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1.
Int J Mol Sci ; 21(6)2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32244989

RESUMO

The Center of Biomedical Research Excellence in Matrix Biology strives to improve our understanding of extracellular matrix at molecular, cellular, tissue, and organismal levels to generate new knowledge about pathophysiology, normal development, and regenerative medicine. The primary goals of the Center are to i) support junior investigators, ii) enhance the productivity of established scientists, iii) facilitate collaboration between both junior and established researchers, and iv) build biomedical research infrastructure that will support research relevant to cell-matrix interactions in disease progression, tissue repair and regeneration, and v) provide access to instrumentation and technical support. A Pilot Project program provides funding to investigators who propose applying their expertise to matrix biology questions. Support from the National Institute of General Medical Sciences at the National Institutes of Health that established the Center of Biomedical Research Excellence in Matrix Biology has significantly enhanced the infrastructure and the capabilities of researchers at Boise State University, leading to new approaches that address disease diagnosis, prevention, and treatment. New multidisciplinary collaborations have been formed with investigators who may not have previously considered how their biomedical research programs addressed fundamental and applied questions involving the extracellular matrix. Collaborations with the broader matrix biology community are encouraged.

2.
Mar Drugs ; 17(12)2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31795126

RESUMO

KTM is a 16 amino acid peptide with the sequence WCCSYPGCYWSSSKWC. Here, we present the nuclear magnetic resonance (NMR) structure and bioactivity of this rationally designed α-conotoxin (α-CTx) that demonstrates potent inhibition of rat α3ß2-nicotinic acetylcholine receptors (rα3ß2-nAChRs). Two bioassays were used to test the efficacy of KTM. First, a qualitative PC12 cell-based assay confirmed that KTM acts as a nAChR antagonist. Second, bioactivity evaluation by two-electrode voltage clamp electrophysiology was used to measure the inhibition of rα3ß2-nAChRs by KTM (IC50 = 0.19 ± 0.02 nM), and inhibition of the same nAChR isoform by α-CTx MII (IC50 = 0.35 ± 0.8 nM). The three-dimensional structure of KTM was determined by NMR spectroscopy, and the final set of 20 structures derived from 32 distance restraints, four dihedral angle constraints, and two disulfide bond constraints overlapped with a mean global backbone root-mean-square deviation (RMSD) of 1.7 ± 0.5 Å. The structure of KTM did not adopt the disulfide fold of α-CTx MII for which it was designed, but instead adopted a flexible ribbon backbone and disulfide connectivity of C2-C16 and C3-C8 with an estimated 12.5% α-helical content. In contrast, α-CTx MII, which has a native fold of C2-C8 and C3-C16, has an estimated 38.1% α-helical secondary structure. KTM is the first reported instance of a Framework I (CC-C-C) α-CTx with ribbon connectivity to display sub-nanomolar inhibitory potency of rα3ß2-nAChR subtypes.

3.
Toxins (Basel) ; 11(12)2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31757080

RESUMO

A pheochromocytoma of the rat adrenal medulla derived (a.k.a. PC12) cell-based assay for dopamine measurement by luminescence detection was customized for the qualitative evaluation of agonists and antagonists of nicotinic acetylcholine receptors (nAChRs). The assay mechanism begins with ligand binding to transmembrane nAChRs, altering ion flow into the cell and inducing dopamine release from the cell. Following release, dopamine is oxidized by monoamine oxidase generating hydrogen peroxide that catalyzes a chemiluminescence reaction involving luminol and horseradish peroxidase, thus producing a detectable response. Results are presented for the action of nAChR agonists (acetylcholine, nicotine, and cytisine), and antagonists (α-conotoxins (α-CTxs) MII, ImI, LvIA, and PeIA) that demonstrate a luminescence response correlating to the increase or decrease of dopamine release. A survey of cell growth and treatment conditions, including nerve growth factor, nicotine, ethanol, and temperature, led to optimal assay requirements to achieve maximal signal intensity and consistent response to ligand treatment. It was determined that PC12 cells treated with a combination of nerve growth factor and nicotine, and incubated at 37 °C, provided favorable results for a reduction in luminescence signal upon treatment of cells with α-CTxs. The PC12 assay is intended for use as a fast, efficient, and economic qualitative method to assess the bioactivity of molecules that act on nAChRs, in which testing of ligand-nAChR binding hypotheses and computational predictions can be validated. As a screening method for nAChR bioactivity, lead compounds can be assessed for their likelihood of exhibiting desired bioactivity prior to being subjected to more complex quantitative methods, such as electrophysiology or live animal studies.

4.
Fitoterapia ; 137: 104281, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31381957

RESUMO

Veratrum californicum is a rich source of steroidal alkaloids, many of which have proven to be antagonists of the Hedgehog (Hh) signaling pathway that becomes aberrant in over twenty types of cancer. These alkaloids first became known in the 1950's due to their teratogenic properties, which resulted in newborn and fetal lambs developing cyclopia as a result of pregnant ewes consuming Veratrum californicum. It was discovered that the alkaloids in V. californicum were concentrated in the root and rhizome of the plant with much lower amounts of the most active alkaloid, cyclopamine, present in the aerial plant, especially in the late growth season. Inspired by the limitations in analytical instrumentation and methods available to researchers at the time of the original investigation, we have used state-of-the-art instrumentation and modern analytical methods to quantitate four steroidal alkaloids based on study parameters including plant part, harvest location, and growth stage. The results of the current inquiry detail differences in alkaloid composition based on the study parameters, provide a detailed assessment for alkaloids that have been characterized previously (cyclopamine, veratramine, muldamine and isorubijervine), and identify at least six alkaloids that have not been previously characterized. This study provides insight into optimal harvest time, plant growth stage, harvest location, and plant part required to isolate, yet to be characterized, alkaloids of interest for exploration as Hh pathway antagonists with desirable medicinal properties.


Assuntos
Alcaloides/química , Esteroides/química , Veratrum/química , Alcaloides/isolamento & purificação , Proteínas Hedgehog/antagonistas & inibidores , Idaho , Estrutura Molecular , Compostos Fitoquímicos/química , Compostos Fitoquímicos/isolamento & purificação , Componentes Aéreos da Planta/química , Rizoma/química , Estações do Ano , Esteroides/isolamento & purificação , Alcaloides de Veratrum
5.
Toxins (Basel) ; 11(2)2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30781866

RESUMO

Nicotinic acetylcholine receptors (nAChRs) are found throughout the mammalian body and have been studied extensively because of their implication in a myriad of diseases. α-Conotoxins (α-CTxs) are peptide neurotoxins found in the venom of marine snails of genus Conus. α-CTxs are potent and selective antagonists for a variety of nAChR isoforms. Over the past 40 years, α-CTxs have proven to be valuable molecular probes capable of differentiating between closely related nAChR subtypes and have contributed greatly to understanding the physiological role of nAChRs in the mammalian nervous system. Here, we review the amino acid composition and structure of several α-CTxs that selectively target nAChR isoforms and explore strategies and outcomes for introducing mutations in native α-CTxs to direct selectivity and enhance binding affinity for specific nAChRs. This review will focus on structure-activity relationship studies involving native α-CTxs that have been rationally mutated and molecular interactions that underlie binding between ligand and nAChR isoform.


Assuntos
Conotoxinas/genética , Conotoxinas/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Animais , Humanos , Mutagênese , Mutação , Conformação Proteica , Relação Estrutura-Atividade
6.
Molecules ; 23(9)2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30200443

RESUMO

Veratrum californicum is a rich source of steroidal alkaloids such as cyclopamine, a known inhibitor of the Hedgehog (Hh) signaling pathway. Here we provide a detailed analysis of the alkaloid composition of V. californicum by plant part through quantitative analysis of cyclopamine, veratramine, muldamine and isorubijervine in the leaf, stem and root/rhizome of the plant. To determine whether additional alkaloids in the extracts contribute to Hh signaling inhibition, the concentrations of these four alkaloids present in extracts were replicated using commercially available standards, followed by comparison of extracts to alkaloid standard mixtures for inhibition of Hh signaling using Shh-Light II cells. Alkaloid combinations enhanced Hh signaling pathway antagonism compared to cyclopamine alone, and significant differences were observed in the Hh pathway inhibition between the stem and root/rhizome extracts and their corresponding alkaloid standard mixtures, indicating that additional alkaloids present in these extracts are capable of inhibiting Hh signaling.


Assuntos
Alcaloides/farmacologia , Proteínas Hedgehog/metabolismo , Transdução de Sinais/efeitos dos fármacos , Veratrum/química , Animais , Biomassa , Camundongos , Células NIH 3T3 , Extratos Vegetais/farmacologia , Folhas de Planta/química , Raízes de Plantas/química , Caules de Planta/química , Padrões de Referência
7.
Toxins (Basel) ; 9(9)2017 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-28906461

RESUMO

Nine publically available biosafety protocols for safely handling conotoxin peptides were tested to evaluate their decontamination efficacy. Circular dichroism (CD) spectroscopy and mass spectrometry (MS) were used to assess the effect of each chemical treatment on the secondary and primary structure of α-CTx MII (L10V, E11A). Of the nine decontamination methods tested, treatment with 1% (m/v) solution of the enzymatic detergent Contrex™ EZ resulted in a 76.8% decrease in α-helical content as assessed by the mean residue ellipticity at 222 nm, and partial peptide digestion was demonstrated using high performance liquid chromatography mass spectrometry (HPLC-MS). Additionally, treatment with 6% sodium hypochlorite (m/v) resulted in 80.5% decrease in α-helical content and complete digestion of the peptide. The Contrex™ EZ treatment was repeated with three additional α-conotoxins (α-CTxs), α-CTxs LvIA, ImI and PeIA, which verified the decontamination method was reasonably robust. These results support the use of either 1% Contrex™ EZ solution or 6% sodium hypochlorite in biosafety protocols for the decontamination of α-CTxs in research laboratories.


Assuntos
Conotoxinas , Descontaminação/métodos , Cromatografia Líquida de Alta Pressão , Contenção de Riscos Biológicos/normas , Espectrometria de Massas
8.
Bioorg Med Chem ; 24(16): 3752-7, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27338657

RESUMO

Veratrum californicum, commonly referred to as corn lily or Californian false hellebore, grows in high mountain meadows and produces the steroidal alkaloid cyclopamine, a potent inhibitor of the Hedgehog (Hh) signaling pathway. The Hh pathway is a crucial regulator of many fundamental processes during vertebrate embryonic development. However, constitutive activation of the Hh pathway contributes to the progression of various cancers. In the present study, a direct correlation was made between the extraction efficiency for cyclopamine from root and rhizome by eight methods, and the associated biological activity in Shh-Light II cells using the Dual-Glo® Luciferase Assay System. Alkaloid recovery ranged from 0.39 to 8.03mg/g, with ethanol soak being determined to be the superior method for obtaining biologically active cyclopamine. Acidic ethanol and supercritical extractions yielded degraded or contaminated cyclopamine with lower antagonistic activity towards Hh signaling.


Assuntos
Alcaloides de Veratrum/farmacologia , Veratrum/química , Biomassa , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Transdução de Sinais/efeitos dos fármacos , Alcaloides de Veratrum/isolamento & purificação
9.
Nat Prod Commun ; 11(9): 1215-1216, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30807002

RESUMO

Devil's claw is a common ingredient in nutraceutical products for the treatment of inflammation due to arthritis. The secondary root extract of Harpagophymn piocumbens contains bioactive iridoid glycosides known as harpagosides. Recent scrutiny of the nutraceutical industry claims that products listing devil's claw on their labels should refer only to H. procumbens, while the closely related, and less expensive, H. zeyheri is not to be classified as devil's claw. .This assertion is in contrast to botanists who claim that either species of Harpagophytum can be generically referred to as devil's claw. The current research aimed to determine the chemical composition of extracts from H. procumbens and H. zeyheri, with the intent to identify whether the bioactive harpagosides were similarly present between species, and how their presence resembled or deviated from commercially available H. procumbens extracts commonly used in -nutraceutical products. A microwave extraction followed by high performance liquid chromatography analysis of root samples from botanical specimens of H. procimbens and H. zeyheri identified similar quantities of harpagoside, regardless of species. The chemical composition between root extracts for each.species was found to contain varying quantities of non-harpagoside constituents, however their harpagoside content was comparable. These findings are intended to inform policymakers, nutraceutical manufacturers, and the general public of the distinction between myth and reality regarding devil's claw supplements.


Assuntos
Glicosídeos/isolamento & purificação , Harpagophytum/química , Extratos Vegetais/química , Piranos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Suplementos Nutricionais , Harpagophytum/classificação , Raízes de Plantas/química , Especificidade da Espécie
10.
Biochem Mol Biol Educ ; 44(1): 63-7, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26537635

RESUMO

Computational molecular docking is a fast and effective in silico method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The DockoMatic tutorial described herein provides a framework by which instructors can guide students through a drug screening exercise. Using receptor models derived from readily available protein crystal structures, docking programs have the ability to predict ligand binding properties, such as preferential binding orientations and binding affinities. The use of computational studies can significantly enhance complimentary wet chemical experimentation by providing insight into the important molecular interactions within the system of interest, as well as guide the design of new candidate ligands based on observed binding motifs and energetics. In this laboratory tutorial, the graphical user interface, DockoMatic, facilitates docking job submissions to the docking engine, AutoDock 4.2. The purpose of this exercise is to successfully dock a 17-amino acid peptide, α-conotoxin TxIA, to the acetylcholine binding protein from Aplysia californica-AChBP to determine the most stable binding configuration. Each student will then propose two specific amino acid substitutions of α-conotoxin TxIA to enhance peptide binding affinity, create the mutant in DockoMatic, and perform docking calculations to compare their results with the class. Students will also compare intermolecular forces, binding energy, and geometric orientation of their prepared analog to their initial α-conotoxin TxIA docking results.


Assuntos
Bioquímica/educação , Biologia Computacional , Peptídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Estudantes , Sítios de Ligação , Simulação de Acoplamento Molecular
11.
Br J Oral Maxillofac Surg ; 50(6): 500-3, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21940078

RESUMO

Appropriate fluid balance is an important factor in the survival of free flaps, and recently there has been a shift towards more conservative fluid regimens. Several surgical specialties have made extensive use of the relatively non-invasive method of measuring cardiac output (CO) to optimise fluid balance during and after surgery, which has resulted in a shorter hospital stay, but little has been published in head and neck surgery. To ascertain its use in the head and neck we sent a postal questionnaire to the anaesthetic departments of 40 major head and neck units identified from the 2010 database of the British Association of Oral and Maxillofacial Surgeons (BAOMS). Questions were asked about the number of free flaps done in the unit each year, the monitoring of central venous and arterial blood pressure (and inotrope protocols), optimal target variables, and whether CO was monitored (with type of device). Thirty-two units responded (80%). While 26 units (81%) routinely monitored central venous pressure (CVP), CO was monitored in only 3 units (9%). There was a wide range of responses in relation to optimal variables and use of inotropes. As with other specialties, it is likely that CO monitoring will become widely used in head and neck reconstructive surgery. Not only does it enhance fluid optimisation, but it may also reduce hospital stay and morbidity. Appropriate clinical studies are urgently needed to evaluate its use in our specialty.


Assuntos
Débito Cardíaco/fisiologia , Hidratação/métodos , Retalhos de Tecido Biológico , Microcirurgia/métodos , Monitorização Fisiológica/métodos , Procedimentos Cirúrgicos Bucais/métodos , Procedimentos Cirúrgicos Reconstrutivos/métodos , Serviço Hospitalar de Anestesia , Pressão Sanguínea/fisiologia , Cardiotônicos/uso terapêutico , Pressão Venosa Central/fisiologia , Unidades Hospitalares , Humanos , Monitorização Intraoperatória/métodos , Inquéritos e Questionários
12.
Biochemistry ; 47(9): 2826-32, 2008 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-18205318

RESUMO

The M-superfamily, one of eight major conotoxin superfamilies found in the venom of the cone snail, contains a Cys framework with disulfide-linked loops labeled 1, 2, and 3 (-CC (1) C (2) C (3) CC-). M-Superfamily conotoxins can be divided into the m-1, -2, -3, and -4 branches, based upon the number of residues located in the third Cys loop between the fourth and fifth Cys residues. Here we provide a three-dimensional solution structure for the m-1 conotoxin tx3a found in the venom of Conus textile. The 15-amino acid peptide, CCSWDVCDHPSCTCC, has disulfide bonds between Cys (1) and Cys (14), Cys (2) and Cys (12), and Cys (7) and Cys (15) typical of the C1-C5, C2-C4, and C3-C6 connectivity pattern seen in m-1 branch peptides. The tertiary structure of tx3a was determined by two-dimensional (1)H NMR in combination with the combined assignment and dynamics algorithm for nuclear magnetic resonance (NMR) applications CYANA program. Input for structure calculations consisted of 62 inter- and intraproton, five phi angle, and four hydrogen bond constraints. The root-mean-square deviation values for the 20 final structures are 0.32 +/- 0.07 and 0.84 +/- 0.11 A for the backbone and heavy atoms, respectively. Surprisingly, the structure of tx3a has a "triple-turn" motif seen in the m-2 branch conotoxin mr3a, which is absent in mr3e, the only other member of the m-1 branch of the M-superfamily whose structure is known. Interestingly, injection of tx3a into mice elicits an excitatory response similar to that of the m-2 branch peptide mr3a, even though the conotoxins have different disulfide connectivity patterns.


Assuntos
Conotoxinas/química , Caramujo Conus/metabolismo , Peptídeos/química , Sequência de Aminoácidos , Animais , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica
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