Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 16 de 16
Filter
1.
Chinese Journal of School Health ; (12): 733-737, 2023.
Article in Chinese | WPRIM (Western Pacific) | ID: wpr-973978

ABSTRACT

Objective@#To analyze the emotional and behavioral problems and associated factors of the only and non only child, and to provide some clues for further monitoring and intervention of psychological and behavioral development among preschool children.@*Methods@#Using a convenience sampling method, a total of 45 065 children enrolled in 153 kindergartens in 23 districts and counties of Chengdu were selected from May to June 2021 to investigate demographic characteristics and children s psycho behavioral development through online questionnaires filled out by their guardians. The Chi square tests were used to analyze whether the differences in abnormality rates of each dimension were statistically significant between the only and non only children. The emotional and behavioral problems of only children and non only children were analyzed by propensity score measurement.@*Results@#The detection rate of abnormal emotional behavior problems in children was 6.10%, including 6.34% in the only child group and 5.84 % in the non only child group. After matching, total difficulty score, and scores of emotional symptoms, conduct problems, hyperactive attention deficit, peer interaction, and social behavior differed between the only child group and the non only child group ( t =9.91, 8.97, 3.91, 15.57, -5.46, 4.08, P <0.01).@*Conclusion@#In terms of the total score of difficulties, emotional symptoms, moral problems, and hyperactivity attention defects, the non only child is better than the only child,but the opposite is true in terms of peer interaction and social behavior. Mental health conditions among the only child should be paid more attention. Whether or not the only child should be taken as an important consideration for preschool children s mental health care.

2.
Biomed Res Int ; 2020: 5426971, 2020.
Article in English | MEDLINE | ID: mdl-32953883

ABSTRACT

BACKGROUND: Previous studies showed that recanalization and angiogenesis within the infarct region are of vital importance to the survival of myocardial cells during the treatment of acute myocardial infarction (AMI). METHODS: In this study, EdU cell proliferation assay, Transwell assay, scratch wound assay, and tube formation assay were used. Twelve bioinformatics analysis packages were used to predict the target genes of miR-101. Target genes were verified by luciferase reporter generation and assay, fluorescent quantitative PCR, and western blotting. Animal model and treatments were detected by M-mode echocardiography and immunofluorescent staining of CD31, Ki67, and α-SMA. RESULTS: AgomiR-101 significantly enhanced HUVEC proliferation, migration, and tube formation. A double-luciferase reporter assay revealed that the hsa-miR-101 mimic attenuated the activity of the EIF4E3'-UTR-wt type plasmid by 36%. The expression levels of HIF-1α and VEGF-A in the scrambled RNA group were significantly lower than those in the EIF4E3 siRNA and agomiR-101 groups. The left ventricular ejection fraction of the AMI+Adv-miR-101 group was significantly higher than that of the AMI+Adv-null and Sham+Adv-null groups. The proliferation of vessel cells in the peripheral infarcted myocardium was higher in the AMI+Adv-miR-101 group than that in the AMI+Adv-null and Sham+Adv-null groups. CONCLUSION: MiR-101 can promote angiogenesis in the region surrounding the myocardial infarction.


Subject(s)
Human Umbilical Vein Endothelial Cells/metabolism , Hypoxia/metabolism , MicroRNAs/metabolism , Myocardial Infarction/metabolism , Neovascularization, Pathologic/metabolism , Animals , Cells, Cultured , Eukaryotic Initiation Factor-4E/metabolism , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Myocardium/metabolism , Myocytes, Cardiac/metabolism , RNA, Small Interfering/metabolism , Stroke Volume/physiology , Vascular Endothelial Growth Factor A/metabolism , Ventricular Function, Left/physiology
3.
Cell Biol Int ; 44(12): 2416-2426, 2020 Dec.
Article in English | MEDLINE | ID: mdl-32808744

ABSTRACT

Multifactor and multistep processes were elucidated to participate in the progression of non-small-cell lung cancer (NSCLC). Circular RNA 0031250 (circ-PRMT5) was a vital factor in NSCLC. However, the role of circ-PRMT5 in cisplatin (DDP)-resistance needed to be further highlighted. Expression profiles of circ-PRMT5, microRNA (miR)-4458, and EV3-like DNA-directed polymerase ζ catalytic subunit (REV3L) were detected using quantitative real-time polymerase chain reaction. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and transwell assays were performed to determine the half-maximal inhibitory concentration of DDP, cell viability, apoptosis, and invasion in vitro. Besides, the protein levels of REV3L and indicated proteins were examined by adopting western blot. Dual-luciferase reporter assay was performed to analyze the interaction between miR-4458 and circ-PRMT5 or REV3L. The functional role of circ-PRMT5 was explored using a xenograft tumor model. Levels of circ-PRMT5 and REV3L were markedly increased, while miR-4458 was downregulated in resistant tissues and cells. Knockdown of circ-PRMT5 enhanced cell apoptosis, DDP-sensitivity, and declined metastasis in NSCLC with DDP resistance. Besides, miR-4458 inhibition or REV3L upregulation could revert circ-PRMT5 absence-mediated effect on DDP-sensitivity in vitro. Mechanically, circ-PRMT5 was a sponge of miR-4458 to regulate REV3L. Importantly, circ-PRMT5 silencing could interact with DDP treatment expedite the decrease of tumor growth in vivo. Circ-PRMT5 promoted DDP resistance via REV3L by sponging miR-4458 in NSCLC, thus providing a novel therapeutic strategy for patients with NSCLC.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Drug Resistance, Neoplasm/genetics , Protein-Arginine N-Methyltransferases/genetics , A549 Cells , Animals , Apoptosis/drug effects , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/genetics , China , Cisplatin/pharmacology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Drug Resistance, Neoplasm/physiology , Female , Humans , Male , Mice , Mice, Nude , MicroRNAs/genetics , MicroRNAs/metabolism , Protein-Arginine N-Methyltransferases/metabolism , RNA, Circular/genetics , RNA, Circular/metabolism , Xenograft Model Antitumor Assays/methods
4.
Medicine (Baltimore) ; 99(20): e20168, 2020 May.
Article in English | MEDLINE | ID: mdl-32443334

ABSTRACT

BACKGROUND: Traditional coronary angiography (CA) as a main technique has been used to determine the coronary artery anatomy and guide percutaneous coronary intervention (PCI). We mainly focused on whether the new techniques could improve the patients' mortality, major adverse cardiovascular events (MACEs), and myocardial infarction. METHODS: For the network meta-analysis, we searched the trials of different PCI guidances from MEDLINE, Current Contents Connect, Google Scholar, EMBASE, Cochrane Library, PubMed, Science Direct, and Web of Science. The last search date was December 10, 2018. RESULTS: The analyses of all results found that there was no significant difference in mortality among the groups. Randomized clinical trials (RCT) analysis showed that intravascular ultrasound (IVUS)-guided PCI was significantly superior to CA, fractional flow reserve, instantaneous wave-free ratio, optical coherence tomography. However, CA, fractional flow reserve, instantaneous wave-free ratio, and optical coherence tomography showed no difference in reducing mortality. The analyses of all results found that there was no significant difference in the incidence of MACEs among the groups. RCTs analysis showed that IVUS-guided PCI was significantly superior to CA, but there was no significant difference among the other groups. The analyses of all results or RCTs showed that there was no significant difference in myocardial infarction incidence among the groups. CONCLUSION: IVUS-guided PCI is an effective method to decrease all-cause death MACEs.


Subject(s)
Coronary Angiography/methods , Coronary Artery Disease/diagnostic imaging , Coronary Vessels/diagnostic imaging , Percutaneous Coronary Intervention/instrumentation , Cardiovascular Diseases/epidemiology , Cardiovascular Diseases/mortality , Coronary Angiography/standards , Coronary Artery Disease/complications , Coronary Vessels/anatomy & histology , Endovascular Procedures/instrumentation , Fractional Flow Reserve, Myocardial/physiology , Humans , Myocardial Infarction/epidemiology , Myocardial Infarction/mortality , Network Meta-Analysis , Percutaneous Coronary Intervention/methods , Randomized Controlled Trials as Topic , Tomography, Optical Coherence/methods , Tomography, Optical Coherence/statistics & numerical data , Ultrasonography, Interventional/methods , Ultrasonography, Interventional/statistics & numerical data
5.
Int J Cardiol ; 273: 207-212, 2018 Dec 15.
Article in English | MEDLINE | ID: mdl-29960763

ABSTRACT

AIMS: The present network meta-analysis was performed to comprehensively compare the ability of different types of antihypertensive agents to ameliorate arterial stiffness in hypertensive patients. METHODS AND RESULTS: To conduct this network meta-analysis, we searched PubMed, the Embase database, and the https://clinicaltrials.gov/ website for all relevant articles concerning clinical trials on hypertension therapy. The last search date was 10 August 2017. As a result, 28 eligible articles were enrolled in our meta-analysis. According to the included studies, there was no significant difference in pulse wave velocity (PWV) between these treatments. The eight types of antihypertension agents outperformed placebo in controlling systolic blood pressure (SBP). Angiotensin-converting enzyme inhibitor (ACEI) outperformed angiotensin II receptor blocker (ARB) in SBP; and angiotensin receptor-neprilysin inhibitor (ARNI) outperformed diuretic (D)in SBP. CONCLUSIONS: This study found that the eight antihypertensive agents show obvious effect on reducing SBP other than arterial stiffness.


Subject(s)
Antihypertensive Agents/therapeutic use , Hypertension/drug therapy , Hypertension/physiopathology , Vascular Stiffness/drug effects , Antihypertensive Agents/pharmacology , Humans , Hypertension/diagnosis , Randomized Controlled Trials as Topic/methods , Vascular Stiffness/physiology
6.
Oncotarget ; 8(64): 107991-108005, 2017 Dec 08.
Article in English | MEDLINE | ID: mdl-29296218

ABSTRACT

BACKGROUND: To determine the effectiveness and safety of LCZ696 for the clinical treatment of hypertension, we performed a meta-analysis of the previous clinical trials. METHODS: Relevant English articles and randomized controlled trials were searched in Pubmed, Embase, EBSCO, Cochrane base and ClinicalTrials.gov. The last search date was July 20th, 2017. RESULTS: Compared with 20mg olmesartan, 200mg and 400mg LCZ696 outperformed olmesartan in terms of reducing mean sitting systolic blood pressure, mean ambulatory systolic blood pressure, mean sitting diastolic blood pressure and mean ambulatory diastolic blood pressure. Compared with 20mg olmesartan, 200mg and 400mg LCZ696 was better than olmesartan in terms of reducing mean sitting pulse pressure. And these studies showed that 400mg LCZ696 was better than 20mg olmesartan in terms of reducing mean ambulatory pulse pressure, however, there was no significant difference between 200mg LCZ696 and 20mg olmesartan in terms of redducing mean ambulatory pulse pressure. In addition, 200mg and 400mg LCZ696 was better than placebo in terms of reducing blood pressure parameters mentioned above. Compared with placebo or 20 mg olmesartan, LCZ696 showed no superiority in terms of reducing adverse events or serious adverse events. CONCLUSIONS: LCZ696 at 200 mg or 400 mg was better at reducing most of blood pressure parameters than 20 mg olmesartan or placebo. Compared with placebo or 20 mg olmesartan, 200 mg or 400 mg LCZ696 do not result in more adverse events in treating hypertension.

7.
Medicine (Baltimore) ; 95(14): e3245, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27057864

ABSTRACT

Many studies show that ivabradine is effective for stable angina.This meta-analysis was performed to determine the effect of treatment duration and control group type on ivabradine efficacy in stable angina pectoris.Relevant articles in the English language in the PUBMED and EMBASE databases and related websites were identified by using the search terms "ivabradine," "angina," "randomized controlled trials," and "Iva." The final search date was November 2, 2015.Articles were included if they were published randomized controlled trials that related to ivabradine treatment of stable angina pectoris.Patients with stable angina pectoris were included.The patients were classified according to treatment duration (<3 vs ≥3 months) or type of control group (placebo vs beta-receptor blocker). Angina outcomes were heart rate at rest or peak, exercise duration, and time to angina onset.Seven articles were selected. There were 3747 patients: 2100 and 1647 were in the ivabradine and control groups, respectively. The ivabradine group had significantly longer exercise duration when they had been treated for at least 3 months, but not when treatment time was less than 3 months. Ivabradine significantly improved time to angina onset regardless of treatment duration. Control group type did not influence the effect of exercise duration (significant) or time to angina onset (significant).Compared with beta-blocker and placebo, ivabradine improved exercise duration and time to onset of angina in patients with stable angina. However, its ability to improve exercise duration only became significant after at least 3 months of treatment.


Subject(s)
Angina, Stable/drug therapy , Benzazepines/therapeutic use , Cardiovascular Agents/therapeutic use , Humans , Ivabradine , Randomized Controlled Trials as Topic , Treatment Outcome
8.
PLoS One ; 9(9): e108356, 2014.
Article in English | MEDLINE | ID: mdl-25268800

ABSTRACT

BACKGROUND: Several randomized controlled trials (RCTs) have evaluated the effect of intra-aortic balloon counterpulsation pump(IABP) on the mortality of acute myocardial infarction (AMI). OBJECTIVES: To analyze the relevant RCT data on the effect of IABP on mortality and the occurrence of bleeding in AMI. DATA SOURCES: Published RCTs on the treatment of AMI by IABP were retrieved in searches of Medline, EMBASE, Cochrane and other related databases. The last search was conducted on July 20, 2014. STUDY ELIGIBILITY CRITERIA: Randomized clinical trials comparing IABP to controls as treatment for AMI. PARTICIPANTS: Patients with AMI. SYNTHESIS METHODS: The primary endpoint was mortality, and the secondary endpoint was bleeding events. To account for to heterogeneity, a random-effects model was used to analyze the study data. RESULTS: Ten trials with a total population of 973 patients that were included in the analysis showed no significant difference in 2-month mortality between the IABP and the control groups. The 6-month mortality in the IABP group was not significantly lower than in the control group in the four RCTs that enrolled 59 AMI patients with CS. But in the four that enrolled AMI 66 patients without CS, the data showed opposite conclusion. CONCLUSIONS: IABP cannot reduce within 2 months and 6-12 months mortality of AMI patients with CS as well as within 2 months mortality of AMI patients without CS, but can reduce 6-12 months mortality of AMI patients without CS. In addition, IABP can increase the risk of bleeding.


Subject(s)
Hemorrhage/mortality , Intra-Aortic Balloon Pumping/adverse effects , Myocardial Infarction/mortality , Adult , Aged , Female , Hemorrhage/etiology , Hemorrhage/pathology , Hemorrhage/therapy , Humans , Intra-Aortic Balloon Pumping/instrumentation , Male , Middle Aged , Myocardial Infarction/etiology , Myocardial Infarction/pathology , Myocardial Infarction/therapy , Randomized Controlled Trials as Topic , Survival Analysis
9.
FEBS J ; 279(3): 420-36, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22118659

ABSTRACT

Enzymatic catalysis and protein signaling are dynamic processes that involve local and/or global conformational changes occurring across a broad range of time scales. (1) H-(15) N relaxation NMR provides a comprehensive understanding of protein backbone dynamics both in the apo (unliganded) and ligand-bound conformations, enabling both fast and slow internal motions of individual amino acid residues to be observed. We recently reported the structure and nucleotide binding properties of the sulfate transporter and anti-sigma factor antagonist (STAS) domain of Rv1739c, a SulP anion transporter protein of Mycobacterium tuberculosis. In the present study, we report (1) H-(15) N NMR backbone dynamics measurements [longitudinal (T(1) ), transverse (T(2) ) and steady-state ({(1) H}-(15) N) heteronuclear NOE] of the Rv1739c STAS domain, in the absence and presence of saturating concentrations of GTP and GDP. Analysis of measured relaxation data and estimated dynamic parameters indicated distinct features differentiating the binding of GTP and GDP to Rv1739c STAS. The 9.55 ns overall rotational correlation time of Rv1739c STAS increased to 10.48 ns in the presence of GTP, and to 13.25 ns in the presence of GDP, indicating significant nucleotide-induced conformational changes. These conformational changes were accompanied by slow time scale (µs to ms) motions in discrete regions of the protein, as reflected by guanine nucleotide-induced changes in relaxation parameters. The observed nucleotide-specific alterations in the relaxation properties of individual STAS residues reflect an increased molecular anisotropy and/or the emergence of conformational equilibria governing functional properties of the STAS domain.


Subject(s)
Anion Transport Proteins/chemistry , Bacterial Proteins/chemistry , Guanine Nucleotides/chemistry , Molecular Dynamics Simulation , Mycobacterium tuberculosis/chemistry , Anisotropy , Guanosine Diphosphate , Guanosine Triphosphate , Magnetic Resonance Spectroscopy , Protein Binding , Protein Structure, Tertiary
10.
J Biol Chem ; 286(10): 8534-8544, 2011 Mar 11.
Article in English | MEDLINE | ID: mdl-21190940

ABSTRACT

The structure and intrinsic activities of conserved STAS domains of the ubiquitous SulP/SLC26 anion transporter superfamily have until recently remained unknown. Here we report the heteronuclear, multidimensional NMR spectroscopy solution structure of the STAS domain from the SulP/SLC26 putative anion transporter Rv1739c of Mycobacterium tuberculosis. The 0.87-Å root mean square deviation structure revealed a four-stranded ß-sheet with five interspersed α-helices, resembling the anti-σ factor antagonist fold. Rv1739c STAS was shown to be a guanine nucleotide-binding protein, as revealed by nucleotide-dependent quench of intrinsic STAS fluorescence and photoaffinity labeling. NMR chemical shift perturbation analysis partnered with in silico docking calculations identified solvent-exposed STAS residues involved in nucleotide binding. Rv1739c STAS was not an in vitro substrate of mycobacterial kinases or anti-σ factors. These results demonstrate that Rv1739c STAS binds guanine nucleotides at physiological concentrations and undergoes a ligand-induced conformational change but, unlike anti-σ factor antagonists, may not mediate signals via phosphorylation.


Subject(s)
Anion Transport Proteins/chemistry , Bacterial Proteins/chemistry , Computer Simulation , Models, Molecular , Mycobacterium tuberculosis/chemistry , Anion Transport Proteins/genetics , Anion Transport Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity Relationship
11.
Biomol NMR Assign ; 3(1): 99-102, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19636956

ABSTRACT

We report (1)H(N), (15)N, and (13)C resonance assignments for the 15.6 kDa STAS domain of the putative sulfate transporter of Mycobacterium tuberculosis, Rv1739c, using heteronuclear, multidimensional NMR spectroscopy. Rv1739c is a SulP anion permease, related in structure to the SLC26 gene family of metazoan anion exchangers and anion channels.


Subject(s)
Anion Transport Proteins/chemistry , Magnetic Resonance Spectroscopy/methods , Mycobacterium tuberculosis/chemistry , Amino Acid Sequence , Carbon Isotopes/chemistry , Molecular Sequence Data , Nitrogen Isotopes/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits , Protons
12.
Ann Pharmacother ; 42(10): 1416-24, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18780805

ABSTRACT

BACKGROUND: Patients with chronic obstructive pulmonary disease (COPD) are often given admixtures of nebulizable drugs to minimize the time of administration in treatment regimens. OBJECTIVE: To evaluate the physicochemical compatibility and aerodynamic characteristics of formoterol fumarate 20 microg/2 mL when mixed or sequentially nebulized with budesonide inhalation suspension 0.5 mg/2 mL, ipratropium bromide 0.5 mg/2.5 mL, cromolyn sodium 20 mg/2 mL, or acetylcysteine 10% (100 mg/mL). METHODS: The admixtures were prepared in triplicate and analyzed for physicochemical compatibility at 0, 15, 30, and 60 minutes after mixing at room temperature. Physical compatibility was determined by visual examination and measurements of pH, osmolality, and turbidity. Chemical stability was evaluated using compendial or in-house-validated high-performance liquid chromatography (HPLC) assay methods. The aerodynamic characteristics of the admixtures or sequentially nebulized drugs were determined from aerosols generated from a Pari LC Plus nebulizer, using an 8-stage cascade impactor followed by HPLC analysis of the deposited drug. RESULTS: The admixtures remained clear, colorless solutions with no precipitation, except for cloudiness observed in the formoterol/budesonide combination due to budesonide suspension. The pH, osmolality, and turbidity for all admixtures were within the initial values (< or = 3%), and there were no significant changes (< or = 2%) in potency of the active components throughout the 1-hour study period. Due to increased drug volume or reconcentration in the nebulizer cup, the respirable fraction/delivered dose increased significantly (p < 0.05) for the mixed or sequentially nebulized drug. However, the fine particle fraction (FPF), mass median aerodynamic diameter, and geometric standard deviation generally remained unchanged for all admixtures, with the exception of FPF for the formoterol/budesonide combination. CONCLUSIONS: Our results indicate that admixtures of formoterol with budesonide, ipratropium, cromolyn, or acetylcysteine are physically and chemically compatible. However, admixing or sequential nebulization significantly increased the amount of drug delivered compared with single drug nebulization. The clinical implications of the in vitro data in patients with COPD have not been determined.


Subject(s)
Anti-Asthmatic Agents/chemistry , Bronchodilator Agents/chemistry , Ethanolamines/chemistry , Acetylcysteine/administration & dosage , Acetylcysteine/chemistry , Aerosols , Anti-Asthmatic Agents/administration & dosage , Bronchodilator Agents/administration & dosage , Budesonide/chemistry , Chromatography, High Pressure Liquid , Cromolyn Sodium/administration & dosage , Cromolyn Sodium/chemistry , Drug Incompatibility , Drug Stability , Ethanolamines/administration & dosage , Expectorants/administration & dosage , Expectorants/chemistry , Formoterol Fumarate , Humans , Hydrogen-Ion Concentration , Ipratropium/chemistry , Nebulizers and Vaporizers , Osmolar Concentration , Particle Size , Pulmonary Disease, Chronic Obstructive/drug therapy , Time Factors
13.
Biochemistry ; 47(21): 5709-17, 2008 May 27.
Article in English | MEDLINE | ID: mdl-18452311

ABSTRACT

We constructed a single-cysteine panel encompassing TM5 of the oxalate transporter, OxlT. The 25 positions encompassed by TM5 were largely tolerant of mutagenesis, and functional product was recovered for 21 of the derived variants. For these derivatives, thiol-directed MTS-linked agents (MTSEA, MTSCE, and MTSES) were used as probes of transporter function, yielding 11 mutants that responded to probe treatment, as indicated by effects on oxalate transport. Further study identified three biochemical phenotypes among these responders. Group 1 included seven mutants, exemplified by G151C, displaying substrate protection against probe inhibition. Group 2 was comprised of a single mutant, P156C, which had unexpected behavior. In this case, we observed increased activity if weak acid/base or neutral probes were used, while exposure to probes introducing a fixed charge led to decreased function. In both instances, the presence of substrate prevented the observed response. Group 3 contained three mutants (e.g., S143C) in which probe sensitivity was increased by the presence of substrate. The finding of substrate-protectable probe modification in groups 1 and 2 suggests that TM5 lies on the permeation pathway, as do its structural counterparts, TM2, TM8, and TM11. In addition, we speculate that substrate binding facilitates TM5 conformational changes that allow new regions to become accessible to MTS-linked probes (group 3). These biochemical data are consistent with the recently developed OxlT homology model.


Subject(s)
Bacterial Proteins/genetics , Cysteine/chemistry , Membrane Transport Proteins/chemistry , Oxalobacter formigenes/metabolism , Bacterial Proteins/chemistry , Biological Transport , Kinetics , Membrane Transport Proteins/genetics , Models, Biological , Molecular Conformation , Mutagenesis , Mutation , Oligonucleotide Probes/chemistry , Oxalobacter formigenes/genetics , Phenotype , Protein Conformation , Protein Structure, Tertiary
14.
J Bacteriol ; 189(19): 7089-97, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17660287

ABSTRACT

The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of L-aspartate (Asp) with release of L-alanine (Ala) and CO(2). The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an L-aspartate-beta-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.


Subject(s)
Alanine/metabolism , Antiporters/metabolism , Aspartic Acid/metabolism , Lactobacillaceae/metabolism , Antiporters/chemistry , Antiporters/genetics , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Lactobacillaceae/genetics , Models, Biological , Models, Molecular , Mutagenesis, Site-Directed , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
15.
Proc Natl Acad Sci U S A ; 102(24): 8513-8, 2005 Jun 14.
Article in English | MEDLINE | ID: mdl-15932938

ABSTRACT

Using the x-ray structure of the glycerol 3-phosphate transporter (GlpT), we devised a model for the distantly related oxalate transporter, OxlT. The model accommodates all earlier biochemical information on OxlT, including the idea that Lys-355 lies on the permeation pathway, and predicts that Lys-355 and a second positive center, Arg-272, comprise the binding site for divalent oxalate. Study of R272K, R272A, and R272Q derivatives verifies that Arg-272 is essential, and comparisons with GlpT show that both anion transporters bind substrates within equivalent domains. In 22 single-cysteine variants in TM7 and TM8, topology as marked by accessibility to Oregon green maleimide is predicted by the model, with similar concordance for 52 positions probed earlier. The model also reconciles cross-linking of a cysteine pair placed near the periplasmic ends of TM2 and TM7, and retrospective study of TM2 and TM11 confirms that positions supporting disulfide trapping lie at a helical interface. Our work describes a pathway to the modeling of OxlT and other transporters in the major facilitator superfamily and outlines simple experimental tests to evaluate such proposals.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Models, Molecular , Oxalobacter formigenes/metabolism , Fluorescence , Maleimides , Mutagenesis , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Sequence Analysis, DNA
16.
J Biol Chem ; 277(23): 20372-8, 2002 Jun 07.
Article in English | MEDLINE | ID: mdl-11919184

ABSTRACT

We constructed a single cysteine panel encompassing transmembrane helix two (TM2) of OxlT, the oxalate/formate antiporter of Oxalobacter formigenes. Among the 21 positions targeted, cysteine substitution identified one (phenylalanine 59) as essential to OxlT expression and three (glutamine 56, glutamine 66, and serine 69) as potentially critical to OxlT function. By probing membranes with a bulky hydrophilic probe (Oregon Green maleimide) we also located a central inaccessible core of at least eight residues in length, extending from leucine 61 to glycine 68. Functional assays based on reconstitution of crude detergent extracts showed that of single cysteine mutants within the TM2 core only the Q63C variant was substantially (> or =95%) inhibited by thiol-specific agents (carboxyethyl methanethiosulfonate and ethylsulfonate methanethiosulfonate). Subsequent analytical work using the purified Q63C protein showed that inhibition by ethylsulfonate methanethiosulfonate was blocked by substrate and that the concentration dependence of such substrate protection occurred with a binding constant of 0.16 mm oxalate, comparable with the Michaelis constant observed for oxalate transport (0.23 mm). These findings lead us to conclude that position 63 lies on the OxlT translocation pathway. Our conclusion is strengthened by the finding that position 63, along with most other positions relevant to TM2 function, is found on a helical face that can be cross-linked to the pathway-facing surface of TM11 (Fu, D., Sarker, R. I., Bolton, E., and Maloney, P. C. (2001) J. Biol. Chem. 276, 8753-8760).


Subject(s)
Bacterial Proteins , Carrier Proteins/metabolism , Formates/metabolism , Membrane Proteins/metabolism , Membrane Transport Proteins , Oxalates/metabolism , Biological Transport , Carrier Proteins/chemistry , Carrier Proteins/genetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mutagenesis, Site-Directed , Structure-Activity Relationship
SELECTION OF CITATIONS
SEARCH DETAIL
...