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1.
Adv Exp Med Biol ; 1131: 7-26, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646505

RESUMO

Measuring free Ca2+ concentration ([Ca2+]) in the cytosol or organelles is routine in many fields of research. The availability of membrane permeant forms of indicators coupled with the relative ease of transfecting cell lines with biological Ca2+ sensors have led to the situation where cellular and subcellular [Ca2+] is examined by many non-specialists. In this chapter, we evaluate the most used Ca2+ indicators and highlight what their major advantages and disadvantages are. We stress the potential pitfalls of non-ratiometric techniques for measuring Ca2+ and the clear advantages of ratiometric methods. Likely improvements and new directions for Ca2+ measurement are discussed.


Assuntos
Cálcio , Citosol , Organelas , Animais , Cálcio/metabolismo , Técnicas Citológicas , Citosol/química , Citosol/metabolismo , Humanos , Organelas/química , Organelas/metabolismo
2.
Results Probl Cell Differ ; 67: 233-250, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31435798

RESUMO

Morphology of Golgi apparatus changes frequently and diversely depending on various cellular conditions and these changes correlate with the balance between membrane inflow and outflow at the Golgi via vesicular transports. In a previous study, we introduced a semi-intact cell system suitable for the reconstitution of morphological changes that organelles undergo as well as the vesicular transport between them. Semi-intact cells are cells that have undergone plasma membrane permeabilization by the cholesterol-dependent pore-forming cytolysin, streptolysin O (SLO). Permeabilization enables the introduction of various molecules into the cells, as well as the substitution of the original cytosol with an exogenously made cytosol prepared from cells in various stages of cell cycle, differentiation, and disease progression. Coupled with a green fluorescent protein(GFP)-visualization technique, this cell-based system enables the analysis of the molecular mechanisms underlying biological processes that are highly dependent on the integrity of the intracellular architecture. In this chapter, we present a variety of reconstitution assays concerning biological reactions pertaining to the Golgi apparatus.


Assuntos
Permeabilidade da Membrana Celular , Complexo de Golgi/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Citosol/química , Citosol/efeitos dos fármacos , Citosol/metabolismo , Complexo de Golgi/efeitos dos fármacos
3.
Biophys Chem ; 252: 106195, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31195340

RESUMO

Astrocytes, the most common type of glial cell, are critical to the health of the central nervous system. Evidence implies that changes in the astrocyte's cytosolic calcium concentration is part of a central mechanism by which information is passed and processed in the cell, and it is linked to both external stimuli impacting the cell as well as downstream events such as metabolism and neurotransmitter release. This work proposes a novel chemical model to further the understanding of how extracellular signals could affect intracellular calcium dynamics and metabolic processes within the cell.


Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Glucose/metabolismo , Algoritmos , Animais , Astrócitos/química , Citosol/química , Citosol/metabolismo , Humanos , Modelos Biológicos
4.
MBio ; 10(3)2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064825

RESUMO

The mitochondrial Ca2+ uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used Trypanosoma cruzi to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca2+ uptake. T. cruzi MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca2+ uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca2+ concentrations ([Ca2+]cyt), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles.IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca2+ sensors and gatekeepers of the MCU, preventing Ca2+ uptake under resting conditions and favoring it at high cytosolic Ca2+ concentrations ([Ca2+]cyt). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca2+]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/genética , Adaptação Fisiológica , Transporte Biológico , Sistemas CRISPR-Cas , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions , Citosol/química , Citosol/metabolismo , Técnicas de Inativação de Genes , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/patogenicidade
5.
Parasitol Res ; 118(6): 1785-1797, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31062084

RESUMO

We report the complete coding sequences of mitochondrial thioredoxin (TsTrx2) and glutaredoxin (TsGrx1) from the cysticerci of T. solium. The full-length DNA of the TsTrx2 gene shows two introns of 88 and 77 bp and three exons. The TsTrx2 gene contains a single ORF of 423 bp, encoding 140 amino acid residues with an estimated molecular weight of 15,560 Da. A conserved C64NPC67 active site and a 30-amino acid extension at its N-terminus were identified. An insulin reduction reaction was used to determine whether it was a functional recombinant protein. The full-length DNA of the TsGrx1 gene shows one intron of 39 bp and a single ORF of 315 bp, encoding 105 amino acid residues with an estimated molecular weight of 12,582 Da. Sequence analysis revealed a conserved dithiol C34PYC37 active site, GSH-binding motifs (CXXC, Lys and Gln/Arg, TVP, and CXD), and a conserved Gly-Gly motif. The r-TsGrx1 kinetic constants for glutathione (GSH) and 2-hydroxyethyl disulfide (HED) were determined. In addition, cytosolic thioredoxin (TsTrx1), as reported by (Jiménez et al., Biomed Res Int 2015:453469, 2015), was cloned and expressed, and its catalytic constants were obtained along with those of the other two reductases. Rabbit-specific antibodies showed immune cross-reactions between TsTrx1 and TsTrx2 but not with TsGrx1. Both TsTGRs as reported by (Plancarte and Nava, Exp Parasitol 149:65-73, 2015) were biochemically purified to obtain and compare the catalytic constants for their natural substrates, r-TsTrx1, and r-TsTrx2, compared to those for Trx-S2E. coli. In addition, we determined the catalytic differences between the glutaredoxin activity of the TsTGRs compared with r-TsGrx1. These data increase the knowledge of the thioredoxin and GSH systems in T. solium, which is relevant for detoxification and immune evasion.


Assuntos
Citosol/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/isolamento & purificação , Mitocôndrias/metabolismo , Taenia solium/genética , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificação , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cysticercus/genética , Cysticercus/isolamento & purificação , Cysticercus/metabolismo , Citosol/química , Dissulfetos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/análogos & derivados , Etanol/metabolismo , Glutarredoxinas/química , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Cinética , Mitocôndrias/química , Mitocôndrias/genética , Fases de Leitura Aberta , Coelhos , Taenia solium/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo
6.
Curr Microbiol ; 76(6): 659-665, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30937514

RESUMO

In the present study, three strains (ChDC F213T, ChDC F251, and ChDC F267) were classified as novel species of genus Fusobacterium based on average nucleotide identity (ANI) and genome-to-genome distance (GGD) analysis and chemotaxonomic characterization. 16S rDNA sequences of strains ChDC F213T, ChDC F251, and ChDC F267 were highly similar to that of F. periodonticum ATCC 33693T (99.6, 99.4, and 99.4%, respectively). ANI and GGD values of the three isolates with F. periodonticum ATCC 33693T ranged from 92.5 to 92.6% and 47.7 to 48.2%, respectively. Considering that threshold of ANI and GGD values for bacterial species discrimination are 95-96% and 70%, respectively, these results indicate that the three isolates represent a novel Fusobacterium species. DNA G + C contents of the three isolates were 28.0 mol% each. Cellular fatty acid analysis of these strains revealed that C14:0, C16:0, and C16:1 ω6c/C16:1 ω7c were major fatty acids. Therefore, these three strains are novel species belonging to genus Fusobacterium. Strain ChDC F213T (= KCOM 1259T = KCTC 5677T = JCM 33009T) is the type strain of a novel species of genus Fusobacterium, for which a name of Fusobacterium pseudoperiodonticum sp. nov. is proposed.


Assuntos
Fusobacterium/classificação , Fusobacterium/isolamento & purificação , Boca/microbiologia , Composição de Bases , Análise por Conglomerados , Citosol/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Fusobacterium/química , Fusobacterium/genética , Humanos , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico
7.
Chemosphere ; 226: 132-139, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30925405

RESUMO

Hydroxylated bromodiphenyl ethers (OH-BDEs) can arise from monooxygenation of anthropogenic BDEs or through natural biosynthetic processes in marine organisms, and several OH-BDEs have been shown to be toxic. OH-BDEs are expected to form sulfate and glucuronide conjugates that are readily excreted, however there is little information on these pathways. We examined the human hepatic glucuronidation and sulfonation of 6-OH-BDE47, 2-OH-BDE68, 4-OH-BDE68 and 2-OH-6'methoxy-BDE68. Human liver microsomes and cytosol were from de-identified female and male donors aged 31 to 75 under an exempt protocol. Recombinant human SULT1A1, 1B1, 1E1 and 2A1 enzymes were prepared from bacterial expression systems. Sulfonation and glucuronidation of each OH-BDE were studied using radiolabeled co-substrates, 3'phosphoadenosine-5'phospho-35S-sulfate or uridine diphospho-ß-D-14C-glucuronic acid in order to quantify the sulfated or glucuronidated products. The OH-BDEs studied were more efficiently glucuronidated than sulfonated. Of the compounds studied, 2-OH-BDE68 was the most readily conjugated, and exhibited an efficiency (Vmax/KM) of glucuronidation of 0.274 ±â€¯0.125 mL/min/mg protein, mean ±â€¯S.D., n = 3, while that for sulfonation was 0.179 ±â€¯0.030 mL/min/mg protein. For both pathways, all Km values were in the low µM range. Studies with human SULT enzymes showed that sulfonation of these four substrates was readily catalyzed by SULT1B1 and SULT1E1. Much lower activity was found with SULT1A1 and SULT2A1. Assuming that the glucuronide and sulfate conjugates are non-toxic and readily excreted, as is the case for most such conjugates, these studies suggest that OH-BDEs should not accumulate in people to the same extent as the parent BDEs.


Assuntos
Éteres/química , Glucuronídeos/química , Fígado/metabolismo , Bifenil Polibromatos/química , Sulfatos/química , Adulto , Idoso , Citosol/química , Feminino , Humanos , Hidroxilação , Fígado/ultraestrutura , Masculino , Microssomos Hepáticos/metabolismo , Pessoa de Meia-Idade
8.
Nat Commun ; 10(1): 711, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755613

RESUMO

Adenosine 5' triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR1.0 responds to relevant ATP concentrations (30 µM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.


Assuntos
Trifosfato de Adenosina/química , Membrana Celular/química , Citosol/química , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas/metabolismo , Trifosfato de Adenosina/genética , Bacillus/citologia , Bacillus/genética , Bacillus/metabolismo , Proteínas de Bactérias/genética , Expressão Gênica , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Processamento de Imagem Assistida por Computador , Cinética , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica
9.
J Bacteriol ; 201(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30617242

RESUMO

Cyclic di-AMP is a recently identified second messenger exploited by a number of Gram-positive bacteria to regulate important biological processes. Here, we studied the phenotypic alterations induced by the increased intracellular c-di-AMP levels in Streptococcus gallolyticus, an opportunistic pathogen responsible for septicemia and endocarditis in the elderly. We report that an S. gallolyticus c-di-AMP phosphodiesterase gdpP knockout mutant, which displays a 1.5-fold higher intracellular c-di-AMP levels than the parental strain UCN34, is more sensitive to osmotic stress and is morphologically smaller than the parental strain. Unexpectedly, we found that a higher level of c-di-AMP reduced biofilm formation of S. gallolyticus on abiotic surfaces and reduced adherence and cell aggregation on human intestinal cells. A genome-wide transcriptomic analysis indicated that c-di-AMP regulates many biological processes in S. gallolyticus, including the expression of various ABC transporters and disease-associated genes encoding bacteriocin and Pil3 pilus. Complementation of the gdpP in-frame deletion mutant with a plasmid carrying gdpP in trans from its native promoter restored bacterial morphology, tolerance to osmotic stress, biofilm formation, adherence to intestinal cells, bacteriocin production, and Pil3 pilus expression. Our results indicate that c-di-AMP is a pleiotropic signaling molecule in S. gallolyticus that may be important for S. gallolyticus pathogenesis.IMPORTANCE Streptococcus gallolyticus is an opportunistic pathogen responsible for septicemia and endocarditis in the elderly and is also strongly associated with colorectal cancer. S. gallolyticus can form biofilms, express specific pili to colonize the host tissues, and produce a specific bacteriocin allowing killing of commensal bacteria in the murine colon. Nevertheless, how the expression of these colonization factors is regulated remains largely unknown. Here, we show that c-di-AMP plays pleiotropic roles in S. gallolyticus, controlling the tolerance to osmotic stress, cell size, biofilm formation on abiotic surfaces, adherence and cell aggregation on human intestinal cells, expression of Pil3 pilus, and production of bacteriocin. This study indicates that c-di-AMP may constitute a key regulatory molecule for S. gallolyticus host colonization and pathogenesis.


Assuntos
Aderência Bacteriana , Biofilmes/crescimento & desenvolvimento , Citosol/química , Fosfatos de Dinucleosídeos/análise , Pressão Osmótica , Streptococcus gallolyticus subspecies gallolyticus/fisiologia , 3',5'-AMP Cíclico Fosfodiesterases/deficiência , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Animais , Linhagem Celular , Células Epiteliais/microbiologia , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Humanos , Camundongos , Streptococcus gallolyticus subspecies gallolyticus/química , Streptococcus gallolyticus subspecies gallolyticus/citologia
10.
Nat Commun ; 10(1): 340, 2019 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-30664642

RESUMO

Microorganisms adapt their biophysical properties in response to changes in their local environment. However, quantifying these changes at the single-cell level has only recently become possible, largely relying on fluorescent labeling strategies. In this work, we utilize yeast (Saccharomyces cerevisiae) to demonstrate label-free quantification of changes in both intracellular osmolarity and macromolecular concentration in response to changes in the local environment. By combining a digital holographic microscope with a millifluidic chip, the temporal response of cellular water flux was successfully isolated from the rate of production of higher molecular weight compounds, in addition to identifying the produced compounds in terms of the product of their refractive index increment [Formula: see text] and molar mass. The ability to identify, quantify and temporally resolve multiple biophysical processes in living cells at the single cell level offers a crucial complement to label-based strategies, suggesting broad applicability in studies of a wide-range of cellular processes.


Assuntos
Citosol/metabolismo , Saccharomyces cerevisiae/química , Análise de Célula Única/métodos , Água/metabolismo , Transporte Biológico , Citosol/química , Citosol/ultraestrutura , Holografia , Dispositivos Lab-On-A-Chip , Concentração Osmolar , Pressão Osmótica , Refratometria , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Análise de Célula Única/instrumentação , Água/química
11.
MBio ; 10(1)2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670615

RESUMO

Correlations between gene transcription and the abundance of high-energy purine nucleotides in Saccharomyces cerevisiae have often been noted. However, there has been no systematic investigation of this phenomenon in the absence of confounding factors such as nutrient status and growth rate, and there is little hard evidence for a causal relationship. Whether transcription is fundamentally responsive to prevailing cellular energetic conditions via sensing of intracellular purine nucleotides, independently of specific nutrition, remains an important question. The controlled nutritional environment of chemostat culture revealed a strong correlation between ATP and GTP abundance and the transcription of genes required for growth. Short pathways for the inducible and futile consumption of ATP or GTP were engineered into S. cerevisiae, permitting analysis of the transcriptional effect of an increased demand for these nucleotides. During steady-state growth using the fermentable carbon source glucose, the futile consumption of ATP led to a decrease in intracellular ATP concentration but an increase in GTP and the guanylate energy charge (GEC). Expression of transcripts encoding proteins involved in ribosome biogenesis, and those controlled by promoters subject to SWI/SNF-dependent chromatin remodelling, was correlated with these nucleotide pool changes. Similar nucleotide abundance changes were observed using a nonfermentable carbon source, but an effect on the growth-associated transcriptional programme was absent. Induction of the GTP-cycling pathway had only marginal effects on nucleotide abundance and gene transcription. The transcriptional response of respiring cells to glucose was dampened in chemostats induced for ATP cycling, but not GTP cycling, and this was primarily associated with altered adenine nucleotide levels.IMPORTANCE This paper investigates whether, independently of the supply of any specific nutrient, gene transcription responds to the energy status of the cell by monitoring ATP and GTP levels. Short pathways for the inducible and futile consumption of ATP or GTP were engineered into the yeast Saccharomyces cerevisiae, and the effect of an increased demand for these purine nucleotides on gene transcription was analyzed. The resulting changes in transcription were most consistently associated with changes in GTP and GEC levels, although the reprogramming in gene expression during glucose repression is sensitive to adenine nucleotide levels. The results show that GTP levels play a central role in determining how genes act to respond to changes in energy supply and that any comprehensive understanding of the control of eukaryotic gene expression requires the elucidation of how changes in guanine nucleotide abundance are sensed and transduced to alter the global pattern of transcription.


Assuntos
Citosol/química , Expressão Gênica , Guanosina Trifosfato/análise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Genética , Trifosfato de Adenosina/metabolismo , Glucose/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento
12.
mSphere ; 3(6)2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30567900

RESUMO

Sepsis caused by Gram-negative bacteria is the consequence of an unrestrained infection that continuously releases lipopolysaccharide (LPS) into the bloodstream, which triggers an uncontrolled systemic inflammatory response leading to multiorgan failure and death. After scrutinizing the immune modulation exerted by a recombinant Fasciola hepatica fatty acid binding protein termed Fh15, our group demonstrated that addition of Fh15 to murine macrophages 1 h prior to LPS stimulation significantly suppresses the expression of proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL1-ß). The present study aimed to demonstrate that Fh15 could exert a similar anti-inflammatory effect in vivo using a mouse model of septic shock. Among the novel findings reported in this article, (i) Fh15 suppressed numerous serum proinflammatory cytokines/chemokines when injected intraperitoneally 1 h after exposure of animals to lethal doses of LPS, (ii) concurrently, Fh15 increased the population of large peritoneal macrophages (LPMs) in the peritoneal cavity (PerC) of LPS-injected animals, and (iii) Fh15 downregulated the expression on spleen macrophages of CD38, a cell surface ectoenzyme with a critical role during inflammation. These findings present the first evidence that the recombinant parasitic antigen Fh15 is an excellent modulator of the PerC cell content and in vivo macrophage activation, endorsing Fh15's potential as a drug candidate against sepsis-related inflammatory response.IMPORTANCE Sepsis is a potentially life-threatening complication of an infection. Sepsis is mostly the consequence of systemic bacterial infections leading to exacerbated activation of immune cells by bacterial products, resulting in enhanced release of inflammatory mediators. Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, is a critical factor in the pathogenesis of sepsis, which is sensed by Toll-like receptor 4 (TLR4). The scientific community highly pursues the development of antagonists capable of blocking the cytokine storm by blocking TLR4. We report here that a recombinant molecule of 14.5 kDa belonging to the Fasciola hepatica fatty acid binding protein (Fh15) is capable of significantly suppressing the LPS-induced cytokine storm in a mouse model of septic shock when administered by the intraperitoneal route 1 h after a lethal LPS injection. These results suggest that Fh15 is an excellent candidate for drug development against endotoxemia.


Assuntos
Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Proteínas de Ligação a Ácido Graxo/administração & dosagem , Proteínas de Helminto/administração & dosagem , Lipopolissacarídeos/imunologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/fisiologia , Choque Séptico/patologia , ADP-Ribosil Ciclase 1/análise , Animais , Células Cultivadas , Citosol/química , Modelos Animais de Doenças , Ácidos Graxos/análise , Injeções Intravenosas , Glicoproteínas de Membrana/análise , Camundongos , Proteínas Recombinantes/administração & dosagem , Baço/imunologia
13.
APMIS ; 126(9): 746-754, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30160021

RESUMO

Use of archived formalin-fixed paraffin-embedded (FFPE) tissue is a standard method for evaluation of proposed prognostic and predictive tumour markers. However, little is known of the preservation of biomarker expression in old FFPE tumour blocks. We investigate the quality of immunohistochemical (IHC) oestrogen (ER) and progesterone receptor (PR) evaluation in FFPE tissue over time (1978-2000) using a large breast cancer tissue microarray (N = 573) with access to receptor analyses in cytosol (CYT) at diagnosis, coexpression of other biomarkers and follow-up data. We found a good correlation between ER analysed with CYT at diagnosis and ER analysed with IHC in archived FFPE tissue from the same tumour. ER evaluation did not seem to be affected by tissue storage time. Nor was there any time-dependent difference in ERIHC correlation with other biomarkers (HER2, Ki67) or survival. Discordant cases were more often classified as ER-positive with IHC than with CYT. For PR, however, we found an increased correlation between methods in more recent time periods. This may possibly be explained by more reliable PRIHC results in newer samples, although other explanations may also contribute. Our results indicate stable ER expression in FFPE tissue archived for up to 40 years.


Assuntos
Neoplasias da Mama/química , Receptores Estrogênicos/análise , Receptores de Progesterona/análise , Neoplasias da Mama/mortalidade , Citosol/química , Feminino , Humanos , Imuno-Histoquímica , Antígeno Ki-67/análise , Inclusão em Parafina , Receptor ErbB-2/análise , Receptores Estrogênicos/imunologia , Receptores de Progesterona/imunologia
14.
Proc Natl Acad Sci U S A ; 115(36): 8966-8971, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30126985

RESUMO

Helicobacter pylori is a human pathogen that infects the stomach, where it experiences variable pH. To survive the acidic gastric conditions, H. pylori produces large quantities of urease, a nickel enzyme that hydrolyzes urea to ammonia, which neutralizes the local environment. One of the regulators of urease expression in H. pylori is HpNikR, a nickel-responsive transcription factor. Here we show that HpNikR also regulates urease expression in response to changes in pH, linking acid adaptation and nickel homeostasis. Upon measuring the cytosolic pH of H. pylori exposed to an external pH of 2, similar to the acidic shock conditions that occur in the human stomach, a significant drop in internal pH was observed. This decrease in internal pH resulted in HpNikR-dependent activation of ureA transcription. Furthermore, analysis of a slate of H. pylori genes encoding other acid adaptation or nickel homeostasis components revealed HpNikR-dependent regulation in response to acid shock. This regulation was consistent with pH-dependent DNA binding to the corresponding promoter sequences observed in vitro with purified HpNikR. These results demonstrate that HpNikR can directly respond to changes in cytosolic pH during acid acclimation and illustrate the exquisitely coordinated regulatory networks that support H. pylori infections in the harsh environment of the human stomach.


Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Helicobacter pylori , Proteínas Repressoras , Transcrição Genética/fisiologia , Urease , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citosol/química , Citosol/metabolismo , Helicobacter pylori/química , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Níquel/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Urease/biossíntese , Urease/química , Urease/genética
15.
J Microbiol ; 56(8): 542-548, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30047082

RESUMO

A novel Gram-stain-negative, motile by means of gliding, and short rod-shaped bacterium, designated HS916T, was isolated from soil polluted by sewer water in Cheonan-si, South Korea. Growth occurred at 10-35°C (optimum 30°C), pH 6.0-8.0 (optimum pH 7.0), and 0-1% sodium chloride (NaCl, w/v). Based on similarities of 16S rRNA gene sequences, strain HS916T was closely related to members of the genus Flavobacterium, exhibiting the highest sequence similarities with Flavobacterium glycines Gm-149T (96.4%), followed by F. granuli Kw05T (96.3%), F. fluminis 3R17T (96.3%), F. aquicola TMd3a3T (96.2%), and F. nitratireducens N1T (96.2%). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain HS916T was placed in a monophyletic cluster with F. nitratireducens N1T and F. fluminis 3R17T. The predominant fatty acids (> 5% of the total) of strain HS916T were iso-C15:0, anteiso-C15:0, iso-C15:0 3-OH, C17:1ω6с, C16:0 3-OH, iso-C17:0 3-OH, and summed feature 3 (C16:1ω7с and/or C16:1ω6с). The major polar lipids of the strain comprised phosphatidylethanolamine, unidentified aminolipids, and five unidentified lipids. The predominant respiratory quinone and the major polyamine were menaquinone-6 (MK-6) and symhomospermidine, respectively. The DNA G + C content of strain HS916T was 34.9 mol%. Based on polyphasic analyses, strain HS916T represents a novel species belonging to the genus Flavobacterium, for which the name Flavobacterium parvum sp. nov. is proposed. The type strain is HS916T (= KACC 19448T = JCM 32368T).


Assuntos
Flavobacterium/classificação , Flavobacterium/isolamento & purificação , Esgotos/microbiologia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Composição de Bases , Análise por Conglomerados , Citosol/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Flavobacterium/genética , Flavobacterium/fisiologia , Concentração de Íons de Hidrogênio , Coreia (Geográfico) , Locomoção , Fosfolipídeos/análise , Filogenia , Quinonas/análise , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Cloreto de Sódio/metabolismo , Temperatura Ambiente , Vitamina K 2/análise
16.
J Microbiol ; 56(8): 549-555, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30047083

RESUMO

SYP-B2174T is a yellow-pigmented, Gram-positive, non-motile, and rod-shaped actinobacterium isolated from the rhizospheric soil of Aquilegia viridiflora Pall. collected from the Xinjiang uygur autonomous region of China. The strain's growth temperature ranges from 1 to 35°C, with an optimal growth being observed at 28°C. Growth occurs from 0 to 5% NaCl and at pH 6-8, with optimal growth being observed in 1% NaCl at pH 7. Comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species Leifsonia kafniensis JCM 17021T and Leifsonia psychrotolerans DSM 22824T with similarities of 97.8 and 97.6%, respectively. The DNA-DNA hybridization values of the strain SYP-B2174T to its closest phylogenetic neighbors were significantly lower than 35.7%. The strain was identified as a novel species of the genus Leifsonia judging by the coryneform morphology, peptidoglycans based upon 2,4-diaminobutyric acid, principal phospholipids phosphatidylglycerol and diphosphatidylglycerol, major menaquinone MK-11, predominant fatty acids of anteiso-C15:0, anteiso-C17:0, and iso-C16:0, and a DNA G + C base composition of 68.7 mol%, for which the name Leifsonia flava sp. nov. is proposed. The type strain is SYP-B2174T (= CGMCC 1.15856T = DSM 105144T = KCTC 39963T).


Assuntos
Actinobacteria/classificação , Actinobacteria/isolamento & purificação , Aquilegia/crescimento & desenvolvimento , Rizosfera , Microbiologia do Solo , Actinobacteria/genética , Actinobacteria/fisiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , Parede Celular/química , China , Análise por Conglomerados , Citosol/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Concentração de Íons de Hidrogênio , Hibridização de Ácido Nucleico , Peptidoglicano/análise , Fosfolipídeos/análise , Filogenia , Pigmentos Biológicos/análise , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Cloreto de Sódio/metabolismo , Temperatura Ambiente , Vitamina K 2/análise
17.
J Trace Elem Med Biol ; 48: 202-212, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29773183

RESUMO

Intracellular labile (free) Zn2+-level ([Zn2+]i) is low and increases markedly under pathophysiological conditions in cardiomyocytes. High [Zn2+]i is associated with alterations in excitability and ionic-conductances while exact mechanisms are not clarified yet. Therefore, we examined the elevated-[Zn2+]i on some sarcolemmal ionic-mechanisms, which can mediate cardiomyocyte dysfunction. High-[Zn2+]i induced significant changes in action potential (AP) parameters, including depolarization in resting membrane-potential and prolongations in AP-repolarizing phases. We detected also the time-dependent effects such as induction of spontaneous APs at the time of ≥ 3 min following [Zn2+]i increases, a manner of cellular ATP dependent and reversible with disulfide-reducing agent dithiothreitol, DTT. High-[Zn2+]i induced inhibitions in voltage-dependent K+-channel currents, such as transient outward K+-currents, Ito, steady-state currents, Iss and inward-rectifier K+-currents, IK1, reversible with DTT seemed to be responsible from the prolongations in APs. We, for the first time, demonstrated that lowering cellular ATP level induced significant decreaeses in both Iss and IK1, while no effect on Ito. However, the increased-[Zn2+]i could induce marked activation in ATP-sensitive K+-channel currents, IKATP, depending on low cellular ATP and thiol-oxidation levels of these channels. The mRNA levels of Kv4.3, Kv1.4 and Kv2.1 were depressed markedly with increased-[Zn2+]i with no change in mRNA level of Kv4.2, while the mRNA level of IKATP subunit, SUR2A was increased significantly with increased-[Zn2+]i, being reversible with DTT. Overall we demonstrated that high-[Zn2+]i, even if nanomolar levels, alters cardiac function via prolonged APs of cardiomyocytes, at most, due to inhibitions in voltage-dependent K+-currents, although activation of IKATP is playing cardioprotective role, through some biochemical changes in cellular ATP- and thiol-oxidation levels. It seems, a well-controlled [Zn2+]i can be novel therapeutic target for cardiac complications under pathological conditions including oxidative stress.


Assuntos
Trifosfato de Adenosina/metabolismo , Arritmias Cardíacas/metabolismo , Citosol/química , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Compostos de Sulfidrila/metabolismo , Zinco/metabolismo , Animais , Citosol/metabolismo , Ventrículos do Coração/citologia , Masculino , Miócitos Cardíacos/citologia , Oxirredução , Ratos , Ratos Wistar , Zinco/química
18.
PLoS Negl Trop Dis ; 12(5): e0006493, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29768419

RESUMO

Trichomonas vaginalis is a causative agent of Trichomoniasis, a leading non-viral sexually transmitted disease worldwide. In the current study, we show Heat shock protein 90 is essential for its growth. Upon genomic analysis of the parasite, it was found to possess seven ORFs which could potentially encode Hsp90 isoforms. We identified a cytosolic Hsp90 homolog, four homologs which can align to truncated cytosolic Hsp90 gene products along with two Grp94 homologs (ER isoform of Hsp90). However, both Grp94 orthologs lacked an ER retention motif. In cancer cells, it is very well established that Hsp90 is secreted and regulates key clients involved in metastases, migration, and invasion. Since Trichomonas Grp94 lacks ER retention motif, we examined the possibility of its secretion. By using cell biology and biochemical approaches we show that the Grp94 isoform of Hsp90 is secreted by the parasite by the classical ER-Golgi pathway. This is the first report of a genome encoded secreted Hsp90 in a clinically important parasitic protozoan.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Protozoários/metabolismo , Tricomoníase/parasitologia , Trichomonas vaginalis/metabolismo , Motivos de Aminoácidos , Citosol/química , Citosol/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Humanos , Transporte Proteico , Proteínas de Protozoários/genética , Trichomonas vaginalis/química , Trichomonas vaginalis/classificação , Trichomonas vaginalis/genética
19.
Med Microbiol Immunol ; 207(3-4): 211-225, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29687353

RESUMO

Although colorectal cancer is the third leading cause of death in Morocco, there are no studies of the microbiome changes associated with the disease in the Moroccan population. The aim of our study was to compare the stool microbiome of Moroccan cancer patients with healthy individuals. We analyzed the microbiome composition of samples from 11 CRC patients and 12 healthy individuals by 16S rRNA amplicon sequencing. Principal coordinate analysis of samples revealed defined cancer versus healthy clusters. Our findings showed that cancer samples had higher proportions of Firmicutes (T = 50.5%; N = 28.4%; p = 0.04), specifically of Clostridia (T = 48.3%; N = 19.0%; p = 0.002), and Fusobacteria (T = 0.1%; N = 0.0%; p = 0.02), especially of Fusobacteriia (T = 0.1%; N = 0.0%; p = 0.02), while Bacteroidetes were enriched in healthy samples (T = 35.1%; N = 62.8%; p = 0.06), particularly the class Bacteroidia (T = 35.1%; N = 62.6%; p = 0.06). Porphyromonas, Clostridium, Ruminococcus, Selenomonas, and Fusobacterium were significantly overrepresented in diseased patients, similarly to other studies. Predicted functional information showed that bacterial motility proteins, flagellar assembly, and fatty acid biosynthesis metabolism were significantly overrepresented in cancer patients, while amino acid metabolism and glycan biosynthesis were overrepresented in controls. This suggests that involvement of these functional metagenomes is similar and relevant in the carcinogenesis process, independent of the origin of the samples. Results from this study allowed identification of bacterial taxa relevant to the Moroccan population and encourages larger studies to facilitate population-directed therapeutic approaches.


Assuntos
Neoplasias Colorretais/complicações , Neoplasias Colorretais/microbiologia , Disbiose , Microbioma Gastrointestinal , Microbiota , Adulto , Idoso , Análise por Conglomerados , Citosol/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Feminino , Humanos , Masculino , Metagenômica , Pessoa de Meia-Idade , Marrocos , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Inquéritos e Questionários , Adulto Jovem
20.
Science ; 359(6382): 1403-1407, 2018 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-29567713

RESUMO

Lymph node metastases in cancer patients are associated with tumor aggressiveness, poorer prognoses, and the recommendation for systemic therapy. Whether cancer cells in lymph nodes can seed distant metastases has been a subject of considerable debate. We studied mice implanted with cancer cells (mammary carcinoma, squamous cell carcinoma, or melanoma) expressing the photoconvertible protein Dendra2. This technology allowed us to selectively photoconvert metastatic cells in the lymph node and trace their fate. We found that a fraction of these cells invaded lymph node blood vessels, entered the blood circulation, and colonized the lung. Thus, in mouse models, lymph node metastases can be a source of cancer cells for distant metastases. Whether this mode of dissemination occurs in cancer patients remains to be determined.


Assuntos
Vasos Sanguíneos/patologia , Linfonodos/patologia , Metástase Linfática/patologia , Inoculação de Neoplasia , Animais , Carcinoma de Células Escamosas , Linhagem Celular Tumoral , Movimento Celular , Rastreamento de Células/métodos , Citosol/química , Feminino , Proteínas Luminescentes/análise , Pulmão/patologia , Melanoma Experimental , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Neoplásicas Circulantes
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