bluuebunny/crossref_metadata_2025_split huggingface dataset:
Commonly Abused Drugs Differentially Increase the Frequency and Amplitude of Accumbal Transient Dopamine Release Events
Authors: Everett Thomas , Leman Ryan , Lopez‐Beltran Hendrick , Chaman Oniza , Isaacs Dominic , Oleson Erik B 
Published: June 2021

The rewarding and reinforcing effects of abused drugs are thought to be mediated by their ability to increase brain dopamine levels. The canon holds that all drugs of abuse increase dopamine release in a brain region called the nucleus accumbens (NAc) but, due to the temporal constraints of previously available neurochemical techniques, it remains unknown how distinct drugs of abuse alter dopamine release in real time. A real time assessment is important due to the nature by which dopamine neurons fire. In awake and behaving rats, dopamine neurons fire in two distinct patterns. At ‘rest,’ dopamine neurons fire in a low‐frequency pacemaker fashion that is thought to produce a tone on high‐affinity dopamine receptors in the NAc. When presented with motivationally relevant stimuli, dopamine neurons fire in high‐frequency bursts. The phasic bursts of dopamine neural activity contribute to the generation of subsecond release events in the NAc that are sufficient in concentration to occupy low‐affinity dopamine receptors. As the activation of these low‐affinity receptors is thought to be particularly important in strengthening action‐outcome associations, we are especially interested in investigating how drugs of abuse alter their patterns of release. Fast‐scan cyclic voltammetry (FSCV) allows for the detection of dopamine release events with a temporal resolution of milliseconds, making it an ideal tool with which to examine how drugs of abuse alter transient dopamine release events in real time. We obtain two primary dependent measures of dopamine release, the frequency at which they occur and the total concentration of each event.Here, we are using FSCV to compare how different drugs of abuse alter the frequency and amplitude of dopamine release events in the awake and behaving rat. Drugs of investigation include: ethanol, diazepam, zolpidem, WIN55,212‐2, methamphetamine, methylenedioxymethamphetamine (MDMA) and heroin. Preliminary data show that the sedative‐hypnotics ethanol, diazepam and zolpidem all increase the frequency but decrease the amplitude of dopamine release events; although the ethanol amplitude effect is biphasic. Heroin, the synthetic cannabinoid WIN55,212‐2, and the psychostimulant methamphetamine increase both the frequency and amplitude of dopamine release events. The psychostimulant and tryptamine psychedelic MDMA increases the amplitude of dopamine release events but not their frequency. Together, these data reveal that the effects of abused drugs on transient dopamine release events varies across drug classes.Support or Funding InformationFunding for this work was provided by NSF grant IOS‐1557755, NIH grant R03DA038734, Boettcher Young Investigator Award and NARSAD Young Investigator Award to EBOThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bulk Material Properties of Beta–Bulge Peptide Hydrogels For Tissue Engineering
Authors: Dreher Stephanie Marie , Salick Daphne A. , Haines‐Butterick Lisa , Schneider Joel P. 
Published: June 2021

Hydrogels are porous, physically rigid, three dimensional matrices primarily composed of water which show promise as scaffolds to aid in tissue regeneration.1,2 The Schneider laboratory has developed a class of peptides that adopt β‐hairpin structure and self‐assemble into a hydrogel in response to specific stimuli.3,4,5,6 The peptide MAX8 consists of alternating valine and lysine residues on each β‐strand flanking a type II′ β‐turn (‐VDPPT‐) with a glutamic acid at position 15. The kinetics of self‐assembly for the MAX8 under cellular encapsulation conditions are fast enough to result in homogeneous gel‐cell constructs.7MAX8 must be prepared by chemical synthesis due to the unnatural amino acid DProline in the turn region. A new sequence, BBH8, was designed to contain all natural amino acids which can possibly be overexpressed. BBH8 was first chemically synthesized and its bulk material properties characterized under physiological and cell culture conditions. Data show that BBH8 behaves analogously to MAX8. Preliminary data also demonstrate that chondrocytes can be encapsulated with homogeneous cellular distribution and remain viable in the network for at least 3 hours. This suggests that BBH8 is an excellent candidate for tissue engineering and possible production by fermentation. Funding provided by NSF CHEM0348323, NIH R01 DE016386‐01, and the Howard Hughes Medical Institute.
Characterization of a benzoylformate decarboxylase and a NAD+/NADP+‐dependent benzaldehyde dehydrogenase from Pseudomonas stutzeri ST‐201
Authors: Saehuan Choedchai , Rojanarata Theerasak , Wiyakrutta Suthep , Meevootisom Vithaya , McLeish Michael J. 
Published: June 2021

Both D‐phenylglycine aminotransferase and benzoylformate decarboxylase (BFDC) activity were detected in cultures of P. stutzeri ST‐201 following induction with D‐phenylglycine. Conversely, induction with benzoylformate enhanced only BFDC activity. Colony hybridization was used to identify two genomic DNA fragments which, when combined, contained two open reading frames whose gene products were characterized as a benzoylformate decarboxylase and a NAD+/NADP+‐dependent benzaldehyde dehydrogenase. The kinetic properties of the BFDC was similar to those of the BFDC found in the mandelate pathway of P. putida ATCC 12633. However, the Km values for NAD(P)+ of the benzaldehyde dehydrogenase were significantly higher than those of the P. putida enzyme. Based on sequence and structural alignments this was likely to be due to a change from leucine to arginine at position 175. Given that P. stutzeri ST‐201 was unable to grow on either isomer of mandelate, and that sequencing indicated that dpgB did not form part of an operon, it appears that the two enzymes form part of a D‐phenylglycine, rather than mandelate, degrading pathway.This work is supported in part by the CHED and Faculty of Graduate Studies, Mahidol University, and granted by TRF through the RGJ‐Ph.D. Program No. Ph.D/0023/2548. The work was also supported by NSF EF 0425719.
Exploring the Strength of Subunit Interfaces Using Guanidine Hydrochloride and Dynamic Light Scattering
Authors: Marion Jimmy , Bell Ellis 
Published: June 2021

Dynamic light scattering is used to determine changes in mass and mean diameter of particles. Through the combination of varying concentrations of guanidine hydrochloride with either monomeric or multimeric protein samples we have developed a novel approach using Dynamic Light Scattering that has allowed us to test not only the strength of a protein's subunit interface but also to test the stability of its tertiary structure. Model systems, such as monomeric Bovine Serum Albumin (BSA) and tetrameric hemoglobin, with a known dissociation pattern, are used as fundamental examples to this light scattering approach. Watermelon Glyoxasomal Malate Dehydrogenase (dimer), E. coli 3Phosphoglycerate Dehydrogenase (tetramer) and Bovine Glutamate Dehydrogenase (hexamer) are used as complex systems to further investigate the approach. By measuring DLS at different guanidine hydrochloride concentrations in the range 0–2M dissociation of the oligomers can be followed where little change in tertiary structure occurs. By including ligands in the guanidine hydrochloride solution this approach is shown to be sensitive enough to detect and quantitate ligand induced changes in the strength of subunit interactions.Supported by NSF Grant MCB 0448905 to Ellis Bell
Inhibition of heat shock protein 90 (Hsp90) and degradation of client proteins associated with oncogenesis using analogs of Novobiocin
Authors: Higgins Michelle , Vielhauer George , Weir Scott , Holzbeierlein Jeff , Blagg Brian 
Published: June 2021

Prostate cancer remains a lethal disease with over 30,000 men dying per year. Thus, there is a critical need to develop novel therapies that translate to improved clinical outcomes. The objective of these studies is to design C‐terminal inhibitors of the molecular chaperone hsp90 resulting in client protein degradation and inhibition of multiple signaling pathways causing cancer cell death of in vitro and in vivo. C‐terminal inhibitors of hsp90 were tested in a dose response manner using the androgen independent PC‐3 prostate cancer cell line by Western blot analysis and examined degradation of client proteins (AKT, HER2, and BCL‐2). Additionally, one inhibitor, KU32, was used in an in vivo orthotopic murine tumor model and examined for reduction of prostate serum antigen (PSA) and degradation of client protein in treated and untreated animals. Prostate cancer cells treated with these inhibitors demonstrated the hallmarks of hsp90 inhibition, specifically, the increased expression of hsp90 and decreased expression of client proteins in a dose dependent fashion. The in vivo studies are currently ongoing. These data suggest that C‐terminal inhibitors may debilitate cancer cell function and slow tumor cell proliferation.These studies were supported by funding from the National Cancer Institute (5 U01 CA120458‐02) and the Department of Defense (W81XWH‐05‐1‐0594).
Single fiber muscle contractile properties in mobility‐limited older adults
Authors: Fielding Roger A. , Krivickas Lisa S. , Gavin Brian P. , Reid Kieran F. , Carabello Robert J. , Phillips Edward M. , Frontera Walter R. 
Published: June 2021

Mobility limitations in older adults are associated with reductions in whole muscle size, strength and power. We examined whether alterations in isolated single muscle fiber contractile properties contribute to the observed reductions in whole muscle function in older adults. Percutaneous needle biopsies of the vastus lateralis muscle were obtained from 20 middle aged healthy (MH, 40‐55y, 9 male), 19 older healthy (OH, 70‐85y, 15 male), and 13 older mobility‐limited (OML, 70‐85y, 7 male, SPPB ≤9) adults. Contractile properties of permeabilized single muscle fibers were evaluated. Data from isolated type I fibers revealed no significant differences in force or power generating properties from muscle obtained from MH, OH, or OML.These data suggest that differences in type I muscle fiber contractile properties cannot explain the deficits in force and power generating capacity observed in mobility‐limited older adults.Supported by USDA 58‐1950‐7‐707 and NIA RO1‐AG‐18844
Diurnal pattern in endogenous insulin secretion persists in severely injured patients
Authors: Pidcoke Heather F , Wolf Steven E , Jones John A , Holcomb John B , Wade Charles E 
Published: June 2021

Intensive insulin therapy decreases mortality due to critical illness but glucose and insulin variability pose a challenge to tight glucose control. We previously demonstrated that circadian rhythms in glucose and exogenous insulin requirement are preserved, and hypothesized that similar patterns in endogenous insulin secretion are also present.Patients in burn and surgical ICUs at a single center receiving intensive insulin therapy were admitted to the study. Blood samples were collected at 4 am and 5 pm. An enzyme‐linked immunosorbent assay (ELISA) for C‐peptide quantified endogenous insulin. Data were analyzed with paired Student's t‐test.C‐peptide levels from fourteen patients admitted to the study were averaged (mean #values/subject was 30±31, range: 6–125). Morning C‐peptide levels (5.1 ng/mL ± 3.4 ng/mL, range: 1.6–11.3 ng/mL) were lower compared to evening levels (6.5 ng/mL ± 4.2 ng/mL, range: 1.7–15.9 ng/mL, p=0.016).Endogenous insulin secretion demonstrates diurnal variation in critically injured patients, with higher evening levels relative to morning values. This study contributes further evidence that the circadian rhythms in hormones of glycemic regulation are preserved after severe trauma. Performance of intensive insulin protocols may improve if glucose targets are adjusted to reflect 24 hour periodicity. Grant support from the NIH, JDRF, and MRMC.
Starting and Sustaining Undergraduate Research
Authors: Bell Ellis 
Published: June 2021

Research is accepted as a necessary part of the education of undergraduates and this is particularly true in the molecular life sciences. The phrase “undergraduate research” implies that there is something distinct about “undergraduate research” and is often interpreted to mean research that is less challenging [ie more suitable for undergraduates] or less demanding of facilities and time, which translate into not the type of research that you see at a research intensive institution. Successful research by undergraduates has all the characteristics of successful research by anyone: it is challenging and addresses important problems, using the most appropriate techniques. Initiating and sustaining such research in a primarily undergraduate institution is the focus of this workshop.Participants will develop a plan to initiate and sustain such research in their own laboratory or institution and will focus on “best practices” for developing a project, recruiting and retaining students, managing a research group from proposal to publication, integrating teaching and research, making appropriate collaborations, and obtaining funding. The workshop will be led by experienced researchers from undergraduate institutions who will be available after the workshop to mentor participants throughout the coming year.The work is supported by National Science Foundation Grant MCB 0448905 to Ellis Bell
Locomotor hand postures and the functional anatomy of the hominid wrist
Authors: Orr Caley Michael 
Published: June 2021

The African apes have stable wrists with limited mobility—probably for energetic efficiency while using the hand as a rigid, propulsive lever during knuckle‐walking (KW). In contrast, the Asian apes have mobile wrists to facilitate a variety of arboreal hand postures. Testing the KW ancestor hypothesis for hominins requires understanding mechanisms of wrist stability in African apes and subsequent identification of related anatomy in the fossil record.A CT‐based method to track 3D carpal kinematics was used to study midcarpal joint mechanics in Pan troglodytes and Pongo pygmaeus. Midcarpal extension in Pan is distinguished by scaphoid supination on the capitate head, which rapidly close‐packs the midcarpus. Coupled with scaphoid‐centrale fusion and a centrale‐capitate ligament, this limits extension and ensures that the scaphoid and capitate act as a rigid unit. Scaphoid‐centrale fusion and a Pan‐like scaphocapitate joint in early hominins suggest a similar midcarpal mechanism, which may indicate a KW ancestor; alternatively, the mechanism may be a more generalized quadrupedal adaptation. Studying carpal kinematics in quadrupedal monkeys will test this alternative hypothesis. Modifications to the scaphocapitate joint and loss of the centrale‐capitate ligament in later hominins probably resulted in the more mobile modern human wrist.Funding: NSF BCS‐622515, Wenner‐Gren Foundation, Sigma Xi, ASU
Extracellular control of muscle regeneration
Authors: Simon Hans‐Georg , Calve Sarah 
Published: June 2021

In urodele amphibian limb regeneration, the muscle provides a reservoir for uncommitted stem‐like blastema cells that will participate in the replacement of the lost structure. However, it is currently not clear what signals activate muscle‐associated satellite cells and/or dedifferentiation of multinucleate myofibers. Cultures of newt muscle fibers allow us to follow these physiological changes at the cellular level using time‐lapse video microscopy.Our studies on extracellular matrix (ECM) distribution during blastema formation demonstrate upregulation of hyaluronic acid (HA), fibronectin (FN), and Tenascin C, suggesting dynamic changes in the ECM composition. Contemporaneous with ECM reorganization, histological analyses of regenerating blastemas reveal increased satellite cell numbers and migration along with myofiber fragmentation. In order to gain a better understanding of the signaling properties of the ECM in regeneration, we are investigating various ECMs and their effects on cell physiology and morphology. Our data indicate that HA induces fragmentation of myotubes and limits adhesion whereas FN or laminin‐containing matrigel stimulates formation of myotubes.The establishment of a physiologically relevant environment and its manipulation in vitro will provide new insights into the control of the differentiated state of the cell by its ECM.Supported by DARPA, NIH, Searle Funds
Dissecting protein complexes with small molecules
Authors: Mapp Anna K 
Published: June 2021

Altered transcriptional patterns are associated with all human diseases, either as a cause of the disease or as an effect. For this reason, molecules that interfere with or promote protein‐protein interactions within the transcriptional regulatory network are attractive targets for therapeutic development and as mechanistic probes. Despite the complexity of the transcriptional regulatory system, the design principles for the identification of small molecules that stimulate assembly of the transcriptional machinery complex are evidently straightforward. We find that several structurally distinct amphipathic molecular scaffolds can function in vitro and in human cells as transcriptional activation domains. In addition, structural studies of these molecules in complex with transcriptional machinery proteins reveal that they target the binding surfaces used by natural transcriptional activators and do so with similar affinity and kinetic profile. Finally, straightforward structural modifications of the molecules can be used to convert them into potent inhibitors of transcriptional machinery assembly.Financial support was provided by the American Cancer Society (RSG 05‐195‐01), the NSF, GSK and Novartis.
Human hepatic stellate cells and hepatocyte co‐cultures maintain differentiation in vitro and in vivo
Authors: Harding Martha J , Barnaeva Elena , Bustamante Marcia , Lepus Christin M , Rahner Christoph , Pober Jordan S , Sjogren Maria H , Rojkind Marcos 
Published: June 2021

Lack of long‐term cultures of mature human hepatocytes (Hep)that maintain liver‐specific functions have hampered investigations of the effects of chronic drug toxicity and liver pathogens. We investigated if matrix‐producing hepatic stellate cells (HSC) sustained Hep survival and differentiation in vitro and in vivo. Co‐cultures (CC) survived 2 months in vitro, maintained expression of albumin, CYP2E1, CD81, and CK19 mRNAs, began to express α‐fetoprotein and secreted α1‐antitrypsin and fibrinogen. CC proliferated and required passaging every 3–4 weeks. CC prepared with Hep from newborn subjects expressed and sustained the hepatoblast markers Thy1 and Sca 1 in vitro. Transplanted Hep survived in CC for at least 8 weeks when engrafted in vascularized protein gels on the abdominal wall of SCID/bg mice. EM revealed that Hep ultrastructure and polarization was preserved and groups of epithelial cells formed bile duct‐like structures in vivo. Our findings suggest that Hep‐HSC CC sustain normal Hep morphology and differentiation. Accordingly, these CC could be used in studies of liver repopulation, hepatitis infections and long‐term toxicity of drugs and alcohol. Supported by AGA RSA (MJH), NIH‐R01HL085416 (JSP), USUHS MDA905‐03‐1‐0006 (MHS) and NIH‐RO1AA10541(MR) grants.
Alcohol Dehydrogenase Dynamics and Catalysis
Authors: Plapp Bryce V. 
Published: June 2021

The catalytic mechanism of horse liver alcohol dehydrogenase has been extensively studied. The enzyme changes conformation when coenzyme and substrates bind. Release of a proton from the catalytic zinc‐water via the proton relay system controls the conformational change and suggest that changes in the coordination of the zinc are involved in substrate binding. Substitutions of various amino acid residues in the active site activate the enzyme by increasing the rate‐limiting release of coenzyme product and affect the conformational change, but the rates of hydride transfer decrease modestly. The hydride ion is transferred with quantum mechanical tunneling, implying that the reacting substrates move closer together at the transition state. The contribution of protein motions is being assessed with studies of various mutated forms of the enzyme. High resolution X‐ray crystallography shows that the reduced nicotinamide ring is puckered in ternary complexes with NADH. X‐ray data can also provide information on protein motions that are correlated with the hydride transfer. Protein dynamics are important for catalysis by liver alcohol dehydrogenase.(Supported by grants from NSF and NIH)
Exploring the Energy Barriers Involved in Allosteric Transitions in Glutamate Dehydrogenase
Authors: Jarman Erika , Bell Ellis 
Published: June 2021

Glutamate Dehydrogenase exhibits negative cooperativity, allosteric activation [by ADP], inhibition [by GTP] and substrate inhibition by glutamate. Each is thought to involve subunit interactions. Ligand induced changes in conformation occur with an energy barrier that must be overcome by energy from ligand binding. In negative cooperativity induced by NAD(P)+ binding, activity goes up but affinity for ligand goes down. We hypothesized that if the increased activity results from enhanced flexibility, raising the thermal energy of the system should overcome the energy barrier resulting in a single form of the enzyme with high activity and low affinity rather than the negative cooperativity. The temperature dependence of the non‐linear Lineweaver Burke plots resulting from negative cooperativity shows that the cooperativity disappears at temperatures above 40°C giving a form with high catalytic efficiency and low affinity, consistent with our hypothesis. At low temperature [15°C or below] the cooperativity also disappears suggesting the energy barrier to trigger the negative cooperativity is too high for ligand derived energy input to overcome. This approach has been extended to examination of both substrate inhibition and heterotropic allosteric regulation in this system to give information about associated energy barriers.Supported by NSF Grant MCB 0448905 to Ellis Bell
The Role of Dynamics in the Activity and Regulation of Oligomeric Dehydrogenases
Authors: Bell Ellis 
Published: June 2021

Three oligomeric dehydrogenases, each catalyzing similar reactions have been studied to examine the role of protein dynamics in both catalysis and allosteric regulation. Global stability has been assessed using DSC or Guanidine Hydrochloride unfolding approaches. Limited proteolysis and H/D exchange approaches have been used to examine the effects of ligands on local flexibility. A novel technique, combining guanidine hydrochloride and dynamic light scattering approaches is used to assess the stability of subunit interfaces. These techniques, combined with site directed mutagenesis and the use of alternative substrates and allosteric activators or inhibitors reveal a pattern of flexibility effects which indicate that efficient substrates or efficient enzymes have significantly more flexible local structure and subunit interface interactions than less efficient substrates or enzymes. Similarly allosteric activators increase both global and local flexibility while allosteric inhibitors decrease flexibility. Overall these studies show that flexibility plays a critical role in catalytic efficiency and that allosteric regulation can be achieved by ligand induced modulations of flexibility rather than simple induced switching from one conformation to another.The work is supported by National Science Foundation Grant MCB 0448905 to Ellis Bell