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Protein Abundance (protein + abundance)

Distribution by Scientific Domains

Life Sciences	55%
Chemistry	27%
Medical Sciences	17%

Distribution within Life Sciences

Neuroscience	23%
Cell & Molecular Biology	18%

Selected Abstracts

Akt2/PKB,-sensitive regulation of renal phosphate transport

ACTA PHYSIOLOGICA, Issue 1 2010
D. S. Kempe
Abstract Aim:, The protein kinase B (PKB)/Akt is known to stimulate the cellular uptake of glucose and amino acids. The kinase is expressed in proximal renal tubules. The present study explored the influence of Akt/PKB on renal tubular phosphate transport. Methods:, The renal phosphate transporter NaPi-IIa was expressed in Xenopus oocytes with or without PKB/Akt and Na+ phosphate cotransport determined using dual electrode voltage clamp. Renal phosphate excretion was determined in Akt2/PKB, knockout mice (akt2,/,) and corresponding wild-type mice (akt2+/+). Transporter protein abundance was determined using Western blotting and phosphate transport by 32P uptake into brush border membrane vesicles. Results:, The phosphate-induced current in NaPi-IIa-expressing Xenopus oocytes was significantly increased by the coexpression of Akt/PKB. Phosphate excretion [,mol per 24 h per g BW] was higher by 91% in akt2,/, than in akt2+/+ mice. The phosphaturia of akt2,/, mice occurred despite normal transport activity and expression of the renal phosphate transporters NaPi-IIa, NaPi-IIc and Pit2 in the brush border membrane, a significantly decreased plasma PTH concentration (by 46%) and a significantly enhanced plasma 1,25-dihydroxyvitamin D3 concentration (by 46%). Moreover, fractional renal Ca2+ excretion was significantly enhanced (by 53%) and bone density significantly reduced (by 11%) in akt2,/, mice. Conclusions:, Akt2/PKB, plays a role in the acute regulation of renal phosphate transport and thus contributes to the maintenance of phosphate balance and adequate mineralization of bone. [source]

MicroRNA expression during chick embryo development

DEVELOPMENTAL DYNAMICS, Issue 11 2006
Diana K. Darnell
Abstract MicroRNAs (miRNAs) are small, abundant, noncoding RNAs that modulate protein abundance by interfering with target mRNA translation or stability. miRNAs are detected in organisms from all domains and may regulate 30% of transcripts in vertebrates. Understanding miRNA function requires a detailed determination of expression, yet this has not been reported in an amniote species. High-throughput whole mount in situ hybridization was performed on chicken embryos to map expression of 135 miRNA genes including five miRNAs that had not been previously reported in chicken. Eighty-four miRNAs were detected before day 5 of embryogenesis, and 75 miRNAs showed differential expression. Whereas few miRNAs were expressed during formation of the primary germ layers, the number of miRNAs detected increased rapidly during organogenesis. Patterns highlighted cell-type, organ or structure-specific expression, localization within germ layers and their derivatives, and expression in multiple cell and tissue types and within sub-regions of structures and tissues. A novel group of miRNAs was highly expressed in most tissues but much reduced in one or a few organs, including the heart. This study presents the first comprehensive overview of miRNA expression in an amniote organism and provides an important foundation for investigations of miRNA gene regulation and function. Developmental Dynamics 235:3156,3165, 2006. © 2006 Wiley-Liss, Inc. [source]

Maternal hypoxia increases the activity of MMPs and decreases the expression of TIMPs in the brain of neonatal rats

DEVELOPMENTAL NEUROBIOLOGY, Issue 3 2010
Wenni Tong
Abstract A recent study has shown that increased activity of matrix metalloproteinases-2 and metalloproteinases-9 (MMP-2 and MMP-9) has detrimental effect on the brain after neonatal hypoxia. The present study determined the effect of maternal hypoxia on neuronal survivability and the activity of MMP-2 and MMP-9, as well as the expression of tissue inhibitors of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2) in the brain of neonatal rats. Pregnant rats were exposed to 10.5% oxygen for 6 days from the gestation day 15 to day 21. Pups were sacrificed at day 0, 4, 7, 14, and 21 after birth. Body weight and brain weight of the pups were measured at each time point. The activity of MMP-2 and MMP-9 and the protein abundance of TIMP-1 and TIMP-2 were determined by zymography and Western blotting, respectively. The tissue distribution of MMPs was examined by immunofluorescence staining. The neuronal death was detected by Nissl staining. Maternal hypoxia caused significant decreases in body and brain size, increased activity of MMP-2 at day 0, and increased MMP-9 at day 0 and 4. The increased activity of the MMPs was accompanied by an overall tendency towards a reduced expression of TIMPs at all ages with the significance observed for TIMPs at day 0, 4, and 7. Immunofluorescence analysis showed an increased expression of MMP-2, MMP-9 in the hippocampus at day 0 and 4. Nissl staining revealed significant cell death in the hippocampus at day 0, 4, and 7. Functional tests showed worse neurobehavioral outcomes in the hypoxic animals. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2010 [source]

Acetaminophen prevents aging-associated hyperglycemia in aged rats: effect of aging-associated hyperactivation of p38-MAPK and ERK1/2

DIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 3 2009
Miaozong Wu
Abstract Background Aging-related hyperglycemia is associated with increased oxidative stress and diminished muscle glucose transporter-4 (Glut4) that may be regulated, at least in part, by the mitogen-activated protein kinases (MAPK). Methods To test the possibility that aging-related hyperglycemia can be prevented by pharmacological manipulation of MAPK hyperactivation, aged (27-month old) Fischer 344/NNiaHSD × Brown Norway/BiNia F1 (F344BN) rats were administered acetaminophen (30 mg/kg body weight/day) for 6 months in drinking water. Results Hepatic histopathology, serum aspartate aminotransferase and alanine aminotransferase analyses suggested that chronic acetaminophen did not cause hepatotoxicity. Compared with adult (6-month) and aged (27-month) rats, very aged rats (33-month) had higher levels of blood glucose, phosphorylation of soleus p38-MAPK and extracellular-regulated kinase 1/2 (ERK1/2), superoxide and oxidatively modified proteins (p < 0.05), and these changes were associated with decreased soleus Glut4 protein abundance (p < 0.05). Chronic acetaminophen treatment attenuated age-associated increase in blood glucose by 61.3% (p < 0.05) and increased soleus Glut4 protein by 157.2% (p < 0.05). These changes were accompanied by diminished superoxide levels, decrease in oxidatively modified proteins (,60.8%; p < 0.05) and reduced p38-MAPK and ERK1/2 hyperactivation (,50.4% and , 35.4%, respectively; p < 0.05). Conclusions These results suggest that acetaminophen may be useful for the treatment of age-associated hyperglycemia. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Growth-induced changes in the proteome of Helicobacter pylori

ELECTROPHORESIS, Issue 5-6 2006
Christina Uwins
Abstract Helicobacter pylori is a major human pathogen that is responsible for a number of gastrointestinal infections. We have used 2-DE to characterise protein synthesis in bacteria grown either on solid agar-based media or in each of two broth culture media (Brucella and brain heart infusion (BHI) broth). Significant differences were observed in the proteomes of bacteria grown either on agar-based or in broth media. Major changes in protein abundance were identified using principal component analysis (PCA), which delineated the profiles derived for the three key growth conditions (i.e. agar plates, Brucella and BHI broth). Proteins detected across the gel series were identified by peptide mass mapping and Edman sequencing. A number of proteins associated with protein synthesis in general as well as specific amino acid synthesis were depressed in broth-grown bacteria compared to plate-grown bacteria. A similar reduction was also observed in the abundance of proteins involved in detoxification. Two of the most abundant spots, identified as UreB and GroEL, in plate-grown bacteria showed a >140-fold drop in abundance in bacteria grown in Brucella broth compared to bacteria grown on agar plates. Two protein spots induced in bacteria grown in broth culture were both identified as glyceraldehyde 3-phosphate dehydrogenase based on their N -terminal amino acid sequences derived by Edman degradation. The underlying causes of the changes in the proteins abundance were not clear, but it was likely that a significant proportion of the changes were due to the alkaline pH of the broth culture media. [source]

Hormones as epigenetic signals in developmental programming

EXPERIMENTAL PHYSIOLOGY, Issue 6 2009
Abigail L. Fowden
In mammals, including man, epidemiological and experimental studies have shown that a range of environmental factors acting during critical periods of early development can alter adult phenotype. Hormones have an important role in these epigenetic modifications and can signal the type, severity and duration of the environmental cue to the developing feto-placental tissues. They affect development of these tissues both directly and indirectly by changes in placental phenotype. They act to alter gene expression, hence the protein abundance in a wide range of different tissues, which has functional consequences for many physiological systems both before and after birth. By producing an epigenome specific to the prevailing condition in utero, hormones act as epigenetic signals in developmental programming, with important implications for adult health and disease. This review examines the role of hormones as epigenetic signals by considering their responses to environmental cues, their effects on phenotypical development and the molecular mechanisms by which they programme feto-placental development, with particular emphasis on the glucocorticoids. [source]

Glucose sensing in the intestinal epithelium

FEBS JOURNAL, Issue 16 2003
Jane Dyer
Dietary sugars regulate expression of the intestinal Na+/glucose cotransporter, SGLT1, in many species. Using sheep intestine as a model, we showed that lumenal monosaccharides, both metabolisable and nonmetabolisable, regulate SGLT1 expression. This regulation occurs not only at the level of transcription, but also at the post-transcriptional level. Introduction of d -glucose and some d -glucose analogues into ruminant sheep intestine resulted in >,50-fold enhancement of SGLT1 expression. We aimed to determine if transport of sugar into the enterocytes is required for SGLT1 induction, and delineate the signal-transduction pathways involved. A membrane impermeable d -glucose analogue, di(glucos-6-yl)poly(ethylene glycol) 600, was synthesized and infused into the intestines of ruminant sheep. SGLT1 expression was determined using transport studies, Northern and Western blotting, and immunohistochemistry. An intestinal cell line, STC-1, was used to investigate the signalling pathways. Intestinal infusion with di(glucos-6-yl)poly(ethylene glycol) 600 led to induction of functional SGLT1, but the compound did not inhibit Na+/glucose transport into intestinal brush-border membrane vesicles. Studies using cells showed that increased medium glucose up-regulated SGLT1 abundance and SGLT1 promoter activity, and increased intracellular cAMP levels. Glucose-induced activation of the SGLT1 promoter was mimicked by the protein kinase A (PKA) agonist, 8Br-cAMP, and was inhibited by H-89, a PKA inhibitor. Pertussis toxin, a G-protein (Gi)-specific inhibitor, enhanced SGLT1 protein abundance to levels observed in response to glucose or 8Br-cAMP. We conclude that lumenal glucose is sensed by a glucose sensor, distinct from SGLT1, residing on the external face of the lumenal membrane. The glucose sensor initiates a signalling pathway, involving a G-protein-coupled receptor linked to a cAMP,PKA pathway resulting in enhancement of SGLT1 expression. [source]

,-catenin mediates insulin-like growth factor-I actions to promote cyclin D1 mRNA expression, cell proliferation and survival in oligodendroglial cultures

GLIA, Issue 9 2010
Ping Ye
Abstract By promoting cell proliferation, survival and maturation insulin-like growth factor (IGF)-I is essential to the normal growth and development of the central nervous system. It is clear that IGF-I actions are primarily mediated by the type I IGF receptor (IGF1R), and that phosphoinositide 3 (PI3)-Akt kinases and MAP kinases signal many of IGF-I-IGF1R actions in neural cells, including oligodendrocyte lineage cells. The precise downstream targets of these signaling pathways, however, remain to be defined. We studied oligodendroglial cells to determine whether ,-catenin, a molecule that is a downstream target of glycogen synthase kinase-3, (GSK3,) and plays a key role in the Wnt canonical signaling pathway, mediates IGF-I actions. We found that IGF-I increases ,-catenin protein abundance within an hour after IGF-I-induced phosphorylation of Akt and GSK3,. Inhibiting the PI3-Akt pathway suppressed IGF-I-induced increases in ,-catenin and cyclin D1 mRNA, while suppression of GSK3, activity simulated IGF-I actions. Knocking-down ,-catenin mRNA by RNA interference suppressed IGF-I-stimulated increases in the abundance of cyclin D1 mRNA, cell proliferation, and cell survival. Our data suggest that ,-catenin is an important downstream molecule in the PI3-Akt-GSK3, pathway, and as such it mediates IGF-I upregulation of cyclin D1 mRNA and promotion of cell proliferation and survival in oligodendroglial cells. © 2010 Wiley-Liss, Inc. [source]

Cover Picture: J. Basic Microbiol.

JOURNAL OF BASIC MICROBIOLOGY, Issue 1 2009
1/200
Two dimensional gel electrophoresis of heparin-bound proteins from Chlamydomonas reinhardtii. Proteins were extracted from cells harvested during night-phase and subjected to heparin-affinity chromatography. Proteins with a high affinity to heparin were eluted and applied to two-dimensional gel electrophoresis. The resulting pattern can be compared to, e.g., protein abundance during day-phase (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source]

PTHrP Signaling Targets Cyclin D1 and Induces Osteoblastic Cell Growth Arrest,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2005
Nabanita S Datta PhD
Abstract PTHrP control of the MC3T3-E1 cell cycle machinery showed that, during differentiation, PTHrP induced G1 growth arrest. Cyclin D1 was a critical mediator as a downstream effector of cAMP, PKC, and MAPK signaling, and the process was PKA-independent. The involvement of JunB has been found critical for PTHrP effects. Introduction: PTH-related protein (PTHrP) has been implicated in the control of bone cell turnover, but the mechanisms underlying its effect on osteoblast proliferation and differentiation have not been clearly defined. The mechanisms by which PTHrP impacts cell cycle proteins and the role of signaling pathways in differentiated osteoblasts were studied. Materials and Methods: To elucidate the role of PTHrP, flow cytometric analyses were performed using MC3T3-E1 and primary mouse calvarial cells. Relative protein abundance (Western blot), physical association of partners (immunoprecipitation), and kinase activities (in vitro kinase assays using either GST-Rb or H1-histone as substrates) of cell cycle-associated proteins in vehicle and PTHrP-treated 7-day differentiated cells were determined. ELISA and/or Northern blot analyses were done to evaluate JunB and cyclin D1 expression. SiRNA-mediated gene silencing experiments were performed to silence JunB protein. Finally, inhibitors of cAMP, protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) were used to determine involvement of different signaling pathways. Results: PTHrP inhibited cyclin D1 protein expression 7-fold in a dose- and time-dependent manner and increased the level of p16 protein in differentiated osteoblasts. Additionally, PTHrP reduced cyclin D1-CDK4/CDK6 and CDK1 kinase activities. Forskolin, a cAMP agonist, mimicked PTHrP action, and the PKC inhibitor, GF109203X, slightly blocked downregulation of cyclin D1, implying involvement of both cAMP and PKC. U0126, a MAPK inhibitor, alone decreased cyclin D1 protein, suggesting that the basal cyclin D1 protein is MAPK dependent. H-89, a PKA inhibitor, did not alter the effect of PTHrP on cyclin D1, suggesting a PKA-independent mechanism. Finally, expression of JunB, an activating protein-1 transcription factor, was significantly upregulated, and silencing JunB (siRNA) partially reversed the cyclin D1 response, implying involvement of JunB in the PTHrP-mediated growth arrest of MC3T3-E1 cells. Conclusion: PTHrP upregulates JunB and reduces cyclin D1 expression while inducing G1 cell cycle arrest in differentiated osteoblasts. Such regulation could be an important determinant of the life span and bone-forming activity of osteoblasts. [source]

Insulin restores glucose inhibition of adenosine transport by increasing the expression and activity of the equilibrative nucleoside transporter 2 in human umbilical vein endothelium

JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2006
Gonzalo Muñoz
L -Arginine transport and nitric oxide (NO) synthesis (L -arginine/NO pathway) are stimulated by insulin, adenosine or elevated extracellular D -glucose in human umbilical vein endothelial cells (HUVEC). Adenosine uptake via the human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) has been proposed as a mechanism regulating adenosine plasma concentration, and therefore its vascular effects in human umbilical veins. Thus, altered expression and/or activity of hENT1 or hENT2 could lead to abnormal physiological plasma adenosine level. We have characterized insulin effect on adenosine transport in HUVEC cultured in normal (5 mM) or high (25 mM) D -glucose. Insulin (1 nM) increased overall adenosine transport associated with higher hENT2-, but lower hENT1-mediated transport in normal D -glucose. Insulin increased hENT2 protein abundance in normal or high D -glucose, but reduced hENT1 protein abundance in normal D -glucose. Insulin did not alter the reduced hENT1 protein abundance, but blocked the reduced hENT1 and hENT2 mRNA expression induced by high D -glucose. Insulin effect on hENT1 mRNA expression in normal D -glucose was blocked by NG -nitro- L -arginine methyl ester (L-NAME, NO synthase inhibitor) and mimicked by S -nitroso- N -acetyl- L,D -penicillamine (SNAP, NO donor). L-NAME did not block insulin effect on hENT2 expression. In conclusion, insulin stimulation of overall adenosine transport results from increased hENT2 expression and activity via a NO-independent mechanism. These findings could be important in hyperglycemia-associated pathological pregnancies, such as gestational diabetes, where plasma adenosine removal by the endothelium is reduced, a condition that could alter the blood flow from the placenta to the fetus affecting fetus growth and development. J. Cell. Physiol. 209: 826,835, 2006. © 2006 Wiley-Liss, Inc. [source]

Using enrichment index for quality control of secretory protein sample and identification of secretory proteins

JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2009
Yong Chen
Abstract Analysis of secretory proteins is an important area in proteomic research. We propose that a good secretory protein sample should be enriched with known secretory proteins, and a secretory protein should be enriched in the secretory protein sample compared with its corresponding soluble cell lysate. Positive identifications of proteins were subjected to quantitation of spectral counts, which reflect relative protein abundance. Enrichment index of the sample (EIS) and the enrichment index for protein (EIP) were obtained by comparing proteins identified in the secretory protein sample and those in the soluble cell lysate sample. The quality of the secretory protein sample can be represented by EIS. EIP was used to identify the secretory proteins. The secretory proteins from mouse dendritic cell sarcoma (DCS) were analyzed by MS. The EISs of two samples were 75.4 and 84.65, respectively. 72 proteins were significantly enriched in secretory protein samples, of which 42 proteins were either annotated in Swiss-Prot and/or predicted by signal peptides to be secretory. In the remaining 30 proteins, 12 and 15 proteins were positively predicted by SecretomeP and ProP, respectively, and 5 proteins were positive by both methods. Furthermore, 11 proteins were found to be present in exosome in other studies that involved mice dendritic cell lines. We suggest that this assessment method is helpful for systemic research of secretory proteins and biomarker discovery for diseases such as cancer. Copyright © 2008 John Wiley & Sons, Ltd. [source]

Long-lasting up-regulation of orexin receptor type 2 protein levels in the rat nucleus accumbens after chronic cocaine administration

JOURNAL OF NEUROCHEMISTRY, Issue 1 2007
Guo-Chi Zhang
Abstract Hypothalamic orexin (hypocretin) neurons project to the key structures of the limbic system and orexin receptors, both orexin receptor type 1 (OXR1) and type 2 (OXR2), are expressed in most limbic regions. Emerging evidence suggests that orexin is among important neurotransmitters that regulate addictive properties of drugs of abuse. In this study, we examined the effect of psychostimulant cocaine on orexin receptor protein abundance in the rat limbic system in vivo. Intermittent administration of cocaine (20 mg/kg, i.p., once daily for 5 days) caused a typical behavioral sensitization response to a challenge cocaine injection at a 14-day withdrawal period. Repeated cocaine administration at the same withdrawal time also increased OXR2 protein levels in the nucleus accumbens while repeated cocaine had no effect on OXR1 and orexin neuropeptide (both orexin-A and orexin-B) levels in this region. In contrast to the nucleus accumbens, OXR2 levels in the frontal cortex, the ventral tegmental area, the hippocampus, and the dorsal striatum (caudate putamen) were not altered by cocaine. Remarkably, the up-regulated OXR2 levels in the nucleus accumbens showed a long-lasting nature as it persisted up to 60 days after the discontinuation of repeated cocaine treatments. In contrast to chronic cocaine administration, an acute cocaine injection was insufficient to modify levels of any orexin receptor and peptide. Our data identify the up-regulation of OXR2 in the nucleus accumbens as an enduring molecular event that is correlated well with behavioral plasticity in response to chronic psychostimulant administration. This OXR2 up-regulation may reflect a key adaptation of limbic orexinergic transmission to chronic drug exposure and may thus be critical for the expression of motor plasticity. [source]

Post-translational regulation of EAAT2 function by co-expressed ubiquitin ligase Nedd4-2 is impacted by SGK kinases

JOURNAL OF NEUROCHEMISTRY, Issue 4 2006
Christoph Boehmer
Abstract The human excitatory amino acid transporter (EAAT)2 is the major glutamate carrier in the mammalian CNS. Defective expression of the transporter results in neuroexcitotoxicity that may contribute to neuronal disorders such as amyotrophic lateral sclerosis (ALS). The serum and glucocorticoid inducible kinase (SGK) 1 is expressed in the brain and is known to interact with the ubiquitin ligase Nedd4-2 to modulate membrane transporters and ion channels. The present study aimed to investigate whether SGK isoforms and the related kinase, protein kinase B (PKB), regulate EAAT2. Expression studies in Xenopus oocytes demonstrated that glutamate-induced inward current (IGLU) was stimulated by co-expression of SGK1, SGK2, SGK3 or PKB. IGLU is virtually abolished by Nedd4-2, an effect abrogated by additional co-expression of either kinase. The kinases diminish the effect through Nedd4-2 phosphorylation without altering Nedd4-2 protein abundance. SGKs increase the transporter maximal velocity without significantly affecting substrate affinity. Similar to glutamate-induced currents, [3H] glutamate uptake and cell surface abundance of the transporter were increased by the SGK isoforms and down-regulated by the ubiquitin ligase Nedd4-2. In conclusion, all three SGK isoforms and PKB increase EAAT2 activity and plasma membrane expression and thus, may participate in the regulation of neuroexcitability. [source]

Aberrant protein expression is associated with decreased developmental potential in porcine cumulus,oocyte complexes

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2010
Melissa Paczkowski
Oocyte developmental competence is progressively obtained during pubertal development in females. Poor developmental potential in oocytes derived from prepubertal females suggests that essential processes required for oocyte development have not been fulfilled. The objective of this experiment was to analyze the protein profiles of porcine cumulus,oocyte complexes (COC) derived from cyclic and prepubertal females to identify alterations in protein abundance that correlate with developmental potential. COC complexes, aspirated from prepubertal and cyclic ovaries, were pooled into three replicates of 400 COCs each per treatment in ,100,µl SOF-HEPES medium. Protein samples were extracted and analyzed by two-dimensional differential in gel electrophoresis (2D-DIGE). Over 1,600 proteins were resolved on each of the three replicate gels. Sixteen protein spots were identified by mass spectrometry, representing 14 unique, differentially expressed proteins (volume ratio greater than 1.3). Glutathione- S -transferase and pyruvate kinase 3 were more abundant in COCs derived from cyclic females, whereas soluble epoxide hydrolase and transferrin were more abundant in prepubertal derived COCs. Abundance of several glycolytic enzymes (enolase 1, pyruvate kinase 3, and phosphoglycerate kinase) was increased in COCs derived from cyclic females, suggesting glucose metabolism is decreased in prepubertal derived COCs. We conclude that the abundance of proteins involved in metabolism and oxidative stress regulation is significantly altered in prepubertal derived COCs and may play a role in the mechanisms resulting in developmental competence. Mol. Reprod. Dev. 77: 51,58, 2010. © 2009 Wiley-Liss, Inc. [source]

Immunohistological intensity measurements as a tool to assess sarcolemma-associated protein expression

NEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 4 2010
V. Arechavala-Gomeza
V. Arechavala-Gomeza, M. Kinali, L. Feng, S. C. Brown, C. Sewry, J. E. Morgan and F. Muntoni (2010) Neuropathology and Applied Neurobiology36, 265,274 Immunohistological intensity measurements as a tool to assess sarcolemma-associated protein expression Aims: The quantification of protein levels in muscle biopsies is of particular relevance in the diagnostic process of neuromuscular diseases, but is difficult to assess in cases of partial protein deficiency, particularly when information on protein localization is required. The combination of immunohistochemistry and Western blotting is often used in these cases, but is not always possible if the sample is scarce. We therefore sought to develop a method to quantify relative levels of sarcolemma-associated proteins using digitally captured images of immunolabelled sections of skeletal muscle. Methods: To validate our relative quantification method, we labelled dystrophin and other sarcolemmal proteins in transverse sections of muscle biopsies taken from Duchenne muscular dystrophy and Becker muscular dystrophy patients, a manifesting carrier of Duchenne muscular dystrophy and normal controls. Results: Using this method to quantify relative sarcolemmal protein abundance, we were able to accurately distinguish between the different patients on the basis of the relative amount of dystrophin present. Conclusions: This comparative method adds value to techniques that are already part of the diagnostic process and can be used with minimal variation of the standardized protocols, without using extra amounts of valuable biopsy samples. Comparative quantification of sarcolemmal proteins on immunostained muscle sections will be of use to establish both the abundance and localization of the protein. Moreover, it can be applied to assess the efficacy of experimental therapies where only partial restoration or upregulation of the protein may occur. [source]

ReSASC: A resampling-based algorithm to determine differential protein expression from spectral count data

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2010
Kristina M. Little
Abstract Label-free methods for MS/MS quantification of protein expression are becoming more prevalent as instrument sensitivity increases. Spectral counts (SCs) are commonly used, readily obtained, and increase linearly with protein abundance; however, a statistical framework has been lacking. To accommodate the highly non-normal distribution of SCs, we developed ReSASC (resampling-based significance analysis for spectral counts), which evaluates differential expression between two conditions by pooling similarly expressed proteins and sampling from this pool to create permutation-based synthetic sets of SCs for each protein. At a set confidence level and corresponding p -value cutoff, ReSASC defines a new p -value, p,, as the number of synthetic SC sets with p>pcutoff divided by the total number of sets. We have applied ReSASC to two published SC data sets and found that ReSASC compares favorably with existing methods while being easy to operate and requiring only standard computing resources. [source]

Secretome analysis of differentially induced proteins in rice suspension-cultured cells triggered by rice blast fungus and elicitor

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2009
Sun Tae Kim
Abstract Secreted proteins were investigated in rice suspension-cultured cells treated with rice blast fungus Magnaporthe grisea and its elicitor using biochemical and 2-DE coupled with MS analyses followed by their in planta mRNA expression analysis. M. grisea and elicitor successfully interacted with suspension-cultured cells and prepared secreted proteins from these cultures were essentially intracellular proteins free. Comparative 2-D gel analyses identified 21 differential protein spots due to M. grisea and/or elicitor over control. MALDI-TOF-MS and ,LC-ESI-MS/MS analyses of these protein spots revealed that most of assigned proteins were involved in defense such as nine chitinases, two germin A/oxalate oxidases, five domain unknown function 26 (DUF 26) secretory proteins, and ,-expansin. One chitin binding chitinase protein was isolated using chitin binding beads and strong enzymatic activity was identified in an in-gel assay. Interestingly, their protein abundance correlated well at transcript levels in elicitor-treated cultures as judged by semi-quantitative RT-PCR. Each identified differentially expressed protein group was compared at transcript levels in rice leaves inoculated with incompatible (KJ401) and compatible (KJ301) races of M. grisea. Time-course profiling revealed their inductions were stronger and earlier in incompatible than compatible interactions. Identified secreted proteins and their expression correlation at transcript level in suspension-cultured cells and also in planta suggest that suspension-cultured cells can be useful to investigate the secretome of rice blast,pathogen interactions. [source]

Quantitative assessment of the structural bias in protein,protein interaction assays

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 22 2008
Åsa K. Björklund
Abstract With recent publications of several large-scale protein,protein interaction (PPI) studies, the realization of the full yeast interaction network is getting closer. Here, we have analysed several yeast protein interaction datasets to understand their strengths and weaknesses. In particular, we investigate the effect of experimental biases on some of the protein properties suggested to be enriched in highly connected proteins. Finally, we use support vector machines (SVM) to assess the contribution of these properties to protein interactivity. We find that protein abundance is the most important factor for detecting interactions in tandem affinity purifications (TAP), while it is of less importance for Yeast Two Hybrid (Y2H) screens. Consequently, sequence conservation and/or essentiality of hubs may be related to their high abundance. Further, proteins with disordered structure are over-represented in Y2H screens and in one, but not the other, large-scale TAP assay. Hence, disordered regions may be important both in transient interactions and interactions in complexes. Finally, a few domain families seem to be responsible for a large part of all interactions. Most importantly, we show that there are method-specific biases in PPI experiments. Thus, care should be taken before drawing strong conclusions based on a single dataset. [source]

Characterisation of organellar proteomes: A guide to subcellular proteomic fractionation and analysis

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 21 2006
Edwin Ho
Abstract Subcellular fractionation is being widely used to increase our understanding of the proteome. Fractionation is often coupled with 2-DE, thus allowing the visualisation of proteins and their subsequent identification and characterisation by MS. Whilst this strategy should be effective, to date, there has been little or no consideration given to differences in the mass, pI, hydropathy or abundance of proteins in the organelles and how analytical strategies can be tailored to match the idiosyncrasies of proteins in each particular compartment. To address this, we analysed 3962 Saccharomyces cerevisiae proteins, previously localised to one or more of 22 subcellular compartments. Different compartments showed significantly different distributions of protein pI and hydropathy. Mitochondrial and ER proteins showed the most dramatic differences to other organelles, in their protein pIs and hydropathy, respectively. We show that organelles can be clustered by similarities in these physicochemical protein characteristics. Interestingly, the distribution of protein abundance was also significantly different between many organelles. Our results show that to fully explore subcellular fractions of the proteome, specific analytical strategies should be employed. We outline strategies for all 22 subcellular compartments. [source]

Reversal of diabetes-evoked changes in mitochondrial protein expression of cardiac left ventricle by treatment with a copper(II)-selective chelator

PROTEOMICS - CLINICAL APPLICATIONS, Issue 4 2007
Mia Jüllig
Abstract Cardiac disease is the commonest cause of death amongst diabetic patients. Diabetic cardiomyopathy, which has a poor prognosis, is characterized by left ventricular hypertrophy and impaired cardiac function and mitochondrial damage is said to contribute to its development. We recently showed that treatment with the CuII -selective chelator, triethylenetetramine (TETA), improved cardiac structure, and function in diabetic subjects without modifying hyperglycemia. Thus, TETA has potential utility for the treatment of heart disease. To further understand the molecular mechanism by which it causes these effects, we have conducted the first study of the effect of oral TETA on protein abundance in the cardiac left ventricle of rats with severe streptozotocin-induced diabetes. Proteomic methods showed that of 211 proteins changed in diabetes, 33 recovered after treatment. Through MS, 16 proteins were identified which may constitute major targets of drug action. Remarkably, most of these were mitochondrial proteins with roles in energy metabolism. In addition to components of the mitochondrial respiratory chain and enzymes involved in fatty acid oxidation, TETA treatment normalized both myocardial expression and enzymatic activity of carnitine palmitoyltransferase 2. These findings indicate that mitochondria constitute major targets in the mechanism by which TETA restores cardiac structure and function in diabetes. [source]

Evidence for downregulation of calcium signaling proteins in advanced mouse adenocarcinoma

THE PROSTATE, Issue 2 2005
Viola C. Ruddat
Abstract BACKGROUND Prostate cancer (PCa) is the leading cancer related death in America. Gleason grading is currently the predominant method for prediction, with only few biomarkers available. More biomarkers, especially as they relate to cancer progression are desirable. METHODS The abundance of several important proteins in prostate tissue was compared between wild-type mouse dorsal prostate and well-differentiated transgenic adenocarcinoma mouse prostate (TRAMP) mouse dorsal prostates, and between wild-type mouse dorsal prostate and poorly-differentiated TRAMP mouse tumor tissue. 2DIGE method in conjunction with MALDI-ToF and Western blots was used to determine differential expression. RESULTS In TRAMP dorsal prostates with well-differentiated adenocarcinoma, there were few significant changes in the protein abundances compared to wild-type dorsal prostates, with the exception of increases in proliferating cell nuclear antigen (PCNA) and beta tubulin, two proteins implicated in cell proliferation, and a more than 2-fold increase in Hsp60, a protein involved in the suppression of apoptosis. In the poorly-differentiated tumors, the changes in protein abundance were substantial. While some of those changes could be related to the disappearance of stromal tissue or the appearance of epithelial tissue, other changes in protein abundance were more significant to the cancer development itself. Most notable was the overall decrease in calcium homeostasis proteins with a 10-fold decrease in calreticulin and Hsp70 and a 40-fold decrease in creatine kinase bb in the cancerous tissue. CONCLUSIONS Proteomics of TRAMP mice provide an excellent method to observe changes in protein abundance, revealing changes in pathways during cancer progression. © 2005 Wiley-Liss, Inc. [source]

How do synonymous mutations affect fitness?

BIOESSAYS, Issue 6 2007
Joanna L. Parmley
While it has often been assumed that, in humans, synonymous mutations would have no effect on fitness, let alone cause disease, this position has been questioned over the last decade. There is now considerable evidence that such mutations can, for example, disrupt splicing and interfere with miRNA binding. Two recent publications suggest involvement of additional mechanisms: modification of protein abundance most probably mediated by alteration in mRNA stability1 and modification of protein structure and activity,2 probably mediated by induction of translational pausing. These case histories put a further nail into the coffin of the assumption that synonymous mutations must be neutral. BioEssays 29:515,519, 2007. © 2007 Wiley Periodicals, Inc. [source]

Dynamic analysis of GS-NS0 cells producing a recombinant monoclonal antibody during fed-batch culture

BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2007
Scott H. Stansfield
Abstract In this study we have analyzed the dynamic covariation of the mammalian cell proteome with respect to functional phenotype during fed-batch culture of NS0 murine myeloma cells producing a recombinant IgG4 monoclonal antibody. GS-NS0 cells were cultured in duplicate 10 L bioreactors (36.5°C, 15% DOT, pH 7.0) for 335 h and supplemented with a continuous feed stream after 120 h. Cell-specific growth rate declined continuously after 72 h of culture. Cell-specific recombinant monoclonal antibody production rate (qP) varied sixfold through culture. Whilst qP correlated with relative recombinant heavy chain mRNA abundance up to 216 h, qP subsequently declined, independent of recombinant heavy chain or light chain mRNA abundance. GS-NS0 cultures were sampled at 48 h intervals between 24 and 264 h of culture for proteomic analyses. Total protein abundance and nascent polypeptide synthesis was determined by 2D PAGE of unlabeled proteins visualized by SYPRO® Ruby and autoradiography of 35S-labeled polypeptides, respectively. Covariation of nascent polypeptide synthesis and abundance with biomass-specific cell growth, glucose and glutamate consumption, lactate and Mab production rates were then examined using two partial least squares regression models. Most changes in polypeptide synthesis or abundance for proteins previously identified by mass spectrometry were positively correlated with biomass-specific growth rate. We conclude that the substantial transitions in cell physiology and qP that occur during culture utilize a relatively constant complement of the most abundant host cell machines that vary primarily with respect to induced changes in cell growth rate. Biotechnol. Bioeng. 2007;97: 410,424. © 2006 Wiley Periodicals, Inc. [source]

Evidence for post-translational regulation of NrtA, the Aspergillus nidulans high-affinity nitrate transporter

NEW PHYTOLOGIST, Issue 4 2007
Ye Wang
Summary ,,Here, influx and efflux of , and net fluxes of and , were measured in Aspergillus nidulans mutants niaD171 and niiA5, devoid of nitrate reductase (NR) and nitrite reductase (NiR) activities, respectively. ,,Transcript and protein abundances of NrtA, the A. nidulans principal high-affinity transporter, were determined using semiquantitative reverse transcription-polymerase chain reaction and western blots, respectively. , influx in niaD171 was negligible relative to wild-type values, whereas efflux to influx ratios increased nine-fold. Nevertheless, NrtA mRNA and NrtA protein were expressed at levels more than two-fold and three-fold higher, respectively, in niaD171 than in the wild-type strain. ,,This is the first demonstration of diminished high-affinity influx associated with elevated transporter levels, providing evidence that, in addition to transcriptional regulation, control of NrtA expression operates at the post-translational level. This mechanism allows for rapid control of transport at the protein level, reduces the extent of futile cycling of that would otherwise represent a significant energy drain when influx exceeds the capacity for assimilation or storage, and may be responsible for the rapid switching between the on and off state that is associated with simultaneous provision of to mycelia absorbing . [source]

Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: The human mammary epithelial cell proteome

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2005
Tao Liu
Abstract Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14,416 confidently identified peptides covering 4294 different proteins with an estimated 10%,gene coverage of the human genome. By using the high efficiency CPE, an additional 1096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1390,proteins were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased with regard to protein Mr,, pI, cellular location, or biological functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems. [source]

In vacuo isotope coded alkylation technique (IVICAT); an N-terminal stable isotopic label for quantitative liquid chromatography/mass spectrometry proteomics,

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2006
Brigitte L. Simons
We present a new isotopic labeling strategy to modify the N-terminal amino group of peptides in a quantifiable reaction without the use of expensive reagents or solvents. The In Vacuo Isotope Coded Alkylation Technique (IVICAT) is a methylation reaction, carried out at low pressure (<100,mTorr), that results in a stable quaternary trimethylammonium group, thus adding a permanent positive charge at the N-terminus of peptides without modifying the , -amino groups of lysine. The methylation reaction increases the signal intensity of modified peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and liquid chromatography (LC)/MS and the isotopic peak pair differs by 9 mass units which can be easily resolved by either instrument. N-terminally trimethylated peptides exhibit collision-induced dissociation (CID) mass spectra that differ from their unmodified analogues by an enhanced b -ion series in MS2 spectra due to the fixed positive charge. Using LC/MS/MS with an LTQ mass analyzer for quantification, the experimentally determined ratios of H9 - to D9 -trimethyl-labeled peptides of , -casein provided accurate estimates of the actual ratios with low % error. IVICAT labeling also accurately quantified proteins in rat kidney inner medullary collecting duct cell types, as judged by comparison with relative quantification by subsequent immunoblotting experiments. IVICAT labeling, when used in conjunction with the new proteomics software QUIL, can accurately report relative protein abundances and increase the sequence coverage of proteins of tissue proteomes. Published in 2006 by John Wiley & Sons, Ltd. [source]