参考文献

Perhexiline maleate,originally classified as a calcium antagonist,is in use as an antianginal agent. The mechanism of its protective effect is unknown,but there is speculation that it involves a modification of myocardial substrate utilization,in which glycolytic sources are used rather than fatty acids. This hypothesis was tested by employing [13C]NMR isotopomer analysis to measure substrate selection in the working rat heart. Substrate utilization was measured from a mixture of substrates present at their physiological concentration,as follows: acetoacetate,glucose,lactate and long-chain fatty acids. Control perfusions were compared with those perfused with perhexiline. It was found that perhexiline increased lactate utilization,which reduced the extent of fatty acid and endogenous substrate oxidation. There was also a significant increase in cardiac output for a small and insignificant increase in oxygen consumption,which suggested an improvement in myocardial efficiency. Thus,it was confirmed by direct measurement that this drug does modify substrate oxidation,which suggests that further investigations of the role that this agent can play in the management of ischemic heart disease would be beneficial. View Publication

Retinal pigment epithelium (RPE) performs numerous functions critical to retinal health and visual function. RPE senescence is a hallmark of aging and degenerative retinal disease development. Here,we evaluated the temporal expression of key nicotinamide adenine dinucleotide (NAD+)-biosynthetic genes and associated levels of NAD+,a principal regulator of energy metabolism and cellular fate,in mouse RPE. NAD+ levels declined with age and correlated directly with decreased nicotinamide phosphoribosyltransferase (NAMPT) expression,increased expression of senescence markers (p16INK4a,p21Waf/Cip1,ApoJ,CTGF and $\beta$-galactosidase) and significant reductions in SIRT1 expression and activity. We simulated in vitro the age-dependent decline in NAD+ and the related increase in RPE senescence in human (ARPE-19) and mouse primary RPE using the NAMPT inhibitor FK866 and demonstrated the positive impact of NAD+-enhancing therapies on RPE cell viability. This,we confirmed in vivo in the RPE of mice injected sub-retinally with FK866 in the presence or absence of nicotinamide mononucleotide. Our data confirm the importance of NAD+ to RPE cell biology normally and in aging and demonstrate the potential utility of therapies targeting NAMPT and NAD+ biosynthesis to prevent or alleviate consequences of RPE senescence in aging and/or degenerative retinal diseases in which RPE dysfunction is a crucial element. View Publication

Older organs represent an untapped potential to close the gap between demand and supply in organ transplantation but are associated with age-specific responses to injury and increased immunogenicity,thereby aggravating transplant outcomes. Here we show that cell-free mitochondrial DNA (cf-mt-DNA) released by senescent cells accumulates with aging and augments immunogenicity. Ischemia reperfusion injury induces a systemic increase of cf-mt-DNA that promotes dendritic cell-mediated,age-specific inflammatory responses. Comparable events are observed clinically,with the levels of cf-mt-DNA elevated in older deceased organ donors,and with the isolated cf-mt-DNA capable of activating human dendritic cells. In experimental models,treatment of old donor animals with senolytics clear senescent cells and diminish cf-mt-DNA release,thereby dampening age-specific immune responses and prolonging the survival of old cardiac allografts comparable to young donor organs. Collectively,we identify accumulating cf-mt-DNA as a key factor in inflamm-aging and present senolytics as a potential approach to improve transplant outcomes and availability. View Publication

Interleukin-4 (IL-4) suppresses the development of multiple sclerosis in a murine model of experimental autoimmune encephalomyelitis (EAE). Here,we show that,in mice with EAE,the accumulation and persistence in the lymph nodes and spleen of a systemically administered serum albumin (SA)-IL-4 fusion protein leads to higher efficacy in preventing disease development than the administration of wild-type IL-4 or of the clinically approved drug fingolimod. We also show that the SA-IL-4 fusion protein prevents immune-cell infiltration in the spinal cord,decreases integrin expression in antigen-specific CD4+ T cells,increases the number of granulocyte-like myeloid-derived suppressor cells (and their expression of programmed-death-ligand-1) in spinal cord-draining lymph nodes and decreases the number of T helper 17 cells,a pathogenic cell population in EAE. In mice with chronic EAE,SA-IL-4 inhibits immune-cell infiltration into the spinal cord and completely abrogates immune responses to myelin antigen in the spleen. The SA-IL-4 fusion protein may be prophylactically and therapeutically advantageous in the treatment of multiple sclerosis. View Publication

Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression,invasion,metastasis,drug resistance,and patient mortality,highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor,R428,that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events,including Akt phosphorylation,breast cancer cell invasion,and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study,R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival,{\textgreater}80 days compared with 52 days; P {\textless} 0.05) mouse models of breast cancer metastasis. Additionally,R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors. View Publication

The recent emergence of the novel,pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here,we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally,we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention. View Publication

Gamma-glutamylcysteine synthetase (gammaGCS),a rate-limiting enzyme in glutathione biosynthesis,plays a central role in glutathione homeostasis and is a target for development of potential therapeutic agents against parasites and cancer. We have determined the crystal structures of Escherichia coli gammaGCS unliganded and complexed with a sulfoximine-based transition-state analog inhibitor at resolutions of 2.5 and 2.1 A,respectively. In the crystal structure of the complex,the bound inhibitor is phosphorylated at the sulfoximido nitrogen and is coordinated to three Mg2+ ions. The cysteine-binding site was identified; it is formed inductively at the transition state. In the unliganded structure,an open space exists around the representative cysteine-binding site and is probably responsible for the competitive binding of glutathione. Upon inhibitor binding,the side chains of Tyr-241 and Tyr-300 turn,forming a hydrogen-bonding triad with the carboxyl group of the inhibitor's cysteine moiety,allowing this moiety to fit tightly into the cysteine-binding site with concomitant accommodation of its side chain into a shallow pocket. This movement is caused by a conformational change of a switch loop (residues 240-249). Based on this crystal structure,the cysteine-binding sites of mammalian and parasitic gammaGCSs were predicted by multiple sequence alignment,although no significant sequence identity exists between the E. coli gammaGCS and its eukaryotic homologues. The identification of this cysteine-binding site provides important information for the rational design of novel gammaGCS inhibitors. View Publication

Previous study demonstrated that the low survival of human embryonic stem cells (hESC) under conventional slow-cooling cryopreservation protocols is predominantly due to apoptosis rather than cellular necrosis. Hence,this study investigated whether a synthetic broad-spectrum irreversible inhibitor of caspase enzymes,Z-VAD-FMK can be used to enhance the post-thaw survival rate of hESC. About 100 mM Z-VAD-FMK was supplemented into either the freezing solution,the post-thaw culture media or both. Intact and adherent hESC colonies were cryopreserved so as to enable subsequent quantitation of the post-thaw cell survival rate through the MTT assay,which can only be performed with adherent cells. Exposure to 100 mM Z-VAD-FMK in the freezing solution alone did not significantly enhance the post-thaw survival rate (10.2{\%} vs. 9.9{\%},p {\textgreater} 0.05). However,when 100 mM Z-VAD-FMK was added to the post-thaw culture media,there was a significant enhancement in the survival rate from 9.9{\%} to 14.4{\%} (p {\textless} 0.05),which was further increased to 18.7{\%} when Z-VAD-FMK was also added to the freezing solution as well (p {\textless} 0.01). Spontaneous differentiation of hESC after cryopreservation was assessed by morphological observations under bright-field microscopy,and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. The results demonstrated that exposure to Z-VAD-FMK did not significantly enhance the spontaneous differentiation of hESC within post-thaw culture. View Publication

The bleomycin group antitumor antibiotics have long been of interest as a consequence of their efficacy in the treatment of certain tumors,not to mention their unique structures and properties in mediating dioxygen activation and sequence selective degradation of DNA. At a chemical level,the structure originally assigned to bleomycin was subsequently reassigned and the new structure has been confirmed by total synthesis. Through the elaboration of structurally modified bleomycin congeners and fragments,synthetic efforts have also facilitated an understanding of the contribution of individual structural domains in bleomycin to sequence selective DNA binding and cleavage,and have also provided insights into the nature of the chemical processes by which DNA degradation takes place. Within the last several years,it has also become apparent that bleomycin can mediate the oxidative degradation of all major classes of cellular RNAs; it seems entirely plausible that RNA may also represent an important locus of action for this class of antitumor agent. In parallel with ongoing synthetic and mechanistic efforts using classical methods,the study of bleomycins attached to solid supports has been shown to provide important mechanistic insights,and the actual elaboration of modified bleomycins by solid phase synthesis constitutes a logical extension of such efforts. View Publication

Metastases kill 90{\%} of cancer patients. It is thus a major challenge in cancer therapy to inhibit the spreading of tumor cells from primary tumor sites to those particular organs where metastases are likely to occur. Whereas the actin cytoskeleton is a key component involved in cell migration,agents targeting actin dynamics have been relatively poorly investigated. Consequently,valuable in vitro pharmacological tools are needed to selectively identify this type of agent. In response to the absence of any standardized process,the present work aims to develop a multi-assay strategy for screening actin-affecting drugs with anti-migratory potentials. To validate our approach,we used two cancer cell lines (MCF7 and A549) and three actin-affecting drugs (cytochalasin D,latrunculin A,and jasplakinolide). We quantified the effects of these drugs on the kinetics of actin polymerization in tubes (by means of spectrofluorimetry) and on the dynamics of actin cytoskeletons within whole cells (by means of fluorescence microscopy). Using quantitative videomicroscopy,we investigated the actual effects of the drugs on cell motility. Finally,the combined drug effects on cell motility and cell growth were evaluated by means of a scratch-wound assay. While our results showed concordant drug-induced effects on actin polymerization occurring in vitro in test tubes and within whole cells,the whole cell assay appeared more sensitive than the tube assay. The inhibition of actin polymerization induced by cytochalasin D was paralleled by a decrease in cell motility for both cell types. In the case of jasplakinolide,which induces actin polymerization,while it significantly enhanced the locomotion of the A549 cells,it significantly inhibited that of the MCF-7 ones. All these effects were confirmed by means of the scratch-wound assay except of the jasplakinolide-induced effects on MCF-7 cell motility. These later seemed compensated by an additional effect occurring during wound recolonization (possibly acting on the cell growth features). In conclusion,the use of multi-assays with different levels of sophistication and biological relevance is recommended in the screening of new actin-affecting drugs with potentially anti-migratory effects. View Publication