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Articles by Y Zhao
Total Records ( 22 ) for Y Zhao
  Q Wang , J Li , J Gu , B Huang , Y Zhao , D Zheng , Y Ding and L. Zeng
 

The green tea constituent, (–)-epigallocatechin-3-gallate (EGCG), has chemopreventive and anticancer effects. This is partially because of the selective ability of EGCG to induce apoptosis and death in cancer cells without affecting normal cells. In the present study, the activity of EGCG against the myeloma cell line, KM3, was examined. Our results demonstrated, for the first time, that the treatment of the KM3 cell line with EGCG inhibits cell proliferation and induces apoptosis, and there is a synergistic effect when EGCG and bortezomib are combined. Further experiments showed that this effect involves the NF-B pathway. EGCG inhibits the expression of the P65 mRNA and P65/pP65 protein, meanwhile it downregulates pIB expression and upregulates IB expression. EGCG also activates caspase-3, -8, cleaved caspase-9, and poly-ADP-ribose polymerase (PARP) and subsequent apoptosis. These findings provided experimental evidence for efficacy of EGCG alone or in combination with bortezomib in multiple myeloma therapy.

  N Wu , Y Zhao , Y Yin , Y Zhang and J. Luo
 

Our previous studies have demonstrated that bone morphogenetic protein 9 (BMP-9) is one of the most efficacious BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). However, the molecular mechanism underlying the BMP-9-induced osteogenic differentiation of MSCs remains to be fully elucidated. In this study, dominant negative (DN) type II TGF-β receptors were constructed and introduced into C3H10T1/2 stem cells, then in vitro and in vivo assays were carried out to analyze and identify the type II TGF-β receptors required for BMP-9-induced osteogenesis. We found that three DN type II TGF-β receptors, DN-BMPRII, DN-ActRII, and DN-ActRIIB, diminished BMP-9-induced alkaline phosphatase (ALP) activity, led to a decrease in BMP-9-induced Smad binding element (SBE)-controled reporter activity, reduced BMP-9-induced expressions of Smad6 and Smad7, and decreased BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, finally resulted in decreased bone masses and immature osteogenesis. These findings strongly suggested that three wild-type II TGF-β receptors, BMPRII, ActRII and ActRIIB, may play a functional role in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells. However, C3H10T1/2 stem cells can express BMPRII and ActRII, but not ActRIIB. Using RNA interference (RNAi), we found that luciferase reporter activity and ALP activity induced by BMP-9 were accordingly inhibited along with the knockdown of BMPRII and ActRII. Taken together, our results demonstrated that BMPRII and ActRII are the functional type II TGF-β receptors in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells.

  Y Zhao , J Liu , Q Hong , C Yang , L Chen , Y Chen , Q Wang , K Zhao and W. Jin
 

Overexpression of multidrug resistance 1 (MDR1) in cancer remains one of the major causes for the failure of chemotherapy. In the present study, we found that MyoD and PEA3 could activate P-glycoprotein (P-gp) expression in SGC7901 cells. Knockdown of MyoD and PEA3 attenuated MDR1 expression and increased the sensitivity of multidrug resistant cancer cells to cytotoxic drugs that were transported by P-gp in SGC7901/VCR cells. MyoD or PEA3 could bind to the E-box and PEA3 sites on the MDR1 promoter and activate its transcription. The regulation of MDR1 expression by MyoD and PEA3 may provide potential ways to overcome MDR in cancer treatment.

  D. J Espiritu , Z. H Huang , Y Zhao and T. Mazzone
 

Endogenous adipocyte apolipoprotein E (apoE) plays an important role in adipocyte lipoprotein metabolism and lipid flux. A potential role for hyperglycemia in regulating adipocyte apoE expression and triglyceride metabolism was examined. Exposure of adipocytes to high glucose or advanced glycosylation end product-BSA significantly suppressed apoE mRNA and protein levels. This suppression was significantly attenuated by antioxidants or inhibitors of the NF-B transcription pathway. Hyperglycemia in vivo led to adipose tissue oxidant stress and significant reduction in adipose tissue and adipocyte apoE mRNA level. Incubation with antioxidant in organ culture completely reversed this suppression. Hyperglycemia also reduced adipocyte triglyceride synthesis, and this could be completely reversed by adenoviral-mediated increases in apoE. To more specifically evaluate an in vivo role for adipocyte apoE expression on organismal triglyceride distribution in vivo, WT or apoE knockout (EKO) adipose tissue was transplanted in EKO recipient mice. After 12 wk, WT adipocytes transplanted in EKO mice accumulated more triglyceride compared with transplanted EKO adipocytes. In addition, EKO recipients of WT adipose tissue had reduced hepatic triglyceride content compared with EKO recipients transplanted with EKO adipose tissue. Our results demonstrate that hyperglycemia and advanced glycosylation end products suppress the expression of adipocyte apoE in vitro and in vivo and thereby reduce adipocyte triglyceride synthesis. In vivo results using adipose tissue transplantation suggest that reduction of adipocyte apoE, and subsequent reduction of adipocyte triglyceride accumulation, could influence lipid accumulation in nonadipose tissue.

  M Hoekstra , S. J. A Korporaal , Z Li , Y Zhao , M Van Eck and T. J. C. Van Berkel
 

Lipoprotein-associated cholesterol has been suggested to make a significant contribution to adrenal steroidogenesis in vivo. To determine whether lipoproteins indeed contribute to optimal adrenal steroidogenesis in mice, in the current study we have determined the effect of relative lipoprotein deficiency on adrenal steroidogenesis in C57BL/6 wild-type mice. Feeding C57BL/6 mice the lipid-lowering drug probucol (0.25% wt/wt) for 2 wk induced a 90% decrease in plasma high-density lipoprotein (HDL) cholesterol levels and a 77% reduction in low-density lipoprotein (LDL) cholesterol levels. Neutral lipid stores were depleted upon probucol treatment specifically in the glucocorticoid-producing zona fasciculata of the adrenal, leading to a 44% decreased plasma corticosterone level under basal conditions. Exposure to lipopolysaccharide (LPS) induced a 37% increase in the adrenal uptake of HDL cholesteryl esters. Probucol-treated mice could induce only a relatively minor corticosterone response upon a LPS challenge compared with controls, which coincided with an approximately twofold increased hepatic expression level of interleukin-6 and tumor necrosis factor (TNF) and an 89% higher TNF response in plasma. Furthermore, a compensatory two- to fivefold upregulation of LDL receptor (cholesterol uptake) and HMG-CoA reductase (cholesterol synthesis) expression was noticed in the adrenals of probucol-treated mice. In conclusion, we have shown that lipoprotein deficiency in mice as a result of probucol feeding is associated with decreased adrenal cortex cholesterol levels, a lower basal and stress-induced plasma glucocorticoid level, and an increased susceptibility to LPS-induced inflammation. Therefore, it is suggested that plasma lipoproteins are required for optimal adrenal steroidogenesis and protection against endotoxemia in mice.

  D. G Kent , M. R Copley , C Benz , S Wohrer , B. J Dykstra , E Ma , J Cheyne , Y Zhao , M. B Bowie , M Gasparetto , A Delaney , C Smith , M Marra and C. J. Eaves
 

Hematopoietic stem cells (HSCs) are generally defined by their dual properties of pluripotency and extensive self-renewal capacity. However, a lack of experimental clarity as to what constitutes extensive self-renewal capacity coupled with an absence of methods to prospectively isolate long-term repopulating cells with defined self-renewal activities has made it difficult to identify the essential components of the self-renewal machinery and investigate their regulation. We now show that cells capable of repopulating irradiated congenic hosts for 4 months and producing clones of cells that can be serially transplanted are selectively and highly enriched in the CD150+ subset of the EPCR+CD48CD45+ fraction of mouse fetal liver and adult bone marrow cells. In contrast, cells that repopulate primary hosts for the same period but show more limited self-renewal activity are enriched in the CD150 subset. Comparative transcriptome analyses of these 2 subsets with each other and with HSCs whose self-renewal activity has been rapidly extinguished in vitro revealed 3 new genes (VWF, Rhob, Pld3) whose elevated expression is a consistent and selective feature of the long-term repopulating cells with durable self-renewal capacity. These findings establish the identity of a phenotypically and molecularly distinct class of pluripotent hematopoietic cells with lifelong self-renewal capacity.

  J. C Mavropoulos , W. C Buschemeyer , A. K Tewari , D Rokhfeld , M Pollak , Y Zhao , P. G Febbo , P Cohen , D Hwang , G Devi , W Demark Wahnefried , E. C Westman , B. L Peterson , S. V Pizzo and S. J. Freedland
 

Purpose: Numerous dietary factors elevate serum levels of insulin and insulin-like growth factor I (IGF-I), both potent prostate cancer mitogens. We tested whether varying dietary carbohydrate and fat, without energy restriction relative to comparison diets, would slow tumor growth and reduce serum insulin, IGF-I, and other molecular mediators of prostate cancer in a xenograft model.

Experimental Design: Individually caged male severe combined immunodeficient mice (n = 130) were randomly assigned to one of three diets (described as percent total calories): very high-fat/no-carbohydrate ketogenic diet (NCKD: 83% fat, 0% carbohydrate, 17% protein), low-fat/high-carbohydrate diet (LFD: 12% fat, 71% carbohydrate, 17% protein), or high-fat/moderate-carbohydrate diet (MCD: 40% fat, 43% carbohydrate, 17% protein). Mice were fed to maintain similar average body weights among groups. Following a preliminary feeding period, mice were injected with 1 x 106 LNCaP cells (day 0) and sacrificed when tumors were ≥1,000 mm3.

Results: Two days before tumor injection, median NCKD body weight was 2.4 g (10%) and 2.1 g (8%) greater than the LFD and MCD groups, respectively (P < 0.0001). Diet was significantly associated with overall survival (log-rank P = 0.004). Relative to MCD, survival was significantly prolonged for the LFD (hazard ratio, 0.49; 95% confidence interval, 0.29-0.79; P = 0.004) and NCKD groups (hazard ratio, 0.59; 95% confidence interval, 0.37-0.93; P = 0.02). Median serum insulin, IGF-I, IGF-I/IGF binding protein-1 ratio, and IGF-I/IGF binding protein-3 ratio were significantly reduced in NCKD relative to MCD mice. Phospho-AKT/total AKT ratio and pathways associated with antiapoptosis, inflammation, insulin resistance, and obesity were also significantly reduced in NCKD relative to MCD tumors.

Conclusions: These results support further preclinical exploration of carbohydrate restriction in prostate cancer and possibly warrant pilot or feasibility testing in humans.

  X Zheng , X. X Cui , Z Gao , Y Zhao , Y Lin , W. J Shih , M. T Huang , Y Liu , A Rabson , B Reddy , C. S Yang and A. H. Conney
 

Epidemiology studies suggest that statins and nonsteroidal anti-inflammatory drugs reduce the risk of prostate cancer. In the present study, LNCaP cells were cultured in regular medium containing fetal bovine serum or in medium supplemented with charcoal-stripped fetal bovine serum to mimic androgen deprivation treatment. We found that atorvastatin (Lipitor) or celecoxib (Celebrex) treatment of LNCaP cells cultured in regular or androgen-depleted medium inhibited growth and stimulated apoptosis. A combination of atorvastatin and celecoxib was more effective than either agent alone. In animal studies, severe combined immunodeficient mice were injected s.c. with LNCaP cells in Matrigel. After 4 to 6 weeks, mice with LNCaP tumors (about 0.6 cm wide and 0.6 cm long) were surgically castrated and received daily i.p. injections of vehicle, atorvastatin (10 µg/g body weight/d), celecoxib (10 µg/g/d), or a combination of atorvastatin (5 µg/g/d) and celecoxib (5 µg/g/d) for 42 days. In all groups, the androgen-dependent LNCaP tumors regressed initially in response to castration, but the tumors eventually progressed to androgen independence and started to grow. Treatment of the mice with atorvastatin or celecoxib alone suppressed the regrowth of LNCaP tumors after castration. A combination of low doses of atorvastatin and celecoxib had a more potent effect in inhibiting the growth and progression of LNCaP tumors to androgen independence than a higher dose of either agent alone. Our results indicate that administration of a combination of atorvastatin and celecoxib may be an effective strategy for the prevention of prostate cancer progression from androgen dependence to androgen independence. Cancer Prev Res; 3(1); 114–24

  J Ju , S. C Picinich , Z Yang , Y Zhao , N Suh , A. N Kong and C. S. Yang
 

The cancer-preventive activity of vitamin E has been studied. Whereas some epidemiological studies have suggested a protective effect of vitamin E against cancer formation, many large-scale intervention studies with -tocopherol (usually large doses) have not demonstrated a cancer-preventive effect. Studies on -tocopherol in animal models also have not demonstrated robust cancer prevention effects. One possible explanation for the lack of demonstrable cancer-preventive effects is that high doses of -tocopherol decrease the blood and tissue levels of -tocopherols. It has been suggested that -tocopherol, due to its strong anti-inflammatory and other activities, may be the more effective form of vitamin E in cancer prevention. Our recent results have demonstrated that a -tocopherol-rich mixture of tocopherols inhibits colon, prostate, mammary and lung tumorigenesis in animal models, suggesting that this mixture may have a high potential for applications in the prevention of human cancer. In this review, we discuss biochemical properties of tocopherols, results of possible cancer-preventive effects in humans and animal models and possible mechanisms involved in the inhibition of carcinogenesis. Based on this information, we propose that a -tocopherol-rich mixture of tocopherols is a very promising cancer-preventive agent and warrants extensive future research.

  Y Zhao , D. A Howatt , F Gizard , T Nomiyama , H. M Findeisen , E. B Heywood , K. L Jones , O. M Conneely , A Daugherty and D. Bruemmer
  Rationale:

The orphan nuclear receptor NOR1 is a member of the evolutionary highly conserved and ligand-independent NR4A subfamily of the nuclear hormone receptor superfamily. Members of this subfamily have been characterized as early response genes regulating essential biological processes including inflammation and proliferation; however, the role of NOR1 in atherosclerosis remains unknown.

Objective:

The goal of the present study was to determine the causal contribution of NOR1 to atherosclerosis development and to identify the mechanism by which this nuclear receptor participates in the disease process.

Methods and Results:

In the present study, we demonstrate expression of NOR1 in endothelial cells of human atherosclerotic lesions. In response to inflammatory stimuli, NOR1 expression is rapidly induced in endothelial cells through a nuclear factor B–dependent transactivation of the NOR1 promoter. Overexpression of NOR1 in human endothelial cells increased the expression of vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule-1, whereas NOR1 deficiency altered adhesion molecule expression in response to inflammatory stimuli. Transient transfection experiments and chromatin immunoprecipitation assays revealed that NOR1 induces VCAM-1 promoter activity by binding to a canonical response element for NR4A receptors in the VCAM-1 promoter. Further functional studies confirmed that NOR1 mediates monocyte adhesion by inducing VCAM-1 and intercellular adhesion molecule-1 expression in endothelial cells. Finally, we demonstrate that NOR1 deficiency reduces hypercholesterolemia-induced atherosclerosis formation in apoE–/– mice by decreasing the macrophage content of the lesion.

Conclusions:

In concert, these studies identify a novel pathway underlying monocyte adhesion and establish that NOR1 serves a previously unrecognized atherogenic role in mice by positively regulating monocyte recruitment to the vascular wall.

  Y Zhao , M Pennings , R. B Hildebrand , D Ye , L Calpe Berdiel , R Out , M Kjerrulf , E Hurt Camejo , A. K Groen , M Hoekstra , W Jessup , G Chimini , T. J. C Van Berkel and M. Van Eck
  Rationale:

Macrophages cannot limit the uptake of lipids and rely on cholesterol efflux mechanisms for maintaining cellular cholesterol homeostasis. Important mediators of macrophage cholesterol efflux are ATP-binding cassette transporter 1 (ABCA1), which mediates the efflux of cholesterol to lipid-poor apolipoprotein AI, and scavenger receptor class B type I (SR-BI), which promotes efflux to mature high-density lipoprotein.

Objective:

The aim of the present study was to increase the insight into the putative synergistic roles of ABCA1 and SR-BI in foam cell formation and atherosclerosis.

Methods and Results:

Low-density lipoprotein receptor knockout (LDLr KO) mice were transplanted with bone marrow from ABCA1/SR-BI double knockout mice, the respective single knockouts, or wild-type littermates. Serum cholesterol levels were lower in ABCA1/SR-BI double knockout transplanted animals, as compared to the single knockout and wild-type transplanted animals on Western-type diet. Despite the lower serum cholesterol levels, massive foam cell formation was found in macrophages from spleen and the peritoneal cavity. Interestingly, ABCA1/SR-BI double knockout transplanted animals also showed a major increase in proinflammatory KC (murine interleukin-8) and interleukin-12p40 levels in the circulation. Furthermore, after 10 weeks of Western-type diet feeding, atherosclerotic lesion development in the aortic root was more extensive in the LDLr KO mice reconstituted with ABCA1/SR-BI double knockout bone marrow.

Conclusions:

Deletion of ABCA1 and SR-BI in bone marrow–derived cells enhances in vivo macrophage foam cell formation and atherosclerotic lesion development in LDLr KO mice on Western diet, indicating that under high dietary lipid conditions, both macrophage ABCA1 and SR-BI contribute significantly to cholesterol homeostasis in the macrophage in vivo and are essential for reducing the risk for atherosclerosis.

  Z. Q Chen , Y Zhao , Z. H Lu , X. Y Li , H. J Shi , J Sun and R. Patrick
  Background

A large number of sudden cardiac arrests occur annually, but the worldwide survival rate is less than 1%. Early initiation of bystander Cardiopulmonary resuscitation (CPR) would improve the survival rate of out-of-hospital sudden cardiac arrests. Students play an important role as bystanders on and off campus both now and in the future. So we wanted to investigate the awareness and attitudes towards CPR of Chinese students, in order to improve the dissemination of bystander CPR in China.

Methods

The survey was conducted by questionnaire in November 2007. We had chosen 3500 students from the city of Wuhan in China randomly according to the stratified cluster sampling technique.

Results

There were 3248 questionnaires answered, and 2763 questionnaires were considered valid. Few respondents reported that they had heard (28%) and studied (27%) of CPR, and only 3% of the respondents had attended a CPR course. The two major sources of information about CPR for Chinese students were television and books. Most respondents expressed a desire to learn CPR (77%), and were willing to disseminate CPR (73%).

Conclusion

Dissemination of CPR among Chinese students has not been executed satisfactorily. The finding highlights the importance of CPR dissemination and efforts should be made to provide more convenient, effective and attractive ways for the Chinese public, especially students, to learn CPR.

  S Sawatsubashi , T Murata , J Lim , R Fujiki , S Ito , E Suzuki , M Tanabe , Y Zhao , S Kimura , S Fujiyama , T Ueda , D Umetsu , T Ito , K. i Takeyama and S. Kato
 

Chromatin reorganization is essential for transcriptional control by sequence-specific transcription factors. However, the molecular link between transcriptional control and chromatin reconfiguration remains unclear. By colocalization of the nuclear ecdysone receptor (EcR) on the ecdysone-induced puff in the salivary gland, Drosophila DEK (dDEK) was genetically identified as a coactivator of EcR in both insect cells and intact flies. Biochemical purification and characterization of the complexes containing fly and human DEKs revealed that DEKs serve as histone chaperones via phosphorylation by forming complexes with casein kinase 2. Consistent with the preferential association of the DEK complex with histones enriched in active epigenetic marks, dDEK facilitated H3.3 assembly during puff formation. In some human myeloid leukemia patients, DEK was fused to CAN by chromosomal translocation. This mutation significantly reduced formation of the DEK complex, which is required for histone chaperone activity. Thus, the present study suggests that at least one histone chaperone can be categorized as a type of transcriptional coactivator for nuclear receptors.

  A. S Morrissy , R. D Morin , A Delaney , T Zeng , H McDonald , S Jones , Y Zhao , M Hirst and M. A. Marra
 

We describe a new method, Tag-seq, which employs ultra high-throughput sequencing of 21 base pair cDNA tags for sensitive and cost-effective gene expression profiling. We compared Tag-seq data to LongSAGE data and observed improved representation of several classes of rare transcripts, including transcription factors, antisense transcripts, and intronic sequences, the latter possibly representing novel exons or genes. We observed increases in the diversity, abundance, and dynamic range of such rare transcripts and took advantage of the greater dynamic range of expression to identify, in cancers and normal libraries, altered expression ratios of alternative transcript isoforms. The strand-specific information of Tag-seq reads further allowed us to detect altered expression ratios of sense and antisense (S-AS) transcripts between cancer and normal libraries. S-AS transcripts were enriched in known cancer genes, while transcript isoforms were enriched in miRNA targeting sites. We found that transcript abundance had a stronger GC-bias in LongSAGE than Tag-seq, such that AT-rich tags were less abundant than GC-rich tags in LongSAGE. Tag-seq also performed better in gene discovery, identifying >98% of genes detected by LongSAGE and profiling a distinct subset of the transcriptome characterized by AT-rich genes, which was expressed at levels below those detectable by LongSAGE. Overall, Tag-seq is sensitive to rare transcripts, has less sequence composition bias relative to LongSAGE, and allows differential expression analysis for a greater range of transcripts, including transcripts encoding important regulatory molecules.

  B. G Hoffman , G Robertson , B Zavaglia , M Beach , R Cullum , S Lee , G Soukhatcheva , L Li , E. D Wederell , N Thiessen , M Bilenky , T Cezard , A Tam , B Kamoh , I Birol , D Dai , Y Zhao , M Hirst , C. B Verchere , C. D Helgason , M. A Marra , S. J. M Jones and P. A. Hoodless
 

The liver and pancreas share a common origin and coexpress several transcription factors. To gain insight into the transcriptional networks regulating the function of these tissues, we globally identify binding sites for FOXA2 in adult mouse islets and liver, PDX1 in islets, and HNF4A in liver. Because most eukaryotic transcription factors bind thousands of loci, many of which are thought to be inactive, methods that can discriminate functionally active binding events are essential for the interpretation of genome-wide transcription factor binding data. To develop such a method, we also generated genome-wide H3K4me1 and H3K4me3 localization data in these tissues. By analyzing our binding and histone methylation data in combination with comprehensive gene expression data, we show that H3K4me1 enrichment profiles discriminate transcription factor occupied loci into three classes: those that are functionally active, those that are poised for activation, and those that reflect pioneer-like transcription factor activity. Furthermore, we demonstrate that the regulated presence of H3K4me1-marked nucleosomes at transcription factor occupied promoters and enhancers controls their activity, implicating both tissue-specific transcription factor binding and nucleosome remodeling complex recruitment in determining tissue-specific gene expression. Finally, we apply these approaches to generate novel insights into how FOXA2, PDX1, and HNF4A cooperate to drive islet- and liver-specific gene expression.

  I. L. K Wong , K. F Chan , Y Zhao , T. H Chan and L. M. C. Chow
  Objectives

The aim of this study was to investigate the synergistic effect of quinacrine and a novel apigenin dimer (compound 9d) on reversing pentamidine resistance of Leishmania parasites.

Methods

Pentamidine-resistant cell lines, LePentR50 and LdAG83PentR50, were generated by gradually increasing pentamidine pressure on wild-type promastigotes. We tested the effects of different combinations of quinacrine and an apigenin dimer on modulating the pentamidine resistance levels of LePentR50 and LdAG83PentR50 using an MTS proliferation assay. We then measured the accumulation level of pentamidine using HPLC. The fractional inhibitory concentration index (FICI) method was used to evaluate the interaction between quinacrine and the apigenin dimer on reversing pentamidine resistance in Leishmania.

Results

LePentR50 and LdAG83PentR50 promastigotes were ~8.6- and 4.6-fold more resistant to pentamidine than their wild-type parents. Amastigotes derived from LePentR50 and LdAG83PentR50 were also pentamidine-resistant. We found that quinacrine can increase the susceptibility of Leishmania to pentamidine. Quinacrine, when used at 6 µM, can increase the IC50 of pentamidine by 3.8-, 3.4-, 3.5- and 6.3-fold in wild-type Leishmania enriettii Le, LePentR50, wild-type Leishmania donovani LdAG83 and LdAG83PentR50, respectively. Quinine, quinidine and verapamil did not show any sensitizing effect. The sensitizing effect of quinacrine was: (i) dose-dependent; (ii) not associated with an increase in pentamidine accumulation; and (iii) only observed in pentamidine-resistant but not sodium stibogluconate-resistant or vinblastine-resistant parasites. Other than quinacrine, we also found that an apigenin dimer (compound 9d), previously shown to be able to inhibit ABCB1-mediated cancer drug resistance in mammalian cells, can also increase the pentamidine susceptibility of Leishmania. 9d, when used at 6 µM, can increase the IC50 of pentamidine by 2.5-, 4.2-, 1.6- and 1.9-fold in Le, LePentR50, LdAG83 and LdAG83PentR50, respectively. Unlike quinacrine, sensitization by 9d was accompanied by an increase in pentamidine accumulation, presumably due to the inhibition of an ABC transporter. Using the FICI method, we found that quinacrine and 9d can act synergistically. When they are used in a 1:1 ratio, they sensitize LePentR50 to pentamidine by 19-fold, with an FICI of 0.48 (P < 0.005), indicating that they might act synergistically.

Conclusions

Our findings support the notion that the pentamidine susceptibility of Leishmania is mediated by multiple targets. Quinacrine and apigenin dimer 9d, each inhibiting its own target, can have a synergistic effect when used together to sensitize Leishmania to pentamidine.

  M Hoekstra , D Ye , R. B Hildebrand , Y Zhao , B Lammers , M Stitzinger , J Kuiper , T. J. C Van Berkel and M. Van Eck
 

Impaired scavenger receptor class B type I (SR-BI)-mediated uptake of HDL-cholesterol esters (HDL-CE) induces adrenal insufficiency in mice. Humans contain an alternative route of HDL-CE clearance, namely through the transfer by cholesteryl ester transfer protein (CETP) to apolipoprotein B lipoproteins for subsequent uptake via the LDL receptor. In this study, we determined whether CETP can compensate for loss of adrenal SR-BI. Transgenic expression of human CETP (CETP Tg) in SR-BI knockout (KO) mice increased adrenal HDL-CE clearance from 33–58% of the control value. SR-BI KO/CETP Tg and SR-BI KO mice displayed adrenal hypertrophy due to equally high plasma adrenocorticotropic hormone levels. Adrenal cholesterol levels and plasma corticosterone levels were 38–52% decreased in SR-BI KO mice with and without CETP expression. SR-BI KO/CETP Tg mice also failed to increase their corticosterone level after lipopolysaccharide challenge, leading to an identical >4-fold increased tumor necrosis factor- response compared with controls. These data indicate that uptake of CE via other routes than SR-BI is not sufficient to generate the cholesterol pool needed for optimal adrenal steroidogenesis. In conclusion, we have shown that CETP-mediated transfer of HDL-CE is not able to reverse adrenal insufficiency in SR-BI knockout mice. Thus, SR-BI-mediated uptake of serum cholesterol is essential for optimal adrenal function.

  K Nagao , Y Zhao , K Takahashi , Y Kimura and K. Ueda
 

ABCA1 plays a major role in HDL metabolism. Cholesterol secretion by ABCA1 is dependent on the presence of extracellular acceptors, such as lipid-free apolipoprotein A-I (apoA-I). However, the importance of the direct interaction between apoA-I and ABCA1 in HDL formation remains unclear. In contrast, ABCB4 mediates the secretion of phospholipids and cholesterol in the presence of sodium taurocholate (NaTC) but not in the presence of apoA-I. In this study, we analyzed apoA-I binding and NaTC-dependent lipid efflux by ABCA1. ABCA1 mediated the efflux of cholesterol and phospholipids in the presence of NaTC as well as in the presence of apoA-I in an ATP-dependent manner. The Tangier disease mutation W590S, which resides in the extracellular domain and impairs apoA-I-dependent lipid efflux, greatly decreased NaTC-dependent cholesterol and phospholipid efflux. However, the W590S mutation did not impair apoA-I binding and, conversely, retarded the dissociation of apoA-I from ABCA1. These results suggest that the W590S mutation impairs ATP-dependent lipid translocation and that lipid translocation or possibly lipid loading, facilitates apoA-I dissociation from ABCA1. NaTC is a good tool for analyzing ABCA1-mediated lipid efflux and allows dissection of the steps of HDL formation by ABCA1.

  C Zhang , S Zheng , Y Wang , Y Zhao , J Zhu and L. Ge
 

Cleidocranial dysplasia (CCD) is a dominantly inherited skeletal dysplasia caused by mutations in the osteoblast-specific transcription factor-encoding gene, RUNX2. To correlate different RUNX2 mutations with CCD clinical spectrum, we studied six independent Chinese CCD patients. In five patients, mutations were detected in the coding region of the RUNX2 gene, including two frameshift mutations and three missense mutations. Of these mutations, four were novel and one had previously been reported. All the detected mutations were exclusively clustered within the Runt domain that affected conserved residues in the Runt domain. In vitro green fluorescent protein fusion studies showed that the three mutations—R225L, 214fs and 172fs—interfered with nuclear accumulation of RUNX2 protein, while T200I mutation had no effect on the subcellular distribution of RUNX2. There was no marked phenotypic difference between patients in craniofacial and clavicles features, while the expressivity of supernumerary teeth in our patient cohort had a striking variation, even among family members. The occurrence of intrafamilial clinical variability raises the view that hypomorphic effects and genetic modifiers may alter the clinical expressivity of these mutations. Our results provide new genetic evidence that mutations involved in RUNX2 contribute to CCD.

  T Yu , W. G Junger , C Yuan , A Jin , Y Zhao , X Zheng , Y Zeng and J. Liu
 

Shockwaves elicited by transient pressure disturbances are used to treat musculoskeletal disorders. Previous research has shown that shockwave treatment affects T-cell function, enhancing T-cell proliferation and IL-2 expression by activating p38 mitogen-activated protein kinase (MAPK) signaling. Here we investigated the signaling pathway by which shockwaves mediate p38 MAPK phosphorylation. We found that shockwaves at an intensity of 0.18 mJ/mm2 induce the release of extracellular ATP from human Jurkat T-cells at least in part by affecting cell viability. ATP released into the extracellular space stimulates P2X7-type purinergic receptors that induce the activation of p38 MAPK and of focal adhesion kinase (FAK) by phosphorylation on residues Tyr397 and Tyr576/577. Elimination of released ATP with apyrase or inhibition of P2X7 receptors with the antagonists KN-62 or suramin significantly weakens FAK phosphorylation, p38 MAPK activation, IL-2 expression, and T-cell proliferation. Conversely, addition of exogenous ATP causes phosphorylation of FAK and p38 MAPK. Silencing of FAK expression also reduces these cell responses to shockwave treatment. We conclude that shockwaves enhance p38 MAPK activation, IL-2 expression, and T-cell proliferation via the release of cellular ATP and feedback mechanisms that involve P2X7 receptor activation and FAK phosphorylation.

  A Dessein , C Chevillard , V Arnaud , X Hou , A. A Hamdoun , H Dessein , H He , S. A Abdelmaboud , X Luo , J Li , A Varoquaux , A Mergani , M Abdelwahed , J Zhou , A Monis , M. G.R Pitta , N Gasmelseed , S Cabantous , Y Zhao , A Prata , C Brandt , N. E Elwali , L Argiro and Y. Li
 

Abnormal fibrosis occurs during chronic hepatic inflammations and is the principal cause of death in hepatitis C virus and schistosome infections. Hepatic fibrosis (HF) may develop either slowly or rapidly in schistosome-infected subjects. This depends, in part, on a major genetic control exerted by genes of chromosome 6q23. A gene (connective tissue growth factor [CTGF]) is located in that region that encodes a strongly fibrogenic molecule. We show that the single nucleotide polymorphism (SNP) rs9402373 that lies close to CTGF is associated with severe HF (P = 2 x 10–6; odds ratio [OR] = 2.01; confidence interval of OR [CI] = 1.51–2.7) in two Chinese samples, in Sudanese, and in Brazilians infected with either Schistosoma japonicum or S. mansoni. Furthermore, SNP rs12526196, also located close to CTGF, is independently associated with severe fibrosis (P = 6 x 10–4; OR = 1.94; CI = 1.32–2.82) in the Chinese and Sudanese subjects. Both variants affect nuclear factor binding and may alter gene transcription or transcript stability. The identified variants may be valuable markers for the prediction of disease progression, and identify a critical step in the development of HF that could be a target for chemotherapy.

  X Huang , X Bai , Y Cao , J Wu , M Huang , D Tang , S Tao , T Zhu , Y Liu , Y Yang , X Zhou , Y Zhao , M Wu , J Wei , D Wang , G Xu , S Wang , D Ma and J. Zhou
 

Angiogenesis is increasingly recognized as an important prognosticator associated with the progression of lymphoma and as an attractive target for novel modalities. We report a previously unrecognized mechanism by which lymphoma endothelium facilitates the growth and dissemination of lymphoma by interacting with circulated T cells and suppresses the activation of CD4+ T cells. Global gene expression profiles of microdissected endothelium from lymphoma and reactive lymph nodes revealed that T cell immunoglobulin and mucin domain–containing molecule 3 (Tim-3) was preferentially expressed in lymphoma-derived endothelial cells (ECs). Clinically, the level of Tim-3 in B cell lymphoma endothelium was closely correlated to both dissemination and poor prognosis. In vitro, Tim-3+ ECs modulated T cell response to lymphoma surrogate antigens by suppressing activation of CD4+ T lymphocytes through the activation of the interleukin-6–STAT3 pathway, inhibiting Th1 polarization, and providing protective immunity. In a lymphoma mouse model, Tim-3–expressing ECs promoted the onset, growth, and dissemination of lymphoma by inhibiting activation of CD4+ T cells and Th1 polarization. Our findings strongly argue that the lymphoma endothelium is not only a vessel system but also a functional barrier facilitating the establishment of lymphoma immune tolerance. These findings highlight a novel molecular mechanism that is a potential target for enhancing the efficacy of tumor immunotherapy and controlling metastatic diseases.

 
 
 
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