RESUMEN
RNA polymerase III (RNAP III) holoenzyme activity and the processing of its products have been linked to several metabolic dysfunctions in lower and higher eukaryotes. Alterations in the activity of RNAP III-driven synthesis of non-coding RNA cause extensive changes in glucose metabolism. Increased RNAP III activity in the S. cerevisiae maf1Δ strain is lethal when grown on a non-fermentable carbon source. This lethal phenotype is suppressed by reducing tRNA synthesis. Neither the cause of the lack of growth nor the underlying molecular mechanism have been deciphered, and this area has been awaiting scientific explanation for a decade. Our previous proteomics data suggested mitochondrial dysfunction in the strain. Using model mutant strains maf1Δ (with increased tRNA abundance) and rpc128-1007 (with reduced tRNA abundance), we collected data showing major changes in the TCA cycle metabolism of the mutants that explain the phenotypic observations. Based on 13C flux data and analysis of TCA enzyme activities, the present study identifies the flux constraints in the mitochondrial metabolic network. The lack of growth is associated with a decrease in TCA cycle activity and downregulation of the flux towards glutamate, aspartate and phosphoenolpyruvate (PEP), the metabolic intermediate feeding the gluconeogenic pathway. rpc128-1007, the strain that is unable to increase tRNA synthesis due to a mutation in the C128 subunit, has increased TCA cycle activity under non-fermentable conditions. To summarize, cells with non-optimal activity of RNAP III undergo substantial adaptation to a new metabolic state, which makes them vulnerable under specific growth conditions. Our results strongly suggest that balanced, non-coding RNA synthesis that is coupled to glucose signaling is a fundamental requirement to sustain a cell's intracellular homeostasis and flexibility under changing growth conditions. The presented results provide insight into the possible role of RNAP III in the mitochondrial metabolism of other cell types.
Asunto(s)
Mitocondrias , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Mitocondrias/metabolismo , Homeostasis , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN no Traducido/metabolismoRESUMEN
The evolutional development of the RNA translation process that leads to protein synthesis based on naturally occurring amino acids has its continuation via synthetic biology, the so-called rational bioengineering. Genetic code expansion (GCE) explores beyond the natural translational processes to further enhance the structural properties and augment the functionality of a wide range of proteins. Prokaryotic and eukaryotic ribosomal machinery have been proven to accept engineered tRNAs from orthogonal organisms to efficiently incorporate noncanonical amino acids (ncAAs) with rationally designed side chains. These side chains can be reactive or functional groups, which can be extensively utilized in biochemical, biophysical, and cellular studies. Genetic code extension offers the contingency of introducing more than one ncAA into protein through frameshift suppression, multi-site-specific incorporation of ncAAs, thereby increasing the vast number of possible applications. However, different mediating factors reduce the yield and efficiency of ncAA incorporation into synthetic proteins. In this review, we comment on the recent advancements in genetic code expansion to signify the relevance of systems biology in improving ncAA incorporation efficiency. We discuss the emerging impact of tRNA modifications and metabolism in protein design. We also provide examples of the latest successful accomplishments in synthetic protein therapeutics and show how codon expansion has been employed in various scientific and biotechnological applications.
Asunto(s)
Aminoácidos/metabolismo , ARN de Transferencia/genética , Biología Sintética/métodos , Evolución Molecular , Código Genético , Biosíntesis de Proteínas , Ribosomas/metabolismoRESUMEN
KfrC proteins are encoded by the conjugative broad-host-range plasmids that also encode alpha-helical filament-forming KfrA proteins as exemplified by the RA3 plasmid from the IncU incompatibility group. The RA3 variants impaired in kfrA, kfrC, or both affected the host's growth and demonstrated the altered stability in a species-specific manner. In a search for partners of the alpha-helical KfrC protein, the host's membrane proteins and four RA3-encoded proteins were found, including the filamentous KfrA protein, segrosome protein KorB, and the T4SS proteins, the coupling protein VirD4 and ATPase VirB4. The C-terminal, 112-residue dimerization domain of KfrC was involved in the interactions with KorB, the master player of the active partition, and VirD4, a key component of the conjugative transfer process. In Pseudomonas putida, but not in Escherichia coli, the lack of KfrC decreased the stability but improved the transfer ability. We showed that KfrC and KfrA were involved in the plasmid maintenance and conjugative transfer and that KfrC may play a species-dependent role of a switch between vertical and horizontal modes of RA3 spreading.
Asunto(s)
Proteínas Bacterianas/química , Especificidad del Huésped , Plásmidos/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Conjugación Genética , Escherichia coli/genética , Modelos Biológicos , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios Proteicos , Eliminación de SecuenciaRESUMEN
Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.
Asunto(s)
Benchmarking , Laboratorios , Calorimetría , Reproducibilidad de los Resultados , TemperaturaRESUMEN
KfrA, encoded on the broad-host-range RA3 plasmid, is an alpha-helical DNA-binding protein that acts as a transcriptional autoregulator. The KfrARA3 operator site overlaps the kfrA promoter and is composed of five 9-bp direct repeats (DRs). Here, the biological properties of KfrA were studied using both in vivo and in vitro approaches. Localization of the DNA-binding helix-turn-helix motif (HTH) was mapped to the N29-R52 region by protein structure modeling and confirmed by alanine scanning. KfrA repressor ability depended on the number and orientation of DRs in the operator, as well as the ability of the protein to oligomerize. The long alpha-helical tail from residues 54 to 355 was shown to be involved in self-interactions, whereas the region from residue 54 to 177 was involved in heterodimerization with KfrC, another RA3-encoded alpha-helical protein. KfrA also interacted with the segrosome proteins IncC (ParA) and KorB (ParB), representatives of the class Ia active partition systems. Deletion of the kfr genes from the RA3 stability module decreased the plasmid retention in diverse hosts in a species-dependent manner. The specific interactions of KfrA with DNA are essential not only for the transcriptional regulatory function but also for the accessory role of KfrA in stable plasmid maintenance.IMPORTANCE Alpha-helical coiled-coil KfrA-type proteins are encoded by various broad-host-range low-copy-number conjugative plasmids. The DNA-binding protein KfrA encoded on the RA3 plasmid, a member of the IncU incompatibility group, oligomerizes, forms a complex with another plasmid-encoded, alpha-helical protein, KfrC, and interacts with the segrosome proteins IncC and KorB. The unique mode of KfrA dimer binding to the repetitive operator is required for a KfrA role in the stable maintenance of RA3 plasmid in distinct hosts.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Plásmidos , Bacterias/genéticaRESUMEN
Protein biosynthesis is energetically costly, is tightly regulated and is coupled to stress conditions including glucose deprivation. RNA polymerase III (RNAP III)-driven transcription of tDNA genes for production of tRNAs is a key element in efficient protein biosynthesis. Here we present an analysis of the effects of altered RNAP III activity on the Saccharomyces cerevisiae proteome and metabolism under glucose-rich conditions. We show for the first time that RNAP III is tightly coupled to the glycolytic system at the molecular systems level. Decreased RNAP III activity or the absence of the RNAP III negative regulator, Maf1 elicit broad changes in the abundance profiles of enzymes engaged in fundamental metabolism in S. cerevisiae In a mutant compromised in RNAP III activity, there is a repartitioning towards amino acids synthesis de novo at the expense of glycolytic throughput. Conversely, cells lacking Maf1 protein have greater potential for glycolytic flux.
Asunto(s)
Glucólisis , ARN Polimerasa III/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Perfilación de la Expresión Génica , Genes Fúngicos , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Glucólisis/genética , Redes y Vías Metabólicas , Modelos Biológicos , Vía de Pentosa Fosfato/genética , Mutación Puntual , Proteoma/genética , Proteoma/metabolismo , ARN Polimerasa III/química , ARN Polimerasa III/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Saccharomyces cerevisiae responds to glucose availability in the environment, inducing the expression of the low-affinity transporters and high-affinity transporters in a concentration dependent manner. This cellular decision making is controlled through finely tuned communication between multiple glucose sensing pathways including the Snf1-Mig1, Snf3/Rgt2-Rgt1 (SRR) and cAMP-PKA pathways. RESULTS: We demonstrate the first evidence that RNA Polymerase III (RNAP III) activity affects the expression of the glucose transporter HXT2 (RNA Polymerase II dependent-RNAP II) at the level of transcription. Down-regulation of RNAP III activity in an rpc128-1007 mutant results in a significant increase in HXT2 mRNA, which is considered to respond only to low extracellular glucose concentrations. HXT2 expression is induced in the mutant regardless of the growth conditions either at high glucose concentration or in the presence of a non-fermentable carbon source such as glycerol. Using chromatin immunoprecipitation (ChIP), we found an increased association of Rgt1 and Tup1 transcription factors with the highly activated HXT2 promoter in the rpc128-1007 strain. Furthermore, by measuring cellular abundance of Mth1 corepressor, we found that in rpc128-1007, HXT2 gene expression was independent from Snf3/Rgt2-Rgt1 (SRR) signaling. The Snf1 protein kinase complex, which needs to be active for the release from glucose repression, also did not appear perturbed in the mutated strain. CONCLUSIONS/SIGNIFICANCE: These findings suggest that the general activity of RNAP III can indirectly affect the RNAP II transcriptional machinery on the HXT2 promoter when cellular perception transduced via the major signaling pathways, broadly recognized as on/off switch essential to either positive or negative HXT gene regulation, remain entirely intact. Further, Rgt1/Ssn6-Tup1 complex, which has a dual function in gene transcription as a repressor-activator complex, contributes to HXT2 transcriptional activation.
Asunto(s)
Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Glucosa/metabolismo , ARN Polimerasa III/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Regulación hacia Arriba , Western Blotting , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Inmunoprecipitación , Mutación , Reacción en Cadena en Tiempo Real de la Polimerasa , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
Yeast Fba1 (fructose 1,6-bisphosphate aldolase) is a glycolytic enzyme essential for viability. The overproduction of Fba1 enables overcoming of a severe growth defect caused by a missense mutation rpc128-1007 in a gene encoding the C128 protein, the second largest subunit of the RNA polymerase III complex. The suppression of the growth phenotype by Fba1 is accompanied by enhanced de novo tRNA transcription in rpc128-1007 cells. We inactivated residues critical for the catalytic activity of Fba1. Overproduction of inactive aldolase still suppressed the rpc128-1007 phenotype, indicating that the function of this glycolytic enzyme in RNA polymerase III transcription is independent of its catalytic activity. Yeast Fba1 was determined to interact with the RNA polymerase III complex by coimmunoprecipitation. Additionally, a role of aldolase in control of tRNA transcription was confirmed by ChIP experiments. The results indicate a novel direct relationship between RNA polymerase III transcription and aldolase.
Asunto(s)
Fructosa-Bifosfato Aldolasa/metabolismo , ARN Polimerasa III/metabolismo , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/genética , Transcripción Genética , Northern Blotting , Western Blotting , Núcleo Celular/metabolismo , Cromatina/genética , Inmunoprecipitación de Cromatina , Citoplasma/metabolismo , Técnica del Anticuerpo Fluorescente , Fructosa-Bifosfato Aldolasa/genética , Inmunoprecipitación , Mutagénesis Sitio-Dirigida , Mutación/genética , ARN Polimerasa III/genética , ARN Mensajero/genética , ARN de Transferencia/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismoRESUMEN
Monolayers of KfrA, a protein assisting in bacteria plasmid segregation, on polystyrene latex particles were produced in controlled self-assembling under diffusion-controlled conditions. The coverage of the protein was quantitatively determined as a function of ionic strength (up to 0.15 M, NaCl) via micro-electrophoretic measurements and concentration depletion with the aid of AFM imaging. The maximum monolayer coverage of KfrA monotonically increased with ionic strength attaining 2.0 mg m(-2) for 0.15 M, NaCl that corresponds to the dimensionless coverage of 0.48. This is in accordance with theoretical calculations derived from the random sequential adsorption modeling assuming a tetrameric aggregation state. A high stability of the monolayers in pH cycling experiments was confirmed, which proves the irreversibility of protein adsorption on latex. The acid base properties and the electrokinetic charge of monolayers were also determined via the electrophoretic mobility measurements carried out for various ionic strength. In this way the isoelectric point of the protein of 4.8 was determined, which is prohibitive via bulk measurements. It was concluded that the procedure used in our work is reliable and efficient for characterizing physicochemical properties if minor amounts of a protein are available.
Asunto(s)
Proteínas Bacterianas/química , Adsorción , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Electroquímica , Electroforesis en Gel de Poliacrilamida , Punto Isoeléctrico , Látex , Luz , Concentración Osmolar , Plásmidos/genética , Multimerización de Proteína , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Dispersión de Radiación , Propiedades de SuperficieRESUMEN
Physicochemical characteristics of the plasmid KfrA protein in electrolyte solutions were done using a combination of dynamic light scattering (DLS), atomic force microscopy (AFM) and electrokinetic methods. The size of the protein was determined via the diffusion coefficient measurements using DLS. It was revealed from these measurements that the protein exists in an aggregated state composed of four molecules. The size of the protein was also determined via AFM imaging of single molecules adsorbed on mica from dilute solutions at pH=3.5. It was 10.6 nm in accordance with the value predicted for an aggregate composed of four monomers in a hexagonal configuration. The aggregation number was also confirmed by kinetics measurements carried out under diffusion controlled transport using AFM imaging of proteins. Further characteristics were acquired via KfrA adsorption on polystyrene latex particles (average size of 820 nm). The electrophoretic mobility of the latex and its zeta potential were determined as a function of the coverage of the protein. The maximum monolayer coverage for pH=3.5 was 1.2 mgm(-2). Additionally, from these measurements the effective charge of KfrA tetramer equal to 12 e (elementary charges) was predicted. The KfrA monolayer on latex was used to determine the isoelectric point of the protein, which was pH=4.5. As concluded, the procedures used in our work proved advantageous for a direct determination of aggregation processes and the effective charge if minor amounts of a protein are available.
Asunto(s)
Proteínas Bacterianas/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Luz , Microscopía de Fuerza Atómica/métodos , Plásmidos/metabolismo , Dispersión de Radiación , Adsorción , Silicatos de Aluminio/química , Proteínas Bacterianas/química , Fenómenos Químicos , Humanos , Punto Isoeléctrico , Multimerización de Proteína , Albúmina Sérica/metabolismo , Electricidad EstáticaRESUMEN
The success rate of introducing new functions into a living species is still rather unsatisfactory. Much of this is due to the very essence of the living state, i.e. its robustness towards perturbations. Living cells are bound to notice that metabolic engineering is being effected, through changes in metabolite concentrations. In this study, we asked whether one could engage in such engineering without changing metabolite concentrations. We have illustrated that, in silico, one can do so in principle. We have done this for the case of substituting the yeast glucose transporter plus hexokinase for the Lactococcus lactis phosphotransferase system, in an L. lactis network, this engineering is 'silent' in terms of metabolite concentrations and almost all fluxes.
Asunto(s)
Proteínas Facilitadoras del Transporte de la Glucosa/genética , Hexoquinasa/genética , Lactococcus lactis/genética , Ingeniería Metabólica/métodos , Fosfotransferasas/genética , Biología de Sistemas/métodos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Simulación por Computador , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Hexoquinasa/metabolismo , Lactococcus lactis/metabolismo , Lactococcus lactis/fisiología , Redes y Vías Metabólicas/fisiología , Modelos Biológicos , Fosfotransferasas/metabolismo , Piruvato Quinasa/metabolismoRESUMEN
BACKGROUND: Thyrotoxicosis is more frequent in postmenopausal women than in the general population, effectively accelerating bone turnover. Interleukin-6 has been shown to be involved in the pathogenesis of bone disorders. Thus, the aim of the present study was to assess the role of IL-6 and its soluble receptor in the pathogenesis of thyrotoxicosis-related disturbances of bone turnover in oestrogen-deficient women. MATERIAL AND METHODS: The study was carried out in 40 subjects with toxic nodular goitre in three groups: Group 1 - 13 premenopausal females, mean age 36 ± 15 years (PremTxâPremEu); Group 2 - 12 postmenopausal females, mean age 66 ± 14 years (PostTxâPostEu); and Group 3 - 15 males, mean age 45 ± 21 years (MTxâMEu). Overt thyrotoxicosis and euthyreosis after treatment with thyrostatics were confirmed by thyrotropin, free thyroxine and free triiodothyronin concentrations. Serum levels of bone turnover markers: TRACP5b and osteocalcin as well as serum IL-6 and IL-6sR were determined using ELISA kits. RESULTS: TRACP5b/osteocalcin quotient was significantly elevated in the PostTx females compared to the PremTx women (p < 0.02). There was a positive correlation between serum TRACP5b and osteocalcin in the studied patients (R = 0.45, p < 0.001). Levels of serum IL-6 values were significantly elevated in PostTx: 3.0 (2.14-6.40) and MTx: 2.24 (1.60-5.10), compared to PremTx females: 1.39 (0.96-2.14) (p < 0.01 and p < 0.05 respectively). There were significant positive correlations between IL-6 and IL-6sR concentrations (R = 0.22, p < 0.05) and between IL-6sR and TRACP5b serum levels (R = 0.23, p < 0.05). CONCLUSIONS: The results of our study suggest that interleukin-6 plays a considerable role in the pathogenesis of thyrotoxicosis-related disturbances of bone turnover in oestrogen-deficient women.
Asunto(s)
Enfermedades Óseas/etiología , Huesos/metabolismo , Interleucina-6/sangre , Osteoporosis Posmenopáusica/etiología , Tirotoxicosis/etiología , Fosfatasa Ácida/sangre , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Densidad Ósea/fisiología , Enfermedades Óseas/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Isoenzimas/sangre , Masculino , Persona de Mediana Edad , Osteocalcina/sangre , Osteoporosis Posmenopáusica/sangre , Receptores de Interleucina-6/sangre , Factores Sexuales , Fosfatasa Ácida Tartratorresistente , Tirotoxicosis/sangre , Adulto JovenRESUMEN
In vitro enzymatic assays of cell-free extracts offer an opportunity to assess in vivo enzyme concentrations. If performed under conditions that resemble the conditions in vivo, they may also reveal some of the capacities and properties of the same enzymes in vivo; we shall call this the ex vivo approach. The kinetic characterization of purified enzymes has yet a different utility for systems biology, as does the in vivo determination of enzyme activities. All these approaches are different, and it is becoming important that the appropriate approach be used for the intended purpose. Here, we therefore discuss five approaches to the measurement of enzyme activity in terms of the source of the enzyme activity, the identity of the assay medium, and the purpose of the assay.
Asunto(s)
Pruebas de Enzimas/métodos , Algoritmos , Animales , Extractos Celulares/química , Medios de Cultivo , Enzimas/química , Enzimas/aislamiento & purificación , Enzimas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Humanos , Cinética , Biología de Sistemas , Levaduras/crecimiento & desarrollo , Levaduras/metabolismoRESUMEN
Biology and medicine have become 'big science', even though we may not always like this: genomics and the subsequent analysis of what the genomes encode has shown that interesting living organisms require many more than 300 gene products to interact. We once thought that somewhere in this jungle of interacting macromolecules was hidden the molecule that constitutes the secret of Life, and therewith of health and disease. Now we know that, somehow, the secret of Life is the jungle of interactions. Consequently, we need to find the Rosetta Stones, i.e. interpretations of this jungle of systems biology. We need to find, perhaps convoluted, paths of understanding and intervention. Systems biochemistry is a good place to start, as it has the foothold that what goes in must come out. In the present paper, we review two strategies, which look at control and regulation. We discuss the difference between control and regulation and prove a relationship between them.
Asunto(s)
Bioquímica/métodos , Modelos Biológicos , Biología de Sistemas/métodos , Animales , HumanosRESUMEN
Systems Biology has a mission that puts it at odds with traditional paradigms of physics and molecular biology, such as the simplicity requested by Occam's razor and minimum energy/maximal efficiency. By referring to biochemical experiments on control and regulation, and on flux balancing in yeast, we show that these paradigms are inapt. Systems Biology does not quite converge with biology either: Although it certainly requires accurate 'stamp collecting', it discovers quantitative laws. Systems Biology is a science of its own, discovering own fundamental principles, some of which we identify here.
Asunto(s)
Vida , Biología de Sistemas , Termodinámica , Bioquímica , Genoma , Física , Levaduras/genética , Levaduras/fisiologíaRESUMEN
Multidrug-resistant variants of the opportunistic human pathogen Enterococcus have recently emerged as leading agents of nosocomial infection. The acquisition of plasmid-borne resistance genes is a driving force in antibiotic-resistance evolution in enterococci. The segregation locus of a high-level gentamicin-resistance plasmid, pGENT, in Enterococcus faecium was identified and dissected. This locus includes overlapping genes encoding PrgP, a member of the ParA superfamily of segregation proteins, and PrgO, a site-specific DNA binding homodimer that recognizes the cenE centromere upstream of prgPO. The centromere has a distinctive organization comprising three subsites, CESII separates CESI and CESIII, each of which harbors seven TATA boxes spaced by half-helical turns. PrgO independently binds both CESI and CESIII, but with different affinities. The topography of the complex was probed by atomic force microscopy, revealing discrete PrgO foci positioned asymmetrically at the CESI and CESIII subsites. Bending analysis demonstrated that cenE is intrinsically curved. The organization of the cenE site and of certain other plasmid centromeres mirrors that of yeast centromeres, which may reflect a common architectural requirement during assembly of the mitotic apparatus in yeast and bacteria. Moreover, segregation modules homologous to that of pGENT are widely disseminated on vancomycin and other resistance plasmids in enterococci. An improved understanding of segrosome assembly may highlight new interventions geared toward combating antibiotic resistance in these insidious pathogens.
Asunto(s)
Centrómero , Farmacorresistencia Microbiana/genética , Resistencia a Múltiples Medicamentos/genética , Enterococcus faecium/genética , Genes Bacterianos , Datos de Secuencia MolecularRESUMEN
INTRODUCTION: The aim of this study was to estimate the influence of corticosteroids on soluble MMP-2, MMP-9 and VCAM-1 in patients with Graves ophthalmopathy (GO) in order to assess their usefulness as a guideline in a therapeutic approach. MATERIAL AND METHODS: Serum gelatinases and VCAM-1 were detected in three groups of subjects: 20 patients with GO (CAS > or = 3, anamnesis of GO > or = 1 yr), 12 patients with no clinical symptoms of ophthalmopathy (Gd) and 10 healthy volunteers. Corticosteroid therapy consisted of intravenous infusions (2 series, 3 grams each time) of methylprednisolone (MP) and subsequent treatment with oral prednisone (60 mg per day) in a tapering schedule. The serum samples were collected 24 hours before MP, 24 hours after MP, after 14 days of treatment with prednisone and at the end of corticosteroid therapy. The levels of soluble MMP-2, MMP-9 and VCAM-1 were determined by the ELISA method. RESULTS: We have found no differences in serum MMP-2 between the groups studied and a significant reduction after MP only in corticosteroid-resistant GO patients. Soluble MMP-9 was highest in the GO group compared with both the Gd and control individuals. Moreover serum MMP-9 decreased in corticosteroid-responsive GO patients after MP and remained at the lower level at the end of the study. Positive correlations between MMP-2 and MMP-9 before and after MP administration were observed. Serum VCAM-1 was significantly elevated both in GO and Gd subjects and pre-treatment VCAM-1 levels were elevated in corticosteroid-responders compared with non-responders. CONCLUSIONS: Our results suggest that serum VCAM-1 may serve as a marker predicting the efficacy of corticosteroids and that soluble MMP-9 may be helpful in monitoring corticosteroid administration and in decision-making with regard to further GO treatment.
Asunto(s)
Glucocorticoides/uso terapéutico , Oftalmopatía de Graves/sangre , Oftalmopatía de Graves/tratamiento farmacológico , Metaloproteinasa 2 de la Matriz/sangre , Metaloproteinasa 9 de la Matriz/sangre , Molécula 1 de Adhesión Celular Vascular/sangre , Adulto , Biomarcadores/sangre , Femenino , Oftalmopatía de Graves/patología , Humanos , Masculino , Metilprednisolona/uso terapéutico , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Prednisona/uso terapéuticoRESUMEN
UV spectroscopy demonstrated that chicken mononucleosomes bind Co(II) and Zn(II) ions at submicromolar concentrations in a tetrahedral mode, at a conserved zinc finger-like site, composed of Cys110 and His113 residues of both H3 molecules. Neither of these metal ions substituted for another, indicating a limited binding reversibility. Molecular modeling indicated that the tetrahedral site is formed by unhindered rotations around Calpha-Cbeta bonds in the side chains of the zinc binding residues. The resulting local rearrangement of the protein structure shields the bound metal ion from the solvent, explaining the observed lack of reversibility of the binding. Consequences of these findings for zinc homeostasis, metal toxicology and nucleosomal regulation are discussed.
Asunto(s)
Histonas/química , Modelos Moleculares , Nucleosomas/química , Dedos de Zinc , Zinc/química , Animales , Sitios de Unión , Pollos , Cobalto/química , Cisteína/química , Histidina/química , Conformación Proteica , Espectrofotometría UltravioletaRESUMEN
Contents of trace elements (copper, zinc, iron, and manganese) were determined in melted cheeses, mold-ripening semi-hard and soft types purchased in 2005 at local shops. The AAS technique was used. Material for study was produced by different Dairy Centers all over Poland in Lublin, Mazowsze, Opole, Podlasie, Silesia, and Wielkopolska regions. Zinc and iron levels in melted cheeses were significantly higher (p < or = 0.05) as compared to mold-ripening soft and semi-hard cheeses. The highest zinc and iron concentrations were found in the samples of Sertop and Jal cheese. The lowest contents were found in Mabelle cheese. Rycki Edam cheese was characterized by highest level of manganese, and Mabelle--by the lowest. However, copper, iron, and manganese contents in all cheese samples could be considered as low and not hazardous for consumer's.