RESUMO
BACKGROUND: The number of migrant older adults with children (MOAC) in China has been increasing in recent years, and most of them are women. This study aimed to explore the mediating effect of social support between social integration and loneliness among the female MOAC in Jinan, China. METHODS: In this study, 418 female MOAC were selected using multi-stage cluster random sampling in Jinan, Shandong Province, China. Loneliness was measured by the eight-item version of the University of California Los Angeles Loneliness Scale (ULS-8), and social support was measured by The Social Support Rating Scale (SSRS). Descriptive analyses, t-tests, ANOVA, and structural equation modeling (SEM) were used to illustrate the relationship between social integration, social support, and loneliness. RESULTS: The average scores of ULS-8 and SSRS were 12.9 ± 4.0 and 39.4 ± 5.9 among female MOAC in this study. Social integration and social support were found to be negatively related to loneliness, and the standardized direct effect was -0.20 [95% CI: -0.343 to -0.068] and -0.39 [95% CI: -0.230 to -0.033], respectively. Social support mediated the relationship between social integration and loneliness, and the indirect effect was -0.16 [95% CI: -0.252 to -0.100]. CONCLUSION: The female MOAC's loneliness was at a relatively lower level in this study. It was found that social integration was negatively associated with loneliness, and social support mediated the relationship between them. Helping female MOAC integrate into the inflow city and improving their social support could be beneficial for alleviating their loneliness.
Assuntos
Migrantes , Humanos , Feminino , Idoso , Masculino , Solidão , Apoio Social , Projetos de Pesquisa , Integração Social , China/epidemiologiaRESUMO
Localization based on single-line lidar is widely used in various robotics applications, such as warehousing, service, transit, and construction, due to its high accuracy, cost-effectiveness, and minimal computational requirements. However, challenges such as LiDAR degeneration and frequent map changes persist in hindering its broader adoption. To address these challenges, we introduce the Contribution Sampling and Map-Updating Localization (CSMUL) algorithm, which incorporates weighted contribution sampling and dynamic map-updating methods for robustness enhancement. The weighted contribution sampling method assigns weights to each map point based on the constraints within degenerate environments, significantly improving localization robustness under such conditions. Concurrently, the algorithm detects and updates anomalies in the map in real time, addressing issues related to localization drift and failure when the map changes. The experimental results from real-world deployments demonstrate that our CSMUL algorithm achieves enhanced robustness and superior accuracy in both degenerate scenarios and dynamic map conditions. Additionally, it facilitates real-time map adjustments and ensures continuous positioning, catering to the needs of dynamic environments.
RESUMO
LacY catalyzes accumulation of galactosides against a concentration gradient by coupling galactoside and H+ transport (i.e., symport). While alternating access of sugar- and H+-binding sites to either side of the membrane is driven by binding and dissociation of sugar, the electrochemical H+ gradient ([Formula: see text]) functions kinetically by decreasing the Km for influx 50- to 100-fold with no change in Kd The affinity of protonated LacY for sugar has an apparent pK (pKapp) of â¼10.5, due specifically to the pKa of Glu325, a residue that plays an irreplaceable role in coupling. In this study, rates of lactose/H+ efflux were measured from pH 5.0 to 9.0 in the absence or presence of a membrane potential (ΔΨ, interior positive), and the effect of the imposed ΔΨ on the kinetics of efflux was also studied in right-side-out membrane vesicles. The findings reveal that [Formula: see text] induces an asymmetry in the transport cycle based on the following observations: 1) the efflux rate of WT LacY exhibits a pKapp of â¼7.2 that is unaffected by the imposed ΔΨ; 2) ΔΨ increases the rate of efflux at all tested pH values, but enhancement is almost 2 orders of magnitude less than observed for influx; 3) mutant Glu325 - Ala does little or no efflux in the absence or presence of ΔΨ, and ambient pH has no effect; and 4) the effect of ΔΨ (interior positive) on the Km for efflux is almost insignificant relative to the 50- to 100-fold decrease in the Km for influx driven by ΔΨ (interior negative).
Assuntos
Galactosídeos/metabolismo , Potenciais da Membrana/fisiologia , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Prótons , Sítios de Ligação , Transporte Biológico , Concentração de Íons de Hidrogênio , Transporte de Íons , Cinética , Lactose/metabolismo , Proteínas de Membrana Transportadoras/genética , Modelos MolecularesRESUMO
BACKGROUND: With the accelerated urbanization and aging population in China, more and more migrant older with children (MOC) moved to new cities. Previous studies mainly explored the acculturation of MOC, yet few focused on the health conditions of this vulnerable group. This study aimed to investigate the effects of oral health and social support on health-related quality of life (HRQOL) of MOC in Weifang, China. METHOD: This study was a cross-sectional study and participants were selected by multi-stage cluster random sampling in Weifang, China. The HRQOL was assessed via the 12-item Short-Form Health Survey (SF-12) which included the mental component summary (MCS) and the physical component summary (PCS). The oral health was evaluated by the Geriatric Oral Health Assessment Index (GOHAI). The social support was administered using the Social Support Rating Scale (SSRS). Descriptive analysis was used to describe participants' sociodemographic variables, oral health and social support. Univariate analysis and binary logistic regression analysis was used to investigate the association between the social support, oral health and HRQOL. RESULTS AND DISCUSSION: It was found that 25.0% of MOC were defined as MCS poor and PCS poor, respectively. Those participants with average and low monthly household income compared to those around them, average and poor oral health, and low levels of social support were more likely to have poor PCS. Those with temporary residence permits, fair and poor oral health, and medium and low levels of social support were more likely to report poor MCS. CONCLUSION: Results indicated that better social support and oral health led to higher HRQOL of MOC. Implications for the government, communities and families of MOC were given to improve their HRQOL.
Assuntos
Qualidade de Vida , Migrantes , Idoso , Criança , China , Estudos Transversais , Humanos , Saúde Bucal , Apoio Social , Inquéritos e QuestionáriosRESUMO
BACKGROUND: Driven by population aging and the rapid urbanization in China, many migrant elderly following children (MEFC) moved to big cities to care for their grandchildren. The purpose of this study is to clarify the mediating effect of social support on the relationship between socioeconomic status (SES) and self-reported oral health status among the MEFC in Weifang, China. METHODS: Multistage cluster random sampling was used to select the participants and finally 613 MEFC were included in the survey. The Social Support Rating Scale (SSRS) and the Chinese version of the Geriatric Oral Health Assessment Index (GOHAI) scale were used for data collection. Descriptive analysis, Rao-Scott test, t-test and structural equation modeling (SEM) were conducted in this study. RESULTS: Mean score of GOHAI of the MEFC was 54.95 ± 6.47. The SES of MEFC exerted positive direct effect both on social support (standardized coefficient = 0.15) and self-reported oral health status (standardized coefficient = 0.22); social support exerted positive direct effect on self-reported oral health status (standardized coefficient = 0.17). Social support partially mediated the association between SES and self-reported oral health status [95% confidence interval (CI) 0.003-0.064, P < 0.05], and the mediating effect of social support accounted for 12.0% of the total effect. CONCLUSIONS: Higher GOHAI score of MEFC indicated their better self-reported oral health status. MEFCs' SES could exert positive effect both on social support and self-reported oral health status, while the mediating effect of social support between SES and self-reported oral health status of MEFC was established.
Assuntos
Saúde Bucal , Migrantes , Criança , Humanos , Idoso , Estudos Transversais , Autorrelato , Classe Social , China/epidemiologia , Apoio SocialRESUMO
Binding kinetics of α-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) of Escherichia coli by FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation (kon = 4-20 µM-1·s-1), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants (koff) increase with the size of the aglycone so that Kd values also increase but remain in the micromolar range for each homolog. Phe27 (helix I) forms an apparent constriction in the pathway for sugar by protruding into the periplasmic cavity. However, replacement of Phe27 with a bulkier Trp does not create an obstacle in the pathway even for large ligands, since binding kinetics remain unchanged. High accessibility of the binding site is also observed in a LacY/nanobody complex with partially blocked periplasmic opening. Remarkably, E. coli expressing WT LacY catalyzes transport of α- or ß-galactopyranosides with oversized aglycones such as bodipy or Aldol518, which may require an extra space within the occluded intermediate. The results confirm that LacY specificity is strictly directed toward the galactopyranoside ring and also clearly indicate that the opening on the periplasmic side is sufficiently wide to accommodate the large galactoside derivatives tested here. We conclude that the actual pathway for the substrate entering from the periplasmic side is wider than the pore diameter calculated in the periplasmic-open X-ray structures.
Assuntos
Proteínas de Escherichia coli/metabolismo , Galactosídeos/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Simportadores/metabolismo , Sítios de Ligação , Transporte Biológico Ativo , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Corantes Fluorescentes , Galactose/química , Galactose/metabolismo , Galactosídeos/química , Cinética , Ligantes , Modelos Moleculares , Estrutura Molecular , Proteínas de Transporte de Monossacarídeos/química , Periplasma/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Simportadores/químicaRESUMO
The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane transport protein, catalyzes galactoside/H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar-binding. Camelid nanobodies were made against a double-mutant Gly46 â Trp/Gly262 â Trp (LacYWW) that produces an outward-open conformation, as opposed to the cytoplasmic open-state crystal structure of WT LacY. Nanobody 9047 (Nb9047) stabilizes WT LacY in a periplasmic-open conformation. Here, we describe the X-ray crystal structure of a complex between LacYWW, the high-affinity substrate analog 4-nitrophenyl-α-d-galactoside (NPG), and Nb9047 at 3-Å resolution. The present crystal structure demonstrates that Nb9047 binds to the periplasmic face of LacY, primarily to the C-terminal six-helical bundle, while a flexible loop of the Nb forms a bridge between the N- and C-terminal halves of LacY across the periplasmic vestibule. The bound Nb partially covers the vestibule, yet does not affect the on-rates or off-rates for the substrate binding to LacYWW, which implicates dynamic flexibility of the Nb-LacYWW complex. Nb9047-binding neither changes the overall structure of LacYWW with bound NPG, nor the positions of side chains comprising the galactoside-binding site. The current NPG-bound structure exhibits a more occluded periplasmic vestibule than seen in a previous structure of a (different Nb) apo-LacYWW/Nb9039 complex that we argue is caused by sugar-binding, with major differences located at the periplasmic ends of transmembrane helices in the N-terminal half of LacY.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Transporte de Monossacarídeos/química , Anticorpos de Domínio Único/química , Simportadores/química , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Estrutura Quaternária de Proteína , Simportadores/genéticaRESUMO
The amounts and characteristics of polychlorinated naphthalenes (PCNs) emitted by a secondary copper smelter were investigated. Differences in the amounts and characteristics of PCNs emitted during different smelting stages were investigated, and the main stage during which PCNs were emitted was identified. PCN concentrations in stack gases emitted during secondary copper smelting were 477.0-762.5â¯ng/m3 (4.4-8.3â¯pg toxic equivalents/m3). The contributions of the different stages to total PCN emissions decreased in the order feeding-fusion stage (65% of total PCN emissions)â¯>â¯oxidation stage (27%)â¯>â¯deoxidation stage (8%). The main contributor to PCN emissions during secondary copper smelting was the feeding-fusion stage. PCN concentrations and profiles in stack gas, fly ash, and deposit ash collected during different smelting stages were determined. PCNs in stack gases were mainly less-chlorinated homologs, and fly ash and deposit ash were dominated by highly-chlorinated homologs. These results will help improve strategies for decreasing and eliminating PCN emissions during secondary copper production.
Assuntos
Poluentes Atmosféricos/análise , Cobre , Monitoramento Ambiental/métodos , Hidrocarbonetos Clorados/análise , Metalurgia , Naftalenos/análise , China , Cinza de Carvão/análise , Gases/análise , OxirreduçãoRESUMO
The lactose permease of Escherichia coli (LacY), a highly dynamic membrane protein, catalyzes symport of a galactopyranoside and an H(+) by using an alternating access mechanism, and the transport cycle involves multiple conformational states. Single-domain camelid nanobodies (Nbs) developed against a LacY mutant immobilized in an outward (periplasmic)-open conformation bind to the flexible WT protein and stabilize the open-outward conformation(s). Here, we use site-directed, distance-dependent Trp quenching/unquenching of fluorescent probes inserted on opposite surfaces of LacY to assess the conformational states of the protein complexed with each of eight unique Nbs that bind exclusively to the periplasmic side and block transport, but increase the accessibility of the sugar-binding site. Nb binding involves conformational selection of LacY molecules with exposed binding epitopes. Each of eight Nbs induces quenching with three pairs of cytoplasmic Trp/fluorophore probes, indicating closing of cytoplasmic cavity. In reciprocal fashion, the same Nbs induce unquenching of fluorescence in three pairs of periplasmic probes due to opening of the periplasmic cavity. Because the extent of fluorescence change with various Nbs differs and the differences correlate with changes in the rate of sugar binding, it is also concluded that the Nbs stabilize several different outward-open conformations of LacY.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Transporte de Monossacarídeos/química , Anticorpos de Domínio Único/química , Simportadores/química , Conformação ProteicaRESUMO
Galactoside/H+ symport by the lactose permease of Escherichia coli (LacY) involves reciprocal opening and closing of periplasmic and cytoplasmic cavities so that sugar- and H+-binding sites become alternatively accessible to either side of the membrane. After reconstitution into proteoliposomes, LacY with the periplasmic cavity sealed by cross-linking paired-Cys residues does not bind sugar from the periplasmic side. However, reduction of the S-S bond restores opening of the periplasmic cavity and galactoside binding. Furthermore, nanobodies that stabilize the double-Cys mutant in a periplasmic-open conformation and allow free access of galactoside to the binding site do so only after reduction of the S-S bond. In contrast, when cross-linked LacY is solubilized in detergent, galactoside binding is observed, indicating that the cytoplasmic cavity is patent. Sugar binding from the cytoplasmic side exhibits nonlinear stopped-flow kinetics, and analysis reveals a two-step process in which a conformational change precedes binding. Because the cytoplasmic cavity is spontaneously closing and opening in the symporter with a sealed periplasmic cavity, it is apparent that an asymmetrical conformational transition controls access of sugar to the binding site.
Assuntos
Cisteína/química , Dissulfetos/química , Proteínas de Escherichia coli/química , Galactose/química , Proteínas de Transporte de Monossacarídeos/química , Proteolipídeos/química , Prótons , Simportadores/química , Sítios de Ligação , Transporte Biológico , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Galactose/metabolismo , Expressão Gênica , Cinética , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/metabolismo , Oxirredução , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Proteolipídeos/metabolismo , Simportadores/metabolismo , TermodinâmicaRESUMO
Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the major pathogens that pose a big challenge to the sericulture industry. Growing evidences have shown that microRNAs play key roles in the regulations of host-pathogen interactions in insects. MicroRNAs have been found in silkworms, whether and how they affect the silkworm-BmCPV interactions are still unknown. Here we investigate the effect of miR-274-3p on the BmCPV replication in the BmCPV-infected silkworm larvae. In our study, BmCPV Nonstructural protein 5 (NS5) was identified to be the target of miR-274-3p based on bioinformatics analysis and luciferase reporter assay. The abundance of NS5 was significantly increased in the presence of miR-274-3p inhibitor based on the qRT-PCR and Western blotting results. Further, qRT-PCR results revealed that the expression of polyhedrin gene of BmCPV in the larvae after applying miR-274-3p inhibitor was significantly increased comparing with that of larvae with negative control. Our results suggest that inhibition of miR-274-3p could facilitate BmCPV replication by up-regulating BmCPV NS5 gene expression and are insightful for further exploring the interactions between silkworm and BmCPV.
Assuntos
Bombyx/metabolismo , Bombyx/virologia , Interações Hospedeiro-Patógeno , MicroRNAs/genética , Reoviridae/fisiologia , Proteínas não Estruturais Virais/genética , Replicação Viral , Animais , Bombyx/genética , Regulação Viral da Expressão Gênica , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , MicroRNAs/metabolismo , Reoviridae/genética , Reoviridae/crescimento & desenvolvimento , Proteínas não Estruturais Virais/metabolismoRESUMO
The generation of and extent to which chlorinated and brominated polycyclic aromatic hydrocarbons (Cl/Br-PAHs) are formed and released from secondary copper smelters remain unknown. This field study, to our knowledge, is the first to identify secondary copper smelters as new sources of Cl/Br-PAHs. Mass concentrations of ∑19Cl-PAHs and ∑19Br-PAHs ranged from 5.8 to 271 ng Nm-3 and 0.59 to 52.4 ng Nm-3, respectively. A comparison of Cl/Br-PAH concentrations in stack gas and fly ash from secondary copper smelters indicated that the use of scrap copper as raw material or the addition of coal or heavy oil as reductant may contribute to the elevated formation and emission of Cl/Br-PAHs. Congener profiles of Cl/Br-PAHs in stack gas and fly ash from secondary copper smelters were different with those of Cl/Br-PAHs from waste incinerations and other previously reported sources, thus could be used as possible fingerprints and source apportionments of environmental Cl/Br-PAHs. Atmospheric levels of Cl/Br-PAHs in the workplace or smelter surroundings were determined and potential exposures were assessed. Although chlorination of PAHs was previously recognized as an important formation pathway of Cl/Br-PAHs, it was not verified to be the major formation pathway for Cl/Br-PAHs from secondary copper smelters in this study.
Assuntos
Cobre , Incineração , Hidrocarbonetos Policíclicos Aromáticos , Cinza de Carvão , Monitoramento Ambiental , HalogenaçãoRESUMO
The lactose permease of Escherichia coli (LacY), a highly dynamic polytopic membrane protein, catalyzes stoichiometric galactoside/H(+) symport by an alternating access mechanism and exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a LacY mutant in an outward (periplasmic)-open conformation to stabilize this state of the WT protein. Twelve purified Nbs inhibit lactose transport in right-side-out membrane vesicles, indicating that the Nbs recognize epitopes on the periplasmic side of LacY. Stopped-flow kinetics of sugar binding by WT LacY in detergent micelles or reconstituted into proteoliposomes reveals dramatic increases in galactoside-binding rates induced by interaction with the Nbs. Thus, WT LacY in complex with the great majority of the Nbs exhibits varied increases in access of sugar to the binding site with an increase in association rate constants (kon) of up to â¼ 50-fold (reaching 10(7) M(-1) â s(-1)). In contrast, with the double-Trp mutant, which is already open on the periplasmic side, the Nbs have little effect. The findings are clearly consistent with stabilization of WT conformers with an open periplasmic cavity. Remarkably, some Nbs drastically decrease the rate of dissociation of bound sugar leading to increased affinity (greater than 200-fold for lactose).
Assuntos
Transporte Biológico Ativo/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Complexos Multiproteicos/metabolismo , Periplasma/metabolismo , Simportadores/metabolismo , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Lactose/química , Lactose/genética , Lactose/metabolismo , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutação , Periplasma/química , Periplasma/genética , Ligação Proteica , Estabilidade Proteica , Simportadores/química , Simportadores/genéticaRESUMO
Camelid nanobodies (Nbs) raised against the outward-facing conformer of a double-Trp mutant of the lactose permease of Escherichia coli (LacY) stabilize the permease in outward-facing conformations. Isothermal titration calorimetry is applied herein to dissect the binding thermodynamics of two Nbs, one that markedly improves access to the sugar-binding site and another that dramatically increases the affinity for galactoside. The findings presented here show that both enthalpy and entropy contribute favorably to binding of the Nbs to wild-type (WT) LacY and that binding of Nb to double-Trp mutant G46W/G262W is driven by a greater enthalpy at an entropic penalty. Thermodynamic analyses support the interpretation that WT LacY is stabilized in outward-facing conformations like the double-Trp mutant with closure of the water-filled cytoplasmic cavity through conformational selection. The LacY conformational transition required for ligand binding is reflected by a favorable entropy increase. Molecular dynamics simulations further suggest that the entropy increase likely stems from release of immobilized water molecules primarily from the cytoplasmic cavity upon closure.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Anticorpos de Domínio Único , Calorimetria , Escherichia coli/enzimologia , Simulação de Dinâmica Molecular , Especificidade por Substrato , TermodinâmicaRESUMO
On the periplasmic side of LacY, two conserved Gly-Gly pairs in helices II and XI (Gly46 and Gly370, respectively) and helices V and VIII (Gly159 and Gly262, respectively) allow close packing of each helix pair in the outward (periplasmic)-closed conformation. Previous studies demonstrate that replacing one Gly residue in each Gly-Gly pair with Trp leads to opening of the periplasmic cavity with abrogation of transport activity, but an increased rate of galactoside binding. To further investigate the role of the Gly-Gly pairs, 11 double-replacement mutants were constructed for each pair at positions 46 (helix II) and 262 (helix VIII). Replacement with Ala or Ser results in decreased but significant transport activity, while replacements with Thr, Val, Leu, Asn, Gln, Tyr, Trp, Glu, or Lys exhibit very little or no transport. Remarkably, however, the double mutants bind galactoside with affinities 10-20-fold higher than that of the pseudo-WT or WT LacY. Moreover, site-directed alkylation of a periplasmic Cys replacement indicates that the periplasmic cavity becomes readily accessible in the double-replacement mutants. Molecular dynamics simulations with the WT and double-Leu mutant in the inward-open/outward-closed conformation provide support for this interpretation.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/genética , Simportadores/química , Simportadores/genética , Alquilação , Sequência de Aminoácidos , Substituição de Aminoácidos , Transporte Biológico Ativo , Sequência Conservada , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicilglicina/química , Glicilglicina/genética , Lactose/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutagênese Sítio-Dirigida , Nitrofenilgalactosídeos/metabolismo , Periplasma/metabolismo , Conformação Proteica , Conformação Proteica em alfa-Hélice , Simportadores/metabolismoRESUMO
Heterogeneous reactions mediated by fly ash are important to polychlorinated dibenzo-p-dioxin and dibenzofuran (PCDD/Fs) formation. However, the formation of polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) through heterogeneous reactions is not yet well understood. Experiments were performed to investigate the thermochemical formation of PBDD/Fs at 150-450 °C through heterogeneous reactions on fly ash from a secondary copper smelter. The maximum PBDD/F concentration was 325 times higher than the initial PBDD/F concentration in the fly ash. The PBDD/F concentration after the experiment at 150 °C was five times higher than the initial concentration. PBDD/Fs have not previously been found to form at such a low temperature. Secondary-copper-smelter fly ash clearly promoted PBDD/F formation, and this conclusion was supported by the low activation energies that were found in Arrhenius's law calculations. Thermochemical formation of PBDD/Fs mediated by fly ash deposited in industrial facilities could explain "memory effects" that have been found for PCDD/Fs and similar compounds released from industrial facilities. Abundant polybrominated diphenyl ethers (PBDEs) that were formed through fly ash-mediated reactions could be important precursors for PBDD/Fs also formed through fly ash-mediated reactions. The amounts of PBDEs that formed through fly ash-mediated reactions suggested that secondary copper smelters could be important sources of reformed PBDEs.
Assuntos
Cinza de Carvão , Cobre , Dibenzofuranos , Monitoramento Ambiental , Éteres Difenil HalogenadosRESUMO
Few side chains in the galactoside/H(+) symporter LacY (lactose permease of Escherichia coli) are irreplaceable for an alternating access mechanism in which sugar binding induces closing of the cytoplasmic cavity and reciprocal opening of a periplasmic cavity. In this study, each irreplaceable residue was mutated individually, and galactoside-induced opening or closing of periplasmic or cytoplasmic cavities was probed by site-directed alkylation. Mutation of Glu126 (helix IV) or Arg144 (helix V), which are essential for sugar binding, completely blocks sugar-induced periplasmic opening as expected. Remarkably, however, replacement of Glu269 (helix VIII), His322 (helix X), or Tyr236 (helix VII) causes spontaneous opening of the periplasmic cavity in the absence of sugar and decreased closing of the cytoplasmic cavity in the presence of galactoside. In contrast, mutation of Arg302 (helix IX) or Glu325 (helix X) has no such effect, and sugar binding induces normal opening and closing of periplasmic and cytoplasmic cavities. Possibly, Glu269, His322, and Tyr236 act in concert to coordinate opening and closing of the cavities by binding water, which also acts as a cofactor in H(+) translocation. Mutation of the triad results in loss of the bound water, which destabilizes LacY, and the cavities open and close in an uncoordinated manner. Thus, the triad mutants exhibit poor affinity for sugar, and galactoside/H(+) symport is abolished as well.
Assuntos
Citoplasma/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lactose/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutação de Sentido Incorreto , Periplasma/metabolismo , Simportadores/metabolismo , Substituição de Aminoácidos , Transporte Biológico/fisiologia , Citoplasma/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Lactose/genética , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/genética , Periplasma/genética , Estrutura Secundária de Proteína , Simportadores/química , Simportadores/genéticaRESUMO
LacY mutant Cys154 â Gly exhibits a periplasmic-closed crystal structure identical to the WT, but is periplasmic-open in the membrane. The mutant hardly catalyzes transport, but binds galactosides from either side of the membrane with the same affinity and is resistant to site-directed proteolysis relative to the pseudo-WT. Site-directed alkylation was also applied to 11 single-Cys mutants in Cys154 â Gly LacY in right-side-out membrane vesicles or after solubilization and purification in dodecyl-ß-D-maltopyranoside (DDM). Unlike the pseudo-WT, Cys replacements on the periplasmic side of the Cys154 â Gly mutant label rapidly in the membrane without sugar, but labeling decreases markedly after the mutant proteins are purified. Thus, Cys154 â Gly LacY likely favors a higher-energy intermediate periplasmic-open conformation in situ, but collapses to a lower-energy periplasmic-closed conformation in DDM after purification. Notably, branched-chain or neopentyl glycol maltoside detergents stabilize Cys154 â Gly LacY in the membrane-embedded form.
Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Simportadores/metabolismo , Carboidratos/química , Membrana Celular/metabolismo , Cristalografia por Raios X/métodos , Cisteína/química , Citoplasma/metabolismo , Detergentes/farmacologia , Relação Dose-Resposta a Droga , Escherichia coli/genética , Ligantes , Maltose/análogos & derivados , Maltose/química , Proteínas de Membrana Transportadoras/química , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
Although an X-ray crystal structure of lactose permease (LacY) has been presented with bound galactopyranoside, neither the sugar nor the residues ligating the sugar can be identified with precision at ~3.5 Å. Therefore, additional evidence is important for identifying side chains likely to be involved in binding. On the basis of a clue from site-directed alkylation suggesting that Asn272, Gly268, and Val264 on one face of helix VIII might participate in galactoside binding, molecular dynamics simulations were conducted initially. The simulations indicate that Asn272 (helix VIII) is sufficiently close to the galactopyranosyl ring of a docked lactose analogue to play an important role in binding, the backbone at Gly268 may be involved, and Val264 does not interact with the bound sugar. When the three side chains are subjected to site-directed mutagenesis, with the sole exception of mutant Asn272 â Gln, various other replacements for Asn272 either markedly decrease affinity for the substrate (i.e., high KD) or abolish binding altogether. However, mutant Gly268 â Ala exhibits a moderate 8-fold decrease in affinity, and binding by mutant Val264 â Ala is affected only minimally. Thus, Asn272 and possibly Gly268 may comprise additional components of the galactoside-binding site in LacY.
Assuntos
Proteínas de Escherichia coli/metabolismo , Galactosídeos/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Simportadores/metabolismo , Sítios de Ligação , Proteínas de Escherichia coli/química , Galactosídeos/química , Proteínas de Transporte de Monossacarídeos/química , Estrutura Secundária de Proteína , Simportadores/químicaRESUMO
Iron foundries have been identified as dioxin sources in previous field investigations. Similar formation mechanisms between dioxins and unintentional polychlorinated naphthalenes (PCNs) have led us to speculate that iron foundries are also potential PCN sources. In this study, PCNs in stack gas and fly ash samples representing atmospheric and residue emissions from 13 typical iron foundry plants were analyzed. The average emission factor of ∑(2-8)PCNs to residue was calculated to be 61 µg t(-1), with a range of 10-107 µg t(-1). The emission factors of ∑(2-8)PCNs to air in two case plants were 267 and 1472 µg t(-1). The derived emission factors might be useful for estimating annual emissions and understanding the contribution of PCNs from iron foundries. The possible formation mechanisms of PCNs, based on the PCN profiles, are discussed. Successive reductions in the abundance of homologues were observed to occur with the increase in chlorine substituted numbers. Abundances of congeners containing more ß-position chlorines in the naphthalene skeleton were much higher than those of congeners containing more α-position chlorines for penta-, hexa-, and hepta- homologues, which suggests that the ß-positions are favored for chlorination. Potential chlorination pathways from tetra- to octa- homologues are proposed.