Opportunities and challenges for ribosome-inactivating proteins in traditional Chinese medicine plants.

Ribosome-inactivating proteins (RIPs) are a class of cytotoxic rRNA N-glycosylase, which widely exist in higher plants in different taxonomy, including many traditional Chinese medicinal materials and vegetables and fruits. In this paper, the traditional Chinese medicinal plants containing RIPs protein were sorted out, and their pharmacological effects and clinical applications were analyzed. Since many RIPs in traditional Chinese medicine plants exhibit antiviral and antitumor activities and show great clinical application potential, people's interest in these proteins is on the rise. This paper summarizes the possible mechanism of RIPs's anti-virus and anti-tumor effects, and discusses its potential problems and risks, laying a foundation for subsequent research on how to exert its anti-virus and anti-tumor effects.

USP18 enhances the resistance of BRAF-mutated melanoma cells to vemurafenib by stabilizing cGAS expression to induce cell autophagy.

This study aims to discern the possible molecular mechanism of the effect of ubiquitin-specific peptidase 18 (USP18) on the resistance to BRAF inhibitor vemurafenib in BRAF V600E mutant melanoma by regulating cyclic GMP-AMP synthase (cGAS). The cancer tissues of BRAF V600E mutant melanoma patients before and after vemurafenib treatment were collected, in which the protein expression of USP18 and cGAS was determined. A BRAF V600E mutant human melanoma cell line (A2058R) resistant to vemurafenib was constructed with its viability, apoptosis, and autophagy detected following overexpression and depletion assays of USP18 and cGAS. Xenografted tumors were transplanted into nude mice for in vivo validation. Bioinformatics analysis showed that the expression of cGAS was positively correlated with USP18 in melanoma, and USP18 was highly expressed in melanoma. The expression of cGAS and USP18 was up-regulated in cancer tissues of vemurafenib-resistant patients with BRAF V600E mutant melanoma. Knockdown of cGAS inhibited the resistance to vemurafenib in A2058R cells and the protective autophagy induced by vemurafenib in vitro. USP18 could deubiquitinate cGAS to promote its protein stability. In vivo experimentations confirmed that USP18 promoted vemurafenib-induced protective autophagy by stabilizing cGAS protein, which promoted resistance to vemurafenib in BRAF V600E mutant melanoma cells. Collectively, USP18 stabilizes cGAS protein expression through deubiquitination and induces autophagy of melanoma cells, thereby promoting the resistance to vemurafenib in BRAF V600E mutant melanoma.

Danhong injection alleviates cerebral ischemia-reperfusion injury by inhibiting autophagy through miRNA-132-3p/ATG12 signal axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Danhong injection (DHI) is a renowned traditional Chinese medicine often used clinically to treat cardiovascular and cerebrovascular diseases. Studies have shown that DHI can significantly alter microRNA (miRNA) expression in the brain tissue. Therefore, exploring specific miRNAs' regulatory mechanisms during treatment with DHI is essential. AIM OF THE STUDY: To investigate DHI's regulatory mechanism on cerebral autophagy in rats with cerebral ischemia-reperfusion injury (CIRI). MATERIAL AND METHODS: Rats were randomly divided into the sham, middle cerebral artery occlusion (MCAO) model, and DHI-treatment groups. The extent of brain damage was evaluated using triphenyl tetrazolium chloride and hematoxylin-eosin staining. Hippocampal cell autophagy was observed using transmission electron microscopy. Autophagy-related proteins were analyzed using western blotting. Differentially expressed miRNAs were screened using high-throughput and real-time quantitative reverse transcription PCR. The relationship between miR-132-3p and ATG12 was confirmed using a dual-luciferase assay. The miR-132-3p mimics and inhibitors were transfected into PC12 cells subjected to oxygen-glucose deprivation (OGD) in vitro and MCAO model rats in vivo. RESULTS: DHI significantly altered the miRNA expression profile in rat brain tissues. The pathological changes in the brain tissues were improved, and the autophagic hippocampal cell vehicles were significantly reduced after DHI treatment. miRNA-132-3p, one of the miRNAs with a significantly different expression, was screened. Kyoto Encyclopedia of Genes and Genomes signal pathway analysis showed that its target genes were closely related to autophagy. Western blotting revealed that the p-PI3K, p-AKT, and mTOR expression increased significantly; AMPK, ULK1, ATG12, ATG16L1, and LC3II/I were downregulated in the DHI group. Dual-luciferase reporter gene experiments showed that miRNA-132-3p could target the ATG12 3'-UTR region directly. In vitro, miRNA-132-3p had a protective effect on OGD/R-induced oxidative stress injury in PC12 cells, improving cell viability, and affecting the expression of autophagy pathway-related proteins. In vivo transfection experiments showed that miR-132-3p could regulate ATG12 expression in CIRI rats' lateral brain tissue, affecting the autophagy signaling pathway. miR-132-3p overexpression reduces CIRI-induced autophagy and protects neurons. CONCLUSION: This study showed that DHI inhibits neuronal autophagy after cerebral ischemia-reperfusion. This may have resulted from miR-132-3p targeting ATG12 and regulating the autophagy signaling pathway protein expression.

BRD4 inhibitor GNE987 exerts anti-cancer effects by targeting super-enhancers in neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is a common extracranial malignancy with high mortality in children. Recently, super-enhancers (SEs) have been reported to play a critical role in the tumorigenesis and development of NB via regulating a wide range of oncogenes Thus, the synthesis and identification of chemical inhibitors specifically targeting SEs are of great urgency for the clinical therapy of NB. This study aimed to characterize the activity of the SEs inhibitor GNE987, which targets BRD4, in NB. RESULTS: In this study, we found that nanomolar concentrations of GNE987 markedly diminished NB cell proliferation and survival via degrading BRD4. Meanwhile, GNE987 significantly induced NB cell apoptosis and cell cycle arrest. Consistent with in vitro results, GNE987 administration (0.25 mg/kg) markedly decreased the tumor size in the xenograft model, with less toxicity, and induced similar BRD4 protein degradation to that observed in vitro. Mechanically, GNE987 led to significant downregulation of hallmark genes associated with MYC and the global disruption of the SEs landscape in NB cells. Moreover, a novel candidate oncogenic transcript, FAM163A, was identified through analysis of the RNA-seq and ChIP-seq data. FAM163A is abnormally transcribed by SEs, playing an important role in NB occurrence and development. CONCLUSION: GNE987 destroyed the abnormal transcriptional regulation of oncogenes in NB by downregulating BRD4, which could be a potential therapeutic candidate for NB.

[Preparation of a recombinant tumor-targeting ribosome inactivating protein luffin-α-NGR and evaluation of its antitumor activity].

Loofah seeds ribosome inactivating protein luffin-α was fused with a tumor-targeting peptide NGR to create a recombinant protein, and its inhibitory activity on tumor cells and angiogenesis were assessed. luffin-α-NGR fusion gene was obtained by PCR amplification. The fusion gene was ligated with pGEX-6p-1 vector to create a recombinant plasmid pGEX-6p-1/luffin-α-NGR. The plasmid was transformed into E. coli BL21, and the target protein was isolated and purified by GST affinity chromatography. The luffin-α-NGR fusion gene with a full length of 849 bp was successfully obtained, and the optimal soluble expression of the target protein was achieved under the conditions of 16 ℃, 0.5 mmol/L IPTG after 16 h induction. SDS-PAGE and Western blotting confirmed the recombinant protein has an expected molecular weight of 56.6 kDa. Subsequently, the recombinant protein was de-tagged by precision protease digestion. The inhibitory effects of the recombinant protein on liver tumor cells HepG2 and breast cancer cells MDA-MB-231 were significantly stronger than that of luffin-α. The Transwell and CAM experiment proved that the recombinant protein luffin-α-NGR also had a significant inhibitory effect on tumor cells migration and neovascularization. The inhibitory activity on tumor cells and angiogenesis of the recombinant luffin-α-NGR protein lays a foundation for the development of subsequent recombinant tumor-targeting drugs.

WNT signaling modulates chemoresistance to temozolomide in p53-mutant glioblastoma multiforme.

Glioblastoma multiforme (GBM) has been characterized by the high incidence, therapy tolerance and relapse. The molecular events controlling GBM resistant to chemotherapy temozolomide (TMZ) remain to be elusive. Here, we identified WNT signaling was amplified by TMZ and mediated drug response in GBM. We found O6-methylguanine DNA methyltransferase (MGMT) was redundant to WNT-mediated chemoresistance, which was highly associated with p53 mutation status. In GBM with p53 mutation, loss of function of p53 downregulated miR-34a expression, which represses transcription of WNT ligand 6 (WNT6) by directly binding to 3' UTR of WNT6 mRNA, leading to activation of WNT signaling, and the eventual WNT-mediated chemoresistance to TMZ. Combined treatment of TMZ with WNT inhibitor or miR34a mimic induced drug sensitivity of p53-mutant GBM cells and extended survival in xenograft mice in vivo. Our findings provide insight into understanding the molecular mechanism of GBM chemoresistance to TMZ and facilitating to develop novel treatment strategy to combat p53-mutant GBM by targeting miR-34a/WNT6 axis.

Carboplatin activates the cGAS-STING pathway by upregulating the TREX-1 (three prime repair exonuclease 1) expression in human melanoma.

Human melanoma is a highly aggressive type of cancer, causing significant mortalities despite the advances in treatment. Carboplatin is a cisplatin analog necessary for the treatment of various cancers and can also be used to treat human melanoma. We assessed the effects and mechanisms leading to inhibited proliferation and induced apoptosis of human melanoma after carboplatin therapy in vitro and in vivo. TREX1, cGAS/STING, and apoptotic protein expressions were determined through RT-qPCR and western blot assays. Cell proliferation was validated through MTT assays. The study used SK-MEL-1 and SK-HEP-1 tumor cell line inoculations along with carboplatin in nude mice to validate the results. The TREX1 levels were down-regulated in human melanoma cell lines. TREX1 overexpression-induced apoptosis and decreased proliferation in the human melanoma cell lines. TREX1 overexpression also activated the cGAS/STING pathway to induce apoptosis and decrease cell growth. Carboplatin activated TREX1, induced apoptosis, and decreased proliferation in the human melanoma cancerous cell lines. Finally, carboplatin reduced the in-vivo tumor size and weight. In conclusion, the study revealed that carboplatin activated TREX1 and cGAS/STING pathways to upregulate apoptosis. The work also provides in vitro and in vivo evidence to understand the effects of TREX overexpression on tumor suppression. Targeting of TREX1/cGAS/STING pathway could be an effective therapeutic alternative to human melanoma.

Expression and purification of a recombinant ELRL-MAP30 with dual-targeting anti-tumor bioactivity.

MAP30 (Momordica antiviral protein 30kD) is a single-chain Ⅰ-type ribosome inactivating protein with a variety of biological activities, including anti-tumor ability. It was reported that MAP30 would serve as a novel and relatively safe agent for prophylaxis and treatment of liver cancer. To determine whether adding two tumor targeting peptides could improve the antitumor activities of MAP30, we genetically modified MAP30 with an RGD motif and a EGFRi motif, which is a ligand with high affinity for αvß3 integrins and with high affinity for EGFR. The recombinant protein ELRL-MAP30 (rELRL-MAP30) containing a GST-tag was expressed in E. coli. The rELRL-MAP30 was highly expressed in the soluble fraction after induction with 0.15 mM IPTG for 20 h at 16 °C. The purified rELRL-MAP30 appeared as a band on SDS-PAGE. It was identified by western blotting. Cytotoxicity of recombinant protein to HepG2, MDA-MB-231, HUVEC and MCF-7 cells was detected by MTT analysis. Half maximal inhibitory concentration (IC50) values were 54.64 µg/mL, 70.13 µg/mL, 146 µg/mL, 466.4 µg/mL, respectively. Proliferation inhibition assays indicated that rELRL-MAP30 could inhibit the growth of Human liver cancer cell HepG2 effectively. We found that rELRL-MAP30 significantly induced apoptosis in liver cancer cells, as evidenced by nuclear staining of DAPI. In addition, rELRL-MAP30 induced apoptosis in human liver cancer HepG2 cells by up-regulation of Bax as well as down-regulation of Bcl-2. Migration of cell line were markedly inhibited by rELRL-MAP30 in a dose-dependent manner compared to the recombinant MAP30 (rMAP30). In summary, the fusion protein displaying extremely potent cytotoxicity might be highly effective for tumor therapy.

Mesenchymal stem cells-derived and siRNAs-encapsulated exosomes inhibit osteonecrosis of the femoral head.

Osteonecrosis of the femoral head (ONFH) is a progressive, obstinate and disabling disease. At present, the treatment of ONFH is still a global medical problem. We aim to explore the role of bone mesenchymal stem cells (BMSCs)-derived and siRNAs-encapsulated exosomes (siRNAs-encapsulated BMSCexos) in ONFH. We first isolated BMSCexos and screened siRNAs of 6 ONFH-related genes for siRNAs-encapsulated BMSCexo. The expression of these 6 ONFH-related genes in dexamethasone (DXM)-treated MC3T3-E1 cell, cell model of ONFH, was detected by RT-qPCR and Western blot analysis. And then, we performed CCK-8 assay, angiogenesis assay and HE staining analysis to test the promotion role of the siRNAs-encapsulated BMSCexo for angiogenesis during ONFH repair. The results suggest that the obtained particles were BMSCexos. The screened effective siRNAs could effectively knock down their expression in VECs. Moreover, siRNAs-encapsulated BMSCexo could effectively knock down the expression of these genes in VECs. In addition, siRNAs-encapsulated BMSCexo promote angiogenesis during ONFH repair. In conclusion, we found siRNAs-encapsulated BMSCexos could promote ONFH repair by angiogenesis, and indicated exosome as the new siRNA carrier is of great significance to improve the efficiency of RNAi.

Gadolinium-doped mesoporous calcium silicate/chitosan scaffolds enhanced bone regeneration ability.

Chitosan (CTS) and mesoporous calcium silicate (MCS) have been developed for bone defect healing; however, their bone regeneration capacity still does not satisfy the patients with bone diseases. Gadolinium (Gd) is accumulated in human bones, and plays a beneficial role in regulating cell performance and bone regeneration. We firstly constructed Gd-doped MCS/CTS (Gd-MCS/CTS) scaffolds by a lyophilization technology. The interconnected arrangement of CTS films lead to forming macropores by using ice crystals as templates during the lyophilization procedure, and the Gd-MCS nanoparticles dispersed uniformly on the macropore walls. The biocompatible chemical components and hierarchical pores facilitated the attachment and spreading of rat bone marrow-derived mesenchymal stem cells (rBMSCs). Interestingly, the Gd dopants in the scaffolds effectively activated the Wnt/ß-catenin signaling pathway, resulting in excellent cell proliferation and osteogenic differentiation capacities. The osteogenic-related genes such as alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and collagen type1 (COL-1) were remarkably up-regulated by Gd-MCS scaffolds as compared with MCS scaffolds, and their expression levels increased in a positive correlation with Gd doping amounts. Moreover, in vivo rat cranial defect tests further confirmed that Gd-MCS/CTS scaffolds significantly stimulated collagen deposition and new bone formation. The exciting finding suggested the beneficial effects of Gd3+ ions on osteogenic differentiation and new bone regeneration, and Gd-MCS/CTS scaffolds can be employed as a novel platform for bone defect healing.

Logistic regression analysis of risk factors for femoral head osteonecrosis after healed intertrochanteric fractures.

PURPOSE: To evaluate the potential risk factors of the development of femoral head osteonecrosis after healed intertrochanteric fractures. METHODS: We retrospectively reviewed all patients who were operated upon with closed reduction and internal fixation for intertrochanteric fractures by our medical group from December 1993 to December 2012. Patients with healed fractures were identified. Age, gender, comorbidities favouring osteonecrosis, causes of injuries, fracture patterns, the location of the primary fracture line, time from injury to surgery, fixation methods, and the development of femur head osteonecrosis of these patients were summarised. Univariate and multivariate logistic regression analysis were performed to evaluate the correlation between potential risk factors and the development of femoral head osteonecrosis. RESULTS: A total of 916 patients with healed intertrochanteric fractures were identified. Femoral head osteonecrosis was found in 8 cases (0.87%). According to the results of univariate logistic regression, a more proximal fracture line, fixation with dynamic hip screws and age were found to be statistically significant factors. The results of multivariate logistic regression analysis indicated that the statistically significant predictors of femoral head osteonecrosis were younger age (odds ratio [OR] = 17.103; 95% confidence interval [CI], 1.988-147.111), a more proximal fracture line (OR = 31.439; 95% CI, 3.700-267.119) and applying dynamic hip screw as the internal fixation (OR = 11.114; 95% CI, 2.064-59.854). CONCLUSIONS: Regular follow-up is commended in young patients with a proximal fracture line who underwent closed reduction and internal fixation with dynamic hip screw, even though the bone had healed.

Continuous hypoxia regulates the osteogenic potential of mesenchymal stem cells in a time-dependent manner.

The effects of hypoxia on the osteogenic potential of mesenchymal stem cells (MSCs) have been previously reported. From these studies, possible factors affecting the association between hypoxia and the osteogenic differentiation of MSCs have been suggested, including hypoxia severity, cell origin and methods of induction. The effect of the duration of hypoxia, however, remains poorly understood. The aim of the present study was to investigate the effect of continuous hypoxia on the induced osteogenesis of MSCs. Rat MSCs were isolated and cultured in vitro. Once the cells had been cultured to passage three, they were switched to 1% oxygen and cultured either with or without osteogenic medium, while cells in the control groups were cultured under normoxia in corresponding conditions. Four osteogenic differentiation biomarkers, runt-related transcription factor 2, osteopontin, osteocalcin and alkaline phosphatase, were analyzed by quantitative polymerase chain reaction and western blotting at defined intervals throughout the culture period. In addition, Alizarin Red staining was used to assess changes in mineralization. The results showed that 1% hypoxia was able to enhance and accelerate the osteogenic ability of the MSCs during the initial phases of differentiation, and the protein expression of certain associated biomarkers was upregulated. However, continuous hypoxia was shown to impair osteogenesis in the latter stages of differentiation. These findings suggest that hypoxia can regulate the osteogenesis of MSCs in a time-dependent manner.

Protective effect of N-acetylcysteine (NAC) on renal ischemia/reperfusion injury through Nrf2 signaling pathway.

The aim of this study was to investigate whether N-acetylcysteine (NAC), a known antioxidant, can protect kidney against ischemic injury through regulating Nrf2 signaling pathway. The expression of Nrf2, HO-1 and cleaved caspase 3 were analyzed by Western blot analysis. Apoptosis of renal tubular epithelial cells was assessed by the TUNEL method. Malondialdehyde (MDA) levels were measured by the thiobarbituric acid reaction. Blood serum creatinine and blood urea nitrogen levels were measured with an Olympus automatic multi-analyzer. We found that NAC significantly increased Nrf2 and downstream HO-1 expression. Furthermore, NAC significantly decreased cleaved caspase 3, p53 and renal epithelial tubular cell apoptosis. In addition, NAC reduced the MDA level. These findings suggest that the protective action of NAC on ischemia renal injury is associated closely with Nrf2 signaling pathway.

Injury-to-surgery interval does not affect postfracture osteonecrosis of the femoral head in young adults: a systematic review.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is a common and severe complication following femoral neck fractures in young adults. Despite significant advances in surgical techniques, radiological evaluation and comprehensive treatment for the prevention of ONFH, the incidence of traumatic ONFH has remained unchanged at approximately 20% in recent decades. The injury-to-surgery interval is considered as a principal factor affecting the occurrence of ONFH, and traditionally, femoral neck fractures are treated emergently. However, the relationship between the injury-to-surgery interval and ONFH occurrence is poorly understood, and previous reviews have not provided a precise explanation due to the lack of strict selection criteria for studies. METHODS: We reviewed previously published articles and included in current systematic review those studies with accurate multivariate analyses that included age, fracture type, operation method, follow-up, ONFH occurrence and injury-to-surgery interval. RESULTS: Six case studies were included and reevaluated. The studies included 263 hips for final analysis, with an overall incidence of postfracture ONFH of 17.5%. Patients were categorized into groups of less/more than 8 h, less/more than 24 h, less/more than 48 h and less/more than 3 weeks based on the individual injury-to-surgery interval. The postfracture ONFH incidence ranged from 13.3% (<8 weeks) to 21.7% (>3 weeks). Operations performed within 3 weeks of injury resulted in a lower ONFH incidence compared with operations performed after 3 weeks; however, this difference was not statistically significant. The ONFH incidence remained relatively stable when the operations were performed within 3 weeks of injury. CONCLUSIONS: The injury-to-surgery interval did not significantly affect the incidence of postoperative ONFH.

[The effect and mechanism of Yiqi Tongluo Jiedu capsule against cerebral ischemia reperfusion injury].

To observe the effect and mechanism of Yiqi Tongluo Jiedu capsule aganist cerebral ischemia reperfusion injury, the SD rats were randomly divided into following groups: sham-operated group, model group, the group of low, medium and high dose of Yiqi Tongluo Jiedu capsule, and nimodipine group. Using focal middle cerebral artery embolization (MCAO) model, following items were observed: symptoms of neurological deficit score; infarct volume; activity of SOD, content of MDA and NO, activity of NOS of ischemic brain tissue; Bcl-2 and Bax protein expression; content of IL-1beta, IL-6 and TNFalpha in serum; IL-1beta mRNA expression of ischemic brain tissue. Results showed that Yiqi Tongluo Jiedu capsule could significantly reduce the symptoms of neurological deficits, promote the recovery symptoms of neurological deficits; narrow infarct volume of brain tissue obviously, reduce the percentage of infarct volume; raise activity of SOD, reduce content of MDA and NO, reduce activity of NOS; increase Bcl-2 protein, reduce Bax expression; reduce content of IL-1beta, IL-6 and TNFa in serum; reduce IL-1beta mRNA expression of ischemic brain tissue. Yiqi Tongluo Jiedu capsule has significant protective effects against ischemic brain injury, it has significant anti-apoptotic, antioxidant and anti-inflammatory effects.

Injury-to-surgery interval does not affect the occurrence of osteonecrosis of the femoral head: a prospective study in a canine model of femoral neck fractures.

BACKGROUND: It is controversial whether an early reduction and internal fixation can reduce the occurrence of femoral neck fracture-induced osteonecrosis of the femoral head (ONFH). This prospective study was designed to reflect the relationship between injury-to-surgery interval (ISI) and traumatic ONFH based on a canine model of femoral neck fractures. MATERIAL/METHODS: Twenty-four dogs were equally divided randomly into 3 groups. A lateral L-shape approach centered left great trochanter was used for exposure of the femoral neck. A low-speed drill was used for making displaced fractures in the narrow femoral neck, with the femoral head kept in situ with ligamentum teres intact. In Group A, the fracture was immediately reduced and fixed with 3 parallel pins; while the operation was done 3 days later in Group B, and 3 weeks later in Group C. Another 2 dogs had their fractures untreated. Postoperatively, all dogs were fed separately and received regular x-ray examination. Left femoral heads were harvested for histological examination with a postoperative follow-up of 3.5 months. RESULTS: The canine model of femoral neck fractures could be achieved successfully. Radiological signs of post-fracture ONFH could not be detected at intervals of 2 weeks, 4 weeks, 1 month and 2 months. Histologically, there were 2 cases with ONFH in Group A, 1 case in Group B, and 2 cases in Group C. The difference had no statistical significance. For untreated fractures, obvious ONFH could be found radiologically. CONCLUSIONS: A shorter ISI may not reduce the incidence of fracture-induced ONFH, which suggests that intrinsic factors play an important role in the occurrence of ONFH.

Roles of Kruppel-associated Box (KRAB)-associated Co-repressor KAP1 Ser-473 Phosphorylation in DNA Damage Response.

The Kruppel-associated box (KRAB)-associated co-repressor KAP1 is an essential nuclear co-repressor for the KRAB zinc finger protein superfamily of transcriptional factors. Ataxia telangiectasia mutated (ATM)-Chk2 and ATM- and Rad3-related (ATR)-Chk1 are two primary kinase signaling cascades activated in response to DNA damage. A growing body of evidence suggests that ATM and ATR phosphorylate KAP1 at Ser-824 in response to DNA damage and regulate KAP1-dependent chromatin condensation, DNA repair, and gene expression. Here, we show that, depending on the type of DNA damage that occurs, KAP1 Ser-473 can be phosphorylated by ATM-Chk2 or ATR-Chk1 kinases. Phosphorylation of KAP1 at Ser-473 attenuated its binding to the heterochromatin protein 1 family proteins and inhibited its transcriptional repression of KRAB-zinc finger protein (KRAB-ZFP) target genes. Moreover, KAP1 Ser-473 phosphorylation induced by DNA damage stimulated KAP1-E2F1 binding. Overexpression of heterochromatin protein 1 significantly inhibited E2F1-KAP1 binding. Elimination of KAP1 Ser-473 phosphorylation increased E2F1-targeted proapoptotic gene expression and E2F1-induced apoptosis in response to DNA damage. Furthermore, loss of phosphorylation of KAP1 Ser-473 led to less BRCA1 focus formation and slower kinetics of loss of γH2AX foci after DNA damage. KAP1 Ser-473 phosphorylation was required for efficient DNA repair and cell survival in response to DNA damage. Our studies reveal novel functions of KAP1 Ser-473 phosphorylation under stress.

Pain relief following osteonecrosis of the femoral head treated by free vascularized fibular grafting.

BACKGROUND: Pain is the core and basic problem in the treatment of osteonecrosis of the femoral head (ONFH). However, it is unclear about the status of pain relief following ONFH treated by free vascularized fibular grafting (FVFG) and the level of pain relief contributed to clinical results. Therefore, we designed a consecutive and prospective study to investigate post-operative pain relief in the treatment of osteonecrosis of the femoral head by FVFG. METHODS: One hundred and fifty-one patients with unilateral osteonecrosis of the femoral head were enrolled consecutively for current prospective study from January to August of 2006. Patients were managed by modified technique of free vascularized fibular grafting. Pre-operative, post-operative Harris hip score (HHS) and Harris pain score (HPS) were recorded and compared statistically, meanwhile, correlation between disease severity and Harris hip score, Harris pain score were revealed. RESULTS: All patients had an average follow-up of 54.3 months. Post-operative Harris hip score could be improved from 73.7 to 83.5 averagely in stage-II patients, 64.6 to 78.9 in stage-III, and 53.6 to 72.4 in stage-IV. As for Harris pain score, it was elevated from 28.8 to 38.6 in stage-II patients, 25.5 to 36.6 in stage-III, and 21.8 to 34.2 in stage-IV. Taken together, HHS was improved from 67.7 to 80.3 (ΔHHS = 12.6), and HPS was improved from 26.6 to 37.3 (ΔHPS = 10.7) averagely. CONCLUSIONS: Harris pain score could be employed to monitor prognosis of osteonecrosis of the femoral head treated by free vascularized fibular grafting. Improvement of HPS was the heaviest contributor to elevation of HHS, and both of them conceived of a close relationship with disease severity.

Synergistic protective effect of astragaloside IV-tetramethylpyrazine against cerebral ischemic-reperfusion injury induced by transient focal ischemia.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragaloside IV and tetramethylpyrazine have been extensively used in the cardio-cerbrovascular diseases of medicine as a chief ingredient of glycoside or alkaloid formulations for the treatment of stroke and myocardial ischemia diseases. AIM OF THE STUDY: To investigate the effects of astragaloside IV (ASG IV) and tetramethylpyrazine (TMPZ) on cerebral ischemia-reperfusion (IR) injury model in rat model. MATERIALS AND METHODS: Rats were randomly divided into the following five groups: sham group, IR group and treatment group including ASG IV, ASG IV-TMPZ and nimodipine treatment. The therapeutic effect was evaluated by micro-positron emission tomography (Micro-PET) using (18)F-fluoro-2-deoxy-d-glucose. The neurological examination, infarct volume and the levels of oxidative stress- and cell apoptosis-related molecules were assessed. RESULTS: Micro-PET imaging showed that glucose metabolism in the right hippocampus was significantly decreased in the IR group compared to the sham group (P<0.01). ASG IV and ASG IV-TMPZ treatments reversed the decreased glucose metabolism in the model group (P<0.05 and P<0.01, respectively). IR induced the increase of Caspase-3 mRNA levels, MDA content and iNOS activity, but it caused the decrease of SOD activity and Bcl-2 expression compared the sham group (P<0.01). ASG IV-TMPZ and ASG IV reversed the IR-induced changes of these parameters, i.e. the down regulation of Caspase-3 mRNA, MDA content and iNOS activity, and the up regulation of SOD activity and Bcl-2 expression (P<0.05). CONCLUSION: This study showed that ASG IV-TMPZ played a pivotal synergistic protective role against focal cerebral ischemic reperfusion damage in a rat experimental model.