Traumatic brain injury (TBI) is associated with enhanced osteogenesis. The aim of this study was to investigate the effect of serum from TBI rats on fracture healing. Results from this study showed that the serum from TBI rats enhanced the expression of bone gamma carboxyglutamate protein (BGLAP), and promoted in vitro proliferation of MC3T3-E1 cells, a mouse osteoblastic cell line. Furthermore, gas chromatography/mass spectrometry (GC/MS) coupled with multivariate statistical analysis was used to identify the changes in global serum metabolites after TBI. We found that arachidonic acid (AA) was significantly enhanced in serum metabolites in TBI subjects, while hydroxybutyric acid, leucine, malic acid, 5-oxyproline, isocitric acid, mannose, and uric acid were reduced. Finally, we examined the effects of AA on BGLAP expression and cell proliferation in MC3T3-E1 cells. We found that BGLAP expression and proliferation of osteoblasts were positively regulated in the presence of AA. These findings suggest that the increased AA in serum after TBI may play a key role in enhancing the speed of fracture healing.
Pulmonary embolism (PE) is a common, potentially fatal disease and its diagnosis is challenging because clinical signs and symptoms are nonspecific. In this study, to investigate protein alterations of a rat PE model, total serum proteins collected at different time points were separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Bioinformatics analysis of 24 differentially expressed proteins showed that 20 had corresponding protein candidates in the database. According to their properties and obvious alterations after PE, changes of serum concentrations of Hp, Fn, DBP, RBP, and TTR were selected to be reidentified by western blot analysis. Semiquantitative RT−PCR showed DBP, RBP, and TTR to be down-regulated at mRNA levels in livers but not in lung tissues. The low serum concentrations of DBP, RBP, and TTR resulted in the up-regulation of 25(OH)D3, vitamin A, and FT4 (ligands of DBP, RBP, and TTR) after acute PE in rat models. The serum levels of Hp and Fn were detected in patients with DVT/PE and controls to explore their diagnostic prospects in acute PE because the mRNA levels of Hp and Fn were found to be up-regulated both in lung tissues and in livers after acute PE. Our data suggested that the concentration of serum Fn in controls was 79.42 ± 31.57 μg/L, whereas that of PE/DVT patients was 554.43 ± 136.18 μg/L (P < 0.001), and that the concentration of serum Hp in controls was 824.37 ± 235.24 mg/L, whereas that of PE/DVT patients was 2063.48 ± 425.38 mg/L (P < 0.001). The experimental PE rat model selected in this study was more similar to the clinical process than the other existing PE animal models, and the findings indicated instant changes of serum proteins within 48 h after acute PE. The exploration of these differentially expressed proteins or their combination with existent markers such as D-dimer may greatly improve the accuracy of the diagnosis of acute PE, but diagnostic tests are still needed to evaluate the sensitivity and specificity of these markers and also the number of false positives and false negatives. Keywords: comparative proteomics • two dimensional gel electrophoresis • mass spectrometry • pulmonary embolism • haptoglobin • ferritin
Abstract Unmatched masses are often observed in the experimental peptide mass spectra when database searching is performed with the ProFound program. Comparison between theoretical and experimental mass spectra of standard proteins shows that contamination accounts for most of the unmatched masses. In this retrospective analysis, the top 100 most probable contaminating masses, as listed in order of their probability, are statistically filtered out from 118 different experimental peptide mass fingerprinting (PMF) maps. Most of the interfering masses originate from trypsin autolysis and human keratins. Subtraction of known contaminants from raw data and using cleaner masses for searching can enhance protein identification by PMF.
Spinal cord injury and regeneration related protein No. 69 (SCIRR69) is a rat transmembrane bZIP transcription factor homologous to mice and human transcription factor CREB3L2. Previous work demonstrated the N-terminal region plays a critical role in its transcriptional activity. In this study, a peptide containing 18 amino acids (5-22aa) at the N-terminus of rat SCIRR69 was synthesized and coupled to the carrier protein as immunogen. One hybridoma cell line was obtained by standard cell fusion technique, followed by enzyme-linked immunosorbent assay (ELISA) and Western blot screening. The newly developed monoclonal antibody (MAb) was designated 4B4, the isotype of which was IgG2a. Immunofluorescence and Western blotting results showed that MAb 4B4 could recognize the SCIRR69 protein in both native and denatured forms. 4B4 will be a useful tool for the functional research of SCIRR69 in future studies.
In order to explore if mature neurons derived from neural stem cells have the potentiality to divide, we utilized the chemical digestion method to disperse the adult rat brain tissue into single cells, and culture them in serum-free medium. After being cultured for about eight days in vitro, the neural stem cells were induced to differentiate into neurons. The neurons were further induced to divide. Utilizing the method of serial photograph and NF-160 immunocytochemistry, the processes of division of some neurons were recorded. At the same time, PCNA+NF-160 (or Chat, GAB A, GAD) double label were used to investigate if the dividing-neurons were mature ones. After the neural stem cells were induced to differentiate in vitro for eight days, they possessed the shape and character of mature neurons. The differentiated neuron had a big nucleus and one or two distinct nucleolus in the nuclear. Within the perikaryon, there were a large amount of dense and Nissl body-like structure. Several long processes emerged from various locations of the cell body. Then, EGF and bFGF were added into the medium to induce division. After two days of induced-division, neuron-like cells were observed to divide; moreover, the number of neuron-like cells in the region increased continually. Immunocytochemistry demonstrated these cells were NF-160-positive. Serial photographs of dividing-process of neuron-like cells were obtained and their daughter cells were also NF-160-positive. After PCNA+NF-160 (or Chat, GABA, GAD) double label, some cells showed brown cell plasma and black nucleus. The above-mentioned results indicate that neurons, which were previously thought to be end-differentiated, can be re-called into cell cycle under appropriate conditions. Mature neurons still have the potential to divide, proliferate and self-renew.
This study investigated the proteomic changes at different time points in the precipitated pellets of rat spinal cords after applying complete spinal cord transection. By two-dimensional electrophoresis, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, MALDI-TOF/TOF and peptide mass fingerprinting analysis, 44 proteins were identified, most of which are membrane and/or organellar proteins. They are mainly involved in metabolic processes (75%), developmental processes (30%), or responses to stimuli (30%), playing negative or positive roles. In particular, decreases of pyruvate dehydrogenase beta, aconitase 2, fumarate hydratase 1, and ATP synthase subunit 6 can lead to ATP depletion by crippling tricarboxylic acid cycle and oxidative phosphorylation. Decreases of several antioxidant proteins such as catalase, peroxiredoxin 1, Parkinson disease 7, and stress-induced phosphoprotein 1 can contribute to the secondary injury of spinal cord. Decreases of development-related 3-phosphoglycerate dehydrogenase and stathmin 1 may be not propitious for spinal cord regeneration. On the other hand, increases of isocitrate dehydrogenase 3 alpha/gamma and glutamate dehydrogenase 1 can help compensate the impaired energy metabolism. Increases of sirtuin 2, crystallin alpha B (CRYAB), and heat shock 27-kDa protein 1 can help resist stresses induced by injury. Increases of adenylate cyclase-associated protein 1 and galactose binding lectin 3 can help regeneration by replaying their roles in neural development. To our knowledge, this is the first case of characterization of the proteomic changes seen in the precipitated fraction of injured spinal cord. Most of the identified proteins were found for the first time to be differentially expressed after spinal cord injury, which may provide new clues about the molecular mechanisms of spinal cord injury and repair.
Abstract The inability of the CNS to regenerate in adult mammals propels us to reveal associated proteins involved in the injured CNS. In this paper, either thoracic laminectomy (as sham control) or thoracic spinal cord transection was performed on male adult rats. Five days after surgery, the whole spinal cord tissue was dissected and fractionated into water‐soluble (dissolved in Tris buffer) and water‐insoluble (dissolved in a solution containing chaotropes and surfactants) portions for 2‐DE. Protein identification was performed by MS and further confirmed by Western blot. As a result, over 30 protein spots in the injured spinal cord were shown to be up‐regulated no less than 1.5‐fold. These identified proteins possibly play various roles during the injury and repair process and may be functionally categorized as several different groups, such as stress‐responsive and metabolic changes, lipid and protein degeneration, neural survival and regeneration. In particular, over‐expression of 11‐zinc finger protein and glypican may be responsible for the inhibition of axonal growth and regeneration. Moreover, three unknown proteins with novel sequences were found to be up‐regulated by spinal cord injury. Further characterization of these molecules may help us come closer to understanding the mechanisms that underlie the inability of the adult CNS to regenerate.
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