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Potatoes are a dietary staple consumed by a significant portion of the world, providing valuable carbohydrates and vitamins. However, most commercially produced potatoes have a high content of highly branched amylopectin starch, which generally results in a high glycemic index (GI). Consumption of foods with high levels of amylopectin elicit a rapid spike in blood glucose levels, which is undesirable for individuals who are pre-diabetic, diabetic, or obese. Some cultivars of potatoes with lower amylopectin levels have previously been identified and are commercially available in niche markets in some countries, but they are relatively unavailable in the United States and Latin America. The high glycemic index of widely available potatoes presents a problematic "consumer's dilemma" for individuals and families that may not be able to afford a better-balanced, more favorable diet. Some native communities in the Andes (Bolivia, Chile, and Peru) reportedly embrace a tradition of providing low glycemic tubers to people with obesity or diabetes to help people mitigate what is now understood as the negative effects of high blood sugar and obesity. These cultivars are not widely available on a global market. This study examines 60 potato cultivars to identify potatoes with low amylopectin. Three independent analyses of potato starch were used: microscopic examination of granule structure, water absorption, and spectrophotometric analysis of iodine complexes to identify potato cultivars with low amylopectin Differences among cultivars tested were detected by all three types of analyses. The most promising cultivars are Huckleberry Gold, Muru, Multa, Green Mountain, and an October Blue x Colorado Rose cross. Further work is necessary to document the ability of these low amylopectin cultivars to reduce blood glucose spike levels in human subjects.
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Solanum tuberosum , Almidón , Humanos , Almidón/química , Amilopectina/química , Solanum tuberosum/química , Glucemia , ObesidadRESUMEN
After age, polymorphisms of the Apolipoprotein E (APOE) gene are the biggest risk factor for the development of Alzheimer's disease (AD). During our investigation to discovery biomarkers in plasma, using 2D gel electrophoresis, we found an individual with and unusual apoE isoelectric point compared to APOE É2, É3, and É4 carriers. Whole exome sequencing of APOE from the donor confirmed a single nucleotide polymorphism (SNP) in exon 4, translating to a rare Q222K missense mutation. The apoE É4 (Q222K) mutation did not form dimers or complexes observed for apoE É2 & É3 proteins.
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BACKGROUND: The Australian Imaging and Biomarker Lifestyle (AIBL) study of aging is designed to aid the discovery of biomarkers. The current study aimed to discover differentially expressed plasma proteins that could yield a blood-based screening tool for Alzheimer's disease. METHODS: The concentration of proteins in plasma covers a vast range of 12 orders of magnitude. Therefore, to search for medium to low abundant biomarkers and elucidate mechanisms of AD, we immuno-depleted the most abundant plasma proteins and pre-fractionated the remaining proteins by HPLC, prior to two-dimensional gel electrophoresis. The relative levels of approximately 3400 protein species resolved on the 2D gels were compared using in-gel differential analysis with spectrally resolved fluorescent protein detection dyes (Zdyes™). Here we report on analysis of pooled plasma samples from an initial screen of a sex-matched cohort of 72 probable AD patients and 72 healthy controls from the baseline time point of AIBL. RESULTS: We report significant changes in variants of apolipoprotein E, haptoglobin, α1 anti-trypsin, inter-α trypsin inhibitor, histidine-rich glycoprotein, and a protein of unknown identity. α1 anti-trypsin and α1 anti-chymotrypsin demonstrated plasma concentrations that were dependent on APOE ε4 allele dose. Our analysis also identified an association with the level of Vitamin D binding protein fragments and complement factor I with sex. We then conducted a preliminary validation study, on unique individual samples compared to the discovery cohort, using a targeted LC-MS/MS assay on a subset of discovered biomarkers. We found that targets that displayed a high degree of isoform specific changes in the 2D gels were not changed in the targeted MS assay which reports on the total level of the biomarker. CONCLUSIONS: This demonstrates that further development of mass spectrometry assays is needed to capture the isoform complexity that exists in theses biological samples. However, this study indicates that a peripheral protein signature has potential to aid in the characterization of AD.
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Epilepsy is a common neurological disorder, which is not well understood at the molecular level. Exactly why some brain regions produce epileptic discharges and others do not is not known. Patients who fail to respond to antiseizure medication (refractory epilepsy) can benefit from surgical removal of brain regions to reduce seizure frequency. The tissue removed in these surgeries offers an invaluable resource to uncover the molecular and cellular basis of human epilepsy. Here, we report a proteomic study to determine whether there are common proteomic patterns in human brain regions that produce epileptic discharges. We analyzed human brain samples, as part of the Systems Biology of Epilepsy Project (SBEP). These brain pieces are in vivo electrophysiologically characterized human brain samples withdrawn from the neocortex of six patients with refractory epilepsy. This study is unique in that for each of these six patients the comparison of protein expression was made within the same patient: a more epileptic region was compared to a less epileptic brain region. The amount of epileptic activity was defined for each patient as the frequency of their interictal spikes (electric activity between seizures that is a parameter strongly linked to epilepsy). Proteins were resolved from three subcellular fractions, using a 2D differential gel electrophoresis (2D-DIGE), revealing 31 identified protein spots that changed significantly. Interestingly, glial fibrillary acidic protein (GFAP) was found to be consistently down regulated in high spiking brain tissue and showed a strong negative correlation with spike frequency. We also developed a two-step analysis method to select for protein species that changed frequently among the patients and identified these proteins. A total of 397 protein spots of interest (SOI) were clustered by protein expression patterns across all samples. These clusters were used as markers and this analysis predicted proteomic changes due to both histological differences and molecular pathways, revealed by examination of gene ontology clusters. Our experimental design and proteomic data analysis predicts novel glial changes, increased angiogenesis, and changes in cytoskeleton and neuronal projections between high and low interictal spiking regions. Quantitative histological staining of these same tissues for both the vascular and glial changes confirmed these findings, which provide new insights into the structural and functional basis of neocortical epilepsy.
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Vasos Sanguíneos/metabolismo , Epilepsia/metabolismo , Neocórtex/metabolismo , Neuroglía/metabolismo , Proteómica , Adolescente , Adulto , Preescolar , Epilepsia/genética , Epilepsia/patología , Epilepsia/fisiopatología , Femenino , Perfilación de la Expresión Génica , Humanos , Lactante , MasculinoRESUMEN
Quantum biology is the study of quantum effects on biochemical mechanisms and biological function. We show that the biological production of reactive oxygen species (ROS) in live cells can be influenced by coherent electron spin dynamics, providing a new example of quantum biology in cellular regulation. ROS partitioning appears to be mediated during the activation of molecular oxygen (O2) by reduced flavoenzymes, forming spin-correlated radical pairs (RPs). We find that oscillating magnetic fields at Zeeman resonance alter relative yields of cellular superoxide (O2â¢-) and hydrogen peroxide (H2O2) ROS products, indicating coherent singlet-triplet mixing at the point of ROS formation. Furthermore, the orientation-dependence of magnetic stimulation, which leads to specific changes in ROS levels, increases either mitochondrial respiration and glycolysis rates. Our results reveal quantum effects in live cell cultures that bridge atomic and cellular levels by connecting ROS partitioning to cellular bioenergetics.
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Metabolismo Energético , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Teoría Cuántica , Especies Reactivas de Oxígeno/metabolismo , Simulación por Computador , Humanos , Campos Magnéticos , Análisis Numérico Asistido por Computador , Quinonas/química , Quinonas/metabolismo , Superóxidos/metabolismoRESUMEN
Direct dynamics simulations, utilizing the RM1 semiempirical electronic structure theory, were performed to study the thermal dissociation of the doubly protonated tripeptide threonine-isoleucine-lysine ion, TIK(H+)2, for temperatures of 1250-2500 K, corresponding to classical energies of 1778-3556 kJ/mol. The number of different fragmentation pathways increases with increase in temperature. At 1250 K there are only three fragmentation pathways, with one contributing 85% of the fragmentation. In contrast, at 2500 K, there are 61 pathways, and not one dominates. The same ion is often formed via different pathways, and at 2500 K there are only 14 m/z values for the product ions. The backbone and side-chain fragmentations occur by concerted reactions, with simultaneous proton transfer and bond rupture, and also by homolytic bond ruptures without proton transfer. For each temperature the TIK(H+)2 fragmentation probability versus time is exponential, in accord with the Rice-Ramsperger-Kassel-Marcus and transition state theories. Rate constants versus temperature were determined for two proton transfer and two bond rupture pathways. From Arrhenius plots activation energies Ea and A-factors were determined for these pathways. They are 62-78 kJ/mol and (2-3) × 1012 s-1 for the proton transfer pathways and 153-168 kJ/mol and (2-4) × 1014 s-1 for the bond rupture pathways. For the bond rupture pathways, the product cation radicals undergo significant structural changes during the bond rupture as a result of hydrogen bonding, which lowers their entropies and also their Ea and A parameters relative to those for C-C bond rupture pathways in hydrocarbon molecules. The Ea values determined from the simulation Arrhenius plots are in very good agreement with the reaction barriers for the RM1 method used in the simulations. A preliminary simulation of TIK(H+)2 collision-induced dissociation (CID), at a collision energy of 13 eV (1255 kJ/mol), was also performed to compare with the thermal dissociation simulations. Though the energy transferred to TIK(H+)2 in the collisions is substantially less than the energy for the thermal excitations, there is substantial fragmentation as a result of the localized, nonrandom excitation by the collisions. CID results in different fragmentation pathways with a significant amount of short time nonstatistical fragmentation. Backbone fragmentation is less important, and side-chain fragmentation is more important for the CID simulations as compared to the thermal simulations. The thermal simulations provide information regarding the long-time statistical fragmentation.
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Toxoplasma gondii is a ubiquitous protozoan parasite with approximately one-third of the worlds' population chronically infected. In chronically infected individuals, the parasite resides primarily in cysts within neurons in the central nervous system. The chronic infection in immunocompetent individuals has been considered to be asymptomatic but increasing evidence indicates the chronic infection can lead to neuropsychiatric disorders such as Schizophrenia, prenatal depression and suicidal thoughts. A better understanding of the mechanism(s) by which the parasite exerts effects on human behavior is limited due to lack of suitable human neuronal models. In this paper, we report the use of human neurons derived from normal cord blood CD34+ cells generated via genetic reprogramming, as an in vitro model for the study T. gondii in neurons. This culture method resulted in a relatively pure monolayer of induced human neuronal-like cells that stained positive for neuronal markers, MAP2, NFL, NFH and NeuN. These induced human neuronal-like cells (iHNs) were efficiently infected by the Prugniad strain of the parasite and supported replication of the tachyzoite stage and development of the cyst stage. Infected iHNs could be maintained through 5 days of infection, allowing for formation of large cysts. This induced human neuronal model represents a novel culture method to study both tachyzoite and bradyzoite stages of T. gondii in human neurons.
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Células Madre Pluripotentes Inducidas/fisiología , Modelos Biológicos , Modelos Teóricos , Neuronas/microbiología , Neuronas/patología , Toxoplasmosis Cerebral/microbiología , Toxoplasmosis Cerebral/patología , Células Cultivadas , HumanosRESUMEN
Although epilepsy is associated with a variety of abnormalities, exactly why some brain regions produce seizures and others do not is not known. We developed a method to identify cellular changes in human epileptic neocortex using transcriptional clustering. A paired analysis of high and low spiking tissues recorded in vivo from 15 patients predicted 11 cell-specific changes together with their 'cellular interactome'. These predictions were validated histologically revealing millimetre-sized 'microlesions' together with a global increase in vascularity and microglia. Microlesions were easily identified in deeper cortical layers using the neuronal marker NeuN, showed a marked reduction in neuronal processes, and were associated with nearby activation of MAPK/CREB signalling, a marker of epileptic activity, in superficial layers. Microlesions constitute a common, undiscovered layer-specific abnormality of neuronal connectivity in human neocortex that may be responsible for many 'non-lesional' forms of epilepsy. The transcriptional clustering approach used here could be applied more broadly to predict cellular differences in other brain and complex tissue disorders.
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Encéfalo/patología , Epilepsia/patología , Transcripción Genética , Adolescente , Adulto , Biomarcadores , Niño , Preescolar , Análisis por Conglomerados , Electroencefalografía , Epilepsia/cirugía , Femenino , Humanos , Lactante , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Neocórtex/patología , Procedimientos Neuroquirúrgicos , ARN/genética , Adulto JovenRESUMEN
Coxiella burnetii is an obligate intracellular bacterial pathogen and the causative agent of Q fever. Chronic Q fever can produce debilitating fatigue and C. burnetii is considered a significant bioterror threat. C. burnetii occupies the monocyte phagolysosome and although prior work has explained features of the host-pathogen interaction, many aspects are still poorly understood. We have conducted a proteomic investigation of human Monomac I cells infected with the Nine Mile Phase II strain of C. burnetii and used the results as a framework for a systems biology model of the host response. Our principal methodology was multiplex differential 2D gel electrophoresis using ZDyes, a new generation of covalently linked fluorescent protein detection dyes under development at Montana State University. The 2D gel analysis facilitated the detection of changes in posttranslational modifications on intact proteins in response to infection. The systems model created from our data a framework for the design of experiments to seek a deeper understanding of the host-pathogen interactions.
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Monocitos/inmunología , Proteómica/métodos , Fiebre Q/inmunología , Biología de Sistemas , Aldehído Deshidrogenasa/metabolismo , Aldehído Deshidrogenasa Mitocondrial , Calgranulina A/metabolismo , Chaperonina 60/metabolismo , Biología Computacional , Coxiella burnetii , Citocinas/metabolismo , Electroforesis en Gel Bidimensional , Enoil-CoA Hidratasa/metabolismo , Humanos , Leucil Aminopeptidasa/metabolismo , Lisosomas/metabolismo , Espectrometría de Masas , Proteínas Mitocondriales/metabolismo , Monocitos/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Procesamiento Proteico-Postraduccional , Pirofosfatasas/metabolismo , Superóxido Dismutasa/metabolismo , Factores de Tiempo , Transaldolasa/metabolismo , Vimentina/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in proteomic analysis, using two-dimensional difference gel electrophoresis (2D-DIGE). Protein labeling with CyDyes is hampered by protein precipitation and gel smearing when used above minimal labeling. The solubility of labeled protein may be improved by introducing water solubilizing groups on the dye such as cysteic acids. However, addition of a negatively charged functionality will have the undesired effect of shifting the pI in relation to the unlabeled protein. These limitations have been addressed through the synthesis of highly water-soluble and pI balancing zwitterionic CyDye fluorophores (Z-CyDyes). The new dyes feature a cysteic acid motif, a titratable amine functionality and a NHS activated ester group. In side by side 2D-DIGE comparisons of Z-CyDyes and CyDyes, the new dyes significantly enhanced protein spot volume and the number of spots that were detected. Z-CyDyes have the potential to enhance the depth of proteome coverage and provide a general strategy for improving the performance of protein tagging reagents.
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Proteínas Arqueales/análisis , Cisteína/análogos & derivados , Electroforesis en Gel Bidimensional/métodos , Colorantes Fluorescentes/análisis , Proteómica/métodos , Sulfolobus solfataricus/química , Carbocianinas/análisis , Solubilidad , Coloración y Etiquetado/métodos , Agua/químicaRESUMEN
Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (â¼34,000 Mr) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu(312)-Arg(322) and Arg(323)-Lys(350)) and extracellular FPR1 peptide (Ile(191)-Arg(201)) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala(323)-Lys(350) only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr(325), Ser(328), Thr(329), Thr(331), Ser(332), Thr(334), and Thr(339). No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC50 = 33 ± 8 to 74 ± 21 nM. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1-phosphospecific antibodies.
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N-Formilmetionina Leucil-Fenilalanina/farmacología , Activación Neutrófila/efectos de los fármacos , Neutrófilos/metabolismo , Receptores de Formil Péptido/metabolismo , Citocalasina B/farmacología , Humanos , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Activación Neutrófila/fisiología , Neutrófilos/citología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Estructura Terciaria de Proteína , Receptores de Formil Péptido/agonistas , Receptores de Lipoxina/metabolismoRESUMEN
The origin and evolutionary relationship of viruses is poorly understood. This makes archaeal virus-host systems of particular interest because the hosts generally root near the base of phylogenetic trees, while some of the viruses have clear structural similarities to those that infect prokaryotic and eukaryotic cells. Despite the advantageous position for use in evolutionary studies, little is known about archaeal viruses or how they interact with their hosts, compared to viruses of bacteria and eukaryotes. In addition, many archaeal viruses have been isolated from extreme environments and present a unique opportunity for elucidating factors that are important for existence at the extremes. In this article we focus on virus-host interactions using a proteomics approach to study Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2. Using cultures grown from the ATCC cell stock, a single cycle of STIV infection was sampled six times over a 72 h period. More than 700 proteins were identified throughout the course of the experiments. Seventy one host proteins were found to change their concentration by nearly twofold (p < 0.05) with 40 becoming more abundant and 31 less abundant. The modulated proteins represent 30 different cell pathways and 14 clusters of orthologous groups. 2D gel analysis showed that changes in post-translational modifications were a common feature of the affected proteins. The results from these studies showed that the prokaryotic antiviral adaptive immune system CRISPR-associated proteins (CAS proteins) were regulated in response to the virus infection. It was found that regulated proteins come from mRNAs with a shorter than average half-life. In addition, activity-based protein profiling (ABPP) profiling on 2D-gels showed caspase, hydrolase, and tyrosine phosphatase enzyme activity labeling at the protein isoform level. Together, this data provides a more detailed global view of archaeal cellular responses to viral infection, demonstrates the power of quantitative two-dimensional differential gel electrophoresis and ABPP using 2D gel compatible fluorescent dyes.
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Securinine, a GABA(A) receptor antagonist, has been reported to enhance monocyte cell killing of Coxiella burnetii without obvious adverse effects in vivo. We employed multiplex 2D gel electrophoresis using Zdyes, a new generation of covalently linked fluorescent differential protein detection dyes to analyze changes in the monocyte proteome in response to Securinine. Securinine antagonism of GABA(A) receptors triggers the activation of p38. We used the differential protein expression results to guide a search of the literature and network analysis software to construct a systems biology model of the effect of Securinine on monocytes. The model suggests that various metabolic modulators (fatty acid binding protein 5, inosine 5'-monophosphate dehydrogenase, and thioredoxin) are at least partially reshaping the metabolic landscape within the monocytes. The actin bundling protein L-plastin, and the Ca(2+) binding protein S100A4 also appear to have important roles in the immune response stimulated by Securinine. Fatty acid binding protein 5 (FABP5) may be involved in effecting lipid raft composition, inflammation, and hormonal regulation of monocytes, and the model suggests that FABP5 may be a central regulator of metabolism in activated monocytes. The model also suggests that the heat shock proteins have a significant impact on the monocyte immune response. The model provides a framework to guide future investigations into the mechanisms of Securinine action and with elaboration may help guide development of new types of immune adjuvants.
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Adyuvantes Inmunológicos/farmacología , Azepinas/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Lactonas/farmacología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Piperidinas/farmacología , Proteoma , Receptores de GABA-A/metabolismo , Presentación de Antígeno/inmunología , Biología Computacional/métodos , Electroforesis en Gel Bidimensional , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de Anillo en Puente , Redes y Vías Metabólicas , Modelos Biológicos , Monocitos/inmunología , Proteómica , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycling, and disease has driven research on their cellular and molecular biology. Knowledge exists for a wide range of viruses; however, a major exception are viruses with archaeal hosts. Archaeal virus-host systems are of great interest because they have similarities to both eukaryotic and bacterial systems and often live in extreme environments. Here we report the first proteomics-based experiments on archaeal host response to viral infection. Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2 was studied using 1D and 2D differential gel electrophoresis (DIGE) to measure abundance and redox changes. Cysteine reactivity was measured using novel fluorescent zwitterionic chemical probes that, together with abundance changes, suggest that virus and host are both vying for control of redox status in the cells. Proteins from nearly 50% of the predicted viral open reading frames were found along with a new STIV protein with a homologue in STIV2. This study provides insight to features of viral replication novel to the archaea, makes strong connections to well-described mechanisms used by eukaryotic viruses such as ESCRT-III mediated transport, and emphasizes the complementary nature of different omics approaches.
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Proteínas Arqueales/análisis , Virus de Archaea/metabolismo , Proteómica/métodos , Sulfolobus solfataricus/metabolismo , Sulfolobus solfataricus/virología , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Virus de Archaea/genética , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Interacciones Huésped-Patógeno , Datos de Secuencia Molecular , Alineación de Secuencia , Sulfolobus solfataricus/química , Espectrometría de Masas en Tándem , Replicación ViralRESUMEN
The structures of protein antigen-antibody (Ag-Ab) interfaces contain information about how Ab recognize Ag as well as how Ag are folded to present surfaces for Ag recognition. As such, the Ab surface holds information about Ag folding that resides with the Ab-Ag interface residues and how they interact. In order to gain insight into the nature of such interactions, a data set comprised of 53 non-redundant 3D structures of Ag-Ab complexes was analyzed. We assessed the physical and biochemical features of the Ag-Ab interfaces and the degree to which favored interactions exist between amino acid residues on the corresponding interface surfaces. Amino acid compositional analysis of the interfaces confirmed the dominance of TYR in the Ab paratope-containing surface (PCS), with almost two fold greater abundance than any other residue. Additionally TYR had a much higher than expected presence in the PCS compared to the surface of the whole antibody (defined as the occurrence propensity), along with aromatics PHE, TRP, and to a lesser degree HIS and ILE. In the Ag epitope-containing surface (ECS), there were slightly increased occurrence propensities of TRP and TYR relative to the whole Ag surface, implying an increased significance over the compositionally most abundant LYS>ASN>GLU>ASP>ARG. This examination encompasses a large, diverse set of unique Ag-Ab crystal structures that help explain the biological range and specificity of Ag-Ab interactions. This analysis may also provide a measure of the significance of individual amino acid residues in phage display analysis of Ag binding.
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Aminoácidos/química , Anticuerpos/química , Complejo Antígeno-Anticuerpo/química , Antígenos/química , Muramidasa/química , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Antígenos/inmunología , Sitios de Unión de Anticuerpos/inmunología , Pollos/metabolismo , Bases de Datos de Proteínas , Humanos , Datos de Secuencia Molecular , Muramidasa/inmunología , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Unión Proteica , Conformación Proteica , Codorniz/metabolismoRESUMEN
The NADPH oxidase of phagocytic leukocytes generates superoxide that plays a critical role in innate immunity and inflammatory responses. The integral membrane protein flavocytochrome b (Cyt b, a.k.a. cytochrome b(558/559)) is the catalytic core of the complex and serves as a prototype for homologs important in regulating signaling networks in a wide variety of animal and plant cells. Our analysis identifies a naturally-occurring Tyr72/His72 polymorphism (p.Y72H) in the p22(phox) subunit of Cyt b at the protein level that has been recognized at the nucleotide level (c.214T > C, formerly C242T) and implicated in cardiovascular disease. In the present study, Cyt b was isolated from human neutrophils and reacted with chemical crosslinkers for subsequent structure analysis by MALDI mass spectrometry. Following mild chemical modification of Cyt b with two pairs of isotopically-differentiated lysine crosslinkers: BS(2)G-d(0)/d(4) and BS(3)-d(0)/d(4), the reaction mixtures were digested with trypsin and purified on C(18)ZipTips to generate samples for mass analysis. MALDI analysis of tryptic digests from each of the above reactions revealed a series of masses that could be assigned to p22(phox) residues 68-85, assuming an intra-molecular crosslink between Lys71 and Lys78. In addition to the 30 ppm mass accuracy obtained with internal mass calibration, increased confidence in the assignment of the crosslinks was provided by the presence of the diagnostic mass patterns resulting from the isotopically-differentiated crosslinking reagent pairs and the Tyr72/His72 p22(phox) polymorphisms in the crosslinked peptides. This work identifies a novel, low-resolution distance constraint in p22(phox) and suggests that the medically-relevant p.Y72H polymorphism has an invariant structural motif in this region. Because position 72 in p22(phox) lies outside regions identified as interactive with other oxidase components, the structural invariance also provides additional support for maturational differences as the source of the wide variation in observed reactive oxygen species production by cells expressing p.Y72H.
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Grupo Citocromo b/química , Espectrometría de Masas/métodos , NADPH Oxidasas/química , NADPH Oxidasas/genética , Neutrófilos/metabolismo , Polimorfismo Genético , Reactivos de Enlaces Cruzados/química , Grupo Citocromo b/metabolismo , Humanos , NADPH Oxidasas/metabolismo , Especies Reactivas de OxígenoRESUMEN
Protein-protein interactions control signaling, specific adhesion, and many other biological functions. The three-dimensional structures of the interfaces and bound ligand can be approached with transferred nuclear Overhauser effect spectroscopy NMR, which can be applied to much larger proteins than conventional NMR and requires less concentrated protein. However, it is not clear how accurately the structures of protein-bound peptides can be determined by transferred nuclear Overhauser effect spectroscopy. We studied the structure of a biotin mimetic peptide (FSHPQNT) bound to streptavidin, because the X-ray structure of the complex is available to 1.74 Å resolution, and we found that conditions could be adjusted so that the off-rates were fast enough for transferred nuclear Overhauser effect spectroscopy NMR. The off-rate was determined with (19)F NMR, using a para-fluoro-phenylalanine analog of the peptide. A new criterion for a lower limit on kinetic off-rate was found, which allowed accurate structure determination at a slower off-rate. Non-specific binding of the peptide to streptavidin was not significant, because biotin blocked the peptide transferred nuclear Overhauser effect spectroscopy. Protein mediation for the long-range peptide transferred nuclear Overhauser effect spectroscopy cross-peaks was corrected by a transferred nuclear Overhauser effect spectroscopy/ROESY averaging procedure. The protein-bound structure of the peptide was determined by transferred nuclear Overhauser effect spectroscopy constrained and simulated annealing. The structure deduced from the NMR was close to the X-ray structure.
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Biotina/química , Modelos Moleculares , Imitación Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Estreptavidina/metabolismo , Secuencia de Aminoácidos , Biotina/metabolismo , Cromatografía Líquida de Alta Presión , Cinética , Unión Proteica , Difracción de Rayos XRESUMEN
BACKGROUND: Energy intake from snacks has been increasing in the American diet, but insulin and glucose responses to foods are generally reported for meal-sized portions (800-1200 kJ). Established methods for insulin determination routinely use indwelling catheters and radioimmunoassay (RIA). The aim of the present study was to develop a more facile method, collecting fingerstick blood samples and measuring insulin with precise ELISA, and then applying this method to determine responses to snack-sized food portions. METHODS: Six healthy, fasting adult volunteers consumed seven different snack foods on separate days, containing approximately 400 kJ/portion. Insulin was measured by ELISA and glucose was measured with the hexokinase procedure in samples collected by fingerstick at 0, 30, and 60 min after consumption of the snack food. RESULTS: A portion of doughnut (half a glazed doughnut) led to marked changes in insulin and glucose; skim milk, an apple, and oatmeal changed insulin significantly; wrinkled peas resulted in a lower glucose response than smooth peas; and walnuts led to non-significant changes in both insulin and glucose over a 60-min period. CONCLUSIONS: The fingerstick sampling and insulin measurement procedure is simple, economical, and more precise than established RIA. The method can be applied to children and adults to monitor insulin responses following food consumption, as well as during therapeutic assessments or intervention trials. Public health advisories regarding snacks that minimize increases in insulin are desirable for individuals trying to reduce or maintain their weight, because elevated insulin stimulates carbohydrate conversion to fat and suppresses the mobilization of stored triglycerides for energy generation.
Asunto(s)
Glucemia/análisis , Ingestión de Alimentos/fisiología , Dedos/irrigación sanguínea , Insulina/sangre , Agujas , Periodo Posprandial/fisiología , Adulto , Capilares , Niño , Ensayo de Inmunoadsorción Enzimática/métodos , Índice Glucémico , HumanosRESUMEN
High-yielding cereals and other staples have produced adequate calories to ward off starvation for much of the world over several decades. However, deficiencies in certain amino acids, minerals, vitamins and fatty acids in staple crops, and animal diets derived from them, have aggravated the problem of malnutrition and the increasing incidence of certain chronic diseases in nominally well-nourished people (the so-called diseases of civilization). Enhanced global nutrition has great potential to reduce acute and chronic disease, the need for health care, the cost of health care, and to increase educational attainment, economic productivity and the quality of life. However, nutrition is currently not an important driver of most plant breeding efforts, and there are only a few well-known efforts to breed crops that are adapted to the needs of optimal human nutrition. Technological tools are available to greatly enhance the nutritional value of our staple crops. However, enhanced nutrition in major crops might only be achieved if nutritional traits are introduced in tandem with important agronomic yield drivers, such as resistance to emerging pests or diseases, to drought and salinity, to herbicides, parasitic plants, frost or heat. In this way we might circumvent a natural tendency for high yield and low production cost to effectively select against the best human nutrition. Here we discuss the need and means for agriculture, food processing, food transport, sociology, nutrition and medicine to be integrated into new approaches to food production with optimal human nutrition as a principle goal.
Asunto(s)
Agricultura , Productos Agrícolas/genética , Trastornos Nutricionales/prevención & control , Plantas Comestibles/genética , Biotecnología , Cruzamiento , Productos Agrícolas/química , Productos Agrícolas/normas , Abastecimiento de Alimentos , Tecnología de Alimentos , Alimentos Fortificados , Humanos , Valor Nutritivo , Plantas Comestibles/químicaRESUMEN
The N-formyl peptide receptor (FPR), a G protein-coupled receptor that binds proinflammatory chemoattractant peptides, serves as a model receptor for leukocyte chemotaxis. Recombinant histidine-tagged FPR (rHis-FPR) was purified in lysophosphatidyl glycerol (LPG) by Ni(2+)-NTA agarose chromatography to >95% purity with high yield. MALDI-TOF mass analysis (>36% sequence coverage) and immunoblotting confirmed the identity as FPR. The rHis-FPR served as an immunogen for the production of 2 mAbs, NFPR1 and NFPR2, that epitope map to the FPR C-terminal tail sequences, 305-GQDFRERLI-313 and 337-NSTLPSAEVE-346, respectively. Both mAbs specifically immunoblotted rHis-FPR and recombinant FPR (rFPR) expressed in Chinese hamster ovary cells. NFPR1 also recognized recombinant FPRL1, specifically expressed in mouse L fibroblasts. In human neutrophil membranes, both Abs labeled a 45-75 kDa species (peak M(r) approximately 60 kDa) localized primarily in the plasma membrane with a minor component in the lactoferrin-enriched intracellular fractions, consistent with FPR size and localization. NFPR1 also recognized a band of M(r) approximately 40 kDa localized, in equal proportions to the plasma membrane and lactoferrin-enriched fractions, consistent with FPRL1 size and localization. Only NFPR2 was capable of immunoprecipitation of rFPR in detergent extracts. The recognition of rFPR by NFPR2 is lost after exposure of cellular rFPR to f-Met-Leu-Phe (fMLF) and regained after alkaline phosphatase treatment of rFPR-bearing membranes. In neutrophils, NFPR2 immunofluorescence was lost upon fMLF stimulation. Immunoblotting approximately 60 kDa species, after phosphatase treatment of fMLF-stimulated neutrophil membranes, was also enhanced. We conclude that the region 337-346 of FPR becomes phosphorylated after fMLF activation of rFPR-expressing Chinese hamster ovary cells and neutrophils.