RGS5 augments astrocyte activation and facilitates neuroinflammation via TNF signaling.

Astrocytes contribute to chronic neuroinflammation in a variety of neurodegenerative diseases, including Parkinson's disease (PD), the most common movement disorder. However, the precise role of astrocytes in neuroinflammation remains incompletely understood. Herein, we show that regulator of G-protein signaling 5 (RGS5) promotes neurodegenerative process through augmenting astrocytic tumor necrosis factor receptor (TNFR) signaling. We found that selective ablation of Rgs5 in astrocytes caused an inhibition in the production of cytokines resulting in mitigated neuroinflammatory response and neuronal survival in animal models of PD, whereas overexpression of Rgs5 had the opposite effects. Mechanistically, RGS5 switched astrocytes from neuroprotective to pro-inflammatory property via binding to the receptor TNFR2. RGS5 also augmented TNFR signaling-mediated pro-inflammatory response by interacting with the receptor TNFR1. Moreover, interrupting RGS5/TNFR interaction by either RGS5 aa 1-108 or small molecular compounds feshurin and butein, suppressed astrocytic cytokine production. We showed that the transcription of astrocytic RGS5 was controlled by transcription factor early B cell factor 1 whose expression was reciprocally influenced by RGS5-modulated TNF signaling. Thus, our study indicates that beyond its traditional role in G-protein coupled receptor signaling, astrocytic RGS5 is a key modulator of TNF signaling circuit with resultant activation of astrocytes thereby contributing to chronic neuroinflammation. Blockade of the astrocytic RGS5/TNFR interaction is a potential therapeutic strategy for neuroinflammation-associated neurodegenerative diseases.

Discovery of 4,6-Disubstituted Pyrimidine Derivatives as Novel Dual VEGFR2/FGFR1 Inhibitors.

Abnormalities in the FGFRs signaling pathway and VEGFR2 amplification often occur in a variety of tumors, and they synergistically promote tumor angiogenesis. Studies have shown that the up-regulation of FGF-2 is closely related to the resistance of VEGFR2 inhibitors. Activation of the FGFRs signal is a signal of compensatory angiogenesis after VEGFR2 resistance. Dual VEGFR2/FGFR1 inhibitors contribute to overcoming the resistance of VEGFR2 inhibitors and inhibit tumor growth significantly. Based on this, we designed and synthesized a series of 4,6-disubstituted pyrimidine derivatives as dual VEGFR2/FGFR1 inhibitors by the molecular hybridization strategy. 3-(2,6-Dichloro-3,5-dimethoxyphenyl)-1-{6-[(4-methoxyphenyl)amino]pyrimidin-4-yl}-1-methylurea (8b) had the best inhibitory activities against VEGFR2 and FGFR1 at 10 µM (82.2 % and 101.0 %, respectively), it showed moderate antiproliferative activities against A549 and KG-1 cell lines as well. Besides, molecular docking was also carried out to study the binding mode of 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-{6-[(4-methoxyphenyl)-amino]-pyrimidin-4-yl}-1-methylurea (8b) with VEGFR2 and FGFR1. These studies reveal that this series of compounds deserve further optimization.

Hemodynamic changes in superficial arteriovenous malformation surgery measured by intraoperative ICG fluorescence videoangiography with FLOW 800 software.

BACKGROUND: Arteriovenous malformation(AVM) have long-term "blood stealing" characteristics, which result in complicated hemodynamic features. To analyze the application of intraoperative indocyanine green angiography with FLOW 800 software in AVM surgeries. METHODS: Data on 17 patients undergoing surgery with ICG fluorescence were collected in Beijing Tiantan Hospital. To analyze the hemodynamic features of AVM and the influence on the peripheral cortex of AVM resection, we assessed the following hemodynamic parameters: maximum intensity, slope of rise, time to half-maximal fluorescence, and transit time from arteries to veins. RESULTS: In the 17 superficial AVMs studied, the time-delay color mode of the FLOW 800 software was superior to the traditional playback mode for identifying feeding arteries, draining veins, and their relation to normal cortical vessels. The maximum fluorescence intensity and slope of the ICG fluorescence curve of feeder arteries and draining veins were higher than those of normal peripheral vessels (P < 0.05). The transit times in AVMs were significantly shorter than those in normal peripheral vessels (P < 0.05). After AVM resection, cerebral flow increased in the cortex, and local cycle time becomes longer, although the differences were not significant (P > 0.05). CONCLUSIONS: Hemodynamic parameter analysis provided quality guidance for the resection of AVMs and could also be used in estimating changes in blood flow in the local cortex to identify abnormal hyperperfusion and residual nidus.

Recent developments and advances of FGFR as a potential target in cancer.

FGFs and their receptors (FGFRs) are critical for many biologic processes, including angiogenesis, wound healing and tissue regeneration. Aberrations in FGFR signaling are common in cancer, making FGFRs a promising target in antitumor studies. To date, many FGFR inhibitors are being detected in clinical studies, and resistance to some inhibitors has emerged. Understanding the mechanisms of resistance is a fundamental step for further implementation of targeted therapies. In this review, we will describe the basic knowledge regarding FGF/FGFR signaling and categorize the clinical FGFR inhibitors. The mechanisms of resistance to FGFR inhibitors and corresponding strategies of overcoming drug resistance will also be discussed.

Increased expression of serum gelsolin in patients with osteosarcoma.

BACKGROUND: Identification of potential serum biomarkers of osteosarcoma to aid in its early diagnosis and in the discovery of possible therapeutic targets is an area of increasing interest. METHODS: Two-dimensional difference-in-gel electrophoresis was used to assess multiple serum samples in patients with osteosarcoma. In addition, differential expression of protein biomarkers was characterized in osteosarcoma serum by using matrix-assisted desorption/ionization time-of-flight mass spectrometry coupled with database interrogation. Serum samples from four individuals with osteosarcoma and four age- and sex-matched healthy control subjects were compared. RESULTS: Fifty-eight significant protein spot features in the osteosarcoma sera were found. These spot features were excised, digested with trypsin, and analyzed with mass spectrometry. Gelsolin was down-regulated only in osteosarcoma. Furthermore, Western blotting and enzyme linked immunosorbent assay (ELISA) confirmed decreased levels of gelsolin in the osteosarcoma serum samples. CONCLUSIONS: These results indicated that gelsolin might have great potential as a biomarker of osteosarcoma and as a potential target for gene therapy.

[Proteomic analysis between keloid and normal skin].

OBJECTIVE: To investigate and search correlative proteins of keloid by comparing the results of differential proteomic analysis between keloid and normal skin. METHODS: From January 2010 to June 2010 two-dimensional gel electrophoresis was used to define patterns of protein expression in keloid skin from 8 patients and matched normal skin from 3 patients. Differential expression protein spots were showed and analyzed by matrix-assisted laser desorption ionization-time of flying/time of flying (MALDI-TOF/TOF) mass spectrometry. RESULTS: This study succeeded to provide a two-dimensional protein profiling comparison between normal skin and keloid. Gel-analysis software identified an average of 2978 spots in keloid while 3053 spots in normal skin and statistical filtering yielded 40 spots of a 4-fold change, 32 of which were identified by using mass spectrometry, 20 were up-regulated and 12 were down-regulated. Functional analysis revealed that these proteins could be fractionated to carrier proteins (3 proteins), signal transduction proteins (4 proteins), proliferation and apoptosis related proteins (2 proteins), cytoskeleton proteins (6 proteins), extracellular matrix proteins (8 proteins), immunity related proteins (3 proteins), tumor related proteins (2 proteins), and function unknown protein (4 proteins). CONCLUSIONS: Proteomic analysis can identify the proteins with variance of keloid versus normal skin. The further research to these differential proteins may help reveal the pathogenesis of keloid and provide new treatments for keloid.

Profiling protein markers associated with lymph node metastasis in prostate cancer by DIGE-based proteomics analysis.

Current predictive tools and imaging modalities are not accurate enough for preoperative diagnosis of lymph node metastatic prostate cancer (LNM PCa). Proteomic analysis is introduced to screen potential biomarkers for early detection of LNM PCa. In our initial study, protein samples from localized and LNM PCa as well as benign prostatic hyperplasia tissues were analyzed using two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) coupled with MALDI-TOF/TOF MS. We identified 58 proteins that were differentially expressed in the LNM PCa group relative to the localized PCa group. Six of these proteins, e-FABP5, MCCC2, PPA2, Ezrin, SLP2, and SM22, are functionally relevant to cancer metastasis. Expression of these proteins was therefore further validated in tissue samples from the original cohort and also from a larger, independent cohort of patients using real time PCR, Western blotting, and immunohistochemistry staining. In addition, the serum levels of e-FABP5 were also examined by ELISA. Relative to localized PCa tissues, LNM PCa tissues had increased expression of e-FABP5, MCCC2, PPA2, Ezrin, and SLP2 and decreased expression of SM22. Patients with LNM PCa had significantly higher levels of serum e-FABP5. This study presents evidence that increased expression of e-FABP5, MCCC2, PPA2, Ezrin, and SLP2 and decreased expression of SM22 are useful diagnostic markers for the existence of LNM PCa.

[Expression of CD45 in the serum of patients with Crohn's disease].

OBJECTIVE: To search for differentially expressed proteins in the serum of patients with Crohn's disease. METHODS: Serum protein samples obtained from 4 patients with Crohn's disease and 4 normal adults were cross-labeled with different CyDyes and underwent two-dimensional differential in-gel electrophoresis (2-D DIGE) and imaging analysis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to identify the differentially expressed proteins. RESULTS: 2-D DIGE revealed that the protein on spot 973 was overexpressed by 2.55 folds in the serum of patients with Crohn's disease compared with that in normal adults (P<0.05). The protein was identified as CD45 using mass spectrometry. CONCLUSION: CD45 overexpression in the serum of patients with Crohn's disease may play a role in the disequilibrium of the immune system.

Identification of cancer-associated proteins by proteomics and downregulation of ß-tropomyosin expression in colorectal adenoma and cancer.

Elucidating the molecular mechanism underlying the development of adenoma, the major precursor lesion of colorectal cancer (CRC), would provide a basis for early detection, prevention as well as treatment of CRC. Using the highly sensitive 2-D DIGE method coupled with MS, we identified 24 differentially expressed proteins in adenoma tissues compared with matched normal colonic mucosa and CRC tissues. Fifteen proteins were downregulated and three proteins were upregulated in adenoma tissues when compared with individual-matched normal colonic mucosa. Five proteins were downregulated, while one protein was upregulated in adenoma tissues when compared with matched CRC tissues. A protein, ß-tropomyosin (TM-ß), recently suggested to be a biomarker of esophageal squamous carcinoma, was downregulated in both adenoma and CRC tissues. Additionally, the reduction in the level of TM-ß in adenoma and CRC tissues was further validated by Western blotting (p<0.05) and RT-PCR (p<0.001). Our findings suggest that downregulation of TM-ß is involved in the early development of CRC and that differentially expressed proteins might serve as potential biomarkers for detection of CRC.

[Detection of serum biomakers of osteosarcoma by proteomic profiling].

OBJECTIVE: To detect differentially expressed proteins in serum of patient with osteosarcoma. METHODS: 8 serum protein samples were recruited (4 cases of osteosarcoma and 4 cases of normal adults), cross-labeled with variant CyDye, followed by two-dimensional differential in-gel electrophoresis (2-D DIGE), image analysis, and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). RESULTS: 24 protein spot-features were significantly increased, and 34 were significantly decreased in the serum from patients with osteosarcoma relative to the controls. The mass spectrometry analysis revealed 18 unique proteins that were increased, and 25 unique proteins decreased in the serum of patients with osteosarcoma. Gelsolin was down-regulated in osteosarcoma, and Western blotting also confirmed a decreased level of gelsolin in the serum of patients with osteosarcoma. CONCLUSION: Our results indicate that gelsolin may have great potential as a biomarker of osteosarcoma and as a potential target for therapy. These preliminary data suggest that incorporation of more samples and new datasets will permit the identification of serum biomarkers for osteosarcoma.

[Serum proteomic variation study in patients with Crohn disease].

OBJECTIVE: To search differentially expressed proteins in serum of patients with Crohn disease. METHODS: Serum protein samples from 4 patients with Crohn disease and 8 healthy adults were recruited cross-labeled with variant CyDye, and then followed by two-dimensional differential in-gel electrophoresis (2-D DIGE), image analysis, and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). RESULTS: The 2-D electrophoresis results were compared between the Crohn disease patients and the healthy adults. The spot 1058 expression in serum of Crohn disease patients increased by 1.68 folds as compared with healthy adults (P<0.05). The protein was identified as haptoglobin by mass spectrometry. CONCLUSION: Up-regulating expression of haptoglobin in serum of Crohn disease patients may play a role in disequilibrium of immunity system.

Comparative proteomic analysis of human osteosarcoma and SV40-immortalized normal osteoblastic cell lines.

AIM: Comparative proteomics provide a powerful approach in screening for alterations in protein levels and post-translational modifications that are associated with tumors. In the present study, we aimed to identify candidate biomarkers to distinguish osteosarcoma (OS) cells from normal osteoblastic cells. METHODS: We employed 3 OS cell lines (U2OS, IOR/OS9, and SaOS-2), and used the SV40-immortalized normal osteoblastic cell line (hFOB1.19) as the control. The differential protein levels in OS and osteoblastic cells were identified using 2-D gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry analyses. Two proteins of interest, the levels of which were significantly increased in OS cells, were further characterized by Western blot analyses. RESULTS: Twenty-six proteins were identified, the expression level of which was either significantly increased or decreased in the OS cells as compared to the control cells. The expression level of the activator of 90 kDa shock protein ATPase homolog 1 (AHA1), was enhanced 12.4-, 24.1-, and 23.8-fold in SaOS-2, IOR/OS9, and U2OS cells, respectively, and the level of the stomatin-like protein 2 (SLP-2) was increased by 10.4- and 7.8-fold in IOR/OS9 and U2OS cells, respectively, as compared to normal osteoblastic cells. Those observations were confirmed by Western blot analyses. CONCLUSION: A differential proteomic analysis was successfully used to identify AHA1 and SLP-2 that were significantly overproduced in OS cells as compared to normal osteoblastic cells, suggesting that those proteins among others may be effective biomarker candidates for the identification of OS cells.

Proteomic identification of differently expressed proteins responsible for osteoblast differentiation from human mesenchymal stem cells.

Human mesenchymal stem cells (hMSC) are a population of multipotent cells that can differentiate into osteoblasts, chondrocytes, adipocytes, and other cells. The exact mechanism governing the differentiation of hMSC into osteoblasts remains largely unknown. Here, we analyzed protein expression profiles of undifferentiated as well as osteogenic induced hMSC using 2-D gel electrophoresis (2-DE), mass spectrometry (MS), and peptide mass fingerprinting (PMF) to investigate the early gene expression in osteoblast differentiation. We have generated proteome maps of undifferentiated hMSC and osteogenic induced hMSC on day 3 and day 7. 2-DE revealed 102 spots with at least 2.0-fold changes in expression and 52 differently expressed proteins were successfully identified by MALDI-TOF-MS. These proteins were classified into 7 functional categories: metabolism, signal transduction, transcription, calcium-binding protein, protein degradation, protein folding and others. The expression of some identified proteins was confirmed by further RT-PCR analyses. This study clarifies the global proteome during osteoblast differentiation. Our results will play an important role in better elucidating the underlying molecular mechanism in hMSC differentiation into osteoblasts.

2-D DIGE and MALDI-TOF-MS analysis of the serum proteome in human osteosarcoma.

We performed 2-D DIGE on proteins prepared from serum obtained from patients with osteosarcoma (OS) and controls, to identify differentially expressed proteins that might serve as serum biomarkers for OS prognosis. Proteins found to be differentially expressed were identified by MALDI-TOF mass spectrometric analysis, coupled with database interrogation. We compared serum samples from four individuals with OS to four age- and sex-matched healthy controls. We identified 24 protein spot-features that were significantly increased, and 34 that were significantly decreased in serum from patients with OS relative to the controls. The MS analysis revealed 18 unique proteins that were increased, and 25 unique proteins that were decreased in OS serum samples. Western blot and ELISA analysis confirmed increased levels of amyloid-related serum protein (SAA) in the OS serum samples. The increased expression levels of SAA were decreased after using MTX and cisplatin combination chemotherapy, and were further decreased after operation. Moreover, increased expression levels of sera SAA were seen in the relapsed patients. Our results suggested that the determination of serum SAA in OS patients might be utilized as a marker for relapse and in evaluation of the efficacy of therapy.

Proteomic analysis of differential protein expression in early process of pancreatic regeneration in pancreatectomized rats.

AIM: A broad-range proteomic approach was applied to investigate the complexity of the mechanisms involved in pancreatic regeneration for identification of new targets of diabetes treatment and potential markers of pancreatic stem cells. METHODS: A regeneration pancreatic model was induced by 90% partial pancreatectomy (Px) in rats. Changes in the protein expression in regenerating rat pancreas on the third day after Px, as compared with rats that received sham surgery, were analyzed by using 2-D gel electrophoresis (2-DE), mass spectrometry (MS), and mass fingerprinting. RESULTS: 2-DE revealed 91 spots with at least 1.5-fold increases in expression at 3 d after pancreatectomy and 53 differentially expressed proteins that were identified by peptide mass fingerprinting (PMF). These included cell growth-related, lipid and energy metabolism-related, protein and amino acid metabolism-related proteins, and signal transduction proteins. Vimentin, CK8, L-plastin, hnRNP A2/B1, and AGAT are associated with embryogenesis and cell differentiation, and may be new potential pancreatic stem cells markers. CONCLUSION: The proteome profiling technique provided a broad-based and effective approach for the rapid assimilation and identification of adaptive protein changes during pancreas regeneration induced by pancreatectomy. Our data clarify the global proteome during the pancreatic proliferation and differentiation processes, which is important for better understanding of pancreatic regeneration and for discovering of protein biomarkers for pancreatic stem cells.

[Study of altered expression of annexin IV and human endometrial receptivity].

OBJECTIVE: To explore the association of altered expression of annexin IV in human endometrium during the implantation window and endometrial receptivity. METHODS: A comparative proteomic strategy, in a combination of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), was adopted to search for proteome alternations of pre-receptive (day LH + 2) versus receptive (LH + 7) endometria. The location and abundance of the identified differentially expressed protein- annexin IV were analyzed by immunostaining and western blot. RESULTS: By comparing protein profiles of LH + 2 and LH + 7 samples, we found a protein up-regulated 2.12 times in LH + 7 samples, with a relative molecular weight of 36,000 and an isoelectric point near pH 5.8. It was characterized using mass spectrometry and was identified as annexin IV. Immunohistochemical analysis revealed an altered localization of annexin IV--in the epithelia on day LH + 2, and both in the epithelia and stroma cells on day LH + 7. Protein levels of annexin IV were up-regulated on day LH + 7 compared with that on day LH + 2 by Western blot. Integrated optical density of the object (OPTDI) was 46.249 +/- 32.376 and 249.507 +/- 31.959, respectively (P = 0.004). CONCLUSIONS: Our study indicates endometrial samples obtained by microbiopsy are available for proteomics studies. It seems possible that the increased expression of annexin IV during the implantation window plays an important role in the morphological differentiation of the uterus to the receptive state.

Roles of CaMKII, PKA, and PKC in the induction and maintenance of LTP of C-fiber-evoked field potentials in rat spinal dorsal horn.

Long-term potentiation (LTP) of C-fiber-evoked field potentials in spinal dorsal horn may be relevant to hyperalgesia, an increased response to noxious stimulation. The mechanism underlying this form of synaptic plasticity is, however, still unclear. Considerable evidence has shown that calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase A (PKA), and protein kinase C (PKC) are important for LTP in hippocampus. In this study, the roles of these three protein kinases in the induction and maintenance of LTP of C-fiber-evoked field potentials were evaluated by application of specific inhibitors of CaMKII (KN-93 and AIP), PKA (Rp-CPT-cAMPS), and PKC (chelerythrine and Gö 6983) at the recording segments before and after LTP induction in urethane-anesthetized Sprague-Dawley rats. We found both KN-93 and AIP, when applied at 30 min prior to tetanic stimulation, completely blocked LTP induction. At 30 min after LTP induction, KN-93 and AIP reversed LTP completely, and at 60 min after LTP induction, they depressed spinal LTP in most rats tested. Three hours after LTP induction, however, KN-93 or AIP did not affect the spinal LTP. Rp-CPT-cAMPS, chelerythrine, and Gö 6983 blocked the spinal LTP when applied at 30 min before tetanic stimulation and reversed LTP completely at 15 min after LTP induction. In contrast, at 30 min after LTP induction, the drugs never affected the spinal LTP. These results suggest that activation of CaMKII, PKA, and PKC may be crucial for the induction and the early-phase but not for the late-phase maintenance of the spinal LTP.

Mitochondrial Mechanisms of Bcl-2 on Ionizing Radiation-induced Apoptosis.

Low dose radiation induces apoptosis and Bcl-2 can inhibit apoptosis induced by a variety of stimuli. The effects and possible mechanisms of Bcl-2 on 4 Gy X-ray-induced apoptosis in Bcl-2 transfected and untransfected cells were studied. The data present here suggested that overexpression of Bcl-2 significantly inhibited X-ray-induced apoptosis in CHO cells within 72 h. Further investigation demonstrated that Bcl-2 prevented the loss of mitochondrial membrane potential (deltapsim) induced by X-ray irradiation. It appears that overexpression of Bcl-2 blocked the ionizing radiation-induced apoptosis. The maintenance of deltapsim plays an important role in the protective mechanism of Bcl-2 on ionizing radiation-induced apoptosis.