Alpha-lipoic acid supplementation protects enzymes from damage by nitrosative and oxidative stress.

BACKGROUND: S-nitrosylation of mitochondrial enzymes involved in energy transfer under nitrosative stress may result in ATP deficiency. We investigated whether α-lipoic acid, a powerful antioxidant, could alleviate nitrosative stress by regulating S-nitrosylation, which could result in retaining the mitochondrial enzyme activity. METHODS: In this study, we have identified the S-nitrosylated forms of subunit 1 of dihydrolipoyllysine succinyltransferase (complex III), and subunit 2 of the α-ketoglutarate dehydrogenase complex by implementing a fluorescence-based differential quantitative proteomics method. RESULTS: We found that the activities of these two mitochondrial enzymes were partially but reversibly inhibited by S-nitrosylation in cultured endothelial cells, and that their activities were partially restored by supplementation of α-lipoic acid. We show that protein S-nitrosylation affects the activity of mitochondrial enzymes that are central to energy supply, and that α-lipoic acid protects mitochondrial enzymes by altering S-nitrosylation levels. CONCLUSIONS: Inhibiting protein S-nitrosylation with α-lipoic acid seems to be a protective mechanism against nitrosative stress. GENERAL SIGNIFICANCE: Identification and characterization of these new protein targets should contribute to expanding the therapeutic power of α-lipoic acid and to a better understanding of the underlying antioxidant mechanisms.

Polymerase I and transcript release factor (PTRF) regulates adipocyte differentiation and determines adipose tissue expandability.

Impaired adipogenesis renders an adipose tissue unable to expand, leading to lipotoxicity and conditions such as diabetes and cardiovascular disease. While factors important for adipogenesis have been studied extensively, those that set the limits of adipose tissue expansion remain undetermined. Feeding a Western-type diet to apolipoprotein E2 knock-in mice, a model of metabolic syndrome, produced 3 groups of equally obese mice: mice with normal glucose tolerance, hyperinsulinemic yet glucose-tolerant mice, and prediabetic mice with impaired glucose tolerance and reduced circulating insulin. Using proteomics, we compared subcutaneous adipose tissues from mice in these groups and found that the expression of PTRF (polymerase I and transcript release factor) associated selectively with their glucose tolerance status. Lentiviral and pharmacologically overexpressed PTRF, whose function is critical for caveola formation, compromised adipocyte differentiation of cultured 3T3-L1cells. In human adipose tissue, PTRF mRNA levels positively correlated with markers of lipolysis and cellular senescence. Furthermore, a negative relationship between telomere length and PTRF mRNA levels was observed in human subcutaneous fat. PTRF is associated with limited adipose tissue expansion underpinning the key role of caveolae in adipocyte regulation. Furthermore, PTRF may be a suitable adipocyte marker for predicting pathological obesity and inform clinical management.

Cardiac mitochondrial proteomic expression in inbred rat strains divergent in survival time after hemorrhage.

We have previously identified inbred rat strains differing in survival time to a severe controlled hemorrhage (StaH). In efforts to identify cellular mechanisms and ultimately genes that are important contributors to enhanced STaH, we conducted a study to characterize potential differences in cardiac mitochondrial proteins in these rats. Inbred rats from three strains [Brown Norway/Medical College of Wisconsin (BN); Dark Agouti (DA), and Fawn Hooded Hypertensive (FHH)] with different StaH (DA = FHH > BN) were assigned to one of three treatment groups (n = 4/strain): nonoperated controls, surgically catheterized rats, or rats surgically catheterized and hemorrhaged 24 h postsurgery. Rats were euthanized 30 min after handling or 30 min after initiation of a 26 min hemorrhage. After euthanasia, hearts were removed and mitochondria isolated. Differential protein expression was determined using 2D DIGE-based Quantitative Intact Proteomics and proteins identified by MALDI/TOF mass spectrometry. Hundreds of proteins (791) differed among inbred rat strains (P ≤ 0.038), and of these 81 were identified. Thirty-eight were unique proteins and 43 were apparent isoforms. For DA rats (longest STaH), 36 proteins increased and 30 decreased compared with BN (shortest STaH). These 81 proteins were associated with lipid (e.g., acyl CoA dehydrogenase) and carbohydrate (e.g., fumarase) metabolism, oxidative phosphorylation (e.g., ubiquinol-cytochrome C reductase), ATP synthesis (F1 ATPase), and H2S synthesis (3-mercaptopyruvate sulfurtransferase). Although we cannot make associations between these identified mitochondrial proteins and StaH, our data do provide evidence for future candidate proteins with which to consider such associations.

CLN5 and CLN8 protein association with ceramide synthase: biochemical and proteomic approaches.

Four patients with juvenile neuronal ceroid lipofuscinoses, a childhood neurodegenerative disorder that was previously described as CLN9 variant, are reclassified as CLN5 disease. CLN5-deficient (CLN5(-/-) ) fibroblasts demonstrate adhesion defects, increased growth, apoptosis, and decreased levels of ceramide, sphingomyelin, and glycosphingolipids. The CLN8 protein (CLN8p) corrects growth and apoptosis in CLN5(-/-) cells. Related proteins containing a Lag1 motif (CerS1/2/4/5/6) partially corrected these deficits, with CerS1, which is primarily expressed in brain, providing the best complementation, suggesting CLN5p activates CerS1 and may co-immunoprecipitate with it. CLN8p complements CLN5-deficient cells, consolidating the interrelationship of CLN5p/CLN8p, whose potential roles are explored as activators of (dihydro)ceramide synthases. Homozygosity mapping using microarray technology led to identification of CLN5 as the culprit gene in previously classified CLN9-defective cases. Similar to CLN5(-/-) cells, ceramide synthase activity, C16/C18:0/C24:0/C24:1 ceramide species, measured by MS is decreased in CLN8(-/-) cells. Comparison of normal versus CLN5(-/-) cell CerS1-bound proteins by immunoprecipitation, differential gel electrophoresis, and MS revealed absence of γ-actin in CLN5(-/-) cells. The γ-actin gene sequence is normal in CLN5(-/-) derived DNA. The γ-actin-bound proteins, vimentin and histones H2Afz/H3F3A/Hist1H4, were absent from the γ-actin protein complex in CLN5(-/-) cells. The function of CLN5p may require vimentin and the histone proteins to bind γ-actin. Defective binding could explain the CLN5(-/-) cellular phenotype. We explore the role of the CLN5/CLN8 proteins in ceramide species specific sphingolipid de novo synthesis, and suggest that CLN5/CLN8 proteins are more closely related than previously believed.

Proteomic analysis of mice expressing human ApoE demonstrates no differences in global protein solubility between APOE 3 and APOE 4 young mice.

Apolipoprotein E (ApoE) is a major lipid carrier protein. In humans, ApoE is expressed in three polymorphic isoforms, which are encoded by three different alleles APOE2, APOE3, and APOE4. In the brains of Alzheimer's disease (AD) patients, each one of these three allelic isoforms is found in several "isoelectric" protein isoforms (qPI), i.e. protein isoforms resulting from PTMs altering the net charge (q) of the polypeptide. AD is a complex disease in which multiple causes and several risk factors affect the onset and disease outcome. A major risk factor for AD is ApoE4; therefore, it is important to characterize the different ApoE qPIs. We have implemented a detergent-based method for isolation and quantitation of protein isoforms, and we found differences in the solubility of protein isoforms depending on the type of solvent used. In this manuscript, we describe these methods and applied them to young human-ApoE targeted replacement mice. Our results indicate that there are no significant differences in the hippocampus proteome of these mice as a function of the APOE genotype.

Acute phase response, inflammation and metabolic syndrome biomarkers of Libby asbestos exposure.

Identification of biomarkers assists in the diagnosis of disease and the assessment of health risks from environmental exposures. We hypothesized that rats exposed to Libby amphibole (LA) would present with a unique serum proteomic profile which could help elucidate epidemiologically-relevant biomarkers. In four experiments spanning varied protocols and temporality, healthy (Wistar Kyoto, WKY; and F344) and cardiovascular compromised (CVD) rat models (spontaneously hypertensive, SH; and SH heart failure, SHHF) were intratracheally instilled with saline (control) or LA. Serum biomarkers of cancer, inflammation, metabolic syndrome (MetS), and the acute phase response (APR) were analyzed. All rat strains exhibited acute increases in α-2-macroglobulin, and α1-acid glycoprotein. Among markers of inflammation, lipocalin-2 was induced in WKY, SH and SHHF and osteopontin only in WKY after LA exposure. While rat strain- and age-related changes were apparent in MetS biomarkers, no LA effects were evident. The cancer marker mesothelin was increased only slightly at 1 month in WKY in one of the studies. Quantitative Intact Proteomic profiling of WKY serum at 1 day or 4 weeks after 4 weekly LA instillations indicated no oxidative protein modifications, however APR proteins were significantly increased. Those included serine protease inhibitor, apolipoprotein E, α-2-HS-glycoprotein, t-kininogen 1 and 2, ceruloplasmin, vitamin D binding protein, serum amyloid P, and more 1 day after last LA exposure. All changes were reversible after a short recovery regardless of the acute or long-term exposures. Thus, LA exposure induces an APR and systemic inflammatory biomarkers that could have implications in systemic and pulmonary disease in individuals exposed to LA.

Simultaneous detection of changes in protein expression and oxidative modification as a function of age and APOE genotype.

To better elucidate temporal changes in protein oxidation resulting from aging and the Alzheimer's disease-associated Apolipoprotein E (ApoE), we developed a 2D-DIGE-based method for simultaneously detecting differential expression and carbonyl oxidation of proteins. Specifically, we examined changes in the levels of oxidation and total protein expression in hippocampi from young-adult (25-30 weeks) and old (76-97 weeks) mice transgenic for the human Apolipoprotein E gene (APOE, APOE3, APOE4) isoforms, APOE3 or APOE4. Protein samples were labeled with either a fluorescent aminooxyacetamide (Alexa Fluor 488) to detect carbonyl modifications or with NHS-Cy3 to detect total protein expression. A protein sample used as an internal control was labeled with NHS-Cy5 and run on each gel. DIGE analysis revealed 38 differentially oxidized and 100 differentially expressed protein spots with significantly different levels (P < 0.05). For oxidized proteins, principal component analysis revealed two distinct clusters: one in which oxidation increased with age independent of APOE genotype, and the second in which oxidation was dependent on APOE genotype. For total protein expression, principal component analysis revealed a large overlap between changes with overall aging and between APOE genotypes. The use of a fluorescent tag to label oxidized proteins, in combination with a NHS-Cy3 to label total protein, makes it possible to determine changes in both protein oxidation and protein expression levels in a single experiment. These studies reveal that the expression levels of peroxiredoxin protein family members Prdx2, 3, and 6 are modified by age, APOE genotype, or both.

Aroclor 1254, a developmental neurotoxicant, alters energy metabolism- and intracellular signaling-associated protein networks in rat cerebellum and hippocampus.

The vast literature on the mode of action of polychlorinated biphenyls (PCBs) indicates that PCBs are a unique model for understanding the mechanisms of toxicity of environmental mixtures of persistent chemicals. PCBs have been shown to adversely affect psychomotor function and learning and memory in humans. Although the molecular mechanisms for PCB effects are unclear, several studies indicate that the disruption of Ca(2+)-mediated signal transduction plays significant roles in PCB-induced developmental neurotoxicity. Culminating events in signal transduction pathways include the regulation of gene and protein expression, which affects the growth and function of the nervous system. Our previous studies showed changes in gene expression related to signal transduction and neuronal growth. In this study, protein expression following developmental exposure to PCB is examined. Pregnant rats (Long Evans) were dosed with 0.0 or 6.0mg/kg/day of Aroclor-1254 from gestation day 6 through postnatal day (PND) 21, and the cerebellum and hippocampus from PND14 animals were analyzed to determine Aroclor 1254-induced differential protein expression. Two proteins were found to be differentially expressed in the cerebellum following PCB exposure while 18 proteins were differentially expressed in the hippocampus. These proteins are related to energy metabolism in mitochondria (ATP synthase, sub unit ß (ATP5B), creatine kinase, and malate dehydrogenase), calcium signaling (voltage-dependent anion-selective channel protein 1 (VDAC1) and ryanodine receptor type II (RyR2)), and growth of the nervous system (dihydropyrimidinase-related protein 4 (DPYSL4), valosin-containing protein (VCP)). Results suggest that Aroclor 1254-like persistent chemicals may alter energy metabolism and intracellular signaling, which might result in developmental neurotoxicity.

Participation of valine 171 in alpha-Helix 5 of Bacillus thuringiensis Cry1Ab delta-endotoxin in translocation of toxin into Lymantria dispar midgut membranes.

The Cry1Ab δ-endotoxin V171C mutant protein exhibits a 25-fold increase in toxicity against Lymantria dispar, which correlates with a faster rate of partitioning into the midgut membrane and slightly decreased protein stability. This is an insect-specific mechanism; similar results were not observed in Manduca sexta, another Cry1Ab δ-endotoxin-susceptible insect.

Proteomic and immunologic analyses of brain tumor exosomes.

Brain tumors are horrific diseases with almost universally fatal outcomes; new therapeutics are desperately needed and will come from improved understandings of glioma biology. Exosomes are endosomally derived 30-100 nm membranous vesicles released from many cell types into the extracellular milieu; surprisingly, exosomes are virtually unstudied in neuro-oncology. These microvesicles were used as vaccines in other tumor settings, but their immunological significance is unevaluated in brain tumors. Our purpose here is to report the initial biochemical, proteomic, and immunological studies on murine brain tumor exosomes, following known procedures to isolate exosomes. Our findings show that these vesicles have biophysical characteristics and proteomic profiles similar to exosomes from other cell types but that brain tumor exosomes have unique features (e.g., very basic isoelectric points, expressing the mutated tumor antigen EGFRvIII and the putatively immunosuppressive cytokine TGF-beta). Administration of such exosomes into syngeneic animals produced both humoral and cellular immune responses in immunized hosts capable of rejecting subsequent tumor challenges but failed to prolong survival in established orthotopic models. Control animals received saline or cell lysate vaccines and showed no antitumor responses. Exosomes and microvesicles isolated from sera of patients with brain tumors also possess EGFR, EGFRvIII, and TGF-beta. We conclude that exosomes released from brain tumor cells are biochemically/biophysically like other exosomes and have immune-modulating properties. They can escape the blood-brain barrier, with potential systemic and distal signaling and immune consequences.

Activation of glycolysis and apoptosis in glycogen storage disease type Ia.

The deficiency of glucose-6-phosphatase (G6Pase) underlies glycogen storage disease type Ia (GSD-Ia, von Gierke disease; MIM 232200), an autosomal recessive disorder of metabolism associated with life-threatening hypoglycemia, growth retardation, renal failure, hepatic adenomas, and hepatocellular carcinoma. Liver involvement includes the massive accumulation of glycogen and lipids due to accumulated glucose-6-phosphate and glycolytic intermediates. Proteomic analysis revealed elevations in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and other enzymes involved in glycolysis. GAPDH was markedly increased in murine G6Pase-deficient hepatocytes. The moonlighting role of GAPDH includes increasing apoptosis, which was demonstrated by increased TUNEL assay positivity and caspase 3 activation in the murine GSD-Ia liver. These analyses of hepatic involvement in GSD-Ia mice have implicated the induction of apoptosis in the pathobiology of GSD-Ia.

Ser170 of Bacillus thuringiensis Cry1Ab delta-endotoxin becomes anchored in a hydrophobic moiety upon insertion of this protein into Manduca sexta brush border membranes.

BACKGROUND: Three spin-labeled mutant proteins, mutated at the beginning, middle, and end of alpha-helix 5 of the Bacillus thuringiensis Cry1Ab delta-endotoxin, were used to study the involvement of these specific amino acid residues in ion transport and to determine conformational changes in the vicinity of these residues when the protein was translocated into a biological membrane. RESULTS: Amino acid residue leucine 157, located in the N-terminal portion of alpha-helix 5, showed no involvement in ion transport, and the environment that surrounds the residue did not show any change when transferred into the biological membrane. Serine 170, located in the middle of the alpha-helix, showed no involvement in ion transport, but our findings indicate that in the membrane-bound state this residue faces an environment that makes the spin less mobile, as opposed to the mobility observed in an aqueous environment. Serine 176, located in the C-terminal end of the alpha-helix 5 is shown to be involved in ion transport activity. CONCLUSION: Ion transport data for L157, S170, and S176, along with the mobility of the spin-labels, structural characterization of the resulting proteins, and toxicity assays against a target insect, suggest that the toxin undergoes conformational changes upon protein translocation into the midgut membrane. These conformational changes result in the midregion of the alpha-helix 5 being exposed to a hydrophobic-like environment. The location of these three residues in the toxin suggests that the entire alpha-helix becomes inserted in the insect midgut membrane.

Profiling of experience-regulated proteins in the songbird auditory forebrain using quantitative proteomics.

Auditory and perceptual processing of songs are required for a number of behaviors in songbirds such as vocal learning, territorial defense, mate selection and individual recognition. These neural processes are accompanied by increased expression of a few transcription factors, particularly in the caudomedial nidopallium (NCM), an auditory forebrain area believed to play a key role in auditory learning and song discrimination. However, these molecular changes are presumably part of a larger, yet uncharacterized, protein regulatory network. In order to gain further insight into this network, we performed two-dimensional differential in-gel expression (2D-DIGE) experiments, extensive protein quantification analyses, and tandem mass spectrometry in the NCM of adult songbirds hearing novel songs. A subset of proteins was selected for immunocytochemistry in NCM sections to confirm the 2D-DIGE findings and to provide additional quantitative and anatomical information. Using these methodologies, we found that stimulation of freely behaving birds with conspecific songs did not significantly impact the NCM proteome 5 min after stimulus onset. However, following 1 and 3 h of stimulation, a significant number of proteins were consistently regulated in NCM. These proteins spanned a range of functional categories that included metabolic enzymes, cytoskeletal molecules, and proteins involved in neurotransmitter secretion and calcium binding. Our findings suggest that auditory processing of vocal communication signals in freely behaving songbirds triggers a cascade of protein regulatory events that are dynamically regulated through activity-dependent changes in calcium levels.

Blocking binding of Bacillus thuringiensis Cry1Aa to Bombyx mori cadherin receptor results in only a minor reduction of toxicity.

BACKGROUND: Bacillus thuringiensis Cry1Aa insecticidal protein is the most active known B. thuringiensis toxin against the forest insect pest Lymantria dispar (gypsy moth), unfortunately it is also highly toxic against the non-target insect Bombyx mori (silk worm). RESULTS: Surface exposed hydrophobic residues over domains II and III were targeted for site-directed mutagenesis. Substitution of a phenylalanine residue (F328) by alanine reduced binding to the Bombyx mori cadherin by 23-fold, reduced biological activity against B. mori by 4-fold, while retaining activity against Lymantria dispar. CONCLUSION: The results identify a novel receptor-binding epitope and demonstrate that virtual elimination of binding to cadherin BR-175 does not completely remove toxicity in the case of B. mori.

Experiencias de aprendizaje metacognitivo en estudiantes de enfermería: revisión de alcance / Metacognitive Learning Experiences in Nursing Students: Scoping Review / Experiências de aprendizagem metacognitiva em estudantes de enfermagem: Uma revisão do escopo

Introducción: la metacognición es comprendida como el conocimiento de los propios procesos cognitivos y su autorregulación por parte de los estudiantes. Objetivo: conocer las tendencias de la investigación en torno a la metacognición en los procesos de enseñanza y aprendizaje en programas de Enfermería. Método: se llevó a cabo una revisión de alcance en torno a la metacognición como una estrategia de reflexión de los procesos de enseñanza aprendizaje al interior de los programas de Enfermería. El proceso de búsqueda se realizó en la base de datos Web of Science en el período comprendido entre 2015-2021; la selección de estudios cumplió con los criterios de inclusión. Resultados: el análisis de la información permitió identificar cuatro tendencias: a) la metacognición en procesos de enseñanza y aprendizaje con algunas didácticas grupales, b) la metacognición en procesos de aprendizaje con simulación clínica, c) el aprendizaje reflexivo como estrategia de regulación metacognitiva aplicada a los procesos de evaluación, d) la metacognición en los planes curriculares de los programas académicos en salud. Conclusiones: la revisión evidencia la implementación de metodologías y estrategias metacognitivas en los espacios de formación académica en los programas de Enfermería, que trascienden un modelo educativo tradicional centrado en los contenidos y se dirigen a un modelo centrado en la reflexión consciente y participativa de los estudiantes en el proceso de aprendizaje.

Introduction: Metacognition is understood as the students' knowledge of their own cognitive processes and their self-regulation. Objective: Aknowledge research trends of metacognition in teaching and learning processes in Nursing programs. Method: A scoping review was carried out on metacognition as a strategy for reflection of the teaching-learning processes within Nursing programs. The search process was carried out in the Web of Science database in the period 2015-2021; the selection of studies met the inclusion criteria. Results: The analysis of information allowed the identification of four trends a) metacognition in teaching and learning processes with some group didactics, b) metacognition in learning processes with clinical simulation, c) reflective learning as a metacognitive regulation strategy applied to evaluation processes, d) metacognition in the curricular plans of academic health programs. Conclusions: The review evidences the implementation of metacognitive methodologies and strategies in academic training spaces in Nursing programs, which transcend a traditional educational model focused on contents and moves to a model centered on students' conscious and participative reflection in the learning process.

Introdução: a metacognição é entendida como o conhecimento que os estudantes têm dos seus próprios processos cognitivos e da sua autorregulação. Objetivo: conhecer as tendências da investigação sobre a metacognição nos processos de ensino e aprendizagem nos programas de enfermagem. Método: foi realizada uma revisão de escopo sobre a metacognição como estratégia de reflexão sobre os processos de ensino-aprendizagem nos programas de enfermagem. O processo de busca foi realizado na base de dados Web of Science no período de 2015-2021; Na seleção dos estudos foram atendidos os critérios de inclusão. Resultados: a análise da informação permitiu identificar quatro tendências a) a metacognição nos processos de ensino e aprendizagem com algumas didácticas de grupo, b) a metacognição nos processos de aprendizagem com simulação clínica, c) a aprendizagem reflexiva como estratégia de regulação metacognitiva aplicada aos processos de avaliação, d) a metacognição nos planos curriculares dos programas académicos de saúde.Conclusões: a revisão evidencia a implementação de metodologias e estratégias metacognitivas em espaços de formação académica em programas de Enfermagem, que transcendem de um modelo educativo tradicional centrado nos conteúdos para um modelo centrado na reflexão consciente e participativa dos estudantes no processo de aprendizagem.

Developmental exposure to a commercial PBDE mixture: effects on protein networks in the cerebellum and hippocampus of rats.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) are structurally similar to polychlorinated biphenyls (PCBs) and have both central (learning and memory deficits) and peripheral (motor dysfunction) neurotoxic effects at concentrations/doses similar to those of PCBs. The cellular and molecular mechanisms for these neurotoxic effects are not fully understood; however, several studies have shown that PBDEs affect thyroid hormones, cause oxidative stress, and disrupt Ca2+-mediated signal transduction. Changes in these signal transduction pathways can lead to differential gene regulation with subsequent changes in protein expression, which can affect the development and function of the nervous system. OBJECTIVE: In this study, we examined the protein expression profiles in the rat cerebellum and hippocampus following developmental exposure to a commercial PBDE mixture, DE-71. METHODS: Pregnant Long-Evans rats were dosed perinatally with 0 or 30.6 mg/kg/day of DE-71 from gestation day 6 through sampling on postnatal day 14. Proteins from the cerebellum and hippocampus were extracted, expression differences were detected by two-dimensional difference gel electrophoresis, and proteins were identified by tandem mass spectrometry. Protein network interaction analysis was performed using Ingenuity® Pathway Analysis, and the proteins of interest were validated by Western blotting. RESULTS: Four proteins were significantly differentially expressed in the cerebellum following DE-71 exposure, whereas 70 proteins were significantly differentially expressed in the hippocampus. Of these proteins, 4 from the cerebellum and 47 from the hippocampus, identifiable by mass spectrometry, were found to have roles in mitochondrial energy metabolism, oxidative stress, apoptosis, calcium signaling, and growth of the nervous system. CONCLUSIONS: Results suggest that changes in energy metabolism and processes related to neuroplasticity and growth may be involved in the developmental neurotoxicity of PBDEs.

Increased yield of endothelial cells from peripheral blood for cell therapies and tissue engineering.

AIM: Peripheral blood-derived endothelial cells (pBD-ECs) are an attractive tool for cell therapies and tissue engineering, but have been limited by their low isolation yield. We increase pBD-EC yield via administration of the chemokine receptor type 4 antagonist AMD3100, as well as via a diluted whole blood incubation (DWBI). MATERIALS & METHODS: Porcine pBD-ECs were isolated using AMD3100 and DWBI and tested for EC markers, acetylated LDL uptake, growth kinetics, metabolic activity, flow-mediated nitric oxide production and seeded onto titanium tubes implanted into vessels of pigs. RESULTS: DWBI increased the yield of porcine pBD-ECs 6.6-fold, and AMD3100 increased the yield 4.5-fold. AMD3100-mobilized ECs were phenotypically indistinguishable from nonmobilized ECs. In porcine implants, the cells expressed endothelial nitric oxide synthase, reduced thrombin-antithrombin complex systemically and prevented thrombosis. CONCLUSION: Administration of AMD3100 and the DWBI method both increase pBD-EC yield.

Proteomic identification of brainstem cytosolic proteins in a neuropathic pain model.

Neuropathic pain involves co-regulation of many genes and their translational products in both peripheral and central nervous system. We used proteomics approaches to investigate expressional changes in cytosolic protein levels in rat brainstem tissues following ligation of lumbar 5 and 6 (L5, L6) spinal nerves, which generates a model of peripheral neuropathic pain (NP). Proteins from brainstem tissue homogenates of NP and SHAM animals were fractionated by two-dimensional (2-DE) gel electrophoresis to produce a high-resolution map of the brainstem soluble proteins. Proteins showing altered expression levels between NP and SHAM were selected. Isolated proteins were in-gel trypsin-digested and the resulting peptides were analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Using the mass spectrometric data, we were able to identify 17 proteins of interest through searches of the Swiss-Prot and NCBi nonredundant protein sequence database. Several of the identified proteins, including fatty acid binding protein-brain (FABP-B), major histocompatibility complex (MHC) class 1, T-cell receptor (TCR) alpha chain, and interleukin-1 (IL-1), showed significantly higher levels in the NP rat brainstem. Proteomic analysis has identified several proteins with differential expression levels in NP as compared to SHAM. However, the function of the proteins identified is postulated; therefore, further experiments are required to determine the true role of each protein in NP.

PPARα activation induces N(ε)-Lys-acetylation of rat liver peroxisomal multifunctional enzyme type 1.

Peroxisomes are ubiquitous subcellular organelles that participate in metabolic and disease processes, with few of its proteins undergoing posttranslational modifications. As the role of lysine-acetylation has expanded into the cellular intermediary metabolism, we used a combination of differential centrifugation, organelle isolation by linear density gradient centrifugation, western blot analysis, and peptide fingerprinting and amino acid sequencing by mass spectrometry to investigate protein acetylation in control and ciprofibrate-treated rat liver peroxisomes. Organelle protein samples isolated by density gradient centrifugation from PPARα-agonist treated rat liver screened with an anti-N(ε)-acetyl lysine antibody revealed a single protein band of 75 kDa. Immunoprecipitation with this antibody resulted in the precipitation of a protein from the protein pool of ciprofibrate-induced peroxisomes, but not from the protein pool of non-induced peroxisomes. Peptide mass fingerprinting analysis identified the protein as the peroxisomal multifunctional enzyme type 1. In addition, mass spectrometry-based amino acid sequencing resulted in the identification of unique peptides containing 4 acetylated-Lys residues (K¹55, K¹7³, K¹9°, and K58³). This is the first report that demonstrates posttranslational acetylation of a peroxisomal enzyme in PPARα-dependent proliferation of peroxisomes in rat liver.

Differentially charged isoforms of apolipoprotein E from human blood are potential biomarkers of Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) is the major cause of dementia among the elderly. Finding blood-based biomarkers for disease diagnosis and prognosis is urgently needed. METHODS: We studied protein distributions in brain tissues, cerebrospinal fluid (CSF), and blood of AD patients by using proteomics and a new proteomic method that we call "2D multiplexed Western blot" (2D mxWd). This method allows us to determine in multiple samples the electrophoretic patterns of protein isoforms with different isoelectric points. RESULTS: Apolipoprotein E (ApoE) displays a unique distribution of electrophoretic isoforms in the presence of AD and also a unique pattern specific to the APOE genotype. CONCLUSIONS: The isoelectric distribution of differentially charged ApoE isoforms was used to determine the presence of AD in a small group of samples. Further studies are needed to validate their use as predictors of disease onset and progression, and as biomarkers for determining the efficacy of therapeutic treatments.