RESUMO
The general secretory, or Sec, system is a primary protein export pathway from the cytosol of Escherichia coli and all eubacteria. Integral membrane protein complex SecDF is a translocation factor that enhances polypeptide secretion, which is driven by the Sec translocase, consisting of translocon SecYEG and ATPase SecA. SecDF is thought to utilize a proton gradient to effectively pull precursor proteins from the cytoplasm into the periplasm. Working models have been developed to describe the structure and function of SecDF, but important mechanistic questions remain unanswered. Atomic force microscopy (AFM) is a powerful technique for studying the dynamics of single-molecule systems including membrane proteins in near-native conditions. The sharp tip of the AFM provides direct access to membrane-external protein conformations. Here, we acquired AFM images and kymographs (â¼100 ms resolution) to visualize SecDF protrusions in near-native supported lipid bilayers and compared the experimental data to simulated AFM images based on static structures. When studied in isolation, SecDF exhibited a stable and compact conformation close to the lipid bilayer surface, indicative of a resting state. Interestingly, upon SecYEG introduction, we observed changes in both SecDF conformation and conformational dynamics. The population of periplasmic protrusions corresponding to an intermediate form of SecDF, which is thought to be active in precursor protein handling, increased more than ninefold. In conjunction, our dynamics measurements revealed an enhancement in the transition rate between distinct SecDF conformations when the translocon was present. Together, this work provides a novel vista of basal-level SecDF conformational dynamics in near-native conditions.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Canais de Translocação SEC , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Bicamadas Lipídicas/química , Transporte Proteico , Canais de Translocação SEC/química , Conformação ProteicaRESUMO
A number of peptides are known to bind lipid bilayer membranes and cause these natural barriers to leak in an uncontrolled manner. Though membrane permeabilizing peptides play critical roles in cellular activity and may have promising future applications in the therapeutic arena, significant questions remain about their mechanisms of action. The atomic force microscope (AFM) is a single molecule imaging tool capable of addressing lipid bilayers in near-native fluid conditions. The apparatus complements traditional assays by providing local topographic maps of bilayer remodeling induced by membrane permeabilizing peptides. The information garnered from the AFM includes direct visualization and statistical analyses of distinct bilayer remodeling modes such as highly localized pore-like voids in the bilayer and dispersed thinned membrane regions. Colocalization of distinct remodeling modes can be studied. Here we examine recent work in the field and outline methods used to achieve precise AFM image data. Experimental challenges and common pitfalls are discussed as well as techniques for unbiased analysis including the Hessian blob detection algorithm, bootstrapping, and the Bayesian information criterion. When coupled with robust statistical analyses, high precision AFM data is poised to advance understanding of an important family of peptides that cause poration of membrane bilayers.
Assuntos
Bicamadas Lipídicas , Peptídeos , Teorema de Bayes , Bicamadas Lipídicas/química , Microscopia de Força Atômica/métodos , Peptídeos/química , Imagem Individual de MoléculaRESUMO
Three novel carbon monoxide-oxidizing Halobacteria were isolated from Bonneville Salt Flats (Utah, USA) salt crusts and nearby saline soils. Phylogenetic analysis of 16S rRNA gene sequences revealed that strains PCN9T, WSA2T and WSH3T belong to the genera Halobacterium, Halobaculum and Halovenus, respectively. Strains PCN9T, WSA2T and WSH3T grew optimally at 40 °C (PCN9T) or 50 °C (WSA2T, WSH3T). NaCl optima were 3 M (PCN9T, WSA2T) or 4 M NaCl (WSH3T). Carbon monoxide was oxidized by all isolates, each of which contained a molybdenum-dependent CO dehydrogenase. G+C contents for the three respective isolates were 66.75, 67.62, and 63.97 mol% as derived from genome analyses. The closest phylogenetic relatives for PCN9T, WSA2T and WSH3T were Halobacterium noricense A1T, Halobaculum roseum D90T and Halovenus aranensis EB27T with 98.71, 98.19 and 95.95â% 16S rRNA gene sequence similarities, respectively. Genome comparisons of PCN9T with Halobacterium noricense A1T yielded an average nucleotide identity (ANI) of 82.0% and a digital DNA-DNA hybridization (dDDH) value of 25.7â%; comparisons of WSA2T with Halobaculum roseum D90T yielded ANI and dDDH values of 86.34 and 31.1â%, respectively. The ANI value for a comparison of WSH3T with Halovenus aranensis EB27T was 75.2â%. Physiological, biochemical, genetic and genomic characteristics of PCN9T, WSA2T and WSH3T differentiated them from their closest phylogenetic neighbours and indicated that they represent novel species for which the names Halobaculum bonnevillei, Halobaculum saliterrae and Halovenus carboxidivorans are proposed, respectively. The type strains are PCN9T (=JCM 32472=LMG 31022=ATCC TSD-126), WSA2T (=JCM 32473=ATCC TSD-127) and WSH3T (=JCM 32474=ATCC TSD-128).
Assuntos
Halobacteriaceae/classificação , Halobacterium/classificação , Filogenia , Salinidade , Microbiologia do Solo , Monóxido de Carbono/metabolismo , DNA Arqueal/genética , Halobacteriaceae/isolamento & purificação , Halobacterium/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Solo/química , UtahRESUMO
A novel member of Acidobacteria was isolated from a microbial mat growing on a geothermally heated dead tree trunk in Hawai'i Volcanoes National Park (HI, USA). The rod-shaped, Gram-negative capsulated cells of strain PMMR2T were non-motile and catalase and oxidase negative. Growth occurred aerobically from 15 to 55 °C (optimum, 40 °C) and at pH values from 4.5 to 7.0 (optimum, 6.5). A limited range of sugars and organic acids supported growth. However, results of a genomic analysis suggested that various polysaccharides might be hydrolysed as carbon sources, and evidence for pectin degradation was observed in liquid cultures. A genomic analysis also revealed genes for a Group 1f uptake hydrogenase; assays with liquid cultures confirmed hydrogen consumption, including uptake at sub-atmospheric concentrations. Nitrate was not dissimilated to nitrite. Major membrane fatty acids included iso-C15 : 0 and iso-C17 : 0. The G+C content was 57.2mol%. A comparative genome analysis revealed an average nucleotide identity of 72.2 % between PMMR2T and its nearest cultured phylogenetic neighbour, Acidobacterium capsulatum ATCC 51196T (=JCM 7670T); analysis of the 16S rRNA gene revealed a 96.8 % sequence identity with Acidobacterium capsulatum ATCC 51196T. These results and other phenotypic differences indicated that strain PMMR2T represents a novel species in the genus Acidobacterium, for which the name Acidobacterium ailaaui sp. nov. is proposed. The type strain, PMMR2T (=DSM 27394T=LMG 28340T), is the second formal addition to the genus Acidobacterium.
Assuntos
Acidobacteria/classificação , Consórcios Microbianos , Filogenia , Acidobacteria/genética , Acidobacteria/isolamento & purificação , Técnicas de Tipagem Bacteriana , Composição de Bases , Metabolismo dos Carboidratos , DNA Bacteriano/genética , Ácidos Graxos/química , Havaí , Temperatura Alta , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Two thermophilic, Gram-stain-positive, rod-shaped, non-spore-forming bacteria (strains KI3(T) and KI4(T)) were isolated from geothermally heated biofilms growing on a tumulus in the Kilauea Iki pit crater on the flank of Kilauea Volcano (Hawai'i, USA). Strain KI3(T) grew over an examined temperature range of 50-70 °C (no growth at 80 °C) and a pH range of 6.0-9.0, with optimum growth at 70 °C and pH 7.0. Strain KI4(T) grew at temperatures of 55-70 °C and a pH range of 5.8-8.0, with optimum growth at 65 °C and pH 6.7-7.1. The DNA G+C contents of strains KI3(T) and KI4(T) were 66.0 and 60.7 mol%, respectively. The major fatty acid for both strains was 12-methyl C(18â:â0). Polar lipids in strain KI3(T) were dominated by glycolipids and phosphatidylinositol, while phosphatidylinositol and phosphoglycolipids dominated in strain KI4(T). Strain KI3(T) oxidized carbon monoxide [6.7±0.8 nmol CO h(-1) (mg protein)(-1)], but strain KI4(T) did not. 16S rRNA gene sequence analyses determined that the strains belong to the class Thermomicrobia, and that strains KI3(T) and KI4(T) are related most closely to Thermomicrobium roseum DSM 5159(T) (96.5 and 91.1% similarity, respectively). 16S rRNA gene sequence similarity between strain KI3(T) and strain KI4(T) was 91.4%. Phenotypic features and phylogenetic analyses supported the affiliation of strain KI3(T) to the genus Thermomicrobium, while results of chemotaxonomic, physiological and biochemical assays differentiated strains KI3(T) and KI4(T) from Thermomicrobium roseum. Strain KI3(T) (â=âDSM 27067(T)â=âATCC BAA-2535(T)) is thus considered to be the type strain of a novel species, for which the name Thermomicrobium carboxidum sp. nov. is proposed. Additionally, the characterization and phylogenetic position of strain KI4(T) showed that it represents a novel species of a new genus, for which the name Thermorudis peleae gen. nov., sp. nov. is proposed. The type strain of Thermorudis peleae is KI4(T) (â=âDSM 27169(T)â=âATCC BAA-2536(T)).
Assuntos
Biofilmes , Monóxido de Carbono/metabolismo , Chloroflexi/classificação , Filogenia , Composição de Bases , Chloroflexi/genética , Chloroflexi/isolamento & purificação , DNA Bacteriano/genética , Ácidos Graxos/química , Glicolipídeos/química , Havaí , Temperatura Alta , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Erupções VulcânicasRESUMO
A thermophilic, aerobic, Gram-stain-positive bacterium (strain PM5(T)), which formed mycelia of irregularly branched filaments and produced multiple exospores per cell, was isolated from a geothermally heated biofilm. Strain PM5(T) grew at 40-65 °C and pH 4.1-8.0, with optimal growth at 55 °C and pH 6.0. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain PM5(T) belonged to the class Ktedonobacteria, and was related most closely to Thermogemmatispora onikobensis ONI-1(T) (97.7â% similarity) and Thermogemmatispora foliorum ONI-5(T) (96.1â%). Morphological features and fatty acid profiles (major fatty acids: iso-C17â:â0, iso-C19â:â0 and 12,17-dimethyl C18â:â0) supported the affiliation of strain PM5(T) to the genus Thermogemmatispora. Strain PM5(T) oxidized carbon monoxide [CO; 10±1 nmol h(-1) (mg protein)(-1)], but did not grow with CO as a sole carbon and energy source. Results from analyses of related strains indicated that the capacity for CO uptake occurred commonly among the members of the class Ktedonobacteria; 13 of 14 strains tested consumed CO or harboured coxL genes that potentially enabled CO oxidation. The results of DNA-DNA hybridization and physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain PM5(T) from the two recognized species of the genus Thermogemmatispora. Strain PM5(T) differed from Thermogemmatispora onikobensis ONI-1(T) in its production of orange pigment, lower temperature optimum, hydrolysis of casein and starch, inability to grow with mannitol, xylose or rhamnose as sole carbon sources, and utilization of organic acids and amino acids. Strain PM5(T) is therefore considered to represent a novel species, for which the name Thermogemmatispora carboxidivorans sp. nov. is proposed. The type strain is PM5(T) (â=âDSM 45816(T)â=âATCC BAA-2534(T)).
Assuntos
Biofilmes , Monóxido de Carbono/metabolismo , Chloroflexi/classificação , Filogenia , Composição de Bases , Chloroflexi/genética , Chloroflexi/isolamento & purificação , DNA Bacteriano/genética , Ácidos Graxos/química , Genes Bacterianos , Havaí , Temperatura Alta , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Oxirredução , Pigmentação , RNA Ribossômico 16S/genéticaRESUMO
Many facultative and obligate anaerobes reduce perchlorate. Likewise, carbon monoxide (CO) oxidation has been documented in many aerobes, facultative anaerobes, and obligate anaerobes. A molybdenum-dependent CO dehydrogenase (Mo-CODH) and a nickel-dependent CO dehydrogenase (Ni-CODH) distinguish the former from the latter. Some Mo-dependent CO oxidizers (Mo-COX) couple CO oxidation to perchlorate reduction, but only at low concentrations of both under conditions that do not support growth in cultures. In contrast, CO-coupled perchlorate reduction has not been documented in Ni-dependent CO oxidizers (Ni-COX). To assess the potential for Ni-COX to reduce perchlorate, a model, obligately anaerobic homoacetogen, Moorella glycerini DSM 11254T, was cultivated with or without perchlorate, usiing CO or glycerol as its sole carbon and energy source. It grew with glycerol with or without perchlorate, and its maximum cell densities were only weakly affected by the perchlorate. However, when CO (at a 30% headspace concentration) was used as a carbon and energy source, perchlorate reduction supported greater cell densities and more rapid growth rates. The stoichiometry of CO uptake, perchlorate reduction, and chloride production were consistent with the cryptic pathway for perchlorate reduction with chlorite as an end product. Chloride production occurred abiologically in the medium due to a reaction between chlorite and the sulfide used as a reducing agent. These results provide the first demonstration of CO-coupled perchlorate reduction supporting growth in Ni-COX, and they provide constraints on the potential for perchlorate-coupled, anaerobic CO oxidation in engineered systems as well as terrestrial systems and hypothetical, sub-surface, serpentinite-hosted systems on Mars.
RESUMO
Behavioral traits such as anxiety and depression have been linked to diversity of the gut microbiome in humans, domesticated animals, and lab-bred model species, but the extent to which this link exists in wild animals, and thus its ecological relevance, is poorly understood. We examined the relationship between a behavioral trait (neophobia) and the cloacal microbiome in wild house sparrows (Passer domesticus, n = 22) to determine whether gut microbial diversity is related to personality in a wild animal. We swabbed the cloaca immediately upon capture, assessed neophobia phenotypes in the lab, and then swabbed the cloaca again after several weeks in captivity to additionally test whether the microbiome of different personality types is affected disparately by captivity, and characterized gut microbiomes using 16S rRNA gene amplicon sequencing. We did not detect differences in cloacal alpha or beta microbial diversity between neophobic and non-neophobic house sparrows, and diversity for both phenotypes was negatively impacted by captivity. Although our results suggest that the adult cloacal microbiome and neophobia are not strongly linked in wild sparrows, we did detect specific OTUs that appeared more frequently and at higher abundances in neophobic sparrows, suggesting that links between the gut microbiome and behavior may occur at the level of specific taxa. Further investigations of personality and the gut microbiome are needed in more wild species to reveal how the microbiome-gut-brain axis and behavior interact in an ecological context.
RESUMO
Relatively little is known about the distribution and diversity of CO-oxidizing bacteria during succession on volcanic deposits even though they are among the primary colonists. We surveyed CO-oxidizing communities across a vegetation gradient on a 1959 cinder deposit using coxL (large subunit gene of carbon monoxide dehydrogenase) sequences. Sequences most closely related to a coxL sequence from Ktedonobacter racemifer, dominated unvegetated cinders, while Proteobacteria-like sequences dominated vegetated sites. The number of coxL operational taxonomic units (OTUs) increased threefold with increased vegetation, and correlated most strongly with the increased beta-Proteobacteria richness (r = 0.987). These compositional shifts were also reflected in overall bacterial community compositions as determined by 16S rRNA gene analysis. Notably, coxL OTU:16S rRNA OTU ratios increased with increased vegetation, indicating that CO oxidizers became a larger fraction of total bacterial richness during succession. Results from most probable number estimates and maximum potential CO uptake activity assays indicate that increased richness is paralleled by increased CO oxidizer abundance, which likely results from increased vegetation and organic carbon content. Collectively, results suggest that in contrast to patterns observed for plant succession, a versatile bacterial functional group that is important during early colonization and succession can remain important in later stages of succession, irrespective of dramatic environmental changes.
Assuntos
Bactérias/classificação , Monóxido de Carbono/metabolismo , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Biodiversidade , Contagem de Colônia Microbiana , DNA Bacteriano/química , Havaí , Dados de Sequência Molecular , Oxirredução , Filogenia , RNA Ribossômico 16S/química , Análise de Sequência de DNARESUMO
Isolation of multiple carbon monoxide (CO)-oxidizing Burkholderia strains and detection by culture-independent approaches suggest that Burkholderia may be an important component of CO-oxidizing communities in Hawaiian volcanic deposits. The absolute and relative abundance of the bacteria in these communities remains unknown, however. In this study, a quantitative PCR (Q-PCR) approach has been developed to enumerate Burkholderia coxL genes (large subunit of carbon monoxide dehydrogenase). This represents the first attempt to enumerate coxL genes from CO oxidizers in environmental samples. coxL copy numbers have been determined for samples from three sites representing a vegetation gradient on a 1959 volcanic deposit that included unvegetated cinders (bare), edges of vegetated sites (edge), and sites within tree stands (canopy). Q-PCR has also been used to estimate copy numbers of Betaproteobacteria 16S rRNA gene copy numbers and total Bacteria 16S rRNA. coxL genes could not be detected in the bare site (detection limit, > or = 4.7 x 10(3) copies per reaction) but average 1.0 x 10(8) + or - 2.4 x 10(7) and 8.6 x 10(8) + or - 7.6 x 10(7) copies g(-1) (dry weight) in edge and canopy sites, respectively, which differ statistically (P = 0.0007). Average Burkholderia coxL gene copy numbers, expressed as a percentage of total Bacteria 16S rRNA gene copy numbers, are 6.2 and 0.7% for the edge and canopy sites, respectively. Although the percentage of Burkholderia coxL is lower in the canopy site, significantly greater gene copy numbers demonstrate that absolute abundance of coxL increases in vegetated sites and contributes to the expansion of CO oxidizer communities during biological succession on volcanic deposits.
Assuntos
Proteínas de Bactérias/genética , Burkholderia/genética , Reação em Cadeia da Polimerase/métodos , Microbiologia do Solo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Burkholderia/metabolismo , Monóxido de Carbono/metabolismo , Análise por Conglomerados , Contagem de Colônia Microbiana/métodos , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Havaí , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
P-glycoprotein (Pgp) is an ATP-dependent efflux transporter and plays a major role in anti-cancer drug resistance by pumping a chemically diverse range of cytotoxic drugs from cancerous tumors. Despite numerous studies with the transporter, the molecular features that drive anti-cancer drug efflux are not well understood. Even subtle differences in the anti-cancer drug molecular structure can lead to dramatic differences in their transport rates. To unmask these structural differences, this study focused on two closely-related anthracycline drugs, daunorubicin (DNR), and doxorubicin (DOX), with mouse Pgp. While only differing by a single hydroxyl functional group, DNR has a 4 to 5-fold higher transport rate than DOX. They both non-competitively inhibited Pgp-mediated ATP hydrolysis below basal levels. The Km of Pgp-mediated ATP hydrolysis extracted from the kinetics curves was lower for DOX than DNR. However, the dissociation constants (KDs) for these drugs determined by fluorescence quenching were virtually identical. Acrylamide quenching of Pgp tryptophan fluorescence to probe the tertiary structure of Pgp suggested that DNR shifts Pgp to a "closed" conformation, while DOX shifts Pgp to an "intermediate" conformation. The effects of these drugs on the Pgp conformational distributions in a lipid bilayer were also examined by atomic force microscopy (AFM). Analysis of AFM images revealed that DNR and DOX cause distinct and significant shifts in the conformational distribution of Pgp. The results were combined to build a conformational distribution model for anthracycline transport by Pgp.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antraciclinas/farmacologia , Antibióticos Antineoplásicos/farmacologia , Daunorrubicina/farmacologia , Doxorrubicina/farmacologia , Animais , Relação Dose-Resposta a Droga , Camundongos , Conformação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologiaRESUMO
It is proposed that differences in plant height and productivity of the salt-marsh cordgrass Spartina alterniflora are the result of a dynamic interaction among tidal water movement, dissolved iron and sulfide concentrations in marsh soils, and bacterial sulfate reduction. Tidal water movement regulates the input of iron into marsh soils and the drainage of sulfide-containing interstitial water, and thereby controls the concentration of dissolved sulfide formed as a result of bacterial sulfate reduction. Near tidal creeks, where water movement and plant height and production are greatest, sulfide concentrations are lowest; in more elevated regions of marsh, where water movement andplant production are least, sulfide concentrations are highest. Plant height and productivity may be limited by the effects of sulfide on nutrient uptake.
RESUMO
The membrane-bound P-glycoprotein (Pgp) transporter plays a major role in human disease and drug disposition because of its ability to efflux a chemically diverse range of drugs through ATP hydrolysis and ligand-induced conformational changes. Deciphering these structural changes is key to understanding the molecular basis of transport and to developing molecules that can modulate efflux. Here, atomic force microscopy (AFM) is used to directly image individual Pgp transporter molecules in a lipid bilayer under physiological pH and ambient temperature. Analysis of the Pgp AFM images revealed "small" and "large" protrusions from the lipid bilayer with significant differences in protrusion height and volume. The geometry of these "small" and "large" protrusions correlated to the predicted extracellular (EC) and cytosolic (C) domains of the Pgp X-ray crystal structure, respectively. To assign these protrusions, simulated AFM images were produced from the Pgp X-ray crystal structures with membrane planes defined by three computational approaches, and a simulated 80â¯Å AFM cantilever tip. The theoretical AFM images of the EC and C domains had similar heights and volumes to the "small" and "large" protrusions in the experimental AFM images, respectively. The assignment of the protrusions in the AFM images to the EC and C domains was confirmed by changes in protrusion volume by Pgp-specific antibodies. The Pgp domains showed a considerable degree of conformational dynamics in time resolved AFM images. With this information, a model of Pgp conformational dynamics in a lipid bilayer is proposed within the context of the known Pgp X-ray crystal structures.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Bicamadas Lipídicas/química , Animais , Lipossomos , Camundongos , Microscopia de Força Atômica , Conformação ProteicaRESUMO
Two dissimilatory nitrate-reducing (Burkholderia xenovorans LB400 and Xanthobacter sp. str. COX) and two denitrifying isolates (Stappia aggregata IAM 12614 and Bradyrhizobium sp. str. CPP), previously characterized as aerobic CO oxidizers, consumed CO at ecologically relevant levels (<100 ppm) under anaerobic conditions in the presence, but not absence, of nitrate. None of the isolates were able to grow anaerobically with CO as a carbon or energy source, however, and nitrate-dependent anaerobic CO oxidation was inhibited by headspace concentrations >100-1000 ppm. Surface soils collected from temperate, subtropical and tropical forests also oxidized CO under anaerobic conditions with no lag. The observed activity was 25-60% less than aerobic CO oxidation rates, and did not appear to depend on nitrate. Chloroform inhibited anaerobic but not aerobic activity, which suggested that acetogenic bacteria may have played a significant role in forest soil anaerobic CO uptake.
Assuntos
Alphaproteobacteria/metabolismo , Monóxido de Carbono/metabolismo , Bacilos e Cocos Aeróbios Gram-Negativos/metabolismo , Nitratos/metabolismo , Microbiologia do Solo , Aerobiose/fisiologia , Anaerobiose/fisiologia , Bradyrhizobium/metabolismo , Burkholderia/metabolismo , Oxirredução , Árvores , Xanthobacter/metabolismoRESUMO
The phylogenetic affiliations of organisms responsible for aerobic CO oxidation in hypersaline soils and sediments were assessed using media containing 3.8 M NaCl. CO-oxidizing strains of the euryarchaeotes, Haloarcula, Halorubrum, Haloterrigena and Natronorubrum, were isolated from the Bonneville Salt Flats (UT) and Atacama Desert salterns (Chile). A halophilic euryarchaeote, Haloferax strain Mke2.3(T), was isolated from Hawai'i Island saline cinders. Haloferax strain Mke2.3(T) was most closely related to Haloferax larsenii JCM 13917(T) (97.0% 16S rRNA sequence identity). It grew with a limited range of substrates, and oxidized CO at a headspace concentration of 0.1%. However, it did not grow with CO as a sole carbon and energy source. Its ability to oxidize CO, its polar lipid composition, substrate utilization and numerous other traits distinguished it from H. larsenii JCM 13917(T), and supported designation of the novel isolate as Haloferax namakaokahaiae Mke2.3(T), sp. nov (= DSM 29988, = LMG 29162). CO oxidation was also documented for 'Natronorubrum thiooxidans' HG1 (Sorokin, Tourova and Muyzer 2005), N. bangense (Xu, Zhou and Tian 1999) and N. sulfidifaciens AD2(T) (Cui et al. 2007). Collectively, these results established a previously unsuspected capacity for extremely halophilic aerobic CO oxidation, and indicated that the trait might be widespread among the Halobacteriaceae, and occur in a wide range of hypersaline habitats.
Assuntos
Monóxido de Carbono/metabolismo , Haloferax , Salinidade , Cloreto de Sódio/metabolismo , Microbiologia do Solo , Aerobiose , Chile , DNA Ribossômico/genética , Genoma Arqueal/genética , Sedimentos Geológicos/microbiologia , Haloferax/genética , Haloferax/isolamento & purificação , Haloferax/metabolismo , Oxirredução , Filogenia , RNA Ribossômico 16S/genética , Solo/químicaRESUMO
The Deepwater Horizon oil spill in the northern Gulf of Mexico represents the largest marine accidental oil spill in history. It is distinguished from past spills in that it occurred at the greatest depth (1,500 m), the amount of hydrocarbon gas (mostly methane) lost was equivalent to the mass of crude oil released, and dispersants were used for the first time in the deep sea in an attempt to remediate the spill. The spill is also unique in that it has been characterized with an unprecedented level of resolution using next-generation sequencing technologies, especially for the ubiquitous hydrocarbon-degrading microbial communities that appeared largely to consume the gases and to degrade a significant fraction of the petroleum. Results have shown an unexpectedly rapid response of deep-sea Gammaproteobacteria to oil and gas and documented a distinct succession correlated with the control of the oil flow and well shut-in. Similar successional events, also involving Gammaproteobacteria, have been observed in nearshore systems as well.
Assuntos
Hidrocarbonetos/toxicidade , Poluição por Petróleo/análise , Água do Mar , Microbiologia da Água , Poluentes Químicos da Água/toxicidade , Áreas Alagadas , Biodegradação Ambiental , Gammaproteobacteria/efeitos dos fármacos , Gammaproteobacteria/crescimento & desenvolvimento , Gammaproteobacteria/metabolismo , Sedimentos Geológicos/microbiologia , Golfo do México , Hidrocarbonetos/análise , Hidrocarbonetos/metabolismo , Metagenômica , Água do Mar/química , Água do Mar/microbiologia , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/metabolismoRESUMO
In an experimental model of lung metastasis we have observed that more metastatic tumors are located on the pleura of the lung than in the parenchyma. To study possible reasons for this differential pattern we have now related the initial distribution of injected tumor cells to the later location and growth rate of metastases in different regions of the lung in C57bl/6 mice. It was found that labeled murine fibrosarcoma cells were evenly distributed throughout the lungs 24 h after intravenous injection into controls and animals previously treated with bleomycin or by exposure to hyperoxia. These treatments, known to induce pulmonary endothelial injury, were associated with increased tumor cell localization in the lung. In all cases, using morphometric methods, we found that after 2 weeks, approximately 75 per cent of metastatic tumors were located at the pleura. By [3H]thymidine labeling in autoradiographs, pleural tumors in all experimental groups had a growth rate 14 times the growth rate of tumors located in the internal regions of the lung. In vitro, the fibrosarcoma cells proliferated more rapidly on connective tissue matrices prepared from normal pleuras than they did on matrices from the remainder of the lung. Protease digestion of these matrices indicated differences in composition with more insoluble collagen, probably type I collagen, present at the pleura. These data suggest that, in spite of the initial random distribution and localization of tumor cells in the lung, there is preferential growth of metastatic tumors at the pleura which may be related to regional differences in the composition of the extracellular matrix.
Assuntos
Fibrossarcoma/secundário , Neoplasias Pulmonares/secundário , Neoplasias Pleurais/secundário , Animais , Colágeno/análise , Tecido Conjuntivo/patologia , Feminino , Fibrossarcoma/patologia , Hidroxiprolina/análise , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células Neoplásicas Circulantes , Neoplasias Pleurais/patologia , Timidina/metabolismoRESUMO
Postnatal lung development was examined in rats born with smaller than normal lungs after either prenatal exposure to glucocorticoid or to an inhibitor of collagen synthesis. At birth, treated animals had lower than normal lung weights, lung to body weight ratios, hydroxyproline (HYP) levels, total DNA; and rates of DNA synthesis. Rats exposed to steroid showed a rapid recovery in growth during the normal postnatal cell proliferative phase from 4 to 11 days, though collagen levels did not return to normal until 3 weeks of age. Rats exposed to a prenatal proline analog showed a much slower rise in lung weight and total DNA, and these levels were still much below normal at 2-3 weeks when the cell proliferation phase was completed. Levels of disaturated phosphatidylcholine were significantly below normal up to 11 days, whereas total HYP was significantly reduced and less fibrillar collagen was seen in the lung throughout the study. The results indicate that the smaller but mature lungs at birth after antenatal steroid show a growth rebound and quickly become structurally normal. In contrast, inhibition of fibroblast growth and collagen deposition produces small lungs at birth, which continue to show inhibited growth and development at least up to 3 weeks of age, when the cell division phase is over.
Assuntos
Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Animais , Ácido Azetidinocarboxílico/farmacologia , Colágeno/metabolismo , Replicação do DNA , Dexametasona/farmacologia , Feminino , Maturidade dos Órgãos Fetais/efeitos dos fármacos , Hidroxiprolina/metabolismo , Pulmão/ultraestrutura , Tamanho do Órgão , Fosfatidilcolinas/metabolismo , Gravidez , Surfactantes Pulmonares/metabolismo , Ratos , Ratos EndogâmicosRESUMO
The effects of injecting dexamethasone to pregnant and newborn rats on the subsequent production of macrophages in the lung and on their phagocytic activity and lysosomal enzyme content were evaluated from late gestation to postnatal day 10 using an organ culture system to collect macrophages. Pieces of lung tissue cultured 6 days on cover glasses produced a halo of macrophages adherent to the glass around the explants. Thymidine labeling showed that the macrophages were derived from dividing precursors in the pulmonary interstitium. DNA synthesis was reduced after steroid treatment, and fewer cells were obtained on cover glasses, particularly just after birth when the macrophage number usually increases. Phagocytic function was also diminished in cells collected after dexamethasone injection, particularly when derived from neonatal animals. In contrast, intracellular levels of non-specific esterase and glucosaminidase were increased, probably indicative of lower phagolysosome formation and lower enzyme secretion. These results demonstrate that steroid administration to fetal or newborn animals subsequently reduces the number and phagocytic activity of macrophages in the lungs. This could reduce the defense mechanisms of the neonate and increase susceptibility to infection.
Assuntos
Animais Recém-Nascidos , Dexametasona/farmacologia , Macrófagos/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , DNA/biossíntese , Esterases/metabolismo , Feminino , Feto/efeitos dos fármacos , Hexosaminidases/metabolismo , Pulmão/citologia , Pulmão/efeitos dos fármacos , Macrófagos/enzimologia , Técnicas de Cultura de Órgãos , Fagocitose/efeitos dos fármacos , Gravidez , Ratos , Ratos EndogâmicosRESUMO
The findings of past research using d-tubocurarine chloride as a control procedure in heart rate conditioning have been equivocal. The present experiment compared classical heart rate conditioning under neuromuscular blockade by d-tubocurarine chloride, and a pharmacologically more specific blocker, carbolonium bromide. Both groups were compared to a placebo condition. The results indicated that carbolonium bromide and placebo treated animals acquired deceleratory cardiac conditional responses, but animals treated with d-tubocurarine chloride did not. Unconditional acceleratory responses were found in groups. The findings were discussed in relation to classical conditioning and the psychopharmacology of neuromuscular blockade.