Publications by authors named "Marcos A Oliveira"

38 Publications

Molecular characterization and expression analysis of anti-Müllerian hormone in common carp (Cyprinus carpio) adult testes.

Gene Expr Patterns 2021 Jun 3;40:119169. Epub 2021 Mar 3.

Reproductive and Molecular Biology Group, Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil. Electronic address:

Anti-Müllerian hormone (Amh) is a member of the transforming growth factor-β (Tgf-β) superfamily required in the regression of Müllerian ducts during gonadal sex differentiation of higher vertebrates. Teleost fish lack Müllerian ducts, but identified Amh orthologs have been shown to exert crucial functions during sex determination and differentiation of several species of teleosts. However, the function of Amh during gametogenesis in adult fish remains poorly investigated. Therefore, to expand present knowledge on the role of Amh in teleosts, the present study aimed to isolate and clone full-length amh cDNA in the common carp, Cyprinus carpio, and examine its expression levels throughout the male reproductive cycle and in response to different hormone treatments of testicular explants. Molecular cloning and characterization showed that the common carp Amh precursor amino acid sequence shared common features to other fish Amh precursors, including a conserved C-terminus (Tgf-β domain) and a double proteolytic cleavage site (R-X-X-R-X-X-R) upstream to the Tgf-β domain. Expression analysis showed amh dimorphic expression in the adult gonads with higher expression in the testes than ovaries. In testes, amh mRNA was detected in Sertoli cells contacting different types of germ cells, although the expression was greatest in Sertoli cells associated with type A undifferentiated spermatogonia. Expression analysis during the reproductive cycle showed that amh transcripts were down-regulated during the developing phase, which is characterized by an increased proliferation of type A undifferentiated spermatogonia and Sertoli cells and appearance of spermatocytes (meiosis) in the testes. Furthermore, ex vivo experiments showed that a 7 day exposure to Fsh or estrogens was required to decrease amh mRNA levels in common carp testicular explants. In summary, this study provided information on the molecular characterization and transcript abundance of amh in common carp adult testes. Altogether, these data will be useful for further investigations on sex determination and differentiation in this species, and also to improved strategies for improved carp aquaculture, such as inhibiting precocious maturation of males.
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http://dx.doi.org/10.1016/j.gep.2021.119169DOI Listing
June 2021

RADSex: A computational workflow to study sex determination using restriction site-associated DNA sequencing data.

Mol Ecol Resour 2021 Jul 9;21(5):1715-1731. Epub 2021 Mar 9.

Physiological Chemistry, Biocenter, University of Wuerzburg, Wuerzburg, Germany.

The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish.
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http://dx.doi.org/10.1111/1755-0998.13360DOI Listing
July 2021

Influence of lysosomal protease sensitivity in the immunogenicity of the antitumor biopharmaceutical asparaginase.

Biochem Pharmacol 2020 12 23;182:114230. Epub 2020 Sep 23.

Departamento de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, São Paulo, Brazil. Electronic address:

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.
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http://dx.doi.org/10.1016/j.bcp.2020.114230DOI Listing
December 2020

Surface-enhanced Raman scattering sensing platform for detecting amyloid-β peptide interaction with an aggregation inhibitor.

Appl Opt 2020 Sep;59(25):7490-7495

Soluble, small amyloid- oligomers () are recognized as significant contributors to the pathology of Alzheimer's disease (AD). Although drugs for treating AD symptoms have been approved, no therapy targeting amyloid- () capable of modifying the course of the disease is available. In an effort to develop a label-free method for screening new anti-AD therapeutic agents, we show the use of a surface-enhanced Raman scattering (SERS) active substrate for detecting the interactions between peptides and spin-labeled fluorine (SLF), a peptide aggregation inhibitor. Changes in the peak positions and intensity ratios of two spectral peaks near 1600 and 2900 can be used to monitor the molecular interactions between SLF and . This study demonstrates the potential of SERS spectroscopy for rapidly screening and identifying new anti- therapeutic agents.
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http://dx.doi.org/10.1364/AO.399307DOI Listing
September 2020

Evolutionary-driven C-MYC gene expression in mammalian fibroblasts.

Sci Rep 2020 07 6;10(1):11056. Epub 2020 Jul 6.

Department of Veterinary Medicine, Federal Rural University of Pernambuco-UFRPE, Recife, Brazil.

The extent to which mammalian cells share similar transcriptomes remains unclear. Notwithstanding, such cross-species gene expression inquiries have been scarce for defined cell types and most lack the dissection of gene regulatory landscapes. Therefore, the work was aimed to determine C-MYC relative expression across mammalian fibroblasts (Ovis aries and Bos taurus) via cross-species RT-qPCR and comprehensively explore its regulatory landscape by in silico tools. The prediction of transcription factor binding sites in C-MYC and its 2.5 kb upstream sequence revealed substantial variation, thus indicating evolutionary-driven re-wiring of cis-regulatory elements. C-MYC and its downstream target TBX3 were up-regulated in Bos taurus fibroblasts. The relative expression of C-MYC regulators [RONIN (also known as THAP11), RXRβ, and TCF3] and the C-MYC-associated transcript elongation factor CDK9 did not differ between species. Additional in silico analyses suggested Bos taurus-specific C-MYC exonization, alternative splicing, and binding sites for non-coding RNAs. C-MYC protein orthologs were highly conserved, while variation was in the transactivation domain and the leucine zipper motif. Altogether, mammalian fibroblasts display evolutionary-driven C-MYC relative expression that should be instructive for understanding cellular physiology, cellular reprogramming, and C-MYC-related diseases.
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http://dx.doi.org/10.1038/s41598-020-67391-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338511PMC
July 2020

Hyperspectral Raman microscopy can accurately differentiate single cells of different human thyroid nodules.

Biomed Opt Express 2019 Sep 5;10(9):4411-4421. Epub 2019 Aug 5.

Department of Pathology & Laboratory Medicine, Univ. of California Davis, Sacramento, CA 95817, USA.

We report on the use of line-scan hyperspectral Raman microscopy in combination with multivariate statistical analyses for identifying and classifying single cells isolated from clinical samples of human thyroid nodules based on their intrinsic Raman spectral signatures. A total of 248 hyperspectral Raman images of single cells from benign thyroid (n = 127) and classic variant of papillary carcinoma (n = 121) nodules were collected. Spectral differences attributed to phenylalanine, tryptophan, proteins, lipids, and nucleic acids were identified for benign and papillary carcinoma cells. Using principal component analysis and linear discriminant analysis, cells were identified with 97% diagnostic accuracy. In addition, preliminary data of cells from follicular adenoma (n = 20), follicular carcinoma (n = 25), and follicular variant of papillary carcinoma (n = 18) nodules suggest the feasibility of further discrimination of subtypes. Our findings indicate that hyperspectral Raman microscopy can potentially be developed into an objective approach for analyzing single cells from fine needle aspiration (FNA) biopsies to enable the minimally invasive diagnosis of "indeterminate" thyroid nodules and other challenging cases.
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http://dx.doi.org/10.1364/BOE.10.004411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6757446PMC
September 2019

Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set.

PLoS One 2019 14;14(8):e0221170. Epub 2019 Aug 14.

Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Pernambuco, Brazil.

Quantitative reverse transcription PCR (RT-qPCR) remains as an accurate approach for gene expression analysis but requires labor-intensive validation of reference genes using species-specific primers. To ease such demand, the aim was to design and test a multi-species primer set to validate reference genes for inter-genus RT-qPCR gene expression analysis. Primers were designed for ten housekeeping genes using transcript sequences of various livestock species. All ten gene transcripts were detected by RT-PCR in Bos taurus (cattle), Bubalus bubalis (buffaloes), Capra hircus (goats), and Ovis aries (sheep) cDNA. Primer efficiency was attained for eight reference genes using B. taurus-O. aries fibroblast cDNA (95.54-98.39%). The RT-qPCR data normalization was carried out for B. taurus vs. O. aries relative gene expression using Bestkeeper, GeNorm, Norm-finder, Delta CT method, and RefFinder algorithms. Validation of inter-genus RT-qPCR showed up-regulation of TLR4 and ZFX gene transcripts in B. taurus fibroblasts, irrespectively of normalization conditions (two, three, or four reference genes). In silico search in mammalian transcriptomes showed that the multi-species primer set is expected to amplify transcripts of at least two distinct loci in 114 species, and 79 species would be covered by six or more primers. Hence, a multi-species primer set allows for inter-genus gene expression analysis between O. aries and B. taurus fibroblasts and further reveals species-specific gene transcript abundance of key transcription factors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0221170PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693880PMC
March 2020

Global analysis of erythroid cells redox status reveals the involvement of Prdx1 and Prdx2 in the severity of beta thalassemia.

PLoS One 2018 6;13(12):e0208316. Epub 2018 Dec 6.

Universidade Federal de São Carlos (UFSCar), Departamento de Genética e Evolução, São Carlos, Brazil.

β-thalassemia is a worldwide distributed monogenic red cell disorder, characterized by an absent or reduced beta globin chain synthesis. The unbalance of alpha-gamma chain and the presence of pathological free iron promote severe oxidative damage, playing crucial a role in erythrocyte hemolysis, exacerbating ineffective erythropoiesis and decreasing the lifespan of red blood cells (RBC). Catalase, glutathione peroxidase and peroxiredoxins act together to protect RBCs from hydrogen peroxide insult. Among them, peroxiredoxins stand out for their overall abundance and reactivity. In RBCs, Prdx2 is the third most abundant protein, although Prdxs 1 and 6 isoforms are also found in lower amounts. Despite the importance of these enzymes, Prdx1 and Prdx2 may have their peroxidase activity inactivated by hyperoxidation at high hydroperoxide concentrations, which also promotes the molecular chaperone activity of these proteins. Some studies have demonstrated the importance of Prdx1 and Prdx2 for the development and maintenance of erythrocytes in hemolytic anemia. Now, we performed a global analysis comparatively evaluating the expression profile of several antioxidant enzymes and their physiological reducing agents in patients with beta thalassemia intermedia (BTI) and healthy individuals. Furthermore, increased levels of ROS were observed not only in RBC, but also in neutrophils and mononuclear cells of BTI patients. The level of transcripts and the protein content of Prx1 were increased in reticulocyte and RBCs of BTI patients and the protein content was also found to be higher when compared to beta thalassemia major (BTM), suggesting that this peroxidase could cooperate with Prx2 in the removal of H2O2. Furthermore, Prdx2 production is highly increased in RBCs of BTM patients that present high amounts of hyperoxidized species. A significant increase in the content of Trx1, Srx1 and Sod1 in RBCs of BTI patients suggested protective roles for these enzymes in BTI patients. Finally, the upregulation of Nrf2 and Keap1 transcription factors found in BTI patients may be involved in the regulation of the antioxidant enzymes analyzed in this work.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0208316PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6283586PMC
May 2019

Edema Induced by a Venom Serine Protease (Cdtsp 2) Involves the PAR Pathway and PKC and PLC Activation.

Int J Mol Sci 2018 Aug 15;19(8). Epub 2018 Aug 15.

Institute of Biosciences, Coastal Campus, BIOMOLPEP, São Paulo State University (UNESP), 11330-900 São Paulo, Brazil.

Snake venom serine proteases (SVSPs) represent an essential group of enzymatic toxins involved in several pathophysiological effects on blood homeostasis. Some findings suggest the involvement of this class of enzymatic toxins in inflammation. In this paper, we purified and isolated a new gyroxin isoform from the (Cdt) venom, designated as Cdtsp 2, which showed significant proinflammatory effects in a murine model. In addition, we performed several studies to elucidate the main pathway underlying the edematogenic effect induced by Cdtsp 2. Enzymatic assays and structural analysis (primary structure analysis and three-dimensional modeling) were closely performed with pharmacological assays. The determination of edematogenic activity was performed using Cdtsp 2 isolated from snake venom, and was applied to mice treated with protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, dexamethasone (Dexa), antagonists for protease-activated receptors (PARs), or saline (negative control). Additionally, we measured the levels of cyclooxygenase 2 (COX-2), malondialdehyde (MDA), and prostaglandin E2 (PGE2). Cdtsp 2 is characterized by an approximate molecular mass of 27 kDa, an isoelectric point (pI) of 4.5, and significant fibrinolytic activity, as well as the ability to hydrolyze Nα-benzoyl-l-arginine 4-nitroanilide (BAPNA). Its primary and three-dimensional structures revealed Cdtsp 2 as a typical snake venom serine protease that induces significant edema via the metabolism of arachidonic acid (AA), involving PARs, PKC, PLC, and COX-2 receptors, as well as inducing a significant increase in MDA levels. Our results showed that Cdtsp 2 is a serine protease with significant enzymatic activity, and it may be involved in the degradation of PAR1 and PAR2, which activate PLC and PKC to mobilize AA, while increasing oxidative stress. In this article, we provide a new perspective for the role of SVSPs beyond their effects on blood homeostasis.
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http://dx.doi.org/10.3390/ijms19082405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121655PMC
August 2018

Structural insights on the efficient catalysis of hydroperoxide reduction by Ohr: Crystallographic and molecular dynamics approaches.

PLoS One 2018 21;13(5):e0196918. Epub 2018 May 21.

Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.

Organic hydroperoxide resistance (Ohr) enzymes are highly efficient Cys-based peroxidases that play central roles in bacterial response to fatty acid hydroperoxides and peroxynitrite, two oxidants that are generated during host-pathogen interactions. In the active site of Ohr proteins, the conserved Arg (Arg19 in Ohr from Xylella fastidiosa) and Glu (Glu51 in Ohr from Xylella fastidiosa) residues, among other factors, are involved in the extremely high reactivity of the peroxidatic Cys (Cp) toward hydroperoxides. In the closed state, the thiolate of Cp is in close proximity to the guanidinium group of Arg19. Ohr enzymes can also assume an open state, where the loop containing the catalytic Arg is far away from Cp and Glu51. Here, we aimed to gain insights into the putative structural switches of the Ohr catalytic cycle. First, we describe the crystal structure of Ohr from Xylella fastidiosa (XfOhr) in the open state that, together with the previously described XfOhr structure in the closed state, may represent two snapshots along the coordinate of the enzyme-catalyzed reaction. These two structures were used for the experimental validation of molecular dynamics (MD) simulations. MD simulations employing distinct protonation states and in silico mutagenesis indicated that the polar interactions of Arg19 with Glu51 and Cp contributed to the stabilization of XfOhr in the closed state. Indeed, Cp oxidation to the disulfide state facilitated the switching of the Arg19 loop from the closed to the open state. In addition to the Arg19 loop, other portions of XfOhr displayed high mobility, such as a loop rich in Gly residues. In summary, we obtained a high correlation between crystallographic data, MD simulations and biochemical/enzymatic assays. The dynamics of the Ohr enzymes are unique among the Cys-based peroxidases, in which the active site Arg undergoes structural switches throughout the catalytic cycle, while Cp remains relatively static.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196918PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5962072PMC
August 2018

Characterization of vasa homolog in a neotropical catfish, Jundiá (Rhamdia quelen): Molecular cloning and expression analysis during embryonic and larval development.

Gene 2018 May 14;654:116-126. Epub 2018 Feb 14.

Reproductive and Molecular Biology Group, Department of Morphology, Institute of Bioscience of Botucatu, São Paulo State University, Botucatu, São Paulo, Brazil. Electronic address:

We have characterized the full-length vasa cDNA from Jundiá, Rhamdia quelen (Heptapteridae, Siluriformes). vasa encodes a member of the DEAD-box protein family of ATP-dependent RNA helicases. This protein is highly conserved among different organisms and its role is associated with RNA metabolism. In the majority of the investigated species, vasa is restricted to the germ cell lineage and its expression has been used to study germline development in many organisms, including fish. The deduced R. quelen vasa amino acid sequence displayed high similarity with Vasa protein sequences from other organisms, and did not cluster with PL10 or P68 DEAD-box protein subfamilies. We also reported that there is no other isoform for vasa mRNA in R. quelen gonads. Expression analysis by RT-PCR and qPCR showed vasa transcripts exclusively expressed in the germ cells of R. quelen gonads. R. quelen vasa mRNA was maternally inherited, and was detected in the migrating primordial germ cells (PGCs) until 264 h post-fertilization during embryonic and larval development. This work has characterized for the first time the full-length R. quelen vasa cDNA, and describes its expression patterns during R. quelen embryonic and larval development. Our results will contribute to the basic reproductive biology of this native species, and will support studies using vasa as a germ cell marker in different biotechnological studies, such as germ cell transplantation.
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http://dx.doi.org/10.1016/j.gene.2018.02.029DOI Listing
May 2018

Incidence of apoptosis after retinoids and insulin-like growth factor-I (IGF-I) supplementation during goat in vitro embryo production.

Zygote 2016 Dec 17;24(6):808-813. Epub 2016 Jun 17.

Laboratório de Biotécnicas Reprodutivas do Departamento de Medicina Veterinária da Universidade Federal Rural de Pernambuco (UFRPE).Av. Dom Manoel de Medeiros s/n,Dois Irmãos,CEP 52171-900 Recife-PE/Brasil.

The addition of growth factors and vitamins enhances goat embryonic development in vitro. However, few attempts have been reported trying to identify supplementation regimens for oocyte maturation or embryo culture with additive properties. The present report was aimed to evaluate if retinoids [0.3 μM retinyl acetate (RAc) and 0.5 μM 9-cis-retinoic acid (RA)] supplementation during goat oocyte maturation and retinoids and/or 50 ng mL-1 IGF-I during embryo culture synergically enhanced embryonic development while diminishing the incidence of apoptosis. All combinations of RAc and RA treatment produced blastocysts with similar efficiencies, while IGF-I enhanced embryos yields irrespectively of retinoid addition. Moreover, retinoids and IGF-I supplementation showed similar caspase activity or DNA fragmentation indexes in blastocysts. In conclusion, supplementation with retinoids and IGF-I during goat embryo culture enhances blastocysts development without synergic reduction of apoptosis.
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http://dx.doi.org/10.1017/S0967199416000125DOI Listing
December 2016

Catalytic Thr or Ser Residue Modulates Structural Switches in 2-Cys Peroxiredoxin by Distinct Mechanisms.

Sci Rep 2016 09 15;6:33133. Epub 2016 Sep 15.

Instituto de Biociências, Campus do Litoral Paulista, Universidade Estadual Paulista Júlio de Mesquita Filho, São Vicente, São Paulo, 11330-900, Brazil.

Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (CP), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-π bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states.
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http://dx.doi.org/10.1038/srep33133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024103PMC
September 2016

Damage induced in red blood cells by infrared optical trapping: an evaluation based on elasticity measurements.

J Biomed Opt 2016 07;21(7):75012

Federal University of Pernambuco, Laboratory of Biomedical Optics and Imaging, Avenida da Arquitetura, s/n, Cidade Universitária, Recife, Pernambuco 50740-530, Brazil.

We evaluated the damage caused to optically trapped red blood cells (RBCs) after 1 or 2 min of exposure to near-infrared (NIR) laser beams at 785 or 1064 nm. Damage was quantified by measuring cell elasticity using an automatic, real-time, homemade, optical tweezer system. The measurements, performed on a significant number (hundreds) of cells, revealed an overall deformability decrease up to ∼104% after 2 min of light exposure, under 10 mW optical trapping for the 785-nm wavelength. Wavelength dependence of the optical damage is attributed to the light absorption by hemoglobin. The results provided evidence that RBCs have their biomechanical properties affected by NIR radiation. Our findings establish limits for laser applications with RBCs.
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http://dx.doi.org/10.1117/1.JBO.21.7.075012DOI Listing
July 2016

Special Measures to Do De Vega Procedure.

Ann Thorac Surg 2016 Jun;101(6):2431-2

Faculty of Medicine of Sao Jose do Rio Preto (FAMERP), Rua Republica do Libano, 2700, casa 80, Jardim Tarraf II, Sao Jose do Rio Preto, Brazil, CEP 15092-440.

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http://dx.doi.org/10.1016/j.athoracsur.2015.12.067DOI Listing
June 2016

Use of retinoids during oocyte maturation diminishes apoptosis in caprine embryos.

Acta Vet Hung 2015 Jun;63(2):234-42

Department of Veterinary Medicine, Federal Rural University of Pernambuco , Av. Dom Manoel de Medeiros, s/n, Dois Irmãos, CEP 52171-900 Recife, Pernambuco , Brazil.

Exposure of caprine oocytes and embryos to retinoids enhances embryonic development, but the mechanisms governing this phenomenon have not been characterised. The aim of the present study was to evaluate if the incidence of apoptosis is affected by the addition of retinyl acetate (RAc) and 9-cis-retinoic acid (RA) during in vitro maturation (IVM) of caprine oocytes. Embryonic development was recorded on days 3 and 8 post-fertilisation, and apoptosis was measured by caspase activity and DNA fragmentation (TUNEL assay). Control zygotes had lower capacity to cleave and reach the blastocyst stage (24.45 ± 2.32 and 5.32 ± 0.81, respectively) than those of RAc- (29.96 ± 1.62 and 7.94 ± 0.93, respectively) and RA-treated groups (30.12 ± 1.51 and 7.36 ± 1.02, respectively). Oocytes and blastocysts positive for TUNEL assay were more frequent, respectively, in the controls (8.20 ± 0.78, 8.70 ± 1.05) than in RAc (5.60 ± 0.52, 4.80 ± 0.51) and RA (6.40 ± 0.69, 5.40 ± 0.69). Caspase activity did not differ between control oocytes (7.20 ± 0.91), RAc (6.60 ± 0.68) and RA (7.30 ± 0.67), but it was reduced in RAc- (5.05 ± 0.62) and RA-treated blastocysts (5.75 ± 0.22) compared to controls (8.35 ± 0.71). These results indicate that the addition of retinoids during IVM increases the developmental potential of goat embryos with a concomitant reduction in apoptosis rates.
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http://dx.doi.org/10.1556/004.2015.021DOI Listing
June 2015

How pH modulates the dimer-decamer interconversion of 2-Cys peroxiredoxins from the Prx1 subfamily.

J Biol Chem 2015 Mar 9;290(13):8582-90. Epub 2015 Feb 9.

From the Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas/SP, 13083-970,

2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H2O2 and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His(113)) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp(76) from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H2O2 can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H2O2 and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress.
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http://dx.doi.org/10.1074/jbc.M114.619205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4375507PMC
March 2015

Comparison of vitrification and conventional freezing for cryopreservation of caprine embryos.

Zygote 2015 Aug 25;23(4):594-602. Epub 2014 Jun 25.

Laboratório de Biotécnicas Aplicadas a Reprodução Animal,Departamento de Medicina Veterinária,Universidade Federal Rural de Pernambuco (UFRPE),Rua Dom Manoel de Medeiros s/n,Dois Irmãos,CEP 52171-900,Recife-PE,Brasil.

The experiment aimed to compare conventional freezing and different vitrification protocols for cryopreservation of caprine embryos at morphological, ultrastructural, and functional levels. Caprine embryos produced in vivo were allocated randomly to three groups: (1) conventional freezing with ethylene glycol (EG); (2) dimethyl sulfoxide + EG (DMSO/EG) vitrification; and (3) dimethylformamide + EG (DMF/EG) vitrification. All groups were scored for cell viability (propidium iodide staining and ultrastructural levels) and re-expansion rate after thawing or warming. Embryos subjected to DMSO/EG vitrification showed higher cell viability (73.33%), compared with DMF/EG vitrification and conventional freezing group embryos (40.00 and 66.66%, respectively). The ultrastructural study revealed that vitrified embryos had greater preservation of cellular structure than embryos from conventional freezing with EG. DMSO/EG vitrification resulted in higher rates of re-expansion in vitro (47.36%) than DMF/EG vitrification (31.58%), and conventional freezing (25.00%). In conclusion, caprine embryos produced in vivo are better cryopreserved after vitrification than conventional freezing, therefore we conclude that DMSO/EG vitrification is the most effective protocol for cryopreservation.
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http://dx.doi.org/10.1017/S0967199414000215DOI Listing
August 2015

Perceptions of tenure and tenure reform in academic pharmacy.

Am J Pharm Educ 2014 May;78(4):75

School of Pharmacy, Chapman University, Orange, California.

Objectives: To determine the academic pharmacy community's perceptions of and recommendations for tenure and tenure reform.

Methods: A survey instrument was administered via either a live interview or an online survey instrument to selected members of the US academic pharmacy community.

Results: The majority of respondents felt that tenure in academic pharmacy was doing what it was intended to do, which is to provide academic freedom and allow for innovation (59.6%). Respondents raised concern over the need for faculty mentoring before and after achieving tenure, whether tenure adequately recognized service, and that tenure was not the best standard for recognition and achievement. The majority (63%) agreed that tenure reform was needed in academic pharmacy, with the most prevalent recommendation being to implement post-tenure reviews. Some disparities in opinions of tenure reform were seen in the subgroup analyses of clinical science vs basic science faculty members, public vs private institutions, and administrators vs nonadministrators.

Conclusions: The majority of respondents want to see tenure reformed in academic pharmacy.
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http://dx.doi.org/10.5688/ajpe78475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4028584PMC
May 2014

Disulfide biochemistry in 2-cys peroxiredoxin: requirement of Glu50 and Arg146 for the reduction of yeast Tsa1 by thioredoxin.

J Mol Biol 2012 Nov 15;424(1-2):28-41. Epub 2012 Sep 15.

Departamento de Biologia, Universidade Estadual Paulista Júlio de Mesquita Filho, Campus do Litoral Paulista São Vicente, São Paulo, Brazil.

2-Cys peroxiredoxin (Prx) enzymes are ubiquitously distributed peroxidases that make use of a peroxidatic cysteine (Cys(P)) to decompose hydroperoxides. A disulfide bond is generated as a consequence of the partial unfolding of the α-helix that contains Cys(P). Therefore, during its catalytic cycle, 2-Cys Prx alternates between two states, locally unfolded and fully folded. Tsa1 (thiol-specific antioxidant protein 1 from yeast) is by far the most abundant Cys-based peroxidase in Saccharomyces cerevisiae. In this work, we present the crystallographic structure at 2.8Å resolution of Tsa1(C47S) in the decameric form [(α(2))(5)] with a DTT molecule bound to the active site, representing one of the few available reports of a 2-Cys Prx (AhpC-Prx1 subfamily) (AhpC, alkyl hydroperoxide reductase subunit C) structure that incorporates a ligand. The analysis of the Tsa1(C47S) structure indicated that Glu50 and Arg146 participate in the stabilization of the Cys(P) α-helix. As a consequence, we raised the hypothesis that Glu50 and Arg146 might be relevant to the Cys(P) reactivity. Therefore, Tsa1(E50A) and Tsa1(R146Q) mutants were generated and were still able to decompose hydrogen peroxide, presenting a second-order rate constant in the range of 10(6)M(-1)s(-1). Remarkably, although Tsa1(E50A) and Tsa1(R146Q) were efficiently reduced by the low-molecular-weight reductant DTT, these mutants displayed only marginal thioredoxin (Trx)-dependent peroxidase activity, indicating that Glu50 and Arg146 are important for the Tsa1-Trx interaction. These results may impact the comprehension of downstream events of signaling pathways that are triggered by the oxidation of critical Cys residues, such as Trx.
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http://dx.doi.org/10.1016/j.jmb.2012.09.008DOI Listing
November 2012

Upconversion ultraviolet random lasing in Nd3+ doped fluoroindate glass powder.

Opt Express 2011 Mar;19(6):5620-6

Departamento de Física, Universidade Federal de Pernambuco, 50670-901 Recife, PE, Brazil.

An upconversion random laser (RL) operating in the ultraviolet is reported for Nd3+ doped fluoroindate glass powder pumped at 575 nm. The RL is obtained by the resonant excitation of the Nd3+ state 2G7/2 followed by energy transfer among two excited ions such that one ion in the pair decays to a lower energy state and the other is promoted to state 4D7/2 from where it decays emitting light at 381 nm. The RL threshold of 30 kW/cm2 was determined by monitoring the photoluminescence intensity as a function of the pump laser intensity. The RL pulses have time duration of 29 ns that is 50 times smaller than the decay time of the upconversion signal when the sample is pumped with intensities below the RL laser threshold.
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http://dx.doi.org/10.1364/OE.19.005620DOI Listing
March 2011

Polyamines are required for the expression of key Hms proteins important for Yersinia pestis biofilm formation.

Environ Microbiol 2010 Jul 19;12(7):2034-47. Epub 2010 Apr 19.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA.

We previously showed that mutations in the genes encoding the two main biosynthetic enzymes responsible for polyamine production, arginine decarboxylase (SpeA) and ornithine decarboxylase (SpeC) cause a loss of biofilm formation in Yersinia pestis. In Y. pestis the development of a biofilm is dependent on 6 Hms (hemin storage) proteins (HmsH, F, R, S, T and P) grouped into 3 operons; hmsHFRS, hmsT and hmsP. In this article we show that polyamines are necessary to maintain the levels of key Hms proteins. In the absence of polyamines there is an approximately 93%, approximately 43% and approximately 90% reduction in protein levels of HmsR, HmsS and HmsT respectively. Overexpression of hmsR and hmsT from plasmids alone can restore biofilm formation to a SpeA(-)SpeC(-) mutant. Addition of exogenous putrescine also restores normal levels of HmsR, HmsS, HmsT and biofilm production. Analyses using transcriptional reporters and quantitative RT-PCR indicate that the initiation of transcription and mRNA stability are not reduced by polyamine deficiency. Instead, translational reporters indicate that polyamines function at least in part by modulating the translation of HmsR and HmsT. Although construction of a consensus Shine-Dalgarno sequence upstream of hmsT modestly reduced the stimulation of translation by putrescine, additional mechanisms likely contribute to the polyamine-dependent expression of HmsT. Finally, we have shown that polyamines play a role in bubonic plague.
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http://dx.doi.org/10.1111/j.1462-2920.2010.02219.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039482PMC
July 2010

Insights into the specificity of thioredoxin reductase-thioredoxin interactions. A structural and functional investigation of the yeast thioredoxin system.

Biochemistry 2010 Apr;49(15):3317-26

Departamento de Biologia, Universidade Estadual Paulista, São Vicente, Brazil.

The enzymatic activity of thioredoxin reductase enzymes is endowed by at least two redox centers: a flavin and a dithiol/disulfide CXXC motif. The interaction between thioredoxin reductase and thioredoxin is generally species-specific, but the molecular aspects related to this phenomenon remain elusive. Here, we investigated the yeast cytosolic thioredoxin system, which is composed of NADPH, thioredoxin reductase (ScTrxR1), and thioredoxin 1 (ScTrx1) or thioredoxin 2 (ScTrx2). We showed that ScTrxR1 was able to efficiently reduce yeast thioredoxins (mitochondrial and cytosolic) but failed to reduce the human and Escherichia coli thioredoxin counterparts. To gain insights into this specificity, the crystallographic structure of oxidized ScTrxR1 was solved at 2.4 A resolution. The protein topology of the redox centers indicated the necessity of a large structural rearrangement for FAD and thioredoxin reduction using NADPH. Therefore, we modeled a large structural rotation between the two ScTrxR1 domains (based on the previously described crystal structure, PDB code 1F6M ). Employing diverse approaches including enzymatic assays, site-directed mutagenesis, amino acid sequence alignment, and structure comparisons, insights were obtained about the features involved in the species-specificity phenomenon, such as complementary electronic parameters between the surfaces of ScTrxR1 and yeast thioredoxin enzymes and loops and residues (such as Ser(72) in ScTrx2). Finally, structural comparisons and amino acid alignments led us to propose a new classification that includes a larger number of enzymes with thioredoxin reductase activity, neglected in the low/high molecular weight classification.
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http://dx.doi.org/10.1021/bi901962pDOI Listing
April 2010

[Ants in a hospital environment and its importance as vector of bacteria].

Neotrop Entomol 2008 Jul-Aug;37(4):472-7

Lab. Ecologia, Univ. Estadual de Goiás.

The external ant community of Hospital Municipal de Morrinhos, in Morrinhos, Goiás State, was characterized by the low rates of richness, diversity, dominance and equity of species abundance. Pheidole sp.1, a polygynic species was numerically dominant in this environment, although it coexists with potentially competitive species. This ant species prevailed within all hospital departments and its space-time distribution was a little aggregated (variance/mean ratio = 1.102, chi2 = 29.38, P < 0.01). Escherichia, Salmonella, Aeromonas, Enterococcus, Staphylococcus and Klebsiella were the bacteria associated to this ant species in nearly all hospital annexes. The unicolonialism of Pheidole sp.1 tends to increase the contamination and dissemination process of infecto-contagious agents. The control and management of this ant species must be followed by practices that reduce the colonization process by other queens and the quantity of site nidification within the hospital.
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http://dx.doi.org/10.1590/s1519-566x2008000400017DOI Listing
May 2009

Polyamines in bacteria: pleiotropic effects yet specific mechanisms.

Adv Exp Med Biol 2007 ;603:106-15

Department of Pharmaceutical Sciences, University of Kentucky, Lexington, USA.

Extensive data in a wide range of organisms point to the importance of polyamine homeostasis for growth. The two most common polyamines found in bacteria are putrescine and spermidine. The investigation of polyamine function in bacteria has revealed that they are involved in a number of functions other than growth, which include incorporation into the cell wall and biosynthesis of siderophores. They are also important in acid resistance and can act as a free radical ion scavenger. More recently it has been suggested that polyamines play a potential role in signaling cellular differentiation in Proteus mirabilis. Polyamines have also been shown to be essential in biofilm formation in Yersinia pestis. The pleiotropic nature of polyamines has made their investigation difficult, particularly in discerning any specific effect from more global growth effects. Here we describe key developments in the investigation of the function of polyamines in bacteria that have revealed new roles for polyamines distinct from growth. We describe the bacterial genes necessary for biosynthesis and transport, with a focus on Y. pestis. Finally we review a novel role for polyamines in the regulation of biofilm development in Y. pestis and provide evidence that the investigation of polyamines in Y. pestis may provide a model for understanding the mechanism through which polyamines regulate biofilm formation.
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http://dx.doi.org/10.1007/978-0-387-72124-8_9DOI Listing
December 2007

Goal-directed fluid management based on pulse pressure variation monitoring during high-risk surgery: a pilot randomized controlled trial.

Crit Care 2007 ;11(5):R100

Department of Anesthesia and Critical Care, Santa Casa de Misericórdia de Passos, 164 rua Santa Casa, 37900-020, Passos, MG, Brazil.

Introduction: Several studies have shown that maximizing stroke volume (or increasing it until a plateau is reached) by volume loading during high-risk surgery may improve post-operative outcome. This goal could be achieved simply by minimizing the variation in arterial pulse pressure (deltaPP) induced by mechanical ventilation. We tested this hypothesis in a prospective, randomized, single-centre study. The primary endpoint was the length of postoperative stay in hospital.

Methods: Thirty-three patients undergoing high-risk surgery were randomized either to a control group (group C, n = 16) or to an intervention group (group I, n = 17). In group I, deltaPP was continuously monitored during surgery by a multiparameter bedside monitor and minimized to 10% or less by volume loading.

Results: Both groups were comparable in terms of demographic data, American Society of Anesthesiology score, type, and duration of surgery. During surgery, group I received more fluid than group C (4,618 +/- 1,557 versus 1,694 +/- 705 ml (mean +/- SD), P < 0.0001), and deltaPP decreased from 22 +/- 75 to 9 +/- 1% (P < 0.05) in group I. The median duration of postoperative stay in hospital (7 versus 17 days, P < 0.01) was lower in group I than in group C. The number of postoperative complications per patient (1.4 +/- 2.1 versus 3.9 +/- 2.8, P < 0.05), as well as the median duration of mechanical ventilation (1 versus 5 days, P < 0.05) and stay in the intensive care unit (3 versus 9 days, P < 0.01) was also lower in group I.

Conclusion: Monitoring and minimizing deltaPP by volume loading during high-risk surgery improves postoperative outcome and decreases the length of stay in hospital.

Trial Registration: NCT00479011.
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http://dx.doi.org/10.1186/cc6117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2556743PMC
May 2008

Investigating the geminal diamine intermediate of Yersinia pestis arginine decarboxylase with substrate, product, and inhibitors using single wavelength stopped-flow spectroscopy.

Biochemistry 2007 Jan;46(2):379-86

Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, USA.

The reaction mechanism of Yersinia pestis arginine decarboxylase has been investigated using a series of substrate, product, and inhibitors. Using single wavelength stopped-flow spectroscopy, novel mechanistic features were noted in the presence of the product, agmatine. By focusing on the excitation and emission wavelengths of the geminal diamine intermediate, we were able to monitor the formation and decay of two different geminal diamine species. Experiments revealed that the enzyme exists in two different conformational states--one that binds ligand and one that does not. The on and off rates for the conversion between the two conformational states was determined to be 390 s-1 and 880 s-1, respectively. The KD for agmatine binding was 6 mM. In addition, experiments revealed a pH-dependent conversion between two states of the enzyme. The deprotonated form of the enzyme binds ligand more slowly than the protonated form. The rates for the formation of the geminal diamine and external aldimine in this pathway were determined to be 25 and 4 s-1, respectively. There is also a slow interconversion between the protonated and deprotonated enzymes that has a pKa of approximately 8.0. Finally, the formation of the geminal diamine was determined to be Mg2+-dependent.
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http://dx.doi.org/10.1021/bi061260hDOI Listing
January 2007

Structural insights into enzyme-substrate interaction and characterization of enzymatic intermediates of organic hydroperoxide resistance protein from Xylella fastidiosa.

J Mol Biol 2006 Jun 7;359(2):433-45. Epub 2006 Apr 7.

Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo 05508-900, Brazil.

Organic hydroperoxide resistance proteins (Ohr) belong to a family of proteins that possess thiol-dependent peroxidase activity endowed by reactive cysteine residues able to reduce peroxides. The crystal structure of Ohr from Xylella fastidiosa in complex with polyethylene glycol, providing insights into enzyme-substrate interactions is described herein. In addition, crystallographic studies, molecular modeling and biochemical assays also indicated that peroxides derived from long chain fatty acids could be the biological substrates of Ohr. Because different oxidation states of the reactive cysteine were present in the Ohr structures from X. fastidiosa, Pseudomonas aeruginosa and Deinococcus radiodurans it was possible to envisage a set of snapshots along the coordinate of the enzyme-catalyzed reaction. The redox intermediates of X. fastidiosa Ohr observed in the crystals were further characterized in solution by electrospray ionization mass spectrometry and by biochemical approaches. In this study, the formation of an intramolecular disulfide bond and oxidative inactivation through the formation of a sulfonic acid derivative was unequivocally demonstrated for the first time. Because Ohr proteins are exclusively present in bacteria, they may represent promising targets for therapeutical drugs. In this regard, the structural and functional analyses of Ohr presented here might be very useful.
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http://dx.doi.org/10.1016/j.jmb.2006.03.054DOI Listing
June 2006

Polyamines are essential for the formation of plague biofilm.

J Bacteriol 2006 Apr;188(7):2355-63

Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.

We provide the first evidence for a link between polyamines and biofilm levels in Yersinia pestis, the causative agent of plague. Polyamine-deficient mutants of Y. pestis were generated with a single deletion in speA or speC and a double deletion mutant. The genes speA and speC code for the biosynthetic enzymes arginine decarboxylase and ornithine decarboxylase, respectively. The level of the polyamine putrescine compared to the parental speA+ speC+ strain (KIM6+) was depleted progressively, with the highest levels found in the Y. pestis DeltaspeC mutant (55% reduction), followed by the DeltaspeA mutant (95% reduction) and the DeltaspeA DeltaspeC mutant (>99% reduction). Spermidine, on the other hand, remained constant in the single mutants but was undetected in the double mutant. The growth rates of mutants with single deletions were not altered, while the DeltaspeA DeltaspeC mutant grew at 65% of the exponential growth rate of the speA+ speC+ strain. Biofilm levels were assayed by three independent measures: Congo red binding, crystal violet staining, and confocal laser scanning microscopy. The level of biofilm correlated to the level of putrescine as measured by high-performance liquid chromatography-mass spectrometry and as observed in a chemical complementation curve. Complementation of the DeltaspeA DeltaspeC mutant with speA showed nearly full recovery of biofilm to levels observed in the speA+ speC+ strain. Chemical complementation of the double mutant and recovery of the biofilm defect were only observed with the polyamine putrescine.
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http://dx.doi.org/10.1128/JB.188.7.2355-2363.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1428407PMC
April 2006

Crystallization and X-ray diffraction properties of Baeyer-Villiger monooxygenase MtmOIV from the mithramycin biosynthetic pathway in Streptomyces argillaceus.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2005 Nov 28;61(Pt 11):1023-6. Epub 2005 Oct 28.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.

The Baeyer-Villiger monooxygenase MtmOIV from Streptomyces argillaceus is a 56 kDa FAD-dependent and NADPH-dependent enzyme that is responsible for the key frame-modifying step in the biosynthesis of the natural product mithramycin. Crystals of MtmOIV were flash-cooled and diffracted to 2.69 A resolution using synchrotron radiation on beamline SER-CAT 22-ID at the Advanced Photon Source. Crystals of MtmOIV are monoclinic and light-scattering data reveal that the enzyme forms dimers in solution. The rotation function suggests the presence of two dimers in the asymmetric unit. L-Selenomethionine-incorporated MtmOIV has been obtained. Structural solution combining molecular-replacement phases and anomalous phases from selenium is in progress.
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http://dx.doi.org/10.1107/S1744309105033221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1978135PMC
November 2005