High-resolution Two-dimensional Electrophoresis Research Articles

Comparative Proteomics of Potato Cultivars with a Variable Dormancy Period.

The control of the duration of the dormancy phase is a significant challenge in the potato industry and for seed producers. However, the proteome landscape involved in the regulation of the length of the dormancy period over potato cultivars remains largely unexplored. In this study, we performed for the first time a comparative proteome profiling of potato cultivars with differential duration of tuber dormancy. More specifically, the proteome profiling of Agata, Kennebec and Agria commercial potato varieties with short, medium and medium-long dormancy, respectively, was assessed at the endodormancy stage using high-resolution two-dimensional electrophoresis (2-DE) coupled to reversed-phase liquid chromatography–tandem mass spectrometry (LC-TripleTOF MS/MS). A total of 11 proteins/isoforms with statistically significant differential abundance among cultivars were detected on 2-DE gels and confidently identified by LC-TripleTOF MS/MS. Identified proteins have known functions related to tuber development, sprouting and the oxylipins biosynthesis pathway. Fructokinase, a mitochondrial ADP/ATP carrier, catalase isozyme 2 and heat shock 70 kDa were the proteins with the strongest response to dormancy variations. To the best of our knowledge, this study reports the first candidate proteins underlying variable dormancy length in potato cultivars.

Proteomic analysis of blood serum - a new approach to the search for diagnostic markers of bronchial asthma in children.

Currently, bronchial asthma (BA) is one of the most pressing medical and social problems, the molecular aspects of the formation and development of BA are insufficiently studied and the diagnosis is not perfect. Carrying out proteomic analysis of BA will not only reveal new biomarkers specific to this disease, but also bring us closer to understanding its pathogenetic mechanisms. The purpose of the study: to study the proteomic profile of blood serum of children with BA to identify proteins associated with this disease A comprehensive clinical and laboratory examination of children suffering from BA and control group patients was performed. Proteomic analysis of depleted blood serum included high-resolution two-dimensional electrophoresis (1 direction: immobiline strips 17cm, pH 3-10, 2 direction: denaturing electrophoresis in 12.5% polyacrylamide gel), protein staining on gels with fluorescent dye Flamingo, protein identification by MALDI-TOF mass spectrometry using the search algorithm Mascot and the Swiss-Prot database. Comparison of the proteomic profile of BA serum and the control group patients serum allowed us to establish that the production of a number of proteins is reduced in this pathology. Among them, proteins in the molecular weight range of 16-33 kDa (p<0.05) were identified: glutathione peroxidase 3, transtyretin, complement components C4b and C3. Research shows that changes in the children's serum proteome occur in BA, affecting proteins that play an important role in immune responses, ligand transport, and antioxidant protection. Special attention should be paid to the differences identified in the course of this work (glutathione peroxidase, transtyretin, C3 and C4 fragments of the complement system) or their combinations. Studying the features of their expression will expand our understanding of the molecular mechanisms underlying chronic inflammation of this disease.

Изучение протеомного спектра околоплодных вод и идентификация белков – источников газотрансмиттеров в качестве маркеров осложненной гестации и прогнозирования постнатальной патологии

Objective. To study the amniotic fluid proteome, to identify gasotransmitter-producing proteins and to evaluate their role as markers of pre-eclampsia (PE) and prediction of the early neonatal period course. Patients and methods. Fifty-eight women were examined. The study group included 28 women who had pregnancy complicated by PE; the control group consisted of 30 women with normal pregnancy. The protein composition of amniotic fluid was determined by high resolution two-dimensional electrophoresis and time-of-flight mass spectrometry. Sulfur-containing proteins, which are the sources of gasotransmitter SH2, were identified according to standard normal tables. Results. It was found that the development of PE is accompanied by significant changes in the amniotic fluid proteome. Four sulfur-containing proteins were found to be absent: retinol-binding protein (S-S- and SH-groups); fatty-acid-binding protein (S-S-groups); carbonyl reductase NAD+ (SH-groups), and haptoglobin (SH-groups). These proteins are involved in cell protection against oxidative and carbonyl stress, in maintaining the integrity of biomembranes and providing the fetus with necessary nutrients. Two additional membrane-bound proteins with high level secretion were found in women with PE, which also contribute negatively to the development of this obstetric pathology due to their pro-inflammatory and pro-oxidant properties. Conclusion. The revealed protein imbalance may be one of the primary factors of molecular disorders in the fetoplacental complex in PE. Key words: gasotransmitters, amniotic fluid, pre-eclampsia, proteomic composition, sulfur-containing proteins

Proteomic Analysis of Pigeonpea (Cajanus cajan) Seeds Reveals the Accumulation of Numerous Stress-Related Proteins.

Pigeonpea is one of the major sources of dietary protein for more than a billion people living in South Asia. This hardy legume is often grown in low-input and risk-prone marginal environments. Considerable research effort has been devoted by a global research consortium to develop genomic resources for the improvement of this legume crop. These efforts have resulted in the elucidation of the complete genome sequence of pigeonpea. Despite these developments, little is known about the seed proteome of this important crop. Here, we report the proteome of pigeonpea seed. To enable the isolation of maximum number of seed proteins, including those that are present in very low amounts, three different protein fractions were obtained by employing different extraction media. High-resolution two-dimensional (2-D) electrophoresis followed by MALDI-TOF-TOF-MS/MS analysis of these protein fractions resulted in the identification of 373 pigeonpea seed proteins. Consistent with the reported high degree of synteny between the pigeonpea and soybean genomes, a large number of pigeonpea seed proteins exhibited significant amino acid homology with soybean seed proteins. Our proteomic analysis identified a large number of stress-related proteins, presumably due to its adaptation to drought-prone environments. The availability of a pigeonpea seed proteome reference map should shed light on the roles of these identified proteins in various biological processes and facilitate the improvement of seed composition.

Proteomic analysis of continuous 900-MHz radiofrequency electromagnetic field exposure in testicular tissue: a rat model of human cell phone exposure.

Although cell phones have been used worldwide, some adverse and toxic effects were reported for this communication technology apparatus. To analyze in vivo effects of exposure to radiofrequency-electromagnetic field (RF-EMF) on protein expression in rat testicular proteome, 20 Sprague-Dawley rats were exposed to 900MHz RF-EMF for 0, 1, 2, or 4h/day for 30 consecutive days. Protein content of rat testes was separated by high-resolution two-dimensional electrophoresis using immobilized pH gradient (pI 4-7, 7cm) and 12% acrylamide and identified by MALDI-TOF/TOF-MS. Two protein spots were found differentially overexpressed (P<0.05) in intensity and volume with induction factors 1.7 times greater after RF-EMF exposure. After 4h of daily exposure for 30 consecutive days, ATP synthase beta subunit (ASBS) and hypoxia up-regulated protein 1 precursor (HYOU1) were found to be significantly up-regulated. These proteins affect signaling pathways in rat testes and spermatogenesis and play a critical role in protein folding and secretion in the endoplasmic reticulum. Our results indicate that exposure to RF-EMF produces increases in testicular proteins in adults that are related to carcinogenic risk and reproductive damage. In light of the widespread practice of men carrying phones in their pockets near their gonads, where exposures can exceed as-tested guidelines, further study of these effects should be a high priority.

Proteome and Peptidome of Vipera berus berus Venom.

Snake venom is a rich source of peptides and proteins with a wide range of actions. Many of the venom components are currently being tested for their usefulness in the treatment of many diseases ranging from neurological and cardiovascular to cancer. It is also important to constantly search for new proteins and peptides with properties not yet described. The venom of Vipera berus berus has hemolytic, proteolytic and cytotoxic properties, but its exact composition and the factors responsible for these properties are not known. Therefore, an attempt was made to identify proteins and peptides derived from this species venom by using high resolution two-dimensional electrophoresis and MALDI ToF/ToF mass spectrometry. A total of 11 protein classes have been identified mainly proteases but also l-amino acid oxidases, C-type lectin like proteins, cysteine-rich venom proteins and phospholipases A2 and 4 peptides of molecular weight less than 1500 Da. Most of the identified proteins are responsible for the highly hemotoxic properties of the venom. Presence of venom phospholipases A2 and l-amino acid oxidases cause moderate neuro-, myo- and cytotoxicity. All successfully identified peptides belong to the bradykinin-potentiating peptides family. The mass spectrometry data are available via ProteomeXchange with identifier PXD004958.

Evaluating two-dimensional electrophoresis profiles of the protein phaseolin as markers of genetic differentiation and seed protein quality in common bean (Phaseolus vulgaris L.).

High-resolution two-dimensional electrophoresis (2-DE) profiles of the protein phaseolin, the major seed storage protein of common bean, display great number of spots with differentially glycosylated and phosphorylated α- and β-type polypeptides. This work aims to test whether these complex profiles can be useful markers of genetic differentiation and seed protein quality in bean populations. The 2-DE phaseolin profile and the amino acid composition were examined in bean seeds from 18 domesticated and wild accessions belonging to the Mesoamerican and Andean gene pools. We found that proteomic distances based on 2-DE profiles were successful in identifying the accessions belonging to each gene pool and outliers distantly related. In addition, accessions identified as outliers from proteomic distances showed the highest levels of methionine content, an essential amino acid deficient in bean seeds. These findings suggest that 2-DE phaseolin profiles provide valuable information with potential of being used in common bean genetic improvement.

Two-Dimensional Gel Electrophoresis and the Beginning of Proteomics

Featured article: O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 1975;250:4007–21.2 No one had heard of proteomics in 1971. The invention of the word was 25 years away (1). At the time, doing molecular biology meant studying bacteria or their viruses, the bacteriophage. But as a starting graduate student in Boulder, Colorado, I wanted to study eukaryotic embryonic development. I joined the laboratory of Jacques Pene, which was focused on bacteriophage replication, because he was interested in starting a new system for study. He suggested the colonial algae, Volvox, which undergoes a simple embryogenesis to make two types of cells and a beautifully structured colony. Based on the bacteriophage paradigm, I chose to isolate mutants altered at different points in development. I assessed how the mutants interrupted or derailed morphogenesis, and then I wanted to compare this to how they influenced the changing pattern of gene expression, which, at the time, was best defined by changes in the proteins expressed. The most …

Evidence for phosphorylation of the major seed storage protein of the common bean and its phosphorylation-dependent degradation during germination

Phaseolin is the major seed storage protein of common bean, Phaseolus vulgaris L., accounting for up to 50% of the total seed proteome. The regulatory mechanisms responsible for the synthesis, accumulation and degradation of phaseolin in the common bean seed are not yet sufficiently known. Here, we report on a systematic study in dormant and 4-day germinating bean seeds from cultivars Sanilac (S) and Tendergreen (T) to explore the presence and dynamics of phosphorylated phaseolin isoforms. High-resolution two-dimensional electrophoresis in combination with the phosphoprotein-specific Pro-Q Diamond phosphoprotein fluorescent stain and chemical dephosphorylation by hydrogen fluoride-pyridine enabled us to identify differentially phosphorylated phaseolin polypeptides in dormant and germinating seeds from cultivars S and T. Phosphorylated forms of the two subunits of type α and β that compose the phaseolin were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MALDI-TOF/TOF tandem MS. In addition, we found that the levels of phosphorylation of the phaseolin changed remarkably in the seed transition from dormancy to early germination stage. Temporal changes in the extent of phosphorylation in response to physiological and metabolic variations suggest that phosphorylated phaseolin isoforms have functional significance. In particular, this prospective study supports the hypothesis that mobilization of the phaseolin in germinating seeds occurs through the degradation of highly phosphorylated isoforms. Taken together, our results indicate that post-translational phaseolin modifications through phosphorylations need to be taken into consideration for a better understanding of the molecular mechanisms underlying its regulation.

Blood plasma protein and lipid profile changes in calves during the first week of life

The present study was undertaken to determine blood plasma protein and lipid profile changes in healthy Polish Holstein-Fresian calves of Black-and-White variety. Blood was drawn immediately after birth, before first colostrum intake and at the 3rd, 6th, 12th, 24th, 36th, 48th and 72nd hour of life. Subsequent four blood samples were collected at 24 hour intervals until the 7th day of life. Plasma proteins within the isoelectric point ranging from 3.0 to 10.0 were separated using high resolution two-dimensional electrophoresis. Among the 74 protein spots detected and analyzed, 16 were significantly altered during the first week of life. Differentially expressed spots were excised from the gels and subjected to peptide mass fingerprinting using MALDI-TOF MS. In total, 12 spots were successfully identified, which correspond to three proteins, namely: apolipoprotein A-I, apolipoprotein A-IV and fibrinogen gamma-B chain. A gradual increase in plasma triglyceride, total cholesterol, HDL and LDL cholesterol values was shown during the first seven days of calves life. The lowest concentration of these indicators were observed at birth and was followed by a rapid increase during the first week of postnatal life. These changes appear to be related to the transition in energy sources, from a maternal nutrient supply comprising mainly carbohydrates and amino acids to a diet which was rich in fat--colostrum and milk. This was reflected by the intense up-regulation of plasma proteins related with lipid transport and lipoprotein metabolism during the first week of life.

Novel potential biomarkers for ST-elevation myocardial infarction identified by proteomic analysis of plasma-derived microparticles

Purpose: Microparticles (MP) are cell-derived membrane vesicles of less than a micron known to be increased in the blood of patients with acute coronary syndromes. They derive from various cells, most notably platelets. Our objective was to compare the proteome of plasma-derived microparticles from ST-elevation myocardial infarction (STEMI) patients and stable coronary artery disease (SCAD) controls, and to detect MP proteins differentially regulated between both groups. Methods: Plasma MP were isolated from 10 STEMI patients and 10 SCAD matched controls. We followed a standardized procedure to guarantee the purity of the microparticle population. MPs were characterized by FACS and by electron microscopy. Proteome analysis was based on high-resolution two-dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Validations were by 1D- and 2D-western blotting in an independent cohort of patients. Results: For the proteomic analysis, samples were pooled for each condition (STEMI and SCAD), and four gels (technical replicates) were run. 1228 spots were detected per gel. The number of differentially regulated spots was of 223 (fold change ≥ 1.5 and p < 0.05). We focused on the analysis of 115 differentially regulated spots with a fold change ≥ 2 and p < 0.05. From those, 71 were up-regulated in MP from STEMI patients, whereas 44 were up-regulated in SCAD controls. Some of the differentially regulated proteins identified play a central role in the activation of the classical pathway of the complement system and are involved in inflammatory response (e.g. complement C3 and C4a). Others are important in the maintenance of hemostasis and participate in platelet activation (e.g. von Willebrand factor, fibrinogen, fibronectin). Another protein identified was alpha-2-macroglobulin, which plays an important role in thrombogenesis and has been recently found to be up-regulated in plasma MP from deep venous thrombosis patients. Interestingly, cardiotrophin-like cytokine factor 1 (CLCF1) was found to be up-regulated in STEMI samples. This is a cytokine with B-cell stimulating capability that we recently found to be present in platelet-derived MP. Conclusions: We performed the first high-resolution proteome analysis of plasma-derived MP from STEMI and SCAD patients. We provide a panel of proteins that vary between both conditions and that might constitute a promising source of biomarkers for STEMI.

Cytosolic Carboxypeptidase 1 Is Involved in Processing α- and β-Tubulin

The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.

Buffer Optimization for Cynodon dactylon Proteome

Two-dimensional (2D) gel electrophoresis is a powerful technique used for the differential protein expression analysis. High-resolution two-dimensional electrophoresis is still the main applied separation technique in proteomics. A successful 2D gel electrophoresis depends on the proper optimized conditions most importantly an optimized buffer. In this study we checked different factors affecting 2D gel results including rehydration time, reducing agents, chaotrophic agents, and detergents. We chose Cynodon dactylon as an experimental plant. Proteins were extracted and purified. For the proper solubilization of the protein sample different rehydration buffers of pH 3-10 were applied having different amount of chaotrophic agents like urea and thiourea, detergents (NP-40, Triton X-100 and CHAPS) and reducing agents (β-merceptoethanol and DTT). To further improve the protein profile on 2D gel, solubilized proteins were re-extracted, washed and re-solubilized in a fresh rehydration buffer. Our modified protocol showed a high increase in protein solubility and improvement in protein 2D gel profile for C. dactylon proteome. In conclusion, we have successfully optimized the conditions for C. dactylon proteome analysis by 2D gel electrophoresis. These optimized conditions will lay the basis for further profound studies in the field of proteomics.

The problems of proteinuria measurement in urine with presence of Bence Jones protein

Objectives Protein concentration measurement in the urine can be problematic in the presence of Bence Jones protein. We have carried out an external quality control assessment with the participation of 79 clinical biochemistry laboratories from the Czech Republic and Slovakia. Design and methods The laboratories received a reference urine sample obtained from a patient with multiple myeloma and lambda free light chain proteinuria and were asked to type the paraprotein using immunofixation and to measure total urinary protein using their established method, most commonly turbidimetry, pyrogallol red assay, and biuret assay. Results There was a very wide inter-laboratory variability in the protein concentration readouts with up to three-fold difference in some cases. High-resolution two-dimensional electrophoresis and linear mass spectrometry showed that a high proportion of the urinary paraprotein was composed of lambda light chain fragments with molecular weight of 12 kDa. Conclusions Our results highlight the challenges of reliable and reproducible measurement of urinary protein concentration in the presence of Bence Jones protein.

Influence of RET/PTC1 and RET/PTC3 oncoproteins in radiation-induced papillary thyroid carcinomas on amounts of cytoskeletal protein species

Radiation-induced human papillary thyroid carcinomas (PTCs) show a high prevalence of fusions of the RET proto-oncogene to heterologous genes H4 (RET/PTC1) and ELE1 (RET/PTC3), respectively. In contrast to the normal membrane-bound RET protein, aberrant RET fusion proteins are constitutively active oncogenic cytosolic proteins that can lead to malignant transformation of thyroid epithelia. To detect specific tumor-associated protein changes that reflect the effect of RET/PTC fusion proteins, we analyzed normal thyroid tissues, thyroid tumors of the RET/PTC1 and RET/PTC3 type and their respective lymph node metastases by a combination of high-resolution two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-mass spectrometry. PTCs without RET rearrangements served as controls. Several cytoskeletal protein species showed quantitative changes in tumors and lymph node metastases harboring RET/PTC1 or RET/PTC3. We observed prominent C-terminal actin fragments assumedly generated by protease cleavages induced due to enhanced amounts of the active actin-binding protein cofilin-1. In addition, three truncated vimentin species, one of which was proven to be headless, were shown to be highly abundant in tumors and metastases of both RET/PTC types. The observed protein changes are closely connected with the constitutive activation of RET-rearranged oncoproteins and reflect the importance to elucidate disease-related typical signatures on the protein species level.

Comparative proteome study of apoptosis induced by As 4S 4 in retinoid acid resistant human acute promyelocytic leukemia NB4-R1 cells

Tetra-arsenic tetra-sulfide (As 4S 4), with improved toxicity profiles relative to arsenic trioxide, is the essential component of the new oral arsenic formulation which is highly effective and safe in the treatment of both newly diagnosed acute promyelocytic leukemia (APL) and more importantly relapsed/refractory APL. Although it is investigated that the therapeutic action of As 4S 4 is closely associated with its induced cells apoptosis, the definitive systematic molecular mechanism of action of As 4S 4 in APL therapy is still remained unknown. In this study, a serial of assays in vitro about the cytotoxicity of As 4S 4 and cellular apoptotic evidences were done, then a proteomic investigation with the high-resolution two-dimensional electrophoresis system and mass spectrometry were performed to obtain for the first time systematic identification and characterization of the global proteome of apoptosis induced by As 4S 4 in retinoic acid (RA)-resistant cells. Among them, expressional and functional regulations of target proteins SET, RPP2 and PHB might be the potential novel effective therapeutic strategies for RA-resistant APL. This study will not only facilitate to understand the signal transduction of apoptosis of RA-resistant APL cells induced by As 4S 4 as a whole, but also is important to screen for drug targets as a new therapeutic strategy for hematopoietic malignant tumors.

The current state of the art in high-resolution two-dimensional electrophoresis

The study of the “proteomes” of human cells, tissues, and body fluids is a big challenge, and several highly sophisticated workflow approaches are pursued to achieve as comprehensive information as possible. Initially proteome analysis was exclusively based on the gel-based workflow, employing two-dimensional electrophoresis of protein extracts followed by mass spectrometry of the tryptic peptide digests of protein spots. Meanwhile several additional proteomics workflows are applied, which are mostly based on separation and analysis of tryptic peptides without separating the protein mixture. However, direct information on quantitative and qualitative changes of protein expressions can only be obtained by methods operating on the protein level, no other method can replace two-dimensional electrophoresis. In this review we compile the different techniques of high-resolution two-dimensional electrophoresis and their further developments to increase the degree of reliance of the method.

Identification of nuclear structural protein alterations associated with seminomas

Currently, there are no specific markers available for the early detection and for monitoring testicular cancer. Based upon an approach that targets nuclear structure, we have identified a set of proteins that are specific for seminomas, which may then have clinical utility for the disease. Utilizing samples obtained from men with no evidence of testicular cancer (n = 5) as well as those with seminomas (n = 6), nuclear matrix proteins were extracted and separated using a high-resolution two-dimensional electrophoresis gel system. The proteins were identified by mass spectrometry analysis. These analyses revealed seven nuclear matrix proteins associated with the normal testes, which did not appear in the seminomas. In the seminomas, four nuclear matrix proteins were identified to be associated with the disease that were absent in the normal testes. Mass spectrometric and immunoblot analyses of these proteins revealed that one of the proteins identified in the normal testes appears to be StAR-related lipid transfer protein 7 (StARD7). In the non-seminoma tissues, one of the identified proteins appears to be cell division protein kinase 10 (CDK10). Both StarD7 and CDK10 could potentially be involved in cell differentiation and growth, and thus may serve as potential targets for therapy of prognostication of seminomas. This is the first study to examine the role of nuclear structural proteins as potential biomarkers in testicular cancer. We are currently examining the roles of some of the identified proteins as potential biomarkers for the disease.

Similarity of electrophoretic dissolved protein spectra from coastal to pelagic seawaters

Dissolved proteins in seawater samples collected from a coastal area of Tokyo Bay, Sagami Bay and a location off the Kuroshio Current were investigated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high resolution two-dimensional electrophoresis (2-DE). Four to nine protein bands were detected in SDS-PAGE in the apparent molecular weight (MW) range from 12 kilo Dalton (kDa) to 49 kDa. The 2-DE technique distinguished 10 to 46 protein spots exhibiting isoelectric point (pI)/MW ranging 4.3–9.2/12–63 kDa. The elecrophoretic patterns were similar between the coastal and pelagic samples, as well as previously reported patterns from various pelagic areas. The close similarity of electrophoretic mobility on both SDS-PAGE and 2-DE gels indicates the compositional homogeneity of dissolved proteins in seawater throughout a broad range of marine environments. Proteinaceous dissolved organic matter (DOM) that was unresolved and smeared staining characteristics on both SDS-PAGE and 2-DE gels was first observed in Tokyo Bay waters in the present study and its possible sources are discussed. Although the two protein species, 48 kDa and 39 kDa proteins, have been identified as homologues of Porin P and low molecular weight-alkaline phosphatase of Pseudomonas aeruginosa PAO1, respectively, four strains of P. aeruginosa and two species of Pseudomonas spp. have been newly identified as the source organisms of these proteins using the N-terminal amino acid sequence data determined in previous studies.

Insights into Mesenchymal Stem Cell Aging: Involvement of Antioxidant Defense and Actin Cytoskeleton

Progenitor cells such as mesenchymal stem cells (MSCs) have elicited great hopes for therapeutic augmentation of physiological regeneration processes, e.g., for bone fracture healing. However, regeneration potential decreases with age, which raises questions about the efficiency of autologous approaches in elderly patients. To elucidate the mechanisms and cellular consequences of aging, the functional and proteomic changes in MSCs derived from young and old Sprague-Dawley rats were studied concurrently. We demonstrate not only that MSC concentration in bone marrow declines with age but also that their function is altered, especially their migratory capacity and susceptibility toward senescence. High-resolution two-dimensional electrophoresis of the MSC proteome, under conditions of in vitro self-renewal as well as osteogenic stimulation, identified several age-dependent proteins, including members of the calponin protein family as well as galectin-3. Functional annotation clustering revealed that age-affected molecular functions are associated with cytoskeleton organization and antioxidant defense. These proteome screening results are supported by lower actin turnover and diminished antioxidant power in aged MSCs, respectively. Thus, we postulate two main reasons for the compromised cellular function of aged MSCs: (a) declined responsiveness to biological and mechanical signals due to a less dynamic actin cytoskeleton and (b) increased oxidative stress exposure favoring macromolecular damage and senescence. These results, along with the observed similar differentiation potentials, imply that MSC-based therapeutic approaches for the elderly should focus on attracting the cells to the site of injury and oxidative stress protection, rather than merely stimulating differentiation.