RESUMEN
Many fields of basic and applied science require efficiently exploring complex systems with high dimensionality. An example of such a challenge is optimising the performance of plasma fusion experiments. The highly-nonlinear and temporally-varying interaction between the plasma, its environment and external controls presents a considerable complexity in these experiments. A further difficulty arises from the fact that there is no single objective metric that fully captures both plasma quality and equipment constraints. To efficiently optimise the system, we develop the Optometrist Algorithm, a stochastic perturbation method combined with human choice. Analogous to getting an eyeglass prescription, the Optometrist Algorithm confronts a human operator with two alternative experimental settings and associated outcomes. A human operator then chooses which experiment produces subjectively better results. This innovative technique led to the discovery of an unexpected record confinement regime with positive net heating power in a field-reversed configuration plasma, characterised by a >50% reduction in the energy loss rate and concomitant increase in ion temperature and total plasma energy.
RESUMEN
Bovine dentin phosphophoryn (BDP), a protein rich in aspartyl (Asp) and O-phosphoseryl (Ser[P]) residues, is synthesized by odontoblasts and is believed to be involved in matrix-mediated biomineralization of dentin. We have purified BDP, using selective precipitation and ion exchange chromatography, from an EDTA soluble dentin extract and converted the Ser(P) residues to S-propylcysteinyl residues that are stable to Edman degradation, facilitating the determination of the amino acid sequence of the N-terminal 38 residues. After the initial Asp-Ser(P)-Pro-Asn-Ser(P)-Ser(P)-Asp-Glu-Ser(P)-Asn-Gly-, the sequence contained the repeated motifs Asp-Ser(P) and Asp-Ser(P)-Ser(P). Purified BDP migrated as a single band on gradient SDS-PAGE with an apparent molecular weight of 156 kDa. This value was consistent with the molecular weight of the dephosphorylated protein of 105 kDa determined by means of MALDI mass spectrometry.
Asunto(s)
Dentina/química , Fosfoproteínas/química , Análisis de Secuencia de Proteína/métodos , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Bovinos , Cromatografía por Intercambio Iónico , Cisteína/análogos & derivados , Cisteína/química , Electroforesis en Gel de Poliacrilamida , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Fosforilación , Subunidades de Proteína , Serina/químicaRESUMEN
Glutamine transport in glucose-energized cells of Streptococcus mutans Ingbritt exhibited Michaelis-Menten-type kinetics with a Vmax of 13.4 nmol/mg dry weight/min and a Kt of 4.1 microM. Diffusion of glutamine into de-energized cells of S. mutans displayed similar type kinetics, with a Kt of 6.8 microM but with a markedly reduced Vmax of 53.9 pmol/mg dry weight/min. Glutamine transport in S. mutans is not proton motive force-driven, as the intracellular accumulation of glutamine by energized cells far exceeded the thermodynamic limits of the proton motive force, and the dissipation of this proton motive force by gramicidin in a high K+ medium did not decrease the intracellular glutamine concentration. Glutamine transport is therefore likely to be energized by ATP hydrolysis. The activity of the transporter was maximal between pH 6.0 and 7.0 and decreased rapidly above pH 7.0. The transport of glutamine was not competitively inhibited by asparagine, glutamate or aspartate, indicating a specific glutamine transport system. Reversed-phase high-pressure liquid chromatography of cell extracts revealed that approximately 26% of the glutamine taken into the cell was converted to glutamate within 10 min. The results are consistent with transported glutamine being converted to glutamate and ammonia by the action of an intracellular glutaminase. Glutamine therefore may be an important source of nitrogen for the cell.
Asunto(s)
Glutamina/metabolismo , Streptococcus mutans/metabolismo , Transportadoras de Casetes de Unión a ATP , Adenosina Trifosfato/metabolismo , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/fisiología , Glucosa , Glutamatos/metabolismo , Glucólisis/fisiología , Gramicidina/farmacología , Concentración de Iones de Hidrógeno , Cinética , Nitrógeno/metabolismo , Potasio/farmacología , Bombas de Protones , Fuerza Protón-Motriz , Streptococcus mutans/efectos de los fármacosRESUMEN
Casein phosphopeptides (CPP) stabilize calcium phosphate through the formation of casein-phosphopeptide amorphous calcium-phosphate complexes (CPP-CP). The ability of CPP-CP to reduce caries activity was investigated by use of specific-pathogen-free rats inoculated with Streptococcus sobrinus. The animals consumed a defined cariogenic diet free of dairy products. Solutions (100 microL) of the CPP-CP (0.1, 0.2, 0.5, 1.0% w/v) were applied to the animals' molar teeth twice daily. Other groups of animals received solutions containing 500 ppm F, the non-phosphorylated peptides of a casein tryptic digest (0.5% w/v), or the calcium-phosphate complex of a synthetic octapeptide, Ac-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe, corresponding to the common sequence in the CPP. The CPP-CP significantly reduced caries activity in a dose-response fashion, with 1.0% CPP-CP producing 55% and 46% reductions in smooth surface and fissure caries activity, respectively, being similar to that of 500 ppm F. The anticariogenic effects of CPP-CP and fluoride were additive, since animals receiving 0.5% CPP-CP plus 500 ppm F had significantly lower caries activity than those animals receiving either CPP-CP or fluoride alone. The tryptic digest of casein with the phosphopeptides selectively removed showed no anticariogenic activity. The synthetic octapeptide-calcium phosphate complex significantly reduced caries activity, confirming that this calcium-phosphate-stabilizing portion of the casein phospho-peptides is associated with anticariogenicity. The CPP-CP did not significantly affect the level of S. sobrinus in fissure plaque.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Fosfatos de Calcio/farmacocinética , Cariostáticos/farmacología , Caseínas/farmacología , Fosfopéptidos/farmacología , Secuencia de Aminoácidos , Análisis de Varianza , Animales , Fosfatos de Calcio/uso terapéutico , Cariostáticos/química , Caseínas/uso terapéutico , Caries Dental/prevención & control , Placa Dental/metabolismo , Dieta Cariógena , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Fluoruros/uso terapéutico , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos , Péptidos/química , Péptidos/uso terapéutico , Fosfopéptidos/uso terapéutico , Ratas , Ratas Sprague-Dawley , Organismos Libres de Patógenos Específicos , Estadísticas no Paramétricas , Streptococcus sobrinus , Remineralización Dental/métodosRESUMEN
Multiple phosphoseryl-containing sequences of proteins stabilize amorphous calcium phosphate and have been implicated in the regulation of biomineralization, protein structure, and enzyme activity. To facilitate studies on the identification and characterization of multiple phosphoseryl-containing sequences of proteins we have developed a simple and efficient purification procedure involving precipitation of Ca2+/ethanol-induced aggregates of the multiple phosphoseryl-containing peptides from enzymic digests. The multiple phosphoseryl-containing peptides of a tryptic digest of casein were selectively precipitated using Ca2+ (20 mol/mol protein) and 50% (v/v) ethanol at pH 3.5, 4.6, and 8.0. The individual peptides of the precipitates were purified using anion-exchange fast-performance liquid chromatography and reversed-phase HPLC and then identified by solid-phase sequence analysis and amino acid composition analysis after vapor-phase hydrolysis. Prior to sequence analysis the phosphopeptides were covalently coupled to arylamine membranes and the phosphoseryl residues converted to S-ethylcysteinyl residues by calcium-ion-catalyzed beta-elimination in the presence of ethanethiol. The modified peptides were sequenced using an Applied Biosystems Inc. automated protein sequencer fitted with a membrane cartridge. Only peptides containing the cluster sequence -Ser(P)-Ser(P)-Ser(P)- were precipitated by Ca2+/ethanol at pH 3.5. The pH 4.6 precipitate contained all the cluster peptides plus two diphosphorylated peptides containing -Ser(P)-Glu-Ser(P)- and -Ser(P)-Thr-Ser(P)-. At pH 8.0, a monophosphorylated peptide containing -Ser(P)-Glu-Glu- was also present in the precipitate with the diphosphorylated and cluster peptides. The recoveries of the peptides in the pH 8.0 selective precipitate ranged from 83 to 95% of that present in the hydrolysate.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Péptidos/análisis , Péptidos/aislamiento & purificación , Fosfoserina/análisis , Fosfoserina/aislamiento & purificación , Secuencia de Aminoácidos , Precipitación Química , Técnicas de Química Analítica , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Concentración de Iones de Hidrógeno , Hidrólisis , Datos de Secuencia Molecular , Análisis de Secuencia/métodosRESUMEN
The heat-stable phosphocarrier protein (HPr) of Streptococcus mutans was extracted from whole cells using sodium lauroylsarcosinate/EDTA and purified to homogeneity by a single-step, ion-exchange chromatographic procedure. The complete amino acid sequence of the protein was determined from peptides generated by trypsin, alpha-chymotrypsin, endoproteinase Glu-C, and cyanogen bromide treatment. The HPr from S. mutans contains 86 or 87 amino acyl residues, depending on removal of the N-terminal Met and the protein shows high sequence homology with HPr from other Gram-positive bacteria. The predicted tertiary structure of the S. mutans HPr, from model building by homology, is an open-faced beta-sandwich consisting of two alpha-helices and a four-stranded antiparallel beta-sheet.
Asunto(s)
Proteínas Bacterianas/química , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Conformación Proteica , Streptococcus mutans/metabolismo , Secuencia de Aminoácidos , Cromatografía por Intercambio Iónico , Quimotripsina , Bromuro de Cianógeno , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/aislamiento & purificación , Homología de Secuencia de Aminoácido , TripsinaRESUMEN
Multiple phosphoseryl-containing sequences of peptides and proteins stabilize amorphous calcium phosphate at neutral and alkaline pH and have been implicated in the nucleation/regulation of biomineralization. In an approach to analyze these peptides using capillary zone electrophoresis (CZE) we have attempted to relate the absolute electrophoretic mobility of various casein phosphopeptides to their physicochemical properties. Multiple phosphoseryl-containing peptides were selectively precipitated from enzymic digests of sodium caseinate and further purified using RP-HPLC and anion-exchange fast protein liquid chromatography. Purified fractions were then analyzed by CZE. Absolute electrophoretic mobilities of 13 peptides were determined by measurement of migration times relative to that of a neutral marker, mesityl oxide. A linear relationship (r2 = 0.993) was obtained between absolute electrophoretic mobility and q/M(r)2/3 where q is the net negative charge of the peptide calculated using relevant pKa values and M(r) is the molecular mass. M(r)2/3 is a measure of the surface area of a sphere that has a volume proportional to the M(r) of the peptide and relates to the frictional drag exerted on the peptide during electrophoretic migration. As absolute electrophoretic mobility is influenced by charge and size CZE can be used to monitor peptide phosphorylation, dephosphorylation, deamidation and truncation. This technique therefore would be suitable for quantitative analysis of peptide substrates in kinase and phosphatase studies. In conclusion CZE is a rapid and efficient technique for the resolution of multiple phosphoseryl-containing peptides from enzymic digests of casein.
Asunto(s)
Caseínas/análisis , Fosfopéptidos/análisis , Fosfoserina/análisis , Secuencia de Aminoácidos , Fenómenos Químicos , Química Física , Electroforesis , Hidrólisis , Datos de Secuencia Molecular , Páncreas/enzimología , TripsinaRESUMEN
Proteins of known composition and structural characteristics were incubated (1.0 mg/mL) with re-suspended salivary sediment (2.5% v/v) in a lactate-salt medium with an initial pH of 5.2 for two hr at 37 degrees C. Hydrolysis of the proteins was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Hydrogen ion, amines, and ammonia were measured by use of a combined pH electrode, high-performance liquid chromatography, and glutamate dehydrogenase, respectively. Of the proteins studied, the caseins alpha s1, beta, and kappa and the histones H1 and H3 were extensively hydrolyzed by the salivary-sediment bacteria. The hydrolysis of these proteins was attributed to their relative lack of tertiary (folded) structure. The only amine detected was the polyamine putrescine arising from the catabolism of arginine following the hydrolysis of the arginine-rich histone H3. None of the other proteins extensively hydrolyzed by salivary sediment, although containing arginyl and lysyl residues, served as substrates for putrescine or cadaverine production. Pre-hydrolysis of the arginine-rich histone H3 and poly-L-arginine with trypsin resulted in a marked increase in putrescine produced, suggesting that the salivary-sediment proteolytic activity was not "trypsin-like". Incubation of salivary-sediment bacteria with the caseins and the histone H3 resulted in an increase in ammonium ion concentration and an associated decrease in hydrogen ion concentration. The increase in ammonium ion concentration not attributed to arginine hydrolysis was correlated with the content of glutaminyl plus asparaginyl residues of the proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Bacterias/metabolismo , Proteínas/metabolismo , Saliva/microbiología , Adulto , Aminas/análisis , Amoníaco/análisis , Electroforesis en Gel de Poliacrilamida , Humanos , Proteínas/análisisRESUMEN
A chromatography column containing hydroxyapatite beads was used to study the effect of different proteins on the rate of hydroxyapatite dissolution. The four phosphoproteins tested (phosvitin, alpha sl-casein, beta-casein and kappa-casein) markedly reduced the rate of hydroxyapatite dissolution. Three nonphosphorylated proteins had a relatively smaller effect. The effect of the protein in reducing the hydroxyapatite dissolution rate has been attributed to protein binding to the surface of hydroxyapatite. The reduction in dissolution rate, expressed as the change in nmol calcium released per min per nmol of phosphoprotein bound to hydroxyapatite, increased with increasing number of phosphoserine residues of the protein. The results are consistent with the proposition that phosphoproteins have a regulatory role in mineralization processes and could provide a mechanism by which dietary and salivary phosphoproteins exert an anticariogenic effect.