The transcriptional repressor protein NsrR senses nitric oxide directly via a [2Fe-2S] cluster

The regulatory protein NsrR, a member of the Rrf2 family of transcription repressors, is specifically dedicated to sensing nitric oxide (NO) in a variety of pathogenic and non-pathogenic bacteria. It has been proposed that NO directly modulates NsrR activity by interacting with a predicted [Fe-S] cluster in the NsrR protein, but no experimental evidence has been published to support this hypothesis. Here we report the purification of NsrR from the obligate aerobe Streptomyces coelicolor. We demonstrate using UV-visible, near UV CD and EPR spectroscopy that the protein contains an NO-sensitive [2Fe-2S] cluster when purified from E. coli. Upon exposure of NsrR to NO, the cluster is nitrosylated, which results in the loss of DNA binding activity as detected by bandshift assays. Removal of the [2Fe-2S] cluster to generate apo-NsrR also resulted in loss of DNA binding activity. This is the first demonstration that NsrR contains an NO-sensitive [2Fe-2S] cluster that is required for DNA binding activity

Hearing, speech, language and communicative participation in patients with Apert syndrome: analysis of correlation with Fibroblast Growth Factor Receptor 2 mutation

Apert syndrome (AS) is caused by the heterozygous presence of 1 of 2 specific missense mutations of the fibroblast growth factor receptor 2 (FGFR2) gene. The 2 adjacent substitutions, designated p.Ser252Trp (S252W) and p.Pro253Arg (P253R), account for more than 98% of cases. Previous research has identified elevated hearing difficulties and incidence of cleft palate in this population. However, the influence of FGFR2 genotype on the speech, language, and communicative participation of children with AS has yet to be examined. Methods: A retrospective case note analysis was completed for all patients with a genetically-confirmed Apert mutation who attended the Oxford Craniofacial Unit over a 43-year period (1978–2020). Medical records were analyzed for speech, language, hearing, and communication data in detail. The therapy outcome measures, based on the World Health Organization International Classification of Functioning, Disability, and Health was used to classify patient's communicative participation. Results: The authors identified 55 AS patients with genetically-confirmed mutation of the FGFR2 gene. One patient with a S252F mutation was excluded. There were 31 patients with the S252W mutation (male = 14; female = 17), age range of last hearing assessment (1–18 years), 64% (18/28) of patients had a cleft palate (including bifid uvula), 15 patients had conductive hearing loss, 1 patient had mixed hearing loss, 18 had otitis media with effusion (4 of whom had a cleft palate); 88% (21/24) of patients had receptive language difficulties, 88% (22/25) of patients had expressive language difficulties, 96% (27/28) of patients had a speech sound disorder. There were 23 patients with the P253R mutation (male = 13; female = 10); age range of last hearing assessment (1–13 years), 35% (8/23) patients had a cleft palate (including bifid uvula), 14 patients had a conductive hearing loss, 17 had otitis media with effusion (2 of whom had a cleft palate). Results indicated that 85% (17/20) of patients had receptive language difficulties, 80% (16/20) had expressive language difficulties, 100% (21/21) had a speech sound disorder. The S252W mutation was significantly-associated with the presence of cleft palate (including bifid uvula) (P = 0.05). Data about the cumulative impact of all of these factors for communicative participation using the therapy outcome measures were available for 47 patients: (30 S252W; 17 P253R). Patients with a S252W mutation had significantly more severe difficulties with communicative participation when compared to individuals with a P253R mutation (P = 0.0005) Cochran-Armitage trend test. Conclusions: Speech, language, communicative participation, and hearing difficulties are pervasive in patients with AS. The severity and functional impact of these difficulties are magnified in patients with the S252W mutation. Results reinforce the importance of considering patients with AS according to genotype

The Transmembrane Domain of CEACAM1-4S Is a Determinant of Anchorage Independent Growth and Tumorigenicity

CEACAM1 is a multifunctional Ig-like cell adhesion molecule expressed by epithelial cells in many organs. CEACAM1-4L and CEACAM1-4S, two isoforms produced by differential splicing, are predominant in rat liver. Previous work has shown that downregulation of both isoforms occurs in rat hepatocellular carcinomas. Here, we have isolated an anchorage dependent clone, designated 253T-NT that does not express detectable levels of CEACAM1. Stable transfection of 253-NT cells with a wild type CEACAM1-4S expression vector induced an anchorage independent growth in vitro and a tumorigenic phenotype in vivo. These phenotypes were used as quantifiable end points to examine the functionality of the CEACAM1-4S transmembrane domain. Examination of the CEACAM1 transmembrane domain showed N-terminal GXXXG dimerization sequences and C-terminal tyrosine residues shown in related studies to stabilize transmembrane domain helix-helix interactions. To examine the effects of transmembrane domain mutations, 253-NT cells were transfected with transmembrane domain mutants carrying glycine to leucine or tyrosine to valine substitutions. Results showed that mutation of transmembrane tyrosine residues greatly enhanced growth in vitro and in vivo. Mutation of transmembrane dimerization motifs, in contrast, significantly reduced anchorage independent growth and tumorigenicity. 253-NT cells expressing CEACAM1-4S with both glycine to leucine and tyrosine to valine mutations displayed the growth-enhanced phenotype of tyrosine mutants. The dramatic effect of transmembrane domain mutations constitutes strong evidence that the transmembrane domain is an important determinant of CEACAM1-4S functionality and most likely by other proteins with transmembrane domains containing dimerization sequences and/or C-terminal tyrosine residues

A comparison of nicotine dose estimates in smokers between filter analysis, salivary cotinine, and urinary excretion of nicotine metabolites

RATIONALE: Nicotine uptake during smoking was estimated by either analyzing the metabolites of nicotine in various body fluids or by analyzing filters from smoked cigarettes. However, no comparison of the filter analysis method with body fluid analysis methods has been published. OBJECTIVES: Correlate nicotine uptake estimates between filter analysis, salivary cotinine, and urinary excretion of selected nicotine metabolites to determine the suitability of these methods in estimating nicotine absorption in smokers of filtered cigarettes. MATERIALS AND METHODS: A 5-day clinical study was conducted with 74 smokers who smoked 1–19 mg Federal Trade Commission tar cigarettes, using their own brands ad libitum. Filters were analyzed to estimate the daily mouth exposure of nicotine. Twenty-four-hour urine samples were collected and analyzed for nicotine, cotinine, and 3′-hydroxycotinine plus their glucuronide conjugates. Saliva samples were collected daily for cotinine analysis. RESULTS: Each method correlated significantly (p < 0.01) with the other two. The best correlation was between the mouth exposure of nicotine, as estimated by filter analysis, and urinary nicotine plus metabolites. Multiple regression analysis implies that saliva cotinine and urinary output are dependent on nicotine mouth exposure for multiple days. Creatinine normalization of the urinary metabolites degrades the correlation with mouth exposure. CONCLUSIONS: The filter analysis method was shown to correlate with more traditional methods of estimating nicotine uptake. However, because filter analysis is less complicated and intrusive, subjects can collect samples easily and unsupervised. This should enable improvements in study compliance and future study designs

Internalization Dissociates β2-Adrenergic Receptors

G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that β2-adrenergic receptors (β2ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β2ARs between subcellular compartments. BRET between β2ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between β2ARs and endosome markers increases. Energy transfer between β2ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled β2ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that β2ARs associate transiently with each other in the plasma membrane, or that β2AR dimers or oligomers are actively disrupted during internalization

Field trial on glucose-induced insulin and metabolite responses in Estonian Holstein and Estonian Red dairy cows in two herds

<p>Abstract</p> <p>Background</p> <p>Insulin secretion and tissue sensitivity to insulin is considered to be one of the factors controlling lipid metabolism <it>post partum</it>. The objective of this study was to compare glucose-induced blood insulin and metabolite responses in Estonian Holstein (EH, n = 14) and Estonian Red (ER, n = 14) cows.</p> <p>Methods</p> <p>The study was carried out using the glucose tolerance test (GTT) performed at 31 ± 1.9 days <it>post partum</it> during negative energy balance. Blood samples were obtained at -15, -5, 5, 10, 20, 30, 40, 50 and 60 min relative to infusion of 0.15 g/kg BW glucose and analysed for glucose, insulin, triglycerides (TG), non-esterified fatty acids (NEFA), cholesterol and β-hydroxybutyrate (BHB). Applying the MIXED Procedure with the SAS System the basal concentration of cholesterol, and basal concentration and concentrations at post-infusion time points for other metabolites, area under the curve (AUC) for glucose and insulin, clearance rate (CR) for glucose, and maximum increase from basal concentration for glucose and insulin were compared between breeds.</p> <p>Results</p> <p>There was a breed effect on blood NEFA (<it>P </it>< 0.05) and a time effect on all metabolites concentration (<it>P </it>< 0.01). The following differences were observed in EH compared to ER: lower blood insulin concentration 5 min after glucose infusion (<it>P </it>< 0.05), higher glucose concentration 20 (<it>P </it>< 0.01) and 30 min (<it>P </it>< 0.05) after infusion, and higher NEFA concentration before (<it>P </it>< 0.01) and 5 min after infusion (P < 0.05). Blood TG concentration in ER remained stable, while in EH there was a decrease from the basal level to the 40^thmin nadir (<it>P </it>< 0.01), followed by an increase to the 60^thmin postinfusion (<it>P </it>< 0.01).</p> <p>Conclusion</p> <p>Our results imply that glucose-induced changes in insulin concentration and metabolite responses to insulin differ between EH and ER dairy cows.</p

Signaling of Human Frizzled Receptors to the Mating Pathway in Yeast

Frizzled receptors have seven membrane-spanning helices and are considered as atypical G protein-coupled receptors (GPCRs). The mating response of the yeast Saccharomyces cerevisiae is mediated by a GPCR signaling system and this model organism has been used extensively in the past to study mammalian GPCR function. We show here that human Frizzled receptors (Fz1 and Fz2) can be properly targeted to the yeast plasma membrane, and that they stimulate the yeast mating pathway in the absence of added Wnt ligands, as evidenced by cell cycle arrest in G1 and reporter gene expression dependent on the mating pathway-activated FUS1 gene. Introducing intracellular portions of Frizzled receptors into the Ste2p backbone resulted in the generation of constitutively active receptor chimeras that retained mating factor responsiveness. Introducing intracellular portions of Ste2p into the Frizzled receptor backbone was found to strongly enhance mating pathway activation as compared to the native Frizzleds, likely by facilitating interaction with the yeast Gα protein Gpa1p. Furthermore, we show reversibility of the highly penetrant G1-phase arrests exerted by the receptor chimeras by deletion of the mating pathway effector FAR1. Our data demonstrate that Frizzled receptors can functionally replace mating factor receptors in yeast and offer an experimental system to study modulators of Frizzled receptors

Dimerization of Receptor Protein-Tyrosine Phosphatase alpha in living cells

BACKGROUND: Dimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined. RESULTS: In order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET) between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPα, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPα -CFP and -YFP fusion proteins, and thus that RPTPα dimerized in living cells. RPTPα dimerization was constitutive, extensive and specific. RPTPα dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization. CONCLUSIONS: We demonstrate here that RPTPα dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs

Structural Alterations in a Component of Cytochrome c Oxidase and Molecular Evolution of Pathogenic Neisseria in Humans

Three closely related bacterial species within the genus Neisseria are of importance to human disease and health. Neisseria meningitidis is a major cause of meningitis, while Neisseria gonorrhoeae is the agent of the sexually transmitted disease gonorrhea and Neisseria lactamica is a common, harmless commensal of children. Comparative genomics have yet to yield clear insights into which factors dictate the unique host-parasite relationships exhibited by each since, as a group, they display remarkable conservation at the levels of nucleotide sequence, gene content and synteny. Here, we discovered two rare alterations in the gene encoding the CcoP protein component of cytochrome cbb3 oxidase that are phylogenetically informative. One is a single nucleotide polymorphism resulting in CcoP truncation that acts as a molecular signature for the species N. meningitidis. We go on to show that the ancestral ccoP gene arose by a unique gene duplication and fusion event and is specifically and completely distributed within species of the genus Neisseria. Surprisingly, we found that strains engineered to express either of the two CcoP forms conditionally differed in their capacity to support nitrite-dependent, microaerobic growth mediated by NirK, a nitrite reductase. Thus, we propose that changes in CcoP domain architecture and ensuing alterations in function are key traits in successive, adaptive radiations within these metapopulations. These findings provide a dramatic example of how rare changes in core metabolic proteins can be connected to significant macroevolutionary shifts. They also show how evolutionary change at the molecular level can be linked to metabolic innovation and its reversal as well as demonstrating how genotype can be used to infer alterations of the fitness landscape within a single host