RESUMO
Entamoeba histolytica virulence results from complex host-parasite interactions implicating multiple amoebic components (e.g., Gal/GalNAc lectin, cysteine proteinases, and amoebapores) and host factors (microbiota and immune response). UG10 is a strain derived from E. histolytica virulent HM-1:IMSS strain that has lost its virulence in vitro and in vivo as determined by a decrease of hemolytic, cytopathic, and cytotoxic activities, increased susceptibility to human complement, and its inability to form liver abscesses in hamsters. We compared the transcriptome of nonvirulent UG10 and its parental HM-1:IMSS strain. No differences in gene expression of the classical virulence factors were observed. Genes downregulated in the UG10 trophozoites encode for proteins that belong to small GTPases, such as Rab and AIG1. Several protein-coding genes, including iron-sulfur flavoproteins and heat shock protein 70, were also upregulated in UG10. Overexpression of the EhAIG1 gene (EHI_180390) in nonvirulent UG10 trophozoites resulted in augmented virulence in vitro and in vivo. Cocultivation of HM-1:IMSS with E. coli O55 bacteria cells reduced virulence in vitro, and the EhAIG1 gene expression was downregulated. In contrast, virulence was increased in the monoxenic strain UG10, and the EhAIG1 gene expression was upregulated. Therefore, the EhAIG1 gene (EHI_180390) represents a novel virulence determinant in E. histolytica.
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The presence of pollutants in soil and water has given rise to diverse analytical and biological approaches to detect and measure contaminants in the environment. Using bacterial cells as reporter strains represents an advantage for detecting pollutants present in soil or water samples. Here, an Escherichia coli reporter strain expressing a chromoprotein capable of interacting with soil or water samples and responding to DNA damaging compounds is validated. The reporter strain generates a qualitative signal and is based on the expression of the coral chromoprotein AmilCP under the control of the recA promoter. This strain can be used simply by applying soil or water samples directly and rendering activation upon DNA damage. This reporter strain responds to agents that damage DNA (with an apparent detection limit of 1 µg of mitomycin C) without observable response to membrane integrity damage, protein folding or oxidative stress generating agents, in the latter case, DNA damage was observed. The developed reporter strain reported here is effective for the detection of DNA damaging agents present in soils samples. In a proof-of-concept analysis using soil containing chromium, showing activation at 15.56 mg/L of Cr(VI) present in soil and leached samples and is consistent with Cr(III) toxicity at high concentrations (130 µg). Our findings suggest that chromogenic reporter strains can be applied for simple screening, thus reducing the number of samples requiring analytical techniques.
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BACKGROUND: Marine sessile organisms display a color palette that is the result of the expression of fluorescent and non-fluorescent proteins. Fluorescent proteins have uncovered transcriptional regulation, subcellular localization of proteins, and the fate of cells during development. Chromoproteins have received less attention until recent years as bioreporters. Here, we studied the properties of aeBlue, a a 25.91 kDa protein from the anemone Actinia equina. OBJECTIVE: To assess the properties of aeBlue chromoprotein under different physicochemical conditions. METHODS: In this article, during the purification of aeBlue we uncovered that it suffered a color shift when frozen. We studied the color shift by different temperature incubation and physicochemical conditions and light spectroscopy. To assess the possible structural changes in the protein, circular dichroism analysis, size exclusion chromatography and native PAGE was performed. RESULTS: We uncover that aeBlue chromoprotein, when expressed from a synthetic construct in Escherichia coli, showed a temperature dependent color shift. Protein purified at 4 °C by metal affinity chromatography exhibited a pinkish color and shifts back at higher temperatures to its intense blue color. Circular dichroism analysis revealed that the structure in the pink form of the protein has reduced secondary structure at 4 °C, but at 35 °C and higher, the structure shifts to a native conformation and Far UV- vis CD spectra revealed the shift in an aromatic residue of the chromophore. Also, the chromophore retains its properties in a wide range of conditions (pH, denaturants, reducing and oxidants agents). Quaternary structure is also maintained as a tetrameric conformation as shown by native gel and size exclusion chromatography. CONCLUSION: Our results suggest that the chromophore position in aeBlue is shifted from its native position rendering the pink color and the process to return it to its native blue conformation is temperature dependent.
Assuntos
Corantes/química , Proteínas Luminescentes/química , Pigmentos Biológicos/química , Proteínas/química , Anêmonas-do-Mar/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cor , Corantes/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Luz , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Oxirredução , Pigmentos Biológicos/metabolismo , Conformação Proteica , Desnaturação Proteica , Proteínas/metabolismo , Espectrofotometria , TemperaturaRESUMO
Entamoeba histolytica is a pathogen that during its infective process confronts the host defenses, which damages the amoebic plasma membrane (PM), resulting in the loss of viability. However, it is unknown whether amoebic trophozoites are able to repair their PM when it is damaged. Acid sphingomyelinases (aSMases) have been reported in mammalian cells to promote endocytosis and removal of PM lesions. In this work, six predicted amoebic genes encoding for aSMases were found to be transcribed in the HM1:IMSS strain, finding that the EhaSM6 gene is the most transcribed in basal growth conditions and rendered a functional protein. The secreted aSMase activity detected was stimulated by Mg+2 and inhibited by Co+2. Trophozoites that overexpress the EhaSM6 gene (HM1-SM6HA) exhibit an increase of 2-fold in the secreted aSMase activity. This transfectant trophozoites exposed to pore-forming molecules (SLO, Magainin, ß-Defensin 2 and human complement) exhibited an increase from 6 to 25-fold in the secreted aSMase activity which correlated with higher amoebic viability in a Ca+2 dependent process. However, other agents that affect the PM such as hydrogen peroxide also induced an increase of secreted aSMase, but to a lesser extent. The aSMase6 enzyme is N- and C-terminal processed. Confocal and transmission electron microscopy showed that trophozoites treated with SLO presented a migration of lysosomes containing the aSMase towards the PM, inducing the formation of membrane patches and endosomes in the control strain. These cellular structures were increased in the overexpressing strain, indicating the involvement of the aSMase6 in the PM injury repair. The pore-forming molecules induced an increase in the expression of EhaSM1, 2, 5 and 6 genes, meanwhile, hydrogen peroxide induced an increase in all of them. In all the conditions evaluated, the EhaSM6 gene exhibited the highest levels of induction. Overall, these novel findings show that the aSMase6 enzyme from E. histolytica promotes the repair of the PM damaged with pore-forming molecules to prevent losing cell integrity. This novel system could act when encountered with the lytic defense systems of the host.
Assuntos
Membrana Celular/fisiologia , Entamoeba histolytica/enzimologia , Entamebíase/parasitologia , Esfingomielina Fosfodiesterase/metabolismo , Trofozoítos/metabolismo , Cálcio/metabolismo , Entamebíase/metabolismo , Humanos , Esfingomielina Fosfodiesterase/genética , Trofozoítos/crescimento & desenvolvimentoRESUMO
Oxidative stress is a key regulator in many cellular processes but also an important burden for living organisms. The source of oxidative damage usually is difficult to measure and assess with analytical tools or chemical indicators. One major limitation is to discriminate the presence of secondary oxidant molecules derived from the cellular metabolism after exposure to the oxidant or the scavenging capacity of reactive oxygen species by cells. Using a whole-cell reporter system based on an optimized HyPer2 protein for Escherichia coli expression, we demonstrate that, as previously shown for eukaryotic organisms, the effect at the transcriptional level of hydrogen peroxide can be monitored in vivo using flow cytometry of bacterial cells without the need of a direct analytical measurement. In this approach, we generated two different HyPer2 expression systems, one that is induced by IPTG and a second one that is induced by oxidative stress responsive promoters to control the expression of the HyPer2 protein and the exposure of higher H2O2 concentrations that has been shown to activate oxidative response genes. Both systems showed that the pathway that leads to the generation of H2O2 in vivo can be traced from H2O2 exposure. Our results indicate that hydrogen peroxide pulses can be readily detected in E. coli cells by a defined fluorescence signature that is H2O2 concentration-dependent. Our findings indicate that although less sensitive than purified protein or expressed in eukaryotic cells, HyPer2 is a good bacterial sensor for H2O2. As proof of concept, this system was used to trace the oxidative capacity of Toluidine Blue O showing that oxidative stress and redox imbalance is generated inside the cell. This system is expanding the repertoire of whole cell probes available for tracing cellular stress in bacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Fluorometria/métodos , Proteínas Luminescentes/metabolismo , Estresse Oxidativo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Genes Reporter/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Amoebiasis is a worldwide health problem caused by the pathogen Entamoeba histolytica. Several virulence factors have been implicated in host invasion, immune evasion, and tissue damage. There are still new factors that remain to be elucidated and characterized. In this work, we obtained amoebic transfectants overexpressing three of the neutral sphingomyelinase enzymes encoded in the E. histolytica genome. The EhnSM3 overexpression induced an increase in hemolytic and cytotoxic activities, besides an increase in gene expression of amoebapore A, B, and C. Meanwhile the EhnSM1 and EhnSM2 overexpression caused an increase in cytopathic activity. In all the neutral sphingomyelinases overexpressing strains, the gene expression levels for cysteine proteinase 5, adhesin 112 and, heavy and light Gal/GalNAc lectin subunits were not affected. We propose that the increase of cytotoxic and lytic effect of EhnSM3 overexpressed strain can be related to the sum of the effect of EhnSM3 plus amoebapores, in a process cell contact-dependent or as mediator by inducing the gene expression of amoebapores enabling a link between EhnSM3 with the virulence phenotype in E. histolytica. Our results suggest a differential role for neutral sphingomyelinases in E. histolytica virulence.
Assuntos
Entamoeba histolytica/patogenicidade , Esfingomielina Fosfodiesterase/metabolismo , Animais , Cães , Entamoeba histolytica/enzimologia , Entamoeba histolytica/genética , Eritrócitos/metabolismo , Expressão Gênica , Genoma de Protozoário , Hemólise , Humanos , Células Madin Darby de Rim Canino , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/isolamento & purificação , Esfingomielinas/metabolismo , Transfecção , VirulênciaRESUMO
Entamoeba histolytica genetic organization and genome structure is complex and under intense research. The genome is fully sequenced, and several tools have been developed for the molecular study of this organism. Nevertheless, good protein tracking tags that are easy to measure and image, like the fluorescent proteins are lacking. In this report, we codon-optimized the red fluorescent protein from the coral Discosoma striata (DsRFP) for its use in E. histolytica and demonstrated functionality in vivo. We envision that this protein can be widely used for the development of transcriptional reporter systems and protein-tagging applications.
Assuntos
Entamoeba histolytica/metabolismo , Substâncias Luminescentes/metabolismo , Proteínas Luminescentes/metabolismo , Animais , Antozoários/química , Clonagem Molecular , Códon/fisiologia , Entamoeba histolytica/genética , Entamoeba histolytica/patogenicidade , Citometria de Fluxo , Expressão Gênica , Proteínas Luminescentes/genética , Microscopia Confocal , Plasmídeos/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Esfingomielina Fosfodiesterase/genética , Virulência , Proteína Vermelha FluorescenteRESUMO
In the past twenty years, molecular genetics has created powerful tools for genetic manipulation of living organisms. Whole genome sequencing has provided necessary information to assess knowledge on gene function and protein networks. In addition, new tools permit to modify organisms to perform desired tasks. Gene function analysis is speed up by novel approaches that couple both high throughput data generation and mining. Synthetic biology is an emerging field that uses tools for generating novel gene networks, whole genome synthesis and engineering. New applications in biotechnological, pharmaceutical and biomedical research are envisioned for synthetic biology. In recent years these new strategies have opened up the possibilities to study gene and genome editing, creation of novel tools for functional studies in virus, parasites and pathogenic bacteria. There is also the possibility to re-design organisms to generate vaccine subunits or produce new pharmaceuticals to combat multi-drug resistant pathogens. In this review we provide our opinion on the applicability of synthetic biology strategies for functional studies of pathogenic organisms and some applications such as genome editing and gene network studies to further comprehend virulence factors and determinants in pathogenic organisms. We also discuss what we consider important ethical issues for this field of molecular biology, especially for potential misuse of the new technologies.
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Bactérias/genética , Biologia Sintética/métodos , Bactérias/metabolismo , Humanos , MicrobiologiaRESUMO
In vitro studies to fourteen previously synthesized chromone-tetrazoles and four novel fluorine-containing analogs were conducted against pathogenic protozoan (Entamoeba histolytica), pathogenic bacteria (Pseudomonas aeruginosa, and Staphylococcus aureus), and human fungal pathogens (Sporothrix schenckii, Candida albicans, and Candida tropicalis), which have become in a serious health problem, mainly in tropical countries.
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Antibacterianos/farmacologia , Antifúngicos/farmacologia , Antiprotozoários/farmacologia , Cromonas/farmacologia , Tetrazóis/farmacologia , Antibacterianos/síntese química , Antifúngicos/síntese química , Antiprotozoários/síntese química , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/crescimento & desenvolvimento , Candida tropicalis/patogenicidade , Cromonas/síntese química , Entamoeba histolytica/efeitos dos fármacos , Entamoeba histolytica/crescimento & desenvolvimento , Entamoeba histolytica/patogenicidade , Flúor/química , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Sporothrix/efeitos dos fármacos , Sporothrix/crescimento & desenvolvimento , Sporothrix/patogenicidade , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/patogenicidade , Tetrazóis/síntese químicaRESUMO
Bacterial reporter assays are powerful tools used to study the effect of different compounds that affect the physiology of cellular processes. Most bacterial reporters are luciferase based and can be monitored in real time. In the present study we designed and implemented two sets of Escherichia coli bacterial reporter assays, using a multicopy plasmid system. Each reporter strain was constructed using either green fluorescent protein or ß-galactosidase (LacZ) proteins. The designed reporter strains are capable of responding in a specific manner to molecules that either oxidative stress, or membrane, protein, or DNA damage. In order to respond to the desired stimulus, promoter sequences from E. coli were used. These sequences correspond to the promoter of the major catalase (KatG) activated with cellular oxidative damage, the promoter of the ß-hydroxydecanoyl-ACP dehydrase (FabA) which is activated with membrane perturbation, the promoter of DNA recombinase (RecA) which is activated by DNA lesions. For protein misfolding, the promoter of the heat-shock responsive chaperon (DnaK) was used. Our constructs displayed activation to damage from specific stimuli, and low response to nonspecific stimuli was detected. Our results suggest that these types of bacterial reporter strains can be used in semiquantitative (fluorometric) and qualitative (ß-galactosidase activity) studies of different xenobiotic substances and pollutants.
Assuntos
Técnicas Biossensoriais , Colorimetria/métodos , Escherichia coli , Proteínas de Fluorescência Verde , Plasmídeos , Sequência de Bases , Dano ao DNA/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Estresse Oxidativo/fisiologia , Regiões Promotoras Genéticas , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Antimicrobial peptides are widely distributed in nature; they play important roles in several aspects of innate immunity and may provide a basis for the design of novel therapeutic agents. In this study, C-amidated tritrpticin, a 13 amino acid tryptophan-rich antimicrobial peptide derived from a porcine cathelicidin, was tested against Trichomonas vaginalis, a protozoan that causes a serious non-viral sexually transmitted disease associated with preterm birth, low birth weight, and high risk of HIV-1 infection. Tritrpticin was selected due to its reasonably easy synthesis and because analogs with lower toxicity may be designed. Our results show that tritrpticin-NH(2) at either 100 or 200 µg/ml (52.5 or 105 µM) clearly reduces the viability and growth of Trichomonas vaginalis. Together with tritrpticin-NH(2), sodium bicarbonate further limited trichomonad growth. Additionally, a low concentration of metronidazole (5.8 µM), the most commonly used medication for Trichomonas vaginalis, was more effective against the growth of the parasite when it was combined with tritrpticin-NH(2).
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antiprotozoários/farmacologia , Oligopeptídeos/farmacologia , Trichomonas vaginalis/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Metronidazol/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Testes de Sensibilidade Parasitária , Bicarbonato de Sódio/farmacologia , Trichomonas vaginalis/crescimento & desenvolvimento , Trichomonas vaginalis/fisiologiaRESUMO
A novel neutral sphingomyelinase (nSMase) was characterized in Entamoeba histolytica trophozoites. SMase, a sphingomyelin-specific form of phospholipase C, catalyzes the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Three amebic putative nSMase genes were found to be actively transcribed. Mg(2+)-independent nSMase activity in the soluble fraction of the trophozoites was stimulated by Mn(2+) and partially inhibited by Zn(2+). nSMase activity of the recombinant protein EhnSM1, increased 4.5-fold in the presence of 0.5mM Mn(2+), and abolished by 5mM Zn(2+). A dose-dependent inhibition of rEhnSM1 was observed with scyphostatin, a specific inhibitor of nSMases. The EhnSM1 and EhnSM3 were detected in the soluble fraction of the amebic lysate as 35-37kDa proteins by western blot analysis. Immunofluorescence assay showed that the overexpressed HA-tagged EhnSM1 and EhnSM3 were localized to the cytosol. The biological role of these novel E. histolytica nSMases described in this work remains to be determined.
Assuntos
Entamoeba histolytica/enzimologia , Entamoeba histolytica/genética , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Amidas/farmacologia , Sequência de Aminoácidos , Western Blotting , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Técnica Indireta de Fluorescência para Anticorpo , Dados de Sequência Molecular , Pironas/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Trofozoítos/enzimologiaRESUMO
Monoxenic cultivation of pathogenic Entamoeba histolytica trophozoites with Escherichia coli serotype 055 which binds strongly to the Gal/GalNAc amoebic lectin, markedly improved the growth of E. histolytica and produced a significant decrease in cysteine proteinase activity and a lower cytopathic activity on monolayer cells after 3 months of monoxenic culture. However, after long term monoxenic culture (12 months) the proteolytic and cytopathic activities were recovered and the amoebic growth reached the maximum yield. Employing the GeneFishing(R) technology and DNA macroarrays we detected differentially gene expression related to the amoebic interaction with bacteria. A number of differentially expressed genes encoding metabolic enzymes, ribosomal proteins, virulence factors and proteins related with cytoskeletal and vesicle trafficking were found. These results suggest that E. coli 055 has a nutritional role that strongly supports the amoebic growth, and is also able to modulate some biological activities related with amoebic virulence.
Assuntos
Entamoeba histolytica/crescimento & desenvolvimento , Entamoeba histolytica/patogenicidade , Escherichia coli/fisiologia , Animais , Linhagem Celular , Técnicas de Cocultura , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Primers do DNA/química , DNA Complementar/biossíntese , DNA de Protozoário/química , Entamoeba histolytica/genética , Escherichia coli/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VirulênciaRESUMO
Serine proteases are one of the biologically most important and widely distributed enzyme families. A protease capable of degrading the substrate Suc-AAF-AMC was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified by ion-exchange chromatography and electroelution, and appeared on 2D-PAGE as a spot of 60 kDa and pI of 4.65. Data obtained from zymogram suggest the active protease is present either as homodimer (130 kDa) or homotetramer (250 kDa). The optimal temperature of the enzyme was 37 degrees C, and it exhibited activity over a broad pH range. The protease was strongly inhibited by TPCK and chelating agents. The enzymatic activity was restored upon addition of calcium. BLAST analysis with the sequence of internal peptides of the protein revealed two open reading frames within the genome of E. histolytica, homologous to members of the family S28, clan SC of serine proteases.
Assuntos
Entamoeba histolytica/enzimologia , Serina Endopeptidases/isolamento & purificação , Sequência de Aminoácidos , Animais , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Entamoeba histolytica/patogenicidade , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologiaRESUMO
Entamoeba histolytica trophozoites are able to degrade human erythrocytes; the loss of erythrocyte cellular matrix and degradation of plasma membrane were observed, along with the decrease in the average size of digestive vacuoles. Ninety-six percent of hemoglobin ingested was hydrolyzed by trophozoites within 3h, as evidenced by electrophoresis. Accordingly, X-ray spectroscopy revealed the presence of iron inside vacuoles after erythrophagocytosis, the concentration of which decreased to control levels in a similar period. Quantification of erythrocyte digestion at the early and late periods was determined by a spectrophotometric procedure, with t(1/2)=1.67 h and 35-min for HM-1:IMSS and HK-9:NIH trophozoites, respectively. In the latter, activity was due to the combined action of intracellular enzymatic activity and exocytosis. E-64c and leupeptin totally inhibited erythrocyte digestion within a 3-h period, thereafter hydrolysis took place at lower rate. Our results suggest that erythrocyte digestion in E. histolytica proceeds in different ways in these two amebic strains, and can be blocked by proteinase inhibitors.
Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Entamoeba histolytica/fisiologia , Eritrócitos/metabolismo , Leucina/análogos & derivados , Animais , Eletroforese em Gel de Poliacrilamida , Entamoeba histolytica/efeitos dos fármacos , Entamoeba histolytica/ultraestrutura , Técnica de Fratura por Congelamento , Hemoglobinas/metabolismo , Histocitoquímica , Humanos , Hidrólise , Leucina/farmacologia , Leupeptinas/farmacologia , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Microscopia de Contraste de Fase , Fagocitose/efeitos dos fármacos , Espectrofotometria , Vacúolos/metabolismoRESUMO
The analysis of behavior of man in the field of biology is carried out through bioethics, considered the science of the survival. In the microbiology, there are numerous discoveries related with pathogenic microorganisms, including those that can be used as weapons in a biological war or in an attack considered bioterrorism. The scientist involved in microbiology can participate with his knowledge in the development and improvement of bioweapons, however from the point of view of bioethics it is not acceptable that he works in an investigation related with these topics, because the defense research can evolve in offensive one. The war is an antisurvival activity, therefore it is not acceptable. In the same way, the biological weapons composed with virus, fungi or alive bacteria, or with toxins from them, neither they are morally accepted. After the terrorist attacks with anthrax in the United States in 2001, the world scientific community in the field of microbiology should show against the use of the microorganisms like bioweapons, at the time of promoting the idea that the responsible use for the microorganisms is a moral imperative for all microbiologists around the world, since the biological weapons are a threat for the human life.
Assuntos
Bioética , Guerra Biológica/ética , Microbiologia/ética , Bioterrorismo/ética , Humanos , GuerraRESUMO
PEHPS medium, developed for zxenic cultivation of Entamoeba histolytica and E. invadens, was also capable of supporting the growth of a Trichomonas vaginalis strain, with an inoculum of 1 to 100 trichomonads/ml. The lorithmic growth phase in PEHPS or in TYI-S-33 medium lasted 72 h; yield (3.33 ñ 0.56 x 10 a the 6 trichomonads/ml), duplication time (4.27 h), number of duplications (16.85), or increase ratio (33,328) in PEHPS medium showed no significant differences with those obtained in TYI-S33 under similar culture conditions. Accordingly, PEHPS medium might be used for the axenic cultivation of T. vaginalis
Assuntos
Entamoeba histolytica/crescimento & desenvolvimento , Entamoeba/crescimento & desenvolvimento , Técnicas In Vitro , Trichomonas vaginalis/crescimento & desenvolvimento , Vida Livre de Germes/imunologiaRESUMO
Las mucopolisacaridosis son un grupo de alteraciones genéticas clasificadas dentro de los "Errores Innatos del Metabolismo" (EIM). Estos EIM son informados por varios autores con una incidencia baja (de 1:5000 a 1:320000 nacidos vivos). Sin embargo pueden ser más frecuentes de lo comunicado en la literatura, sobre todo cuando se tienen presentes como causa de enfermedad y son aplicados los estudios de escrutinio neonatal actualmente accesibles. Se detallan algunos de los análisis de laboratorio utilizados para su diagnóstico, y se describe brevemente un caso de mucopolisacaridosis infantil, que resalta la importancia del diagnóstico precoz mediante la aplicación de métodos sencillos. Se sugiere implementar y aplicar estos procedimientos en todo laboratorio clínico, para apoyar el diagnóstico oportuno de dichos desórdenes genéticos
Assuntos
Humanos , Masculino , Pré-Escolar , Técnicas de Laboratório Clínico , Técnicas de Laboratório Clínico/estatística & dados numéricos , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/fisiopatologia , Mucopolissacaridoses/fisiopatologia , Mucopolissacaridoses/urinaRESUMO
The objective of this investigation was to quantitatively evaluate, by using light and transmission electron microscopy, the intracellular digesion of human erythrocytes (HE), ingested by Entamoeba histolytica trophozoites in vitro. Amebas fed 10 min with HE were postincubated 3 - 12 h at 36.5ºC without HE, and fixed with glutaraldehyde. After staining by the benzidine reaction, HE per ameba, and percent of amebas containing at least one HE were determined by light microscopy. Trophozoites ingested an average of eight HE per ameba and 92 percent of them contained HE after the 10 min pulse. Erithrocytes were clearly observed as reddish-brown corpuscles in the amebic cytoplasm. During postphagocytosis incubations progressive loss of HE staining, as well as changes in size of food vacuoles, were observed, thus proving the intracellullar digestion of HE. The hydrolysis of HE was corroborated with the electro microscope, the HE cytoplasmic matrix being the first structure catabolized and later the plasma membrane. Quantitative analysis demostrated decrease of 56 percent of ameba containing HE, and 1.35 HE per ameba on average, after 3 h postphagocytosis. Afterwards, both parameters decreased at a slow rate until HEdisappeared. The t ½ of HE witin amebas was 2 h