Cell-free expression of NO synthase and P450 enzyme for the biosynthesis of an unnatural amino acid L-4-nitrotryptophan

Cell-free system has emerged as a powerful platform with a wide range of in vitro applications and recently has contributed to express metabolic pathways for biosynthesis.Here we report in vitro construction of a native biosynthetic pathway for L-4-nitrotryptophan(L-4-nitro-Trp)synthesis using an Escherichia coli-based cell-free protein synthesis(CFPS)system.Naturally,a nitric oxide(NO)synthase(TxtD)and a cytochrome P450 enzyme(TxtE)are responsible for synthesizing L-4-nitro-Trp,which serves as one substrate for the biosynthesis of a nonribosomal peptide herbicide thaxtomin A.Recombinant coexpression of TxtD and TxtE in a heterologous host like E.coli for L-4-nitro-Trp production has not been achieved so far due to the poor or insoluble expression of TxtD.Using CFPS,TxtD and TxtE were successfully expressed in vitro,enabling the formation of L-4-nitro-Trp.After optimization,the cell-free system was able to synthesize approximately 360μM L-4-nitro-Trp within 16 h.Overall,this work expands the application scope of CFPS for study and synthesis of nitro-containing compounds,which are important building blocks widely used in pharmaceuticals,agrochemicals,and industrial chemicals.

Hyperlipidemia and erectile dysfunction

We have done consecutive studies to investigate the effects of impaired lipid metabolism on the contractile and re-laxation response of cavernous smooth muscles and to elucidate its pathogenesis: 1) incidence of hyperlipidemia in im-potent patients; 2) erection response to intracavemous injection of papaverine in impotent patients with hyperlipidemia;3) relaxation responses of isolated cavemosal smooth muscles to endothelium-independent and endothelium-dependentvasodilators in impotent patients with hypercholesterolemia or hypertriglyceridemia; 4) involvement of superoxide radi-cal in the impaired endothelium-dependent relaxation of cavemous smooth muscle in hypercholesterolemic rabbits; S)effects of isolated lipoproteins and triglyceride, combined oxidized LDL plus triglyceride, and combined oxidized LDLplus HDL on contractile and relaxation response of rabbit cavemous smooth muscles; 6) involvement of e-NOS in theimpaired endothelium-dependent relaxation of cavemous smooth muscle in hypercholesterolemic rabbit. Hypercholes-terolemia may cause impairment of endothelium-dependent relaxation. Oxidized LDL is the major causative cholesterolof the impaired relaxation response. A chain reaction, the production of superoxide radicals and functional impairmentof eNOS may be a major cause of the functional impairment in the early stages of hypercholesterolemia.

Synthesis and Antibacterial Activities of Selenide Derivatives of Benzisoselenazolone

A series of Benzisolselenazolone (BISA) derivatives were synthesized and evaluated for their antibacterial activities againstE coli. by using LKB-2277 bioactivity monitor. Other bioactivities were tested by the method of High Throughput Screening for pharmaceutical activity compounds (HTP) BISA derivatives 3b, at the concentration of 40 μg/mL, showed 100%antibacterial activity and 62%inhibition rate of aldose reductase (at the concentration of 5μg/mL). These new compound structures have determined by IR,1H NMR and MS spectra.

Titanate nanofibers reduce Kruppel-like factor 2(KLF2)-eNOS pathway in endothelial monolayer:A transcriptomic study

Although titanate nanofibers(TiNFs)and titanate nanotubes(TiNTs)have been proposed as relatively biocompatible nanomaterials(NMs),there is currently lacking of systemic studies which investigated the toxicity of TiNFs and TiNTs to endothelium.In this study,we developed endothelial monolayer model by using cell culture inserts,and systemically investigated the toxicity of TiNFs and TiNTs by RNA-seq,with a focus on Kruppel-like factor(KLF)-mediated effects,since KLF are transcription factors(TF)involved in the regulation of vascular biology.It was shown that NMs did not significantly induce cytotoxicity despite substantial internalization.However,the expression of many KLF was altered,and Western blot further confirmed that NMs down-regulated KLF2 proteins.Ingenuity pathway analysis(IPA)revealed that NMs altered the expression of KLF2-targed genes,typically the genes involved in inflammatory responses.KLF2-related Gene Ontology(GO)terms and Kyoto Encyclopedia of Gene and Genomes(KEGG)pathways were also altered,and it should be noticed that NMs altered GO terms and KEGG pathways related with endothelial NO synthase(eNOS).This study further verified that NMs decreased intracellular NO and eNOS proteins.All the observed effects were more obvious for TiNFs compared with TiNTs.Combined,this study showed that TiNFs or TiNTs we re non-cytotoxic to endothelial monolayer model,but TiNFs and more modestly TiNTs decreased KLF2 leading to decreased eNOS proteins and NO production.Our data may provide novel understanding about the toxicity of TiNFs as well as other Ti-based NMs to endothelium.

Negative effects of tumor cell nitric oxide on anti-glioblastoma photodynamic therapy

Glioblastomas are highly aggressive brain tumors that can persist after exposure to conventional chemotherapy or radiotherapy.Nitric oxide(NO)produced by inducible NO synthase(iNOS/NOS2)in these tumors is known to foster malignant cell proliferation,migration,and invasion as well as resistance to chemo-and radiotherapy.Minimally invasive photodynamic therapy(PDT)sensitized by 5-aminolevulinic acid(ALA)-induced protoporphyrin IX(PpIX)is a highly effective anti-glioblastoma modality,but it is also subject to NO-mediated resistance.Studies by the authors have revealed that glioblastoma U87 and U251 cells use endogenous iNOS/NO to not only resist photokilling after an ALA/light challenge,but also to promote proliferation and migration/invasion of surviving cells.Stress-upregulated iNOS/NO was found to play a major role in these negative responses to PDT-like treatment.Our studies have revealed a tight network of upstream signaling events leading to iNOS induction in photostressed cells and transition to a more aggressive phenotype.These events include activation or upregulation of pro-survival/pro-expansion effector proteins such as NF-κB,phosphoinositide-3-kinase(PI3K),protein kinase-B(Akt),p300,Survivin,and Brd4.In addition to this upstream signaling and its regulation,pharmacologic approaches for directly suppressing iNOS at its activity vs.transcriptional level are discussed.One highly effective agent in the latter category is bromodomain and extra-terminal(BET)inhibitor,JQ1,which was found to minimize iNOS upregulation in photostressed U87 cells.By acting similarly at the clinical level,a BET inhibitor such as JQ1 should markedly improve the efficacy of anti-glioblastoma PDT.