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Find video protocols related to scientific articles indexed in Pubmed.
Blocking the janus-activated kinase pathway reduces tumor necrosis factor alpha-induced interleukin-18 bioactivity by caspase-1 inhibition.
Arthritis Res. Ther.
PUBLISHED: 03-25-2014
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Our objective was to examine the role of the janus-activated kinase (JAK) pathway in the modulation of tumor necrosis factor-? (TNF)-induced-IL-18 bioactivity by reduction of caspase-1 function.
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Morphine glucuronidation and glucosidation represent complementary metabolic pathways that are both catalyzed by UDP-glucuronosyltransferase 2B7: kinetic, inhibition, and molecular modeling studies.
J. Pharmacol. Exp. Ther.
PUBLISHED: 01-23-2014
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Morphine 3-?-D-glucuronide (M3G) and morphine 6-?-D-glucuronide (M6G) are the major metabolites of morphine in humans. More recently, morphine-3-?-d-glucoside (M-3-glucoside) was identified in the urine of patients treated with morphine. Kinetic and inhibition studies using human liver microsomes (HLM) and recombinant UGTs as enzyme sources along with molecular modeling were used here to characterize the relationship between morphine glucuronidation and glucosidation. The M3G to M6G intrinsic clearance (C(Lint)) ratio (?5.5) from HLM supplemented with UDP-glucuronic acid (UDP-GlcUA) alone was consistent with the relative formation of these metabolites in humans. The mean C(Lint) values observed for M-3-glucoside by incubations of HLM with UDP-glucose (UDP-Glc) as cofactor were approximately twice those for M6G formation. However, although the M3G-to-M6G C(Lint) ratio remained close to 5.5 when human liver microsomal kinetic studies were performed in the presence of a 1:1 mixture of cofactors, the mean C(Lint) value for M-3-glucoside formation was less than that of M6G. Studies with UGT enzyme-selective inhibitors and recombinant UGT enzymes, along with effects of BSA on morphine glycosidation kinetics, were consistent with a major role of UGT2B7 in both morphine glucuronidation and glucosidation. Molecular modeling identified key amino acids involved in the binding of UDP-GlcUA and UDP-Glc to UGT2B7. Mutagenesis of these residues abolished morphine glucuronidation and glucosidation. Overall, the data indicate that morphine glucuronidation and glucosidation occur as complementary metabolic pathways catalyzed by a common enzyme (UGT2B7). Glucuronidation is the dominant metabolic pathway because the binding affinity of UDP-GlcUA to UGT2B7 is higher than that of UDP-Glc.
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Generation, validation, and application of a P450 homology model.
Curr Top Med Chem
PUBLISHED: 09-20-2013
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In vitro validation of a protein homology model is critical for determining the predictivity of a computationally generated structure. Here we discuss the generation, validation, and application of a homology model for CYP1A1. Validation of the CYP1A1 homology model, generated using the highly homologous crystal template of human CYP1A2 (pdb 2HI4), was achieved using the prototypic substrate 7-ethoxyresorufin (Eres). The model was subsequently applied to generate CYP1A1 mutants with increased catalytic efficiency (Vmax/Km) towards the anticancer prodrug dacarbazine (DTIC). Thirty-three directed CYP1A1 mutants were generated and expressed in E. coli; six of these were generated to rationalise docking data obtained from in silico experiments using Eres. DTIC N-demethylation by the CYP1A1 E161K, E256K, and I458V mutants exhibited Michaelis-Menten kinetics, with decreases in Km that doubled the catalytic efficiency relative to wild-type (P < 0.05). As a chemotherapeutic agent, DTIC has relatively poor clinical activity in human malignancies and exhibits numerous adverse effects, which presumably arise from bioactivation in the liver and other tissues resulting in systemic exposure to the cytotoxic metabolite. The successful generation of CYP1A1 enzymes with catalytically enhanced DTIC activation highlights their potential use as a strategy for P450-based gene directed enzyme prodrug therapy (GDEPT) in the treatment of metastatic malignant melanoma. Moreover, the combination of in vitro kinetic analyses with in silico docking data from a validated homology model has allowed interpretation of the structure-activity relationships of this enzyme-substrate pair.
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The Drosophila BTB domain protein Jim Lovell has roles in multiple larval and adult behaviors.
PLoS ONE
PUBLISHED: 01-01-2013
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Innate behaviors have their origins in the specification of neural fates during development. Within Drosophila, BTB (Bric-a-brac,Tramtrack, Broad) domain proteins such as Fruitless are known to play key roles in the neural differentiation underlying such responses. We previously identified a gene, which we have termed jim lovell (lov), encoding a BTB protein with a role in gravity responses. To understand more fully the behavioral roles of this gene we have investigated its function through several approaches. Transcript and protein expression patterns have been examined and behavioral phenotypes of new lov mutations have been characterized. Lov is a nuclear protein, suggesting a role as a transcriptional regulator, as for other BTB proteins. In late embryogenesis, Lov is expressed in many CNS and PNS neurons. An examination of the PNS expression indicates that lov functions in the late specification of several classes of sensory neurons. In particular, only two of the five abdominal lateral chordotonal neurons express Lov, predicting functional variation within this highly similar group. Surprisingly, Lov is also expressed very early in embryogenesis in ways that suggests roles in morphogenetic movements, amnioserosa function and head neurogenesis. The phenotypes of two new lov mutations that delete adjacent non-coding DNA regions are strikingly different suggesting removal of different regulatory elements. In lov(47) , Lov expression is lost in many embryonic neurons including the two lateral chordotonal neurons. lov(47) mutant larvae show feeding and locomotor defects including spontaneous backward movement. Adult lov(47) males perform aberrant courtship behavior distinguished by courtship displays that are not directed at the female. lov(47) adults also show more defective negative gravitaxis than the previously isolated lov(91Y) mutant. In contrast, lov(66) produces largely normal behavior but severe female sterility associated with ectopic lov expression in the ovary. We propose a negative regulatory role for the DNA deleted in lov(66) .
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Application of homology modeling to generate CYP1A1 mutants with enhanced activation of the cancer chemotherapeutic prodrug dacarbazine.
Mol. Pharmacol.
PUBLISHED: 08-04-2011
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The chemotherapeutic prodrug dacarbazine (DTIC) has limited efficacy in human malignancies and exhibits numerous adverse effects that arise from systemic exposure to the cytotoxic metabolite. DTIC is activated by CYP1A1 and CYP1A2 catalyzed N-demethylation. However, structural features of these enzymes that confer DTIC N-demethylation have not been characterized. A validated homology model of CYP1A1 was employed to elucidate structure-activity relationships and to engineer CYP1A1 enzymes with altered DTIC activation. In silico docking demonstrated that DTIC orientates proximally to Ser122, Phe123, Asp313, Ala317, Ile386, Tyr259, and Leu496 of human CYP1A1. The site of metabolism is positioned 5.6 ? from the heme iron at an angle of 105.3°. Binding in the active site is stabilized by H-bonding between Tyr259 and the N(2) position of the imidazole ring. Twenty-seven CYP1A1 mutants were generated and expressed in Escherichia coli in yields ranging from 9 to 225 pmol P450/mg. DTIC N-demethylation by the E161K, E256K, and I458V mutants exhibited Michaelis-Menten kinetics, with decreases in K(m) (183-249 ?M) that doubled the catalytic efficiency (p < 0.05) relative to wild-type CYP1A1 (K(m), 408 ± 43 ?M; V(max), 28 ± 4 pmol · min(-1) · pmol of P450(-1)). The generation of enzymes with catalytically enhanced DTIC activation highlights the potential use of mutant CYP1A1 proteins in P450-based gene-directed enzyme prodrug therapy for the treatment of metastatic malignant melanoma.
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Homodimerization of UDP-glucuronosyltransferase 2B7 (UGT2B7) and identification of a putative dimerization domain by protein homology modeling.
Biochem. Pharmacol.
PUBLISHED: 07-01-2011
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Although homodimerization of UGT1A proteins is well established, direct evidence for dimerization of UGT2B7, which is arguably the most important enzyme involved in human drug glucuronidation, is currently lacking. This study characterized UGT2B7 homodimerization by co-immunopreciptation and generated a UGT2B7 homology model that identified the dimerization domain. It was demonstrated that co-expressed, solubilized UGT2B7 proteins differentially tagged with hemagglutinin (UGT2B7-HA) and c-MYC (UGT2B7-cMYC) co-immunoprecipitated as active homodimers that catalyzed 4-methylumbelliferone glucuronidation. Substrate binding affinities (assessed as S(50) values) of the tagged and co-expressed tagged proteins were essentially identical to that of native UGT2B7. Co-association was not observed in a mixed UGT2B7-HA and UGT2B7-cMYC protein preparation. Generation of a UGT2B7 homology model established from plant and human templates was achieved using SYBYLX1.2 with all residues energy minimized using the Tripos Force Field. The UGT2B7 model allowed elucidation of a putative protein dimerization domain within the B-C loop of each UGT2B7 monomer. The eighteen amino acid dimerization domain is present in all UGT2B enzymes and comprises a proposed dimerization signature motif (FPPSYVPVVMS). Stabilization of the dimer interface is maintained by the formation of two salt bridges, aromatic ?-? stacking interactions, two S-aromatic (face) interactions, and the presence of proline brackets. The homology model further provides important insights into structure-function relationships of this enzyme and the mechanism responsible for the atypical glucuronidation kinetics for substrates of UGT2B7 and other human UGT enzymes.
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Human telomerase model shows the role of the TEN domain in advancing the double helix for the next polymerization step.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-23-2011
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Telomerases constitute a group of specialized ribonucleoprotein enzymes that remediate chromosomal shrinkage resulting from the "end-replication" problem. Defects in telomere length regulation are associated with several diseases as well as with aging and cancer. Despite significant progress in understanding the roles of telomerase, the complete structure of the human telomerase enzyme bound to telomeric DNA remains elusive, with the detailed molecular mechanism of telomere elongation still unknown. By application of computational methods for distant homology detection, comparative modeling, and molecular docking, guided by available experimental data, we have generated a three-dimensional structural model of a partial telomerase elongation complex composed of three essential protein domains bound to a single-stranded telomeric DNA sequence in the form of a heteroduplex with the template region of the human RNA subunit, TER. This model provides a structural mechanism for the processivity of telomerase and offers new insights into elongation. We conclude that the RNADNA heteroduplex is constrained by the telomerase TEN domain through repeated extension cycles and that the TEN domain controls the process by moving the template ahead one base at a time by translation and rotation of the double helix. The RNA region directly following the template can bind complementarily to the newly synthesized telomeric DNA, while the template itself is reused in the telomerase active site during the next reaction cycle. This first structural model of the human telomerase enzyme provides many details of the molecular mechanism of telomerase and immediately provides an important target for rational drug design.
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PRIDB: a Protein-RNA interface database.
Nucleic Acids Res.
PUBLISHED: 11-11-2010
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The Protein-RNA Interface Database (PRIDB) is a comprehensive database of protein-RNA interfaces extracted from complexes in the Protein Data Bank (PDB). It is designed to facilitate detailed analyses of individual protein-RNA complexes and their interfaces, in addition to automated generation of user-defined data sets of protein-RNA interfaces for statistical analyses and machine learning applications. For any chosen PDB complex or list of complexes, PRIDB rapidly displays interfacial amino acids and ribonucleotides within the primary sequences of the interacting protein and RNA chains. PRIDB also identifies ProSite motifs in protein chains and FR3D motifs in RNA chains and provides links to these external databases, as well as to structure files in the PDB. An integrated JMol applet is provided for visualization of interacting atoms and residues in the context of the 3D complex structures. The current version of PRIDB contains structural information regarding 926 protein-RNA complexes available in the PDB (as of 10 October 2010). Atomic- and residue-level contact information for the entire data set can be downloaded in a simple machine-readable format. Also, several non-redundant benchmark data sets of protein-RNA complexes are provided. The PRIDB database is freely available online at http://bindr.gdcb.iastate.edu/PRIDB.
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The orthodontic-restorative interface: 2. Compensating for variations in tooth number and shape.
Dent Update
PUBLISHED: 05-25-2010
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The aim of this second article in this series of two is to outline a variety of methods which may be used to compensate for variations in tooth shape and number using a combination of orthodontic and restorative approaches. It will also provide an overview of other areas of patient care which necessitate a multi-disciplinary orthodontic/restorative approach. The article will highlight the importance of combined planning from the outset and the close relationship between the different specialties, which must be maintained throughout treatment. The methods of compensating for variations in tooth number and shape will often require contributions from both orthodontist and restorative dentist. It is important that both disciplines are involved in the assessment and treatment planning process so that they know what will be expected of them during the patients care. Treatment planning in isolation may lead to care being delivered which is below the optimum standard which can be achieved. The orthodontist and restorative dentist are likely to liaise with the patients general dental practitioner so that he/she can provide the restorative treatment in some cases.
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The orthodontic-restorative interface: 1. Patient assessment.
Dent Update
PUBLISHED: 04-27-2010
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The first article in this series of two aims to outline the assessment of patients for whom a combined orthodontic-restorative approach would be beneficial. In particular, it will concentrate on the assessment of patients who have hypodontia and tooth size discrepancies. The importance of the aesthetic assessment for these cases will be highlighted. Variations in tooth number and tooth size discrepancy often require a combined treatment planning approach from the orthodontist and restorative dentist. The referring general dental practitioner has a key role in recognizing that this approach may be required and highlighting this in the initial patient referral. It is likely in the more straightforward cases that the GDP will be providing the restorative treatment and so an increased understanding of these cases would be beneficial. In the second paper, treatment options will be presented. Clinical Relevance: For patients who require a combined orthodontic/restorative approach, it is important that orthodontic and restorative disciplines liaise closely in the assessment and treatment planning process so that optimal care may be planned.
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AFM study of the interaction of cytochrome P450 2C9 with phospholipid bilayers.
Chem. Phys. Lipids
PUBLISHED: 03-19-2009
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Cytochromes P450 (CYP) are key enzymes involved in the metabolism of drugs and other lipophilic xenobiotics and endogenous compounds. In this study, atomic force microscopy was applied to characterise the association of CYP2C9 to dimyristoylphosphatidylcholine (DMPC) supported phospholipid bilayers. CYP2C9 was found to exclusively localise in the gel domains of partially melted DMPC bilayers. Despite lacking the N-terminus transmembrane spanning domain, the CYP2C9 protein appeared to partially embed into the membrane bilayer, as evidenced by an increase in melting temperature of surrounding phospholipids. Reversible binding of CYP2C9 via an engineered His tag to a phospholipid bilayer was facilitated using nickel-chelating lipids, presenting potential applications for biosensor technologies.
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Effects of amino acid substitutions at positions 33 and 37 on UDP-glucuronosyltransferase 1A9 (UGT1A9) activity and substrate selectivity.
Biochem. Pharmacol.
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UGT1A9 contributes to the glucuronidation of numerous drugs and xenobiotics. There is evidence to suggest that the Met33Thr substitution, as occurs in the polymorphic variant UGT1A9*3, variably affects xenobiotic glucuronidation. The equivalent position in UGT1A4 is also known to influence enzyme activity, whilst an N-terminal domain histidine (His37 in UGT1A9) is believed to function as the catalytic base in most UGT enzymes. To elucidate the roles of key amino acids and characterise structure-function relationships, we determined the effects of amino acid substitutions at positions 33 and 37 of UGT1A9 on the kinetics of 4-methylumbelliferone (4-MU), mycophenolic acid (MPA), propofol (PRO), sulfinpyrazone (SFZ), frusemide (FSM), (S)-naproxen (NAP) and retigabine (RTB) glucuronidation, compounds that undergo glucuronidation at either a phenolic (4-MU, MPA, PRO), carboxylate (FSM, NAP), acidic carbon (SFZ) or amine (RTB) function. Substitution of Met33 with Val, Ile, Thr, and Gln, as occur in UGT1A1, UGT1A3, UGT1A4 and UGT1A6 respectively, variably affected kinetics and catalytic efficiency. Whilst K(m) values were generally higher and V(max) and CL(int) values were generally lower than for wild-type UGT1A9 with most substrate-mutant pairs, the pattern and the magnitude of the changes in each parameter differed substantially. Moreover, exceptions occurred; CL(int) values for MPA and FSM glucuronidation by the position-33 mutants were the same as or higher than that of UGT1A9. Mutation of His37 abolished activity towards all substrates, except RTB N-glucuronidation. The data confirm the importance of single amino acids for UGT enzyme activity and substrate selectivity, and support a pivotal role for residue-33 in facilitating substrate binding to UGT1A9.
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Identification of residues that confer sugar selectivity to UDP-glycosyltransferase 3A (UGT3A) enzymes.
J. Biol. Chem.
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Recent studies in this laboratory characterized the UGT3A family enzymes, UGT3A1 and UGT3A2, and showed that neither uses the traditional UDP-glycosyltransferase UGT co-substrate UDP-glucuronic acid. Rather, UGT3A1 uses GlcNAc as preferred sugar donor and UGT3A2 uses UDP-Glc. The enzymatic characterization of UGT3A mutants, structural modeling, and multispecies gene analysis have now been employed to identify a residue within the active site of these enzymes that confers their unique sugar preferences. An asparagine (Asn-391) in the UGT signature sequence of UGT3A1 is necessary for utilization of UDP-GlcNAc. Conversely, a phenylalanine (Phe-391) in UGT3A2 favors UDP-Glc use. Mutation of Asn-391 to Phe in UGT3A1 enhances its ability to utilize UDP-Glc and completely inhibits its ability to use UDP-GlcNAc. An analysis of homology models docked with UDP-sugar donors indicates that Asn-391 in UGT3A1 is able to accommodate the N-acetyl group on C2 of UDP-GlcNAc so that the anomeric carbon atom (C1) is optimally situated for catalysis involving His-35. Replacement of Asn with Phe at position 391 disrupts this catalytically productive orientation of UDP-GlcNAc but allows a more optimal alignment of UDP-Glc for sugar donation. Multispecies sequence analysis reveals that only primates possess UGT3A sequences containing Asn-391, suggesting that other mammals may not have the capacity to N-acetylglucosaminidate small molecules. In support of this hypothesis, Asn-391-containing UGT3A forms from two non-human primates were found to use UDP-GlcNAc, whereas UGT3A isoforms from non-primates could not use this sugar donor. This work gives new insight into the residues that confer sugar specificity to UGT family members and suggests a primate-specific innovation in glycosidation of small molecules.
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Protein-RNA interface residue prediction using machine learning: an assessment of the state of the art.
BMC Bioinformatics
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RNA molecules play diverse functional and structural roles in cells. They function as messengers for transferring genetic information from DNA to proteins, as the primary genetic material in many viruses, as catalysts (ribozymes) important for protein synthesis and RNA processing, and as essential and ubiquitous regulators of gene expression in living organisms. Many of these functions depend on precisely orchestrated interactions between RNA molecules and specific proteins in cells. Understanding the molecular mechanisms by which proteins recognize and bind RNA is essential for comprehending the functional implications of these interactions, but the recognition code that mediates interactions between proteins and RNA is not yet understood. Success in deciphering this code would dramatically impact the development of new therapeutic strategies for intervening in devastating diseases such as AIDS and cancer. Because of the high cost of experimental determination of protein-RNA interfaces, there is an increasing reliance on statistical machine learning methods for training predictors of RNA-binding residues in proteins. However, because of differences in the choice of datasets, performance measures, and data representations used, it has been difficult to obtain an accurate assessment of the current state of the art in protein-RNA interface prediction.
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The glycosidation of xenobiotics and endogenous compounds: versatility and redundancy in the UDP glycosyltransferase superfamily.
Pharmacol. Ther.
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The covalent addition of sugars to small organic molecules is mediated by a superfamily of UDP glycosyltransferases (UGTs) found in animals, plants and bacteria. This superfamily evolved by gene duplication and divergence to manage exposure to a changing environment of lipophilic chemicals. The recent characterization of the UGT3A family provides further insights into the origin and evolution of this superfamily in mammals and the role of individual UGTs in the formation of the various chemical glycosides found in body tissues and fluids. Furthermore, the unique UDP-sugar specificities of the two enzymes in this family inform our knowledge of UGT structure relating to catalysis and UDP-sugar specificity. In addition to the UGT3 gene family, three other gene families, UGTs1, 2, and 8, are found in mammalian genomes. The 19 members of the UGT1 and 2 families have a major role in processing lipophilic chemicals due to their capacity to glucuronidate a broad range of structurally-dissimilar substrates. In contrast, the UGT3 enzymes only have a minor role, as their activities are very low in the major drug-metabolic organs, and their N-acetylglucosaminide and glucoside products are only a minor component of circulating and excreted drug metabolites. Although the endogenous role of the UGT3 family is still unknown, participation in the processing of lipophilic chemicals in specific cell types or at specific times during ontogeny cannot be excluded. In contrast to the UGT 1, 2 and 3 families, the single member of the UGT8 family appears to have no role in drug metabolism.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.