Alterations of housekeeping proteins in human aged and diseased hearts.

Pathological remodeling includes alterations of ion channel function and calcium homeostasis and ultimately cardiac maladaptive function during the process of disease development. Biochemical assays are important approaches for assessing protein abundance and post-translational modification of ion channels. Several housekeeping proteins are commonly used as internal controls to minimize loading variabilities in immunoblotting protein assays. Yet, emerging evidence suggests that some housekeeping proteins may be abnormally altered under certain pathological conditions. However, alterations of housekeeping proteins in aged and diseased human hearts remain unclear. In the current study, immunoblotting was applied to measure three commonly used housekeeping proteins (ß-actin, calsequestrin, and GAPDH) in well-procured human right atria (RA) and left ventricles (LV) from diabetic, heart failure, and aged human organ donors. Linear regression analysis suggested that the amounts of linearly loaded total proteins and quantified intensity of total proteins from either Ponceau S (PS) blot-stained or Coomassie Blue (CB) gel-stained images were highly correlated. Thus, all immunoblotting data were normalized with quantitative CB or PS data to calibrate potential loading variabilities. In the human heart, ß-actin was reduced in diabetic RA and LV, while GAPDH was altered in aged and diabetic RA but not LV. Calsequestrin, an important Ca2+ regulatory protein, was significantly changed in aged, diabetic, and ischemic failing hearts. Intriguingly, expression levels of all three proteins were unchanged in non-ischemic failing human LV. Overall, alterations of human housekeeping proteins are heart chamber specific and disease context dependent. The choice of immunoblotting loading controls should be carefully evaluated. Usage of CB or PS total protein analysis could be a viable alternative approach for some complicated pathological specimens.

The relationship between TEM8 and early diagnosis and prognosis of lung cancer.

BACKGROUND: To explore the TEM8 expression in patients with lung cancer and its relationship with clinical pathology and prognosis, and to analyze the diagnostic value of TEM8. METHODS: A total of 204 patients with lung cancer diagnosed and treated in Zhongmeng Hospital Zhalantun and the First Affiliated Hospital of Jinzhou Medical University from March 2013 to February 2016 were enrolled in the study group, and 203 healthy subjects in the control group. qRT-PCR technique was applied to detect the TEM8 expression. Combined with clinical information, the diagnostic value of TEM8 for lung cancer and the correlation of clinical characteristics of TEM8 were analyzed. The 3-year survival curves of patients with low and high TEM8 expressions were compared. RESULTS: The expression in the study group was significantly higher than that in the control group (P<0.05). When the cut-off value was 1.125, the sensitivity, specificity and AUC of TEM8 in the diagnosis of lung cancer were 50.00%, 98.00% and 0.726 respectively. The TEM8 expression also differs when in smoking, lymphatic metastasis, TNM stage, differentiation degree and pleural invasion classification (P<0.050). 132 patients were included in the survival group and 72 patients were included in the death group. There was a difference between the two groups in the effect of TEM8 on the prognosis (P<0.001). CONCLUSIONS: TEM8 showed high expression in the study group. TEM8 had good diagnostic efficacy and was expected to be an excellent indicator for early clinical diagnosis and prognosis of lung cancer.

Reactive oxygen species-regulating proteins peroxiredoxin 2 and thioredoxin, and glyceraldehyde-3-phosphate dehydrogenase are differentially abundant in induced sputum from smokers with lung cancer or asbestos exposure.

Lung cancer is a deadly disease, typically caused by known risk factors, such as tobacco smoke and asbestos exposure. By triggering cellular oxidative stress and altering the antioxidant pathways eliminating reactive oxygen species (ROS), tobacco smoke and asbestos predispose to cancer. Despite easily recognizable high-risk individuals, lung cancer screening and its early detection are hampered by poor diagnostic tools including the absence of proper biomarkers. This study aimed to recognize potential lung cancer biomarkers using induced sputum noninvasively collected from the lungs of individuals in risk of contracting lung cancer. Study groups composed of current and former smokers, who either were significantly asbestos exposed, had lung cancer, or were unexposed and asymptomatic. Screening of potential biomarkers was performed with 52, and five differentially abundant proteins, peroxiredoxin 2 (PRDX2), thioredoxin (TXN), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), extracellular matrix protein 1 (ECM1), and protein S100 A8 (S100A8), were chosen to undergo validation, for their previously known connection with oxidative stress or cancer. Results from the validation in 123 sputa showed that PRDX2, TXN, and GAPDH were differentially abundant in sputa from individuals with lung cancer. TXN had a negative correlation with asbestos exposure, yet a positive correlation with smoking and lung cancer. Thus, tobacco smoking, asbestos exposure, and lung carcinogenesis may disturb the cellular redox state in different ways. A strong correlation was found among PRDX2, TXN, GAPDH, and S100A8, suggesting that these proteins may present a diagnostic biomarker panel to aid recognizing individuals at high risk of contracting lung cancer.

Featured Article: Pyruvate preserves antiglycation defenses in porcine brain after cardiac arrest.

Cardiac arrest (CA) and cardiocerebral resuscitation (CCR)-induced ischemia-reperfusion imposes oxidative and carbonyl stress that injures the brain. The ischemic shift to anaerobic glycolysis, combined with oxyradical inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), provokes excessive formation of the powerful glycating agent, methylglyoxal. The glyoxalase (GLO) system, comprising the enzymes glyoxalase 1 (GLO1) and GLO2, utilizes reduced glutathione (GSH) supplied by glutathione reductase (GR) to detoxify methylglyoxal resulting in reduced protein glycation. Pyruvate, a natural antioxidant that augments GSH redox status, could sustain the GLO system in the face of ischemia-reperfusion. This study assessed the impact of CA-CCR on the cerebral GLO system and pyruvate's ability to preserve this neuroprotective system following CA. Domestic swine were subjected to 10 min CA, 4 min closed-chest CCR, defibrillation and 4 h recovery, or to a non-CA sham protocol. Sodium pyruvate or NaCl control was infused (0.1 mmol/kg/min, intravenous) throughout CCR and the first 60 min recovery. Protein glycation, GLO1 content, and activities of GLO1, GR, and GAPDH were analyzed in frontal cortex biopsied at 4 h recovery. CA-CCR produced marked protein glycation which was attenuated by pyruvate treatment. GLO1, GR, and GAPDH activities fell by 86, 55, and 30%, respectively, after CA-CCR with NaCl infusion. Pyruvate prevented inactivation of all three enzymes. CA-CCR sharply lowered GLO1 monomer content with commensurate formation of higher molecular weight immunoreactivity; pyruvate preserved GLO1 monomers. Thus, ischemia-reperfusion imposed by CA-CCR disabled the brain's antiglycation defenses. Pyruvate preserved these enzyme systems that protect the brain from glycation stress. Impact statement Recent studies have demonstrated a pivotal role of protein glycation in brain injury. Methylglyoxal, a by-product of glycolysis and a powerful glycating agent in brain, is detoxified by the glutathione-catalyzed glyoxalase (GLO) system, but the impact of cardiac arrest (CA) and cardiocerebral resuscitation (CCR) on the brain's antiglycation defenses is unknown. This study in a swine model of CA and CCR demonstrated for the first time that the intense cerebral ischemia-reperfusion imposed by CA-resuscitation disabled glyoxalase-1 and glutathione reductase (GR), the source of glutathione for methylglyoxal detoxification. Moreover, intravenous administration of pyruvate, a redox-active intermediary metabolite and antioxidant in brain, prevented inactivation of glyoxalase-1 and GR and blunted protein glycation in cerebral cortex. These findings in a large mammal are first evidence of GLO inactivation and the resultant cerebral protein glycation after CA-resuscitation, and identify novel actions of pyruvate to minimize protein glycation in postischemic brain.

Riboflavin and ultraviolet illumination affects selected platelet mRNA transcript amounts differently.

BACKGROUND: Pathogen inactivation (PI) techniques use ultraviolet (UV) illumination with or without a photosensitizer to destroy pathogen RNA and DNA. Although lacking a nucleus and innate DNA transcription, platelets (PLTs) contain RNA and can synthesize proteins. The impact of PI on PLT protein synthesis and function is unknown; altered synthesis may affect overall PLT quality. In this study we determine to what extent PLT RNA is affected by PI. STUDY DESIGN AND METHODS: In a pool-and-split design, paired apheresis PLT concentrates were treated with riboflavin and UV illumination or were left untreated. PLT total RNA and mRNA amounts specific for glycoproteins (GP)IIIa, GPIIb, and GPIb; α-granule proteins PLT factor (PF)4; osteonectin and thrombospondin (TSP); and housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using absorbance and quantitative polymerase chain reaction. RESULTS: After treatment, amounts of all analyzed mRNAs were significantly reduced (p < 0.05), but to different degrees. For GAPDH and PF4, transcripts appeared less susceptible to the treatment, with 70% remaining 1 hour after UV illumination. For GPIIIa and TSP, less than 15% remained after treatment. There was a correlation (R(2) = 0.85) between transcript length and amount of mRNA remaining 1 hour after treatment. Total RNA demonstrated a life span equal to the PLT life span of 10 to 11 days. CONCLUSION: This is the first report of the impact of riboflavin and UV illumination on PLT mRNA. Results suggest that all mRNA present in PLTs is affected by the treatment although the degree of the effect varies among transcripts.

Angiogenic response pattern during normal and impaired skin flap re-integration in mice: a comparative study.

BACKGROUND: Distal skin flap necrosis represents a severe complication in surgery. This study investigated angiogenic responses in healthy and impaired pedicled skin flap tissue in normal and diabetic mice. METHODS: Histologic, qRT-PCR, ELISA and immunoblot techniques determined expression and localization of angiogenesis-related growth factors, receptors and cell types upon skin flap re-integration. RESULTS: Skin flap tissue re-integration was severely disturbed in diabetic mice. Impaired skin flap tissue lost early VEGF expression from wound margin keratinocytes and markedly reduced expression of endothelium-specific receptors Tie-2 and FLT-1. Numbers of blood vessels were reduced in impaired flaps. In addition, HIF-1α protein was absent from disturbed skin flap tissue. Reduced VEGF expression and the loss of epithelium in disturbed skin flaps were paralleled by the appearance of VEGF expressing inflammatory infiltrate. CONCLUSION: In summary, our data show a dysregulated spatial and temporal pattern of angiogenic processes during skin flap re-integration in diabetic mice. Our data suggest that reduced expression of angiogenic receptors in skin flap tissue might contribute to a loss of VEGF function in impaired tissue.

Expression profile and subcellular localization of GAPDH in the smooth muscle cells of human atherosclerotic plaque: an immunohistochemical and ultrastructural study with biological therapeutic perspectives.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been considered a classical glycolytic enzyme involved exclusively in cytosolic energy production. Several recent studies, however, have demonstrated that GAPDH is a multifunctional protein whose presence and activity can be regulated by disease states and/or experimental manipulation. Expression levels of GAPDH have been shown to be altered in certain tumors as well as in proliferating and differentiating cells. Since dedifferentiation and proliferation of smooth muscle cells (SMCs) are important features of human atherosclerosis, we have characterized the expression profile of GAPDH in the SMCs of atherosclerotic plaques and its putative interrelationship with the synthetic/proliferative status of these cells utilizing the proliferating cell nuclear antigen (PCNA) antibody, a valuable marker of cell proliferation. Western blot data revealed that GAPDH was significantly upregulated in atherosclerotic plaque specimens. Immunohistochemical stains demonstrated that GAPDH accumulated in the nucleus of dedifferentiated SMCs that also showed positive immunoreactivity for PCNA, but remained cytoplasmatic in the contractile SMCs (PCNA-negative), thus reflecting the proliferative, structural and synthetic differences between them. We suggest that, in human atherosclerotic plaque, GAPDH might exert additional functions that are independent of its well-documented glycolytic activity and might play key roles in development of the disease.

Stain-Free technology as a normalization tool in Western blot analysis.

Western blots are used to specifically measure the relative quantities of proteins of interest in complex biological samples. Quantitative measurements can be subject to error due to process inconsistencies such as uneven protein transfer to the membrane. These non-sample-related variations need to be compensated for by an approach known as normalization. Two approaches to data normalization are commonly employed: housekeeping protein (HKP) normalization and total protein normalization (TPN). In this study, we evaluated the performance of Stain-Free technology as a novel TPN tool for Western blotting experiments in comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a representative of the HKP normalization strategy. The target protein (TP) used for this study was MCM7, a DNA licensing replication factor, which was shown previously to be down-regulated by 20% in irradiated lymphoblastoid cell lines (LCLs). We studied the regulation of MCM7 with a multiplex Western blotting approach based on fluorescently labeled secondary antibodies and found that Stain-Free technology appears to be more reliable, more robust, and more sensitive to small effects of protein regulation when compared with HKP normalization with GAPDH. Stain-Free technology offers the additional advantages of providing checkpoints throughout the Western blotting process by allowing rapid visualization of gel separation and protein transfer.

Group B streptococcus GAPDH is released upon cell lysis, associates with bacterial surface, and induces apoptosis in murine macrophages.

Glyceraldehyde 3-phosphate dehydrogenases (GAPDH) are cytoplasmic glycolytic enzymes that, despite lacking identifiable secretion signals, have been detected at the surface of several prokaryotic and eukaryotic organisms where they exhibit non-glycolytic functions including adhesion to host components. Group B Streptococcus (GBS) is a human commensal bacterium that has the capacity to cause life-threatening meningitis and septicemia in newborns. Electron microscopy and fluorescence-activated cell sorter (FACS) analysis demonstrated the surface localization of GAPDH in GBS. By addressing the question of GAPDH export to the cell surface of GBS strain NEM316 and isogenic mutant derivatives of our collection, we found that impaired GAPDH presence in the surface and supernatant of GBS was associated with a lower level of bacterial lysis. We also found that following GBS lysis, GAPDH can associate to the surface of many living bacteria. Finally, we provide evidence for a novel function of the secreted GAPDH as an inducer of apoptosis of murine macrophages.

Proteomic profiling of human bone marrow mesenchymal stem cells under shear stress.

Mesenchymal stem cells (MSCs) are promising seed cells for tissue engineering of blood vessels. As seed cells, MSCs must endure blood fluid shear stress after transplantation. It has been shown that fluid shear stress can regulate the proliferation and differentiation of MSCs. However, the effects of fluid shear stress on MSCs including the types of proteins modulated are still not well understood. In this study, we exposed human mesenchymal stem cells (HMSCs) to 3 dyn/cm(2) shear stress for 6 h and compared them to a control group using proteomic analysis. Thirteen specific proteins were affected by shear stress, 10 of which were up-regulated. Shear stress especially induced sustained increases in the expression of Annexin A2 and GAPDH, which have been specifically shown to affect HMSCs function. We present here the first comparative proteome analysis of effect of shear stress on HMSCs.

Design and evaluation of a real-time PCR assay for quantification of JAK2 V617F and wild-type JAK2 transcript levels in the clinical laboratory.

The somatic mutation JAK2 V617F is associated with BCR-ABL1-negative myeloproliferative neoplasms. Detection of this mutation aids diagnosis of these neoplasms, and quantification of JAK2 V617F may provide a method to monitor response to therapy. For these reasons, we designed a clinical assay that uses allele-specific PCR and real-time detection with hydrolysis probes for the quantification of JAK2 V617F, wild-type JAK2, and GAPDH transcripts. Mutant and wild-type JAK2 were quantified by using external plasmid standards that contain the relevant JAK2 V617F or JAK2 sequence, respectively. We tested 55 peripheral blood specimens from patients with suspected myeloproliferative neoplasms and 55 peripheral blood specimens from patients not known to have myeloproliferative neoplasms. Low-level, nonspecific amplification was detected in reactions containing a high copy number of plasmid standards and in specimens from patients not known to have myeloproliferative neoplasms, necessitating the use of a laboratory-established mutant to wild-type cutoff. The limit of detection established by using cell line dilutions is 0.1%, and this method identified three JAK2 V617F-positive patients who were not detected by a less sensitive method. The assay characteristics and our initial evaluation indicate this method can be used for the detection and quantification of JAK2 V617F, which should be useful for diagnosis of myeloproliferative neoplasms and potentially for monitoring minimal residual disease in future trials of therapies targeted to myeloproliferative neoplasms.

Identification of major S-nitrosylated proteins in murine experimental autoimmune encephalomyelitis.

Nitrosative stress has been implicated in the pathophysiology of several CNS disorders, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). We have recently shown that protein nitrosothiols (PrSNOs) accumulate in the brain of MS patients, and there is indirect evidence that PrSNO levels are also increased in EAE. In this study we sought to identify the major PrSNOs in the spinal cord of EAE animals prepared by active immunization of C57/BL6 mice with MOG(35-55) peptide. For this purpose, PrSNOs from control and EAE mice at various disease stages were derivatized with HPDP-biotin, and the biotinylated proteins were isolated with streptavidin-agarose. Proteins from total and streptavidin-bound fractions were then analyzed by Western blotting using antibodies against the major S-nitrosylated substrates of CNS tissue. With this approach we found that the proportion of S-nitrosylated neurofilament proteins, NMDA receptors, alpha/beta-tubulin, beta-actin, and GAPDH is increased in EAE. Other potential substrates either were not S-nitrosylated in vivo (HCN3, HSP-72, CRMP-2, gamma-actin, calbindin) or their S-nitrosylation levels were unaltered in EAE (Na/K ATPase, hexokinase, glycogen phosphorylase). We also discovered that neuronal specific enolase is the major S-nitrosylated protein in acute EAE. Given that S-nitrosylation affects protein function, it is likely that the observed changes are significant to the pathophysiology of inflammatory demyelination.

Validation of reference genes for quantitative measurement of immune gene expression in shrimp.

To accurately measure the relative expression of a target gene, mRNA expression data is routinely normalized with reference to an internal control gene. We examined the transcriptional stability of four internal control genes, beta-actin, glyceraldehyde-3 phosphate dehydrogenase (GAPDH), elongation factor1-alpha (EF1-alpha), and 18S ribosomal RNA (18S rRNA) while measuring the mRNA expression of a gene encoding a pattern recognition protein, lipopolysaccharide and glucan binding protein (LGBP) gene, in healthy and white spot syndrome virus (WSSV) infected shrimp (Penaeus stylirostris) before and after (4, 8, 16 and 32 h) challenge using real-time RT-PCR. Here, we describe a method to rank the internal control genes based on a linear regression analysis. This analysis enables us to analyze the multivariate data set, e.g. time course study samples with control and treatment groups. Using the linear regression analysis and the WSSV-challenged time course samples, GAPDH was found to be the most stable internal control gene followed by the genes EF1-alpha, 18S rRNA and beta-actin. Using the program geNorm, GAPDH was also found to be the most stable gene followed by the genes EF1-alpha, beta-actin and 18S rRNA. Using the program NormFinder, the ranking of the internal control genes were in the order of EF1-alpha>GAPDH>18S rRNA>beta-actin. The ability to compare the healthy and WSSV infected samples in parallel by the regression analysis makes this method a very useful approach while identifying the optimal reference gene for gene expression analysis.

Further studies on the hepatoprotective effects of Anoectochilus formosanus.

The purpose of this study was to investigate the hepatoprotective effects of Anoectochilus formosanus effective fraction (AFEF) on chronic liver damage induced by carbon tetrachloride (CCl4) in mice. CCl4 (5%; 0.1 mL/10 g body weight) was given twice a week for 9 weeks, and mice received AFEF throughout the whole experimental period. Plasma GPT, hepatic levels of hydroxyproline and malondialdehyde were significantly lower in mice treated with AFEF compared with those treated with CCl4 only. Liver pathology in the AFEF-treated mice was also improved. RT-PCR analysis showed that AFEF treatment increased the expression of methionine adenosyltransferase 1A and decreased the expression of collagen(alpha1)(I) and transforming growth factor-beta1. These results clearly demonstrated that AFEF reduced the hepatic damage induced by CCl4 in mice.

Proteomics analysis of the tombusvirus replicase: Hsp70 molecular chaperone is associated with the replicase and enhances viral RNA replication.

Plus-strand RNA virus replication occurs via the assembly of viral replicase complexes involving multiple viral and host proteins. To identify host proteins present in the cucumber necrosis tombusvirus (CNV) replicase, we affinity purified functional viral replicase complexes from yeast. Mass spectrometry analysis of proteins resolved by two-dimensional gel electrophoresis revealed the presence of CNV p33 and p92 replicase proteins as well as four major host proteins in the CNV replicase. The host proteins included the Ssa1/2p molecular chaperones (yeast homologues of Hsp70 proteins), Tdh2/3p (glyceraldehyde-3-phosphate dehydrogenase, an RNA-binding protein), Pdc1p (pyruvate decarboxylase), and an unknown approximately 35-kDa acidic protein. Copurification experiments demonstrated that Ssa1p bound to p33 replication protein in vivo, and surface plasmon resonance measurements with purified recombinant proteins confirmed this interaction in vitro. The double mutant strain (ssa1 ssa2) showed 75% reduction in viral RNA accumulation, whereas overexpression of either Ssa1p or Ssa2p stimulated viral RNA replication by approximately threefold. The activity of the purified CNV replicase correlated with viral RNA replication in the above-mentioned ssa1 ssa2 mutant and in the Ssa overexpression strains, suggesting that Ssa1/2p likely plays an important role in the assembly of the CNV replicase.

Differential protein expression in brain capillary endothelial cells induced by hypoxia and posthypoxic reoxygenation.

Cerebral ischemia causes functional alteration of the blood-brain barrier, formed by brain capillary endothelial cells (BCEC). Changes in protein expression and activity of selected differentially expressed enzymes were investigated in BCEC subjected to hypoxia (24 h) alone or followed by a 24-h reoxygenation. BCEC proteins were isolated, separated by 2-DE, and identified by MALDI-MS. Computer-based 2-D gel analysis identified 21 up-regulated proteins and 4 down-regulated proteins after hypoxia alone and 9 proteins that were further up-regulated after posthypoxic reoxygenation. The expression of the majority of hypoxia-induced proteins was reduced toward control levels during reoxygenation. The most prominent changes were identified for glycolytic enzymes (e.g., phosphoglycerate kinase), proteins of the ER (e.g., calreticulin), and cytoskeletal (e.g., vimentin) proteins. The results indicate that BCEC respond to hypoxia/reoxygenation by adaptive up-regulation of proteins involved in the glycolysis, protein synthesis, and stress response.

Glyceraldehyde-3-phosphate dehydrogenase in the extracellular space inhibits cell spreading.

The occurrence and the novel function of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the extracellular space were studied. The extracellular GAPDH with the same molecular mass as the intracellular GAPDH was detected in the conditioned medium of mammalian cultured cell lines such as COS-7, HEK293, MCF-7, HepG2, PC-12, and Neuro-2a cells. Western blot analysis represented the occurrence of GAPDH, but not alpha-tubulin (an intracellular marker protein), in the conditioned medium of COS-7 cells. Furthermore, GAPDH was found in rat serum. These results indicate that GAPDH was secreted outside of the cells. Addition of GAPDH to the cultured medium of COS-7, HEK293, and HepG2 cells allowed cells to undergo morphological changes. In COS-7 cells, the extracellular GAPDH inhibited cell spreading without influencing the cell growth. Western blot and immunofluorescent microscopy analyses revealed that the extracellular GAPDH bound to COS-7 cells in time- and dose-dependent manners. However, a mutant substituting Ser for Cys at position 151 of GAPDH resulted in no binding to the cells, no decreased cell-spreading efficiency and no cell morphological changes. These results indicate that the Cys151 was involved in the binding of GAPDH to cells and the GAPDH-inhibited cell spreading.

Oxidation of glyceraldehyde-3-phosphate dehydrogenase enhances its binding to nucleic acids.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a protein with various activities far from its enzymatic function. Here, we showed that the oxidation of SH-groups of the active site of GAPDH enhanced its binding with total transfer RNA or with total DNA. Both NAD and NADH-the cofactors of GAPDH-inhibited the GAPDH-RNA (DNA) interaction, though NAD was much less effective than NADH in the case of oxidized GAPDH. Oxidation of GAPDH strongly decreased its affinity to NAD but not to NADH. Immobilized tetramers of GAPDH dissociated into dimers during the incubation with total RNA but not DNA. The staining of HeLa cells with monoclonal antibodies specific to dimers, monomers or the denatured form of GAPDH revealed the condensation of non-native forms of GAPDH in the nucleus. The role of oxidation of GAPDH in the regulation of the quaternary structure of the enzyme and in its interaction with nucleic acids is discussed.

Coxsackievirus and adenovirus receptor (CAR)-positive immature osteoblasts as targets of adenovirus-mediated gene transfer for fracture healing.

Adenovirus vectors are expected to be a powerful tool for gene therapy to treat severe fractures. Adenovirus invades cells through binding to the coxsackievirus and adenovirus receptor (CAR) on the cell membrane. CAR expression is low in normal adult animals, but it is induced on regenerating cells in some experimental models. We made a rib fracture model in mice and evaluated the histological changes and CAR mRNA expression by RT-PCR 1, 5, 10, 14, and 21 days after the fracture. CAR mRNA was expressed exclusively in the fractured ribs at each time point, but not in the normal ribs. We detected the CAR protein immunohistochemically in fibroblast-like cells in the fracture callus on days 10 and 14 after fracture. In situ hybridization showed that these fibroblast-like cells expressed mRNA of type I collagen and osteopontin, but not osteocalcin, defining the cells as immature osteoblasts. We then transferred small doses (10(4)-10(8) PFU) of lacZ-expressing adenovirus vector into immature osteoblasts on day 14. beta-galactosidase was detected only on the immature osteoblasts at every dose. Immature osteoblasts play an important role in the matrix replacement step in fracture healing. CAR-mediated gene transfer into immature osteoblasts can be reasonable for adenovirus-mediated treatment of fracture healing.