Biomedical subjects

John M Arthur

Publications and source records attributed to John M Arthur.

12 recordsLinked to original sources

PubMed2006

Proteomics in renal research.

Proteomic technologies are used with increasing frequency in the renal community. In this review, we highlight the use in renal research of a number of available techniques including two-dimensional gel electrophoresis, liquid chromatography/mass spectrometry, surface-enhanced laser desorption/ionization, capillary electrophoresis/mass spectrometry, and antibody and tissue arrays. These techniques have been used to identify proteins or changes in proteins specific to regions of the kidney or associated with renal diseases or toxicity. They have also been used to examine protein expression changes and posttranslational modifications of proteins during signaling. A number of studies have used proteomic methodologies to look for diagnostic biomarkers in body fluids. The rapid rate of development of the technologies along with the combination of classic physiological and biochemical techniques with proteomics will enable new discoveries.

Chromatography, Liquid↗

PubMed2006

Comparison of variability associated with sample preparation in two-dimensional gel electrophoresis of cardiac tissue.

Variability is a major complicating factor in analysis by two-dimensional gel electrophoresis. Improvements in methodologies have focused on improving individual gel quality rather than reproducibility. We homogenized rat cardiac tissue and rehydrated using a matrix of buffers to determine the optimal sample conditions. Six buffers were used to solubilize the proteins. Solubilized proteins were separated by isoelectric focusing using four buffers. Gels were run in triplicate to assess the method of preparation yielding the least variability. Number of spots and variability were different between conditions. Proteins solubilized in a buffer containing 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10, ampholytes, DTT, and protease inhibitors and focused in a buffer containing 9 M urea and 4% NP40 had the lowest coefficient of variation. Variability was compared across isoelectric point ranges and was different. Minimizing technical variability in two-dimensional polyacrylamide gel electrophoresis is critical to identify differences between conditions. Sample preparation should be optimized to minimize variability as well as to maximize the number of spots seen.

Animals↗

PubMed2005

Normalization and analysis of residual variation in two-dimensional gel electrophoresis for quantitative differential proteomics.

Although two-dimensional gel electrophoresis (2-DE) has long been a favorite experimental method to screen proteomes, its reproducibility is seldom analyzed with the assistance of quantitative error models. The lack of models of residual distributions that can be used to assign likelihood to differential expression reflects the difficulty in tackling the combined effect of variability in spot intensity and uncertain recognition of the same spot in different gels. In this report we have analyzed a series of four triplicate two-dimensional gels of chicken embryo heart samples at two distinct development stages to produce such a model of residual distribution. In order to achieve this reference error model, a nonparametric procedure for consistent spot intensity normalization had to be established, and is also reported here. In addition to variability in normalized intensity due to various sources, the residual variation between replicates was observed to be compounded by failure to identify the spot itself (gel alignment). The mixed effect is reflected by variably skewed bimodal density distributions of residuals. The extraction of a global error model that accommodated such distribution was achieved empirically by machine learning, specifically by bootstrapped artificial neural networks. The model described is being used to assign confidence values to observed variations in arbitrary 2-DE gels in order to quantify the degree of over-expression and under-expression of protein spots.

Animals↗

PubMed2005

Prediction of urinary protein markers in lupus nephritis.

BACKGROUND: Lupus nephritis is divided into six classes and scored according to activity and chronicity indices based on histologic findings. Treatment differs based on the pathologic findings. Renal biopsy is currently the only way to accurately predict class and activity and chronicity indices. We propose to use patterns of abundance of urine proteins to identify class and disease indices. METHODS: Urine was collected from 20 consecutive patients immediately prior to biopsy for evaluation of lupus nephritis. The International Society of Nephrology/Renal Pathology Society (ISN/RPS) class of lupus nephritis, activity, and chronicity indices were determined by a renal pathologist. Proteins were separated by two-dimensional gel electrophoresis. Artificial neural networks were trained on normalized spot abundance values. RESULTS: Biopsy specimens were classified in the database according to ISN/RPS class, activity, and chronicity. Nine samples had characteristics of more than one class present. Receiver operating characteristic (ROC) curves of the trained networks demonstrated areas under the curve ranging from 0.85 to 0.95. The sensitivity and specificity for the ISN/RPS classes were class II 100%, 100%; III 86%, 100%; IV 100%, 92%; and V 92%, 50%. Activity and chronicity indices had r values of 0.77 and 0.87, respectively. A list of spots was obtained that provided diagnostic sensitivity to the analysis. CONCLUSION: We have identified a list of protein spots that can be used to develop a clinical assay to predict ISN/RPS class and chronicity for patients with lupus nephritis. An assay based on antibodies against these spots could eliminate the need for renal biopsy, allow frequent evaluation of disease status, and begin specific therapy for patients with lupus nephritis.

Adult↗

PubMed2003

Sodium loading changes urinary protein excretion: a proteomic analysis.

Plasma sodium concentration is maintained even when sodium intake is altered. Sodium homeostasis may involve changes in renal tubular protein expression that are reflected in the urine. We used proteomic analysis to investigate changes in urinary protein excretion in response to acute sodium loading. Rats were given deionized water followed by hypertonic (2.7%) saline for 28 h each. Urinary protein expression was determined during the final 4 h of each treatment. Acute sodium loading increased urinary sodium excretion (4.53 +/- 1.74 vs. 1.70 +/- 0.27 mmol/day, P = 0.029). Urinary proteins were separated by two-dimensional PAGE and visualized by Sypro ruby staining. Differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry followed by peptide mass fingerprinting. The abundance of a total of 45 protein components was changed after acute sodium loading. Neutral endopeptidase, solute carrier family 3, meprin 1alpha, diphor-1, chaperone heat shock protein 72, vacuolar H(+)-ATPase, ezrin, ezrin/radixin/moesin-binding protein, glutamine synthetase, guanine nucleotide-binding protein, Rho GDI-1, and chloride intracellular channel protein 1 were decreased, whereas albumin and alpha-2u globulin were increased. Some of these proteins have previously been shown to be associated with tubular transport. These data indicate that alterations in the excretion of several urinary proteins occur during acute sodium loading.

Amino Acid Sequence↗

PubMed2003

Proteomics.

PURPOSE OF REVIEW: Proteomics encompasses a group of technologies that attempt to separate, identify and characterize a global set of proteins. This review will highlight the technologies available, outline the capabilities, advantages and disadvantages of each and briefly describe applications in nephrology. RECENT FINDINGS: Proteomics provides information about abundance, location, chemical modification and protein-protein interactions that is not available from genomic technologies. Several proteomic approaches are now widely available. Liquid chromatography/mass spectrometry, two-dimensional gel electrophoresis, antibody arrays and protein chips (surface enhanced laser desorption ionization) provide opportunities to identify and compare an abundance of proteins as well as to determine posttranslational modifications, subcellular location and molecular interactions. Recent advances such as multidimensional chromatographic analysis and isotope coded affinity tags have expanded the usefulness of these approaches. SUMMARY: Proteomic technologies are improving and developing rapidly. These techniques will be valuable tools to develop markers for disease, identify and evaluate proteins as drug targets and understand renal physiology at the protein level.

Antibodies↗

PubMed2003

Proteomic identification of a large complement of rat urinary proteins.

The characterization of urinary proteins is an important tool to identify disease-related biomarkers and to better understand renal physiology. Expression of urinary proteins has been previously studied by Western blotting and other immunological methods. The scope of such studies, however, is limited to previously identified proteins for which specific antibodies are existed. We used proteomic analysis to identify proteins and to construct a proteome map for Sprague-Dawley (SD) rat urine isolated by ultracentrifugation. Urinary proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and visualized by silver staining. Proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), followed by peptide mass fingerprinting using the NCBI protein database. A total of 350 protein spots were visualized. From 250 excised spots, 111 protein components were identified including transporters, transport regulators, chaperones, enzymes, signaling proteins, cytoskeletal proteins, pheromone-binding proteins, receptors, and novel gene products. The presence of a number of these identified proteins was unexpected and had not previously been identified in the urine. 2-D Western blot analyses for randomly selected proteins (ezrin, HSP70, beta- and gamma-actin, Rho-GDI, and l-myc) clearly confirmed the proteomic identification. Several potential posttranslational modifications were predicted by bioinformatic analyses. These data indicate that a large complement of expected and unexpected urinary proteins can be simultaneously studied by proteomic analysis. This approach may lead to better understanding of renal physiology and pathophysiology, and to biomarker discovery.

Actins↗

PubMed2002

Efficient adenylyl cyclase activation by a beta2-adrenoceptor-G(i)alpha2 fusion protein.

The G-protein G(i)alpha can activate adenylyl cyclase (AC), but the relevance of this AC activation is unknown. We used receptor-G protein co-expression and receptor-G protein fusion proteins to investigate G(i)alpha(2) regulation of AC in Sf9 cells. G(i)alpha(2) was fused to the beta(2)-adrenoceptor (beta(2)AR), a preferentially G(s)-coupled receptor, or the formyl peptide receptor (FPR), a G(i)-coupled receptor. The FPR co-expressed with, or fused to, G(i)alpha(2), reduced AC activity. In contrast, the beta(2)AR fused to G(i)alpha(2) was a highly efficient AC activator, while the beta(2)AR co-expressed with G(i)alpha(2) was not. Agonist efficiently stimulated incorporation of [alpha-32P]GTP azidoanilide into beta(2)AR-G(i)alpha(2). We explain AC activation by beta(2)AR-G(i)alpha(2) by a model in which there is interaction of the beta(2)AR and AC, preventing tethered G(i)alpha(2) from interacting with the inhibitory G(i)alpha site of AC. The postulated beta(2)AR/AC interaction brings G(i)alpha(2) into close proximity of the G(s)alpha site of AC, enabling G(i)alpha(2) to activate AC.

Adenylyl Cyclases↗

PubMed2002

Proteomic analysis reveals alterations in the renal kallikrein pathway during hypoxia-induced hypertension.

Obstructive sleep apnea syndrome (OSAS), a disorder characterized by episodic hypoxia (EH) during sleep, is associated with systemic hypertension. We used proteomic analysis to examine differences in rat kidney protein expression during EH, and their potential relationship to EH-induced hypertension. Young male Sprague-Dawley rats were exposed to either EH or sustained hypoxia (SH) for 14 (EH14/SH14) and 30 (EH30/SH30) days. Mean arterial blood pressure was significantly increased only in EH30 (p < 0.0002). Kidney proteins were resolved by two-dimensional-PAGE and were identified by MALDI-MS. Renal expression of kallistatin, a potent vasodilator, was down-regulated in all animals. Expression of alpha-1-antitrypsin, an inhibitor of kallikrein activation, was up-regulated in EH but down-regulated in SH. Western blotting showed significant elevation of B(2)-bradykinin receptor expression in all normotensive animals but remained unchanged in hypertensive animals. Proteins relevant to vascular hypertrophy, such as smooth muscle myosin and protein-disulfide isomerase were up-regulated in EH30 but were down-regulated in SH30. These data indicate that EH induces changes in renal protein expression consistent with impairment of vasodilation mediated by the kallikrein-kallistatin pathway and vascular hypertrophy. In contrast, SH-induced changes suggest the kallikrein- and bradykinin-mediated compensatory mechanisms for prevention of hypertension and vascular remodeling. To test the hypothesis suggested by the proteomic data, we measured the effect of EH on blood pressure in transgenic hKLK1 rats that overexpress human kallikrein. Transgenic hKLK1 animals were protected from EH-induced hypertension. We conclude that EH-induced hypertension may result, at least in part, from altered regulation of the renal kallikrein system.

Amino Acid Sequence↗

PubMed2002

Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation.

BACKGROUND: Proteomic techniques have recently become available for large-scale protein analysis. The utility of these techniques in identification of urinary proteins is poorly defined. We constructed a proteome map of normal human urine as a reference protein database by using two differential fractionated techniques to isolate the proteins. METHODS: Proteins were isolated from urine obtained from normal human volunteers by acetone precipitation or ultracentrifugation, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry followed by peptide mass fingerprinting. RESULTS: A total of 67 protein forms of 47 unique proteins were identified, including transporters, adhesion molecules, complement, chaperones, receptors, enzymes, serpins, cell signaling proteins and matrix proteins. Acetone precipitated more acidic and hydrophilic proteins, whereas ultracentrifugation fractionated more basic, hydrophobic, and membrane proteins. Bioinformatic analysis predicted glycosylation to be the most common explanation for multiple forms of the same protein. CONCLUSIONS: Combining two differential isolation techniques magnified protein identification from human urine. Proteomic analysis of urinary proteins is a promising tool to study renal physiology and pathophysiology and to determine biomarkers of renal disease.

Acetone↗

PubMed2002

Differential expression of proteins in renal cortex and medulla: a proteomic approach.

BACKGROUND: Western blotting has previously been used to identify changes in protein expression in renal tissue. However, only a few proteins can be studied in each experiment by Western blot. We have used proteomic tools to construct protein maps of rat kidney cortex and medulla. METHODS: Expression of proteins was determined by silver stain after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein spots were excised and digested with trypsin. Peptide masses were identified by MALDI-TOF mass spectrometry. The Mascot search engine was used to analyze the peptide masses and identify the proteins. RESULTS: Seventy-two proteins were identified (54 unique proteins) out of approximately 1000 spots visualized on each gel. Most of the spots were expressed both in cortex and medulla. Of the identified proteins, three were expressed only in medulla and one only in cortex. Nine proteins were expressed in both regions but to a greater extent in cortex and three proteins were expressed more in medulla. Differential expression was confirmed for three proteins by Western blot. CONCLUSIONS: A large group of proteins and their relative expression levels from cortical and medullary portions of rat kidneys were found. Sixteen proteins are differentially expressed. Proteomics can be used to identify differential expression of proteins in the kidney on a large scale. Proteomics should be useful to detect changes in renal protein expression in response to a large range of physiological and pathophysiological stimuli.

Animals↗

PubMedDate not supplied

Identification of proteins in slow continuous ultrafiltrate by reversed-phase chromatography and proteomics.

Continuous modes of renal replacement therapy (CRRT) are increasingly being utilized in the intensive care unit. The removal of cytokines and other inflammatory proteins during ultrafiltration may be responsible for some of the beneficial effects of CRRT. We used proteomic tools to identify proteins found in the ultrafiltrate from a patient with acute renal failure. Identification of these proteins could help elucidate the mechanism(s) of improved outcome with continuous renal replacement therapy. Protein was loaded on a reversed-phase C4 column and eluted with stepwise isocratic flows starting with 0%, 5%, 10%, 25%, and 50% of acetonitrile. Effluent was collected, pooled, desalted, and separated by two-dimensional gel electrophoresis (2DE). Reversed-phase separation improved the resolution and the number of spots seen on the gels. Protein spots were digested with trypsin and spotted onto MALDI plates. Proteins were identified by either peptide mass fingerprinting using a MALDI-TOF mass spectrometer or by peptide sequencing using a MALDI-TOF/TOF tandem mass spectrometer. From 196 spots cut, 47 were identified, representing multiple charge forms of 10 different proteins. Proteins identified were albumin, apolipoprotein A-IV, beta-2-microglobulin, lithostathine, mannose-binding lectin associated serine protease 2 associated protein, plasma retinol-binding protein, transferrin, transthyretin, vitamin D-binding protein and Zn alpha-2 glycoprotein. Continuous renal replacement therapy is frequently used in acutely ill patients with renal failure. Removal of proteins occurs during this process. The physiological significance of this protein removal is unclear. Identification of these proteins will lead to better understanding of the role of protein removal in continuous renal replacement therapy.

Chromatography, High Pressure Liquid↗