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1.
Blood Adv ; 5(17): 3407-3417, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34495313

RESUMO

Recent compelling evidence showed that innate immune effector cells could recognize allogeneic grafts and prime an adaptive immune response. Signal regulatory protein α (SIRPα) is an immunoglobulin superfamily receptor that is expressed on myeloid cells; the interaction between SIRPα and its ubiquitously expressed ligand CD47 elicits an inhibitory signal that suppresses macrophage phagocytic function. Additional studies showed that donor-recipient mismatch in SIRPα variants might activate monocytic allorecognition, possibly as the result of non-self SIRPα-CD47 interaction. However, the frequency of SIRPα variation and its role in hematopoietic stem cell transplantation (HSCT) remains unexplored. We studied 350 patients with acute myeloid leukemia/myelodysplastic syndrome who underwent HLA-matched related HSCT and found that SIRPα allelic mismatches were present in 39% of transplantation pairs. SIRPα variant mismatch was associated with a significantly higher rate of chronic graft-versus-host disease (GVHD; hazard ratio [HR], 1.5; P = .03), especially de novo chronic GVHD (HR, 2.0; P = .01), after adjusting for other predictors. Those with mismatched SIRPα had a lower relapse rate (HR, 0.6; P = .05) and significantly longer relapse-free survival (RFS; HR, 0.6; P = .04). Notably, the effect of SIRPα variant mismatch on relapse protection was most pronounced early after HSCT and in patients who were not in remission at HSCT (cumulative incidence, 73% vs 54%; HR, 0.5; P = .01). These findings show that SIRPα variant mismatch is associated with HSCT outcomes, possibly owing to innate allorecognition. SIRPα variant matching could provide valuable information for donor selection and risk stratification in HSCT.


Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Imunidade Inata
2.
Prev Vet Med ; 184: 105157, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33002657

RESUMO

Canine brucellosis, caused by Brucella canis, is an infectious disease with implications for canine as well as human health. The identification of infected dogs originating from and around two South Dakota Indian reservations prompted an examination of the seroprevalence of B. canis in stray or owner-surrendered dogs from these communities. Using results from in-clinic screening tests of 3898 dogs over more than 4 years, we determined an overall apparent B. canis seroprevalence of 6.8% (adjusted estimated true prevalence of 29.4%), with rates declining over time. The apparent rate was similar to other surveys of stray dog populations in the US. Older dogs were significantly more likely to be B. canis-positive than younger dogs, as were reproductively intact dogs versus altered dogs (although this difference was not statistically significant). There were geographic differences in seropositive rates as well, with higher rates found in dogs originating from one reservation compared to other locations. Current diagnostic tests lack sensitivity to effectively identify all B. canis-infected dogs, but results from this study are valuable for investigating differences among risk factors for infection. Because of the potential for B. canis to infect other dogs and people, stray dog populations should be screened for B. canis before those animals are placed in adoptive homes.


Assuntos
Brucella canis/isolamento & purificação , Brucelose/veterinária , Doenças do Cão/epidemiologia , Animais , Brucelose/epidemiologia , Brucelose/microbiologia , Doenças do Cão/microbiologia , Cães , Feminino , Masculino , Prevalência , Estudos Soroepidemiológicos , South Dakota/epidemiologia
3.
PLoS Genet ; 16(9): e1009029, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32997655

RESUMO

Stress-induced changes to the dendritic architecture of neurons have been demonstrated in numerous mammalian and invertebrate systems. Remodeling of dendrites varies tremendously among neuron types. During the stress-induced dauer stage of Caenorhabditis elegans, the IL2 neurons arborize to cover the anterior body wall. In contrast, the FLP neurons arborize to cover an identical receptive field during reproductive development. Using time-course imaging, we show that branching between these two neuron types is highly coordinated. Furthermore, we find that the IL2 and FLP arbors have a similar dendritic architecture and use an identical downstream effector complex to control branching; however, regulation of this complex differs between stress-induced IL2 branching and FLP branching during reproductive development. We demonstrate that the unfolded protein response (UPR) sensor IRE-1, required for localization of the complex in FLP branching, is dispensable for IL2 branching at standard cultivation temperatures. Exposure of ire-1 mutants to elevated temperatures results in defective IL2 branching, thereby demonstrating a previously unknown genotype by environment interaction within the UPR. We find that the FOXO homolog, DAF-16, is required cell-autonomously to control arborization during stress-induced arborization. Likewise, several aspects of the dauer formation pathway are necessary for the neuron to remodel, including the phosphatase PTEN/DAF-18 and Cytochrome P450/DAF-9. Finally, we find that the TOR associated protein, RAPTOR/DAF-15 regulates mutually exclusive branching of the IL2 and FLP dendrites. DAF-15 promotes IL2 branching during dauer and inhibits precocious FLP growth. Together, our results shed light on molecular processes that regulate stress-mediated remodeling of dendrites across neuron classes.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Neurônios/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Fatores Quimiotáticos/genética , Fatores Quimiotáticos/metabolismo , Dendritos/fisiologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Larva/citologia , Larva/crescimento & desenvolvimento , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Neurônios/citologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Resposta a Proteínas não Dobradas
4.
J Phys Chem B ; 124(38): 8209-8220, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32838524

RESUMO

Osmosensing transporter ProP detects the increase in cytoplasmic cation concentration associated with osmotically induced cell dehydration and mediates osmolyte uptake into bacteria. ProP is a 12-transmembrane helix protein with an α-helical, cytoplasmic C-terminal domain (CTD) linked to transmembrane helix XII (TM XII). It has been proposed that the CTD helix associates with the anionic membrane surface to lock ProP in an inactive conformation and that the release of the CTD may activate ProP. To investigate this possible activation mechanism, we have built and simulated a structural model in which the CTD was anchored to the membrane by TM XII and the CTD helix was associated with the membrane surface. Molecular dynamics simulations showed specific intrapeptide salt bridges forming when the CTD associated with the membrane. Experiments supported the presence of the salt bridge Lys447-Asp455 and suggested a role for these residues in osmosensing. Simulations performed at different salt concentrations showed weakened CTD-lipid interactions at 0.25 M KCl and gradual stiffening of the membrane with increasing salinity. These results suggest that salt cations may affect CTD release and activate ProP by increasing the order of membrane phospholipids.


Assuntos
Proteínas de Escherichia coli , Simportadores , Sequência de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Lipídeos , Simportadores/metabolismo
6.
J Biol Chem ; 295(4): 981-993, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31826918

RESUMO

Ubiquinone 8 (coenzyme Q8 or Q8) mediates electron transfer within the aerobic respiratory chain, mitigates oxidative stress, and contributes to gene expression in Escherichia coli In addition, Q8 was proposed to confer bacterial osmotolerance by accumulating during growth at high osmotic pressure and altering membrane stability. The osmolyte trehalose and membrane lipid cardiolipin accumulate in E. coli cells cultivated at high osmotic pressure. Here, Q8 deficiency impaired E. coli growth at low osmotic pressure and rendered growth osmotically sensitive. The Q8 deficiency impeded cellular O2 uptake and also inhibited the activities of two proton symporters, the osmosensing transporter ProP and the lactose transporter LacY. Q8 supplementation decreased membrane fluidity in liposomes, but did not affect ProP activity in proteoliposomes, which is respiration-independent. Liposomes and proteoliposomes prepared with E. coli lipids were used for these experiments. Similar oxygen uptake rates were observed for bacteria cultivated at low and high osmotic pressures. In contrast, respiration was dramatically inhibited when bacteria grown at the same low osmotic pressure were shifted to high osmotic pressure. Thus, respiration was restored during prolonged growth of E. coli at high osmotic pressure. Of note, bacteria cultivated at low and high osmotic pressures had similar Q8 concentrations. The protection of respiration was neither diminished by cardiolipin deficiency nor conferred by trehalose overproduction during growth at low osmotic pressure, but rather might be achieved by Q8-independent respiratory chain remodeling. We conclude that osmotolerance is conferred through Q8-independent protection of respiration, not by altering physical properties of the membrane.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Pressão Osmótica , Ubiquinona/farmacologia , Aerobiose/efeitos dos fármacos , Anisotropia , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Fluorescência , Fluidez de Membrana/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Mutação/genética , Concentração Osmolar , Proteolipídeos/metabolismo , Trealose/metabolismo
7.
Biophys J ; 115(11): 2152-2166, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30448037

RESUMO

ProP is a member of the major facilitator superfamily, a proton-osmolyte symporter, and an osmosensing transporter. ProP proteins share extended cytoplasmic carboxyl terminal domains (CTDs) implicated in osmosensing. The CTDs of the best characterized, group A ProP orthologs, terminate in sequences that form intermolecular, antiparallel α-helical coiled coils (e.g., ProPEc, from Escherichia coli). Group B orthologs lack that feature (e.g., ProPXc, from Xanthomonas campestris). ProPXc was expressed and characterized in E. coli to further elucidate the role of the coiled coil in osmosensing. The activity of ProPXc was a sigmoid function of the osmolality in cells and proteoliposomes. ProPEc and ProPXc attained similar activities at the same expression level in E. coli. ProPEc transports proline and glycine betaine with comparable high affinities at low osmolality. In contrast, proline weakly inhibited high-affinity glycine-betaine uptake via ProPXc. The KM for proline uptake via ProPEc increases dramatically with the osmolality. The KM for glycine-betaine uptake via ProPXc did not. Thus, ProPXc is an osmosensing transporter, and the C-terminal coiled coil is not essential for osmosensing. The role of CTD-membrane interaction in osmosensing was examined further. As for ProPEc, the ProPXc CTD co-sedimented with liposomes comprising E. coli phospholipid. Molecular dynamics simulations illustrated association of the monomeric ProPEc CTD with the membrane surface. Comparison with the available NMR structure for the homodimeric coiled coil formed by the ProPEc-CTD suggested that membrane association and homodimeric coiled-coil formation by that peptide are mutually exclusive. The membrane fluidity in liposomes comprising E. coli phospholipid decreased with increasing osmolality in the range relevant for ProP activation. These data support the proposal that ProP activates as cellular dehydration increases cytoplasmic cation concentration, releasing the CTD from the membrane surface. For group A orthologs, this also favors α-helical coiled-coil formation that stabilizes the transporter in an active form.


Assuntos
Betaína/metabolismo , Técnicas Biossensoriais , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Prolina/metabolismo , Simportadores/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Dimerização , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Mutagênese Sítio-Dirigida , Mutação , Concentração Osmolar , Conformação Proteica , Domínios Proteicos , Homologia de Sequência , Especificidade por Substrato , Simportadores/química , Simportadores/genética
9.
Curr Genet ; 64(4): 795-798, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29427078

RESUMO

Cardiolipin (CL) is a key player in bacterial cell biology. CL accumulates at the poles of rod-shaped cells; the polar localization and function of diverse bacterial proteins are CL-dependent. Cardiolipin (CL) is an unusual phospholipid comprised of a glycerol headgroup coupled with two phosphatidate moieties. CL-rich membrane domains are often visualized with the fluorescent indicator 10-N-nonyl-acridine orange (NAO). Recent data show that NAO can also indicate phosphatidylglycerol localization under different experimental conditions, in the absence of CL. The formation of CL-rich membrane domains at bacterial cell poles was predicted to occur spontaneously, by lipid microphase separation arising from the conical CL shape. New data reveal that membrane-anchored cardiolipin synthase A is targeted to the cytoplasmic membrane surface at bacterial cell poles. Thus, localized CL synthesis, interaction of CL with ClsA, and membrane curvature could all contribute to retention of CL at cell poles. These observations provide new insight regarding the mechanism for assembly of CL-rich membrane domains in prokaryotes and eukaryotes.


Assuntos
Cardiolipinas/química , Cardiolipinas/genética , Proteínas de Membrana/genética , Bactérias/genética , Cardiolipinas/biossíntese , Biologia Celular/tendências , Membrana Celular/química , Membrana Celular/genética , Proteínas de Membrana/química
10.
Mol Microbiol ; 107(5): 623-638, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29280215

RESUMO

Osmosensing by transporter ProP is modulated by its cardiolipin (CL)-dependent concentration at the poles of Escherichia coli cells. Other contributors to this phenomenon were sought with the BACterial Two-Hybrid System (BACTH). The BACTH-tagged variants T18-ProP and T25-ProP retained ProP function and localization. Their interaction confirmed the ProP homo-dimerization previously established by protein crosslinking. YdhP, YjbJ and ClsA were prominent among the putative ProP interactors identified by the BACTH system. The functions of YdhP and YjbJ are unknown, although YjbJ is an abundant, osmotically induced, soluble protein. ClsA (CL Synthase A) had been shown to determine ProP localization by mediating CL synthesis. Unlike a deletion of clsA, deletion of ydhP or yjbJ had no effect on ProP localization or function. All three proteins were concentrated at the cell poles, but only ClsA localization was CL-dependent. ClsA was shown to be N-terminally processed and membrane-anchored, with dual, cytoplasmic, catalytic domains. Active site amino acid replacements (H224A plus H404A) inactivated ClsA and compromised ProP localization. YdhP and YjbJ may be ClsA effectors, and interactions of YdhP, YjbJ and ClsA with ProP may reflect their colocalization at the cell poles. Targeted CL synthesis may contribute to the polar localization of CL, ClsA and ProP.


Assuntos
Cardiolipinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Membrana/metabolismo , Osmorregulação , Simportadores/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Citoplasma/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Deleção de Genes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Concentração Osmolar , Conformação Proteica , Multimerização Proteica , Simportadores/química , Simportadores/genética , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética
11.
Mol Microbiol ; 103(3): 469-482, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27802568

RESUMO

Osmosensing transporter ProP protects bacteria from osmotically induced dehydration by mediating the uptake of zwitterionic osmolytes. ProP activity is a sigmoidal function of the osmolality. ProP orthologues share an extended, cytoplasmic C-terminal domain. Orthologues with and without a C-terminal, α-helical coiled-coil domain respond similarly to the osmolality. ProP concentrates at the poles and septa of Escherichia coli cells in a cardiolipin (CL)-dependent manner. The roles of phospholipids and the C-terminal domain in subcellular localization of ProP were explored. Liposome association of peptides representing the C-terminal domains of ProP orthologues and variants in vitro was compared with subcellular localization of the corresponding orthologues and variants in vivo. In the absence of coiled-coil formation, the C-terminal domain bound liposomes and ProP concentrated at the cell poles in a CL-independent manner. The presence of the coiled-coil replaced those phenomena with CL-dependent binding and localization. The effects of amino acid replacements on lipid association of the C-terminal peptide fully recapitulated their effects on the subcellular localization of ProP. These data suggest that polar localization of ProP results from association of its C-terminal domain with the anionic lipid-enriched membrane at the cell poles. The coiled-coil domain present on only some orthologues renders that phenomenon CL-dependent.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Simportadores/metabolismo , Simportadores/fisiologia , Sequência de Aminoácidos , Cardiolipinas/metabolismo , Dimerização , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Concentração Osmolar , Fosfolipídeos/metabolismo , Domínios Proteicos , Simportadores/genética
12.
J Neurosci Nurs ; 48(5): E2-E11, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27579967

RESUMO

OBJECTIVE: The aim of this study was to evaluate the economic and quality outcomes associated with a collaborative advanced practice nurse and hospitalist physician model of care on the inpatient stroke unit as compared with usual hospitalist physician-led care. BACKGROUND: High functioning collaborative teams are anticipated to be essential under value-based reimbursement. METHODS: Hospitalist nurse practitioners were assigned to the stroke unit in collaboration with hospitalist physicians to implement daily hospital management for patients with stroke and transient ischemic attack. To evaluate outcomes associated with the care model, a retrospective cross-sectional design was used with 100 patients in the collaborative advanced practice nurse and hospitalist physician care group and 100 patients in the usual hospitalist physician-led care group. Primary outcome measures were length of stay, 30-day readmissions, stroke core measure documentation, and patient experiences of care. Analysis of demographic characteristics assured that the samples were similar. RESULTS: The collaborative care group performed better on one of five stroke core quality measures and on two of three patient experiences of care measures. Mean length of stay and hospital readmissions were similar between groups. Five patients left the stroke unit against medical advice in the usual hospitalist physician-led care group, whereas there were no discharges against medical advice in the collaborative care group. CONCLUSION: Advanced practice nurse and hospitalist physician collaboration is a promising model for healthcare quality improvement during inpatient stroke care; results are likely generalizable to other adult medicine populations.


Assuntos
Comportamento Cooperativo , Avaliação de Resultados em Cuidados de Saúde , Relações Médico-Enfermeiro , Acidente Vascular Cerebral/terapia , Idoso , Estudos Transversais , Feminino , Médicos Hospitalares/organização & administração , Humanos , Tempo de Internação , Masculino , Modelos Organizacionais , Profissionais de Enfermagem/organização & administração , Avaliação de Resultados em Cuidados de Saúde/economia , Avaliação de Resultados em Cuidados de Saúde/organização & administração , Readmissão do Paciente , Qualidade da Assistência à Saúde/economia , Estudos Retrospectivos
13.
Biochemistry ; 55(9): 1301-13, 2016 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-26871755

RESUMO

Osmosensing transporters mediate osmolyte accumulation to forestall cellular dehydration as the extracellular osmolality increases. ProP is a bacterial osmolyte-H(+) symporter, a major facilitator superfamily member, and a paradigm for osmosensing. ProP activity is a sigmoid function of the osmolality. It is determined by the osmolality, not the magnitude or direction of the osmotic shift, in cells and salt-loaded proteoliposomes. The activation threshold varies directly with the proportion of anionic phospholipid in cells and proteoliposomes. The osmosensory mechanism was probed by varying the salt composition and concentration outside and inside proteoliposomes. Data analysis was based on the hypothesis that the fraction of maximal transporter activity at a particular luminal salt concentration reflects the proportion of ProP molecules in an active conformation. ProP attained the same activity at the same osmolality when diverse, membrane-impermeant salts were added to the external medium. Contributions of Coulombic and/or Hofmeister salt effects to ProP activation were examined by varying the luminal salt cation (K(+) and Na(+)) and anion (chloride, phosphate, and sulfate) composition and then systematically increasing the luminal salt concentration by increasing the external osmolality. ProP activity increased with the sixth power of the univalent cation concentration, independent of the type of anion. This indicates that salt activation of ProP is a Coulombic, cation effect resulting from salt cation accumulation and not site-specific cation binding. Possible origins of this Coulombic effect include folding or assembly of anionic cytoplasmic ProP domains, an increase in local membrane surface charge density, and/or the juxtaposition of anionic protein and membrane surfaces during activation.


Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Osmose/fisiologia , Simportadores/genética , Simportadores/metabolismo , Sequência de Aminoácidos , Proteínas de Escherichia coli/química , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Cloreto de Sódio/metabolismo , Simportadores/química
14.
Biochemistry ; 54(37): 5735-47, 2015 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-26325238

RESUMO

Transporter-mediated osmolyte accumulation stimulates the growth of Escherichia coli in high-osmolality environments. YehZYXW was predicted to be an osmoregulatory transporter because (1) osmotic and stationary phase induction of yehZYXW is mediated by RpoS, (2) the Yeh proteins are homologous to the components of known osmoregulatory ABC transporters (e.g., ProU of E. coli), and (3) YehZ models based on the structures of periplasmic betaine-binding proteins suggested that YehZ retains key betaine-binding residues. The betaines choline-O-sulfate, glycine betaine, and dimethylsulfoniopropionate bound YehZ and ProX with millimolar and micromolar affinities, respectively, as determined by equilibrium dialysis and isothermal titration calorimetry. The crystal structure of the YehZ apoprotein, determined at 1.5 Å resolution (PDB ID: 4WEP ), confirmed its similarity to other betaine-binding proteins. Small and nonpolar residues in the hinge region of YehZ (e.g., Gly223) pack more closely than the corresponding residues in ProX, stabilizing the apoprotein. Betaines bound YehZ-Gly223Ser an order of magnitude more tightly than YehZ, suggesting that weak substrate binding in YehZ is at least partially due to apo state stabilization. Neither ProX nor YehZ bound proline. Assays based on osmoprotection or proline auxotrophy failed to detect YehZYXW-mediated uptake of proline, betaines, or other osmolytes. However, transport assays revealed low-affinity glycine betaine uptake, mediated by YehZYXW, that was inhibited at high salinity. Thus, YehZYXW is a betaine transporter that shares substrate specificity, but not an osmoregulatory function, with homologues like E. coli ProU. Other work suggests that yehZYXW may be an antivirulence locus whose expression promotes persistent, asymptomatic bacterial infection.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Betaína/metabolismo , Proteínas de Escherichia coli/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Cristalografia por Raios X , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/química , Osmorregulação , Proteínas Periplásmicas de Ligação/química , Ligação Proteica , Conformação Proteica
16.
Appl Environ Microbiol ; 80(17): 5366-78, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24951793

RESUMO

Osmolyte accumulation and release can protect cells from abiotic stresses. In Escherichia coli, known mechanisms mediate osmotic stress-induced accumulation of K(+) glutamate, trehalose, or zwitterions like glycine betaine. Previous observations suggested that additional osmolyte accumulation mechanisms (OAMs) exist and their impacts may be abiotic stress specific. Derivatives of the uropathogenic strain CFT073 and the laboratory strain MG1655 lacking known OAMs were created. CFT073 grew without osmoprotectants in minimal medium with up to 0.9 M NaCl. CFT073 and its OAM-deficient derivative grew equally well in high- and low-osmolality urine pools. Urine-grown bacteria did not accumulate large amounts of known or novel osmolytes. Thus, CFT073 showed unusual osmotolerance and did not require osmolyte accumulation to grow in urine. Yeast extract and brain heart infusion stimulated growth of the OAM-deficient MG1655 derivative at high salinity. Neither known nor putative osmoprotectants did so. Glutamate and glutamine accumulated after growth with either organic mixture, and no novel osmolytes were detected. MG1655 derivatives retaining individual OAMs were created. Their abilities to mediate osmoprotection were compared at 15°C, 37°C without or with urea, and 42°C. Stress protection was not OAM specific, and variations in osmoprotectant effectiveness were similar under all conditions. Glycine betaine and dimethylsulfoniopropionate (DMSP) were the most effective. Trimethylamine-N-oxide (TMAO) was a weak osmoprotectant and a particularly effective urea protectant. The effectiveness of glycine betaine, TMAO, and proline as osmoprotectants correlated with their preferential exclusion from protein surfaces, not with their propensity to prevent protein denaturation. Thus, their effectiveness as stress protectants correlated with their ability to rehydrate the cytoplasm.


Assuntos
Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Pressão Osmótica , Estresse Fisiológico , Betaína/metabolismo , Meios de Cultura/química , Escherichia coli/crescimento & desenvolvimento , Metilaminas/metabolismo , Prolina/metabolismo , Cloreto de Sódio/metabolismo , Compostos de Sulfônio/metabolismo , Temperatura , Ureia/metabolismo
17.
J Bacteriol ; 196(6): 1286-96, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24443528

RESUMO

ProQ is a cytoplasmic protein with RNA chaperone activities that reside in FinO- and Hfq-like domains. Lesions at proQ decrease the level of the osmoregulatory glycine betaine transporter ProP. Lesions at proQ eliminated ProQ and Prc, the periplasmic protease encoded by the downstream gene prc. They dramatically slowed the growth of Escherichia coli populations and altered the morphologies of E. coli cells in high-salinity medium. ProQ and Prc deficiencies were associated with different phenotypes. ProQ-deficient bacteria were elongated unless glycine betaine was provided. High-salinity cultures of Prc-deficient bacteria included spherical cells with an enlarged periplasm and an eccentric nucleoid. The nucleoid-containing compartment was bounded by the cytoplasmic membrane and peptidoglycan. This phenotype was not evident in bacteria cultivated at low or moderate salinity, nor was it associated with murein lipoprotein (Lpp) deficiency, and it differed from those elicited by the MreB inhibitor A-22 or the FtsI inhibitor aztreonam at low or high salinity. It was suppressed by deletion of spr, which encodes one of three murein hydrolases that are redundantly essential for enlargement of the murein sacculus. Prc deficiency may alter bacterial morphology by impairing control of Spr activity at high salinity. ProQ and Prc deficiencies lowered the ProP activity of bacteria cultivated at moderate salinity by approximately 70% and 30%, respectively, but did not affect other osmoregulatory functions. The effects of ProQ and Prc deficiencies on ProP activity are indirect, reflecting their roles in the maintenance of cell structure.


Assuntos
Cisteína Endopeptidases/deficiência , Endopeptidases/deficiência , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Proteínas de Membrana Transportadoras/deficiência , Salinidade , Cloreto de Sódio/metabolismo , Betaína/metabolismo , Membrana Celular/efeitos dos fármacos , Cromossomos Bacterianos/efeitos dos fármacos , Meios de Cultura/química , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli , Periplasma/efeitos dos fármacos , Proteínas de Ligação a RNA
18.
J Biol Chem ; 287(33): 27813-22, 2012 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-22740696

RESUMO

H(+) symporter ProP serves as a paradigm for the study of osmosensing. ProP attains the same activity at the same osmolality when the medium outside cells or proteoliposomes is supplemented with diverse, membrane-impermeant solutes. The osmosensory mechanism of ProP has been probed by varying the solvent within membrane vesicles and proteoliposomes. ProP activation was not ion specific, did not require K(+), and could be elicited by large, uncharged solutes polyethylene glycols (PEGS). We hypothesized that ProP is an ionic strength sensor and lumenal macromolecules activate ProP by altering ion activities. The attainable range of lumenal ionic strength was expanded by lowering the phosphate concentration within proteoliposomes. ProP activity at high osmolality, but not the osmolality, yielding half-maximal activity (Π(1/2)/RT), decreased with the lumenal phosphate concentration. This was attributed to acidification of the proteoliposome lumen due to H(+)-proline symport. The ionic strength yielding half-maximal ProP activity was more anion-dependent than Π(1/2)/RT for proteoliposomes loaded with citrate, sulfate, phosphate, chloride, or iodide. The anion effects followed the Hofmeister series. Lumenal bovine serum albumin (BSA) lowered the lumenal ionic strength at which ProP became active. Osmolality measurements documented the non-idealities of solutions including potassium phosphate and other solutes. The impacts of PEGS and BSA on ion activities did not account for their impacts on ProP activity. The effects of the tested solutes on ProP appear to be non-coulombic in nature. They may arise from effects of preferential interactions and macromolecular crowding on the membrane or on ProP.


Assuntos
Proteínas de Escherichia coli/química , Lipossomos/química , Polietilenoglicóis/química , Simportadores/química , Animais , Bovinos , Transporte de Íons/fisiologia , Concentração Osmolar , Soroalbumina Bovina/química
19.
Annu Rev Microbiol ; 65: 215-38, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21663439

RESUMO

To thrive, cells must control their own physical and chemical properties. This process is known as cellular homeostasis. The dilute solutions traditionally favored by experimenters do not simulate the cytoplasm, where macromolecular crowding and preferential interactions among constituents may dominate critical processes. Solutions that do simulate cytoplasmic conditions are now being characterized. Corresponding cytoplasmic properties can be varied systematically by imposing osmotic stress. This osmotic stress approach is revealing how cytoplasmic properties modulate protein folding and protein?nucleic acid interactions. Results suggest that cytoplasmic homeostasis may require adjustments to multiple, interwoven cytoplasmic properties. Osmosensory transporters with diverse structures and bioenergetic mechanisms activate in response to osmotic stress as other proteins inactivate. These transporters are serving as paradigms for the study of in vivo protein-solvent interactions. Experimenters have proposed three different osmosensory mechanisms. Distinct mechanisms may exist, or these proposals may reflect different perceptions of a single, unifying mechanism.


Assuntos
Fenômenos Fisiológicos Bacterianos , Equilíbrio Hidroeletrolítico , Bactérias/genética , Bactérias/metabolismo , Transporte Biológico , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Osmose , Estresse Fisiológico
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