RESUMO
INTRODUCTION: Decubitus ulcers of the sacral region are common conditions in bedridden patients. Deep lesions (Stages III and IV) often require surgical treatment for closure. Flaps of the region are the first choice for treatment. We present our experience in the treatment of these lesions and compare two different approaches: local fasciocutaneous flap and gluteus maximus myocutaneous flap with V-Y advancement. METHOD: From March 2009 to May 2014, 32 patients underwent closure of sacral pressure ulcers by flaps, 17 of them with rotational local fasciocutaneous flaps and 15 with myocutaneous flaps of the gluteus maximus muscle with V-Y advancement. Evolution regarding complications and rate of success after two months was compared between the groups. RESULTS: Out of the 32 operated patients we obtained resolution of lesions after two months in 23 (71.8%), 10 patients in the fasciocutaneous flap group (58.8%) and 13 cases in the myocutaneous flap group (86.6%). The most common complication was partial dehiscence of sutures in 12 patients (37.5%), 8 patients in the fasciocutaneous flap group (47%) and 4 patients in the myocutaneous flap group (26.6%). The group of patients reconstructed with local fasciocutaneous flaps presented 3 cases with seroma, one with hematoma and 6 with partial cutaneous necrosis; these patients also required more drainage time. CONCLUSIONS: Both the local rotational fasciocutaneous flap and the myocutaneous flap of the gluteus maximus muscle in V-Y flap can be used in the surgical treatment of sacral ulcers. In our experience, a reduced success rate and more complications were found in the local fasciocutaneous reconstructive method.
RESUMO
This paper describes a new approach to assay phospholipases which cleave glycosylphosphatidylinositol using a biotinylated protein substrate coupled to 125I-streptavidin and Triton X-114 phase separation. Substrate preparation with variant surface glycoprotein of Trypamosoma brucei, its characterization and solubilization by glycosylphosphatidylinositol-specific phospholipase C and D are reported. Hydrolysis of substrate exhibited first-order kinetics with respect to enzyme concentration, and the rate constant of the reaction is independent both from substrate concentration and reaction time. This assay was compared with the one using 3H-myristoylated variant surface glycoprotein and proved to be equally suitable to quantitate glycosylphosphatidylinositol-specific phospholipases, with the advantage that avoids biosynthetic labeling. Furthermore, it introduces a basic methodology which can be easily adapted to use other glycosylphosphatidylinositol-anchored proteins as substrates.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Fosfolipase D/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Animais , Proteínas de Bactérias , Biotina , Radioisótopos do Iodo , Cinética , Fosfatidilinositol Diacilglicerol-Liase , Ratos , Solubilidade , Estreptavidina , Especificidade por Substrato , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismoRESUMO
A Leishmania (Viannia) braziliensis (Lb) promastigote cDNA library in lambda gt11 was screened with patients' sera with the aim of identifying antigens specifically related to mucocutaneous leishmaniasis (MCL). One of the clones isolated, 133P, consistently reacted with MCL sera; it was sequenced and found to encode the C-terminal three-quarters of a protein belonging to the highly conserved Hsp70 family. Since Hsp70 proteins from different species tend to be less conserved through the C-terminal end, it was predicted that this region would be more antigenic and was likely to bear the discriminatory epitopes. In order to test this hypothesis, the N- and C-terminal halves of the polypeptide encoded by 133P, 133P-N and 133P-C, respectively, were expressed in Escherichia coli. Immunoblotting analysis demonstrated that 133P-C reacted more strongly with a pool of MCL sera than 133P-N, and both recombinant proteins reacted faintly with pools of cutaneous (CL) and visceral (VL) leishmaniasis sera. These results confirmed the predicted epitope location in the C-terminal region. The 133P-C fragment was also expressed as a fusion protein with glutathione-S-transferase (GST-133P-C), affinity-purified with glutathione-agarose and tested by ELISA with individual sera. From 46 Lb-infected patients, 41 sera (89%) were positive, no cross-reactivity was observed with healthy, Trypanosoma cruzior L. amazonensis-infected individuals. Despite a relatively high cross-reactivity with VL sera, the enhanced humoral response of MCL as compared with CL patients might be interesting for studies on disease aggravation.
Assuntos
Anticorpos Antiprotozoários , Antígenos de Protozoários/imunologia , Proteínas de Choque Térmico HSP70/imunologia , Epitopos Imunodominantes/imunologia , Leishmania braziliensis/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Sequência de Bases , Clonagem Molecular , DNA Complementar , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Leishmaniose/sangue , Leishmaniose/imunologia , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Homologia de Sequência de Aminoácidos , SorologiaRESUMO
This study reports, for the first time, the detection of glycosylphosphatidylinositol (GPI) membrane anchors in proteins of a pathogenic fungus, Paracoccidioides brasiliensis. Taking into account that fungal antigens are found in the sera of paracoccidioidiomycosis patients and that cleavage of this glycolipid by phospholipases is a means of selective protein release, the presence of an enzyme with this property has also been investigated. Using a methodological approach in which the proteins were immobilized on nitrocellulose, treated with phospholipase C of Trypanosoma brucei and then probed with antibodies which recognize the 1,2-cyclic-phosphate inositol moiety formed as a reaction product in proteins bearing the glycolipid anchor, it was possible to detect a major glycoprotein in the 80- to 90-kDa range, as well as two other minor species of 66 and 43 kDa. All of them bind to Concanavalin-A and are also substrates of a very potent fungal phospholipase C which is inhibited by p-chloromercuri-phenylsulfonic acid and is insensitive to EDTA. The integrity of glycosylphosphatidylinositol anchors in proteins of P. brasiliensis is impaired by 0.1 M NaOH, a finding indicative of a diacyl glycerolipid moiety which is quite surprising since it is, with the exception of African trypanosomes surface proteins and Torpedo acetylcholinesterase, an uncommon feature among GPIs in general. The present findings may have implications in the pathology of paracoccidiodomycosis.
Assuntos
Glicosilfosfatidilinositóis/isolamento & purificação , Paracoccidioides/química , Fosfolipases Tipo C/isolamento & purificação , Diglicerídeos/química , Proteínas Fúngicas/química , Glicoproteínas/química , Paracoccidioides/enzimologiaRESUMO
The 90-kDa stage-specific 1G7-antigen has been implicated in the invasion of host cells by the metacyclic forms of Trypanosoma cruzi. The antigen is attached to the plasma membrane via glycosylphosphatidylinositol, the partial structure of which was the first to be determined for a protein of this parasite. In this study, the complete structure of the lipid component of the anchor was determined by electrospray mass spectrometry, gas chromatography mass spectrometry, phospholipase sensitivity and high-performance thin-layer chromatography of the diaradylglycerol components after benzoylation. These analyses showed that the lipid moiety of 1G7-antigen is composed essentially of 1-O-hexadecyl-2-O-hexadecanoyl-phosphatidylinositol and 1-O-hexadecyl-2-O-octadecanoyl-phosphatidylinositol. The high sensitivity of the electrospray mass spectrometric analysis unexpectedly revealed the presence of a small proportion of putative inositol-phosphoceramide structures, and confirmed the absence of inositol-acylated species. An interesting finding was that the biosynthetic incorporation of [3H]palmitate labelled solely the acyl position, and not the 1-O-alkyl chain in the 1G7-antigen anchor.
Assuntos
Antígenos de Protozoários/química , Glicosilfosfatidilinositóis/química , Trypanosoma cruzi/química , Animais , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Lipídeos/química , Espectrometria de Massas , Dados de Sequência Molecular , Palmitatos/análise , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/imunologiaRESUMO
Glycosyl-phosphatidylinositol (GPI) membrane anchors are present on a large number of eukaryotic plasma membrane proteins. Some of these anchors can be cleaved with bacterial phosphatidylinositol-specific phospholipases C, and a glycosyl-phosphatidylinositol-specific phospholipase C from Trypanosoma brucei, to reveal an epitope called the cross-reacting determinant. Other glycosyl-phosphatidylinositol anchors are resistant to the action of these enzymes prior to treatment with mild base. A simple method is described for identifying both phospholipase-sensitive and -resistant anchors using anti-cross-reacting determinant antibodies on Western blots. This procedure represents a high-sensitivity general method for the identification of GPI-anchored proteins.
Assuntos
Western Blotting/métodos , Glicosilfosfatidilinositóis/análise , Diester Fosfórico Hidrolases/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Epitopos/análise , Glicosilfosfatidilinositóis/metabolismo , Estrutura Molecular , Fosfatidilinositol Diacilglicerol-Liase , Relação Estrutura-Atividade , Especificidade por Substrato , Trypanosoma brucei brucei/enzimologiaRESUMO
The 18-kDa protein from Mycobacterium leprae is a major target for the immune response in leprosy. We have developed a system to express this antigen in yeast as a fusion protein with the C-terminal region of the yeast membrane protein GAS1, which would render the recombinant protein anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Cells lacking the GAS1 gene and transformed with the hybrid 18-kDa-GAS1 construct express a polypeptide that reacts with an 18-kDa-specific monoclonal antibody. In addition, these cells react with an alpha-CRD antibody after GPI-PLC treatment. The non-transformed cells are negative. These data indicate that our system may be suitable for the expression of foreign proteins in yeast in a GPI-anchored form.
Assuntos
Proteínas de Bactérias/genética , Glicosilfosfatidilinositóis/genética , Mycobacterium leprae/imunologia , Proteínas de Saccharomyces cerevisiae , Proteínas de Bactérias/imunologia , Proteínas Fúngicas/genética , Genes Fúngicos , Vetores Genéticos , Glicosilfosfatidilinositóis/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Mycobacterium leprae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/imunologiaRESUMO
Since glycosylphosphatidylinositol is the most common form of attachment of proteins to membranes in T. cruzi, and that this parasite depends on surface-mediated interactions for survival within the vector and mammalian host, it is probable that a drug which interfers with the metabolism of glycosylphosphatidylinositol (GPI) could be successfully employed in chemotherapy. Over the last few years several groups have been characterizing this mode of attachment in T. cruzi and more recently we have been concentrating our efforts on the identification of candidate precursors for protein anchors in metacyclic trypomastigotes. Previously detected GPI heterogeneity regarding solubilization of a major stage-specific antigen (1G7-Ag) by phospholipase C led us to investigate whether biosynthetic precursors with similar properties could also be identified. Two glycolipid species whose migration properties resemble glycolipids A and C of T. brucei were amenable to biosynthetic radiolabelling with palmitic acid, inositol, ethanolamine, glucosamine and mannose. Following purification, these species were submitted to classical GPI diagnostic treatments. In both cases digestion with GPI-specific phospholipase D (GPIPLD) produced phospatidic acid and treatment with either mild base or phospholipase A2 (PLA2) produced free fatty acid, indicating an acylation at least at position 2 of the glycerol. The glycolipid A-like species proved to be susceptible to solubilization by PIPLC of B. thuringiensis and by GPIPLC of T. brucei and the glycolipid C-like material proved to be fully resistant to both lipases.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Ácidos Graxos/química , Glicolipídeos/química , Glicosilfosfatidilinositóis/química , Precursores de Proteínas/química , Proteínas de Protozoários/química , Trypanosoma cruzi/química , Fosfolipases Tipo C/química , Animais , Cromatografia em Camada Fina , Ácidos Graxos/metabolismo , Glicolipídeos/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
A phospholipase from human serum capable of hydrolyzing glycosylphosphatidylinositol membrane anchors was described and partially characterized by our group some years ago. This activity presented a pH optimum between 5.0 and 6.0 and was inhibited by EDTA, EGTA and 1,10-phenanthroline. Partial purification showed that the enzyme was a glycoprotein with an apparent molecular weight of 140 kDa as judged by gel filtration. Other investigators characterized at the same time a phospholipase D with similar properties but with a pH optimum near 7.5. We now confirm that the human serum enzyme is indeed a phospholipase D capable of hydrolyzing mfVSG and glycolipids A and C from T. brucei. Isoelectric focusing of whole sera suggests the presence of two isoforms, one with a pI of 4.7 which was the form previously purified by our group, and others with pI from 6.2 to 7.4.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Lipase/metabolismo , Fosfolipase D/sangue , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Humanos , Hidrólise , Focalização Isoelétrica , Lipase/sangueRESUMO
Since glycosylphosphatidylinositol is the most common form of attachment of proteins to membranes in T. cruzi, and that this parasite depends on surface-mediated interactions for survival within the vector and mammalian host, it is probable that a drug which interfers with the metabolism of glycosylphosphatidylinositol (GPI) could be successfully employed in chemotherapy. Over the last few years several groups have been characterizing this mode of attachment in T. cruzi and more recently we have been concentrating our efforts on the identification of candidate precursors for protein anchors in metacyclic trypomastigotes. Previously detected GPI heterogeneity regarding solubilization of a major stage-specific antigen (1G7-Ag) by phospholipase C led us to investigate whether biosynthetic precursors with similar properties could also be identified. Two glycolipid species whose migration properties resemble glycolipids A and C of T. brucei were amenable to biosynthetic radiolabelling with palmitic acid, inositol, ethanolamine, glucosamine and mannose. Following purification, these species were submitted to classical GPI diagnostic treatments. In both cases digestion with GPI-specific phospholipase D (GPIPLD) produced phosphatidic acid and treatment with either mild base or phospholipase A2 (PLA2) produced free fatty acid, indicating an acylation at least at position 2 of the glycerol. The glycolipid A-like species proved to be susceptible to solubilization by PIPLC of B. thuriengiensis and by GPIPLC of T. brucei and the glycolipid C-like material proved to be fully resistant to both lipases. Although the glycolipid A-like species indeed presents these and other properties compatible with a precursor for the chemically characterized 1G7-Ag anchor, the PLC-resistant species which is completely insensitive to nitrous acid deamination might be an exception to the general finding of a non-acetylated glucosamine in the GPI moieties so far described
Assuntos
Antígenos de Protozoários , Fosfatidilinositóis/química , Glicolipídeos/química , Trypanosoma cruzi/imunologia , Fosfolipases Tipo C/química , Sequência de Bases , Sequência de Carboidratos , Ácidos Graxos , Dados de Sequência MolecularRESUMO
A phospholipase from human serum capable of hydrolyzing glycosylphosphatidylinositol membrane anchors was described and partially characterized by our group some years ago. This activity presented a pH optimum between 5.0 and 6.0 and was inhibited by EDTA, EGTA and 1,10-phenanthroline. Partial purification showed that the enzyme was a glycoprotein with an apparent molecular weight of 140 kDa as judged by gel filtration. Other investigators characterized at the same time a phospholipase D with similar properties but with a pH optimum near 7.5. We now confirm that the human serum enzyme is indeed a phospholipase D capable of hydrolyzing mfVSG and glycolipids A and C from T. brucei. Isoelectric focusing of whole sera suggests the presence of two isoforms, one with a pI of 4.7 which was the form previously purified by our group, and others with pI from 6.2 to 7.4
Assuntos
Humanos , Fosfatidilinositóis/química , Glicolipídeos/química , Hidrólise , Fosfolipase D , Plasma , Cromatografia em Gel , Lipase , Trypanosoma brucei brucei , Glicoproteínas Variantes de Superfície de TrypanosomaRESUMO
We have previously shown that 35- and 50-kDa glycoconjugates of cultured metacyclic trypomastigotes participate in the attachment of parasites to mammalian cells. Here we show that when metacyclic trypomastigotes are incubated with [3H]sialyllactose, most of the sialic acid is transferred to these 35/50-kDa molecules in a reaction catalyzed by a parasite transsialidase. The sialic acid is incorporated in oligosaccharides of about 10 glucose units in size that are released from the glycoconjugate by mild alkaline hydrolysis. Compositional analysis reveals that the 35/50-kDa molecules are highly glycosylated proteins rich in threonine, galactose, N-acetyl-glucosamine and sialic acid. These glycoproteins can be labeled in vivo with [3H]palmitate, and the labeled fatty acid is released by glycosylphosphatidylinositol specific phospholipases C. This result, associated with the fact that they contain mannose, ethanolamine, myo-inositol, and lipid, indicate that these glycoproteins are anchored to the membrane by glycosylphosphatidylinositol. During cell invasion, these molecules appear to be capped and locally released by the parasite.
Assuntos
Glicoproteínas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Neuraminidase/metabolismo , Proteínas de Protozoários/metabolismo , Ácidos Siálicos/metabolismo , Trypanosoma cruzi/enzimologia , Aminoácidos/análise , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/isolamento & purificação , Glicoproteínas/isolamento & purificação , Células HeLa , Humanos , Mucinas/metabolismo , Ácido N-Acetilneuramínico , Proteínas de Protozoários/isolamento & purificação , Trítio , Trypanosoma cruzi/fisiologiaRESUMO
The presence of GPI anchors and phospholipases capable of solubilizing them in Trypanosoma cruzi has been investigated in epimastigotes, metacyclic trypomastigotes from axenic cultures and tissue culture trypomastigotes. The GPI anchored proteins in epimastigote forms are scarce when compared to their abundance in the parasite forms which can infect mammals, and GPI-solubilizing phospholipases C have been found in all life cycles stages. In epimastigote and metacyclic forms, the activity is found in the soluble fraction upon cell lysis, whereas in tissue cultured trypomastigotes it is membrane bound and, being mostly sensitive to p-chloromercuriphenylsulfonate, resembles closely the GPI specific phospholipase of Trypanosoma brucei. Sequential immunoprecipitations with monoclonal antibodies and anti-CRD indicated the presence of several sub-populations among the surface proteins of metacyclic trypomastigotes, five of these belonging to the GPI-anchored 90 kD family. Among this family, the epitopes recognized by MAb-1G7 are present in three members, one of them also expressing the 3F6 epitope. There are 2 members recognized only by MAb-3F6 but not by MAb-1G7, one of them being probably galactosylated on the GPI since it can be immunoprecipitated by anti-CRD. Very strangely, the epitope recognized by the MAb-WIC29.26 was always present on the gp72, as originally described, but under certain circumstances appeared cryptic on one of the 90 kD species. During epimastigote transformation into metacyclic trypomastigotes in vitro, the ability of the GPI of the 1G7-antigen to be solubilized by phospholipase C and D varies depending on the age of the culture and presence or absence of fetal calf serum. Different patterns of solubilization were also obtained for 1G7-Ag, depending on whether the test is performed with parasite lysates or with antigen affinity purified from them. Our data indicate that the phospholipase C resistance observed does not arise from acylation on the inositol, as previously described for acetylcholinesterase from human erythrocytes, being rather due to factors which either modify the GPI or affect the action of the phospholipases. Previously unreported resistance to glycosylphosphatidylinositol-specific phospholipase D has been observed both to glycosylphosphatidylinositol-specific phospholipase D has been observed both to 1G7-Ag and 10D8-Ag, the GPI-anchored mucynlike protein which is acceptor of sialic acid in metacyclic forms. Our findings are discussed in the light of the presently known structures of GPI in this parasite, and imaginative speculation on biological roles for the GPI phospholipase system in T. cruzi is also provided.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Fosfolipases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Anticorpos Monoclonais , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Humanos , Hidrólise , Testes de Precipitina/métodos , Solubilidade , Trypanosoma brucei brucei/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/imunologia , Fosfolipases Tipo C/metabolismoRESUMO
The 1G7-antigen is expressed by the infective metacyclic trypomastigote stage of the protozoan parasite Trypanosoma cruzi. The 1G7-antigen is a 90-kDa glycoprotein, present at about 40,000 copies/cell, which is anchored in the plasma membrane via a glycosylphosphatidylinositol (GPI) membrane anchor. The glycan of the GPI anchor has been isolated from immunopurified 1G7-antigen and its structure determined using a combination of methylation linkage analysis and exoglycosidase sequencing. The structure of the glycan is Man alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2. The glucosamine residue is in glycosidic linkage to a phosphatidylinositol moiety. The penultimate nonreducing alpha-Man residue is substituted with phosphate, which is most likely part of an ethanolamine phosphate bridge linking the GPI anchor to the 1G7-antigen polypeptide. The glycan sequence was obtained from 1.1 nmol of glycoprotein isolated from a detergent lysate of whole cells. The procedures reported here represent a high sensitivity protocol for determining GPI glycan structures from small quantities of biological material. The structure of the 1G7-antigen GPI anchor is consistent with the conserved core structure of all GPI anchors analyzed to date and is similar to that of the T. cruzi lipopeptidophosphoglycan. The biosynthesis of GPI anchors and lipopeptidophosphoglycan in T. cruzi is discussed in the light of this structural homology.
Assuntos
Antígenos de Protozoários/metabolismo , Glicolipídeos/metabolismo , Fosfatidilinositóis/metabolismo , Polissacarídeos/metabolismo , Trypanosoma cruzi/imunologia , Animais , Antígenos de Protozoários/isolamento & purificação , Sequência de Carboidratos , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Cromatografia Gasosa-Espectrometria de Massas , Glicosilfosfatidilinositóis , Hidrólise , Metilação , Dados de Sequência MolecularRESUMO
We searched for the presence of glycophosphatidylinositol (GPI)-anchored proteins in epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi, by treatment of parasite lysates with the GPI-specific phospholipase C of Trypanosoma brucei. Upon treatment, several proteins (70-90 kDa) in metacyclics, but none in epimastigotes, reacted with antibodies to the cross-reacting determinant (CRD), an epitope revealed on the variant surface glycoproteins of T. brucei following removal of the diacylglycerol moiety from their GPI-anchor. Since these T. cruzi metacyclic proteins also lost their original amphiphilicity, as judged by Triton X-114 phase separation, it is very likely that they are linked to the membrane by GPI. One of these proteins is the 90 kDa protein, the major surface protein of G and Tulahuen strains, recognized by the monoclonal antibody 1G7. A variable portion of the 90 kDa molecules was resistant to solubilization by T. brucei lipase. The reasons for this are not clear but susceptibility appeared to increase with the age of the T. cruzi culture. Enzymes that solubilize GPI-anchored proteins were detected in epimastigotes and metacyclics, but the enzymatic activity in these forms was smaller than the activity detected in the same cell numbers of trypomastigotes of T. cruzi originated from infected mammalian cells or from T. brucei bloodstream forms. A preliminary characterization of these activities indicates that at least two classes of enzymes, one of them inhibited by o-phenanthroline, are present in epimastigotes and metacyclics. None of the reagents tested fully inhibited the phospholipases.
Assuntos
Glicolipídeos/metabolismo , Proteínas de Membrana/análise , Fosfatidilinositóis/metabolismo , Trypanosoma cruzi/análise , Animais , Anticorpos Monoclonais/imunologia , Reações Cruzadas , Epitopos/imunologia , Glicosilfosfatidilinositóis , Imunoensaio , Proteínas de Membrana/metabolismo , Trypanosoma brucei brucei/enzimologia , Trypanosoma cruzi/imunologia , Fosfolipases Tipo C/metabolismoRESUMO
A lipase has been identified in human serum which can convert the membrane form of the variant surface glycoprotein of Trypanosoma brucei to a water soluble form. The conversion can be monitored by loss of [3H] myristic acid incorporated into the diacylglycerol of the glycophosphatidylinositol membrane anchor of the protein, but does not lead to the exposure of the antigenic determinant in the polar head group of the glycolipid. The serum lipase is a glycoprotein, and is optimally active at pH 5.4. Treatment at 62 degrees for one hour does not inactivate the enzyme, which is inhibited by chelating agents.
Assuntos
Lipase/metabolismo , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Animais , Quelantes/farmacologia , Diglicerídeos/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Concentração de Íons de Hidrogênio , Lipase/antagonistas & inibidores , Lipase/sangue , Peso Molecular , Ácido Mirístico , Ácidos Mirísticos/metabolismoRESUMO
Yeast forms of the pathogenic fungus Paracoccidioides brasiliensis secrete into the culture supernatant a 43,000 daltons glycoprotein (Gp43) which can be immunoprecipitated specifically by sera from patients with paracoccidioidomycosis. We show here that following labelling of P. brasiliensis with (35S)methionine, Gp43 was detected as the major component in the culture supernatant fluid as early as 1 hour after addition of the radiolabel. The amount of Gp43, as determined by a competitive radioimmunoassay, or by staining total protein after sodium dodecylsulfate-polyacrylamide gel electrophoresis, progressively increased in the culture supernatant until the culture reached the late exponential phase. It then decreased and continued to do so in the stationary phase. These results indicate that Gp43 is continuously produced and secreted in the medium by actively growing yeasts and that cultures in the exponential phase of growth should be used for a maximal yield of this exocellular antigen.