Publications by authors named "Bingnan Han"

22 Publications

  • Page 1 of 1

Absolute Structure Determination and Kv1.5 Ion Channel Inhibition Activities of New Debromoaplysiatoxin Analogues.

Mar Drugs 2021 Nov 11;19(11). Epub 2021 Nov 11.

Department of Development Technology of Marine Resources, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China.

Potassium channel Kv1.5 has been considered a key target for new treatments of atrial tachyarrhythmias, with few side effects. Four new debromoaplysiatoxin analogues with a 6/6/12 fused ring system were isolated from marine cyanobacterium sp. Their planar structures were elucidated by HRESIMS, 1D and 2D NMR. The absolute configuration of oscillatoxin J () was determined by single-crystal X-ray diffraction, and the absolute configurations of oscillatoxin K (), oscillatoxin L () and oscillatoxin M () were confirmed on the basis of GIAO NMR shift calculation followed by DP4 analysis. The current study confirmed the absolute configuration of the pivotal chiral positions (7S, 9S, 10S, 11R, 12S, 15S, 29R and 30R) at traditional ATXs with 6/12/6 tricyclic ring system. Compound , and exhibited blocking activities against Kv1.5 with IC values of 2.61 ± 0.91 µM, 3.86 ± 1.03 µM and 3.79 ± 1.01 µM, respectively. However, compound exhibited a minimum effect on Kv1.5 at 10 µM. Furthermore, all of these new debromoaplysiatoxin analogs displayed no apparent activity in a brine shrimp toxicity assay.
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http://dx.doi.org/10.3390/md19110630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8622842PMC
November 2021

Natural Products from Actinomycetes Associated with Marine Organisms.

Mar Drugs 2021 Nov 10;19(11). Epub 2021 Nov 10.

Department of Development Technology of Marine Resources, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China.

The actinomycetes have proven to be a rich source of bioactive secondary metabolites and play a critical role in the development of pharmaceutical researches. With interactions of host organisms and having special ecological status, the actinomycetes associated with marine animals, marine plants, macroalgae, cyanobacteria, and lichens have more potential to produce active metabolites acting as chemical defenses to protect the host from predators as well as microbial infection. This review focuses on 536 secondary metabolites (SMs) from actinomycetes associated with these marine organisms covering the literature to mid-2021, which will highlight the taxonomic diversity of actinomycetes and the structural classes, biological activities of SMs. Among all the actinomycetes listed, members of (68%), (6%), and (3%) are dominant producers of secondary metabolites. Additionally, alkaloids (37%), polyketides (33%), and peptides (15%) comprise the largest proportion of natural products with mostly antimicrobial activity and cytotoxicity. Furthermore, the data analysis and clinical information of SMs have been summarized in this article, suggesting that some of these actinomycetes with multiple host organisms deserve more attention to their special ecological status and genetic factors.
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http://dx.doi.org/10.3390/md19110629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8621598PMC
November 2021

Penispirozines A-H, Three Classes of Dioxopiperazine Alkaloids with Spirocyclic Skeletons Isolated from the Mangrove-Derived .

J Nat Prod 2020 09 20;83(9):2647-2654. Epub 2020 Aug 20.

Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People's Republic of China.

Eight new dioxopiperazine alkaloids, penispirozines A-H (-), were discovered from the mangrove-derived fungus HDN13-309. Their structures were elucidated by spectroscopic analysis, TDDFT-ECD calculations, and X-ray diffraction. Compound had an unusual pyrazino[1,2]oxazadecaline coupled with a thiophane ring system, and compound possessed a 6/5/6/5/6 pentacyclic ring system with two rare spirocyclic centers. Interestingly, compounds - were distinguished by not only the existence of a -thiophane or -furan ring system but also the chirality of the pentacyclic moiety. Compounds and increased the expression of the two relevant phase II detoxifying enzymes SOD2 and HO-1 at 10 μM.
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http://dx.doi.org/10.1021/acs.jnatprod.0c00451DOI Listing
September 2020

Deep-Learning-Enabled Fast Optical Identification and Characterization of 2D Materials.

Adv Mater 2020 Jul 9;32(29):e2000953. Epub 2020 Jun 9.

Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Advanced microscopy and/or spectroscopy tools play indispensable roles in nanoscience and nanotechnology research, as they provide rich information about material processes and properties. However, the interpretation of imaging data heavily relies on the "intuition" of experienced researchers. As a result, many of the deep graphical features obtained through these tools are often unused because of difficulties in processing the data and finding the correlations. Such challenges can be well addressed by deep learning. In this work, the optical characterization of 2D materials is used as a case study, and a neural-network-based algorithm is demonstrated for the material and thickness identification of 2D materials with high prediction accuracy and real-time processing capability. Further analysis shows that the trained network can extract deep graphical features such as contrast, color, edges, shapes, flake sizes, and their distributions, based on which an ensemble approach is developed to predict the most relevant physical properties of 2D materials. Finally, a transfer learning technique is applied to adapt the pretrained network to other optical identification applications. This artificial-intelligence-based material characterization approach is a powerful tool that would speed up the preparation, initial characterization of 2D materials and other nanomaterials, and potentially accelerate new material discoveries.
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http://dx.doi.org/10.1002/adma.202000953DOI Listing
July 2020

Asymmetric hot-carrier thermalization and broadband photoresponse in graphene-2D semiconductor lateral heterojunctions.

Sci Adv 2019 Jun 14;5(6):eaav1493. Epub 2019 Jun 14.

Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

The massless Dirac electron transport in graphene has led to a variety of unique light-matter interaction phenomena, which promise many novel optoelectronic applications. Most of the effects are only accessible by breaking the spatial symmetry, through introducing edges, p-n junctions, or heterogeneous interfaces. The recent development of direct synthesis of lateral heterostructures offers new opportunities to achieve the desired asymmetry. As a proof of concept, we study the photothermoelectric effect in an asymmetric lateral heterojunction between the Dirac semimetallic monolayer graphene and the parabolic semiconducting monolayer MoS. Very different hot-carrier cooling mechanisms on the graphene and the MoS sides allow us to resolve the asymmetric thermalization pathways of photoinduced hot carriers spatially with electrostatic gate tunability. We also demonstrate the potential of graphene-2D semiconductor lateral heterojunctions as broadband infrared photodetectors. The proposed structure shows an extreme in-plane asymmetry and provides a new platform to study light-matter interactions in low-dimensional systems.
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http://dx.doi.org/10.1126/sciadv.aav1493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570507PMC
June 2019

Engineering Yarrowia lipolytica to Simultaneously Produce Lipase and Single Cell Protein from Agro-industrial Wastes for Feed.

Sci Rep 2018 01 15;8(1):758. Epub 2018 Jan 15.

Key Lab of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan, 430074, China.

Lipases are scarcely exploited as feed enzymes in hydrolysis of lipids for increasing energy supply and improving nutrient use efficiency. In this work, we performed homologous overexpression, in vitro characterization and in vivo assessment of a lipase from the yeast Yarrowia lipolytica for feed purpose. Simultaneously, a large amount of yeast cell biomass was produced, for use as single cell protein, a potential protein-rich feed resource. Three kinds of low cost agro-industrial wastes were tested as substrates for simultaneous production of lipase and single cell protein (SCP) as feed additives: sugarcane molasses, waste cooking oil and crude glycerol from biodiesel production. Sugarcane molasses appeared as the most effective cheap medium, allowing production of 16420 U/ml of lipase and 151.2 g/L of single cell protein at 10 liter fermentation scale. In vitro characterization by mimicking a gastro-intestinal environment and determination of essential amino acids of the SCP, and in vivo oral feeding test on fish all revealed that lipase, SCP and their combination were excellent feed additives. Such simultaneous production of this lipase and SCP could address two main concerns of feed industry, poor utilization of lipid and shortage of protein resource at the same time.
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http://dx.doi.org/10.1038/s41598-018-19238-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768715PMC
January 2018

The functional characterization and comparison of two single CRD containing C-type lectins with novel and typical key motifs from Portunus trituberculatus.

Fish Shellfish Immunol 2017 Nov 12;70:398-407. Epub 2017 Sep 12.

Key Laboratory of Marine Ecosystem and Biogeochemistry, Second Institute of Oceanography, State Oceanic Administration, Hangzhou 310012, China. Electronic address:

C-type lectins are a superfamily of Ca-dependent carbohydrate-recognition proteins, which play crucial roles in innate immunity including nonself-recognition and pathogen elimination. In the present study, two single-CRD containing C-type lectins were identified from swimming crab Portunus trituberculatus (designated as PtCTL-2 and PtCTL-3). The open reading frame (ORF) of PtCTL-2 encoded polypeptides of 485 amino acids with a signal peptide and a single carbohydrate-recognition domain (CRD), while PtCTL-3's ORF encoded polypeptides of 241 amino acids with a coiled-coil region and a single-CRD. The key motifs determining carbohydrate binding specificity in PtCTL-2 and PtCTL-3 were EPR (Glu-Pro-Arg) and QPD (Gln-Pro-Asp). EPR is a motif being identified for the first time, whereas QPD is a typical motif in C-type lectins. Different PAMPs binding features of the two recombinant proteins - PtCTL-2 (rPtCTL-2) and PtCTL-3 (rPtCTL-3) have been observed in our experiments. rPtCTL-2 could bind three pathogen-associated molecular patterns (PAMPs) with relatively high affinity, including glucan, lipopolysaccharide (LPS) and peptidoglycan (PGN), while rPtCTL-3 could barely bind any of them. However, rPtCTL-2 could bind seven kinds of microbes and rPtCTL-3 could bind six kinds in microbe binding assay. Moreover, rPtCTL-2 and rPtCTL-3 exhibited similar agglutination activity against Gram-positive bacteria, Gram-negative bacteria and fungi in agglutination assay. All these results illustrated that PtCTL-2 and PtCTL-3 could function as important pattern-recognition receptors (PRR) with broad nonself-recognition spectrum involved in immune defense against invaders. In addition, the results of carbohydrate binding specificity showed that PtCTL-2 with novel key motif had broad carbohydrate binding specificity, while PtCTL-3 with typical key motif possessed different carbohydrate binding specificity from the classical binding rule. Furthermore, PtCTL-2 and PtCTL-3 could also function as opsonin to enhance encapsulation of hemocytes against Ni-NTA beads.
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http://dx.doi.org/10.1016/j.fsi.2017.09.029DOI Listing
November 2017

Harnessing biodiesel-producing microbes: from genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway.

Crit Rev Biotechnol 2017 Feb 2;37(1):26-36. Epub 2015 Nov 2.

e Key Laboratory for Marine Drugs, Department of Pharmacy, Renji Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai , P.R. China.

Microbial production routes, notably whole-cell lipase-mediated biotransformation and fatty-acids-derived biosynthesis, offer new opportunities for synthesizing biodiesel. They compare favorably to immobilized lipase and chemically catalyzed processes. Genetically modified whole-cell lipase-mediated in vitro route, together with in vivo and ex vivo microbial biosynthesis routes, constitutes emerging and rapidly developing research areas for effective production of biodiesel. This review presents recent advances in customizing microorganisms for producing biodiesel, via genetic engineering of lipases and metabolic engineering (including system regulation) of fatty-acids-derived pathways. Microbial hosts used include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus oryzae. These microbial cells can be genetically modified to produce lipases under different forms: intracellularly expressed, secreted or surface-displayed. They can be metabolically redesigned and systematically regulated to obtain balanced biodiesel-producing cells, as highlighted in this study. Such genetically or metabolically modified microbial cells can support not only in vitro biotransformation of various common oil feedstocks to biodiesel, but also de novo biosynthesis of biodiesel from glucose, glycerol or even cellulosic biomass. We believe that the genetically tractable oleaginous yeast Yarrowia lipolytica could be developed to an effective biodiesel-producing microbial cell factory. For this purpose, we propose several engineered pathways, based on lipase and wax ester synthase, in this promising oleaginous host.
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http://dx.doi.org/10.3109/07388551.2015.1104531DOI Listing
February 2017

Assembly of lipase and P450 fatty acid decarboxylase to constitute a novel biosynthetic pathway for production of 1-alkenes from renewable triacylglycerols and oils.

Biotechnol Biofuels 2015 26;8:34. Epub 2015 Feb 26.

Key Laboratory of Biofuels, and Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, 266101 Qingdao, Shandong China.

Background: Biogenic hydrocarbons (biohydrocarbons) are broadly accepted to be the ideal 'drop-in' biofuel alternative to petroleum-based fuels due to their highly similar chemical composition and physical characteristics. The biological production of aliphatic hydrocarbons is largely dependent on engineering of the complicated enzymatic network surrounding fatty acid biosynthesis.

Result: In this work, we developed a novel system for bioproduction of terminal fatty alkenes (1-alkenes) from renewable and low-cost triacylglycerols (TAGs) based on the lipase hydrolysis coupled to the P450 catalyzed decarboxylation. This artificial biosynthetic pathway was constituted using both cell-free systems including purified enzymes or cell-free extracts, and cell-based systems including mixed resting cells or growing cells. The issues of high cost of fatty acid feedstock and complicated biosynthesis network were addressed by replacement of the de novo biosynthesized fatty acids with the fed cheap TAGs. This recombinant tandem enzymatic pathway consisting of the Thermomyces lanuginosus lipase (Tll) and the P450 fatty acid decarboxylase OleTJE resulted in the production of 1-alkenes from purified TAGs or natural oils with 6.7 to 46.0% yields.

Conclusion: Since this novel hydrocarbon-producing pathway only requires two catalytically efficient enzymatic steps, it may hold great potential for industrial application by fulfilling the large-scale and cost-effective conversion of renewable TAGs into biohydrocarbons. This work highlights the power of designing and implementing an artificial pathway for production of advanced biofuels.
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http://dx.doi.org/10.1186/s13068-015-0219-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4355466PMC
March 2015

Quantitative LC-MS/MS analysis of arachidonoyl amino acids in mouse brain with treatment of FAAH inhibitor.

Anal Biochem 2013 Jan 5;432(2):74-81. Epub 2012 Oct 5.

Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47904, USA.

An additional class of endogenous lipid amides, N-arachidonoyl amino acids (Ara-AAs), is growing in significance in the field of endocannabinoids. The development, validation, and application of a sensitive and selective method to simultaneously monitor and quantify the level of Ara-AAs along with anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in mouse brain has been established. The linearity of the method over the concentration ranges of 0.2-120 pg/μl for the standards of N-arachidonoyl amino acids, N-arachidonoyl alanine (NAAla), serine (NASer), γ-aminobutyric acid (NAGABA), and glycine (NAGly); 0.7-90 pg/μl for AEA-d(0)/d(8); and 7.5-950 pg/μl for 2-AG was determined with R(2) values of 0.99. Also the effects of the FAAH inhibitor URB 597 on the endogenous levels of these analytes were investigated. AEA and NASer brain levels exhibit a dose-dependent increase after systemic administration of URB 597, whereas NAGly and NAGABA were significantly decreased after treatment. NAAla and 2-AG were not altered after URB 597 treatment. The potential benefit of establishing this assay extends beyond the quantification of the Ara-AAs along with AEA and 2-AG in mouse brain, to reveal a variety of pharmacological effects and physiological roles of these analytes.
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http://dx.doi.org/10.1016/j.ab.2012.09.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3760509PMC
January 2013

Identification of CBX3 and ABCA5 as putative biomarkers for tumor stem cells in osteosarcoma.

PLoS One 2012 3;7(8):e41401. Epub 2012 Aug 3.

Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, Frederick, Maryland, United States of America.

Recently, there has been renewed interest in the role of tumor stem cells (TSCs) in tumorigenesis, chemoresistance, and relapse of malignant tumors including osteosarcoma. The potential exists to improve osteosarcoma treatment through characterization of TSCs and identification of therapeutic targets. Using transcriptome, proteome, immunophenotyping for cell-surface markers, and bioinformatic analyses, heterogeneous expression of previously reported TSC or osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and aldehyde dehydrogenase, among others, was observed in vitro. However, consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma cultures was identified. ABCA5 was identified as a putative biomarker of TSCs and/or osteosarcoma. Lastly, in a high-throughput screen we identified epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase (3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; RHPS4)-targeted therapeutic agents as candidates for TSC ablation in osteosarcoma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041401PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3411700PMC
February 2013

A comparative 'bottom up' proteomics strategy for the site-specific identification and quantification of protein modifications by electrophilic lipids.

J Proteomics 2012 Oct 26;75(18):5724-33. Epub 2012 Jul 26.

Department of Chemistry, Oregon State University, Corvallis, OR 97331, USA.

We report a mass spectrometry-based comparative "bottom up" proteomics approach that combines d(0)/d(4)-succinic anhydride labeling with commercially available hydrazine (Hz)-functionalized beads (Affi-gel Hz beads) for detection, identification and relative quantification of site-specific oxylipid modifications in biological matrices. We evaluated and applied this robust and simple method for the quantitative analysis of oxylipid protein conjugates in cardiac mitochondrial proteome samples isolated from 3- and 24-month-old rat hearts. The use of d(0)/d(4)-succinic anhydride labeling, Hz-bead based affinity enrichment, nanoLC fractionation and MALDI-ToF/ToF tandem mass spectrometry yielded relative quantification of oxylipid conjugates with residue-specific modification information. Conjugation of acrolein (ACR), 4-hydroxy-2-hexenal (HHE), 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-noneal (ONE) to cysteine, histidine and lysine residues were identified. HHE conjugates were the predominant subset of Michael-type adducts detected in this study. The HHE conjugates showed higher levels in mitochondrial preparations from young heart congruent with previous findings by others that the n-3/n-6 PUFA ratio is higher in young heart mitochondrial membranes. Although this study focuses on protein adducts of reactive oxylipids, the method might be equally applicable to protein carbonyl modifications caused by metal catalyzed oxidation reactions.
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http://dx.doi.org/10.1016/j.jprot.2012.07.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3468693PMC
October 2012

Wewakamide A and guineamide G, cyclic depsipeptides from the marine cyanobacteria Lyngbya semiplena and Lyngbya majuscula.

J Microbiol Biotechnol 2011 Sep;21(9):930-6

Department of Ocean Science and Engineering, Zhejiang University, Hangzhou, China 310028.

Two new cyclic depsipeptides wewakamide A (1) and guineamide G (2) have been isolated from the marine cyanobacterium Lyngbya semiplena and Lyngbya majuscula, respectively, collected from Papua New Guinea. The amino and hydroxy acid partial structures of wewakamide A and guineamide G were elucidated through extensive spectroscopic techniques, including HR-FABMS, 1D (1)H and (13)C NMR, as well as 2D COSY, HSQC, HSQCTOCSY, and HMBC spectra. The sequence of the residues of wewakamide A was determined through a combination of ESI-MS/MS, HMBC, and ROESY. Wewakamide A possesses a β-amino acid, 3-amino-2-methylbutanoic acid (Maba) residue, which has only been previously identified in two natural products, guineamide B (3) and dolastatin D (4). Although both new compounds (1,2) showed potent brine shrimp toxicity, only guineamide G displayed significant cytotoxicity to a mouse neuroblastoma cell line with LC(50) values of 2.7 micrometer.
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http://dx.doi.org/10.4014/jmb.1105.05011DOI Listing
September 2011

The structure elucidation of isomalyngamide K from the marine cyanobacterium Lyngbya majuscula by experimental and DFT computational methods.

J Mol Struct 2011 Mar;989(1-3):109-113

Marine Resources Research Institute, Department of Ocean Science and Engineering, Xixi Campus, Zhejiang University, Hangzhou, China 310028.

The 2Z-isomer of malyngamide K has been isolated along with the known compounds malyngamide C, deoxy-C and K, and characterized from a Papua New Guinea field collection of the cyanobacterium Lyngbya majuscula. The planar structure was deduced by 1D and 2D NMR spectroscopic and mass spectral data interpretation. The absolute configurations were determined on the basis of spectroscopic techniques, chemical degradation and DFT theoretical calculations.
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http://dx.doi.org/10.1016/j.molstruc.2011.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3066435PMC
March 2011

A copper chelate of thiosemicarbazone NSC 689534 induces oxidative/ER stress and inhibits tumor growth in vitro and in vivo.

Free Radic Biol Med 2011 Jan 4;50(1):110-21. Epub 2010 Nov 4.

Biological Testing Branch, Developmental Therapeutics Program, SAIC-Frederick, Frederick, MD 21702, USA.

In this study, a Cu(2+) chelate of the novel thiosemicarbazone NSC 689534 was evaluated for in vitro and in vivo anti-cancer activity. Results demonstrated that NSC 689534 activity (low micromolar range) was enhanced four- to fivefold by copper chelation and completely attenuated by iron. Importantly, once formed, the NSC 689534/Cu(2+) complex retained activity in the presence of additional iron or iron-containing biomolecules. NSC 689534/Cu(2+) mediated its effects primarily through the induction of ROS, with depletion of cellular glutathione and protein thiols. Pretreatment of cells with the antioxidant N-acetyl-l-cysteine impaired activity, whereas NSC 689534/Cu(2+) effectively synergized with the glutathione biosynthesis inhibitor buthionine sulfoximine. Microarray analysis of NSC 689534/Cu(2+)-treated cells highlighted activation of pathways involved in oxidative and ER stress/UPR, autophagy, and metal metabolism. Further scrutiny of the role of ER stress and autophagy indicated that NSC 689534/Cu(2+)-induced cell death was ER-stress dependent and autophagy independent. Last, NSC 689534/Cu(2+) was shown to have activity in an HL60 xenograft model. These data suggest that NSC 689534/Cu(2+) is a potent oxidative stress inducer worthy of further preclinical investigation.
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http://dx.doi.org/10.1016/j.freeradbiomed.2010.10.696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3014388PMC
January 2011

Proteomic analysis of nuclei isolated from cancer cell lines treated with indenoisoquinoline NSC 724998, a novel topoisomerase I inhibitor.

J Proteome Res 2010 Aug;9(8):4016-27

Developmental Therapeutics Program, SAIC-Frederick Inc., NCI-Frederick, Frederick, Maryland 21702, USA.

The indenoisoquinoline NSC724998 is a novel topoisomerase I (Top1) inhibitor entering Phase I clinical trials at the National Cancer Institute, USA. In this study, 2-D PAGE analysis was performed on nuclear lysates prepared from HCT-116 and A375 cells treated with 1 microM NSC724998 for 24 h and the differentially regulated spots identified by LC-MS/MS. One-hundred fourteen protein spot differentials were identified, 66 from A375 cells and 48 from HCT-116 cells. Proteins related to apoptosis changed specifically in A375 cells, whereas proteins involved in the ubiquitin-proteasome system were highly enriched in treated HCT-116 cells. Importantly, 12 differentially expressed proteins (ETFA, HCC1, HNRCL, KAP1, NPM, NUCL, PRDX1, PRP19, PSB6, RAE1L, RU2A, and SFRS9) were common to both cell lines. Western blotting and immunocytochemistry confirmed significant nuclear upregulation of both the proteasome subunit PSB6 and the transcriptional repressor KAP1. Interestingly, increased KAP1 polypeptide was accompanied by enhanced phosphorylation at Ser824. Similar to gammaH2AX, KAP1 phosphorylation was consistently enhanced in a panel of 12 cell lines and in A375 xenografts following NSC 724998 treatment. In summary, these data enhance our understanding of protein dynamics in the nucleus following DNA damage and provide an alternate marker (pKAP1) with potential for monitoring clinical responses to Top1 poisons.
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http://dx.doi.org/10.1021/pr100194dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2917484PMC
August 2010

Artemisinin dimer anticancer activity correlates with heme-catalyzed reactive oxygen species generation and endoplasmic reticulum stress induction.

Int J Cancer 2009 Sep;125(6):1266-75

Developmental Therapeutics Program, SAIC-Frederick, NCI-Frederick, Frederick, MD 21702, USA.

Analogs of the malaria therapeutic, artemisinin, possess in vitro and in vivo anticancer activity. In this study, two dimeric artemisinins (NSC724910 and 735847) were studied to determine their mechanism of action. Dimers were >1,000 fold more active than monomer and treatment was associated with increased reactive oxygen species (ROS) and apoptosis induction. Dimer activity was inhibited by the antioxidant L-NAC, the iron chelator desferroxamine and exogenous hemin. Similarly, induction of heme oxygenase (HMOX) with CoPPIX inhibited activity, whereas inhibition of HMOX with SnPPIX enhanced it. These results emphasize the importance of iron, heme and ROS in activity. Microarray analysis of dimer treated cells identified DNA damage, iron/heme and cysteine/methionine metabolism, antioxidant response, and endoplasmic reticulum (ER) stress as affected pathways. Detection of an ER-stress response was relevant because in malaria, artemisinin inhibits pfATP6, the plasmodium orthologue of mammalian sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases (SERCA). A comparative study of NSC735847 with thapsigargin, a specific SERCA inhibitor and ER-stress inducer showed similar behavior in terms of transcriptomic changes, induction of endogenous SERCA and ER calcium mobilization. However, thapsigargin had little effect on ROS production, modulated different ER-stress proteins and had greater potency against purified SERCA1. Furthermore, an inactive derivative of NSC735847 that lacked the endoperoxide had identical inhibitory activity against purified SERCA1, suggesting that direct inhibition of SERCA has little inference on overall cytotoxicity. In summary, these data implicate indirect ER-stress induction as a central mechanism of artemisinin dimer activity.
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http://dx.doi.org/10.1002/ijc.24496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752979PMC
September 2009

Design, synthesis, and application of a hydrazide-functionalized isotope-coded affinity tag for the quantification of oxylipid-protein conjugates.

Anal Chem 2007 May 27;79(9):3342-54. Epub 2007 Mar 27.

Department of Chemistry, and the Linus Pauling Institute, Oregon State University, Corvallis, Oregon 97331, USA.

An isotopically coded affinity probe was developed and evaluated for the characterization and quantification of proteins adducted by 2-alkenals derived from lipid peroxidation (LPO) processes. Lipid-derived 2-alkenals, such as acrolein and 4-hydroxy-2-nonenal (HNE), have the ability to react with cysteine, histidine, and lysine residues in proteins, thus causing protein damage and loss of protein function. Such modifications of proteins are difficult to characterize in biological samples by mass spectrometry due to the complexity of protein extracts and the low abundance of adducted proteins. The novel aldehyde-reactive, hydrazide-functionalized, isotope-coded affinity tag (HICAT) described in this study was found effective for the selective isolation, detection, and quantification of Michael-type adducts of 2-alkenals with proteins using a combination of affinity isolation, nanoLC, and matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS). The chemical and mass spectrometric properties of the new probe are demonstrated on a model protein treated with HNE. The efficacy of HICAT for the analysis of complex samples was tested using preparations of mitochondrial proteins that were modified in vitro with HNE. The potential of the HICAT strategy for the identification, characterization, and quantification of in vivo oxylipid-protein conjugates is demonstrated on cardiac mitochondrial protein preparations, in which, for example, the ADP/ATP translocase 1 was found adducted to the 2-alkenals, acrolein and 4-hydroxy-2-hexenal, at Cys-256.
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http://dx.doi.org/10.1021/ac062262aDOI Listing
May 2007

New role for an old probe: affinity labeling of oxylipid protein conjugates by N'-aminooxymethylcarbonylhydrazino d-biotin.

Anal Chem 2006 Oct;78(19):6847-54

Department of Chemistry, Oregon State University, Corvallis, Oregon 97331, USA.

Free radicals, electrophiles, and endogenous reactive intermediates are generated during normal physiological processes and are capable of modifying DNA, lipids, and proteins. However, elevated levels of oxidative modifications of proteins by reactive species are implicated in the etiology and pathology of oxidative stress-mediated diseases, neurodegeneration, and aging. A mass spectrometry-based approach is reported that aids to the identification and characterization of carbonyl-modified proteins. The method uses N'-aminooxymethylcarbonylhydrazino d-biotin, a biotinylated hydroxylamine derivative that forms an oxime derivative with the aldehyde/keto group found in oxidatively modified proteins. In this paper, the method is demonstrated for one class of carbonyl-modified proteins, namely, oxylipid peptide and protein conjugates formed by Michael addition-type conjugation reactions of alpha,beta-unsaturated aldehydic lipid peroxidation products with nucleophilic peptide side chains. This new application of an "old" probe, which has been used for the detection of abasic sites in DNA strands, introduces a biotin moiety into the oxylipid peptide conjugate. The biotin-modified oxylipid peptide conjugate is then amenable to enrichment using avidin affinity capture. The described method represents an attractive alternative to hydrazine-based derivatization methods for oxidized peptides and proteins because the reduction step necessary for the transformation of the hydrazone bond to the chemically more stable hydrazine bond can be omitted. Tandem mass spectrometry of the labeled oxylipid peptide conjugates indicates that the biotin moiety is at least partially retained on the fragment ion during the collisionally induced dissociation experiments, a prerequisite for the use of automated database searching of uninterpreted tandem mass spectra. The reported approach is outlined for the detection, identification, and characterization of oxylipid peptide conjugates, but the labeling chemistry may also be applicable to other carbonyl-modified proteins.
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http://dx.doi.org/10.1021/ac0607257DOI Listing
October 2006

Aurilides B and C, cancer cell toxins from a Papua New Guinea collection of the marine cyanobacterium Lyngbya majuscula.

J Nat Prod 2006 Apr;69(4):572-5

College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA.

Cytotoxicity-guided fractionation of a strain of the marine cyanobacterium Lyngbya majuscula collected from Papua New Guinea led to the isolation of aurilides B (1) and C (2). The planar structures of 1 and 2 were established by spectroscopic analysis, including HR-FABMS, 1D (1)H and (13)C NMR, and 2D COSY, HSQC, HSQC-TOCSY, and HMBC spectra. The absolute configuration was determined by spectroscopic analysis and chiral HPLC analysis of acid hydrolysates of 1 and 2. Both aurilides B and C showed in vitro cytotoxicity toward NCI-H460 human lung tumor and the neuro-2a mouse neuroblastoma cell lines, with LC(50) values between 0.01 and 0.13 microM. Aurilide B (1) was evaluated in the NCI 60 cell line panel and found to exhibit a high level of cytotoxicity (the mean panel GI(50) concentration was less than 10 nM) and to be particularly active against leukemia, renal, and prostate cancer cell lines.
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http://dx.doi.org/10.1021/np0503911DOI Listing
April 2006

The wewakpeptins, cyclic depsipeptides from a Papua New Guinea collection of the marine cyanobacterium Lyngbya semiplena.

J Org Chem 2005 Apr;70(8):3133-9

College of Pharmacy and Department of Chemistry, Oregon State University, Corvallis, OR 97331, USA.

Four new depsipeptides have been isolated from the marine cyanobacterium Lyngbya semiplena collected from Papua New Guinea. The amino and hydroxy acid partial structures of wewakpeptins A-D (1-4) were elucidated through extensive spectroscopic techniques, including HR-FABMS, 1D (1)H and (13)C NMR, as well as 2D COSY, HSQC, HSQC-TOCSY, and HMBC spectra. The sequence of the residues was determined through a combination of multifaceted approaches including ESI-MS/MS, HMBC, ROESY, and a modified 1D HMBC experiment. The absolute stereochemistry of each residue was determined by chiral HPLC and chiral GC-MS methods. The wewakpeptins represent an unusual arrangement of amino and hydroxy acid subunits relative to known cyanobacterial peptides and possess a bis-ester, a 2,2-dimethyl-3-hydroxy-7-octynoic acid (Dhoya) or 2,2-dimethyl-3-hydroxyoctanoic acid (Dhoaa) residue, and a diprolyl group reminiscent of dolastatin 15. Wewakpeptin A and B were the most cytotoxic among these four depsipeptides with an LC(50) of approximately 0.4 muM to both the NCI-H460 human lung tumor and the neuro-2a mouse neuroblastoma cell lines.
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http://dx.doi.org/10.1021/jo0478858DOI Listing
April 2005

Semiplenamides A-G, fatty acid amides from a Papua New Guinea collection of the marine cyanobacterium Lyngbya semiplena.

J Nat Prod 2003 Oct;66(10):1364-8

College of Pharmacy, Oregon State University, Corvallis, Oregon 97331, USA.

Semiplenamides A (1) to G (7), a series of new anandamide-like fatty acid amides, were isolated from a 1997 Papua New Guinea collection of the marine cyanobacterium Lyngbya semiplena. The planar structures of these lipids were determined using standard 1D and 2D NMR methods. The relative stereochemistry of the aliphatic portion of the new metabolites was deduced from 1D NOE data and (1)H-decoupling experiments, while the absolute stereochemistry of the amino alcohol moieties was assigned by chemical derivatization and chiral GC-MS methods. All of these new metabolites displayed toxicity in the brine shrimp model system, while semiplenamides A, B, and G showed weak affinity for the rat cannabinoid CB(1) receptor and semiplenamide A was a moderate inhibitor (IC(50) = 18.1 muM) of the anandamide membrane transporter (AMT).
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http://dx.doi.org/10.1021/np030242nDOI Listing
October 2003
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