human tissue, exon 2 is the major leader exon, and (C) that rhGH induces tyrosine phosphorylation of 93, 120 and 134 kDa proteins but does not alter IGF-I gene expression. The IM9 cell may form an important model to investigate a GH transduction pathway not coupled to the IGF-I gene.

Cell Line↗
PubMed1994

[Emergency valvuloplasty with a balloon catheter of a tricuspid bioprosthesis in Ebstein's anomaly].

A case of severe stenosis of a tricuspid bioprosthesis was successfully dilated in a 13 year old young male with Ebstein's anomaly. The patient had congestive heart failure and developed cardiogenic shock; angiography revealed absence of emptying of the right atrium. Multiple size balloons (#4 to 23) were sequentially introduced with successful bioprosthesis dilation and shock resolution. Tricuspid bioprosthesis can be dilated even in critical conditions and, although a palliative procedure, balloon valvuloplasty can be a lifesaving procedure in patients with cardiogenic shock.

Adolescent↗
PubMed1994

[Functional anatomy of the junction of the left atrium and the pulmonary veins].

PURPOSE: To study the spatial arrangement of the bundles of myocardial fibers presents in the left atrial-venous junctions and in the wall of the pulmonary veins. METHODS: The study was made on 24 human adult hearts, together with pulmonary vessels, fixed in 10% formaldehyde solution. Each specimen was cleared of remnants of pericardium to expose the myocardial fibers as clearly as possible. Particular attention was paid to the atrial-venous junction and the extension of the myocardial fibers in the pulmonary veins. The specimens were embedded in celloidin and cut serially at a thickness of 70 mu and stained by methods of Azan's trichrome. RESULTS: The myocardial bundles leave the atrial wall around the openings of all pulmonary veins forming a sphincter-like structure. These fibers are continuous in the adventitial coat running in oblique or spiral directions. Some bundles, more external, leaving from the atrial wall, surround the ostium of the veins and return to the atrium, forming true loops. CONCLUSION: The extremities of the pulmonary veins and their junctions with the atrium have a morphological substract which may be of physiological importance in the control of the pulmonary venous pressure and blood flow.

Adult↗
PubMed1993

Angiographic diagnosis of anomalous coronary artery in tetralogy of Fallot.

OBJECTIVE: To obtain angiographic views in tetralogy of Fallot that can show whether or not an anomalous coronary artery passes anterior to the right ventricular outflow tract. DESIGN: (a) A 10 year retrospective review of all patients who underwent repair of tetralogy of Fallot up to December 1990; (b) a prospective study of 30 children undergoing routine cardiac catheterisation. PATIENTS AND METHODS: 295 cases in whom standard angiographic views had been used were reviewed retrospectively. Thirty non-consecutive children with tetralogy of Fallot were studied prospectively, including one child previously studied in whom diagnosis of an unsuspected anomalous coronary artery was made only at operation. The aortogram was performed with > or = 45 degrees caudocranial and 20 degrees-30 degrees left anterior oblique angles. SETTING: Tertiary referral centre. RESULTS: Ten of the 295 cases reviewed were shown to have a coronary vessel traversing the right ventricular outflow tract. In one case the diagnosis was suspected before operation but it was missed in the others. Even in retrospect we could not be certain of the precise anatomy with the use of standard angiographic views. In the prospective study the caudocranial aortogram showed the aortic valve face on in all the patients. The right ventricular outflow tract lay in a left and anterior (seen as superior) position in relation to the aortic root. Thus any vessel crossing the outflow tract could be identified. Identification of the aortic cusps allowed precise definition of the origin of the coronary arteries. All but four had normal origin and course of the coronary arteries. Four had paired left anterior descending arteries (including the restudied patient), in all cases with a large vessel originating from the right coronary artery passing across the right ventricular outflow tract. CONCLUSIONS: Important anomalies of the coronary arteries in tetralogy of Fallot may remain undiagnosed if standard angiographic projections are used. Aortography with > or = 45 degrees caudocranial and 20 degrees-30 degrees left anterior oblique angles allows precise definition of the anatomy and certainty as to whether any major vessel crosses the right ventricular outflow tract. Interpretation, however, can only be correct if the projection is technically adequate with a view of the aortic valve face on. Furthermore, a normal bifurcation of the left main stem does not exclude a second left anterior descending artery crossing the pulmonary outflow tract.

Child↗
PubMed1993

Stimulation of tyrosine phosphorylation in human cells by activation of the growth hormone receptor.

To investigate the possibility that tyrosine phosphorylation of cellular proteins might play a role in GH receptor signaling, we have studied tyrosine phosphorylation induced by GH and by GH mutants in the human lymphocyte line IM-9, a homologous cell system which is known to respond to GH by increased proliferation. IM-9 cells were treated with physiological concentrations of recombinant human GH (rhGH). Protein lysates from these cells were then analyzed by polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with an antibody specific for phosphotyrosine. rhGH stimulated the tyrosine phosphorylation of two proteins having M(r) of approximately 93,000 and 120,000. Tyrosine phosphorylation of these proteins was time and dose dependent. At 2 nM rhGH tyrosine phosphorylation of these two proteins was evident by 5 min, maximal at 15 min, and decreasing by 45 min of treatment. At doses of 10 and 100 nM rhGH, tyrosine phosphorylation was stimulated by 1 min of GH treatment. IM-9 cells were also treated with genetically engineered mutant forms of the GH protein. Previous biophysical analysis of these mutant GH proteins has shown that the GH protein contains two distinct binding sites which interact in a sequential manner with the extracellular domains of two distinct GH receptor molecules, thus forming a dimeric complex. By treating IM-9 cells with these same GH mutants and analyzing tyrosine phosphorylation, we found that tyrosine phosphorylation was inhibited under conditions which prevent receptor dimerization, thus providing evidence that formation of a dimeric GH:(GH receptor)2 complex may be important for intracellular signaling by the GH receptor.

Binding Sites↗
PubMed1993

Human growth hormone (GH) receptor is characterized as the 134-kilodalton tyrosine-phosphorylated protein activated by GH treatment in IM-9 cells.

Activation of the GH receptor (GHR) results in tyrosine phosphorylation of cellular proteins, and this tyrosine phosphorylation is believed to be important in GH action. We have shown previously that GH rapidly stimulates the tyrosine phosphorylation of 134-, 120-, and 93-kilodalton (kDa) proteins in the human IM-9 lymphocyte line. We now provide three lines of evidence indicating that the 134-kDa tyrosine-phosphorylated protein, not the 120- or 93-kDa proteins, is the GHR. 1) A monoclonal antibody that interacts with the extracellular domain of the GHR (Mab263) immunoprecipitated a 134-kDa phosphotyrosine-containing protein, but not a 120- or 93-kDa protein, from GH-treated IM-9 cells. 2) The GHR contains N-linked carbohydrates. When total cell lysates from GH-treated IM-9 cells were treated with endoglycosidases to remove these carbohydrates, the majority of the 134-kDa phosphotyrosine-containing protein was no longer detected. Furthermore, the immunoprecipitated 134-kDa protein was completely deglycosylated and resulted in a single band of approximately 100 kDa. Neither the 120- nor 93-kDa tyrosine-phosphorylated proteins were affected by endoglycosidase treatment. 3) The Mab263 antibody immunoprecipitated a 134-kDa phosphotyrosine-containing protein from GH-treated 293 cells (human embryonic kidney cell line) that stably express the full-length rabbit GHR. This protein was not detected in control cells expressing the neomycin resistance gene alone. We conclude that the 134-kDa protein that is tyrosine phosphorylated upon GH stimulation of IM-9 cells is the GHR.

Antibodies, Monoclonal↗
PubMed1992

The effect of oxidation/reduction on the charge heterogeneity of the human glucocorticoid receptor.

In order to investigate the molecular basis for the charge heterogeneity which has been reported for the glucocorticoid receptor, we have analyzed the [3H]dexamethasone mesylate ([3H]DM)1 affinity labeled receptor from HeLa S3 cells by high resolution two-dimensional gel analysis. The [3H]DM labeled glucocorticoid receptor from HeLa cells exists as a population of 5-6 isoforms which range over approx. 0.6 pI units but have similar molecular weights. This heterogeneity is apparently the result of modification(s) of a single gene product since the affinity labeled receptor from Chinese hamster ovary (CHO) cells transfected with the human glucocorticoid receptor cDNA displays the same pattern of heterogeneity on two-dimensional gels. Since previous one-dimensional gel studies from our laboratory showed that the structure of the [3H]DM labeled glucocorticoid receptor from HeLa cells is highly susceptible to sulfhydryl group modification, we investigated the potential role of the same modifications in the apparent charge heterogeneity of the glucocorticoid receptor. Treatment of the affinity labeled receptor with iodoacetamide which alkylates free sulfhydryl groups and irreversibly prevents the formation of intra- or intermolecular disulfide bonds, reproducibly resulted in the appearance of 5-6 discrete isoforms of the receptor protein. Treatment with dithiothreitol, a reversible reducing reagent, resulted in detection of 3 to 4 isoforms of the glucocorticoid receptor. In marked contrast, treatment with sodium tetrathionate, which induces intramolecular disulfide bond formation, resulted in only one detectable isoform of the [3H]DM labeled glucocorticoid receptor. These data demonstrate that the oxidation/reduction state of sulfhydryl groups within the receptor protein can account for much of the charge heterogeneity of this ligand dependent transcription factor.

Affinity Labels↗
PubMed1991

Etoposide in the treatment of six children with Langerhans cell histiocytosis (histiocytosis X)

Six children received etoposide as the single agent for treatment of Langerhans cell histiocytosis (LCH; histiocytosis X). Five were less than 2 years old at diagnosis. All had multiorgan involvement; one had liver and pulmonary dysfunction. Two infants also had clinical signs of immune deficiency. Complete response was observed in five. There was no major toxicity. Although three of four evaluable patients relapsed, the drug was considered useful in moving the children from a symptomatic to an asymptomatic clinical status. Etoposide may become a "first-line" drug in the treatment of systemic LCH, especially when the side effects of steroid therapy are considered unacceptable.

Child, Preschool↗
PubMed1989

Direct evidence for intra- and intermolecular disulfide bond formation in the human glucocorticoid receptor. Inhibition of DNA binding and identification of a new receptor-associated protein.

We have investigated the potential for the steroid affinity-labeled human glucocorticoid receptor to form both intramolecular and intermolecular disulfide bonds. Glucocorticoid receptors labeled in intact HeLa S3 cells with the covalent affinity label [3H]dexamethasone mesylate ([3H]DM) were analyzed on denaturing 5-12% polyacrylamide gels under both nonreducing and reducing conditions. Under nonreducing conditions the affinity-labeled receptor migrated as a heterogeneous species having an average molecular mass of approximately 96 kDa whereas, under reducing conditions, the receptor migrated as a more discrete form. These data suggest that a reducing environment can influence the structure of the glucocorticoid receptor monomer and further imply that sulfhydryl groups within the affinity-labeled receptor are available for modification. To pursue this observation in greater detail, we tested the effect of oxidizing conditions on the structure of the glucocorticoid receptor. The presence of low concentrations (0.125-0.5 mM) of three oxidizing reagents (sodium tetrathionate, disulfiram, and iodosobenzoate) altered the migration of the affinity-labeled receptor resulting in forms of apparent lower molecular mass (as low as 78 kDa). This altered migration, not seen with most other cytosolic proteins, is consistent with the formation of intramolecular disulfide bonds within the receptor which presumably cause it to assume a folded conformation and migrate faster through the gel. At higher concentrations of these reagents (up to 5.0 mM), we also detect a saturably labeled [3H]DM band which has a higher molecular mass (approximately 140 kDa), indicating the formation of intermolecular disulfide bonds between the [3H]DM-labeled receptor and another closely associated protein(s) having a molecular mass of approximately 40 kDa. The effects which these oxidizing reagents have on glucocorticoid receptor structure are completely reversed upon the addition of dithiothreitol, indicating that the observed changes in migration do not reflect receptor proteolysis but rather a folding and unfolding within the receptor monomeric protein. We have also analyzed the effect of this oxidation/reduction on the function of the glucocorticoid receptor. Oxidation of the [3H]DM-labeled receptor complex with 0.5 mM sodium tetrathionate inhibited activation of receptor to a form capable of binding to DNA-cellulose. This inhibition can be reversed with dithiothreitol at 25 degrees C but not at 0 degrees C, suggesting that these oxidizing reagents are inhibitory at the transformation and/or activation steps.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity Labels↗
PubMed1987

Application of a protein-blotting procedure to the study of human glucocorticoid receptor interactions with DNA.

To exert their effects, glucocorticoid receptor complexes interact selectively with DNA sequences known as glucocorticoid regulatory elements. We have studied the interaction between human glucocorticoid receptors and mouse mammary tumor virus (MMTV) DNA by means of a procedure that permits analysis after immobilization of the receptor on nitrocellulose. Proteins from crude cytosolic or nuclear extracts were electrophoresed on NaDodSO4/PAGE gels, soaked in a urea buffer to remove NaDodSO4, transferred to nitrocellulose, and probed with nick-translated MMTV [32P]DNA in a 5% nonfat dry milk buffer, which minimizes nonselective DNA-protein interactions. We present evidence that MMTV [32P]DNA interacts selectively with the glucocorticoid receptor. These data include comigration of [3H]dexamethasone mesylate-labeled band and bound MMTV [32P]DNA on gel electrophoresis systems; localization of DNA-binding activity in the cytosol of cells incubated with steroid at 0 degrees C and in the nucleus and cytosol of cells incubated at 37 degrees C; binding of the MMTV DNA to highly purified receptor; and absence of MMTV DNA binding activity in extracts from cells whose receptor has been down-regulated. Furthermore, glucocorticoid receptors analyzed under these conditions exhibit selective binding to DNA fragments that contain glucocorticoid regulatory elements.

Cytosol↗
PubMedDate not supplied

[Disseminated cutaneous leishmaniasis due to Leishmania viannia braziliensis in the state of Maranhão, Brazil].

The authors describe a case of a patient from Barreirinhas, MA with disseminated cutaneous leishmaniasis, who presented 58 lesions spread all over his body, with many different aspects, but ulcerative lesions were predominant. They discuss the difficulty of laboratory diagnosis in the beginning of the investigation and its therapeutics implications. The parasite isolated was identified as Leishmania viannia braziliensis using monoclonal antibodies in the fluorescent antibody test. This form of the disease is different from the diffuse cutaneous leishmaniasis found in Maranhão, due to Leishmania (Leishmania) amazonensis which is responsible for most of the cases of cutaneous leishmaniasis in our state. The possible mechanisms of lesions dissemination are discussed.

Adult↗
PubMedDate not supplied

Spontaneous regional healing of extensive skin lesions in diffuse cutaneous Leishmaniasis (DCL).

The authors report a case of diffuse cutaneous leishmaniasis, with longstanding evolution and presenting with diffuse infiltrated lesions rich in amastigotes in the absence of mucosal involvement. In situ characterization with monoclonal antibodies revealed Leishmania amazonensis. Large regional lesions have presented spontaneous healing without specific therapy. Considering that DCL presents with a defect in the cellular immune response, this fact demonstrate that this patient may develop a regional cellular immune response enough to destroy the parasites and to produce clearing of some lesions.

Child↗