bluuebunny/crossref_metadata_2025_split huggingface dataset:
C‐12 Binds to Tubulin at the Colchicine‐binding Site and Target Microtubules by Preferentially Binding to GTP‐bound Tubulin
Authors: Prassanawar Shweta Shyam , Kumari Anuradha , Guchhait Sankar K , Panda Dulal 
Published: June 2021

In eukaryotic cells, microtubules are essential for cell division as they assemble into dynamic mitotic spindles which sequester and segregate the duplicated chromosomes. Microtubule‐targeting agents (MTAs) inhibit the dynamicity of the microtubules, thereby hampering chromosome segregation and ultimately inhibiting cancer cell proliferation. A natural compound combretastatin A‐4 (CA‐4), a potent MTA, is undergoing several clinical trials for the treatment of various cancers. However, its toxicity and low bioavailability present a major challenge for it to be considered as an approved drug. C‐12, a derivative of CA‐4, has been found to be more potent than CA‐4 in inhibiting proliferation of several cancer cell lines. In this work, we examined the binding characteristics of C‐12 and its effect in triple negative breast cancer (TNBC) cells. C‐12 inhibited the assembly of microtubules, in vitro, as observed by a turbidimetry assay and electron microscopy. A molecular docking study suggested that C‐12 interacts with tubulin with a binding energy of −10.53 kcal/mol and prefers the colchicine‐binding site on the tubulin dimer. This was further confirmed in a competitive assay, where podophyllotoxin, a colchicine site binder, strongly inhibited the binding of C‐12 to tubulin with an inhibitory constant (Ki) of 0.6 ± 0.2 μM. The fluorescence intensity of C‐12 increases upon binding to tubulin. Therefore, we analyzed the binding of C‐12 to tubulin using the fluorescence of C‐12. Interestingly, the compound exhibited a relatively greater affinity for GTP‐bound tubulin than the GDP‐bound tubulin. The dissociation constant (Kd) for C‐12 and GTP‐tubulin interaction was found to be 1 ± 0.8 μM which is significantly less than the Kd of C‐12 and GDP‐tubulin (9.2 ± 1.2 μM). This suggested that in cells, C‐12 might preferentially bind to the growing plus end of the microtubules. We also examined the effects of C‐12 on the cultured TNBC cells. Immunofluorescence analysis revealed that C‐12 depolymerized the interphase microtubule network and perturbed the localization of γ‐tubulin in TNBC cells. Moreover, the compound hampered chromosome congression and blocked the cells at mitosis, thereby inhibiting cell division. The results, altogether suggested that C‐12 is a potent microtubule targeting agent that binds to tubulin at the colchicine site and perturbs microtubule function in cells.Support or Funding InformationThis work is supported by TATA Innovation fellowship awarded to D.P. from Department of Biotechnology, Government of India.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis of 1,4‐disubstituted aminoanthraquinone derivatives for purification of lactate dehydrogenase
Authors: Kubista Jordan Louise 
Published: June 2021

Cibacron Blue 3G‐A is a dye commonly used for the purification of various enzymes, in particular, isolating lactate dehydrogenase. Companies have Cibacron Blue available periodically but it is rather expensive. Because of these problems, an alternative dye that can be easily synthesized and has similar properties to Cibacron Blue is desired. Cibacron Blue is a dye designed to mimic natural biological ligands, such as NAD+/NADH. A suitable replacement should exhibit comparable affinity and purifying ability for targeted proteins. Cibacron Blue contains an anthraquinone core, which is a distinct feature due to its versatility. Many different functional groups can be substituted on to the anthraquinone core giving it a multitude of different properties and functions. The goal of this research is to synthesize an anthraquinone derivative that exhibits similar properties and functions as Cibacron Blue. The alternatives include aromatic anilines at the 1,4 positions of the anthraquinone to emulate Cibacron Blue's structure at the anthraquinone core and allow for attachment to a solid support. These were synthesized by treatment of 1,4‐bistosyloxyanthraquinone with two substituted anilino nucleophiles to build a library. These alternatives should mimic the adenine moiety of NAD+, which are accommodated in a hydrophobic crevice formed by five chain regions of lactate dehydrogenase. The left side of the anthraquinone is left unsubstituted since it is believed this is where it binds to the hydrophobic nucleic fold of enzymes. After the synthesis of the small library of aminoanthraquinone derivatives was completed, they are characterized. Binding affinity was tested using Fluorescence quenching. This method allows for comparisons between the binding of lactate dehydrogenase to the synthesized products and binding of lactate dehydrogenase to NADH using Kd values. The Kd value is an equilibrium dissociation constant that is used to evaluate binding affinity.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovering c‐di‐AMP binding proteins in Streptomyces
Authors: Lu Wei‐Shin , Bennett Jennifer A. 
Published: June 2021

Streptomyces is an abundant genus of actinobacteria known for its ability to produce useful antibiotics. Streptomycetes transduce signals through a secondary messenger cyclic di‐AMP (c‐di‐AMP). This more recently discovered molecule is the only known secondary messenger to be essential to some bacteria, regulating many functions such as cell metabolism and DNA repair. Although c‐di‐AMP is an important molecule in the physiology of bacteria such as Streptomyces, not much is known about the potential c‐di‐AMP binding proteins or effector molecules in Streptomyces. The objective of this research was to discover and identify potential c‐di‐AMP binding proteins, particularly in Streptomyces coelicolor and Streptomyces scabies. Protein samples were either incubated with c‐di‐AMP capture compound and streptavidin beads or only with streptavidin beads, which acts as a control. Many isolated binding proteins were identified via trypsin digest followed by mass spectrometry.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selective Inhibitor Design Toward CA IX For Breast Cancer Treatment Using Carbohydrate‐Based Compounds
Authors: Murray Akilah Briana , Lomelino Carrie Lynn , Li Haoxi , Horenstein Nicole , McKenna Robert 
Published: June 2021

Human carbonic anhydrase IX (CA IX) is upregulated in neoplastic tissues and has been studied as a drug target for anticancer chemotherapy. Inhibition of CA IX has been shown to be therapeutically favorable in terms of reducing tumor growth. Recently, it been observed that sweeteners bind and selectively inhibit CA IX over other CA isoforms. It was found that cyclic sulfonamides, such as saccharin, inhibits CA IX with 60‐fold isoform selectivity, while disaccharides, such as sucrose, have been found bound at the opening of the active site. As fragment‐based studies show, the most potent inhibitors result from the combination of small molecules. A chemical linker would provide the spacial requirements to optimize interactions with residues in the selective pocket of the active site, improving selectivity for CA IX and preventing off target inhibition of the ubiquitously expressed isoform, CA II. Such compounds would simultaneously inhibit activity through two mechanisms: displacing the ZBS essential for catalysis and blocking the active site from substrate binding. The “tail‐approach” was done using sweeteners as fragments to develop inhibitors with higher CA IX selectivity by connecting saccharin (zinc‐binding group) to a β glucoside (tail) using a triazole linker. In this study, we present the X‐ray crystal structures of carbohydrate compounds in complex with the cancer‐related and off‐target CA isoforms, as well as the effect these compounds have on a panel of breast cancer cell lines. It was found that the carbohydrate compounds have different binding modes with compounds binding directly to the catalytic zinc in CA IX (mimic) and through a bridging water in CA II, which can account for the increased selectivity for CA IX (mimic) when compared to CA II. It was also found that the carbohydrate compounds decreased the metastatic potential of UFH‐001 breast cancer cells that are representative of triple negative breast cancer cells that have high CA IX expression. In summary, These studies will contribute to the knowledge of treating more aggressive and late stage breast cancers and identify new opportunities to increase the effectiveness of current cancer therapies.Support or Funding InformationABM was supported by training grant T32AI0007110 from the NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Drug Delivery Systems to Overcome Cisplatin‐Resistance in Lung Cancer
Authors: Diaz Anthony Aponte , Correa Clarissa , Delgado Yamixa , Sánchez Anamaris Torres , Rodríguez Melissa Milian 
Published: June 2021

In the US, lung cancer is the second most common cancer and the leading cause of cancer mortality in both men and women. Many cytotoxic agents for cancer chemotherapy are being developed under in vitro and in vivo studies, however, the main problem is the low therapeutic index. Another important aspect is the acquired resistance to drugs (e.g. Cisplatin) by cancer cells. The aim of this research project is the development of drug delivery systems (DDS) using bovine serum albumin (BSA) as the drug's carrier and cisplatin (CisPt) and curcumin (Curc) as the drugs. Heat and ultrasonication were used to promote partial unfolding of the protein in a nanoparticle rearrangement and intercalation of the drug moieties into the carrier protein. The DDS developed include BSA‐CisPt and BSA‐Curc. To characterize the ratio of the conjugation of these systems colorimetric assays were performed. Braford assay was used for BSA, for Curc, its intrinsic absorbance and for CisPt, o‐phenylenediamine/heat assay. The preliminary results of the DDS report a ratio of BSA‐Cisplatin 1:25 and BSA‐Curcumin 1:30. Both DDS demonstrated cytotoxicity on lung cancer cells (A549) of an IC50 in the μM range after 24h. In future experiments the DDS’ size will be determined utilizing a dynamic light scattering technique. In addition, other molecules such as folic acid and polyethylene glycol will be added to improve specificity and stability respectively. These DDS have potential to minimize drug systemic toxicity, increased bioavailability and reduce multidrug resistance.Support or Funding InformationSan Juan Bautista Institutional funds and applying to travel awards.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heat Shock Protein 90 Inhibition in Kasumi‐1 Leukemic Stem Cells
Authors: Rosolen Matthew Jeremy , Smith MacKinzie D. , Bacon Nickolas A. , Sollars Vincent 
Published: June 2021

Heat Shock Protein 90 (HSP90) is a well‐conserved chaperone protein present in all human cell types. In cancers, HSP90 protein expression is often upregulated as a result of the cancer cell's elevated metabolic rate and accumulation of mutations resulting in protein‐folding defects. Inhibition of HSP90 can be a potent anticancer strategy, and drugs that target HSP90 are currently in clinical trials. Luminespib (AUY922) is an HSP90 inhibitor that is currently undergoing clinical trials. This drug is unique in that it is one of the only HSP90 inhibitors known to target both the alpha‐ and beta‐isoforms of the protein. Various dosages of AUY922 were administered to the Kasumi‐1 acute myeloid leukemia cell line to evaluate the potency of AUY922. A Western Blot analysis was performed in three biological replicates to measure expression levels of Heat Shock Protein 70 (HSP70), a molecular partner of HSP90, that is upregulated when HSP90 is inhibited. A three‐colored flow cytometry experiment was conducted with CD34, CD38 and Live/Dead label to determine the effects of increasing dosages of AUY922 on both the overall population and specifically the stem cell population, which is thought to drive relapse. We showed that HSP90 inhibition flushes the cancer stem cell population in a dose‐responsive manner. This work may be important in furthering the discussion of drug development that targets the cancer stem cell population to promote durable remission and to prevent relapse.Support or Funding InformationSupported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and by NIH Grant R15 CA186017.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The A‐kinase anchoring protein b‐synemin binds PKCe
Authors: Russell Mary , Prudner Bethany , Kuneli Melissa , Damron Derek , Chen Yijing 
Published: June 2021

Synemin is a type IV intermediate filament (IF) protein. The two largest isoforms (α and β) are co‐expressed in a variety of cell types including muscle cells. Earlier we found that in muscle cells α‐synemin preferentially localizes to the sarcolemma and intercalated disks while β‐synemin is concentrated at the Z‐disks. Both isoforms also have an unusually long tail domain compared to most IF proteins. This domain is a site for numerous protein interactions, some unique to each isoform and some common to both. For example, in the past we found that both bind protein kinase A (PKA) and are thus also classified as A‐kinase anchoring proteins (AKAPs). AKAPs act to tether PKA to specific subcellular locations to maintain the specificity of the PKA signaling pathway. Excitingly, we have now found evidence that synemin also binds PKCɛ. Specifically, co‐immunoprecipitation studies show that PKCɛ binds to β‐synemin only upon stimulation of the β‐adrenergic pathway with isoproterenol. Additionally, western analysis using cells expressing either wild type or mutant β‐synemin indicates that, upon loss of β‐synemin anchored PKA, there is a decrease in phosphorylation of PKC substrates, including a ~22 kDa protein. Studies are ongoing to determine the identity of this protein. Based on molecular weight and known PKCɛ substrates in muscle cells we predict this protein is either troponin I (TnI) or myosin regulatory light chain 2 (RLC2). In support of RLC2, yeast‐two hybrid analysis revealed that protein‐protein interaction occurs between β‐synemin and RLC2. Thus, β‐synemin is proposed be a site of cross‐talk between PKA and PKCɛ and also may localize these kinases to the sarcomere at the substrate for PKCɛ.Support or Funding InformationNIH R01 HL65701 (DD)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Genetic Screen for Proteins Involved in ASP Invasion of the Wing Disc of Drosophila melanogaster
Authors: Gondal Anas , Salim Hasan , Srivastava Ajay 
Published: June 2021

Fruit flies (D. melanogaster) are ideal genetic model organisms that can closely replicate human genetic anomalies and characteristics. The invasion of the Air Sac Primordia (ASP), an organ analogous to the human lung, into the Wing Imaginal Disc (WID) of larval fruit flies is closely related to tumor metastasis in humans, as both processes use similar mechanisms to break down the basement membrane. By studying genes responsible for ASP invasion, researchers may be able to identify analogous genes in humans responsible for tumor metastasis. A genetic screen, using 100 protein trap lines, was conducted to find genes expressed in ASP and potentially involved in ASP invasion. The protein trap lines tagged endogenous proteins with a Green Fluorescent Protein (GFP) to indicate tissue localization of selected proteins under a fluorescent microscope. WID were isolated from 10 third instar larvae of each line and imaged using fluorescence microscopy. Eleven of the 100 lines screened exhibited GFP expression in the WID, but not in the ASP. Only one of the 65 lines exhibited GFP expression in the ASP. The gene responsible was Apontic (apt). Apontic's role in ASP invasion will be further investigated using RNA interference to knock down the gene.Support or Funding InformationAnas Gondal*, Hasan Salim, Dr. Ajay Srivastava, and Nathan PowersGatton Academy of Math and Science, Department of Biology and Biotechnology Center, Western Kentucky University, Bowling Green, KYKBRIN AREA and IDeA Grants funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health Grant # P20GM103436Gatton Academy Research Internship Grant (RIG)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of Cytoskeleton‐Associated Protein 4 (CKAP4) Expression Enhances α5β1‐mediated Bladder Cancer Cell Adhesion and Migration
Authors: Lobo Sonia , Stackhouse Elizabeth , Majernick Thomas 
Published: June 2021

The transmembrane protein cytoskeleton‐associated protein 4 (CKAP4) plays an essential role in the maintenance of endoplasmic reticulum structure by anchoring the rough ER to microtubules in epithelial cells. CKAP4 has also been identified as a biologically relevant receptor for four unique ligands—tissue plasminogen activator, surfactant protein A, Dickkopf1, and antiproliferative factor. Recent data indicates that loss of CKAP4 expression leads to remodeling of the actin cytoskeleton, including disruption of actin stress fibers, and altered expression of genes known to promote cell motility and dynamic changes in the cytoskeleton. Among these genes, β1‐integrin expression was dramatically increased in response to CKAP4 silencing. Given the importance of tumor cell adhesion and migration for cancer invasion and metastasis, it is important to understand how CKAP4, through modulation of integrin functions, acts to regulate these cellular processes. To this end, we took a loss‐of‐function approach where CKAP4 expression was knocked down by means of siRNA in the human bladder carcinoma cell line T24. Western blot analysis revealed that CKAP4 knockdown induced β1‐integrin protein levels after 24 hours and continued to increase up to 96 hours after transfection with CKAP4 siRNA. Because β1‐integrin associates with α5‐integrin to form the fibronectin receptor, we investigated whether α5‐integrin protein levels are affected by CKAP4 knockdown. Western blot analysis revealed that CKAP4 knockdown induced α5‐integrin protein levels after 24 hours and continued to increase up to 96 hours after transfection with CKAP4 siRNA. Importantly, loss of CKAP4 led to increased surface expression of β1 and α5 integrin and increased cell adhesion and migration on fibronectin as measured by flow cytometry and electric cell‐substrate impedance sensing, respectively. Activation of ER stress and AMPK signaling pathways was also observed in CKAP4‐silenced bladder carcinoma cells. Our data indicate that CKAP4 knockdown mediates adhesion and migration of human bladder cancer cells through transcriptional up‐regulation of α5β1 integrin.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiac fibroblast lineages and extracellular matrix maturation in postnatal heart development
Authors: Yutzey Katherine 
Published: June 2021

During postnatal heart development in mice, the ventricular myocardium undergoes extensive remodeling with increased organization and maturation of cardiomyocytes, fibroblasts and endothelial cells. In addition, the extracellular matrix (ECM), consisting mostly of mature fibrillar collagen is produced, providing necessary mechanical support for increased cardiac output. In adult hearts, the matricellular protein periostin (postn) is induced during cardiac injury and is required for collagen deposition in scar formation and pathologic fibrosis. During postnatal heart remodeling, postn also is expressed in cardiac fibroblasts coincident with collagen 1a1 gene expression. At the same time, fibronectin expression indicative of immature ECM permissive for cardiomyocyte proliferation is decreased. By postnatal day 30, postn expression is decreased and the collagen‐rich ECM surrounding individual cardiomyocytes is apparent. The expression of postn during postnatal heart remodeling and ECM maturation supports shared molecular regulation of cardiac fibroblasts and collagen fibrillogenesis in development and disease. However, the specific contributions of postn‐expressing cardiac fibroblasts to the initial production of the collagen‐rich ECM of the heart or to maturation and hypertrophy of adjacent cardiomyocytes are not yet known.Support or Funding InformationThis work was supported by grants from the NIH/NHLBI (P01HL069779 and R01HL142217)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Setting Up an In Vitro System for Echinoderm Radial Nerve Cord Explants
Authors: Díaz Eduardo Andres Quesada , Figueroa‐Delgado Paola , Castro‐Ruiz Christian , García‐Arrarás José E. 
Published: June 2021

Among animal phyla, some of the least studied nervous systems are those of the phylum Echinodermata. Part of the problem lies in that most of their nervous components are embedded in the body wall that has calcareous skeletal components. We have now developed a dissection technique for successful isolation of the radial nerve cords (RNCs) and an in vitro system where the isolated RNCs can be cultured. This in vitro system is thus amenable to experimental manipulations aimed at characterizing the nervous system of the sea cucumber Holothuria glaberrima and to extend our studies on its regeneration capabilities. In this procedure, the body wall containing the RNCs is surgically excised and digested with collagenase producing RNCs explants that can be kept in culture for up to 2 weeks. We have performed histological studies to determine changes in the RNC anatomy during culture. We have also performed immunohistochemistry using neuronal and collagen markers to identify specific cellular populations. Labeling with a nuclear dye (DAPI) shows the presence of cells throughout the cultured period with a slight increase in nuclear counts in the medial and lateral regions of the RNC neuropile through days 3 to 7 when compared to in vivo RNCs (control). Neuronal markers, RN1 monoclonal antibody and anti‐GFSKLYFamide polyclonal antibody, identified nerve fibers and, neuron‐like cell bodies along the RNC periphery, as has been documented previously in in vivo studies. In addition, anti‐collagen marker IBA2 highlighted the demarcation between the ectoneural and hyponeural subdivisions of RNC explants kept for at least 3 days in culture. Our results showed that the central nervous system components of an echinoderm can be kept alive, maintaining its structure and morphology for various length of time. These RNC explant cultures provide a unique tool to study cellular and molecular basis of processes occurring in nervous systems such as neuronal regeneration and degeneration.Support or Funding InformationRISE Program: 5R25GM061151‐17This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.