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1.
Genes Immun ; 17(2): 128-38, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26821283

ABSTRACT

The B-lymphocyte kinase (BLK) gene is associated genetically with several human autoimmune diseases including systemic lupus erythematosus. We recently described that the genetic risk is given by two haplotypes: one covering several strongly linked single-nucleotide polymorphisms within the promoter of the gene that correlated with low transcript levels, and a second haplotype that includes a rare nonsynonymous variant (Ala71Thr). Here we show that this variant, located within the BLK SH3 domain, is a major determinant of protein levels. In vitro analyses show that the 71Thr isoform is hyperphosphorylated and promotes kinase activation. As a consequence, BLK is ubiquitinated, its proteasomal degradation enhanced and the average life of the protein is reduced by half. Altogether, these findings suggest that an intrinsic autoregulatory mechanism previously unappreciated in BLK is disrupted by the 71Thr substitution. Because the SH3 domain is also involved in protein interactions, we sought for differences between the two isoforms in trafficking and binding to protein partners. We found that binding of the 71Thr variant to the adaptor protein BANK1 is severely reduced. Our study provides new insights on the intrinsic regulation of BLK activation and highlights the dominant role of its SH3 domain in BANK1 binding.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Amino Acid Substitution , Lupus Erythematosus, Systemic/genetics , Membrane Proteins/genetics , Mutation , src-Family Kinases/genetics , Adaptor Proteins, Signal Transducing/immunology , Amino Acid Sequence , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Binding Sites , Cell Line, Tumor , Gene Expression , Half-Life , Haplotypes , Humans , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/pathology , Membrane Proteins/immunology , Models, Molecular , Molecular Sequence Data , Promoter Regions, Genetic , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/immunology , Proteolysis , Sequence Alignment , Ubiquitination , src-Family Kinases/immunology
2.
Parasite Immunol ; 35(5-6): 180-7, 2013.
Article in English | MEDLINE | ID: mdl-23473453

ABSTRACT

The enzyme indoleamine 2,3-dioxigenase (IDO) is critical for the regulation of immune responses in pro-tolerogenic antigen-presenting cell. To address the profile of immune responses associated with Chagas disease, we measured IDO activity of peripheral blood mononuclear cells from 168 chronic patients and 13 healthy donors. We found that IDO activity was increased in patients with Chagas disease when compared with controls. Moreover, the IDO activity of patients with Chagas disease in the symptomatic chronic phase, involving cardiac or digestive alterations, was higher than that detected in asymptomatic patients and correlated with the severity of the symptoms. Furthermore, benznidazole treatment induced a long-lasting decrease in IDO activity in symptomatic patients, reaching levels comparable with those of healthy donors. These results suggest that a pro-tolerogenic state is associated with the severity of Chagas disease and that benznidazole treatment is a valuable tool for breaking the parasite-driven immune tolerance in the symptomatic chronic phase of Chagas disease.


Subject(s)
Chagas Disease/drug therapy , Chagas Disease/enzymology , Indoleamine-Pyrrole 2,3,-Dioxygenase/blood , Nitroimidazoles/therapeutic use , Trypanocidal Agents/therapeutic use , Adult , Chagas Disease/immunology , Female , Humans , Immune Tolerance , Leukocytes, Mononuclear/enzymology , Male , Middle Aged , Young Adult
3.
Haemophilia ; 18(6): 982-9, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22646163

ABSTRACT

The presence of VWF in plasma-derived FVIII (pdFVIII/VWF) products has been pointed out as a key difference with recombinant FVIII (rFVIII) products with regard to immunogenicity. A Surface Plasmon Resonance (SPR) study was designed to characterize in detail the interaction between anti-FVIII (IgGs) from a severe haemophilia A patient, and FVIII from concentrates of different sources. Full-length rFVIII (preincubated or not with purified VWF), B domain-deleted (BDD)-rFVIII and pdFVIII/VWF were analysed. To ensure reproducible conditions for accurate determination of kinetic constants, a capture-based assay format was developed using protein G surfaces for specific and reversible coupling of endogenous anti-FVIII antibodies. Concentration ranges (nm) of FVIII products tested were 9-0.03 (rFVIII) and 6-0.024 (pdFVIII/VWF). The association with antibodies was monitored for 3-5 min, whereas dissociation of the complex was followed for 5-20-240 min. A strong interaction of rFVIII and BDD-rFVIII with patient's IgG was detected with the K (D) values in the low picomolar range (5.9 ± 3.0 and 12.7 ± 6.9 pm, respectively) and very slow dissociation rates, while pdFVIII/VWF showed only marginal binding signals. The VWF complexed rFVIII displayed reduced binding signals compared with uncomplexed rFVIII, but the K (D) was still in the picomolar range (4.1 ± 1.9 pm) indicating insufficient complex formation. rFVIII, alone or bound to exogenously added VWF, showed high affinity for anti-FVIII IgGs from a severe haemophilia A patient whereas pdFVIII/VWF did not. These results are in agreement with those studies that point towards rFVIII concentrates to be more immunogenic than pdFVIII concentrates.


Subject(s)
Factor VIII/metabolism , von Willebrand Factor/metabolism , Animals , Antibodies, Anti-Idiotypic/immunology , Antigen-Antibody Complex , Bacterial Proteins/metabolism , Hemophilia A/pathology , Humans , Immunoglobulin G/immunology , Kinetics , Mice , Severity of Illness Index , Surface Plasmon Resonance
5.
Clin Exp Immunol ; 131(1): 41-7, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12519384

ABSTRACT

Differential aspects of the host immune response generated by Trypanosoma cruzi infection were examined in two different mouse strains, BALB/c (haplotype H2-Kd) which does not overcome the acute phase of the infection and C57BL/6 (haplotype H2-Kb) which survives to the acute phase. After infection an increase in CD3+ T cells was observed in both mouse strains in the peritoneal cavity. However, while the CD3+ T cells from the BALB/c mice showed an increase in the IL-4 cytokine expression level, the same type of cells from the C57BL/6 mice showed an increase in IFN-gamma expression. In addition, only the macrophages from the C57BL/6 mice were activated secreting IL-12 and TNF-alpha and producing, moreover, high levels of nitrites. It was observed that also after parasite infection the expression of macrophage and dendritic cells CD40 and CD86 co-stimulation molecules from the spleen were diminished in BALB/c but not in C57BL/6 mice. In correlation with this observation the macrophages from the spleen of infected BALB/c mice secreted lower concentrations of nitrites than the C57BL/6 mouse cells. Also, the spleen dendritic cells from infected BALB/c mice had a small potential to present alloantigens in contrast to that observed in the infected C57BL/6 mouse cells.


Subject(s)
Antigen-Presenting Cells/immunology , B7-1 Antigen/immunology , CD40 Antigens/immunology , Chagas Disease/immunology , Cytokines/metabolism , Trypanosoma cruzi/immunology , Animals , Antigen-Presenting Cells/metabolism , CD3 Complex/immunology , Cytokines/immunology , Dendritic Cells/immunology , Disease Susceptibility , Interferon-gamma/immunology , Interferon-gamma/metabolism , Interleukin-2/immunology , Interleukin-2/metabolism , Interleukin-4/immunology , Interleukin-4/metabolism , Macrophage Activation , Macrophages/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Nitrites , Species Specificity , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolism
6.
Immunol Cell Biol ; 80(3): 241-7, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12067411

ABSTRACT

We analyse the effect of Trypanosoma cruzi heat-shock protein-70 (HSP70) on the maturation of murine dendritic cells (DC)generated from bone marrow precursor cells. The results obtained show that HSP70, both alone and fused to the KMP11 antigen, as well as a HSP70 fragment, is capable of maturing murine DC. Mature DC have enhanced expression of IL12, TNF-alpha cytokines, costimulation molecules and activation markers, showing a clear increase in the allostimulatory capacity. These findings suggest that T. cruzi HSP70 may be a very useful vehicle for developing DC-based immunoprophylaxis and therapy against infections.


Subject(s)
Antigens, Protozoan/immunology , Dendritic Cells/immunology , HSP70 Heat-Shock Proteins/pharmacology , Membrane Glycoproteins/immunology , Protozoan Proteins/immunology , Trypanosoma cruzi/immunology , Animals , Antigens, Protozoan/genetics , Cells, Cultured , Dendritic Cells/drug effects , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Hematopoietic Stem Cells/physiology , Membrane Glycoproteins/genetics , Mice , Polymyxin B/pharmacology , Protozoan Proteins/genetics , Recombinant Fusion Proteins/pharmacology , Recombinant Proteins/pharmacology
7.
Mol Immunol ; 38(4): 279-87, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11566321

ABSTRACT

Cytotoxic T lymphocyte response against Jurkat-A2/K(b) cells expressing the T. cruzi KMP11 protein has been evaluated after immunization of C57BL/6-A2/K(b) transgenic mice with the KMP11 and KMP11-HSP70 recombinant proteins. The results show that mice immunized with KMP11 covalently fused to the T. cruzi HSP70 protein, but not mice immunized with KMP11 alone, elicit a CTL response against the Jurkat-A2/K(b) cells expressing the KMP11 protein. The data also show that spleen cells from mice immunized with the fusion protein and stimulated with the K1 peptide induce lysis of both the Jurkat-A2/K(b) cells transfected with the KMP11 coding gene and the Jurkat-A2-K(b) cells pulsed with the K1 peptide. Splenocytes stimulated with the K3 peptide induce lysis of the Jurkat-A2/K(b) cells loaded with the K3 peptide but they do not recognize the target cells expressing the KMP11 protein. Similar results were obtained using lymph node from mice immunized with the peptides. Thus, we believe there are two cytotoxic T cell epitopes restricted to the A2 molecule (K1(KMP11) (4-12) and K3(KMP11) (41-50)) in the KMP11 protein, and suggest that the K1 peptide could be considered an immunodominant antigen whilst the K3 peptide may be regarded as a cryptic epitope. The fact that the CTL lines induced in B6-A2/K(b) mice recognize human cells expressing KMP11 protein, indicates that the KMP11 antigen fused to HSP70 could be a good candidate for the induction of immunoprotective cytotoxic responses against T. cruzi natural infection.


Subject(s)
Antigens, Protozoan/immunology , H-2 Antigens/immunology , HLA-A2 Antigen/immunology , HSP70 Heat-Shock Proteins/genetics , T-Lymphocytes, Cytotoxic/immunology , Trypanosoma cruzi/immunology , Animals , Antigens, Protozoan/genetics , Cytotoxicity Tests, Immunologic , Epitopes, T-Lymphocyte/immunology , HLA-A2 Antigen/metabolism , Humans , Immunization , Jurkat Cells , Mice , Mice, Inbred C57BL , Mice, Transgenic , Peptides/metabolism , Recombinant Fusion Proteins/immunology , Transfection
8.
Clin Exp Immunol ; 123(3): 465-71, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11298135

ABSTRACT

The high variability among strains and isolates of Trypanosoma cruzi and the existence of shared antigenic determinants with other pathogens, particularly with members of the Leishmania genus make difficult the specific diagnosis of Chagas' disease. The data reported in this paper show that the T. cruzi KMP11 protein is an immunodominant antigen highly recognized by the sera from chagasic and leishmaniasis patients. By the use of amino- and carboxyl-terminal truncated KMP11 recombinant proteins and synthetic peptides, evidence is provided that while the sera from chagasic patients recognize linear peptides the sera from patients with visceral leishmaniasis must be predominantly directed against conformational epitopes. We found that a particular linear determinant, located in the carboxyl-terminal region of the protein, is recognized with high specificity and sensitivity only by sera from Chagas' disease patients, suggesting it could be a good candidate for differential serodiagnosis of Chagas' disease.


Subject(s)
Antibodies, Protozoan/immunology , Chagas Disease/immunology , Epitope Mapping , Membrane Glycoproteins/immunology , Mitochondria/immunology , Protozoan Proteins/immunology , Adult , Amino Acid Sequence , Antibodies, Protozoan/blood , Binding, Competitive , Epitopes , Humans , Leishmaniasis/immunology , Molecular Sequence Data , Peptide Fragments/immunology
9.
Ars pharm ; 42(1/2): 67-80, ene. 2001. ilus, graf
Article in En | IBECS | ID: ibc-23457

ABSTRACT

El análisis de la capacidad de unión a células T2 realizado con 31 péptidos correspondientes a distintas regiones de la proteína HSP70 de Trypanosoma cruzi, muestra que 14 de estos péptidos tienen una alta o media afinidad por la molécula presentadora A2.1. Interesantemente, el presente manuscrito pone de manifiesto que la inmunización de ratones transgénicos A2/Kb con la proteína recombinante HSP70 de T. cruzi induce CTLs que reconocen células EL4A2/Kb cargadas de forma independiente con tres de los péptidos con afinidad de unión a moléculas A2. Estos péptidos presentan una homología menor del 65 por ciento con sus homólogos de la proteína HSP70 humana. Los resultados obtenidos permiten sugerir la posibilidad de que la HSP70 de T. cruzi pueda ser usada como diana para inducir actividad inmune citotóxica en humanos (AU)


Subject(s)
Animals , Mice , Trypanosoma cruzi/genetics , Epitopes, T-Lymphocyte/genetics , HLA-A2 Antigen/genetics , Trypanosoma cruzi/immunology , Mice, Transgenic/genetics , Mice, Transgenic/immunology , Cytotoxicity Tests, Immunologic , HSP70 Heat-Shock Proteins/genetics
10.
Int Immunol ; 12(12): 1685-93, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11099308

ABSTRACT

The Trypanosoma cruzi HSP70 recombinant protein has the capacity to stimulate splenocytes or lymph node cells from naive mice in a non-haplotype-restricted way. The proliferative response is abolished by proteinase K digestion and by specific anti-HSP70 antibodies. The induced stimulation index was maximal after 24 h of incubation with the protein. This stimulation leads to cell death in a Fas-Fas ligand-independent way. The phenotype of the expanded cells was CD3(+) TCRalphass(+) CD4(+). HSP70-responsive cells express a broad range of cytokines including IFN-gamma, IL-2 and tumor necrosis factor-alpha. After 48 h of incubation with HSP70 there was a significant increase in relative intracellular levels of CD3 TCRalphass receptors. The expanded CD4(+) cell population expressed CD25; however, in contrast to concanavalin A-treated culture, delayed CD44 expression was observed.


Subject(s)
Antigens, Protozoan/pharmacology , Apoptosis , HSP70 Heat-Shock Proteins/pharmacology , Lymphocyte Activation/drug effects , T-Lymphocytes/drug effects , Trypanosoma cruzi/immunology , Animals , CD3 Complex/analysis , CD4 Antigens/analysis , Cells, Cultured , Cytokines/immunology , Endopeptidase K/pharmacology , Fas Ligand Protein , Lymph Nodes/drug effects , Lymph Nodes/immunology , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, Antigen, T-Cell, alpha-beta/analysis , Recombinant Proteins/pharmacology , Spleen/drug effects , Spleen/immunology , T-Lymphocytes/immunology , Time Factors , fas Receptor/metabolism
11.
Biochim Biophys Acta ; 1490(1-2): 1-10, 2000 Jan 31.
Article in English | MEDLINE | ID: mdl-10786612

ABSTRACT

We have recently described that the Trypanosoma cruzi histone H2A genes are actively transcribed as two sized classes of polyadenylated transcripts and that they differ in the 3'-UTRs due to the insertion of a partial SINE sequence in the 3'-end of some of H2A gene units. The expression of the H2A genes in the non-replicative trypomastigote forms is very low, whereas in the replicative forms, there is significant and constitutive transcription of the H2A genes probably regulated in a posttranscriptional way and associated to DNA replication. The data presented in this paper reveal that in epimastigotes, the steady-state levels of the H2A mRNAs are determined by controlling the stability of the messengers in the cytoplasm, most likely mediated by a nuclease attack. The data also indicate that there must be an additional control, associated to the parasite growth phase, which may act at the maturation step of the transcripts. The data suggest, moreover; that the cytoplasmic level of the H2A protein might be involved in the regulation of its own synthesis by controlling translation of existing messengers.


Subject(s)
Genes, Protozoan , Histones/genetics , RNA, Messenger/chemistry , Trypanosoma cruzi/genetics , Animals , Aphidicolin/pharmacology , Cytoplasm/chemistry , Histones/biosynthesis , Hydroxyurea/pharmacology , RNA Processing, Post-Transcriptional , RNA, Messenger/analysis , Time Factors , Transcription, Genetic , Trypanosoma cruzi/drug effects , Trypanosoma cruzi/growth & development
12.
Acta Trop ; 75(2): 203-10, 2000 Mar 25.
Article in English | MEDLINE | ID: mdl-10708660

ABSTRACT

The analysis of three recombinant clones containing the histone H2A locus isolated from a genomic library of Trypanosoma cruzi DNA shows that the H2A gene loci are formed by 1.2 and 0.76 kb long intercalated units organized in a head-to-tail tandem array. The difference in length between the two gene units is due to the presence of a short interspersed nucleotide element (SINE)-like DNA sequence inserted at the 3' end of some of these units. Southern, northern and chromosomal blot analysis of a Brazilian Y strain and six Colombian strains demonstrated the existence of polymorphisms regarding the relative copy number of the H2A gene units, the relative abundance of the H2A transcripts and their chromosomal location. These results show the existence of a dynamic organization in the H2A loci among T. cruzi strains in which a SINE-like sequence may be involved and support the fact that T. cruzi has a high degree of plasticity in its genome.


Subject(s)
Genes, Protozoan , Genome, Protozoan , Histones/genetics , Trypanosoma cruzi/genetics , Animals , Blotting, Northern , Blotting, Southern , Brazil , Cloning, Molecular , Colombia , DNA, Protozoan/analysis , Electrophoresis, Gel, Pulsed-Field , Escherichia coli/metabolism , Gene Dosage , Genetic Vectors , Histones/biosynthesis , Humans , Polymorphism, Genetic , RNA, Protozoan/analysis , Recombinant Proteins/biosynthesis , Short Interspersed Nucleotide Elements
13.
Psicothema (Oviedo) ; 12(1): 1-4, feb. 2000.
Article in Es | IBECS | ID: ibc-14584

ABSTRACT

Durante el desarrollo de la psicología se han elaborado una serie de «mitos» que se han propagado dentro y fuera de los centros de formación. Esos «mitos» han contribuido a desarrollar una enseñanza sesgada y una práctica profesional cargada de prejuicios sobre lo que la psicología científica puede (o debe) ofrecer a la sociedad. Así, a menudo se ha dado por hecho que la psicología no puede hacer ciencia de espalda a las corrientes ideológicas de los científicos que la practican. Contrariamente a lo que pudiera pensarse, esos «mitos» no solamente han circulado fuera de la psicología científica, sino que también han sido alimentados desde dentro. Este artículo revisa alguno de estos «mitos», cuestionando las bases sobre los que se han construido. La meta es contribuir a enterrar los que se podrían considerar «fantasmas del pasado», de modo que la psicología científica pueda ahora trabajar de un modo coordinado para intentar resolver los importantes problemas que le competen (AU)


Some «myths» in psychology: Science and ideology. Several «myths» are running inside and outside the scientific psychology. These «myths» have influenced the teaching of psychology in the classrooms. They also have contributed to a practical exercise overloaded by several bias about what psychology as a science can (or must) do for the benefit of society. Thus, some have suggested that it is impossible to keep apart science and ideology if you are a psychologist. Contrary to what could be supossed, those «myths» are not only outside scientific psychology, but also deep inside. This paper is intended to revisit some of these «myths», debunking their biased backgrounds. The main goal is to send away what can be termed as «the ghost of the past». Doing so, perhaps scientific psychology shall work in a coordinated way from now on (AU)


Subject(s)
Humans , Psychology/trends , Behavioral Sciences/trends , Prejudice , Mythology/psychology , Health Knowledge, Attitudes, Practice
14.
Anal Biochem ; 267(1): 234-5, 1999 Feb 01.
Article in English | MEDLINE | ID: mdl-9918677

ABSTRACT

In this paper, we describe the application of electroporation to deliver phage DNA into bacterial cells in order to recover it as phage particles. The methodology represents a quicker and cheaper alternative to the use of packaging extracts to rescue phage clones stored as naked DNAs. Furthermore, our data demonstrate that there were not rearrangements or recombinations between phage DNAs when a mixture of different DNAs was electroporated, suggesting the use of electroporation as a reliable method for construction of gene libraries.


Subject(s)
Bacteriophage lambda/isolation & purification , DNA, Viral/administration & dosage , Electroporation/methods , Bacteriophage lambda/genetics , DNA, Viral/genetics , Escherichia coli/genetics , Escherichia coli/virology , Gene Library , Gene Rearrangement , Genes, Viral , Genetic Vectors , Recombination, Genetic , Transformation, Genetic
15.
Mol Biochem Parasitol ; 92(2): 313-24, 1998 May 01.
Article in English | MEDLINE | ID: mdl-9657335

ABSTRACT

In a previous report we have described that the T. cruzi histone H2A gene is encoded in two independent gene clusters located in a single chromosome. In the present paper we show that both gene cluster are actively transcribed as two sized classes of polyadenylated mRNAs demonstrating, moreover, the existence of alternative splicing sites and microheterogeneities at the polyadenylation site. We also describe that while the expression of the H2A genes in the non replicative trypomastigote forms is only residual, in the replicative forms there is constitutive transcription of these genes and that the transcription is not associated to DNA replication. The data show, moreover, that in the replicative forms the steady state levels of the H2A mRNAs are controlled at a post-transcriptional level which is associated to DNA replication.


Subject(s)
Gene Expression Regulation , Histones/genetics , Multigene Family , Protozoan Proteins/genetics , Trypanosoma cruzi/genetics , Amino Acid Sequence , Animals , Base Sequence , Cycloheximide/pharmacology , DNA Transposable Elements , DNA, Protozoan/biosynthesis , Molecular Sequence Data , Promoter Regions, Genetic , Protein Synthesis Inhibitors/pharmacology , RNA Splicing , RNA, Messenger/metabolism , Sequence Alignment , Transcription, Genetic/drug effects , Trypanosoma cruzi/drug effects
16.
Parasitol Res ; 83(3): 226-32, 1997.
Article in English | MEDLINE | ID: mdl-9089717

ABSTRACT

In the present paper we describe the identification of an antigenic epitope that appears to be associated with the surface membrane of the Trypanosoma cruzi parasite, probably implicated in infectivity. Anti-TcMe antibodies inhibited the infectivity of fibroblast LLC-MK2 cells by 34% relative to a preimmune serum. The epitope was specifically recognized by 55% of the sera from 80 chagasic patients. The anti-TcMe antibody immunoprecipitated proteins of about 60 and 40 kDa from epimastigote and cell-culture trypomastigote forms, respectively. It is likely that the 60- and 40-kDa proteins are processed from higher-molecular-weight precursors, since the antibody immunoprecipitated protein fractions in the range of 115-150 kDa from in vitro translation products of poly A+ RNA.


Subject(s)
Antigens, Protozoan/immunology , Epitopes/immunology , Trypanosoma cruzi/immunology , Amino Acid Sequence , Animals , Cell Line , Fibroblasts/cytology , Humans , Macaca mulatta , Molecular Sequence Data , Rabbits
17.
J Clin Microbiol ; 34(2): 324-8, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8789008

ABSTRACT

A novel multiprimer PCR method with the potential to identify mycobacteria in clinical samples is presented. The assay relies on the simultaneous amplification of three bacterial DNA genomic fragments by using different sets of oligonucleotide primers. The first set of primers amplifies a 506-bp fragment from the gene for the 32-kDa antigen of Mycobacterium tuberculosis, which is present in most of the species belonging to the genus Mycobacterium. The second set of primers amplifies a 984-bp fragment from the IS6110 insertion sequence of the bacteria belonging to the M. tuberculosis complex. The third set of primers, derived from an M. tuberculosis species-specific sequence named MTP40, amplifies a 396-bp genomic fragment. Thus, while the multiprimer system would render three amplification fragments from the M. tuberculosis genome and two fragments from the Mycobacterium bovis genome, a unique amplification fragment would be obtained from nontuberculous mycobacteria. The results obtained, using reference mycobacterial strains and typed clinical isolates, show that the multiprimer PCR method may be a rapid, sensitive, and specific tool for the differential identification of various mycobacterial strains in a single-step assay.


Subject(s)
Mycobacterium/classification , Mycobacterium/genetics , Polymerase Chain Reaction/methods , Base Sequence , DNA Primers/genetics , DNA Probes/genetics , DNA Transposable Elements , DNA, Bacterial/genetics , Evaluation Studies as Topic , Humans , Molecular Sequence Data , Mycobacterium/isolation & purification , Mycobacterium Infections/diagnosis , Mycobacterium Infections/microbiology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Polymerase Chain Reaction/statistics & numerical data , Sensitivity and Specificity , Tuberculosis, Pulmonary/diagnosis , Tuberculosis, Pulmonary/microbiology
18.
J Mol Biol ; 247(1): 49-59, 1995 Mar 17.
Article in English | MEDLINE | ID: mdl-7534829

ABSTRACT

In the present paper we describe the characterization of a Trypanosoma cruzi cDNA (L1Tc) corresponding to a transcript from a new long terminal repeat (LTR) retrotransposon. This element is present in a high-copy number, and is found dispersed throughout the T. cruzi genome. Northern analysis shows an abundant expression of L1Tc-related sequences with a major band of about 5 kb. The transcript has at its 3' end a fragment of a highly repetitive DNA sequence (E12A), at its 5' end a ribosomal mobile element-like sequence and three putative open reading frames (ORF) in different frames. The ORF2 codes for a protein which has significant homology with the retrotranscriptase-related sequences from non-LTR retrotransposons containing the seven domains present in all the retrotranscriptase and retrotranscriptase-related proteins. The ORF3 codes for a gag-like protein showing unusual cysteine motifs present in all non-LTR trypanosomatid elements, similar to the C2H2 zinc finger family of transcription factors. Interestingly, ORF1 codes for a protein with significant homology to the major human AP endonuclease protein, and maintains in similar positions most of the amino acid domains described for all the Ape family of proteins. The presence of Ape-related sequences, described for the first time in a non-LTR retrotransposon (L1Tc), may have functional relevance for these types of elements.


Subject(s)
DNA, Protozoan/genetics , Retroelements/genetics , Trypanosoma cruzi/genetics , Amino Acid Sequence , Animals , Base Sequence , Consensus Sequence , DNA Repair , DNA, Complementary/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase , DNA-Binding Proteins/genetics , Deoxyribonuclease IV (Phage T4-Induced) , Genes, gag , Genes, pol , Lyases/genetics , Molecular Sequence Data , Open Reading Frames , RNA-Directed DNA Polymerase/genetics , Repetitive Sequences, Nucleic Acid , Sequence Alignment , Sequence Homology, Amino Acid , Transcription, Genetic
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