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1.
Toxicol Appl Pharmacol ; 303: 1-10, 2016 07 15.
Article in English | MEDLINE | ID: mdl-27105553

ABSTRACT

Most studies to evaluate kidney safety biomarkers have been performed in rats. This study was conducted in Cynomolgus monkeys in order to evaluate the potential usefulness of novel biomarkers of nephrotoxicity in this species. Groups of 3 males were given daily intramuscular injections of gentamicin, a nephrotoxic agent known to produce lesions in proximal tubules, at dose-levels of 10, 25, or 50mg/kg/day for 10days. Blood and 16-h urine samples were collected on Days -7, -3, 2, 4, 7, and at the end of the dosing period. Several novel kidney safety biomarkers were evaluated, with single- and multiplex immunoassays and in immunoprecipitation-LC/MS assays, in parallel to histopathology and conventional clinical pathology parameters. Treatment with gentamicin induced a dose-dependent increase in kidney tubular cell degeneration/necrosis, ranging from minimal to mild severity at 10mg/kg/day, moderate at 25mg/kg/day, and to severe at 50mg/kg/day. The results showed that the novel urinary biomarkers, microalbumin, α1-microglobulin, clusterin, and osteopontin, together with the more traditional clinical pathology parameters, urinary total protein and N-acetyl-ß-D-glucosaminidase (NAG), were more sensitive than blood urea nitrogen (BUN) and serum creatinine (sCr) to detect kidney injury in the monkeys given 10mg/kg/day gentamicin for 10days, a dose leading to an exposure which is slightly higher than the desired therapeutic exposure in clinics. Therefore, these urinary biomarkers represent non-invasive biomarkers of proximal tubule injury in Cynomolgus monkeys which may be potentially useful in humans.


Subject(s)
Anti-Bacterial Agents/toxicity , Gentamicins/toxicity , Kidney Diseases/chemically induced , Kidney Diseases/urine , Acetylglucosaminidase/urine , Alanine Transaminase/blood , Alpha-Globulins/urine , Animals , Anti-Bacterial Agents/blood , Anti-Bacterial Agents/pharmacokinetics , Aspartate Aminotransferases/blood , Biomarkers/blood , Biomarkers/urine , Blood Glucose/analysis , Blood Urea Nitrogen , Clusterin/urine , Creatine/blood , Creatine/urine , Gentamicins/blood , Gentamicins/pharmacokinetics , Kidney/drug effects , Kidney/metabolism , Kidney/pathology , Kidney Diseases/blood , Kidney Diseases/pathology , Macaca fascicularis , Male , Necrosis/chemically induced , Osteopontin/urine , Serum Albumin/analysis
2.
Biofouling ; 31(9-10): 699-707, 2015.
Article in English | MEDLINE | ID: mdl-26479780

ABSTRACT

The biofouling affinity of different polymeric surfaces (polypropylene, polysulfone, polyethylene terephthalate, and polyether ether ketone) in comparison to stainless steel (SS) was studied for the model bacterium Escherichia coli K12 DSM 498 and native biofilms originating from Rhine water. The biofilm mass deposited on the polymer surfaces was minimized by several magnitudes compared to SS. The cell count and the accumulated biomass of E. coli on the polymer surfaces showed an opposing linear trend. The promising low biofilm formation on the polymers is attributed to the combination of inherent surface properties (roughness, surface energy and hydrophobicity) when compared to SS. The fouling characteristics of E. coli biofilms show good conformity with the more complex native biofilms investigated. The results can be utilized for the development of new polymer heat exchangers when using untreated river water as coolant or for other processes needing antifouling materials.


Subject(s)
Biofilms/growth & development , Biofouling , Escherichia coli/growth & development , Hot Temperature , Polymers/chemistry , Stainless Steel/chemistry , Equipment Design , Hydrophobic and Hydrophilic Interactions , Models, Theoretical , Polyethylene Glycols/chemistry , Rivers/microbiology , Sulfones/chemistry , Surface Properties , Water Microbiology
3.
J Pharm Biomed Anal ; 115: 552-61, 2015 Nov 10.
Article in English | MEDLINE | ID: mdl-26319748

ABSTRACT

The underlying pharmacokinetic profile of liposomal drug delivery systems is not yet fully known. This is primarily due to a lack of suitable quantitative bioanalytical methodology to simultaneously determine separate liposomal-encapsulated and non-encapsulated drug tissue concentrations in complex biological samples. Here, an LC-MS method was developed which enables the simultaneous quantification of separate liposomal-encapsulated prednisolone phosphate and non-encapsulated prednisolone concentrations in whole blood and liver tissue. Liquid chromatography, negative electrospray ionization and Orbitrap-MS analysis allowed highly accurate and sensitive detection of prednisolone phosphate (PP) and prednisolone (P) in complex matrix. Using dexamethasone phosphate and dexamethasone as internal standards, the quantitative LC-MS method was optimized and validated for high selectivity, sensitivity and quantitative accuracy of PP and P from liposomes. The lower limits of quantitation were 0.99µmol/L blood and 0.53nmol/g liver for PP, and 229nmol/L blood and 0.514nmol/g liver for P. Quantitative accuracies of 84-118% were observed. The intra-run precision was ≤11%. Application of this new LC-MS method will yield the first liposomal pharmacokinetic profile showing accurate encapsulated and non-encapsulated drug tissue concentrations separately. This is also the first quantitative LC-MS method for the simultaneous quantification of the prodrug PP and its parent drug P in whole blood and liver tissue samples.


Subject(s)
Chromatography, Liquid , Glucocorticoids/blood , Liver/metabolism , Prednisolone/analogs & derivatives , Spectrometry, Mass, Electrospray Ionization , Technology, Pharmaceutical/methods , Animals , Calibration , Chemistry, Pharmaceutical , Chromatography, Liquid/standards , Glucocorticoids/administration & dosage , Glucocorticoids/chemistry , Glucocorticoids/pharmacokinetics , Limit of Detection , Liposomes , Male , Mice, Inbred C57BL , Prednisolone/administration & dosage , Prednisolone/blood , Prednisolone/chemistry , Prednisolone/pharmacokinetics , Reference Standards , Reproducibility of Results , Spectrometry, Mass, Electrospray Ionization/standards , Tissue Distribution
4.
Sci Rep ; 5: 8759, 2015 Mar 04.
Article in English | MEDLINE | ID: mdl-25737130

ABSTRACT

Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system.


Subject(s)
Hepatocytes/enzymology , Hepatocytes/metabolism , Mass Spectrometry/methods , Peptides/metabolism , ATP Binding Cassette Transporter, Subfamily B/immunology , ATP Binding Cassette Transporter, Subfamily B/metabolism , Amino Acid Sequence , Antibodies/immunology , Antibody Affinity , Aryl Hydrocarbon Hydroxylases/immunology , Aryl Hydrocarbon Hydroxylases/metabolism , Atorvastatin/pharmacokinetics , Cells, Cultured , Chromatography, Liquid/methods , Cytochrome P-450 CYP3A/immunology , Cytochrome P-450 CYP3A/metabolism , Epitopes/immunology , Epitopes/metabolism , Hepatocytes/cytology , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacokinetics , Peptides/immunology , Pravastatin/pharmacokinetics , Primary Cell Culture , Sequence Homology, Amino Acid
5.
BMC Genomics ; 15: 1154, 2014 Dec 20.
Article in English | MEDLINE | ID: mdl-25528190

ABSTRACT

BACKGROUND: The human neuroblastoma cell line, SH-SY5Y, is a commonly used cell line in studies related to neurotoxicity, oxidative stress, and neurodegenerative diseases. Although this cell line is often used as a cellular model for Parkinson's disease, the relevance of this cellular model in the context of Parkinson's disease (PD) and other neurodegenerative diseases has not yet been systematically evaluated. RESULTS: We have used a systems genomics approach to characterize the SH-SY5Y cell line using whole-genome sequencing to determine the genetic content of the cell line and used transcriptomics and proteomics data to determine molecular correlations. Further, we integrated genomic variants using a network analysis approach to evaluate the suitability of the SH-SY5Y cell line for perturbation experiments in the context of neurodegenerative diseases, including PD. CONCLUSIONS: The systems genomics approach showed consistency across different biological levels (DNA, RNA and protein concentrations). Most of the genes belonging to the major Parkinson's disease pathways and modules were intact in the SH-SY5Y genome. Specifically, each analysed gene related to PD has at least one intact copy in SH-SY5Y. The disease-specific network analysis approach ranked the genetic integrity of SH-SY5Y as higher for PD than for Alzheimer's disease but lower than for Huntington's disease and Amyotrophic Lateral Sclerosis for loss of function perturbation experiments.


Subject(s)
Genomics , Neuroblastoma/pathology , Parkinson Disease/genetics , Cell Line, Tumor , DNA Copy Number Variations , DNA Transposable Elements/genetics , Gene Expression Profiling , Genetic Variation , Humans , INDEL Mutation , Proteomics
6.
Biochim Biophys Acta ; 1844(5): 927-32, 2014 May.
Article in English | MEDLINE | ID: mdl-24060810

ABSTRACT

Mass spectrometry-based (MS) methods are effective tools for discovering protein biomarker candidates that can differentiate between physiological and pathophysiological states. Promising candidates are validated in studies comprising large patient cohorts. Here, targeted protein analytics are used to increase sample throughput. Methods involving antibodies, such as sandwich immunoassays or Western blots, are commonly applied at this stage. Highly-specific and sensitive mass spectrometry-based immunoassays that have been established in recent years offer a suitable alternative to sandwich immunoassays for quantifying proteins. Mass Spectrometric ImmunoAssays (MSIA) and Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA/iMALDI) are two prominent types of MS-based immunoassays in which the capture is done either at the protein or the peptide level. We present an overview of these emerging types of immunoassays and discuss their suitability for the discovery and validation of protein biomarkers. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.


Subject(s)
Biomarkers/analysis , Chromatography, Affinity/methods , Disease , Immunoassay/methods , Mass Spectrometry/methods , Pathology, Molecular , Proteins/metabolism , Humans , Peptide Fragments/analysis
7.
Cell Mol Life Sci ; 70(19): 3709-22, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23739949

ABSTRACT

The Tyrolean Iceman, a Copper-age ice mummy, is one of the best-studied human individuals. While the genome of the Iceman has largely been decoded, tissue-specific proteomes have not yet been investigated. We studied the proteome of two distinct brain samples using gel-based and liquid chromatography-mass spectrometry-based proteomics technologies together with a multiple-databases and -search algorithms-driven data-analysis approach. Thereby, we identified a total of 502 different proteins. Of these, 41 proteins are known to be highly abundant in brain tissue and 9 are even specifically expressed in the brain. Furthermore, we found 10 proteins related to blood and coagulation. An enrichment analysis revealed a significant accumulation of proteins related to stress response and wound healing. Together with atomic force microscope scans, indicating clustered blood cells, our data reopens former discussions about a possible injury of the Iceman's head near the site where the tissue samples have been extracted.


Subject(s)
Brain Chemistry , Mummies , Proteome/genetics , Proteome/metabolism , Biopsy/methods , Brain/metabolism , Genome, Human , Humans
8.
J Child Psychol Psychiatry ; 54(9): 969-76, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23452272

ABSTRACT

BACKGROUND: A deficit in affective rather than cognitive empathy is thought to be central to psychopathic traits. However, empirical evidence for empathy deficits in adolescents with psychopathic traits is limited. We investigated the concurrent and prospective effects of psychopathic traits on affective and cognitive trait empathy in late adolescence. METHODS: A community sample of 107 males and 126 females who were approximately 16-year olds at Time 1 participated in four annual waves. Sex-specific classes of adolescents' psychopathic traits were created using Latent Class Analyses. Subsequently, we investigated class differences in level and development of empathy. RESULTS: For both sexes, Latent Class Analyses produced two classes: one class with low and one with moderate levels of psychopathic traits. Consistent with our hypothesis, for both sexes, adolescents with moderate levels of psychopathic traits reported lower mean levels of affective empathy than adolescents with low levels of psychopathic traits. In addition, female adolescents with moderate psychopathic traits reported lower mean levels of cognitive empathy. Male adolescents showed a trend in this direction. No differences between classes were found in development of empathy, which increased over years. CONCLUSIONS: This is the first study to show that male and female adolescents with higher levels of psychopathic traits have lower levels of affective empathy not only concurrently but also prospectively over a 3-year period. Females additionally showed a similar pattern on cognitive empathy. In this community sample, developmental results suggest that adolescents with higher levels of psychopathic traits have relative rather than absolute empathy deficits.


Subject(s)
Antisocial Personality Disorder/psychology , Empathy , Adolescent , Affect , Age Factors , Cognition , Female , Humans , Male , Prospective Studies , Psychiatric Status Rating Scales , Psychological Tests , Psychology, Adolescent , Sex Factors , Young Adult
9.
PLoS One ; 7(11): e50339, 2012.
Article in English | MEDLINE | ID: mdl-23166843

ABSTRACT

Recent studies have shown that while psychopathy and non-psychopathic antisociality overlap, they differ in the extent to which cognitive impairments are present. Specifically, psychopathy has been related to abnormal allocation of attention, a function that is traditionally believed to be indexed by event-related potentials (ERPs) of the P3-family. Previous research examining psychophysiological correlates of attention in psychopathic individuals has mainly focused on the parietally distributed P3b component to rare targets. In contrast, very little is known about the frontocentral P3a to infrequent novel events in psychopathy. Thus, findings on the P3 components in psychopathy are inconclusive, while results in non-psychopathic antisocial populations are clearer and point toward an inverse relationship between antisociality and P3 amplitudes. The present study adds to extant literature on the P3a and P3b in psychopathy by investigating component amplitudes in psychopathic offenders (N = 20), matched non-psychopathic offenders (N = 23) and healthy controls (N = 16). Also, it was assessed how well each offender group was able to differentially process rare novel and target events. The offender groups showed general amplitude reductions compared to healthy controls, but did not differ mutually on overall P3a/P3b amplitudes. However, the psychopathic group still exhibited normal neurophysiological differentiation when allocating attention to rare novel and target events, unlike the non-psychopathic sample. The results highlight differences between psychopathic and non-psychopathic offenders regarding the integrity of the neurocognitive processes driving attentional allocation, as well as the usefulness of alternative psychophysiological measures in differentiating psychopathy from general antisociality.


Subject(s)
Antisocial Personality Disorder/diagnosis , Antisocial Personality Disorder/physiopathology , Attention/physiology , Cognition/physiology , Criminals/psychology , Event-Related Potentials, P300/physiology , Adult , Diagnosis, Differential , Electroencephalography , Humans , Linear Models , Middle Aged , Netherlands
10.
Rapid Commun Mass Spectrom ; 26(23): 2777-85, 2012 Dec 15.
Article in English | MEDLINE | ID: mdl-23124669

ABSTRACT

RATIONALE: Isobaric labeling strategies (e.g. iTRAQ or TMT) are commonly applied in tandem mass spectrometric (MS/MS) level quantitative proteomics. However, we frequently observed missing isotope reporter ion signals in a large-scale liquid chromatography/matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometric (LC/MALDI-TOF/TOF) quantitative proteomics experiment. To understand this issue, we systematically investigated the processing of MS/MS spectra into peak lists prior to peptide identification and quantification. METHODS: A 15-protein standard, with six proteins in different concentrations, was labeled with iTRAQ 4-plex, iTRAQ 8-plex or TMT 6-plex, tryptic digested and measured using LC/MALDI-TOF/TOF. Three commercially and open-source available peak list generation software tools were compared based on missing reporter ions, peptide identification and quantification. RESULTS: We found that each tool discarded lower-intensity reporter ions, when they followed a higher intensity reporter ion, due to the implemented de-isotoping algorithms. By using the non-de-isotoping setting within TS2Mascot, we found that all reporter ions are exported, yet less peptides were identified with Mascot. Therefore, we developed a strategy merging the de-isotoped and non-de-isotoped outputs from TS2Mascot using the Perl script RICmerge.pl. CONCLUSIONS: With this approach, we correctly quantified all labeled peptides that were identified within the 15-protein standard. This strategy allows improved annotation of isobaric tag labeled peptide MS/MS spectra and improves downstream peptide and protein quantification in proteomics studies.


Subject(s)
Proteins/chemistry , Proteomics/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Animals , Chromatography, High Pressure Liquid/methods , Humans , Peptides/analysis , Tandem Mass Spectrometry/methods
11.
Proteomics ; 12(4-5): 516-29, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22246699

ABSTRACT

Protease-catalyzed hydrolysis of peptide bonds is one of the most pivotal post-translational modifications fulfilling manifold functions in the regulation of cellular processes. Therefore, dysregulation of proteolytic reactions plays a central role in many pathophysiological events. For this reason, understanding the molecular mechanisms in proteolytic reactions, in particular the knowledge of proteases involved in complex processes, expression levels and activity of protease and knowledge of the targeted substrates are an indispensable prerequisite for targeted drug development. The present review focuses on mass spectrometry-based proteomic methods for the analysis of protease cleavage sites, including the identification of the hydrolyzed bonds as well as of the surrounding sequence. Peptide- and protein-centric approaches and bioinformatic tools for experimental data interpretation will be presented and the major advantages and drawbacks of the different approaches will be addressed. The recent applications of these approaches for the analysis of biological function of different protease classes and potential future directions will be discussed.


Subject(s)
Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Proteins/metabolism , Proteomics/methods , Databases, Protein , Mass Spectrometry/methods
12.
PLoS One ; 5(5): e10642, 2010 May 14.
Article in English | MEDLINE | ID: mdl-20498845

ABSTRACT

One motivation of systems biology research is to understand gene functions and interactions from functional genomics data such as that derived from microarrays. Up-to-date structural and functional annotations of genes are an essential foundation of systems biology modeling. We propose that the first essential step in any systems biology modeling of functional genomics data, especially for species with recently sequenced genomes, is gene structural and functional re-annotation. To demonstrate the impact of such re-annotation, we structurally and functionally re-annotated a microarray developed, and previously used, as a tool for disease research. We quantified the impact of this re-annotation on the array based on the total numbers of structural- and functional-annotations, the Gene Annotation Quality (GAQ) score, and canonical pathway coverage. We next quantified the impact of re-annotation on systems biology modeling using a previously published experiment that used this microarray. We show that re-annotation improves the quantity and quality of structural- and functional-annotations, allows a more comprehensive Gene Ontology based modeling, and improves pathway coverage for both the whole array and a differentially expressed mRNA subset. Our results also demonstrate that re-annotation can result in a different knowledge outcome derived from previous published research findings. We propose that, because of this, re-annotation should be considered to be an essential first step for deriving value from functional genomics data.


Subject(s)
Databases, Genetic , Genomics/methods , Models, Genetic , Systems Biology/methods , Animals , Chickens/genetics , Gene Expression Profiling , Gene Expression Regulation , Gene Regulatory Networks/genetics , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/genetics
13.
Health Policy ; 97(1): 44-52, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20347179

ABSTRACT

OBJECTIVES: To assess whether delays to outpatient specialty care can be solved by improving the way supply and demand are matched, without adding capacity. METHODS: A systematic review of the interventions applied by 18 clinics using the model of 'advanced access' and a statistical analysis of the effects of the interventions on their delays. RESULTS: The clinics applied different combinations of interventions aimed at improving the way they match supply and demand, improving the efficiency of the way supply is organised and at reducing unnecessary demand. Fourteen clinics show statistically significant improvements. Two probably significantly improved and two clinics did not. Their access reduced on average 55%, from 47 to 21 days. CONCLUSIONS: It seems that delays in outpatient specialty care can be solved to a large extend by improving the way supply and demand are matched. Policy makers should analyse whether delays are caused by capacity problems or matching problems. For the latter, it appears more effective to invest in the ability to react then the ability to plan. Policy makers should create incentives for clinics to keep access short and remove incentives that stimulate delays.


Subject(s)
Ambulatory Care/organization & administration , Medicine/organization & administration , Ambulatory Care/statistics & numerical data , Health Facility Size/organization & administration , Health Services Accessibility/organization & administration , Health Services Accessibility/statistics & numerical data , Health Services Needs and Demand/organization & administration , Health Services Needs and Demand/statistics & numerical data , Humans , Medicine/statistics & numerical data , Models, Organizational , Time Factors , Waiting Lists
14.
BMC Bioinformatics ; 10 Suppl 11: S9, 2009 Oct 08.
Article in English | MEDLINE | ID: mdl-19811693

ABSTRACT

UNLABELLED: The widespread availability of microarray technology has driven functional genomics to the forefront as scientists seek to draw meaningful biological conclusions from their microarray results. Gene annotation enrichment analysis is a functional analysis technique that has gained widespread attention and for which many tools have been developed. Unfortunately, most of these tools have limited support for agricultural species. Here, we evaluate and compare four publicly available computational tools (Onto-Express, EasyGO, GOstat, and DAVID) that support analysis of gene expression datasets in agricultural species. We use AgBase as the functional annotation reference for agricultural species. The selected tools were evaluated based on i) available features, usage and accessibility, ii) implemented statistical computational methods, and iii) annotation and enrichment performance analysis. Annotation was assessed using a randomly selected test gene annotation set and an experimental differentially expressed gene-set--both from chicken. The experimental set was also used to evaluate identification of enriched functional groups.Comparison of the tools shows that they produce different sets of annotations for the two datasets and different functional groups for the experimental dataset. While DAVID, GOstat and Onto-Express annotate comparable numbers of genes, DAVID provides by far the most annotations per gene. However, many of DAVID's annotations appear to be redundant or are at very high levels in the GO hierarchy. The GOSlim distribution of annotations shows that GOstat, Onto-Express and EasyGO provide similar GO distributions to those found in AgBase while annotations from DAVID show a different GOSlim distribution, again probably due to duplication and many non-specific terms. No consistent trends were found in results of GO term over/under representation analysis applied to the experimental data using different tools. While GOstat, David and Onto-Express could retrieve some significantly enriched terms, EasyGO did not show any significantly enriched terms. There was little agreement about the enriched terms identified by the tools. CONCLUSION: Different tools for functionally annotating gene sets and identifying significantly enriched GO categories differ widely in their results when applied to a test annotation gene set and an experimental dataset from chicken. These results emphasize the need for care when interpreting the results of such analysis and the lack of standardization of approaches.


Subject(s)
Agriculture , Chickens/genetics , Computational Biology/methods , Oligonucleotide Array Sequence Analysis , Animals , Databases, Genetic , Expressed Sequence Tags , Gene Expression Profiling
15.
Rapid Commun Mass Spectrom ; 21(23): 3905-9, 2007.
Article in English | MEDLINE | ID: mdl-17990261

ABSTRACT

Non-electrophoretic methods based on two-dimensional liquid chromatography followed directly by tandem mass spectrometry (2D-LC/MS(2)) have become the preferred method for high-throughput expression proteomics and are widely applied to fresh tissues. Pre-fractionation techniques are also used in combination with 2D-LC/MS(2) to both increase the proteome size and to assign cellular locations. Data from such experiments have become central to systems biology analyses. Here we apply a differential detergent (pre)fractionation (DDF) followed by 2D-LC/MS(2) to frozen archival tissues. Our results show that by using frozen archival tissues, we do not lose proteome coverage or the ability to assign proteins to cellular compartments. In addition, we were able to assign 'biological process' Gene Ontology (GO) annotations, which will facilitate systems biological modeling of our proteomics data.


Subject(s)
Chemical Fractionation/methods , Chromatography, High Pressure Liquid/methods , Detergents/chemistry , Peptide Mapping/methods , Proteome/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Spleen/metabolism , Animals , Chickens , Cryopreservation/methods , Electrophoresis , Organ Culture Techniques , Organ Preservation/methods , Proteome/analysis
16.
J Proteome Res ; 4(2): 316-24, 2005.
Article in English | MEDLINE | ID: mdl-15822906

ABSTRACT

Differential detergent fractionation (DDF), which relies on detergents to sequentially extract proteins from eukaryotic cells, has been used to increase proteome coverage of 2D-PAGE. Here, we used DDF extraction in conjunction with the nonelectrophoretic proteomics method of liquid chromatography and electrospray ionization tandem mass spectrometry. We demonstrate that DDF can be used with 2D-LC ESI MS2 for comprehensive cellular proteomics, including a large proportion of membrane proteins. Compared to some published methods designed to isolate membrane proteins specifically, DDF extraction yields comprehensive proteomes which include twice as many membrane proteins. Two-thirds of these membrane proteins have more than one trans-membrane domain. Since DDF separates proteins based upon their physicochemistry and subcellular localization, this method also provides data useful for functional genome annotation. As more genome sequences are completed, methods which can aid in functional annotation will become increasingly important.


Subject(s)
Detergents/chemistry , Proteins/isolation & purification , Proteomics , Animals , Chickens , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Eukaryotic Cells/chemistry , Spectrometry, Mass, Electrospray Ionization
17.
Biotechnol Bioeng ; 59(5): 525-33, 1998 Sep 05.
Article in English | MEDLINE | ID: mdl-10099368

ABSTRACT

The influence of the pellet morphology of the basidiomycete Cyathus striatus on the production of the antibiotics striatals A, B, and C was investigated. The main operating parameters in fermenters of different sizes were the tip speed and the volumetric power input. Different methods were developed for quantification of morphological characteristics. The apparent viscosity of the suspension was measured with a cylinder rheometer. Sediment density was measured with a sedimentation apparatus. Particle size distributions were recorded with an image analysis system. By means of the presented measuring methods, morphological characteristics could be determined and enabled an early assessment of the fermentation course and the antibiotics production. During the exponential growth phase of the fungus the relative sediment height correlated with the biomass concentration. The pellet morphology at this stage influenced the later production of striatals. The yield of the striatals was markedly influenced by pellet size and sediment density. Since these morphological characteristics determine the rheological properties of the culture the measurement of the apparent viscosity of the culture in the production phase allowed predictions of the production yield.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Basidiomycota/metabolism , Fermentation , Models, Chemical , Models, Statistical , Rheology/instrumentation , Time Factors
18.
J Chromatogr A ; 763(1-2): 49-56, 1997 Feb 28.
Article in English | MEDLINE | ID: mdl-9129314

ABSTRACT

Bovine serum albumin (BSA) was continuously desalted from alkaline salts (a mixture of sodium phosphate, sodium chloride and potassium chloride) using a size-exclusion gel as the stationary phase. An annular chromatograph was used to achieve a continuous mode of operation and therefore a reasonable throughput. Distribution and mass transfer coefficients of the substances as well as bed properties were obtained by batch chromatography. These separations were simulated mathematically applying an approximate linear chromatographic theory. It was shown experimentally and theoretically that the BSA and the salt solution could be recovered continuously in a purity higher than 98%. The influence of rotation rate on the resolution of the individual peaks was investigated.


Subject(s)
Chromatography/methods , Phosphates , Potassium Chloride , Serum Albumin, Bovine/isolation & purification , Sodium Chloride , Chromatography/instrumentation , Chromatography, Gel , Mathematics , Rotation
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