bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement of Muscle, Tendon, Ligament, Cartilage, and Bone Protein Synthesis Rates In Vivo in Humans
Authors: Smeets Joey , Horstman Astrid , Vles George , Emans Pieter , Goessens Joy , Gijsen Annemie , van Kranenburg Janneau , van Loon Luc
Published: June 2021

Introduction

Skeletal muscle plasticity is reflected by a dynamic balance between protein synthesis and breakdown, with basal muscle protein synthesis rates typically ranging between 0.04 and 0.06 %/h. Though it is evident that other musculoskeletal tissues should also express some level of plasticity, protein synthesis rates of most of these tissues have never been assessed. The present study applies contemporary stable isotope methodology to assess basal muscle, tendon, ligament, bone, as well as cartilage protein synthesis rates in vivo in humans.

Methods

Six otherwise healthy patients (62±3 y), scheduled to undergo unilateral total knee arthroplasty, were included in this study. Primed continuous intravenous infusions with L‐[ring‐13C6]‐Phenylalanine were initiated 2.5 h prior to surgery and continued during surgery. Throughout the surgical procedure the following tissue samples were obtained: muscle, tendon, cruciate ligaments, cartilage, bone, menisci, fat and synovium. Tissue‐specific fractional protein synthesis rates (%/h) were assessed by measuring the incorporation of labelled L‐[ring‐13C6]‐Phenylalanine in tissue protein and were compared with muscle tissue protein synthesis rates using a paired t test.

Results

Protein bound L‐[ring‐13C6]‐Phenylalanine enrichments did not differ substantially between the different musculoskeletal tissues and skeletal muscle, except for notch bone tissue (P<0.01). Highest enrichment levels were observed in synovium derived protein (0.029±0.008 MPE) and lowest enrichment levels in patellar bone tissue protein (0.005±0.002 MPE). Tendon, bone, cartilage, fat, anterior cruciate ligament, posterior cruciate ligament, and menisci tissue protein synthesis rates averaged 0.06±0.01, 0.03±0.01, 0.04±0.01, 0.11±0.03, 0.07±0.02, 0.04±0.01, and 0.04±0.01 %/h, respectively, and did not significantly differ from skeletal muscle protein synthesis rates (0.04±0.01 %/h; P>0.05). Synovium derived protein and notch bone tissue protein synthesis rates were respectively higher and lower compared to skeletal muscle protein synthesis rates (P<0.05 and P<0.01, respectively).

Conclusion

Basal protein synthesis rates in various musculoskeletal tissues are within the same range of skeletal muscle protein synthesis rates, with fractional muscle, tendon, bone, cartilage, ligament and menisci tissue protein synthesis rates ranging between 0.02 and 0.13 % per hour.

Support or Funding Information

This study received no specific grant from any funding agency.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deubiquitinase UCHL1 Regulates Myogenesis in Skeletal Muscles
Authors: Gao Hongbo , Wu Penglong , Wang Xuejun , Li Yifan
Published: June 2021

Myogenesis is crucial for skeletal muscle regeneration in injury and disease. This multi‐step process is closely regulated by several myogenic factors. Timely activation and inactivation of these factors require ubiquitin‐proteasome system (UPS)‐mediated protein degradation. In this aspect, deubiquitinases (DUBs) could also play a role in myogenesis. Ubiquitin C‐terminal hydrolase L1 (UCHL1) is a DUB that is highly expressed in the brain. Though undetectable in adult skeletal muscles, UCHL1 protein level is significantly increased in injured muscles. However, the functional significance of UCHL1 in skeletal muscles is unknown. Using in vitro and in vivo approaches, this study tested a hypothesis that UCHL1 can play a role in skeletal muscle myogenesis and regeneration.

Our data show that UCHL1 protein level was gradually downregulated during myoblast C2C12 differentiation. Knockdown of UCHL1 expression significantly reduced C2C12 cell proliferation. In contrast, UCHL1 knockdown markedly accelerated cell differentiation, evidenced by more myotubes in UCHL1 deficient cells, which was accompanied by significant upregulation of myogenic factors.

In mouse muscles, UCHL1 was expressed by type I and IIa muscle fibers. Denervation increased UCHL1 protein levels in muscle fibers, which was completely abolished in muscles from the mice with UCHL1 skeletal muscle specific knockout (smKO). smKO mice showed reduced satellite cells in soleus and declined type I fiber number. Moreover, smKO muscles exhibited right shift of fiber distribution and a remarkable loss of type I muscle fiber upon denervation as compared with wildtype ones. These observations further suggest the potential involvement of UCHL1 in myogenesis for the regeneration of type I fibers.

Support or Funding Information

NIH grants No. 1R15HL118696 and 1R03AG051926 and USD SSOM BBS bridge grant, CBBRe pilot grant, PQCD pilot grant.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Remote Transplantation of Human Adipose‐derived Stem Cells Induces Regression of Cardiac Hypertrophy by Regulating the Macrophage Polarization in Spontaneously Hypertensive Rats
Authors: Lee Tsung‐Ming , Yang Chen‐Chia
Published: June 2021

Left ventricular hypertrophy in hypertension has prognostic significance on cardiovascular mortality and morbidity. Cardioprotection by stem cell therapy can be evoked through extracardiac paracrine approaches. Recently, we have shown that butylidenephthalide (BP) improves adipose‐derived stem cell (ADSC) engraftment via attenuated reactive oxygen species (ROS) production. This prompted us to investigate whether remote transplantation of stem cells confers attenuated left ventricular hypertrophy at an established phase of hypertension. Male spontaneously hypertensive rats (SHRs) aged 12 weeks were randomly allocated to receive hamstring injection of vehicle, ADSCs, and BP‐preconditioned ADSCs for 8 weeks. As compared with untreated SHRs, naïve ADSCs decreased the ratio of left ventricular to body weight, cardiomyocyte cell size, and collagen deposition independent of hemodynamic changes. These changes were accompanied by attenuated myocardial ROS production and increased p‐STAT3 levels. At day 3 after implantation into the right hamstring muscle, SHR treated with vehicle was associated with increased myocardial M1 macrophage infiltration, which can be inhibited by either naïve ADSCs or BP‐preconditioned ADSCs. However, compared with naïve ADSCs, BP‐preconditioned ADSCs provided a further decrease of ROS and left ventricular hypertrophy and an increase of local ADSC engraftment, STAT3 phosphorylation, STAT3 activity, STAT3 nuclear translocation, myocardial IL‐10 levels and the percentage of M2 macrophage infiltration. SIN‐1 or 3SI‐201 reversed the effects of BP‐preconditioned ADSCs increase on myocardial IL‐10 levels. Furthermore, SIN‐1 abolished the phosphorylation of STAT3, whereas superoxide levels were not affected following the inhibition of STAT3. Our results highlighted the feasibility of remote transplantation of ADSCs can be considered as an alternative procedure to reverse cardiac hypertrophy. BP‐pretreated ADSCs polarize macrophages into M2 immunoregulatory cells more efficiently than naïve ADSCs via ROS/STAT3 pathway.

Support or Funding Information

nil

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Conceptual Framework for the Core Concept of Cell Membrane
Authors: Michael Joel , Modell Harold
Published: June 2021

The core concepts of physiology are descriptions of phenomena that have great explanatory power in every area of physiology. They are “big ideas” that are comprised of many smaller ideas. The process of “unpacking” a core concept yields a hierarchically arranged statement of the sub‐ideas that we have called a conceptual framework (CF). Our goal has been to produce CFs that will facilitate teaching and learning physiology.

We have unpacked three of the core concepts (homeostasis, flow down gradients, and cell‐cell communication) and validated the resulting conceptual frameworks with feedback from the physiology teaching community; this work has been reported in Advances in Physiology Education.

Here we present our conceptual framework for the core concept of cell membrane. We have chosen to describe both the structure of the membrane and the functions of the various components making up the membrane. Cells and their membranes are, of course, present in every part of the organism.

Preliminary feedback from a small number of physiology teachers suggest that our unpacking has captured those ideas that can be used as tools by both teachers of physiology and their students.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Spatial Patterning of Scleral Papillae in the Embryonic Chicken Eye
Authors: Giffin Jennifer L. , Hayes Molly , Andrews Daniel DT. , Zinck Nicholas W. , Franz‐Odendaal Tamara A.
Published: June 2021

The anterior portion of the eye in birds is supported by a ring of bony elements within the sclera, constituting the scleral ossicles. These intramembranous bones are induced by a series of transient papillae in the overlying conjunctival epithelium during embryonic development. A well‐defined spatiotemporal pattern of formation is followed, resulting in a complete circle of 13–16 evenly‐spaced papillae per eye in the chicken embryo. The regular distribution and variable number of papillae in this system give it a strong similarity to other reaction‐diffusion models of patterning, such as feather and carapace formation, in which short‐range activation is coupled with long‐range inhibition. Therefore, the objective of this study is to investigate whether scleral papillae are formed through a reaction‐diffusion mechanism in the embryonic chicken eye. To carry out this study, chicken embryos at Hamburger and Hamilton stages 28–34 (5.5–8 days post fertilization) were collected, corresponding to the induction and formation of a complete set of scleral papillae. Quantitative morphometrical analyses were performed on dissected eyes, confirming that while eye size increases throughout development, the spacing between the papillae does not. Computer modelling using the Meinhardt‐Gierer equation indicates that scleral papillae development likely follows the typical Turing mechanism of reaction‐diffusion pattern formation. Quantitative PCR on discrete papillary and interpapillary regions will enable us to study the interactions of known signalling molecules including Hedgehog (Hh), Bone Morphogenic Proteins (BMPs), Fibroblast Growth Factors (FGFs) and Wingless (Wnt). The results of this study provide considerable insight into the spacing pattern of scleral papillae. A reaction‐diffusion mechanism explains the ability of scleral papillae to maintain their even distribution regardless of differences in numbers among species or between left and right eyes in the same individual. Identifying the signalling pathways implicated in scleral papillae spacing would further enhance our understanding of not only the position, but also the size and shape of the scleral ossicles as compensation for gaps in the sclerotic ring is known to occur.

Support or Funding Information

This research was funded by the Natural Sciences and Engineering Research Council of Canada.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional and 3D Ultrastructural Characteristics of a Cement Gland in a Giant Danio (D. cf. malabaricus)
Authors: Coffing Gabrielle C. , Nelson Hannah M. , Hester Kamil , Veillard Maeva , Ostreicher Samantha , Higginbotham Clay , Tomamichel Wendy , Lafontant Pascal
Published: June 2021

Cement glands are transient ectodermally‐derived adhesive organs found in the larval stage of several aquatic species. We have identified a transient structure on the apical aspect of a giant danio (Devario cf. malabaricus) head, a member of the danioninae subfamily of cyprinids. We hypothesize that this structure is a cement gland (CG), the primary adhesive organ of the giant danio (GD), and that its cellular components support its function. Using a novel adhesion assay, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and serial block face‐scanning electron microscopy (SBF‐SEM), we investigated the function of the CG and the structures of the cells that constitute it. Our studies have shown that the gland produces a glycoconjugates‐containing mucus that allows larvae to attach to surfaces in their environments. The larvae remain attached until day 5 when they begin swimming. Lectin staining and SEM revealed that the CG is composed primarily of goblet and epithelial cells organized in a lattice‐like pattern. These studies also show that the CG disappears by week 2. TEM confirms that the CG consists of elongated goblet cells containing granules of varying electron densities. These cells are framed apically by intercalated epithelial cells, and basally by a layer of squamous epithelium. Finally, we used SBF‐SEM and Amira 6 to reconstruct the three dimensional spatial relationship of these cells. To our knowledge, this is the first functional and ultrastructural study of a CG in a danioninae. Our results suggest that the cement gland is an important organ in GD development.

Support or Funding Information

DePauw FDC, SRF, Faculty Fellowship

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anatomy of the Respiratory System of the African Grey Parrot (Psittacus erithacus)
Authors: Lawson Adam B. , Echols M Scott , Schachner Emma R.
Published: June 2021

The morphology and function of the avian respiratory system has been studied and debated for over a century due to its distinct features, such as: unidirectional airflow, aerodynamic valves, and a volume‐constant lung ventilated by non‐vascularized, compliant air sacs. Intra‐ and interspecific variation in the shape, size, and number of avian bronchi and ventilatory air sacs has been described from gross dissections and latex injections, but little has been done to describe the three dimensional (3D) morphology and variation of the avian respiratory system in situ, largely due to the destructive nature of dissection. This research aims to generate a detailed anatomical description and 3D digital models of the lower respiratory system of the African grey parrot (Psittacus erithacus). Four micro‐computed tomography (micro CT) datasets of deceased P. erithacus specimens were segmented to produce 3D tetrahedral surface meshes of the pulmonary tree, lung surfaces, air sac surfaces, and skeleton. The anatomical models were then validated via latex injections. Bronchial tree measurements of P. erithacus (n=8) were collected for intraspecific and interspecific comparisons with two other well‐studied archosaurian taxa: the ostrich (Struthio camelus, n=10) and the American alligator (Alligator mississippiensis, n=10). Similarities in the relationships between the carina and the first four large secondary airways of the bronchial tree suggest a role in maintaining unidirectional airflow patterns in these taxa that would have been present in a common ancestor. In some P. erithacus specimens, the cervical air sacs were found to have a broad, thin, and sub‐sternal expansion that extended caudally to encapsulate more than half the ventral abdominal viscera. In another specimen, the right and left abdominal air sacs merged along the midline to form a single, common abdominal sac. These results suggest that the shape and extent of the air sacs in P. erithacus are more variable than previously described for other avian taxa. The models, which will be made available as a detailed 3D digital anatomical atlas through the Grey Parrot Anatomy Project, will likely become an invaluable resource to avian veterinarians for surgical planning.

Support or Funding Information

Association of Avian Veterinarians (AAV) Research Grant Award

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Average Diameter of Fibrous Bundles Connecting the Optic Nerve to the Optic Sheath Decreases with Increased Intracranial Pressure
Authors: East Lauren , Newell Maegan , Awalgral Parth , Earnest Brittany , Heger Ian , Gibson Robert W. , Lyon Matthew
Published: June 2021

Objective

The objective of this study was to evaluate the effect of increased intracranial pressure (ICP) on the fiber bundles connecting the optic nerve to the optic sheath in a swine model.

Methods

Increased ICP was induced in an anesthetized swine model by passing a catheter through a drilled opening into the epidural space. ICP was monitored continuously. Post euthanasia and enucleation of the swine eyes, a section of optic nerve sheath, with the intact nerve, was removed from both the control and experimental swine model. The tissue was fixed overnight in paraformaldehyde and glutaraldehyde in a sodium cacodylate buffer, postfixed in osmium tetroxide in a sodium cacodylate buffer, dehydrated with a graded ethanol series, and critical point dried with carbon dioxide. The dried control and experimental model sections were mounted and imaged using a scanning electron microscope (SEM). Images of the trabecular meshwork were taken radially around the optic nerve at several magnifications. The images were segmented and analyzed using the ImageJ plug‐in DiameterJ. Segmentation quality was confirmed using image subtraction of the original image from the segmented image and vice versa. All images had false positive and false negative total percentages of less than 5% of all pixels. Image overlays of the segmented and original images confirmed this qualitatively.

Results

Mean and median fiber bundle diameters were recorded for a total of 61 images. 30 (49.1%) of the images analyzed were from the control sample and 31 (50.9%) were from the experimental sample. An analysis of variance found that both the mean (.52 μm) and median (.62 μm) fibrous bundle diameters were significantly different (smaller) for the experimental model as compared to the control (.73 μm and .85 μm) respectively (p<.0001). The relationship between mean and median fibrous bundle diameters and sample (control or experimental) was not significantly correlated to radial location (relative to the optic nerve) or magnification of the image based on analyses of covariance.

Conclusion

This research demonstrates that in a swine model the fibrous bundles of the optic sheath's connections to the optic nerve are statistically different (smaller) after exposure to increased ICP. Future research will evaluate if the thinning is caused by increased ICP, by increasing the sample size and using other methods to induce increased ICP. Future research may impact the current understanding of traumatic brain injury.

Support or Funding Information

Medical Scholars Program, Medical College of Georgia at Augusta University, Summer 2017

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Morphological and morphometric study of the cervical vagus nerve myelinated fibers in acute and chronic experimental diabetes.
Authors: Fontanesi Lucas Baptista , Ferrão Ana Clara Miotello , Neri Letícia Oliveira , Fazan Valéria Paula Sassoli
Published: June 2021

The literature is scarce on pathological studies of the vagus nerve in patients known to have chronic autonomic diabetic neuropathy. Most of the morphological studies of the vagus nerve in experimental diabetes involve the study of the abdominal segment of this nerve or its mesenteric branches. In these studies, the authors demonstrate a presence of diabetic neuropathy and characterize the dying back process of this neuropathy. However, the morphological lesions described by these authors are exclusive of the unmyelinated fibers, since in these segments, the vagus nerve no longer presents myelinated fibers. The objective of the present study was to investigate the existence of morphological and morphometric alterations of the cervical vagus nerve myelinated fibers in acute and chronic experimental diabetes. Male Wistar rats received a single injection of streptozotocin (40mg/kg) 15 days (n = 6) or 12 weeks (n = 6) prior to the experiments. Control rats (n = 6) received equal volume of citrate buffer solution. Proximal and distal segments of the cervical vagus nerves had their fascicle and myelinated fibers and respective axons measured with the aid of computer software. The morphometric data comparison was performed between segments in the same nerve, sides and groups and differences were considered significant when p <0.05. Our results show that there are alterations in the morphology and morphometry of myelinated fibers and axons of the vagus nerve in diabetic animals, especially in their distal segments in both experimental groups. However, in the chronic diabetic group the alterations were more evident and committed the proximal segments. Thus, the results suggest a presence of axonal neuropathy in the vagus nerve due to experimental diabetes, and that alterations were progressive in a time dependent manner.

Support or Funding Information

FAEPA, CAPES and CNPq

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Validation Studies of CRF Receptor 1 as a Target for AD
Authors: Nguyen Kevin Hoang , Ellisman Maya , Patanapirom Jasmine , Sarsoza Floyd , Rissman Robert
Published: June 2021

Alzheimer's disease (AD) is the most common form of dementia and is defined by two neuropathologic hallmarks: senile plaques and neurofibrillary tangles, respectively composed of β‐amyloid (Aβ) and phosphorylated tau protein. Very few incidences of AD are developed from genetic mutations. A majority of cases arise sporadically, and are thought to result from the interplay of genetic and environmental factors. Corticotropin‐releasing factor (CRF) signaling in AD, when exacerbated by stress, has been shown to influence AD pathology. With an aging population of veterans and individuals with stress‐related neuropsychiatric disorders (i.e. PTSD, depression, anxiety, etc.), uncovering the link between this stress pathway and AD pathogenesis can help to develop therapeutics to relieve the emotional and economic burden created by AD. Rodent models of this disease have found that stress exposure and CRF signaling are linked to the pathogenesis of AD. In this study, we explored whether therapeutic targets that modulate the CRF system (i.e. the CRFR1‐selective antagonist R121919) could impact the behavioral and pathological endpoints seen in AD mice. AD mice (PSAPP) were aged to 6 and 12 months in the presence or absence of restraint stress and/or R121919. Drug‐treated PSAPP mice had significantly reduced Aβ levels, plaque accumulation, and cognitive impartment, relative to the control cohorts. Collectively, our data provide evidence that CRFR1 modulation may be mechanistically linked to the neuropathogenesis of AD.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential pattern of expression for endothelial cell markers in the microvessels of different brain regions.
Authors: Mbagwu Smart , Marti Christiane , Blanc Pauline , Mantel Pierre‐Yves , Filgueira Luis
Published: June 2021

Background

The role of microvascular endothelial cells in the brain is of importance in both healthy and diseased conditions. Various studies have reported heterogenicity in the phenotypical, morphological and molecular characteristics of endothelial cells in the different organs of the body. There is still not much known about the heterogenicity of the endothelial cells of the human brain microcirculation. This study was aimed at characterizing qualitatively the pattern of expression for endothelial cell markers for the microvessels within different regions of the brain.

Methods

Paraffin sections from different anatomical regions (precentral and postcentral gyrus, hippocampus, rhinal and visual cortex) of human formalin fixed brains (n=3) were stained immunohistochemically for endothelial cell markers, including CD31, claudin 5, von Willebrand Factor, E‐ selectin and P‐selectin.

Results

The expression patterns of the markers was heterogenous in the different microvessels within the regions of the brain studied. The capillaries had the most positive expression among the different biomarkers especially for VWF and CD31 while the arterioles were most positive for the E‐Selectin marker. The density of of the stained microvessels were more in the white matter compared to the gray matter with the visual, rhinal and hippocampal regions showing high densities of the microvessels.

Conclusion

The expression pattern of the different endothelial markers is heterogenous for the microvasculature within the different anatomical regions of the brain. This results indicate that the heterogenicity in microvascular endothelial cells contribute to functional differences in the different brain regions.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Limited Proteolysis Analysis of a DEAD‐box Protein and its Domain Truncated Variants
Authors: Arnold Megan , Garcia Ivelitza
Published: June 2021

RNA helicases play a crucial role in virtually all aspects of RNA metabolism, and although they share a highly conserved structure, the enzymes exhibit a wide variety of biochemical activity. One family of RNA helicases is DEAD‐box proteins, and while their functions are diverse, the one defining activity of these proteins is their ability to hydrolyze ATP in the presence of single‐stranded or double‐stranded RNA.1 ATPase activity leads to changes in RNA affinity, enabling ligand dissociation and cyclic rebinding to promote correct RNA folding in the cell. For example, Rok1p is a yeast DEAD‐box protein essential in ribosomal RNA folding, and is characterized by a catalytic core where ATP and RNA bind, as well as two peripheral domains.2 The peripheral domains (N‐terminal domain or NTD, and the C‐terminal domain, or CTD), are proposed to regulate ATPase activity as well as provide a region for RNA specificity. Thus, peripheral domains could play a role in regulating the overall structure of the protein and its mechanism of RNA refolding. To monitor peripheral effects, the structural changes of Rok1p in the presence and absence of a ligand were analyzed using limited proteolysis under various conditions. These results were subsequently compared to the truncated variant, Rok1p‐ΔCTD. This comparative analysis determined the structural role of the NTD, and to some extent, the CTD. Time dependent proteolysis suggests that the protein is more dynamic at higher temperatures. In the presence of RNA or a nucleotide, the level of structure fluctuation increases. The observed level of conformational changes is minimal yet has dramatic effects on the ATPase activity of the protein. Specifically, the results show that the protein does not occupy a fully extended core state, which accounts for the unique catalytic activity of Rok1p.

Support or Funding Information

The Thomas Lord Charitable Trust Fellowship

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Non‐Active Site Amino Acid Residues on Inhibitor Selectivity
Authors: Eck Tyler , Gubler Ueli , Goodey Nina
Published: June 2021

Enzyme inhibitors are important in biological processes and as pharmaceutical drugs, the cornerstone of modern medicine. It is necessary to build a comprehensive picture of the binding event between the inhibitor and its enzyme target to effectively design pharmaceutical drugs and predict the binding of known drugs to new targets. It is currently known that inhibitor selectivity is in part determined by the amino acid residues within the “active site”, which is the site/cavity in the enzyme where the inhibitor binds. Less well understood is the role of residues situated away from (distal to) the active site and how they contribute to inhibitor selectivity, which refers to the fact that inhibitor molecules often bind to several members of an enzyme family but not all. By understanding the features that determine inhibitor binding within an enzyme family, it is possible to design better inhibitors and predict possible new targets for a given inhibitor. Using Bacillus stearothermophilus dihydrofolate reductase (Bs DHFR) as a model system, this project studies how amino acids distal from the active site affect inhibitor binding and selectivity. Previous studies in our lab have indeed predicted and experimentally verified three clusters of amino acids in Bs DHFR that are distal to the active site but impact inhibitor binding. Here, random mutations are being introduced individually into these clusters. Mutants will be screened for DHFR activity using a functional assay based on ability to synthesize thymidine. DHFR coding genes in selected colonies will be sequenced. Genes with functional amino acid mutations will be used to produce the corresponding DHFR enzyme variants. To ensure that the variants are active, kcat and KM values will be determined. To investigate the impact of the mutations on inhibitor selectivity, KD values will be determined for multiple inhibitors including Methotrexate (MTX), Trimethoprim (TMP), and Pyrimethamine (PMT). Analysis of these results will show how and how much distal residues affect Bs DHFR's inhibitor selectivity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oxidative Susceptibility of Calmodulin Cardiac Arrhythmia Mutants
Authors: Moen Rebecca Jane , McCarthy Megan , Hinde Amanda , Thomas David Dale
Published: June 2021

The congenital arrhythmia disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) is linked to missense mutations within the ubiquitous calcium‐sensing protein calmodulin (CaM). CaM plays various important roles in cellular and biological processes. In cardiomyocytes, CaM is critical for regulating excitation‐contraction coupling by binding calcium as well as binding a diverse set of targets including the ryanodine receptor (RyR2) to regulate intracellular calcium levels. CaM is a unique protein, not only in its ability to bind four calcium ions through its N‐ and C‐terminal lobes but also due to its high number of methionine amino acid residues within its primary sequence. These methionine residues are solvent‐exposed upon binding calcium and facilitate CaM binding its targets. However, the location of these methionine residues make CaM extremely sensitive to oxidative modification by reactive oxygen species (ROS). ROS can directly interact with CaM, causing reversible oxidation of the methionine side chain to a methionine sulfoxide or irreversible oxidation to methionine sulfone. CaM also contains a potential PEST sequence, an internal signal for rapid proteolytic degradation. We have investigated the oxidative and proteolytic susceptibility of CPVT cardiac arrhythmia CaM mutants with particular focus on methionine oxidation.

Support or Funding Information

NIH R37 AG026160

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crossroad control of cell proliferation and apoptosis by PEA‐15 phosphorylation homeostasis and allosteric regulation of protein conformations and interactions
Authors: Wei Yufeng , Crespo Sergio , Hassan Sherouk
Published: June 2021

Cell fate is determined by the relative tendency of cell proliferation and cell death. PEA‐15 (phosphoprotein enriched in astrocytes, 15 kD), a small, non‐catalytic, death‐effector domain (DED) containing protein, regulates both proliferation and apoptosis through protein‐protein interactions. PEA‐15 is widely expressed in different tissues and highly conserved among mammals, and the overall expression level of PEA‐15 is almost constant in most tissues. PEA‐15 contains two phosphorylation sites on the C‐terminal tail, Ser104 and Ser116, and the phosphorylation states of these two serine can vary significantly depending on cell and tissue types and/or cellular environment and conditions. Unphosphorylated PEA‐15 inhibits extracellular signal‐regulated kinase (ERK)‐dependent cell proliferation, while the doubly phosphorylated protein binds to Fas‐associated death domain (FADD) and blocks death receptor mediated apoptosis. Using advanced nuclear magnetic resonance (NMR) techniques, we previously reported a surprising conformational change of PEA‐15 DED upon interaction with ERK2. In addition, DED conformation is significantly modulated by the phosphorylation of the C‐terminal serine residues, which switches binding specificity from ERK1/2 to FADD. Based on our most recent results, we propose the phosphorylation homeostasis model, in which that the balance between phosphorylated and unphosphorylated PEA‐15 is strictly regulated in different cell types and tissues and controls the cell fate, any disruption of the delicate balance could lead to various diseases, such as cancers and neurodegenerative diseases. To test this hypothesis, we recently conducted quantitative Western blot to detect the phosphorylation states of PEA‐15 in various primary cells and cell lines, as well as the effects of PEA‐15 phosphorylation on the activation of caspases in the cells. We have also transfected wildtype, S104A/S116A (unphosphorylated mimic), and S104D/S116D (phosphorylated mimic) PEA‐15 into these cells. Using novel in‐cell NMR technique, we studied the conformation of these proteins within the cellular contents, where all its natural interactions are present. The effects of transfected proteins on apoptosis and activation of caspases have also been examined.

Support or Funding Information

NIH AREA R15CA179410

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
n‐π* and Other Atomic Level Interactions of Formamides with Nucleobases and Base Analogs in Water
Authors: Karim Rezwana , Cheng Xian , Record M. Thomas
Published: June 2021

To understand the molecular origins of favorable amide‐nucleobase interactions and predict or interpret effects of amides on nucleic acid processes, we quantify the preferential interactions of formamide and N‐methylated formamides with nucleobases and base analogs differing in amount and composition of water‐accessible surface area (ASA) by solubility assays. These formamide series solutes have much larger amide sp2C ASA than the alkylurea solutes studied previously. Using an established additive, ASA‐based analysis, we interpret these thermodynamic results to determine intrinsic strengths of interaction of each amide with unit area of the five types of nucleobase unified atoms (sp2C, sp3C; sp2N, sp3N; sp2O). All formamides interact favorably with all nucleobases and base analogs studied. “One‐way” analysis of results to date indicate favorable interactions of these formamides with all five types of unified atoms of nucleobases, and set the stage for a “two‐way” atom‐atom analysis quantifying interactions of the four atoms of amides (aliphatic sp3C; amide sp2C, sp2N and sp2O) with five atoms on nucleobase (aromatic ring sp2C and sp2N, methyl sp3C, amino sp3N, carbonyl sp2O). In particular, this “two‐way” analysis will quantify the favorable n‐π* interactions of nucleobase carbonyl O with amide sp2C and of formamide sp2O with nucleobase heterocyclic ring sp2C to compare with the very favorable n‐π* interactions of amide sp2O with homocyclic aromatic ring sp2C reported previously. Progress toward these goals will be reported.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
α‐synuclein inhibits Snx3‐retromer‐mediated retrograde recycling of iron transporters in a S. cerevisiae model of Parkinson's disease
Authors: Witt Stephan N. , Nagarajan Sureshbabu N. , Liu Zhengchang , Hemphill Wayne O. , Shi Runhua , Uversky Vladimir N. , Patel Dhaval
Published: June 2021

We probed the role of alpha‐synuclein (α‐syn) in modulating sorting nexin 3 (Snx3)‐retromer‐mediated recycling of iron importers in the budding yeast Saccharomyces cerevisiae. Fet3/Ftr1 comprises the high affinity iron importer in yeast. Fet3 is a membrane‐bound multicopper ferroxidase, whose ferroxidase domain is orthologous to human ceruloplasmin. The ferroxidase domain of Fet3 oxidizes external Fe+2 to Fe+3, and the Fe+3 ions then channel through the Ftr1 permease into the cell. When the concentration of external iron is low (< 1 μM), Fet3/Ftr1 is maintained on the plasma membrane by retrograde endocytic‐recycling; whereas, when the concentration of external iron is high (> 10 μM), Fet3/Ftr1 is endocytosed and sorted to the vacuole for degradation. We discovered that α‐syn expression phenocopies the high iron condition: under the low iron condition (< 1 μM), α‐syn inhibits Snx3‐retromer‐mediated recycling of Fet3/Ftr1 and consequently the complexes are sorted to the vacuole for degradation. Because α‐syn has a propensity to bind to negatively charged lipids and vesicles, we asked whether α‐syn might disrupt recycling by interfering with the association of retromer components with early endosomes. Using fluorescence microscopy, we found that α‐syn blocks the association of retromer components Snx3‐mCherry and Vps17‐mCherry molecules with early endosomes. Early endosomes are enriched in phosphatidylinositol‐3‐monophosphate (PI3P), and Snx3 and Vps17 each possess a phox homology domain that selectively binds to PI3P. We tested the hypothesis that α‐syn blocks the binding of Snx3 to vesicles enriched PI3P in a liposome‐protein flotation assay using synthetic liposomes containing phosphatidylcholine, phosphatidylserine, and PI3P (70:25:5) and found that α‐syn inhibits the association of the purified phox homology domain of Snx3 to the synthetic vesicles. Overall, we show that α‐syn disrupts endocytic recycling by interfering with retromer (Fig. 1), and that α‐syn accelerates the sorting to the vacuole of Fet3/Ftr1 complexes that emanate from the plasma membrane. This work raises the possibility that α‐syn can disrupt iron homeostasis in cells by interfering with the trafficking of iron transporters.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of Protein Phosphatase 2A Substrates
Authors: Wang Xinru , Page Rebecca , Wolfgang Peti
Published: June 2021

The protein ser/thr phosphatases (PSPs) dephosphorylate 1000s of key biological targets, making them attractive therapeutic targets. The serine/threonine protein phosphatases 1 and 2B (PP1; PP2B or calcineurin/CN) bind substrates and regulatory proteins using short functional sequences known as Short Linear Motifs (SLiMs). These SLiMs are found within intrinsically disordered regions (IDRs) of substrates and regulatory proteins and mediate specific protein‐protein interactions. While tremendous progress had been made in identifying where and how SLiMs bind PP1 and CN, very little is known about how substrates are specifically recruited to PP2A, a validated cancer drug target. Here we describe three structures of the first known PP2A‐SLiM interaction (B56:pS‐RepoMan, B56:pS‐BubR1, and B56:pSpS‐BubR1), which reveal that this PP2A‐specific SLiM is defined as LSPIxE. We also show that the affinity of this SLiM for B56 is modulated both by residues immediately adjacent to the SLiM and its phosphorylation, both of which provide the fine‐tuning needed to ensure signaling pathway fidelity. Finally, by using this newly discovered motif, we identify nearly 100 proteins that we predict are novel PP2A:B56 regulators/substrates, substantially expanding the PP2A interactome. We are now leveraging this data to identify new PP2A‐specific SLiMs. Further, we are expanding this approach to discover new SLiMs that are specific for other PSPs, especially PP1 and CN. Together, these data are providing a powerful approach not only for dissecting PSP interaction networks in cells but also for targeting PSP diseases, such as cancer.

Support or Funding Information

NIH: R01GM098482 NIH: R01NS091336

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a cell‐based assay for Tau strain discrimination
Authors: Angeles‐Perez Lidiette M. , Vaquer‐Alicea Jaime , Manon Victor , Diamond Marc
Published: June 2021

Alzheimer's Disease and other related tauopathies are characterized by the accumulation of the microtubule‐associated protein Tau into fibrillary deposits. Interestingly, there is evidence supporting amyloid structures, or strains, formed as a consequence of Tau aggregation are unique to each pathology, suggesting different conformations play an important role in the development of specific tauopathies. The potential critical role of Tau in disease progression, together with the isolation of 19 different Tau strains, led us to work in developing a panel of cell‐based fluorescence resonance energy transfer (FRET) biosensors. Each biosensor carried a different mutation on Tau's aggregation‐prone repeat domain, based on the hypothesis that each mutant would have unique interactions with Tau strains and, thus, alter Tau aggregation kinetics within the cell. We hoped that by studying these differences in FRET signal we would be able to discriminate between distinct amyloid conformations. In order to do this, we first created the biosensors by transducing HEK 293 cells with lentivirus coding Tau's repeat domain with specific mutations, fused to both, cyan florescent protein (CFP) or yellow florescent protein (YFP). These biosensors were later treated with different sources of Tau seeds and FRET flow cytometry was used to describe their seeding activity. Experiments with the 19 strains in our Tau library and the engineered biosensors revealed noticeable differences in percent FRET positivity, such that discrimination was achieved for most of the strains. Interestingly, the highest seeding activity among all the biosensors was obtained from strain 15 (DS15), even at low total protein concentrations. Additionally, biosensors expressing the wild‐type four‐repeat domain of Tau had higher percent FRET positivity only when seeded with strains derived from Corticobasal Degeneration (CBD) patients. Furthermore, treatment of our panel of biosensors with brain homogenates of multiple tauopathy patients correlated with our previous results and revealed more distinct patterns of FRET behavior. Together, these results suggest that measuring proteopathic seeding activity while using our panel of biosensors is a sensitive and reliable tool to discriminate between different amyloid conformations, which could potentially lead the development of improved diagnostic methods for neurodegenerative disorders.

Support or Funding Information

This research project was supported by the NIH‐Maximizing Access to Research Careers (Grant # 5T34GM0072821‐38), the Summer Undergraduate Research Fellowship at UT Southwestern Medical Center and the Center for Alzheimer's and Neurodegenerative Diseases at UT Southwestern Medical Center.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“The Importance of a Phenolic Group in the Substrates of Iodotyrosine Deiodinase”
Authors: Quinones Carla Michelle , Kozyryev Anton , Rokita Steven
Published: June 2021

Iodotyrosine deiodinase (IYD) is an enzyme that is responsible for recycling iodide from monoiodotyrosine (I‐Tyr) and diiodotyrosine (I2‐Tyr), which are formed as side‐products during the biosynthesis of thyroid hormones. Recently, a gene encoding IYD was found in bacterium Haliscomenobacter hydrossis (hhIYD), but the function of hhIYD in this organism is still unknown. This bacterium does not generate thyroid hormone and is not known to require iodide salvage. HhIYD can bind and deiodinate 2‐iodophenol, but not as efficiently as I‐Tyr. We wanted to know how broad the substrate specificity for this enzyme is; thus, the goal of the project is to learn how important the OH‐group of I‐Tyr is for catalysis. Since aryl amine and hydroxyl groups have common properties, we synthesized the aniline derivative of I‐Tyr. Both OH‐ and NH2‐groups are electron donating substituents to the phenyl ring and can participate in hydrogen bonding. Moreover, we can propose a similar mechanism for deiodination of both I‐Tyr and its aniline derivative. We expressed and purified hhIYD. The binding of the aniline derivative to hhIYD was measured using fluorescence quenching, but no binding was observed even at a ligand concentration as high as 1370 μM. We also tested deiodination of the aniline derivative of I‐Tyr by hhIYD, but no deiodination was detected above turnover number of 7.05 × 10−5 s−1. We expect that this project will help to identify the full catalytic potential of IYD and its role in nature.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biosynthesis of oxetanocin: are two cofactors better than one?
Authors: Bridwell‐Rabb Jennifer , Liu Hung‐wen , Zhong Aoshu , Drennan Catherine
Published: June 2021

The biosynthesis of the natural product oxetanocin has been connected to a plasmid‐borne Bacillus megaterium gene cluster that contains two genes involved in oxetanocin production (oxsA and oxsB). Here, we present detailed structural and biochemical analysis that confirms the involvement of the cobalamin (Cbl) dependent S□adenosylmethionine (AdoMet) radical enzyme, OxsB, and an HD‐domain phosphohydrolase enzyme, OxsA, in oxetanocin production. These studies of OxsB provide a framework for understanding the coordination of AdoMet and Cbl in performing chemistry and reveal an HD‐domain phosphohydrolase enzyme with a restructured active site specific for a phosphorylated oxetanocin compound. Our crystallographic endeavors have allowed for elucidation of a biochemical scheme for the production of oxetanocin, expanded the catalytic repertoire of the HD‐domain phosphohydrolase enzyme superfamily, and provided the first structure of a Cbl‐dependent AdoMet radical enzyme.

Support or Funding Information

Support for this work came from National Institute of Health Grants F32‐GM108189 (J.B.‐R.) and GM035906 (H.‐w.L.), and the Welch Foundation grant F‐1511 (H.‐w.L.). C.L.D. is a Howard Hughes Medical Institute Investigator. This work is also based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6M detector on 24‐ID‐C beam line is funded by a NIH‐ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.