bluuebunny/crossref_metadata_2025_split huggingface dataset:
Histology and Organization of the Fascia
Authors: Gordon Marion K 
Published: June 2021

The diameter of collagen fibrils is a contributing factor to the functional properties of fascia. This is demonstrated by pathologies such as Dupuytren's disease, where shortening of the palmar fascia causes contracture of fingers. Cell proliferation plus overproduction of collagen are hallmarks of the disease. Excess collagen production suggests a fibrotic disorder, however Dupuytren's is different from fibrotic pathologies such as lung fibrosis, because deposited fibrils are of smaller than normal diameters, while in lung fibrosis, diameters are larger.The minor fibrillar collagens, types III and V, FACIT collagen XIV, and certain proteoglycans regulate fibril diameters during fibrillogenesis, which occurs in steps: First, short heterotypic collagen fibrils with thin diameters are made; Second, short fibril segments gain length; Third, long fibril segments attain larger diameters by fusion with adjacent fibrils. The underlying problem in Dupuytren's disease may be the initiation of a wound healing state, with synthesis of a provisional matrix, but without the subsequent remodeling that returns the tissue to normal. By comparing the extracellular matrix components of diseased fascia with normal and fibrotic tissues, we gain clues about what molecules play important roles in the function, architecture and structural integrity of healthy fascia.
Human mitochondrial alanyl‐tRNA synthetase: A variant mode of RNA recognition
Authors: Borchert Karen , Anderson David , Natala Nakita , Zhu Yirong , Riley Lucas , Ener Maraia , Brown Julia , Chihade Joseph 
Published: June 2021

Metazoan mitochondria contain tRNAs which are highly diverged in both sequence and structure from those found elsewhere in evolution. These tRNAs are recognized and specifically charged by nuclearly encoded mitochondrial aminoacyl‐tRNA synthetases. Our work is focused on characterizing the interaction between human mitochondrial alanyl‐tRNA synthetase (Hs mt AlaRS) and its tRNA substrate.Mutational analysis of both the enzyme and tRNA substrate estabilished that contacts between the acceptor stem and the N‐terminal domains of the enzyme are relatively unimportant for establishing tRNA identity, in contrast to all previously characterized alanyl‐tRNA synthetases. However, the C‐terminal half of Hs mt AlaRS and the D‐acceptor domain of the tRNA are required for aminoacylation, suggesting that in the human mitochondrial system a second set of tRNA‐AlaRS contacts plays a role in determining tRNAAla identity. Because mitochondrial tRNAs are structurally fragile, we have used in‐line probing to validate RNA secondary and tertiary structure.An important identity element in non‐mitochondrial tRNAAla is a G3:U70 wobble pair. Comparing N‐terminal AlaRS sequences from a variety of mitochondrial and non‐mitochondrial sources, some of which aminoacylate substrates that lack this element, has allowed us to identify residues associated with acceptor stem recognition.
Trafficking of neuroligin mutant proteins associated with autism
Authors: De Jaco Antonella , Miller Meghan , Comoletti Davide , Dubi Noga , Taylor Palmer 
Published: June 2021

Mutations in the synaptic adhesion proteins, neuroligins (NLs) and neurexins have been found to play an important role in the etiology of autistic spectral disorders. We characterized the NL3‐Arg451Cys mutation found in two affected brothers. The exposed Cys causes retention of the protein in the endoplasmic reticulum (ER) reducing processing and delivery to the cell membrane. A new alteration found linked to autism is a deletion of exon 4 in the NL4 gene. We show by confocal fluorescence microscopy that this deletion causes defective intracellular trafficking of the mutated protein that is retained in the ER. To define molecular mechanisms involved in trafficking of NL proteins, we investigated protein‐protein interaction mechanisms between NLs and ER‐resident chaperones by immunoprecipitation. We identify calnexin and calreticulin as mayor players in the retention of NL3‐Arg451Cys protein. The use of glycan processing inhibitors shows that the binding to calnexin is important in the degradation pathway of the NLs. Other candidate partner proteins involved in the trafficking of wild type and mutant proteins are identified by 2D‐electophoresis and mass spectrometry. Metabolic labeling of NL3 has been used to study the half life of wt and mutant proteins and the sequence of association with single chaperones. Understanding the mechanisms by which these proteins are retained within the cell should provide important insights into cellular abnormalities that contribute to autism.This work was supported by NIH R37‐GM18360 and U0‐1 ES10337 to PT and pilot research award to DC.
ERp29: A luminal chaperone that facilitates CFTR trafficking in response to 4‐Phenylbutyrate
Authors: Suaud Laurence , Miller Katelyn , Alvey Lora , Yan Wusheng , Robay Amal , Hubbard Michael , Rubenstein Ronald C 
Published: June 2021

Sodium 4‐Phenylbutyrate (4PBA) improves the aberrant intracellular trafficking of deltaF508‐CFTR in Cystic Fibrosis (CF) epithelial cells. We hypothesized that 4PBA causes a complex cellular adaptation leading to altered expression of molecular chaperones and improved deltaF508 intracellular trafficking. To identify elements of this cellular response, we performed differential display RT‐PCR on RNA extracted from IB3‐1 bronchiolar epithelial cells (genotype deltaF508/W1282X) treated for 0–24 hours with 1 mM 4PBA. We isolated a cDNA clone for ERp29 (29 kDa ER Lumenal Protein) that had a time‐dependant increase in mRNA expression. ERp29 is a ubiquitous, conserved thioredoxin homolog that is prominently expressed in lung. IB3‐1 cells treated with 4PBA had increased ERp29 mRNA expression by ribonuclease protection assays and protein expression by immunoblot. In coimmunoprecipitation experiments, ERp29 was associated with deltaF508‐CFTR in IB3‐1 cell lysates, but not detectably with wt‐CFTR in lysates of T84 intestinal epithelial cells. In oocytes, modest ERp29 overexpression increased the functional expression of wt and deltaF508‐CFTR. This effect was lost at higher levels of ERp29 overexpression. The increased wt‐CFTR functional expression corresponded to increased expression of CFTR at the oocyte surface. These data are consistent with increased ERp29 expression facilitating ΔF508 trafficking and functional expression in CF epithelial cells in response to 4PBA.Supported by grants from NIDDK.
Use of VX and V‐analog compounds to probe the spatial requirements of the active site of wild type and mutant human paraoxonase 1
Authors: Otto Tamara Caviston , Yeung David T , Cerasoli Douglas M , Lenz David E 
Published: June 2021

Human paraoxonase 1 (HuPON1) exhibits catalytic activity toward the organophosphorus nerve agent VX, but the catalytic mechanism mediating this activity is unresolved. To examine the effect of substrate volume on activity, the hydrolysis of five analogs of VX was examined. The analogs were designed such that the O‐ ethyl alkyl group of VX was replaced with O‐propyl (V1), O‐isopropyl (V2) or O‐isobutyl (V3) on the retained group, or the 2‐diisopropyl alkyl groups were replaced with 2‐diethyl (V4) or 2‐dimethyl (V5) on the leaving group. Both Km and kcat values for V1, V2, and V3 decreased as the retained group increased in size. In contrast, reducing the size of the leaving group (V4 and V5) had little effect on Km and progressively decreased kcat. The H1 15W mutant of HuPON1 hydrolyzed V1, V4 and V5, whereas no turnover of V2 or V3 was detected. Further analyses determined that V3 was recognized as a competitive inhibitor of VX hydrolysis by the H115W mutant. The results indicate that the volume of both the retained and leaving groups can affect binding of these substrates to HuPON1 and also suggest that the R‐chain of the retained group may directly interact with residue 115. Together, the results allow an estimate of the size of the HuPON1 binding pocket to be made.This work was supported by NIH Centers of Excellence Grant U‐54 NS 058183‐01 Center for Catalytic Bioscavenger Medical Defense Research. TCO was a NRC research fellow.
Amyloid‐beta peptide toxicity in neuronal‐like, differentiated PC12 cells: calpain‐calpastatin, caspase and membrane damage
Authors: Kosower Nechama S , Vaisid Tali , Elkind Esther , Barnoy Sivia 
Published: June 2021

Amyloid‐beta peptide (Aβ) is central to the pathogenesis of Alzheimer's disease (AD). It is toxic to neurons, possibly via causing initial neuronal membrane and synaptic damage. Although implicated in AD, calpain and caspase interaction or the role of the specific calpain inhibitor, calpastatin, are little understood.Objective:to examine effects of Aβ 25–35 (sAβ) on the proteases, on calpastatin and on the membrane in neuronal‐like cells.Results:sAβ‐treated cells exhibited primarily cell membrane damage (varicosities along neurites, enhanced membrane permeability to propidium iodide (PI), no apparent nuclear changes of apoptosis, and little poly(ADP‐ribose) polymerase (PARP) degradation). The sAβ‐induced damage contrasted with the staurosporine‐induced damage (nuclear apoptotic changes, PARP degradation, no PI permeability). sAβ led to caspase‐8 and calpain activation (with cross‐talk between them), promotion of calpastatin degradation (by caspase‐8 and by calpain), and enhanced degradation of fodrin (mainly by calpain). Calpastatin overexpression attenuated the sAβ‐toxicity (lowering PI permeability and fodrin degradation).Conclusions:Aβ causes primarily neuronal membrane damage, responding to calpain/caspase‐8 activation and calpastatin loss. Increased expression of calpastatin and/or decrease in the proteases may serve to ameliorate early symptoms of AD.
Kinases in Diabetes and Hypertension
Authors: Cobb Melanie H. , Cummings Staci , Wauson Eric , Heise Charles , Raman Malavika , Lawrence Michael , Chen Wei 
Published: June 2021

WNKs (with‐no‐lysine [K]) 1–4 are unique protein‐serine/threonine kinases conserved in multicellular organisms. The ATP‐binding lysine present in all protein kinases is in an atypical position in WNKs. Two of the four WNKs were identified as genes mutated in pseudohypoaldosteronism type II, a genetic syndrome characterized by hypertension and hyperkalemia. All four WNKs contain very large noncatalytic regions with no currently known domain organization that appear to function as scaffolds. WNKs have been implicated not only in regulation of ion balance, but also in cell survival and proliferation, membrane trafficking and embryonic organ development. Mitogen‐activated protein kinases MAPKs transduce signals in a multitude of cellular pathways in response to many ligands and cell stimuli. MAPKs have been implicated in diseases including cancer, inflammatory disease, obesity, and diabetes. The MAPKs ERK1/2 are required for glucose‐regulated insulin gene transcription in pancreatic beta cells. Nutrients and hormones that stimulate or reduce insulin secretion have a parallel effect on ERK1/2 activity in beta cells. ERK1/2 control the function of several transcription factors that mediate glucose‐regulated insulin gene transcription, including at least two factors that are encoded by maturity‐onset diabetes (MODY) genes.Research supported by grants from the National Institutes of Health, R01‐GM53032, R37‐DK034128, R01‐DK055310 and the Robert A. Welch Foundation I‐1243 and the American Diabetes Association Mentor‐based Postdoctoral Fellowship award.
O‐GlcNAc modification of CaMKIV
Authors: Dias Wagner Barbosa , Cheung Win , Wang Zihao , Hart Gerald 
Published: June 2021

Similar to phosphorylation, O‐GlcNAcylation (or simply GlcNAcylation) is an abundant, dynamic, and inducible post‐translational modification. In some cases, GlcNAcylation and phosphorylation occur at the same or adjacent sites, modulating each other. GlcNAcylated proteins are crucial in regulating virtually all cellular processes, including signaling, cell cycle, and transcription, among others. GlcNAcylation affects protein‐protein interactions, activity, stability, and expression. Calcium (Ca2+) is a central secondary messenger involved in regulating cytoskeletal proteins, ion pumps, and activities of enzymes, including Ca2+/calmodulin‐dependent protein kinases (CaMKs). CaMKIV is a multifunctional, serine‐threonine protein kinase that is activated in the presence of increased intracellular Ca2+, acting as a potent mediator of Ca2+‐induced gene expression. Here, we show that CaMKIV is highly GlcNAcylated in vitro and in vivo. In addition, we identify five sites of GlcNAcylation on CaMKIV. Site‐directed mutagenesis of these residues and their influence on activity and function are currently under investigation.This work is supported by NIH grants CA42486 and HD13563. G.W. H. receives a share of royalty received by the university on sales of the CTD 110.6 antibody. Terms of this arrangement are managed by JHU.
Pleiotropic effects of PHLPP on cell signaling
Authors: Reyes Gloria Ximena , Newton Alexandra 
Published: June 2021

The PH domain Leucine Repeat Protein Phosphatases PHLPP1 and PHLPP2 were discovered in a directed search for phosphatases that directly dephosphorylate and inactivate Akt, a key controller of many cellular processes such as cell survival, cell death, proliferation, and motility. Given the relative paucity of phosphatases compared to the abundance of phosphorylated sequences in cells, we asked whether the PHLPP phosphatases controlled additional signaling networks to the Akt pathway. To this end, we used siRNA to deplete PHLPPs from diverse cell lines and analyzed substrates phosphorylated downstream of growth factors such as EGF, PDGF, insulin and TGF‐beta. We found that genetic depletion of PHLPP isoforms dramatically increased agonist‐evoked signaling by each of these agonists. The effects of PHLPP knockdown depended on the tumorigenic state of the cell lines and the growth factor used. Importantly, PHLPP knockdown affected not only Akt phosphorylation, but also the phosphorylation of substrates which are modulated by distinct pathways from Akt, such as those involving the MAPK and SMAD proteins. These findings reveal differential and/or synergistic contributions of PHLPP isoforms to suppressing signaling in numerous cellular pathways, revealing an important pleiotropic role of these phosphatases on cell signaling.This work was funded by NIH GM NIH 067946
Protein Aggregation‐Suppression: Molecular Clusters of Arginine are responsible
Authors: Das Utpal , Srinivasan Alagiri 
Published: June 2021

Aggregation of unfolded proteins occurs through the exposed hydrophobic surfaces. Any mechanism of aggregation‐supression should explain these hydrophobic interactions. The mechanism of action of arginine as aggregation‐suppressor is not understood. We propose a mechanism based on the hydrophobic interactions of arginine.Arginine solutions display hydrotropic effect on pyrene. ANS fluorescence studies indicate the presence of hydrophobic environment. Mass spectroscopic analyses and light scattering studies indicate that arginine exists as clusters in the gas phase and in solution. Arginine changes the RPC elution profile and the hydrodynamic volume of Alzheimer's amyloid beta 1–42 (Aβ1–42). Arginine increases the solubility of Aβ1–42 peptide and decreases the aggregation.Our results show that molecular clusters of arginine in solutions display a hydrophobic surface by the alignment of its three methylene groups. The hydrophobic surfaces of the proteins and the arginine clusters interact. The masking of hydrophobic surface inhibits protein‐protein aggregation. This study was supported by the Department of Biotechnology, Government of India.
Discovering the substrates of β‐1,4‐galactosyltransferase‐1 by use of unnatural UDP‐galactose analogs
Authors: Bond Michelle , Kohler Jennifer 
Published: June 2021

The genome encodes seven galactosyltransferases capable of adding galactose in a β1,4 linkage to terminal GlcNAc; however, they are not interchangeable. Despite the fact that in vitro assays of the enzymes do not reveal specificity differences among them, β1,4 galactosyltransferase family members have different biological roles. β1,4‐galactosyltransferase‐1 has been recognized as both an important Golgi‐resident enzyme and as a lectin. Our goal is to identify the cellular substrates of β1,4‐galactosyltransferase‐1 and thereby determine the molecular basis of its biological roles.To identify the biological substrates of β1,4galactosyltransferase‐1, we are re‐engineering the enzyme to accept an orthogonal and labeled UDP‐galactose analog. We predict that the mutant, but not the wild‐type, enzyme will accept the UDP‐Gal analog. Modified β1,4galactosyltransferase‐1 can then be introduced in place of the wild type enzyme. In this way, we will be able to directly label the substrates of a single β1,4‐galactosyltransferase family member. More generally, we expect that similar methods could be used to decipher the substrates of the over 100 glycosyltransferases identified in the human genome.Sources of support: NSF Graduate Fellowship, March of Dimes
Inhibition of Lysozyme Fibrillation by Natural and Synthetic Melanins
Authors: Holmes Earle W. , Thompson Kenneth D 
Published: June 2021

Amyloidogenesis, a multistep process in which protein mis‐folding results in the formation of aggregates with cross beta sheet structures, is associated with more than 20 diseases including diabetes, AL amyloidosis, Alzheimer's disease, and prion diseases. Hence, there is an ongoing interest in the development of drugs that inhibit amyloid formation or deposition. In this study, we tested the ability of natural and synthetic polyketides to inhibit lysozyme fibrillation in vitro. Solutions of lysozyme (Hen egg, 20 mg/mL) were incubated at 58°C in 20 mM glycine HCl buffer at 58°C for 8 days in the presence of one of the following compounds (concentration tested): 1. Kraft lignin (125 μg/mL), 2. sulfonated Kraft lignin (190 μg/mL), 3. allomelanin extracted from Echinacea purpurea (210 μg/mL), 4. ES‐EM (166 μg/mL), 5. ES‐CAM (190 μg/mL), 6. C11 (130 μg/mL), or 7. C12 (150 μg/mL). Compounds 4–7 are synthetic melanins obtained From BioActive Polymers.com, (La Grange Park, Illinois, 60526). The incubation mixtures were sampled repeatedly and the kinetics of lysozyme fibrillation were evaluated by Thioflavin T fluorescence, native PAGE, and fluorescence and dark field microscopy. Compounds 6, 4, 3, and 7 inhibited the development of THT fluorescence to levels that were 16%, 42%, 52%, and 61%, respectively of the untreated control mixtures. PAGE analysis of the time points taken during the incubation containing C11 showed that this compound decreased the generation of lysozyme oligomers during the first 48 hours of incubation and delayed the progression of the assembly of lysozyme monomers and oligomers into lysozyme fibers and aggregates. The data suggests that natural and synthetic melanins may be of therapeutic value as fibrillogenesis inhibitors.[Supported in part by a gift from the Charles and Lee Finkl Amyloidosis Foundation]
Epithelial γδ T cell‐specific costimulation mediated by JAML‐CAR interactions
Authors: Witherden Deborah A , Verdino Petra , Rieder Stephanie E , Haynes Matthew R , Mills Robyn E , Teyton Luc , Fischer Wolfgang , Wilson Ian A , Havran Wendy L 
Published: June 2021

γδ T cells in murine skin (DETC) represent a prototypical γδ intraepithelial lymphocyte population. They express an invariant γδ T cell receptor, Vγ3Vδ1, and play important roles as effectors and regulators of homeostasis through recognition of antigen expressed on damaged or diseased keratinocytes. DETC do not express many coreceptors important for αβ T cell activation. To date, no γδ‐specific accessory molecules have been described. Our recent focus has been to identify molecules on γδ T cells that play a key role in their activation and function. We have identified the first γδ T cell‐specific costimulatory molecule, Junctional Adhesion Molecule‐Like protein (JAML) and identify Coxsackie and Adenovirus Receptor (CAR) as its ligand. Only costimulation of epithelial γδ T cells occurs through JAML, αβ‐and lymphoid γδ T cells are unresponsive. JAML costimulation leads to cell proliferation and cytokine production through activation of MAP kinase pathways. Our results demonstrate a novel receptor‐ligand pair specific for epithelial γδ T cell function. In addition, the crystal structure of JAML reveals a unique Ig domain prototype that undergoes conformational changes upon ligand binding, suggesting a mechanism for initiating signaling through JAML. Experiments are underway to establish the role of JAML‐CAR interactions in epithelial γδ T cell responses during wound repair and other epithelial challenges.This work was supported by NIH grants AI32751 and AI52257 (W.L.H.), AI042266 and CA58896 (I.A.W.), an Erwin‐Schrödinger Fellowship of the Austrian Science Fund (P.V.) and The Leukemia and Lymphoma Society (S.E.R.).
IFNbeta induces mature DC apoptosis through the caspase 11/caspase 3 pathway
Authors: Yen Jui‐Hung , Ganea Doina 
Published: June 2021

IFNβ has been used in the treatment of multiple sclerosis for more than a decade, although the molecular mechanisms are still not clear. In this study, we report for the first time that IFNβ induces apoptosis in dendritic cells (DC) matured with a cytokine cocktail (TNFα+IL‐1+IL‐6+PGE2). In addition, LPS induces IFNβ during DC maturation and leads to DC apoptosis. LPS induced DC apoptosis is abolished in the presence of neutralizing IFNβ antibodies, indicating the role of IFNβ in the apoptosis of mature DC. IFNβ‐induced apoptosis is mediated through induction of caspase‐11 and subsequent activation of caspase 3. DC matured with LPS or cytokine cocktail + IFNβ express caspase 11 mRNA and are positive for activated caspase 3. STAT‐1 signaling is essential for IFNβ induced apoptosis, since DC from STAT‐1 deficient mice are resistant to IFNβ‐induced apoptosis, and do not express caspase 11 or activated caspase 3. Caspase 11 promoter contains STAT‐1 and NFκB binding sites which are essential for caspase 11 expression. We report that treatment with the cytokine cocktail+IFNβ or with LPS induces increased levels of NFkB p65 and STAT‐1 phosphorylation. We propose that signaling through both NFκB and STAT‐1 leads to caspase 11 expression and caspase 3 activation in mature DC, resulting in apoptosis and subsequent downregulation of the inflammatory process.This work is support by NIH 2RO1AI052306.
Dendritic Epidermal T Cells Promote Wound Healing by Production of Vascular Endothelial Growth Factor Mediated by HIF‐1α Signaling
Authors: Kim Chang‐Hoon , Witherden Deborah A , Havran Wendy L 
Published: June 2021

Local hypoxia is general consequence of an acute skin wound due to vascular disruption and high oxygen consumption by cells at the edge of the wound. The resident γδ T cells in the skin, Dendritic Epidermal T Cells (DETC), are known to support local tissue homeostasis and wound repair by local production of growth factors, such as keratinocyte growth factors (KGFs) and insulin‐like growth factor (IGF)‐1. We have analyzed the functional role of DETC in response to local tissue hypoxia generated by an acute skin wound. The hypoxic status of the skin wound induced HIF‐1α expression in DETC. HIF‐1α signalling during hypoxia induced expression of vascular endothelial growth factor (VEGF) by DETC. Wound healing was impaired after HIF‐1α gene‐silencing in DETC and this impairment was rescued by VEGF administration. Together this data demonstrates that acute skin wound induces HIF‐1α signaling in DETC, which promote VEGF production with the result of enhancing skin wound healing.Grant: NIH‐AI‐36964