Ana Camacho - Academia.edu (original) (raw)

Papers by Ana Camacho

Research paper thumbnail of Description of a novel eukaryotic deoxyuridine 5′-triphosphate nucleotidohydrolase in Leishmania major

Biochemical Journal, 1997

A Leishmaniamajor full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.... more A Leishmaniamajor full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.6.1.23) was isolated from a cDNA expression library by genetic complementation of dUTPase deficiency in Escherichiacoli. The cDNA contained an open reading frame that encoded a protein of 269 amino acid residues with a calculated molecular mass of 30.3 kDa. Although eukaryotic dUTPases exhibit extensive similarity and there are five amino acid motifs that are common to all currently known dUTPase enzymes, the sequence of the protozoan gene differs significantly from its eukaryotic counterparts. None of the characteristic motifs were readily identifiable and the sequence encoded a larger polypeptide. However, the products of the reaction were dUMP and PPi, competition experiments with other deoxyribonucleoside triphosphates showed that the reaction is specific for dUTP, and the protozoan gene complemented dUTPase deficiency in Escherichiacoli. The gene is of single copy; Northern blots in...

Research paper thumbnail of A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi

Biochemical Journal, 1997

We report the isolation and characterization of a genomic clone containing the open reading frame... more We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts. Further examination of the 5′ flanking region by cDNA analysis and establishment of the splice acceptor sites clearly indicated that the corresponding mRNA apparently lacks sequences encoding a membrane N-terminal domain. The reductase gene is a single copy and is located on a chromosome of 1.36 Mb as determined by contour-clamped homogeneous electric field electrophoresis. The overall cellular distribution of enzymic activity was investigated after differential centrifu...

Research paper thumbnail of Use of n-Octyl-beta-D-thioglucopyranoside in the isolation of a bacterial membrane protein: an improved method for immunological applica- tions

Journal of Surface Science and Technology

Surfactants are essential in the isolation of integral membrane biomolecules from biological memb... more Surfactants are essential in the isolation of integral membrane biomolecules from biological membranes of different microorganisms. Our objective is the isolation of the major outer membrane protein (MOMP) of Chlamydia trachomatis, to use it in the preferential and specific detection of immunoglobulins. We have improved a method using an alkylglycoside surfactant, n-Octyl-b-D-thioglucoside (OTG), which is a non-ionic detergent used in membrane solubilization due to its "weak" action preserving the biological and functional properties of solubilized biomolecules. Different solubilization conditions, such as surfactant and salt concentrations, temperature and the presence of additive (di-thiothreitol (DTT)), were tested. To know the influence of the parameters previously indicated on the micellar properties and the solubilizing ability of the surfactant, we have studied the micellization process of OTG under several conditions by using static fluorescence measurements. MOMP ...

Research paper thumbnail of Descripción de inmunógenos de Chlamydia pneumoniae reconocidos por el suero de sujetos con enfermedad arterial periférica

Medicina Clínica, 2006

(grupo de investigación CTS 521 para el «Estudio de los agentes infecciosos relacionados con proc... more (grupo de investigación CTS 521 para el «Estudio de los agentes infecciosos relacionados con procesos clínicos de causa desconocida») y la Universidad de Granada (proyecto «Detección de Chlamydophila pneumoniae en la arteriopatía periférica. El papel del leucocito»).

Research paper thumbnail of A mutant dihydrofolate reductase-thymidylate synthase from Leishmania major as a selectable marker in transfection experiments

Molecular and Biochemical Parasitology, 1996

Research paper thumbnail of Characterization of the transcriptional regulator Rv3124 of Mycobacterium tuberculosis identifies it as a positive regulator of molybdopterin biosynthesis and defines the functional consequences of a non-synonymous SNP in the Mycobacterium bovis BCG orthologue

Microbiology, 2010

A number of single-nucleotide polymorphisms (SNPs) have been identified in the genome of Mycobact... more A number of single-nucleotide polymorphisms (SNPs) have been identified in the genome of Mycobacterium bovis BCG Pasteur compared with the sequenced strain M. bovis 2122/97. The functional consequences of many of these mutations remain to be described; however, mutations in genes encoding regulators may be particularly relevant to global phenotypic changes such as loss of virulence, since alteration of a regulator's function will affect the expression of a wide range of genes. One such SNP falls in bcg3145, encoding a member of the AfsR/DnrI/SARP class of global transcriptional regulators, that replaces a highly conserved glutamic acid residue at position 159 (E159G) with glycine in a tetratricopeptide repeat (TPR) located in the bacterial transcriptional activation (BTA) domain of BCG3145. TPR domains are associated with protein–protein interactions, and a conserved core (helices T1–T7) of the BTA domain seems to be required for proper function of SARP-family proteins. Structur...

Research paper thumbnail of Bacillus subtilis Bacteriophage SPP1 DNA Packaging Motor Requires Terminase and Portal Proteins

Journal of Biological Chemistry, 2003

Initiation of headful packaging of SPP1 DNA concatemers involves the interaction of the terminase... more Initiation of headful packaging of SPP1 DNA concatemers involves the interaction of the terminase, G1P and G2P, and the portal protein, G6P. G1P, which specifically recognizes the non-adjacent pacL and pacR subsites and directs loading of G2P to pacC, interacts with G6P. G2P, which has endonuclease, DNA binding, and ATPase activities, interacts with G1P and does it transiently with G6P. The stoichiometry of G1P on the G1P⅐G2P complex promotes the transition from a G2P endonuclease to an ATPase. G6P does not alter the endonuclease activity of G2P. Both G1P and G6P, which do not have endogenous ATPase activity, synergistically enhance and modulate the ATPase activity of G2P. Based on these results, we propose a model in which the modulation of the ATPase and endonuclease activities of G2P accounts for the role of the terminase in headful packaging.

Research paper thumbnail of Characterization of deoxyuridine 5′-triphosphate nucleotidohydrolase from Trypanosoma cruzi

FEBS Letters, 2002

We report the cloning and kinetic characterization of Trypanosoma cruzi deoxyuridine 5P P-triphos... more We report the cloning and kinetic characterization of Trypanosoma cruzi deoxyuridine 5P P-triphosphate nucleotidohydrolase (dUTPase) whose coding sequence was isolated by genetic complementation in Escherichia coli. The deduced amino acid sequence was similar to Leishmania major dUTPase although it exhibits an amino acid insertion which is sensitive to protease inactivation. The catalytically active species of the enzyme is a dimer and a detailed kinetic characterization showed that it is highly speci¢c for dUTP and dUDP. The general observation that dUTPases from the Trypanosomatidae di¡er in sequence, conformation and substrate speci¢city suggests that a di¡erent family of dUTPases exists in certain organisms, which may be exploited as drug targets against infectious diseases.

Research paper thumbnail of Evaluation of a Novel Enzyme-Linked Immunosorbent Assay To Detect Immunoglobulin G Antibody to Enolase for Serodiagnosis of Invasive Candidiasis

Clinical and Vaccine Immunology, 2007

The performance of a new test to detect antibodies to Candida albicans recombinant enolase was in... more The performance of a new test to detect antibodies to Candida albicans recombinant enolase was investigated in 47 immunocompromised and 51 immunocompetent patients. The sensitivity, specificity, and positive and negative predictive values of the test for the diagnosis of invasive candidiasis were 81.0, 83.9, 79.1, and 85.5%, respectively.

Research paper thumbnail of pSM19035-encoded ζ toxin induces stasis followed by death in a subpopulation of cells

Microbiology, 2006

The toxin–antitoxin operon of pSM19035 encodes three proteins: the ω global regulator, the ε labi... more The toxin–antitoxin operon of pSM19035 encodes three proteins: the ω global regulator, the ε labile antitoxin and the stable ζ toxin. Accumulation of ζ toxin free of ε antitoxin induced loss of cell proliferation in both Bacillus subtilis and Escherichia coli cells. Induction of a ζ variant (ζY83C) triggered stasis, in which B. subtilis cells were viable but unable to proliferate, without selectively affecting protein translation. In E. coli cells, accumulation of free ζ toxin induced stasis, but this was fully reversed by expression of the ε antitoxin within a defined time window. The time window for reversion of ζ toxicity by expression of ε antitoxin was dependent on the initial cellular level of ζ. After 240 min of constitutive expression, or inducible expression of high levels of ζ toxin for 30 min, expression of ε failed to reverse the toxic effect exerted by ζ in cells growing in minimal medium. Under the latter conditions, ζ inhibited replication, transcription and translati...

Research paper thumbnail of Description of a novel eukaryotic deoxyuridine 5′-triphosphate nucleotidohydrolase in Leishmania major

Biochemical Journal, 1997

A Leishmaniamajor full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.... more A Leishmaniamajor full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.6.1.23) was isolated from a cDNA expression library by genetic complementation of dUTPase deficiency in Escherichiacoli. The cDNA contained an open reading frame that encoded a protein of 269 amino acid residues with a calculated molecular mass of 30.3 kDa. Although eukaryotic dUTPases exhibit extensive similarity and there are five amino acid motifs that are common to all currently known dUTPase enzymes, the sequence of the protozoan gene differs significantly from its eukaryotic counterparts. None of the characteristic motifs were readily identifiable and the sequence encoded a larger polypeptide. However, the products of the reaction were dUMP and PPi, competition experiments with other deoxyribonucleoside triphosphates showed that the reaction is specific for dUTP, and the protozoan gene complemented dUTPase deficiency in Escherichiacoli. The gene is of single copy; Northern blots in...

Research paper thumbnail of A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi

Biochemical Journal, 1997

We report the isolation and characterization of a genomic clone containing the open reading frame... more We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts. Further examination of the 5′ flanking region by cDNA analysis and establishment of the splice acceptor sites clearly indicated that the corresponding mRNA apparently lacks sequences encoding a membrane N-terminal domain. The reductase gene is a single copy and is located on a chromosome of 1.36 Mb as determined by contour-clamped homogeneous electric field electrophoresis. The overall cellular distribution of enzymic activity was investigated after differential centrifu...

Research paper thumbnail of Use of n-Octyl-beta-D-thioglucopyranoside in the isolation of a bacterial membrane protein: an improved method for immunological applica- tions

Journal of Surface Science and Technology

Surfactants are essential in the isolation of integral membrane biomolecules from biological memb... more Surfactants are essential in the isolation of integral membrane biomolecules from biological membranes of different microorganisms. Our objective is the isolation of the major outer membrane protein (MOMP) of Chlamydia trachomatis, to use it in the preferential and specific detection of immunoglobulins. We have improved a method using an alkylglycoside surfactant, n-Octyl-b-D-thioglucoside (OTG), which is a non-ionic detergent used in membrane solubilization due to its "weak" action preserving the biological and functional properties of solubilized biomolecules. Different solubilization conditions, such as surfactant and salt concentrations, temperature and the presence of additive (di-thiothreitol (DTT)), were tested. To know the influence of the parameters previously indicated on the micellar properties and the solubilizing ability of the surfactant, we have studied the micellization process of OTG under several conditions by using static fluorescence measurements. MOMP ...

Research paper thumbnail of Descripción de inmunógenos de Chlamydia pneumoniae reconocidos por el suero de sujetos con enfermedad arterial periférica

Medicina Clínica, 2006

(grupo de investigación CTS 521 para el «Estudio de los agentes infecciosos relacionados con proc... more (grupo de investigación CTS 521 para el «Estudio de los agentes infecciosos relacionados con procesos clínicos de causa desconocida») y la Universidad de Granada (proyecto «Detección de Chlamydophila pneumoniae en la arteriopatía periférica. El papel del leucocito»).

Research paper thumbnail of A mutant dihydrofolate reductase-thymidylate synthase from Leishmania major as a selectable marker in transfection experiments

Molecular and Biochemical Parasitology, 1996

Research paper thumbnail of Characterization of the transcriptional regulator Rv3124 of Mycobacterium tuberculosis identifies it as a positive regulator of molybdopterin biosynthesis and defines the functional consequences of a non-synonymous SNP in the Mycobacterium bovis BCG orthologue

Microbiology, 2010

A number of single-nucleotide polymorphisms (SNPs) have been identified in the genome of Mycobact... more A number of single-nucleotide polymorphisms (SNPs) have been identified in the genome of Mycobacterium bovis BCG Pasteur compared with the sequenced strain M. bovis 2122/97. The functional consequences of many of these mutations remain to be described; however, mutations in genes encoding regulators may be particularly relevant to global phenotypic changes such as loss of virulence, since alteration of a regulator's function will affect the expression of a wide range of genes. One such SNP falls in bcg3145, encoding a member of the AfsR/DnrI/SARP class of global transcriptional regulators, that replaces a highly conserved glutamic acid residue at position 159 (E159G) with glycine in a tetratricopeptide repeat (TPR) located in the bacterial transcriptional activation (BTA) domain of BCG3145. TPR domains are associated with protein–protein interactions, and a conserved core (helices T1–T7) of the BTA domain seems to be required for proper function of SARP-family proteins. Structur...

Research paper thumbnail of Bacillus subtilis Bacteriophage SPP1 DNA Packaging Motor Requires Terminase and Portal Proteins

Journal of Biological Chemistry, 2003

Initiation of headful packaging of SPP1 DNA concatemers involves the interaction of the terminase... more Initiation of headful packaging of SPP1 DNA concatemers involves the interaction of the terminase, G1P and G2P, and the portal protein, G6P. G1P, which specifically recognizes the non-adjacent pacL and pacR subsites and directs loading of G2P to pacC, interacts with G6P. G2P, which has endonuclease, DNA binding, and ATPase activities, interacts with G1P and does it transiently with G6P. The stoichiometry of G1P on the G1P⅐G2P complex promotes the transition from a G2P endonuclease to an ATPase. G6P does not alter the endonuclease activity of G2P. Both G1P and G6P, which do not have endogenous ATPase activity, synergistically enhance and modulate the ATPase activity of G2P. Based on these results, we propose a model in which the modulation of the ATPase and endonuclease activities of G2P accounts for the role of the terminase in headful packaging.

Research paper thumbnail of Characterization of deoxyuridine 5′-triphosphate nucleotidohydrolase from Trypanosoma cruzi

FEBS Letters, 2002

We report the cloning and kinetic characterization of Trypanosoma cruzi deoxyuridine 5P P-triphos... more We report the cloning and kinetic characterization of Trypanosoma cruzi deoxyuridine 5P P-triphosphate nucleotidohydrolase (dUTPase) whose coding sequence was isolated by genetic complementation in Escherichia coli. The deduced amino acid sequence was similar to Leishmania major dUTPase although it exhibits an amino acid insertion which is sensitive to protease inactivation. The catalytically active species of the enzyme is a dimer and a detailed kinetic characterization showed that it is highly speci¢c for dUTP and dUDP. The general observation that dUTPases from the Trypanosomatidae di¡er in sequence, conformation and substrate speci¢city suggests that a di¡erent family of dUTPases exists in certain organisms, which may be exploited as drug targets against infectious diseases.

Research paper thumbnail of Evaluation of a Novel Enzyme-Linked Immunosorbent Assay To Detect Immunoglobulin G Antibody to Enolase for Serodiagnosis of Invasive Candidiasis

Clinical and Vaccine Immunology, 2007

The performance of a new test to detect antibodies to Candida albicans recombinant enolase was in... more The performance of a new test to detect antibodies to Candida albicans recombinant enolase was investigated in 47 immunocompromised and 51 immunocompetent patients. The sensitivity, specificity, and positive and negative predictive values of the test for the diagnosis of invasive candidiasis were 81.0, 83.9, 79.1, and 85.5%, respectively.

Research paper thumbnail of pSM19035-encoded ζ toxin induces stasis followed by death in a subpopulation of cells

Microbiology, 2006

The toxin–antitoxin operon of pSM19035 encodes three proteins: the ω global regulator, the ε labi... more The toxin–antitoxin operon of pSM19035 encodes three proteins: the ω global regulator, the ε labile antitoxin and the stable ζ toxin. Accumulation of ζ toxin free of ε antitoxin induced loss of cell proliferation in both Bacillus subtilis and Escherichia coli cells. Induction of a ζ variant (ζY83C) triggered stasis, in which B. subtilis cells were viable but unable to proliferate, without selectively affecting protein translation. In E. coli cells, accumulation of free ζ toxin induced stasis, but this was fully reversed by expression of the ε antitoxin within a defined time window. The time window for reversion of ζ toxicity by expression of ε antitoxin was dependent on the initial cellular level of ζ. After 240 min of constitutive expression, or inducible expression of high levels of ζ toxin for 30 min, expression of ε failed to reverse the toxic effect exerted by ζ in cells growing in minimal medium. Under the latter conditions, ζ inhibited replication, transcription and translati...