Publications by authors named "Alejandra Diaz"

20 Publications

  • Page 1 of 1

Maternal undernutrition during pregnancy and lactation increases transcription factors, ETV5 and GDNF, and alters regulation of apoptosis and heat shock proteins in the testis of adult offspring in the rat.

Reprod Fertil Dev 2021 May;33(7):484-496

Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, L8S 4L8, Canada; and Department of Pediatrics, McMaster University, Hamilton, L8S 4L8, Canada, and Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, L8S 4L8, Canada.

We tested whether changes in Sertoli cell transcription factors and germ cell heat shock proteins (HSPs) are linked to the effects of maternal undernutrition on male offspring fertility. Rats were fed ad libitum with a standard diet (CONTROL) throughout pregnancy and lactation or with 50% of CONTROL intake throughout pregnancy (UNP) or lactation (UNL) or both periods (UNPL). After postnatal Day 21, 10 male pups per group were fed a standard diet ad libitum until postnatal Day 160 when testes were processed for histological, mRNA and immunohistochemical analyses. Compared with CONTROL: caspase-3 was increased in UNP and UNPL (P=0.001); Bax was increased in UNL (P=0.002); Bcl-2 (P<0.0001) was increased in all underfed groups; glial cell line-derived neurotrophic factor (P=0.002) was increased in UNP and UNL; E twenty-six transformation variant gene 5 and HSP70 were increased, and HSP90 was diminished in all underfed groups (P<0.0001). It appears that maternal undernutrition during pregnancy and lactation disrupts the balance between proliferation and apoptosis in germ cells, increasing germ cell production and perhaps exceeding the support capacity of the Sertoli cells. Moreover, fertility could be further compromised by changes in meiosis and spermiogenesis mediated by germ cell HSP90 and HSP70.
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http://dx.doi.org/10.1071/RD20260DOI Listing
May 2021

Identification of Novel Thermosensors in Gram-Positive Pathogens.

Front Mol Biosci 2020 26;7:592747. Epub 2020 Nov 26.

Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Rosario, Argentina.

Temperature is a crucial variable that every living organism, from bacteria to humans, need to sense and respond to in order to adapt and survive. In particular, pathogenic bacteria exploit host-temperature sensing as a cue for triggering virulence gene expression. Here, we have identified and characterized two integral membrane thermosensor histidine kinases (HKs) from Gram-positive pathogens that exhibit high similarity to DesK, the extensively characterized cold sensor histidine kinase from . Through experiments, we demonstrate that SA1313 from and BA5598 from , which likely control the expression of putative ATP binding cassette (ABC) transporters, are regulated by environmental temperature. We show here that these HKs can phosphorylate the non-cognate response regulator DesR, partner of DesK, both and , inducing in the expression of the gene upon a cold shock. In addition, we report the characterization of another DesK homolog from , YvfT, also closely associated to an ABC transporter. Although YvfT phosphorylates DesR , this sensor kinase can only induce expression in when overexpressed together with its cognate response regulator YvfU. This finding evidences a physiological mechanism to avoid cross talk with DesK after a temperature downshift. Finally, we present data suggesting that the HKs studied in this work appear to monitor different ranges of membrane lipid properties variations to mount adaptive responses upon cooling. Overall, our findings point out that bacteria have evolved sophisticated mechanisms to assure specificity in the response to environmental stimuli. These findings pave the way to understand thermosensing mediated by membrane proteins that could have important roles upon host invasion by bacterial pathogens.
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http://dx.doi.org/10.3389/fmolb.2020.592747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726353PMC
November 2020

Novelties in the genus Espejo (Tillandsioideae, Bromeliaceae).

PhytoKeys 2019 2;132:99-110. Epub 2019 Oct 2.

Herbario Metropolitano, Departamento de Biología, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana Unidad Iztapalapa, C.P. 09340, Ciudad de México, México Universidad Autónoma Metropolitana Mexico Mexico.

Based on morphological evidence, we propose to raise Tillandsia mauryana forma secundifolia to species level with the name (Ehlers) Hern.-Cárdenas, Espejo & López-Ferr. can be readily distinguished by the falciform rosettes, the broadly oblong to square, 1-1.2 × 0.8-1.1 cm leaf sheaths and by the 1.8-2 × 0.7-1.2 cm floral bracts. Additionally, we describe and illustrate Hern.-Cárdenas, Espejo & López-Ferr., from the state of Oaxaca, Mexico. The new species is morphologically similar to , but differs by the nearly square leaf sheaths, 1.3-1.5 × 0.4-0.5 cm spikes and by the presence of only one flower per spike. A key to the taxa, morphological descriptions, list of specimens examined, illustrations and a distribution map of the described taxa are included.
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http://dx.doi.org/10.3897/phytokeys.132.36959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785572PMC
October 2019

Mucosal immunization with polymeric antigen BLSOmp31 using alternative delivery systems against Brucella ovis in rams.

Vet Immunol Immunopathol 2019 Mar 2;209:70-77. Epub 2019 Mar 2.

Laboratorio de Inmunología, Departamento de Sanidad Animal y Medicina Preventiva (SAMP), Centro de Investigación Veterinaria Tandil (CIVETAN-CONICET-CICPBA), Facultad de Ciencias Veterinarias (FCV), Universidad Nacional del Centro de la Provincia de Buenos Aires (UNCPBA), Tandil, 7000, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina. Electronic address:

Subcellular vaccines against ovine contagious epididymitis due Brucella ovis can solve some shortcomings associated with the use of Brucella melitensis Rev 1. We have demonstrated that the parenteral immunization with polymeric antigen BLSOmp31 emulsified in oil adjuvant conferred significant protection against B. ovis in rams. In our previous studies, we have characterized chitosan microspheres (ChMs) and a thermoresponsive and mucoadhesive in situ gel (Poloxamer 407-Ch) as two novel formulation strategies for the delivery of BLSOmp31 in nasal as well as conjunctival mucosa. In the present work, we evaluated the immunogenicity and protection conferred by the intranasal and conjunctival immunization with these two mucosal delivery systems against B. ovis in rams. BLSOmp31-ChM administered by intranasal route and BLSOmp31-P407-Ch applied by intranasal or conjunctival routes induced systemic, local and preputial IgG and IgA antibody response. Neither formulation showed interference in the serological diagnosis. Thus, mucosal immunization using either formulation induced significant specific cellular immune responses (in vitro and in vivo) and it prevented the excretion of B. ovis in semen. Although these vaccines did not prevent infection in immunized rams, colonization reduction of infected organs and bacterial distribution differed significantly between vaccinated and unvaccinated rams.
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http://dx.doi.org/10.1016/j.vetimm.2019.02.005DOI Listing
March 2019

Identification and characterization of an M cell marker in nasopharynx- and oropharynx-associated lymphoid tissue of sheep.

Vet Immunol Immunopathol 2019 Feb 18;208:1-5. Epub 2018 Dec 18.

Centre for Animal Biotechnology, Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Victoria, 3010, Australia. Electronic address:

M cells play a pivotal role in the induction of immune responses within the mucosa-associated lymphoid tissues. M cells exist principally in the follicle-associated epithelium (FAE) of the isolated solitary lymphoid follicles as well as in the lymphoid follicles of nasopharynx-associated lymphoid tissue and gut associated lymphoid tissue (GALT). Through lymphatic cannulation it is possible to investigate local immune responses induced following nasal Ag delivery in sheep. Hence, identifying sheep M cell markers would allow the targeting of M cells to offset the problem of trans-epithelial Ag delivery associated with inducing mucosal immunity. Sheep cDNA from the tonsils of the oropharynx and nasopharynx was PCR amplified using Glycoprotein-2 (GP)-specific primers and expressed as a poly-His-tagged recombinant sheep GP (56 kDa) in HEK293 cells. The recombinant GP protein was purified using Ni-NTA affinity chromatography and polyclonal serum against the protein was raised in rats. The antiserum recognized the recombinant sheep GP and purified rat IgG against GP stained M cells in sections of sheep tonsils from nasopharynx and oropharynx. M cells were found to be present in epithelium of the palatine tonsils (oropharynx), pharyngeal tonsils as well as tubal tonsils (nasopharynx). They were also present in the FAE of the scattered lymphoid follicles over the base of the nasopharynx. Thus, GP has been identified to be an important M cell marker of nasopharynx and oropharynx-associated lymphoid tissues in sheep.
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http://dx.doi.org/10.1016/j.vetimm.2018.12.005DOI Listing
February 2019

A genetic screen for mutations affecting temperature sensing in Bacillus subtilis.

Microbiology (Reading) 2019 01 15;165(1):90-101. Epub 2018 Nov 15.

2​Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario and Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Rosario, Argentina.

Two component systems, composed of a receptor histidine kinase and a cytoplasmic response regulator, regulate pivotal cellular processes in microorganisms. Here we describe a new screening procedure for the identification of amino acids that are crucial for the functioning of DesK, a prototypic thermosensor histidine kinase from Bacillus subtilis. This experimental strategy involves random mutagenesis of the membrane sensor domain of the DesK coding sequence, followed by the use of a detection procedure based on changes in the colony morphogenesis that take place during the sporulation programme of B. subtilis. This method permitted us the recovery of mutants defective in DesK temperature sensing. This screening approach could be applied to all histidine kinases of B. subtilis and also to kinases of other bacteria that are functionally expressed in this organism. Moreover, this reporter assay could be expanded to develop reporter assays for a variety of transcriptionally regulated systems.
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http://dx.doi.org/10.1099/mic.0.000741DOI Listing
January 2019

Polymeric antigen BLSOmp31 in aluminium hydroxide induces serum bactericidal and opsonic antibodies against Brucella canis in dogs.

Vet Immunol Immunopathol 2017 Feb 5;184:36-41. Epub 2016 Dec 5.

Laboratorio de Inmunología, Departamento de Sanidad Animal y Medicina Preventiva (SAMP), Centro de Investigación Veterinaria de Tandil (CIVETAN-CONICET-CIC), Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires (UNCPBA), Tandil, Argentina; Consejo Nacional de Investigaciones Científicas (CONICET), Argentina.

Polymeric antigen BLSOmp31 is an immunogenic vaccine candidate that confers protection against Brucella canis in mice. In this preliminary study, the immunogenicity and safety of BLSOmp31 adsorbed to aluminum hydroxide gel (BLSOmp31-AH) were evaluated in Beagle dogs. In addition, the potential to elicit serum antibodies with complement-dependent bactericidal activity and/or to enhance phagocytosis by neutrophils were analyzed. Dogs were immunized three times with BLSOmp31-AH by subcutaneous route, followed by an annual booster. The vaccine elicited specific antibodies 3 weeks after the first immunization. Annual booster induced comparable antibody response as the primary series. Humoral immune response stimulated by BLSOmp31-AH did not interfere with routine agglutination test for canine brucellosis. Antibodies demonstrated a high complement-dependent bactericidal activity against B. canis. Moreover, opsonization by immune serum not only stimulated binding and uptake of the bacteria by neutrophils but effectively enhanced the destruction of B. canis. Specific IgG was detected in 3/4 immunized dogs in preputial secretions. The antibody profile corresponded to a marked Th2 response, since IgG1 prevailed over IgG2 and cellular immune response was not detected in vitro or in vivo. These results require further evaluation in larger field studies to establish the full prophylactic activity of BLSOmp31 against canine brucellosis.
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http://dx.doi.org/10.1016/j.vetimm.2016.11.004DOI Listing
February 2017

Engineered feature used to enhance gardening at a 3800-year-old site on the Pacific Northwest Coast.

Sci Adv 2016 Dec 21;2(12):e1601282. Epub 2016 Dec 21.

Katzie Development Limited Partnership, 10946 Katzie Road, Pitt Meadows, British Columbia V3Y 2G6, Canada.

Humans use a variety of deliberate means to modify biologically rich environs in pursuit of resource stability and predictability. Empirical evidence suggests that ancient hunter-gatherer populations engineered ecological niches to enhance the productivity and availability of economically significant resources. An archaeological excavation of a 3800-year-old wetland garden in British Columbia, Canada, provides the first direct evidence of an engineered feature designed to facilitate wild plant food production among mid-to-late Holocene era complex fisher-hunter-gatherers of the Northwest Coast. This finding provides an example of environmental, economic, and sociopolitical coevolutionary relationships that are triggered when humans manipulate niche environs.
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http://dx.doi.org/10.1126/sciadv.1601282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5176348PMC
December 2016

Immune response induced by conjunctival immunization with polymeric antigen BLSOmp31 using a thermoresponsive and mucoadhesive in situ gel as vaccine delivery system for prevention of ovine brucellosis.

Vet Immunol Immunopathol 2016 Oct 5;178:50-6. Epub 2016 Jul 5.

Laboratorio de Inmunología, Departamento de Sanidad Animal y Medicina Preventiva (SAMP), Centro de Investigación Veterinaria Tandil (CIVETAN-CONICET-CIC), Facultad de Ciencias Veterinarias (FCV), Universidad Nacional del Centro de la Provincia de Buenos Aires (UNCPBA), Tandil, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina. Electronic address:

Control of ovine brucellosis with subcellular vaccines can solve some drawbacks associated with the use of Brucella melitensis Rev.1. Previous studies have demonstrated that the polymeric antigen BLSOmp31 administered by parenteral route was immunogenic and conferred significant protection against B. ovis in rams. Immunization with BLSOmp31 by conjunctival route could be efficient for the induction of mucosal and systemic immune responses. In this work, we evaluated the conjunctival immunization using a thermoresponsive and mucoadhesive in situ gel composed of Poloxamer 407 (P407) and chitosan (Ch) as vaccine delivery system for BLSOmp31 in rams. Serum samples, saliva, lacrimal, preputial and nasal secretions were analyzed to measure specific IgG and IgA antibodies. Cellular immune response was evaluated in vivo and in vitro. Immunization with BLSOmp31-P407-Ch induced high IgG antibody levels in serum and preputial secretions which remained at similar levels until the end of the experiment. Levels of IgG in saliva, lacrimal and nasal secretions were also higher compared to unvaccinated control group but decreased more rapidly. IgA antibodies were only detected in nasal and preputial secretions. BLSOmp31-P407-Ch stimulated a significant cellular immune response in vivo and in vitro. The induction of systemic and local immune responses indicates a promising potential of P407-Ch for the delivery of BLSOmp31 by conjunctival route.
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http://dx.doi.org/10.1016/j.vetimm.2016.07.004DOI Listing
October 2016

Spray dried microspheres based on chitosan: A promising new carrier for intranasal administration of polymeric antigen BLSOmp31 for prevention of ovine brucellosis.

Mater Sci Eng C Mater Biol Appl 2016 May 2;62:489-96. Epub 2016 Feb 2.

Laboratorio de Inmunología, Departamento de Sanidad Animal y Medicina Preventiva (SAMP), Centro de Investigación Veterinaria Tandil (CIVETAN-CONICET-CIC), Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina. Electronic address:

Previous studies have demonstrated that parenteral immunization with polymeric antigen BLSOmp31 induced a strong immune response and conferred protection against Brucella ovis in rams. This work describes the development of a novel formulation strategy for the delivery of BLSOmp31 in the nasal mucosa. Chitosan microparticles were prepared by spray-drying technology processes and recombinant chimera BLSOmp31 was loaded by passive adsorption onto chitosan microspheres, which were characterized by means of the evaluation of size, zeta potential, morphology, and loading and release rate of BLSOmp31. The mucoadhesive properties of microspheres were evaluated by studying the interaction between microparticles and mucin. The antigen BLSOmp31 integrity was investigated by SDS-PAGE. The yield of production of spray-drying process was 68.95%. Microspheres had a good sphericity, 1-10 μm of particle size and had a positive charge. The loading capacity was found to be 45.19%. The initial fast release of BLSOmp31 from chitosan microparticles was 60%. The BLSOmp31 integrity was not affected by passive adsorption (ionic interaction). The amount of mucin adsorbed on the surface of CMs-BLSOmp31 was lower than on the surface of blank CMs at neutral pH. In vivo studies were carried out in rams. Intranasal immunization induced systemic and local antibodies. In conclusion, the use of BLSOmp31-loaded chitosan spray-drying microspheres offers a promising way for nasal mucosal vaccination in sheep against brucellosis.
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http://dx.doi.org/10.1016/j.msec.2016.01.084DOI Listing
May 2016

Pyruvate dehydrogenase deficiency presenting as isolated paroxysmal exercise induced dystonia successfully reversed with thiamine supplementation. Case report and mini-review.

Eur J Paediatr Neurol 2015 Sep 14;19(5):497-503. Epub 2015 May 14.

Unit of Neurology, Dept. of Pediatrics and Dept. of Neurology, Clínica las Condes, Santiago, Chile.

Background: Pyruvate dehydrogenase (PDH) deficiency is a disorder of energy metabolism with variable clinical presentations, ranging from severe infantile lactic acidosis to milder chronic neurological disorders. The spectrum of clinical manifestations is continuously expanding.

Methods And Results: We report on a 19-year-old intelligent female with PDH deficiency caused by a Leu216Ser mutation in PDHA1. She presented with recurrent hemidystonic attacks, triggered by prolonged walking or running, as the unique clinical manifestation that manifested since childhood. Laboratory workup and neuroimages were initially normal but bilateral globus pallidum involvement appeared later on brain MRI. Dystonia completely remitted after high doses of thiamine, remaining free of symptoms after 3 years of follow up. We reviewed the literature for similar observations.

Conclusions: Dystonia precipitated by exercise may be the only symptom of a PDH deficiency, and the hallmark of the disease as high serum lactate or bilateral striatal necrosis at neuroimaging may be absent. A high index of suspicion and follow up is necessary for diagnosis. The clinical presentation of this patient meets the criteria for a Paroxysmal Exercise induced Dystonia, leading us to add this entity as another potential etiology for this type of paroxysmal dyskinesia, which is besides a treatable condition that responds to thiamine supplementation.
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http://dx.doi.org/10.1016/j.ejpn.2015.04.008DOI Listing
September 2015

Evaluation of the efficacy of outer membrane protein 31 vaccine formulations for protection against Brucella canis in BALB/c mice.

Clin Vaccine Immunol 2014 Dec 22;21(12):1689-94. Epub 2014 Oct 22.

Laboratorio de Inmunología, Departamento SAMP, Facultad de Ciencias Veterinarias, Centro de Investigación Veterinaria de Tandil (CIVETAN-CONICET), Universidad Nacional del Centro de la Provincia de Buenos Aires, Tandil, Argentina

Canine brucellosis is an infectious disease caused by the Gram-negative bacterium Brucella canis. Unlike conventional control programs for other species of the genus Brucella, currently there is no vaccine available against canine brucellosis, and preventive measures are simply diagnosis and isolation of infected dogs. New approaches are therefore needed to develop an effective and safe immunization strategy against this zoonotic pathogen. In this study, BALB/c mice were subcutaneously immunized with the following: (i) the recombinant Brucella Omp31 antigen formulated in different adjuvants (incomplete Freund adjuvant, aluminum hydroxide, Quil A, and Montanide IMS 3012 VGPR), (ii) plasmid pCIOmp31, or (iii) pCIOmp31 plasmid followed by boosting with recombinant Omp31 (rOmp31). The immune response and the protective efficacy against B. canis infection were characterized. The different strategies induced a strong immunoglobulin G (IgG) response. Furthermore, spleen cells from rOmp31-immunized mice produced gamma interferon and interleukin-4 (IL-4) after in vitro stimulation with rOmp31, indicating the induction of a mixed Th1-Th2 response. Recombinant Omp31 administered with different adjuvants as well as the prime-boost strategy conferred protection against B. canis. In conclusion, our results suggest that Omp31 could be a useful candidate for the development of a subcellular vaccine against B. canis infection.
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http://dx.doi.org/10.1128/CVI.00527-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248782PMC
December 2014

The vaccine candidate BLSOmp31 protects mice against Brucella canis infection.

Vaccine 2013 Dec 30;31(51):6129-35. Epub 2013 Jul 30.

Laboratorio de Inmunología, Departamento SAMP, Centro de Investigación Veterinaria de Tandil (CIVETAN) (CONICET), Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires, Tandil, Argentina; Consejo Nacional de Investigaciones Científicas (CONICET), Argentina.

Canine brucellosis represents a major reproductive problem worldwide and it is considered a zoonotic disease. New approaches are therefore urgently needed to develop an effective and safe immunization strategy against Brucella canis. In the present study, BALB/c mice were subcutaneously immunized with the recombinant chimera rBLSOmp31 formulated in different adjuvants. The different strategies induced a vigorous immunoglobulin G (IgG) response, with high titers of IgG1 as well as IgG2. Besides, spleen cells from rBLSOmp31-immunized mice produced gamma interferon and IL-4, suggesting the induction of a mixed Th1-Th2. Vaccination with rBLSOmp31-IFA formulation provided the best protection levels comparable with that given by control vaccines. None of the immunization strategies induced serological interference in diagnosis. Hitherto, this is the first report that a recombinant vaccine confers protection against B. canis in mice.
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http://dx.doi.org/10.1016/j.vaccine.2013.07.041DOI Listing
December 2013

Immune response and serum bactericidal activity against Brucella ovis elicited using a short immunization schedule with the polymeric antigen BLSOmp31 in rams.

Vet Immunol Immunopathol 2013 Jul 11;154(1-2):36-41. Epub 2013 Apr 11.

Laboratorio de Inmunología, Depto. SAMP, Centro de Investigación Veterinaria de Tandil (CIVETAN)- CONICET, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires (U.N.C.P.B.A.), Tandil, Buenos Aires, Argentina.

Brucella ovis is the etiologic agent of ovine brucellosis. The control measures for this disease are periodical diagnosis by serological tests and/or bacteriological culture and culling of positive animals. Vaccination with Brucella melitensis Rev 1 is recommended when prevalence is high. This attenuated strain vaccine gives protection against B. ovis but it has important disadvantages associated with the development of antibodies interfering with serodiagnosis, virulence for humans and the prohibition of its use in countries considered free of B. melitensis. Consequently, there is a need for new safe and effective brucellosis vaccines to be developed. We have previously reported that the polymeric subcellular vaccine BLSOmp31 confers protection against experimental challenge with B. ovis when rams are immunized three times. In the present work we evaluated and characterized, along 56 weeks after the first immunization of adult rams, the evolution of the immune response elicited by BLSOmp31 using a short immunization schedule.
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http://dx.doi.org/10.1016/j.vetimm.2013.04.003DOI Listing
July 2013

Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide.

J Bacteriol 2012 Mar 20;194(6):1378-88. Epub 2012 Jan 20.

The Scripps Research Institute, Department of Molecular and Experimental Medicine, La Jolla, California, USA.

Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0F∼P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0F∼P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.
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http://dx.doi.org/10.1128/JB.06747-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294843PMC
March 2012

[Clinical, electrophysiological and molecular study of 26 chilean patients with spinal muscular atrophy].

Rev Med Chil 2011 Feb 11;139(2):197-204. Epub 2011 Jul 11.

Unidad de Neurología, Departamento de Pediatría, Clínica Las Condes, Santiago, Chile.

Background: Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder affecting the anterior horn cells of the spinal cord resulting in muscle weakness and atrophy, linked to the homozygous disruption of the survival motor neuron 1 (SMN1) gene. It is the leading genetic cause of infant death. It has been classified into three types based on the severity of symptoms. Type I SMA is the most severe form with death within the first 2 years of life. Type II and III SMA patients show intermediate and mild forms of the disorder.

Aim: To describe the clinical and electrophysiological findings of 26 Chilean patients with SMA with molecular confirmation.

Patients And Methods: Retrospective multicenter analysis of patients with SMA assessed between 2003 and 2010. The diagnosis was suspected on clinical and electrophysiological criteria. Since 2006 molecular genetics confirmation was implemented in one of our centers.

Results: Twenty-six patients between 2 months and 18 years of age at presentation were analyzed; 15 (58%) were males. SMA I, II and III clinical criteria were observed in 4 (15.4 %), 11 (42.3%) and 11 (42.3%)patients, respectively. All had proximal muscle weakness and atrophy. Electromyography showed features of acute denervation or re-innervation with normal motor and sensory nerve conduction. Nine patients required a muscle biopsy. The genetic confirmation of the disease by PCR technique followed by restriction fragment length polymorphism method disclosed the SMN1 gene deletion in all 26 cases. All patients died secondary to respiratory failure, between eight and 14 months of life.

Conclusions: An adequate clinical and molecular diagnosis of spinal muscular atrophy will help for a better management of these patients.
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http://dx.doi.org//S0034-98872011000200009DOI Listing
February 2011

[Satoyoshi syndrome: report of one case].

Rev Med Chil 2009 Apr 25;137(4):542-6. Epub 2009 Jun 25.

Radiología, Clínica Las Condes, Santiago, Chile.

Satoyoshi syndrome is a rare multisystemic disease of presumed autoimmune etiology characterized by progressive painful intermittent muscle spasms, diarrhea frequently associated with malabsorption, alopecia, skeletal abnormalities and endocrine disorders with a poor long-term prognosis due to early crippling. We report a 14-year-old Chilean girl with clinical and radiological features of the syndrome who has been successfully treated with prednisone and carbamazepine. She remarkably recovered from muscle spasms, alopecia and diarrhea. At follow up, 24 months later, she persists asymptomatic with considerable improvement in her quality of life.
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http://dx.doi.org//S0034-98872009000400013DOI Listing
April 2009

Functional role for a conserved aspartate in the Spo0E signature motif involved in the dephosphorylation of the Bacillus subtilis sporulation regulator Spo0A.

J Biol Chem 2008 Feb 28;283(5):2962-72. Epub 2007 Nov 28.

Department of Molecular and Experimental Medicine, Division of Cellular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Sporulation is a complex developmental system characterizing Gram-positive bacteria of the genus Bacillus and Clostridium. In Bacillus subtilis the phosphorelay signal transduction system regulates the initiation of sporulation by integrating a myriad of positive and negative signals through the action of histidine sensor kinases and aspartyl phosphate phosphatases. The Spo0E family of phosphatases dephosphorylates the Spo0A response regulator and transcription factor of the phosphorelay. In this study we analyzed the role of the Spo0E signature motif in protein activity. This family is characterized by a conserved signature motif centered around the sequence "SQELD." Alanine scanning mutagenesis was carried out on the T(35)IXXSQ ELDCLI(46) residues of B. subtilis Spo0E and in vivo and in vitro activities were analyzed. The ability of the mutant proteins to interact with Spo0A approximately P was assayed by fluorescence resonance energy transfer spectroscopy. The results suggested that aspartate 43 has a critical role in Spo0E catalytic activity, whereas the other residues have a role in protein conformation and/or interaction with Spo0A. Residues Thr(35) and Cys(44) did not seem to have any critical functional or structural role. We propose that Asp(43) of Spo0E may function in a manner similar to the one proposed for the catalytic mechanisms of nucleotidase members of the haloacid dehalogenase family. These proteins use an aspartyl nucleophile as their common catalytic strategy and the active site of haloacid dehalogenase proteins shares a common geometry and identity of conserved amino acids with the active site of response regulators ( Ridder, I. S., and Dijkstra, B. W. (1999) Biochem. J. 339, 223-226 ).
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http://dx.doi.org/10.1074/jbc.M709032200DOI Listing
February 2008

Membrane topology of the acyl-lipid desaturase from Bacillus subtilis.

J Biol Chem 2002 Dec 24;277(50):48099-106. Epub 2002 Sep 24.

Instituto de Biologia Molecular y Celular de Rosario, Universidad Nacional de Rosario, Argentina.

The Bacillus subtilis acyl-lipid desaturase (Delta5-Des) is an iron-dependent integral membrane protein, able to selectively introduce double bonds into long chain fatty acids. Structural information on membrane-bound desaturases is still limited, and the present topological information is restricted to hydropathy plots or sequence comparison with the evolutionary related alkane hydroxylase. The topology of Delta5-Des was determined experimentally in Escherichia coli using a set of nine different fusions of N-terminal fragments of Delta5-Des with the reporter alkaline phosphatase (Delta5-Des-PhoA). The alkaline phosphatase activities of cells expressing the Delta5-Des-PhoA fusions, combined with site-directed mutagenesis of His residues identified in most desaturases, suggest that a tripartite motif of His essential for catalysis is located on the cytoplasmic phase of the membrane. These data, together with surface Lys biotinylation experiments, support a model for Delta5-Des as a polytopic membrane protein with six transmembrane- and one membrane-associated domain, which likely represents a substrate-binding motif. This study provides the first experimental evidence for the topology of a plasma membrane fatty acid desaturase. On the basis of our results and the presently available hydrophobicity profile of many acyl-lipid desaturases, we propose that these enzymes contain a new transmembrane domain that might play a critical role in the desaturation of fatty acids esterified in glycerolipids.
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http://dx.doi.org/10.1074/jbc.M208960200DOI Listing
December 2002
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