bluuebunny/crossref_metadata_2025 huggingface dataset:
Age exacerbates chronic catecholamine‐induced impairments in contractile reserve
Authors: Liles John Thomas , Ida Kevin , Joly Kristin , Chapo Joseph , Peng Yanyu , Plato Craig F
Published: June 2021

Heart failure is more prevalent and impaired contractile reserve develops with advancing age. This study evaluated whether adult, non‐senescent (A) rats (18 wk) are more susceptible to detrimental effects of chronic isoproterenol (ISO) administration than younger (Y) rats (12 wk). Rats received daily injections of ISO (0.1 mg/kg, s.c.) or vehicle (V) for three weeks. V‐Y rats had greater baseline +dP/dtmax compared to V‐A rats, as well as to both ISO‐Y and ISO‐A rats (6812±264 vs. 5841±239, 5089±223, and 5356±197 mmHG/sec, respectively). ISO induced a greater impairment in contractile reserve (Δ +dP/dtmax) in A than in Y to two mechanistically distinct inotropes (digoxin, milrinone; 20 ‐ 200 ul/kg/min), while basal and agonist‐induced changes in heart rate, MAP, or SAP were not different across groups. ISO also induced greater increases in cardiac hypertrophy (LV index: 33±3 % vs. 22±5 %), caspase‐3 activity (34 % vs. 5%), and β‐MHC mRNA expression (3.7 vs. 1.3 fold induction) in A relative to Y rats. Moreover, ISO decreased expression of the calcium handling protein, SERCA2a, by 55% in A relative to Y rats. These results demonstrate adult rats develop greater impairments in systolic performance than younger rats when exposed to chronic catecholamine excess. Reduced contractile reserve may result from increased β‐MHC expression, increased cardiomyocyte apoptosis/caspase activity, and/or calcium dysregulation.
Genesis Of the tendon of the Popliteus muscle
Authors: Ghosh Tamojit
Published: June 2021

Object of Study

The genesis of the tendon of the popliteus muscle

Summary of Results

The genesis of the origin of the popliteus is central to understanding the morphology of the postero‐lateral aspect of the knee joint. The popliteus muscle and its tendon have separate evolutionary origins.They get connected in the course of evolution. The tendon is not present in reptiles. It evolved into its present form in humans by incorporating various structures of the lateral aspect of the knee that disappear resulting in its formation. The most important of these is the femoro‐fibular meniscus. This work provides proof of the genesis of the popliteus tendon and delineates which of its surrounding structures it evolved from. Origins of the arcuate popliteal ligament and the short lateral ligament are also discussed. The disappearance of structures previously existing in the post‐lateral aspect of the knee are also mentioned and the relevance to this work delineated.

Statement of Conclusions

I have provided evidence that the popliteus tendon has evolved from the femoro‐fibular meniscus. The adjoining short lateral ligament and the arcuate ligament are both evolutionarily related to the tendon. Various dissections form part of the evidence chain. The femoro‐fibular meniscus gets incorporated into the tendon of the popliteus while it climbs up from a sub‐genual position to a supra‐genual one in the course of evolution.

The proximal attachment of the popliteus makes possible the 'unlocking action' of the muscle in the upright walking human which is not possible in lower vertebrates where the muscle is sub‐genual. This is the the major teleological explanation of the climb of the popliteus tendon to it's present proximal attachment in the lateral aspect of the intercondylar fossa.

Grant Funding Source

Self
Presence Of Anamalous Extensor Muscles On The Dorsum Of The Hand
Authors: Singh Keerti , Donnahue Dennie , Rao Suresh
Published: June 2021

The presence of accessory muscle bellies and tendons in the hand is of great interest to the hand surgeons. This knowledge can be borne in mind while performing hand surgeries, tendon rerouting or transplants. These extra muscles often present as a ganglion, soft tissue tumor or mass in the hand which could be quite misleading to the surgeon. In the present paper we are reporting two cases of accessory muscles found on the dorsum of hand, which were encountered while performing routine dissections of cadavers for undergraduate students.

Case I: a short muscle was found on the dorsum of the hand which was inserted to the index finger (extensor digitorum manus brevis).

Case II: an extra muscle was found arising deep to the extensor digitorum (EDC) which was inserted to the middle finger (extensor medii propius).

Grant Funding Source

University
Asymmetry of the Cranio‐cervico‐omo‐clavicular complex (CCOCC) in Right‐handed Humans is Functionally Related to Handedness
Authors: Osborn Michelle L. , Homberger Dominique G.
Published: June 2021

In humans, the clavicle and scapula are suspended from the occiput and the uniquely large mastoid process by the upper trapezius and sternocleidomastoid (SCM) muscles, thereby creating the cranio‐cervico‐omo‐clavicular complex (CCOCC). While it is known that the humerus is more robust and the clavicle shorter on the dominant side of individuals, other skeletal characters of the CCOCC have not been analyzed in this respect. When weights are carried or lifted, the contracting upper trapezius muscle offsets the added weight on the arms and stabilizes the shoulder by using the clavicle as a lever arm. The SCM muscle counteracts the action of the upper trapezius on the skull, preventing the skull from being retroflexed. To test these premises using a natural experiment, we analyzed the skeletal asymmetry of 54 right‐handed individuals. 71% had a wider right mastoid process. 90% of a subsample of 9 individuals had a higher right superior nuchal line and a wider right mastoid process, thereby providing larger attachment sites for the larger trapezius and SCM muscles of right‐handed individuals. These results suggest that the upper trapezius and SCM muscles act as synergists to stabilize the head and shoulders when loads are carried. Our inference is supported by the innervation and developmental pattern of the trapezius and SCM muscles.
A STUDY OF CONGENITAL ANOMALIES AT A UNIVERSITY TEACHING HOSPITAL
Authors: McNeil Rosie Thecla , Ekwere E.O. , Pam S. , Afolabi E.O. , Jeminiwa B.O. , Obri S.A.
Published: June 2021

This is a seven (7) years retrospective study of all cases of congenital anomalies that presented at the Jos University Teaching Hospital from January 1998 to December 2005. A total of 200 cases were seen at the Special Care Baby Unit (SCBU) of the Paediatrics Department of the hospital.

The results were as follows: anomalies of the gastrointestinal system had the highest incidence (30.5%), central nervous system abnormalities, 24.5%, skeletal system anomalies, 8.0%, cardiovascular system anomalies, 7.5%, chromosomal anomalies, 7.5,urogenital system anomalies, 5.0%, cutaneus system anomalies, 2.0% and metabolic disorders, 1.5%. 13.5% of cases presented with multiple anomalies.
Regulation of G Protein Signals by Membrane Domains
Authors: Scarlata Suzanne F , Sengupta Parijat
Published: June 2021

Caveolae are membrane domains having caveolin‐1 (Cav‐1) or caveolin‐3 (Cav‐3) as their main structural component. To determine whether caveolae could affect Ca2+ signaling through the Gαq/phospholipase Cβ (PLCβ) pathway, we studied Gαq and Gβγ interactions in Fischer Rat Thyroid cells that lack Cav‐1 (FRTcav−) and a Cav‐1 stable transfection line (FRTcav+) that forms caveolae‐like domains. We find that eCFP‐Gβγ and Gαq‐eYFP are closely associated in both cell lines. Upon stimulation, Gαq‐eYFP and eCFP‐Gβγ association remains high in FRTcav− cells, but decreases completely in FRTcav+ cells suggesting that Cav‐1 is increasing the separation between Gαq‐Gβγ subunits. In FRTcav− cells, a rapid recovery of calcium is observed after stimulation, but FRTcav+ cells show a sustained elevated Ca2+ level. Fluorescence studies show specific interactions between Gαq and Cav‐1 that increase upon stimulation, and that the mobility of eGFP‐Gβγ in FRTcav+ cells, but not FRTcav‐ cells, increases greatly with stimulation. These results suggest that activation allows Gαq to switch association from Gβγ to Cav‐1 releasing Gβγ from caveolae and prolonging the heterotrimer recombination time. In cardiac myocytes, stimulation by carbachol causes a loss in co‐localization between Cav‐3 and Gβ1 suggesting that this mechanism may be responsible for prolonged Ca2+ signals in these cells.
Shear stress modulates TGF‐beta signaling and EMT in endothelial cells
Authors: Egorova Anastasia D , de Ruiter Marco C , van de Pas Simone , ten Dijke Peter , Poelmann Robert E , Hierck Beerend P
Published: June 2021

Blood flow and concomitant shear stress play an instrumental role in the development of the cardiovascular system as endothelial cells (EC) functionally respond to shear. During cardiac development areas of high shear stress, i.e. the outflow and atrioventricular cushions, are characterized by high expression of Krüppel‐like factor 2 (Klf2) and by epithelial to mesenchymal transformation (EMT). In the adult situation exposure of EC to high and pulsatile flow induces a quiescent phenotype associated with high levels of Klf2 and a lack of EMT. Low and disturbed flow renders EC pro‐proliferative, marked by the activation of transforming growth factor‐beta (TGF‐β) signaling. Interestingly, embryonic EMT in areas of high shear is also associated with the activation of the TGF‐β pathway. In this study we investigated the shear stress modulation of TGF‐β signaling in EC. Cultured mouse embryonic EC were subjected to various flow profiles and levels in a shear stress bioreactor. The induced response was assessed by analysis of Klf2, TGF‐β ligands, TGF‐β serine/threonine kinase receptors Alk1 and Alk5, their pathway components, inhibitory Smads and downstream signaling targets. In order to discriminate between Alk1 and Alk5 signaling, Alk5 activity was selectively inhibited. The results indicate an intricate relation between flow induced signaling and the activation of TGF‐beta pathway and EMT in endothelial cells
Differential expression of prawn brain proteins in the context of dominance hierarchies
Authors: Vazquez‐Acevedo Nietzell , Perez Juliana , Melendez Loyda , Duan Fenghai , Rivera Nilsa , Torres Alejandra , Rullan Yarely , Ruiz Eduardo , Sosa Maria
Published: June 2021

Adult prawns (M. rosenbergii) develop through three morphological types (small [SC], yellow [YC], and blue claws [BC]), each representing a level in a group's dominance hierarchy, BCs being the most dominant. We are interested in understanding the role played by peptides in the mechanisms underlying the establishment of dominance hierarchies in this prawn. One possibility is that modulatory peptides are differentially expressed in the three morphotypes. We performed SELDI‐TOF mass spectrometry experiments to study protein expression differences among the brains of BC, YC and SC male prawns. Preliminary analysis identified 13 differentially expressed protein peaks. Nine of them differed between BCs and YCs, six between YCs and SCs, and three between BCs and SCs. Data also revealed that five peaks differed across all three morphotypes. These results suggest that there are proteins that are expressed differently across the brains of the three male morphotypes, suggesting they may correlate with the corresponding dominance status. Experiments using 2D‐DIGE and tandem mass spectrometry are being performed to confirm those differentially expressed proteins to then determine whether they play a role in modulating behaviors associated with dominance status.

Supported by: MBRS SCORE S06GM008224, MRISP MH48190, RCMI G12RR03051, MBRS RISE R25‐GM061838, NINDS SNRP U54NS0430311
Pulmonary expression of mechanogated K2P channels with special reference to the neuroepithelial body micro‐environment
Authors: Lembrechts Robrecht , Brouns Inge , Pintelon Isabel , Schnorbusch Kathy , Timmermans Jean‐Pierre , Adriaensen Dirk
Published: June 2021

The two‐pore‐domain K+ (K2P) channels TREK and TRAAK are gated by mechanical stimuli. Recently, we suggested the potential involvement of neuroepithelial bodies (NEBs) in mechanotransduction from the airways. NEBs are structurally well‐defined airway receptors composed of groups of neuroendocrine airway epithelial cells that receive at least two different populations of myelinated vagal afferent nerve terminals. Physiological evidence, but lack of neurochemical characterization of several classes of vagal mechanosensitive airway receptors, prompted us to focus on the expression of mechanogated K2P ion channels in mouse airways and more in particular in the NEB micro‐environment. TREK‐1 immunoreactivity was found in the lungs but seemed to be restricted to airway smooth muscle cells. TRAAK on the other hand appeared to be mainly expressed in the terminals of vagal myelinated nerves, both intraepithelially in NEBs and in so‐called smooth muscle associated airway receptors (SMARs). The observation that the extensive terminals of vagal myelinated afferents in NEBs reveal intrinsic mechanosensitive properties strengthens our hypothesis that, in addition to SMARs, the NEB micro‐environment likely accounts for subpopulations of the electrophysiologically characterized vagal airway mechanosensors.

Support: FWO grants G.0085.04 and G.0081.08 (DA); UA grants GOA BOF 2007 (DA) and KP BOF 2006 (IB)
Comparative scaling analyses of mammalian eye size and brain size
Authors: Segev Tamar G , Heesy Christopher P
Published: June 2021

Studies of the relative sizes of brain components in mammals suggest that areas responsible for sensory processing, including visual processing, are correlated with aspects of ecology, especially diet and activity pattern. However, mammalian eye shapes and sizes do not display similar adaptations to ecological factors. This disparity in ecological adaptations between eye and brain size is especially surprising given the importance of activity pattern, a correlate of light availability, to eye morphology in most other vertebrate classes. Current data suggest that an isometric scaling relationship best describes eye to brain size size scaling in mammals. In this study, we compile the most taxonomically diverse and extensively sampled dataset on eye size and shape, overall brain volume, and activity pattern, in mammals. Results demonstrate that eye and brain sizes are correlated, although regression slopes do not support an isometric relationship. Anthropoid primates are important outliers, demonstrating adaptive size and shape differences in eye morphology. We conclude that in mammals allometry more strongly affects eye to brain size scaling than do ecological factors.

Grant Funding Source

Midwestern University
The HHMI National Genomics Research Initiative ‐ A Model Laboratory Experience
Authors: Jordan Tuajuanda C.
Published: June 2021

The goal of the HHMI National Genomics Research Initiative (NGRI) is to expose novice undergraduates, primarily freshmen, to the process of doing science in the context of an authentic research experiment. The NGRI is a research collaboration between a lead scientist, college and university faculty across the nation who guide the students through the research experiment, and HHMI staff. The students are engaged in a national collaborative network focused on addressing a single research question while they gain hands‐on experience using techniques and tools in microbiology, molecular biology, and bioinformatics. The NGRI represents a model for training future biomedical scientists ‐ a collaborative, multi‐disciplinary, and computational endeavor serving to enhance students' problem‐solving, critical‐thinking, analytical, and communication skills while advancing scientific knowledge. Preliminary assessment data indicate positive impact of this experience on student engagement and retention.
DNaseI‐hypersensitivity and qRT‐PCR analysis of a chromatin region linked to FSH muscular dystrophy
Authors: TSUMAGARI KOJI , Xu Xueqiung , Crawford Greg , Ehrlich Melanie
Published: June 2021

There is much uncertainty about the initial gene impacted by a shortened D4Z4 array at 4q35 in facioscapulohumeral muscular dystrophy (FSHD) patients. Whether 4q35 FRG1, ANT1, FRG2, or DUX4 are overexpressed in FSHD is controversial. Expression microarrays did not reveal a candidate FSHD gene at 4q35. Using DNase‐chip to map DNaseI‐hypersensitive (DH) sites in chromatin, we looked for evidence of unannotated genes at 4q35 that might be involved in FSHD. DH sites can indicate gene regions that are expressed or poised for expression. DH mapping is not weighted toward known genes, protein‐coding genes, or frequently examined cell types, unlike expression microarrays. DH sites were seen at FRG1 and ANT1 in three FSHD and three control myoblast samples but not at FRG2 or the DUX4‐containing D4Z4. Two DH sites far from known genes were seen preferentially in FSHD myoblasts. Most tested amplicons in 14 subregions near these DH sites, which were in a 4q35.2 gene desert, were positive but exhibited no consistent FSHD or myoblast specificity. They gave RT‐dependent products with Ct values ranging from 28‐38, generally ~6‐12 cycles less than for the HPRT reference. Therefore, there was transcription of many sequences even in this gene desert apparently yielding noncoding RNAs, whose biological relevance remains to be determined. (Supported by NIH Grant NS048859 and FSH Society Grant FSHS‐MGBF‐013)
ROC1 and ROC2: Interactions with the Nuclear Variant of Bmp4
Authors: Cordner Ryan D. , Loos Trina J. , Bridgewater Laura C.
Published: June 2021

Bone morphogenetic protein 4 (Bmp4) is a growth factor involved in many developmental processes including limb patterning, forebrain development, and skeletal development. Our lab has recently discovered a novel nuclear variant of Bmp4 (nBmp4). In an effort to determine the function of this nuclear variant, a yeast two‐hybrid screen was performed. The screen revealed that nBmp4 interacts with Regulator of Cullins 1 and 2 (ROC1 and ROC2). ROC proteins are components of E3 ubiquitin ligase complexes, which help confer substrate specificity to regulate the protein degradation process. ROC1 and ROC2 each have three distinct domains: the N‐terminus, the RING finger, and the C‐terminus. A series of deletion constructs was tested in subsequent yeast‐two hybrid assays, which were performed to elucidate the domains within ROC1 and ROC2 that interact with nBmp4. The N‐terminal domains of ROC proteins bind to the cullin component of E3 ubiquitin ligases, and RING finger domains act as binding sites for E2 ubiquitin conjugating enzymes. The results of the yeast two‐hybrid assays suggest that ROC1and ROC2 interact with nBmp4 through their N‐terminal and RING domains, respectively, suggesting that nBmp4 may either function as an E3 ubiquitin ligase, an E2 ubiquitin conjugating enzyme, or it may inhibit the binding of either to ROC proteins. Supported by NIH grant AR48839 and by the Fulton Family Foundation.
Proteasome inhibition modulates protein synthesis: Interplay with PERK, MAP kinase and JUN kinase
Authors: Zhang Le , Ebenezer Philip J. , Keller Jeffrey N.
Published: June 2021

Proteasomal inhibition occurs during aging as well as in many neurodegenerative disorders. Recent studies from our laboratory and others have elucidated the potential for interplay between the proteasome and protein synthesis machinery. In the present study we report on the effects of proteasome inhibition in neural cells. Our studies demonstrate that proteasome inhibition induces reversible inhibition protein synthesis, and that the degree to which cells recover the ability to synthesize proteins following proteasome inhibition is strongly associated with the ability of neural cells to survive the toxicity of proteasome inhibition. Additionally, our studies indicate important roles for PERK, MAP kinase, and JUN kinase in regulating the effects proteasome inhibition has upon neural cells. In particular we will outline the role these kinases play in mediating the ability of proteasome inhibition to impair specific aspects of the protein synthesis machinery, as well as the role these kinases play in allowing cells to successfully recover from the toxicity associated with proteasome inhibition. Taken together, these data have important implications for understanding the basis for protein synthesis and protein turnover during aging as well as in a variety of neurodegenerative disorders associated with proteasome inhibition.

This work was generously supported by grants from the NIA (AG029885, AG025771) to Jeffrey N. Keller
Thermodynamic and Structural Origins for the Thermostability of Taq DNA Polymerase
Authors: Liu Chin‐Chi , Yang Yanling , LiCata Vince J.
Published: June 2021

The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. We have previously shown that at 25oC, Klentaq polymerase (the large fragment domain of Taq) has a folding free energy (δGfold) of ‐27 kcal/mole, making it one of the most stable monomeric proteins yet characterized. In contrast, Klenow polymerase has a δGfold of only ‐4.7 kcal/mole at 25oC, which is quite low for a protein of its size (68kDa). Here we report full stability curves (δG versus temperature) for Klentaq and Klenow, which reveal that Klentaq's extreme free energy of folding originates from a significantly decreased entropic penalty of folding (δSconf). Structural analyses of the denatured‐state ensembles for the two proteins indicate that the source of these energetics is a significantly smaller and less solvent accessible denatured state for Klentaq relative to Klenow. It is also notable that although the average denatured state diameter for Klenow is approximately 1.4X larger than for Klentaq, they both fall within the range for random‐coil behavior.
The Hace1 E3 ligase: linking misfolded protein load and oncogenesis
Authors: rotblat barak , Zhang Fan , Sorensen poul HB
Published: June 2021

Hace1 is a new member of the HECT family of E3 protein‐ubiquitin ligases. We previously showed that the Hace1 gene is a tumor suppressor that is inactivated in human Wilms' tumor, and that Hace1‐/‐ mice develop spontaneous tumors. Moreover, various stresses dramatically increases tumor incidence in these mice. The goal of this study is to characterize the role Hace1 is playing in cellular stress and more specifically in misfolded protein handling by the cell. We now show that Hace1 decreases the half‐life of GFP‐Huntingtin‐128Q and eliminates its aggregates in a proteasome independent manner. This indicates that Hace1 may mediate mutant Huntingtin degradation via other pathways such as autophagy. Indeed, Hace1‐/‐ cells show decreased autophagy induction. Fluorescence microscopy suggests that Hace1 is localized to a recently described cellular structure known as IPOD‐ Insoluble Protein Deposit. Using fluorescence recovery after photo‐bleaching we determined that GFP‐Hace1 interacts with IPOD in a highly dynamic manner suggesting that Hace1 plays a role in the function of IPOD rather than being transported to them as aggregates. Taken together, these data indicate that the tumor suppressor Hace1 reduces accumulation of misfolded proteins by promoting autophagy, suggesting that the deregulation of non‐proteasomal processes that manage misfolded protein load may promote oncogenesis in the cell.
Amyloid‐forming peptides from fungal adhesins
Authors: Ramsook Caleen B , Soybelman Gregory , Fung Raymond , Lipke Peter N
Published: June 2021

Candida albicans Als adhesins bind peptide ligands and mediate cell aggregation with amyloid properties. A 13‐residue peptide, Als5p322‐334, has a 90% β‐aggregation potential and forms amyloids, as do truncated versions of the Als5 protein (Otoo et al, Euk Cell 7: 776‐782, 2008). We therefore searched in other fungal adhesins for β‐aggregation forming sequences with the TANGO algorithm. Two additional adhesion peptides, from C. albicans Eap1p117‐133 and Saccharomyces cerevisiae Flo1p305‐315 have aggregation potentials of 90% and 42% respectively. Peptide interactions were analyzed by circular dichroism, absorbance and fluorescence spectroscopy to monitor secondary structure and amyloid formation. CD spectra showed random coil for both Eap1 and Flo1 peptides in buffer, changing to β‐aggregate with an ellipticity minimum at 230‐235nm after stirring to induce amyloid formation. The stirred solutions of both Flo1 and Eap1 peptides showed an increase in Congo Red absorbance with a shoulder near 550nm, and also had greatly enhanced thioflavin‐T fluorescence. These results with the Flo1 and Eap1 peptides suggest a change from non‐amyloid characteristics when initially dissolved in buffer to amyloid formation with stirring. Therefore, sequences from two additional adhesins show conformational changes leading to amyloid formation. Supported by SC1 GM083756.
Characterization of endothelial argininosuccinate synthase subcellular localization and protein interactions relevant to the function of the citrulline‐nitric oxide cycle
Authors: Corbin Karen Davidowitz , Pendleton Laura C , Shriver Sandra K , Eichler Duane C
Published: June 2021

Argininosuccinate synthase (AS) is an essential mediator of endothelial health by providing a dedicated source of arginine for nitric oxide (NO) production and promoting endothelial cell viability. Our laboratory has previously demonstrated that AS is present in endothelial caveolar fractions along with endothelial nitric oxide synthase (eNOS) and argininosuccinate lyase (AL), the core components of the citrulline‐NO cycle. To expand on those studies, we utilized immunofluorescence microscopy and showed that AS localized to the Golgi, perinuclear region and plasma membrane in conjunction with eNOS. We also demonstrated that AS co‐localizes with caveolin‐1 and HSP90, key regulators of eNOS function. Co‐immunoprecipitation studies showed that AS interacts with caveolin‐1 and HSP90. In the case of caveolin‐1, a binding motif in the AS protein sequence suggested a direct interaction. To characterize the nitric oxide metabolome from a more global perspective, we utilized co‐immunoprecipitation followed by mass spectrometry to identify several putative AS and eNOS interacting partners that are either novel or understudied in the regulation of NO production. Our work highlights the functional co‐localization and interactions of citrulline‐NO cycle components in vascular endothelial cells, which has important implications for our understanding of vascular biology.
Characterization of HIV‐1 Reverse Transcriptase Nucleotide Specificity by Conformationally Sensitive Fluorescence
Authors: Kellinger Matthew William , Piasecki Shawn , Johnson Kenneth Allen
Published: June 2021

Site‐specific labeling of HIV‐1 Reverse Transcriptase (HIVRT) with a conformationally sensitive fluorophore has been optimized to allow for transient kinetic characterization of steps preceding and following chemistry. The labeling did not affect the kinetics of incorporation. Pre‐steady state Quench‐flow data has been globally fit with Stopped‐flow fluorescence data to support a minimal two‐step model for nucleotide binding. Initial ground state binding is followed by a rate limiting conformational change which results in a 10‐fold decrease in the overall Kd for correct nucleotide. Kinetic partitioning of the closed complex then commits the enzyme to chemical incorporation rather than nucleotide release. This work provides the first comprehensive characterization of an induced fit model for HIVRT, and has been used to further understand mechanisms of resistance of 3TC by the M184V HIVRT mutant.
Adhesion of lysozyme and transferrin to Omafilcon A contact lenses
Authors: Keith Edward O. , Chandrasekaran Neena S. , Solanki Darshan H. , Desai Shan S. , Janoff Andrea M.
Published: June 2021

Human tears contain ~60 different proteins that accumulate on contact lenses. We examine the adhesion of human transferrin to FDA Group II Omafilcon A contact lenses, fabricated from a biomimetic material whose interaction with tear proteins is not well characterized and for which the consequences of protein accumulation are unclear. Omafilcon A lenses were incubated in human transferrin for five days, and protein adhesion was determined by bicinchoninic acid colorimetry on a daily basis. Transferrin adhered to the Omafilcon A lenses to a lesser extent (~10%) than it did to Alphafilcon A lenses (also FDA Group II). Our previous data indicate the same is true for lysozyme adhesion, with Omafilcon A lenses adsorbing less lysozyme (~25%) than Alphafilcon A lenses. Levels of transferrin and lysozyme adhesion to Omafilcon A lenses are lower than their levels of adhesion to any other type of contact lenses in all four FDA groups, suggesting that Omafilcon A lenses are better able to resist protein adhesion than contact lenses fabricated from other materials.

Supported by a NSU President's Faculty Scholarship Award, the NSU Health Professions Division, and the Farquhar College of Arts and Sciences at NSU.
Phosphoinositide‐mimicking peptide sequences are binding targets for PH domains
Authors: Mendrola Jeannine , Lemmon Mark A
Published: June 2021

Pleckstrin homology (PH) domains are the 11th most common domain in the human proteome and are best known as phosphoinositide (PI) targeting modules. However, less than 10% of PH domains recognize PIs with high affinity and specificity raising the question of what is the target of the other 90%. We have identified peptides enriched in acidic or phosphoserine residues as potential protein targets for PH domains. Nearly 40% of all S. cerevisiae PH domains were able to recognize the highly acidic protein Nsr1p in pulldown experiments, making this a more common property than high affinity PI binding. We also showed binding of GST‐fusions of PLC‐δ1 or yeast Boi2p PH domains to a multiphosphorylated peptide of IRBIT with estimated KD values of 0.3μM and 8.4μM respectively. PH domain mutations that disrupted PI binding also abrogated IRBIT binding. We hypothesize that stretches of acidic residues and/or phosphoserines structurally mimic inositol phosphates/phosphoinositide headgroups, and thus bind to at least a subclass of PH domains. Such 'phosphoinositide mimicry' could represent a previously undescribed molecular recognition mechanism for driving specific PH domain/protein interactions.
The Screening of PDZ‐Domain Array to Identify JAM‐B Interacting Proteins
Authors: Samant Ritika , Naik Ulhas
Published: June 2021

Tight junctions (TJs) host complexes that include PDZ domain‐containing proteins. PDZ proteins participate in transmembrane molecule organization and TJ formation. Transmembrane protein Junctional Adhesion Molecule‐A (JAM‐A) is found in TJs and interacts with PDZ proteins AF‐6 and ZO‐1 via its PDZ binding motif. Another JAM family member, JAM‐B displays such a motif but PDZ binding partners are unknown. Because of the similar roles and cytoplasmic regions of JAM‐A and JAM‐B, we hypothesize that JAM‐B also binds to PDZ proteins.

To identify these interactions, HA‐tagged JAM‐B will be used to probe a protein microarray spotted with PDZ domains. A cDNA construct encoding a HA‐tagged GST JAM‐B protein was created. E. coli BL21 cells transformed with this construct were induced by IPTG, and recombinant protein expression and solubility was analyzed by SDS‐PAGE. JAM‐B ‐ PDZ domain interactions on the array will be detected by chemiluminescence. This analysis of JAM‐B and PDZ protein interactions will further our understanding of signaling pathways particularly pertaining to TJ formation and regulation. Supported by an HHMI Undergraduate Science Education Grant, Beckman Foundation and the NIH.
D‐AKAP2 interacts with Rab4 and Rab11 through its RGS domains and regulates transferrin recycling
Authors: Eggers Christopher T. , Schafer Jenny C. , Goldenring James R. , Taylor Susan S.
Published: June 2021

Dual‐specificity A‐Kinase Anchoring Protein 2 (D‐AKAP2/AKAP10) has been linked genetically to heart disease and breast cancer, but its biological function has remained unknown. Besides PKA‐ and PDZ‐binding motifs, D‐AKAP2 has two tandem Regulator of G protein Signaling (RGS) domains with no known binding partners. In HeLa cells, D‐AKAP2 is primarily cytosolic but can localize to membranes of the endocytic recycling compartment where it co‐localizes with endocytosed transferrin. Rab4 and Rab11, two small GTPases known to regulate membrane recycling, co‐localize with D‐AKAP2 and recruit it to endosomes. Co‐expression of D‐AKAP2 and Rab11 causes both proteins to accumulate on enlarged endosomes. Co‐immunoprecipitation and co‐localization studies demonstrated that D‐AKAP2 forms protein complexes through the RGS domains with both Rabs, preferring GTP‐locked Rab4 and wild‐type Rab11. Yeast two‐hybrid analysis confirmed that the tandem RGS domains were sufficient for interactions with both Rabs. Knockdown of D‐AKAP2 by RNA interference causes the Rab11 compartment to become more condensed, tubular, and peripheral. These changes correspond to a significant increase in the rate of transferrin recycling, suggesting that the RGS domains of D‐AKAP2 act as novel Rab effectors that inhibit recycling from the Rab4/Rab11 compartment. This work was funded by the NIH (DK054441) and the American Cancer Society.
Quantitative intensity/area, semi‐automated controls for multiplex immunofluorescence tissue‐based assays
Authors: Hamann Stefan , Vengrenyuk Yevgen , Capodieci Paola , Teverovskiy Mikhail , Mesa‐Tejada Ricardo
Published: June 2021

After developing two 5‐antigen multiplex (Mx) immunofluorescence assays using qualitative tissue controls in formalin‐fixed, paraffin‐embedded (FFPE) prostate cancer (CaP) tissue sections, we proposed to optimize control evaluation by quantification of signal‐derived features. Three CaP cell lines (LNCaP, PC‐3, DU‐145) were used as substrates for a nuclear antigen in each Mx [androgen receptor (AR) and Ki‐67]. Cells suspended in agar were FFPE as arrays (CLA) containing all three cell lines. Mx results are recorded by a computer‐driven microscope system with a Nuance(tm) camera (CRI, Woburn MA) and filters to capture serial images 10 nm apart (wave length range: 520 ‐ 720 nm) including emission spectra for all fluors used. Grey scale images identifying antigen distribution by its spectral profile are created by unmixing fluor signals in each image series. High throughput image analysis scripts and algorithms previously developed to interrogate tissue sections were adapted to measure intensity/area features of AR or Ki‐67 in CLA nuclei (classified as "DAPI objects"), expressed as (1) mean intensity of all cells, and (2) mean and (3) percentage of positive cells above a threshold derived from negative control sections. Results from a test run are thus validated by control metrics within established antigen/cell line ranges, an objective approach surpassing in accuracy and efficiency subjective visual criteria.
Roles of Tyrosine322 and Tyrosine324 in GnRH Receptor Function
Authors: Feng Hao , Leung Po Ki , Hu Lian , Krsmanovic Lazar Z , Catt Kevin J
Published: June 2021

GnRH receptor binding properties and cAMP signaling were analyzed in GT1‐7 cells expressing the endogenous GnRH‐R, and in COS‐7 cells transfected with the mouse GnRH‐R. Agonist stimulation of the wild type GnRH‐R expressed in COS‐7 cells using a pGFP2‐N vector caused a monotonic dose‐dependent increase in cAMP production. Deletion of Tyr322, or change of Tyr322 to Ser, significantly increased GnRH binding capacity of the mutant receptor (3‐5 fold). In such receptors, GnRH‐induced cAMP production was abolished, indicating the absence of Gs activation. GnRH‐activated phosphorylation of mitogen‐activated protein kinases (MAPKs) was also abolished in the mutant receptor, consistent with impairment of the protein kinase C (PKC)‐dependent signaling pathway. The GnRH‐R binding capacity was abolished when Tyr322 was substituted with Phe. Substitution of Tyr324 of the mouse GnRH‐R with non‐aromatic hydroxyl group‐containing Ser caused loss of agonist binding. In contrast, when Tyr324 was replaced with Phe the mutant receptor retained high binding capacity and cAMP signaling comparable to that of the wild type GnRH‐R. These findings demonstrate the importance of both Tyr322 and Tyr324 of the mouse GnRH receptor as crucial residues that determine its agonist binding and signaling properties.
Impact of Vitamin D Receptor‐Mediated Signaling and Epithelial‐Stromal Interactions on Tumor Progression in the LPB‐Tag Mouse Model of Prostate Cancer
Authors: Mordan‐McCombs Sarah Elizabeth , Gaupel Ann‐Christin , Welsh JoEllen , Tenniswood Martin
Published: June 2021

The role of stromal‐epithelial (S‐E) interactions has been well characterized in the developing prostate, and is known to be dependent on the presence of a stromal androgen receptor (AR). We have utilized the LPB‐Tag mouse model of prostate tumor progression to study the role of S‐E interactions that occur in tumor development. We have crossed this transgene onto a vitamin D receptor (VDR) null background to elucidate cross‐talk between the VDR and AR and the role of testosterone in these interactions. Our data suggest that signaling through the VDR abrogates tumor progression in animals with low circulating testosterone; however this regulation is lost in androgen replete mice. To identify components of AR signaling that impinge on VDR‐mediated tumor cell growth, we isolated the stromal and epithelial compartments of the prostate by laser capture microdissection (LCM) in 15 week old mice with mid‐grade prostatic neoplasias. Total RNA was isolated from the LCM samples and subjected to microarray analysis. Bioinformatic analysis of the changes in stromal and epithelial gene expression and alterations in gene expression due to VDR ablation suggest that the VDR modulates the expression of several genes involved in cell cycle arrest (p21), cell proliferation (IGF, IGFR, PCNA), and apoptosis (survivin). This research is supported by R01 CA101114 to JW and MT.
Examination of the structure and dynamics of the calcium‐binding protein S100A5
Authors: Liriano Melissa Ana , Varney Kristen M. , Wright Nathan T. , Wilder Paul T. , Charpentier Thomas H. , Weber David J.
Published: June 2021

The S100 family is a class of small, homodimeric proteins that are often characterized by their Ca2+‐dependent biological effects, which is typically the result of a calcium‐dependent conformational change. To characterize whether S100A5 undergoes a conformational change similar to that observed in other S100 proteins, we will first determine the high resolution structure of this protein by NMR in the absence and presence of calcium. The HSQC spectras we have obtained of S100A5 depicts vast chemical shift pertubations from no calcium to calcium‐loaded S100A5, which is highly suggestive of a conformational change. S100A5 shares approximately 50% sequence homology with all the other S100 proteins yet it binds to Ca2+ with an affinity 10‐100 times greater then any other S100 member. We hypothesize that the high affinity of S100A5 is due to either a structural difference between S100A5 and other S100 proteins or that A5 lacks the dynamic properties observed in the C‐terminal region of other S100 proteins that could contribute to their lesser affinity for Ca2+. In order to elucidate which mechanism is the basis of A5's high affinity for Ca2+, we propose to also solve the Ca2+‐bound S100A5 using X‐ray crystallography to determine if there is an additional Ca2+ ion ligand present. Internal dynamics will also be measured using 15N NMR relaxation methods to prove whether or not S100A5 lacks dynamic properties in the C‐terminal EF‐hand.

Student support provided by an NIGMS Initiative for Maximizing Student Diversity Grant (R25‐GM 55036)
Comparison of the subcellular localization of phosphodiesterase (PDE4) of normal and dystrophic mouse skeletal muscle
Authors: Langdon Casey G. , Bloom Timothy J.
Published: June 2021

There are four genes that encode members of the cyclic nucleotide phosphodiesterase 4 (PDE4) family. Earlier work in the lab has shown that there is a decrease in particulate PDE4 activity between 5 and 15 weeks of age in skeletal muscle from the muscular dystrophy model mdx. Western blot bands in particulate fractions from mdx muscle show an age‐related decrease in PDE4 protein. In this study, PDE4 was examined immunohistochemically to determine whether the expression changes correspond to an alteration in subcellular localization. Skeletal muscle from hind legs was harvested and frozen in Neg‐50 tissue sectioning media, and sectioned in 10 µm thick longitudinal and cross‐sections. Sections were probed with an antiserum specific for PDE4A or an antiserum that recognizes multiple PDE4 families. Bound antibodies were visualized by confocal microscopy using a fluorescently‐tagged secondary antibody. The anti‐PDE4A antibody showed staining at the sarcolemma of 5 week‐old normal and mdx muscle. This staining was retained through 15 weeks. Imaging with the non‐selective PDE4 antiserum showed a stronger signal for PDE4 localized along the sarcolemma in 5 week‐old normal muscle compared to mdx muscle. These observations suggest that a PDE4 other than PDE4A is responsible for the observed age‐related differences in PDE4 expression seen in mdx muscle. This project was supported by NIH Grant 1R15AR050408‐01A2 from NIAMS.
Effects of isoangustone A isolated from hexane/ethanol extract of Glycyrrhiza uralensis (HEGU) on cell cycle progression in DU145 human prostate cancer cells
Authors: Seon MiRa , Hong Ji Eun , Lim Soon Sung , Kim Jong‐Sang , Park Jung Han Yoon
Published: June 2021

Glycyrrhiza uralensis (licorice) has been shown to exhibit various pharmacological effects including antivirus, anti‐allergic and anti‐tumor effects. However, chronic consumption of high amounts of glycyrrhizin in licorice is known to induce undesirable hypermineralocorticoid syndromes, such as hypertension and hypokalemia. We previously prepared a hexane/ethanol extract of Glycyrrhiza uralensis (HEGU) which lacks glycyrrhizin and observed that HEGU induced apoptosis and cell cycle arrest in DU145 human prostate cancer cell growth. This study examined the effects of the active compound isoangustone A in HEGU on cell cycle progression of DU145 cells. Isoangustone A was obtained by subjecting the HEGU to a silica gel column and eluting by n‐hexane‐ethyl acetate gradient systems. Isoangustone A (2.5 ‐ 7.5 mg/L) dose‐dependently decreased the viable cell numbers and induced a G1 phase arrest. Isoangustone A reduced the levels of cyclin‐dependent kinase (CDK) 2, CDK 4, cyclin A, and cyclin D1 proteins. However, isoangustone A did not affect the levels of the cyclin E protein. These results indicate that isoangustone A induces G1 phase arrest in DU145 human prostate cancer cells by inhibiting CDK activities.
A Study on the glucose‐regulating Enzyme Activities of Water Extracts from Korea Medicinal Herbs
Authors: Choe Myeon , Kim Dae Jung , Chung Mi ja , Seo Dong Joo , You Jin Kyoun
Published: June 2021

This study investigated the anti‐diabetic effects of water extracts from Korea medicinal herbs on hepatic glucose‐regulating enzymes, such as acetyl‐CoA carboxylase (ACC) and glucokinase (GK). The hepatic cytosol faction of a type ? diabetic animal (GK rat, Goto‐kakizaki) was used in acetyl‐CoA carboxylase (ACC) and glucokinase (GK) activity assays. Type 2 diabetes generally characterized by non‐insulin‐dependent hyperglycemia in early stages and the therapeutic approach available is by retarding absorption of glucose through the inhibition of α‐glucosidase enzyme in the intestinal. Thus α‐glucosidase inhibition assay was assayed. The Korea medicinal herbs were Salvia miltiorrhiza (DS), , Panax notoginsengs (JC), Lycium chinense (JGP), Dioscorea japonica (SY), R. glutinosa (SJH), Pyrus pyrifolia fructus (YSB), Cucumis melo var. makuwa stem (CWJ), Coix lachrymajobi var. mayuen (YM), Cornus officinalis (SSY), Paeonia suffruticosa (MDP), Cordyceps sinensis (DCH), Acanthopanax senticosus (GSO). The ACC activity in cytosol fraction of liver was higher in the 0.01mg/ml DCH water extract than that of the control and SSY water extract significantly increased the GK activity. YSB, MDP, GSO water extracts had high α‐glucosidase inhibitory activity and the effect was higher than that of acarbose (reference α‐glucosidase inhibitor).

These observations suggest that DCH, SSY, YSB, MDP and GSO water extracts are the effective candidates for preventing type 2 diabetes.
Frozen pickle juice reduces mealtime glycemia in healthy adults
Authors: Johnston Carol S , Appel Christy L
Published: June 2021

The antiglycemic effect of 20 g vinegar ingested at mealtime is well characterized; yet chronic vinegar ingestion is impracticable due to esophageal discomfort and astringent taste. Vinegar is more palatable when incorporated in a food matrix. This trial examined whether a frozen pickle juice product possessed antiglycemic properties. Healthy, non‐diabetic adults (13 F, 3 M; BMI: 25.4±1.4 kg/m2; age: 29.4±2.8 y) consumed frozen pickle juice (56 g, Pickle Sickle.com LLC; Seguin TX) or sugar free popsicles (71 g, Fla·Vor·Ice Light; Jel Sert Company, West Chicago IL) immediately prior to a carbohydrate load (1 buttered bagel and 6 oz fruit juice) in a randomized, crossover fashion with two weeks separating treatments. Fasting and postprandial blood samples were collected for glucose analysis. The incremental peak glucose concentration at 30 min post‐meal was reduced 42% with frozen pickle juice ingestion as compared to control (p=0.05). Postprandial glycemia (incremental area‐under‐the‐curve) was reduced by the pickle juice treatment at 0‐60 and 0‐120 min as compared to control (‐46%, p=0.038 and ‐39%, p=0.074). These data indicate that pickled food products possess antiglycemic effects similar to vinegar in healthy adults. Foods containing vinegar may help pre‐diabetics and diabetics manage their condition and may be considered functional foods. This research was supported by the ASU Nutrition Research Fund.
Are immunoglobulin concentrations associated with body composition of adolescents in Mexico?
Authors: Valdes‐Ramos Roxana , Zúñiga‐Torres Guadalupe M , Marrtínez‐Carrillo Beatriz E , Pardo‐Morales Rosa V , Wârnberg Julia , Marcos Ascensión , Benítez‐Arciniega Alejandra D
Published: June 2021

Objective

To analyze the association between serum immunoglobulins A, G and M, and body composition of adolescents from the State of Mexico.

Methods

Informed consent from parents and assent from adolescents was obtained, for the evaluation of 114 subjects from the Capital city of Toluca and the adjacent semi‐urban county of Lerma de Villada. Height, weight, waist and hip circumference, as well as tricipital and subscapular skinfolds were measured by trained personnel; BMI and waist/hip ratio were calculated, while body fat was calculated from skinfolds and measured by bioimpedance. Immunoglobulin concentrations were determined by nephelometry. Data were analyzed by independent samples t‐test and Pearson correlations.

Results

Subjects were mainly females (71.2%), with a mean age of 16.8 years (+ 0.8). No differences were found between subjects with normal weight or body fat and those with overweight, central obesity or excess body fat. Females had higher immunoglobulin concentrations than males (IgG t=2.24, p= 0.027; IgA t= 2.05, p=0.043; IgM t=4.49, p<0.001). Positive correlations were found for IgA with tricipital skinfold thickness (r= 0.192, p=0.041); for IgM with tricipital skinfold thickness (r= 0.221, p=0.018) and for body fat by bioimpedance (r= 0.243, p=0.009).

Conclusions

Immunoglobulin concentrations were not affected by overweight, obesity, excessive or central body fat in adolescents.