Publications by authors named "Frederick T Greenaway"

25 Publications

  • Page 1 of 1

The potential role of miR-124-3p in tumorigenesis and other related diseases.

Mol Biol Rep 2021 Apr 20. Epub 2021 Apr 20.

Carlson School of Chemistry and Biochemistry, Clark University, Worcester, MA, 01610, USA.

MicroRNAs (miRNAs) are a class of single-stranded noncoding and endogenous RNA molecules with a length of 18-25 nucleotides. Previous work has shown that miR-124-3p leads to malignant progression of cancer including cell apoptosis, migration, invasion, drug resistance, and also recovers neural function, affects adipogenic differentiation, facilitates wound healing through control of various target genes. miR-124-3p has been mainly previously characterized as a tumor suppressor regulating tumorigenesis and progression in several cancers, such as hepatocellular carcinoma (HCC), gastric cancer (GC), bladder cancer, ovarian cancer (OC), and leukemia, as a tumor promotor in breast cancer (BC), and it has been also widely studied in a variety of neurological diseases, like Parkinson's disease (PD), dementia and Alzheimer's disease (AD), and cardiovascular diseases, ulcerative colitis (UC), acute respiratory distress syndrome (ARDS). To lay the groundwork for future therapeutic strategies, in this review we mainly focus on the most recent years of literature on the functions of miR-124-3p in related major cancers, as well as its downstream target genes. Although current work as yet provides an incomplete picture, miR-124-3p is still worthy of more attention as a practical and effective clinical biomarker.
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http://dx.doi.org/10.1007/s11033-021-06347-4DOI Listing
April 2021

CRKL promotes hepatocarcinoma through enhancing glucose metabolism of cancer cells via activating PI3K/Akt.

J Cell Mol Med 2021 Mar 1;25(5):2714-2724. Epub 2021 Feb 1.

Department of Biotechnology, College of Basic Medical Sciences, Dalian Medical University, Dalian, China.

Abnormal glucose metabolism may contribute to cancer progression. As a member of the CRK (v-crk sarcoma virus CT10 oncogene homologue) adapter protein family, CRKL (CRK-like) associated with the development and progression of various tumours. However, the exact role and underlying mechanism of CRKL on energy metabolism remain unknown. In this study, we investigated the effect of CRKL on glucose metabolism of hepatocarcinoma cells. CRKL and PI3K were found to be overexpressed in both hepatocarcinoma cells and tissues; meanwhile, CRKL up-regulation was positively correlated with PI3K up-regulation. Functional investigations revealed that CRKL overexpression promoted glucose uptake, lactate production and glycogen synthesis of hepatocarcinoma cells by up-regulating glucose transporters 1 (GLUT1), hexokinase II (HKII) expression and down-regulating glycogen synthase kinase 3β (GSK3β) expression. Mechanistically, CRKL promoted glucose metabolism of hepatocarcinoma cells via enhancing the CRKL-PI3K/Akt-GLUT1/HKII-glucose uptake, CRKL-PI3K/Akt-HKII-glucose-lactate production and CRKL-PI3K/Akt-Gsk3β-glycogen synthesis. We demonstrate CRKL facilitates HCC malignancy via enhancing glucose uptake, lactate production and glycogen synthesis through PI3K/Akt pathway. It provides interesting fundamental clues to CRKL-related carcinogenesis through glucose metabolism and offers novel therapeutic strategies for hepatocarcinoma.
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http://dx.doi.org/10.1111/jcmm.16303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933966PMC
March 2021

A novel ETV6-miR-429-CRKL regulatory circuitry contributes to aggressiveness of hepatocellular carcinoma.

J Exp Clin Cancer Res 2020 Apr 23;39(1):70. Epub 2020 Apr 23.

Department of Biotechnology, College of Basic Medical Sciences, Dalian Medical University, Dalian, 116044, China.

Background: Tumor metastasis is one of the main causes of the high mortality of hepatocellular carcinoma (HCC). E-Twenty Six variant gene 6 (ETV6) is a strong transcriptional repressor, associated with the development and progression of tumors. However, the exact role and underlying mechanism of ETV6 in HCC remain unclear.

Methods: Western blotting, quantitative real-time PCR and immunohistochemistry were used to detect the expression levels of ETV6, CRKL (v-crk sarcoma virus CT10 oncogene homologue (avian)-like) and miR-429 in HCC tissues and cells; Transwell chamber and F-actin cytoskeleton staining assay to examine the effects of ETV6 and CRKL deregulation on the migration, invasion and cytoskeleton of HCC cells; Co-immunoprecipitation assay to determine the interaction between CRKL and ETV6; Chromatin immunoprecipitation assay to investigate the interaction between ETV6 and miR-429.

Results: We established a novel ETV6-miR-429-CRKL regulatory circuitry contributes to HCC metastasis. ETV6 and CRKL were frequently increased, while miR-429 was downregulated in both hepatocarcinoma tissues and hepatocarcinoma cells. Moreover, ETV6 upregulation was positively correlated with CRKL upregulation, and two negative correlations were also established for ETV6 and CRKL upregulation with miR-429 downregulation in both hepatocarcinoma patients' tumorous tissues and hepatocarcinoma cells. Functional investigations revealed that overexpression and knockdown of ETV6 was remarkably effective in promoting and suppressing HCC cell migration, invasion, cytoskeleton F-actin expression and arrangement, whereas, CRKL overexpression exhibited similar effects to the overexpression of ETV6. Mechanistically, ETV6 negatively regulates miR-429 expression by directly binding to the promoter region of miR-429; miR-429 negatively regulates CRKL expression by selectively targeting CRKL-3'-UTR; ETV6 directly binds to CRKL and positively regulates its expression, which in turn CRKL positively regulates ETV6 expression.

Conclusions: Our data demonstrated that ETV6 promotes migration and invasion of HCC cells by directly binding to promoter region of miR-429 via modulating CRKL expression. The newly identified ETV6-miR-429-CRKL regulatory circuitry contributes to the aggressiveness of HCC, which provides new clues for fundamental research on diagnosis and treatment parameters for HCC.
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http://dx.doi.org/10.1186/s13046-020-01559-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178969PMC
April 2020

Crystal structure of tetra-aqua-bis-(pyrimidin-1-ium-4,6-diolato-κ)manganese(II).

Acta Crystallogr E Crystallogr Commun 2017 Apr 31;73(Pt 4):620-622. Epub 2017 Mar 31.

Carlson School of Chemistry and Biochemistry, Clark University, 950 Main St, Worcester, MA 01610, USA.

The Mn ion in the structure of the mononuclear title compound, [Mn(CHNO)(HO)], is situated on an inversion center and is coordinated by two O atoms from two deprotonated 4,6-di-hydroxy-pyrimidine ligands and by four O atoms from water mol-ecules giving rise to a slightly distorted octa-hedral coordination sphere. The complex includes an intra-molecular hydrogen bond between an aqua ligand and the non-protonated N ring atom. The extended structure is stabilized by inter-molecular hydrogen bonds between aqua ligands, by hydrogen bonds between N and O atoms of the ligands of adjacent mol-ecules, and by hydrogen bonds between aqua ligands and the non-coordinating O atom of an adjacent mol-ecule.
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http://dx.doi.org/10.1107/S2056989017004649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382635PMC
April 2017

Comparing hydrazine-derived reactive groups as inhibitors of quinone-dependent amine oxidases.

J Enzyme Inhib Med Chem 2017 Dec;32(1):496-503

a Carlson School of Chemistry and Biochemistry, Clark University , Worcester , MA , USA.

Lysyl oxidase has emerged as an important enzyme in cancer metastasis. Its activity has been reported to become upregulated in several types of cancer, and blocking its activity has been shown to limit the metastatic potential of various cancers. The small-molecules phenylhydrazine and β-aminopropionitrile are known to inhibit lysyl oxidase; however, issues of stability, toxicity, and poorly defined mechanisms limit their potential use in medical applications. The experiments presented herein evaluate three other families of hydrazine-derived compounds - hydrazides, alkyl hydrazines, and semicarbazides - as irreversible inhibitors of lysyl oxidase including determining the kinetic parameters and comparing the inhibition selectivities for lysyl oxidase against the topaquinone-containing diamine oxidase from lentil seedlings. The results suggest that the hydrazide group may be a useful core functionality that can be developed into potent and selective inhibitors of lysyl oxidase and eventually find application in cancer metastasis research.
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http://dx.doi.org/10.1080/14756366.2016.1265518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6009937PMC
December 2017

Role of annexin A6 in cancer.

Oncol Lett 2015 Oct 15;10(4):1947-1952. Epub 2015 Jul 15.

Department of Biotechnology, Dalian Medical University, Dalian, Liaoning 116044, P.R. China.

Annexin A6 (AnxA6) is a member of a conserved superfamily of Ca-dependent membrane-binding annexin proteins. It participates in membrane and cytoskeleton organization, cholesterol homeostasis, membrane trafficking, cell adhesion and signal transduction. The expression levels of AnxA6 are closely associated with melanoma, cervical cancer, epithelial carcinoma, breast cancer, gastric cancer, prostate cancer, acute lymphoblastic leukemia, chronic myeloid leukemia, large-cell lymphoma and myeloma. AnxA6 exhibits dual functions in cancer, acting either as a tumor suppressor or promoter, depending on the type of cancer and the degree of malignancy. In several types of cancer, AnxA6 acts via Ras, Ras/MAPK and/or FAK/PI3K signaling pathways by mainly mediating PKCα, p120GAP, Bcr-Abl and YY1. In the present review, the roles of AnxA6 in different types of cancer are summarized.
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http://dx.doi.org/10.3892/ol.2015.3498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4579822PMC
October 2015

Annexin A11 in disease.

Clin Chim Acta 2014 Apr 6;431:164-8. Epub 2014 Feb 6.

Carlson School of Chemistry and Biochemistry, Clark University, Worcester, MA 01610, USA.

Ubiquitously expressed in many cell types, annexin A11 (Anxa11) is a member of the multigene family of Ca(2+)-regulated phospholipid-dependent and membrane-binding annexin proteins. Studies have shown that Anxa11 plays an important role in cell division, Ca(2+) signaling, vesicle trafficking and apoptosis. The deregulation and mutation of Anxa11 are involved in systemic autoimmune diseases, sarcoidosis and the development, chemoresistance and recurrence of cancers. Malfunction of Anxa11 may lead to or enhance the metastasis, invasion and drug resistance of cancers through the platelet-derived growth factor receptor (PDGFR) pathway and/or the mitogen-activated protein kinase (MAPK)/p53 pathway. In a variety of diseases, Anxa11 is most commonly reported to function through interactions with apoptosis-linked gene-2 protein (ALG-2) and/or calcyclin (S100A6). Although it has been little studied, Anxa11 is a promising biomarker for the diagnosis, treatment and prognosis of certain diseases. In this review, the associations of Anxa11 with Ca(2+)-regulated exocytosis, cytokinesis, sex differentiation, autoimmune diseases, thrombolysis and cancers are summarized and interpreted.
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http://dx.doi.org/10.1016/j.cca.2014.01.031DOI Listing
April 2014

ACTB in cancer.

Clin Chim Acta 2013 Feb 22;417:39-44. Epub 2012 Dec 22.

Department of Biotechnology, Dalian Medical University, Dalian 116044, China.

Beta-actin (ACTB) has traditionally been regarded as an endogenous housekeeping gene and has been widely used as a reference gene/protein in quantifying expression levels in tumors. However, ACTB is closely associated with a variety of cancers and accumulating evidence indicates that ACTB is de-regulated in liver, melanoma, renal, colorectal, gastric, pancreatic, esophageal, lung, breast, prostate, ovarian cancers, leukemia and lymphoma. ACTB is generally found to be up-regulated in the majority of tumor cells and tissues. The abnormal expression and polymerization of ACTB and the resulting changes to the cytoskeleton are revealed to be associated with the invasiveness and metastasis of cancers. The current review explores relevant mechanisms, integrates current understandings, and provides suggestions for future studies of the roles of ACTB in tumors.
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http://dx.doi.org/10.1016/j.cca.2012.12.012DOI Listing
February 2013

Identification of the copper-binding ligands of lysyl oxidase.

J Neural Transm (Vienna) 2011 Jul 29;118(7):1101-9. Epub 2010 Dec 29.

Carlson School of Chemistry and Biochemistry, Clark University, Worcester, MA 01610, USA.

In order to identify the ligands coordinating with copper in lysyl oxidase, the enzyme was expressed in an E. coli expression system and active enzyme obtained after refolding in the presence of Cu(II). The five histidines found in the putative copper-binding region were sequentially mutated to alanines and the enzymatic activities of the resultant mutants were monitored, together with the copper content, the CD and fluorescence spectra, and the redox-cycling activity. The spectroscopic results show that in all cases the protein folded correctly but that the copper-content, enzymatic activity, and redox-cycling ability depended on the mutation. One mutant was fully functional, and two others completely lacked copper, the lysyl tyrosyl quinone (LTQ) cofactor, and activity. A fourth incorporated copper but lacked LTQ and enzymatic activity. The remaining mutant incorporated copper and had redox-cycling activity but no enzymatic activity. The results suggest that three of the five histidines in the putative copper-binding domain, H292, H294, H296, are the copper ligands and essential to the formation of LTQ. A fourth, H289, is not involved in LTQ formation or activity, while a fifth, H303, is suggested to be a general base in the catalytic mechanism.
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http://dx.doi.org/10.1007/s00702-010-0559-4DOI Listing
July 2011

Identification of the disulfide bonds of lysyl oxidase.

J Neural Transm (Vienna) 2011 Jul 28;118(7):1111-4. Epub 2010 Dec 28.

Carlson School of Chemistry and Biochemistry, Clark University, 950 Main St, Worcester, MA 01610-1477, USA.

Proteolytic digestion of bovine aortic lysyl oxidase followed by tandem mass spectrometry has enabled assignment of all five disulfide bonds. The results indicate that the enzyme has a very stable central core containing three disulfide bonds, the lysyl tyrosyl quinone cross-link and the copper. This core is well isolated from solvent with the result that the oxidized (normal) form of the enzyme is remarkably resistant to proteolysis and is unusually stable at high temperatures and in the presence of denaturants.
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http://dx.doi.org/10.1007/s00702-010-0560-yDOI Listing
July 2011

Purification of high yields of catalytically active lysyl oxidase directly from Escherichia coli cell culture.

Protein Expr Purif 2010 Nov 23;74(1):116-21. Epub 2010 Jun 23.

Department of Chemistry, Pomona College, Claremont, CA 91711, United States.

Lysyl oxidase is a highly insoluble enzyme requiring high concentrations of urea to solubilize. A method to obtain lysyl oxidase in high yields directly from an Escherichia coli culture without the need for refolding of inclusion bodies has been developed using nutrient rich media. pET21b was used to overexpress the lysyl oxidase enzyme and to introduce a C-terminal 6X histidine tag for purification. Lysyl oxidase yields of 10 mg of active and properly folded enzyme per liter of media have been obtained. Purification was achieved via affinity chromatography using a Ni-NTA column. Copper content was found to be 19%. LTQ cofactor formation in LOX is a self-processing event in the presence of copper. LTQ content was determined to be 24% based on reaction with phenylhydrazine to form a phenylhydrazone adduct. Quantification of this adduct was attained using the previously reported extinction coefficient of 15.4 mM(-1)cm(-1). LTQ presence was also verified by redox cycling. Specific enzymatic activity was measured to be 0.31 U/mg, one of the highest activities reported.
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http://dx.doi.org/10.1016/j.pep.2010.06.013DOI Listing
November 2010

Modeling Cu(II) binding to peptides using the extensible systematic force field.

Bioinorg Chem Appl 2010 11;2010:724210. Epub 2010 Mar 11.

Department of Chemistry, Emmanuel College, Boston, MA 02115, USA.

The utility of the extensible systematic force field (ESFF) was tested for copper(II) binding to a 34-amino-acid Cu(II) peptide, which includes five histidine residues and is the putative copper-binding site of lysyl oxidase. To improve computational efficiency, distance geometry calculations were used to constrain all combinations of three histidine ligands to be within bonding distance of the copper and the best results were utilized as starting structures for the ESFF computations. All likely copper geometries were modeled, but the results showed only a small dependence on the geometrical model in that all resulted in a distorted square pyramidal geometry about the copper, some of the imidazole rings were poorly oriented for ligation to the Cu(II), and the copper-nitrogen bond distances were too long. The results suggest that ESFF should be used with caution for Cu(II) complexes where the copper-ligand bonds have significant covalency and when the ligands are not geometrically constrained to be planar.
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http://dx.doi.org/10.1155/2010/724210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2837899PMC
June 2010

A C-terminal hydrophobic, solvent-protected core and a flexible N-terminus are potentially required for human papillomavirus 18 E7 protein functionality.

Biochimie 2010 Jul 6;92(7):901-8. Epub 2010 Mar 6.

Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China.

The oncogenic potential of the high-risk human papillomavirus (HPV) relies on the expression of genes specifying the E7 and E6 proteins. To investigate further the variation in oligomeric structure that has been reported for different E7 proteins, an HPV-18 E7 cloned from a Hispanic woman with cervical intraepithelial neoplasia was purified to homogeneity most probably as a stable monomeric protein in aqueous solution. We determined that one zinc ion is present per HPV-18 E7 monomer by amino acid and inductively coupled plasma-atomic emission spectroscopy analysis. Intrinsic fluorescence and circular dichroism spectroscopic results indicate that the zinc ion is important for the correct folding and thermal stability of HPV-18 E7. Hydroxyl radical mediated protein footprinting coupled to mass spectrometry and other biochemical and biophysical data indicate that near the C-terminus, the four cysteines of the two Cys-X(2)-Cys motifs that are coordinated to the zinc ion form a solvent inaccessible core. The N-terminal LXCXE pRb binding motif region is hydroxyl radical accessible and conformationally flexible. Both factors, the relative flexibility of the pRb binding motif at the N-terminus and the C-terminal metal-binding hydrophobic solvent-protected core, combine together and facilitate the biological functions of HPV-18 E7.
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http://dx.doi.org/10.1016/j.biochi.2010.02.028DOI Listing
July 2010

Biochemical, functional and structural characterization of Akbu-LAAO: a novel snake venom L-amino acid oxidase from Agkistrodon blomhoffii ussurensis.

Biochimie 2010 Apr 25;92(4):343-9. Epub 2010 Jan 25.

Department of Biotechnology, Dalian Medical University, Dalian, China.

An L-amino acid oxidase (Akbu-LAAO) was isolated from the venom of Agkistrodon blomhoffii ussurensis snake using DEAE Sephadex A-50 ion-exchange, Sephadex G-75 gel filtration, and high performance liquid chromatographies. The homogeneity and molecular mass of Akbu-LAAO were analyzed by SDS-PAGE and MALDI-TOF spectrometry. The sequences of ten peptides from Akbu-LAAO were established by HPLC-nESI-MS/MS analysis. Protein sequence alignment indicated that i) that Akbu-LAAO is a new snake venom LAAO, and ii) Akbu-LAAO shares homology with several LAAOs from the venoms of Calloselasma rhodost, Agkistrodon halys, Daboia russellii siamensis, and Trimeresurus stejnegeri. Akbu-LAAO is a homodimer with a molecular mass of approximately 124.4 kDa. It reacts optimally with its enzymatic substrate, Leu, at pH 4.7 with a K(m) of 2.1 mM. ICP-AES measurements showed that Akbu-LAAO contains four Zn(2+) per dimer that are unessential for the hydrolytic activity of the enzyme. The emission fluorescence intensity of Akbu-LAAO decreases by 61% on removal of Zn(2+) indicating that the zinc probably helps maintain the structural integrity of the enzyme. The addition of exogenous metal ions, including Mg(2+), Mn(2+), Ca(2+), Ce(3+), Nd(3+), Co(2+) and Tb(3+), increases the l-Leu hydrolytic activity of the enzyme. Akbu-LAAO shows apparent anti-aggregation effects on human and rabbit platelets. It exhibits a strong bacteriostasis effect on Staphylococcus aureus, eighteen fold that of cephalosporin C under the same conditions. Taken together, the biochemical, proteomic, structural and functional characterizations reveal that Akbu-LAAO is a novel LAAO with promise for biotechnological and medical applications.
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http://dx.doi.org/10.1016/j.biochi.2010.01.013DOI Listing
April 2010

A novel phospholipase A2 from Agkistrodon blomhoffii ussurensis venom: purification, proteomic, functional and structural characterizations.

Biochimie 2009 Apr 6;91(4):558-67. Epub 2009 Feb 6.

Department of Biotechnology, Dalian Medical University, Dalian, 116044, China.

A phospholipase A(2) was isolated from the snake venom of Chinese Agkistrodon blomhoffii Ussurensis by column chromatography using DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration chromatography and Mono Q ion-exchange chromatography, and designated as Akbu-PLA(2). It showed an average molecular mass of 13,980+/-3 amu determined by MALDI TOF mass spectrometry. Protein identification results from HPLC-nESI-MS/MS analysis indicated that the Akbu-PLA(2) was a new snake venom acidic PLA(2). Seven peptides were sequenced from Akbu-PLA(2) by HPLC-nESI-MS/MS analysis. Sequencing alignment indicated that Akbu-PLA(2) shared homolog peptides of phospholipases A(2) from the venoms of Gloydius ussurensis, Gloydius halys, Gloydius halys (halys viper), Deinagkistrodon acutus and Agkistrodon halys Pallas. Akbu-PLA(2) has an optimum hydrolytic activity temperature of approximately 45 degrees C. The intrinsic fluorescences of Tyr and Trp residues of Akbu-PLA(2) showed emission wavelengths red-shifted by 13.6 and 1.6 nm from those of free Tyr and Trp, respectively. Akbu-PLA(2) was shown to contain one Ca(2+) per monomer by ICP-AES measurement. The Ca(2+) ion was found to be critical for both the hydrolytic activity and the structure of Akbu-PLA(2). Ca(2+) increased the emission fluorescence intensity and the hydrophobicity of the environment of Akbu-PLA(2). The hydrolytic activity of Akbu-PLA(2) was accelerated due to the addition of Ca(2+) ion by enhancing the substrate binding. However, a protein component with the molecular weight two-fold relative to that of Akbu-PLA(2) was found to be difficult to eliminate for the purification of Akbu-PLA(2). HPLC-nESI-MS/MS detected the same peptides from it as from Abku-PLA(2), which indicated that it should be a homodimer of Akbu-PLA(2). A proteomic approach, 2D SDS-PAGE coupled to HPLC-nESI-MS/MS, supported the co-existence of the Akbu-PLA(2) monomer and dimer in the crude snake venom. Results from the combination of phosphoprotein and glycoprotein specific stains combined with the HPLC-nESI-MS/MS method indicated that both the Akbu-PLA(2) monomer and dimer were both phosphorylated and glycosylated. The addition of exogenous Ca(2+) ion was found to be able to promote the dimer formation of Akbu-PLA(2). We conclude that a novel PLA(2) was successfully obtained. The systemically biochemical, proteomic, structural and functional characterization results from Akbu-PLA(2) reveal new threads and provide valuable inputs for the study of snake venom phospholipases A(2).
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http://dx.doi.org/10.1016/j.biochi.2009.01.013DOI Listing
April 2009

A novel serine protease from the snake venom of Agkistrodon blomhoffii ussurensis.

Toxicon 2008 Dec 9;52(7):760-8. Epub 2008 Sep 9.

Department of Biochemistry, Dalian Medical University, Dalian 116044, PR China.

A novel serine protease, ABUSV-SPase, was isolated to homogeneity for the first time from Chinese Agkistrodon blomhoffii ussurensis snake venom, and its enzymatic and structural properties were characterized by multiple techniques. ABUSV-SPase is a stable monomeric protein with a molecular mass of 26,752.6a.m.u. It reacts optimally with its substrate Nalpha-tosyl-l-arginine methyl ester (TAME) at pH 7.0 and 41 degrees C. ESI-MS/MS analysis indicates that ABUSV-SPase is a new serine protease, sharing peptide homologies with various snake venom serine proteases, especially the snake venom thrombin-like enzymes of this group, and serine protease precursors. It is a zinc-containing protein, and although zinc is not essential for activity, its replacement by various divalent metal ions, including Mg2+, Mn2+, and Ca2+, increases the TAME hydrolysis activity of the enzyme. The intrinsic fluorescences of Tyr and Trp residues of ABUSV-SPase have emission wavelengths red-shifted by 12.8nm and 3.6nm from those of free Tyr and Trp, respectively. The zinc ion increases the hydrophobicity of the environment of the Trp residues, increases the thermostability of the protein, and affects the protein secondary structure to stabilize the enzyme, but appears to have no direct role in its esterase hydrolysis activity.
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http://dx.doi.org/10.1016/j.toxicon.2008.08.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845635PMC
December 2008

High-performance liquid chromatography/nano-electrospray ionization tandem mass spectrometry, two-dimensional difference in-gel electrophoresis and gene microarray identification of lymphatic metastasis-associated biomarkers.

Rapid Commun Mass Spectrom 2008 Oct;22(20):3172-8

Department of Biochemistry & Molecular Biology, Dalian Medical University, Dalian 116044, PR China.

The potential biomarkers for the lymphatic metastatic process of mouse hepatocarcinoma were investigated by using two-dimensional difference in-gel electrophoresis (2D DIGE), high-performance liquid chromatography/nano-electrospray ionization tandem mass spectrometry (HPLC/nESI-MS/MS) and GeneChip. 2D DIGE was performed to screen and quantify the differentially expressed proteins between two well-established mouse hepatocarcinoma cell lines, Hca-F with 75% and Hca-P with 25% metastasis rate of lymph node potentials. The protein spots in the gel were visualized by the highly sensitive Deep Purple (GE Healthcare) fluorescent stain. Protein identification was obtained for gel spots by HPLC/nESI-MS/MS analysis with high quality. GeneChip microarray was performed to identify genes differentially expressed at the mRNA level. Seventeen genes including the chloride intracellular channel l, caspase 3, fructose bisphosphatase 2, glutamate dehydrogenase 1, V-crk sarcoma virus CT10 oncogene homolog, N-myc downstream regulated gene1, villin2, gelsolin, enoyl coenzyme A hydratase 1, transketolase, vimentin, annexins A5 and A7, keratin complex2 basic gene7 and gene8, lactamase (bata 2) and Ero1-like protein were found abnormally regulated and expressed concordantly both at the protein and mRNA levels between the two cell lines. More than half of these genes were for the first time revealed to be involved directly in hepatocarcinoma due to the lymphatic metastasis. The interdisciplinary combination of HPLC/nESI-MS/MS with 2D DIGE and GeneChip techniques opens up the possibility for the biomarker discovery of disease with high confidence.
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http://dx.doi.org/10.1002/rcm.3725DOI Listing
October 2008

A novel plasminogen activator from Agkistrodon blomhoffii Ussurensis venom (ABUSV-PA): purification and characterization.

Biochem Biophys Res Commun 2006 Oct 7;348(4):1279-87. Epub 2006 Aug 7.

Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, Liaoning 116027, China.

A plasminogen activator with arginine ester hydrolysis activity (ABUSV-PA) has been identified and purified to homogeneity from Chinese Agkistrodon blomhoffii Ussurensis snake venom. ABUSV-PA, a monomeric protein with molecular mass of 27815.2 Da, was purified 180-fold with 0.02% recovery for protein and 3.6% recovery for esterase activity. ABUSV-PA reacts optimally with its substrate N(alpha)-tosyl-l-arginine-methyl ester (TAME) at approximately pH 7.5 and at 51 degrees C. Measurement from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveals that ABUSV-PA is a Zn(2+)-containing protein with a stoichiometry of 1:1 [Zn(2+)]:[ABUSV-PA]. Analyses of esterase hydrolysis and UV absorption and CD spectra indicate that Zn(2+) plays an important role in maintaining the structural integrity rather than the esterase activity of ABUSV-PA. Divalent metal ions, including Ca(2+), Mg(2+), Cu(2+), Ni(2+), Mn(2+), and Co(2+), increase the TAME hydrolysis activity of ABUSV-PA. A red-shift of the emission wavelengths of the synchronous fluorescence of ABUSV-PA, compared to those of free Tyr and Trp, indicates a conformation where the Tyr and Trp residues are in exposed hydrophilic environments. The presence of zinc increases the hydrophobicity of the conformational environments surrounding the Trp residues of ABUSV-PA and affects the secondary structure of ABUSV-PA, as proved by UV absorption and CD spectroscopy.
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http://dx.doi.org/10.1016/j.bbrc.2006.07.183DOI Listing
October 2006

Characterization of a fibrinolytic enzyme (ussurenase) from Agkistrodon blomhoffii ussurensis snake venom: insights into the effects of Ca2+ on function and structure.

Biochim Biophys Acta 2006 Aug 27;1764(8):1340-8. Epub 2006 Jun 27.

Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, Dalian, 116027, China.

Fibrino(geno)lytic enzymes from snake venoms have been identified as high quality therapeutic agents for treatment of blood clots and strokes. They act on fibrinogen and fibrin, leading to defibrinogenation of blood, lysis of fibrin, and a consequent decrease in blood viscosity. In this work, a fibrinolytic enzyme (ussurenase) from China Agkistrodon blomhoffii Ussurensis snake venom, was purified to homogeneity, identified as a stable 23,367.8 Da monomeric protein, and was identified as a new kind of snake venom metalloproteinase. Ussurenase reacts optimally with fibrin clots at pH 7.5-8.3 and a temperature of 33-41 degrees C. Although many fibrinolytic enzymes are known to be zinc-dependent, measurements from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveal that ussurenase is a Ca2+-containing protein with a molar ratio of 1:1 ([Ca2+]:[enzyme]). Ca2+ is crucial to the fibrin clot hydrolysis by ussurenase but also plays an important role in maintaining the structural integrity of the enzyme. The addition of Ca2+ to the apoenzyme induces a conformational change making the environments surrounding the Trp residues of the enzyme more hydrophobic. The presence of Ca2+ also increases the structural stability of ussurenase, so that higher concentrations of the denaturant guanidine hydrochloride are required to denature the native ussurenase compared to the apo-form. UV absorption and CD spectroscopy experiments show that Ca2+ increases the thermostability and changes the secondary structure of ussurenase. All these data suggest that Ca2+ is crucial for the correct folding and activity of ussurenase.
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http://dx.doi.org/10.1016/j.bbapap.2006.06.003DOI Listing
August 2006

Low-temperature (180 K) crystal structures of tetrakis-mu-(niflumato)di(aqua)dicopper(II) N,N-dimethylformamide and N,N-dimethylacetamide solvates, their EPR properties, and anticonvulsant activities of these and other ternary binuclear copper(II)niflumate complexes.

J Inorg Biochem 2005 Feb;99(2):355-67

Laboratoire de Chimie Générale, Faculté de Médecine et de Pharmacie, Poitiers, France.

Two pseudopolymorphs, solvates, of [Cu(2)(II)(niflumate)(4)(H(2)O)(2)] of unknown structure were obtained following solution of [Cu(2)(II)(niflumate)(4)(H(2)O)(2)] in N,N-dimethylacetamide (DMA) or N,N-dimethylformamide (DMF). Low-temperature crystal structures obtained for these solvates revealed that they were ternary aqua DMA and DMF solvates: [Cu(2)(II)(niflumate)(4)(H(2)O)(2)].4DMA and [Cu(2)(II)(niflumate)(4)(H(2)O)(2)].4DMF. Intermolecular hydrogen bonding interactions account for the formation of these stable DMA and DMF solvates. These pseudopolymorphs contain a centrosymmetric binuclear center with Cu-Cu bond distances ranging from 2.6439(7) to 2.6452(9) A; the coordination sphere of Cu(II) is characterized by one long Cu-O (water) bond length of 2.128(3)-2.135(3) A and four short Cu-O (carboxylate) bonds of 1.949(3)-1.977(3) A. Crystal parameters for the DMA pseudopolymorph: a=10.372(1), b=19.625(2), c=17.967(2) A, beta=97.40(1) degrees , V=3626.8(6) A(3); monoclinic system; space group: P2(1)/a and for the DMF pseudopolymorph: a=10.125(2), b=18.647(3), c=19.616(4) A, alpha=74.38(2)(o), beta=88.18(2)(o), gamma=79.28(2)(o), V=3504(1) A(3); triclinic system; space group: P1. EPR spectra of these solids are identical and show strong antiferromagnetic coupling between the copper atoms, similar to the spectrum obtained for [Cu(2)(II)(niflumate)(4)(DMSO)(2)]. The [Cu(2)(II)(niflumate)(4)(H(2)O)(2)], [Cu(2)(II)(niflumate)(4)(H(2)O)(2)].4DMA, [Cu(2)(II)(niflumate)(4)(H(2)O)(2)].4DMF, [Cu(2)(II)(niflumate)(4)(DMF)(2)], and[Cu(2)(II)(niflumate)(4)(DMSO)(2)] evidenced protection against maximal electroshock-induced seizures and Psychomotor seizures at various times after treatment, consistent with the well known antiinflammatory activities of Cu chelates, but failed to protect against Metrazol-induced seizures while evidencing some Rotorod Toxicity consistent with a mechanism of action involving sedative activity.
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http://dx.doi.org/10.1016/j.jinorgbio.2004.10.001DOI Listing
February 2005

Synthesis, crystal structures, and anti-convulsant activities of ternary [Zn(II)(3,5-diisopropylsalicylate)(2)], [Zn(II)(salicylate)(2)] and [Zn(II)(aspirinate)(2)] complexes.

J Inorg Biochem 2004 Nov;98(11):1734-49

Laboratoire de Cristallographie et RMN biologiques, UMR CNRS 8015, Université René Descartes, Paris, France.

Following observations that bis(3,5-diisopropylsalicylato)diaquazinc(II), [Zn(II)(3,5-DIPS)(2)(H(2)O)(2)], had anti-convulsant activity, bis(acetylsalicylate)diaquazinc(II), [Zn(II)(aspirinate)(2)(H(2)O)(2)], and the Zn(II) ternary 1,10-phenanthroline (phen), 2,9-dimethyl-1,10-phenanthroline (neocuproine, NC) or dimethyl sulfoxide (DMSO) complexes of Zn(II)3,5-diisopropylsalicylate, salicylate, and acetylsalicylate were synthesized and spectroscopically characterized. Anti-convulsant and Rotorod toxicity activities of these complexes were determined to examine their anti-convulsant and undesirable central nervous stimulant or depressant activities of these Zn(II) non-steroidal anti-inflammatory agent complexes. Bis(3,5-diisopropylsalicylato)-1,10-phenanthorlinezinc(II), [Zn(II)(3,5-DIPS)(2)(phen)], (1) has one bidentate phen ligand and two mono-deprotonated 3,5-DIPS ligands. One of the carboxylates bonds in an asymmetric chelating mode. The Zn(II) atom exhibits a distorted bicapped rectangular pyramidal environment N(2)O(2)OO (4+1+1 *). In the neocuproine complex, bis(3,5-diisopropylsalicylato)-2,9-dimethyl-1,10-phenanthorlinezinc(II), [Zn(II)(3,5-DIPS)(2)(NC)] (2), the Zn(II) atom has a much more distorted bicapped rectangular pyramidal environment, N(2)O(2)O(2) (4+2 *), compared to 1. The two carboxylate ligands exhibit the same asymmetric coordinating mode with longer metalloelement-oxygen bond distances compared to 1. The space group of [Zn(II)(aspirinate)(2)(H(2)O)(2)] (3), which has been reported as Cc is corrected to C2/c. The zinc atom exhibits a (4+2 *) bicapped square pyramidal environment. While the two ternary phenanthroline-containing complexes, 1 and 2, evidenced weak protection against maximal electroshock (MES)- and subcutaneous Metrazol (scMET) induced seizures, [Zn(II)(3,5-DIPS)(2)(DMSO)(2)], [Zn(II)(aspirinate)(2)(H(2)O)(2)], and bis(salicylato)-1,10-phenanthorlinezinc(II), [Zn(II)(salicylate)(2)(phen)], were found to be particularly useful in protecting against MES and scMET seizures and [Zn(II)(aspirinate)(2)(H(2)O)(2)] and [Zn(II)(salicylate)(2)(phen)] were found to have activity in protecting against Psychomotor seizures, without causing Rotorod toxicity. Activities of these and other Zn(II) complexes of non-steroidal anti-inflammatory agents are consistent with the well-known anti-inflammatory responses of Zn(II)-dependent enzymes. There was also some evidence of Rotorod toxicity consistent with a mechanism of action involving sedative-hypnotic activity of recognized anti-epilepticdrugs.
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http://dx.doi.org/10.1016/j.jinorgbio.2004.07.010DOI Listing
November 2004

A peptide model of the copper-binding region of lysyl oxidase.

J Inorg Biochem 2004 Aug;98(8):1427-35

Department of Chemistry, Emmanuel College, Boston, MA 02115, USA.

The copper-binding site of lysyl oxidase remains extremely poorly characterized and although models have been suggested for copper(II) coordination by three histidine ligands, as has been found for other copper-containing amine oxidases, there has been no experimental confirmation of these suggestions. In this work, two synthetic peptides with 24 and 34-amino acid residues, respectively, were chosen from the highly conserved histidine-rich sequence previously suggested as the copper-binding region of lysyl oxidase. These peptides each bind one equivalent of Cu(II), at the same site in the two peptides. Spectroscopic (NMR, electron paramagnetic resonance (EPR), CD, visible absorption and fluorescence) techniques were employed to investigate the nature of the resulting complexes. The results indicate that at neutral pH three histidine ring nitrogen atoms and one carboxylate oxygen atom coordinate as the in-plane ligands of the copper, which is in an approximately tetragonally-distorted octahedral geometry. Modeling of the copper-peptides using the consistent force field (CFF91) produces a minimum energy configuration with three histidines and one water molecule as the copper ligands. CD, EPR and fluorescence results are reported for lysyl oxidase and compared with results for the peptides.
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http://dx.doi.org/10.1016/j.jinorgbio.2004.04.010DOI Listing
August 2004

Low-temperature (180 K) crystal structure, electron paramagnetic resonance spectroscopy, and propitious anticonvulsant activities of CuII2(aspirinate)4(DMF)2 and other CuII2(aspirinate)4 chelates.

J Inorg Biochem 2003 Aug;96(2-3):375-85

Laboratoire de Chimie Générale, Faculté de Médecine et de Pharmacie, Poitiers, France.

The purpose of this research was to characterize by X-ray crystallography the ternary dimethylformamide (DMF) Cu(II) complex of acetylsalicylic acid (aspirin), in an effort to compare the structure-activity relationships for the anticonvulsant activity of this and other Cu(II)aspirinate chelates. The ternary DMF Cu(II) complex of aspirin was synthesized and crystals grown from a DMF solution were characterized by single crystal X-ray diffraction. This crystalline material was analyzed for anticonvulsant activity in the Maximal Electroshock (MES) Grand Mal and subcutaneous Metrazol (scMET) Petit Mal models of seizure used to detect anticonvulsant activity. The ternary DMF complex was found to be a monomolecular binuclear complex, tetrakis-mu-(acetylsalicylato)bis(dimethylformamido)dicopper(II) [Cu(II)(2)(aspirinate)(4)(DMF)(2)] with the following parameters: monoclinic, space group P2(1)/n, a=12.259 (1), b=10.228 (1), c=16.987 (1) A, beta=92.07 (1) degrees; V=2128.5 (3) A(3); Z=2. The structure was determined at 180 K from 2903 unique reflections (I>1sigma(I)) to the final values of R=0.030 and wR=0.033 using F. This binuclear complex contains four acetylsalicylate bridging ligands which are related to each other in a two by two symmetry center. The four nearest O atoms around each Cu atom form a closely square planar arrangement with the square pyramidal coordination completed by the dimethylformamide oxygen atom occupying an apical position at a distance of 2.154 (1) A. Each Cu atom is displaced towards the DMF ligand by 0.187 A from the plane of the four O atoms. Electron paramagnetic resonance (EPR) spectra of [Cu(II)(2)(aspirinate)(4)(DMF)(2)] crystals show a strong antiferromagnetic coupling of the copper atoms, similar to that observed with other binuclear copper(II)salicylate compounds. Studies used to detect anticonvulsant activity revealed that [Cu(II)(2)(aspirinate)(4)(DMF)(2)] was an effective anticonvulsant in the MES model of seizure but ineffective against scMET-induced seizures. The monomolecular ternary binuclear [Cu(II)(2)(aspirinate)(4)(DMF)(2)] complex is more effective in inhibiting MES-induced seizures than other binuclear or mononuclear Cu(II) chelates of aspirin including: binuclear polymeric [Cu(II)(2)(aspirinate)(4)], [Cu(II)(2)(aspirinate)(4)(H(2)O)], which is anticipated to be less polymeric, and monomolecular ternary [Cu(II)(2)(aspirinate)(4)(DMSO)(2)] and [Cu(II)(aspirinate)(2)(Pyr)(2)]. These and other chelates appear to be more effective in the scMET model of seizure than [Cu(II)(2)(aspirinate)(4)(DMF)(2)]. These structure-activity relationships support the potential efficacy of Cu chelates of aspirin in treating epilepsies.
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http://dx.doi.org/10.1016/s0162-0134(03)00153-3DOI Listing
August 2003

Synthesis, crystal structure, EPR properties, and anti-convulsant activities of binuclear and mononuclear 1,10-phenanthroline and salicylate ternary copper(II) complexes.

J Inorg Biochem 2002 Apr;89(1-2):18-28

Laboratoire de Cristallographie et de Résonance Magnétique Nucléaire Biologiques UMR 8015, Faculté de Pharmacie Paris V, 75006, Paris, France.

Two ternary Cu(II) complexes of 1,10-phenanthroline (phen) and singly (Hsal(-)) or dideprotonated (sal(2-)) salicylate ligands were synthesized, their X-ray crystal structure and electron paramagnetic resonance spectral characteristics determined, and evaluated for anti-convulsant activities in the maximal electroshock (MES) and Metrazol models of seizure and Rotorod toxicity. The X-ray crystal structure of [bis(1,10-phenanthroline)-mu-bis(salicylato-O,O')dicopper(II)] dihydrate, 1, ([Cu(II)(2)(phen)(2)(sal)(2)].2[H(2)O]), shows it to be binuclear. This dimer consists of two centrosymmetrically related pseudo-five coordinate Cu(II) atoms 3.242(2) A apart and bridged by two dideprotonated salicylate ligands. The X-ray crystal structure of [bis(1,10-phenanthroline)(salicylato)copper(II)][salicylate] monohydrate, 2, ([Cu(II)(phen)(2)(Hsal)](+)[Hsal](-)[H(2)O]), shows it to be mononuclear. This complex cation exhibits a highly irregular distorted square pyramidal geometry about the Cu(II) atom, (4+1+1*). Each salicylate is singly deprotonated and one of them is ligand bonded in an asymmetric chelating mode. EPR results for 2 indicate that in concentrated DMF solution phen remains bonded to copper but salicylate is likely monodentate in contrast to the situation for 1. However, in dilute DMF solution, both 1 and 2 form the same species, which accounts for the similarity in anti-convulsant activity of the two compounds. Both 1 and 2 were found to be effective in preventing MES-induced seizures and ineffective in preventing Metrazol-induced seizures. Rotorod toxicity, consistent with central nervous system depression, paralleled the observed anti-convulsant activity. It is suggested that the observed anti-convulsant activity is consistent with central nervous system depression as a physiological mechanism in overcoming MES-induced seizures due to MES-induced brain inflammatory disease.
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http://dx.doi.org/10.1016/s0162-0134(01)00324-5DOI Listing
April 2002