bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of HNO‐Derived Modifications on the Cardiac Protein Phospholamban
Authors: Keceli Gizem , Majumdar Ananya , Thorpe Chevon N. , Mahaney James E. , Paolocci Nazareno , Toscano John P.
Published: November 2023

Congestive heart failure frequently involves reduced cardiac contractility and slowed muscle relaxation. HNO (nitroxyl, azanone), the protonated one electron reduced form of nitric oxide, has received significant attention as a potential heart failure therapeutic. This reactive nitrogen species augments contractility and accelerates relaxation in both normal and failing hearts. In humans, greater than 70% of cytosolic Ca2+ is pumped into the sarco(endo)plasmic reticulum (SR) by SR Ca2+‐ATPase (SERCA2a). The activity of the Ca2+ pump is regulated by a small, transmembrane protein, phospholamban (PLN). Our previous work has shown that HNO modifies PLN and affects SERCA2a activity independent of the β‐adrenergic pathway. We have applied our 15N‐edited NMR method for sulfinamide detection and biochemical approaches to investigate HNO‐derived modifications on PLN. These studies demonstrate that PLN cysteines 46 and 41 are the sites of HNO reactivity. Furthermore, our results indicate that sulfinamide formation is the major HNO‐derived modification in the presence of excess HNO donor, whereas disulfide formation is expected to dominate under physiologically relevant conditions.

Support or Funding Information

National Science Foundationand Cardioxyl Pharmaceuticals
Bacterial Cell Wall Remodeling to Study the Production of Immunoactivating Fragments
Authors: Liang Hai , DeMeester Kristen E , Hou Ching‐Wen , Grimes Catherine Leimkuhler
Published: November 2023

The human innate immune system is the front line of defense to protect the body from bacterial infection. One mechanism of activation involves various immunoactivating fragments derived from bacterial cell wall engaging the appropriate receptor to trigger an inflammatory response. Muramyl dipeptide (MDP), a small fragment of bacterial cell wall has been shown to have immunoactivating properties via the innate immune receptor Nod2. Deficiency in sensing MDP may cause serious inflammatory bowel disease (IBD) such as Crohn's Disease (CD). However, how MDP and other immunostimulatory fragments are generated is not well understood. The discovery of new immunoactivating fragments is critical to understand the interactions between human innate immune system and the bacteria it encounters (commensal or pathogenic). Here a combination of organic synthesis, biochemistry and genetic engineering approaches were implemented to bioorthogonally label bacterial peptidoglycan. We have synthesized a variety of azide functionalized cell wall intermediates, including: D‐form amino acids and UDP‐sugar substrates. The peptidoglycan biosynthetic enzymes were purified and assayed for substrate promiscuity. As these enzymes showed relaxed substrate specificity, bacterial cells were grown in the presence of the azide containing precursors. The cells were then labeled with a bioorthogonal azide‐alkyne click reaction to link a fluorescent molecule onto the cell wall. The bacterial cell walls were successfully visualized. Using this method, we will further study the interaction of human innate immune cells and fluorophore labeled bacterial cells to reveal new immunoactivating molecules.

Support or Funding Information

Funding of this project is provided by Centers of Biomedical Research Excellence (COBRE)
Carbohydrate‐Protein Interactions with Poly‐amido‐saccharides (PASs) as Unique Carbohydrate‐Inspired Polymer Structures
Authors: Chin Stacy L. , Grinstaff Mark W.
Published: November 2023

Natural polysaccharides are of significant interest in biochemistry research because of their structural diversity and essential roles in metabolism, energy storage, cellular signaling, protein recognition, and other physiological processes. However the synthesis of polysaccharides is challenging due to the presence of many similar functional groups and the large number of stereocenters throughout the polymer structure. In response to the need for reliable approaches to prepare polysaccharides, we reported a synthetic method to prepare well‐defined, enantiopure carbohydrate polymers, termed poly‐amido‐saccharides (PASs), using a controlled anionic polymerization of b‐lactam sugar monomers. Unlike natural polysaccharides, in which sugar units are joined by ether linkages, pyranose units in PASs are linked through the 1‐and 2‐positions by an amide group, which affords PASs with unique structural features and interesting chemical properties. We recently found that PASs 1) possess a helical conformation in their secondary structure, 2) are able to maintain its rigid structure under varying pH, temperature, ionic strength, and denaturing conditions, and 3) are non‐cytotoxic and can be endocytosed across different mammalian cell lines.

Our initial investigation indicate that glucose‐derived PAS (glc‐PAS) structures can be recognized by lectin concanavalin A at the same site as glucose through binding activity assays, which suggests the potential for PASs to serve as natural polysaccharide mimics. Based on this preliminary result, we hypothesize that sulfated galactose‐derived PAS (gal‐PAS) structures can potentially interact proteins in the coagulation cascade and posses anticoagulant‐like properties, similarly to heparin. The synthesis and characterization of glc‐PAS, gal‐PAS, and sulfated glc‐PAS polymer structures with via IR, NMR, and GPC will also be discussed.
Metformin Modulation of Dichloroacetate‐Induced Oxidative Stress and its Impact on Mitochondrial Integrity in VM‐M3 Glioblastoma Cells
Authors: Ward Nathan P , Poff Angela M , Koutnik Andrew P , D'Agostino Dominic P
Published: November 2023

There is accumulating evidence that altered mitochondrial function in cancer contributes to a metabolic phenotype that confers a survival advantage towards cancer cells. A hallmark of this phenotype is increased reactive oxygen species (ROS) production often coupled with upregulated antioxidant defense. This redox balance is an intriguing therapeutic target as many traditional chemotherapeutics elicit cytotoxicity through enhanced ROS production. Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, promotes pyruvate flux into the mitochondria and oxidative metabolism consequently enhancing ROS production. As an adjuvant, DCA is shown to enhance cytotoxicity and reverse resistance to a number of chemotherapeutic agents through induction of oxidative stress. Dual targeting of glycolytic and mitochondrial metabolism in cancer may elicit a greater therapeutic response than either alone. Metformin, a first line therapy for type II diabetes mellitus, inhibits complex I of the electron transport chain (ETC), disrupts oxidative phosphorylation and subsequently increases glycolytic metabolism. As such, we investigated the impact of metformin on DCA cytotoxicity in VM‐M3 glioblastoma cells. We propose that metformin insult to the ETC will augment DCA‐induced ROS production and increase oxidative stress, thus enhancing cytotoxicity. We evaluated mitochondrial membrane potential (DYm) and superoxide production through confocal fluorescence microscopy as well as cytochrome c localization through immunofluorescence. We demonstrate that DCA and metformin independently promote dissipation of DYm (p<0.01) and that metformin treatment reduces superoxide production compared to untreated VM‐M3 cells (p<0.01). DCA increases superoxide production in a concentration dependent manner and co‐treatment with metformin reverses its reduction of ROS production (p<0.05). Moreover, the combination exhibits enhanced cytotoxicity towards VM‐M3 cells (p<0.05). These findings support continued investigation of DCA and metformin in combination as a potential anti‐cancer therapy.
Differential Regulation of mTORC1 by Amino Acids
Authors: Jewell Jenna L , Guan Kun‐Liang
Published: November 2023

Organisms sense nutrient fluctuations and respond accordingly to control cell growth. When this process goes awry, the result can be detrimental, often leading to cancer. Central to this process is the mechanistic target of rapamycin (mTOR), a conserved kinase that is the main component of a protein complex termed mTOR complex 1 (mTORC1). mTORC1 activation is frequently observed in human cancers, and therapeutics that target mTORC1 have proven efficacious in clinical trials. Amino acids are the most potent stimuli for mTORC1 activation; however, the detailed molecular mechanisms are obscure, and importantly, the specific amino acids that regulate mTORC1 are unclear. Recently, we discovered that the amino acids, leucine and glutamine activate mTORC1 through two distinct signaling pathways involving the small G‐proteins RagA/B and the adenosine diphosphate ribosylation factor‐1 (Arf1), respectively. Importantly, we identified Arf1 as an essential component that specifically couples glutamine signaling to mTORC1 activation at the lysosome and independent of RagA/B. This is important because cancer cells have a high dependence on glutamine for growth and proliferation. Our results uncover a signaling cascade to mTORC1 activation independent of the Rag GTPases and suggest that mTORC1 is differentially regulated by specific amino acids.

Support or Funding Information

Cancer Prevention & Research Institute of Texas (CPRIT)
PDK2 Mediated Alternative Splicing Switches Bnip3 From Cell Death to Cell Survival
Authors: Kirshenbaum Lorrie , Gang Hongying , Dhingra Rimpy , Lin Junjun , Hai Yan , Margulets Victoria
Published: November 2023

Herein we describe a novel survival pathway that operationally links alternative pre‐mRNA splicing of the hypoxia‐inducible death protein Bcl‐2 19kD Interacting Protein 3 (Bnip3) to the unique glycolytic phenotype in cancer cells. While a full length Bnip3 protein (Bnip3FL) encoded by exons 1–6 was expressed isoform in normal cells and promoted cell death, a truncated spliced variant of Bnip3 mRNA deleted for exon 3 (Bnip3Dex3) was preferentially expressed in several human adenocarcinomas promoted survival. Reciprocal inhibition of the Bnip3Dex3/Bnip3FL isoform ratio by inhibiting pyruvate dehydrogenase kinase isoform 2 (PDK2) in Panc‐1 cells rapidly induced mitochondrial perturbations and cell death. The findings of the present study reveal a novel survival pathway that functionally couples the unique glycolytic phenotype in cancer cells to hypoxia resistance via a PDK2‐ dependent mechanism that switches Bnip3 from cell death to survival. Discovery of the survival Bnip3Dex3 isoform may fundamentally explain how certain cells resist Bnip3 and avert death during hypoxia.
Treadmill Exercise Decreases Aberrant Crypt Foci Lymph Associated in the Large Intestine of Obese Mice
Authors: Kriska Adam , Emmons Russel , Jung Paul , Xu Bianca , De Lisio Michael , Pan Yuan‐Xiang , Chen Hong
Published: November 2023

Colorectal cancer is the third leading cancer for both incidence and mortality. Obesity increases risk of developing colorectal cancer. On the other hand, increased physical activity is associated with a decreased risk of a variety of cancers. However, mechanisms of how physical activity reduces risk of cancer development remain to be illustrated. We hypothesize that exercise reduces the risk of developing colon cancer in obese animals. The aim of our study was to understand how diet and exercise affect metabolism and colon cancer formation in a preclinical model. To test this, C57BL/6N mice were fed either a control diet, AIN‐93M, or a high fat (HF) diet consisting of 45% fat for 9 weeks followed by intraperitoneal injection of carcinogen Azoxymethane (AOM). Mice on HF diet were divided into two groups: high‐fat (HF) and high‐fat plus exercise (HF+EX). A 24‐week progressive treadmill‐training program (1 hr/d, 3 d/wk) was used to exercise the mice in the HF+EX group. At the end of the study, all colons were sampled for histological examination of aberrant crypt foci (ACF) development by methylene blue staining. As expected the HF group had more ACF compared to the control group (5.29 vs 3.93, p < 0.05), but was not statistically different from the HF+EX group (5.29 vs 6.11, p = 0.23). Interestingly, the HF+EX group had more lymph nodules in the colon epithelial surface than the HF and control group (37, 17, and 12, respectively, p < 0.05). This led to the observation that ACF found around lymphatic tissue were significantly less in the exercised HF+EX group compared to the non‐exercised HF group (0.25 vs 0.61, p < 0.05). In addition, qRT‐PCR analysis of gene expression in the liver showed that cMYC expression was lower in the HF+EX group compared to both the HF and control groups. Since cMYC is an oncogene that plays a role in glucose metabolism, mitochondrial biogenesis, lipid synthesis, and ribosome biogenesis, further analysis is done to determine the involvement of the various pathways in this mouse model of colon cancer. In summary, data from this study indicate that exercise may play a protective role in colon cancer development by increasing activity of the immune response through the development of lymphatic system in the colon and reducing the potential of ACF formation.
Reconstructing jaw adductors in plesiadapid plesiadapiforms from Berru, France (Thanetian, ELMA)
Authors: Kristjanson Heather , Perry Jonathan MG
Published: November 2023

Plesiadapiforms are an extinct group of mammals closely related to euprimates (true primates) that existed from the Cretaceous into the Eocene, where they have been recovered from North America, Asia and Europe. In Europe, dental evidence suggests that plesiadapid plesiadapiforms at the Paleocene/Eocene boundary continued to evolve their folivorous diet as a dietary niche in response to the emergence of omnivorous and frugivorous euprimates and rodents. Because non‐dental quantification of the plesiadapiform masticatory apparatus has been rare, the research conducted here tests the dentally‐based hypothesis that the latest‐occurring species of plesiadapid plesiadapiforms, Platychoerops, demonstrates a masticatory apparatus more adapted to a folivorous diet than does the earlier, also folivorous species, Plesiadapis. Strepsirrhine primates will be used as extant analogues for reconstructing the jaw adductors (masseter, temporalis, medial pterygoid) of Plesiadapis tricuspidens and Platychoerops antiquus from new cranial material and mandibles. Osteological proxies for reconstructing physiological cross‐sectional area (PCSA) and muscle mass in strepsirrhines were used to model these parameters in the fossil specimens. As demonstrated in strepsirrhines, the masseter muscle is expected to be relatively larger (as measured by PCSA) than the temporalis in folivores. Results indicate that while the masseter is significantly larger than the temporalis in all specimens (p<0.05), indicating folivory in plesiadapids, it is not significantly larger than the temporalis in Platychoerops than Plesiadapis. This suggests that while the teeth between the two species demonstrate clear differences in folivorous adaptations, teeth are more plastic than muscles, which may be slow to adapt. This research has implications for the disputed evolution of Platychoerops in Europe and the role diet plays in early primate niche occupation.
Avocado Oil Improves Mitochondrial Function and Decreases Oxidative Stress in Kidney Mitochondria of Type‐2 Diabetic Rats
Authors: Ortiz‐Avila Omar , del Consuelo Figueroa‐Garcia Maria , Mejia‐Zepeda Ricardo , Cabrera‐Nuñez Faride , Flores‐Ledesma Claudia G , Hernandez‐Esparza Manjury J , Saavedra‐Molina Alfredo , Cortes‐Rojo Christian
Published: November 2023

Nephropathy is one of the most worrying complications of diabetes because of its direct association with the high mortality rate in diabetic patients. Diabetes is characterized by an increase in ROS generation and impaired mitochondrial dysfunction. Overproduction of mitochondrial ROS has been recognized as the single major event leading to the activation of pathways conducing to diabetic nephropathy. Previously, we have shown that avocado oil prevents kidney mitochondrial dysfunction and decreases ROS production in a rat model of type I diabetes. Avocado oil is a source of MUFA with some potential beneficial effects in health such as improvement of diabetic dyslipidemia, also contains several antioxidants which may modulate ROS production. Despite these antecedents, it is unknown whether avocado oil has the same effects over kidney mitochondrial dysfunction and excessive ROS production in a rat model of type 2 diabetes. To address this issue, avocado oil was administered for 3 and 6 months to Goto‐Kakizaki rats, a lean model of type 2 diabetes. Diabetic rats display a severe impairment in state 3 respiration, leading to 68% and 45.6% decrease in respiratory control ratio (RCR). Avocado oil slightly prevented this effect at 3 months but fully prevent inhibition of RCR at 6 months. Diabetes elicited a 70% decrease in mitochondrial membrane potential at 3 months, with avocado oil slightly preventing this effect. In contrast, at 6 months, there was no difference in this parameter in any experimental group in comparison to the control. Avocado oil led to a decrease of 38% and 70% in ROS production in mitochondria from diabetic rats at 3 and 6 months, respectively, in comparison to untreated diabetic rats. Mitochondria from diabetic rats exhibited a 83% decrease in mitochondrial GSH/GSSG ratio at both 3 and 6 months, indicating a condition of strong oxidative stress; supplementation with avocado oil fully prevented this effect at 3 months while at 6 months, a partial protective effect was seen as mitochondria of diabetic rats supplemented with avocado oil shown a ~40% decrease in this parameter. These results suggest that avocado oil prevented also type 2 diabetes‐induced mitochondrial dysfunction and exacerbated oxidative stress in kidney mitochondria.

Support or Funding Information

Supported by a grant from Programa de Investigación 2016 de la Coordinación de la Investigación Científica de la UMSNH (CIC‐UMSNH) (CCR).
Engineering Clonal Human Cell Lines Stably Expressing a Single p73 Isoform
Authors: Cordova Ricardo A. , Gushterova Irena , Carastro L. Michael
Published: November 2023

The p73 gene is a member of the p53 tumor suppressor family. This gene has two promoters, P1 and P2, which are the transcription initiation sites for the mRNAs encoding TAp73 and DNp73 isoforms, respectively. Recently, we reported that a human colon cancer cell line, Caco‐2, has no detectable expression of either of the two p73 protein isoforms through immunoblotting. In order to facilitate future molecular studies of specific p73 isoform functions as well as to create control samples expressing only one detectable p73 isoform for immunoblotting and other molecular techniques, pCI‐neo mammalian expression vectors encoding one of the four p73 isoform cDNAs (TAp73a, TAp73b, DNp73a, DNp73b) were stably transfected into Caco‐2 cells by electroporation. Clonal populations of transfected cells were selected using antibiotic resistance, isolated and expanded. Clonal cell line populations were used to isolate total RNA using RNeasy Plus Mini Kits (QIAGEN). Total RNA samples were used to synthesize cDNA and perform TaqMan Real Time‐PCR Comparative CT analyses in order to determine the p73 isoforms mRNA expression levels relative to untransfected Caco‐2 cells. Our initial TaqMan Real Time‐PCR data indicate a 6‐fold expression of TAp73 mRNA in clone TAp73a #12 (RQ = 6.1536; RQmin = 2.6054, RQmax = 14.0758), a 25‐fold expression of TAp73 mRNA in clone TAp73b #2 (RQ: 24.9555; RQmin = 23.3701, RQmax = 26.6485), 1‐fold expression of DNp73 mRNA in clone DNp73a #9 (RQ: 1.3434; RQmin = 0.8763, RQmax = 2.0594) and 1800‐fold expression of DNp73 mRNA in clone DNb #2 (RQ: 1850,175; RQmin = 1587.564, RQmax = 2156.226) as compared to untransfected Caco‐2. Based on our TaqMan Real Time‐PCR data, we concluded that these four isolated clonal Caco‐2 cell populations overexpress the transfected p73 mRNA isoform.

Support or Funding Information

We thank The University of Tampa David Delo Research Professor Grant (to L.M.C.) for generous financial support of this project.
Conformational Flexibility of SIKE
Authors: Standen Heather , Bell Ellis , Bell Jessica
Published: November 2023

Suppressor of IKKe (SIKE)inhibits TBK1 mediated phosphorylation of interferon regulatory factor 3 (IRF3) and misregulation of TBK1 is involved in both autoimmune disorders and cancer. Our work (Marion et al, J.Biol.Chem 288, 18612–23, 2013) demonstrated that TBK1 can phosphorylate SIKE in up to 6 positions, altering its ability to inhibit TBK1. Little however is known concerning the three dimensional structure or stability of SIKE or the effects of phosphorylation. SIKE (his tagged) has been expressed and purified using Nickel NTA affinity chromatography and shown to be homogenous by MALDI‐tof mass spectrometry. To determine the effects of phosphorylation on the conformational flexibility of the protein, SIKE and SIKE‐S6E (where the 6 phosphorylatable serines have been mutated to the phosphomimic, glutamate), and to provide support for homology models of the protein created using Phyre2, limited proteolysis experiments, using immobilized trypsin and tandem mass spectrometry identification of the early cleavage sites, and Hydrogen‐Deuterium exchange studies in conjunction with pepsin cleavage after various periods of exchange are being used.

Support or Funding Information

This work was supported by NIH grant R21A1 107447 to JKB
Phosphomimetic Constructs of Akt/PKB do not Mimic Activated Akt in Cells
Authors: Kunkel Maya T. , Newton Alexandra C.
Published: November 2023

Akt/protein kinase B is a serine/threonine kinase of the AGC family of kinases which plays a key role in signaling downstream of phosphatidylinositol 3‐kinase. Akt is activated following localization to PIP3 via its PH domain, and once recruited to the plasma membrane, Akt is phosphorylated at Thr308 within its activation loop by its upstream kinase PDK1 followed by phosphorylation at the C‐terminal hydrophobic site, Ser473. Here we show that phosphomimetic substitutions at these positions do not mimic the global signaling output of phosphorylated Akt. Specifically, we use live cell imaging with a FRET‐based Akt activity reporter, B Kinase Activity Reporter (BKAR), to show that constructs of Akt with Asp at position 308, or Asp at both Thr308 and Ser473, are neither basally active nor activatable following stimulation of COS cells with agonists such as EGF. In contrast, wild‐type Akt is robustly activated following EGF stimulation. Consistent with the lack of activity of Akt‐T308D revealed by live cell imaging, Western blot analysis using a phospho‐specific antibody to Akt consensus substrate phosphorylation sites also reveals no basal or EGF‐stimulated phosphorylation by the phosphomimetic construct. Although some previous reports indicate that substitution with Asp at both sites yields an active kinase, our data reveal that Asp is a poor phosphomimetic at the key regulatory sites of Akt, thus one should exercise caution in the use of such constructs to examine the biology of Akt signaling.

Support or Funding Information

This work was supported by NIH P01 DK5444.
Cancer‐Associated Fusion Proteins Reveal Inhibitory Role of Protein Kinase C's Processing Phosphorylations
Authors: Baffi Timothy R. , Ngoc Van An‐Angela , Antal Corina E. , Newton Alexandra C.
Published: November 2023

Since the discovery that the Philadelphia translocation event produces the oncogenic Bcr‐Abl fusion gene that sustains chronic myelogenous leukemia (CML), gain‐of‐function fusion proteins have emerged as novel cancer drivers and potential therapeutic targets. Recently, several fusion proteins involving translocations in the N‐terminal regulatory region of the tumor suppressor Protein Kinase C (PKC) were identified in cancer. Here, we show that the fusion proteins are not processed by phosphorylation, an event that accompanies the maturation of wild‐type PKC, yet are, surprisingly, constitutively and maximally active, independent of membrane‐targeting second messengers. This constitutive activity is not a result of the various proteins to which PKC is fused, but instead results from deletion of the autoinhibitory pseudosubstrate in the fusion protein: constructs of PKC deleted in just the pseudosubstrate are also not phosphorylated yet constitutively and maximally active in cells. Because unphosphorylated PKC is unstable and sensitive to degradation, it is not clear whether these fusion proteins exist in appreciable levels in the tumors in which their mRNA was identified. Importantly, however, characterization of these fusions reveals the unexpected finding that phosphorylation serves as a mechanism to maintain it in an autoinhibited state. Characterizing the functional consequences of fusion proteins in cancer is not only critical to understanding their contribution to oncogenic signaling, but also to elucidating the underlying biochemical regulatory mechanisms of relevant enzymes such as PKC.

Support or Funding Information

This work was supported by the National Institutes of Health Grant GM43154 and the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752).
Oscillatory Control of Interferon Regulatory Factor 3
Authors: Gregory Shana , Ng Chen Seng , Hoffmann Alexander
Published: November 2023

Innate immune responses to pathogen exposure are coordinated by two transcription factors, Nuclear Factor‐kappaB (NF‐κB) and Interferon Regulatory Factor 3 (IRF3). Prior work has established that NF‐κB activity shows complex temporal dynamics, and several inflammatory stimuli trigger oscillations of its nuclear activity. Indeed, oscillatory control of NF‐κB has been a focus for the research community to understand the underlying negative feedback control by IκBα and its physiological function. To our surprise, recent studies in our laboratory have revealed that IRF3 is also oscillatory in response to lipopolysaccharide (LPS) stimulation. However, those preliminary studies employed an overexpressed GFP‐IRF3 fusion protein. In this study, we sought to determine whether the endogenous IRF3 also shows oscillatory dynamics or whether the prior observation may be an artifact of the exogenous fusion protein. To this end cytosolic and nuclear fractions were collected from macrophages and Western Blots were performed. After improving the reliability of the Western blot protocol our data confirm the single cell results in that IRF3 is rapidly translocated to the nucleus within 30 min following LPS stimulation, is then exported back to the cytoplasm by 90 min, and relocated to the nucleus by 2 hrs. The importance of this finding is that it suggests the hypothesis that different stimuli may trigger different IRF3 oscillatory dynamics that may result in different innate immune responses. Future studies will address that hypothesis.

Support or Funding Information

This investigation was supported in part by a MARC Undergraduate Student Training in Academic Research (U‐STAR) National Research Service Award (NRSA) Institutional Research Training Grant (2 T34 GM008663) from the National Institutes of Health, National Institute for General Medical Sciences.
Sex‐specific cultures of neonatal cardiomyocytes possess differential cellular and biochemical properties in response to induced hypertrophy
Authors: Ross Abigail , Deaton Michael , Waikel Rebekah
Published: November 2023

It is well established that premenopausal women develop cardiovascular disease (CVD) at a lower rate of incidence than men. However, it is not well understood how estrogen and other female‐specific factors prevent the development of CVD, including cardiac hypertrophy and heart failure. Using a sex‐specific primary neonatal rat cardiomyocyte culture system, we sought to define cellular sex‐specific responses to known hypertrophy agents phenylephrine (PE) and angiotensin‐II (AngII) in the presence or absence of estradiol (E2), a known anti‐hypertrophic agent. Postnatal, day‐2 rat pups were sexed based on the presence or absence of ovaries and uterus, and same‐sex hearts were combined for primary cardiomyocyte cultures accompanied by a fibroblast removal step. Sex‐specific cardiomyocyte cultures were established and treated with PE or AngII to induce hypertrophy in the presence or absence of E2, after which cell size and gene expression were assessed. Interestingly, untreated male and female primary cultures exhibited significant differences on the cellular level. Male cardiomyocytes possess a larger base line cell surface area as compared to female cardiomyocytes. Male cultures also proliferate more rapidly, as confirmed by cell counts and BrdU incorporation analysis. Both male and female cultures demonstrated similar increases in cell size in response to hypertrophy agents. Male cultures were found to be more responsive to the attenuating effects of E2. Hypertrophy markers including Brain Natriuretic Peptide (BNP), Atrial Natriuretic Peptide (ANP), MHC‐alpha, and MHC‐beta were also differentially expressed in a sex‐specific manner in response to PE and AngII. Gene expression analysis of proteins known to contribute to the hypertrophy phenotype, such as the Tumor Growth Factor Beta (Tgfβ) family, also demonstrated both estrogen and sex‐specific regulation. These observed variations in sex‐specific cardiomyocyte cultures may be a key to understanding male and female differences in CVD pathogenesis.
Sorting Nexin 9 Differentiates Ligand‐Activated Smad3 from Smad2 for Nuclear Translocation and TGFβ Signaling
Authors: Kang Jeong‐Han , Wilkes Mark C. , Repellin Claire E. , Andrianifahanana Mahefatiana , Yin Xueqian , Leof Edward B.
Published: November 2023

The primary mediator of transforming growth factor β (TGFβ) responses are the Smad proteins. Here, we identify sorting nexin 9 (SNX9) as being required for Smad3 (but not Smad2) nuclear translocation and cell stimulation. SNX9 principally interacts with phosphorylated (p) Smad3 and promotes more rapid nuclear delivery from that observed independent of ligand. Although SNX9 does not bind Imp7, Impb, Nup153 or Nup214, it mediates the association of pSmad3 with Imp8 and the nuclear membrane. This facilitates recruitment of pSmad3/SNX9 to the nuclear pore, heterodimerization of Imp8 with Impb, and nuclear translocation of pSmad3, but not SNX9. The demonstration that Smad3 is regulated in a distinct manner from Smad2 provides the opportunity to develop intervention strategies to enhance or dampen specific aspects of the cellular response to TGFβ.

Support or Funding Information

This work was supported by Public Health Service grants GM‐055816 and GM‐054200 from National Institutes of General Medical Sciences.
Induction of Browning of White Adipocyte Formation by Ganoderma tsugae Extract
Authors: Tseng Hsiu‐Hsueh , Yeh Wan‐Chun , Tu Yun‐Chen , Yang Bi‐Fen , Lai Yen‐Ting , Kao Ming‐Ching , Liu Jah‐Yao
Published: November 2023

Ganoderma, also known as Lingzhi or Reishi, is the top grade of Traditional Chinese Medicine (TCM) and has gained considerable attention in oriental countries for its potential functions of promoting the health benefit, regulating the vital energy, postponing the old and feeble, and extending life span that were mentioned in the TCM books. However, the metabolic health benefits by regulating the vital energy from Ganoderma remain to be experimentally confirmed. Adipose tissue is viewed as one major lipid metabolic and energy‐modulated organ, and is metabolically adaptive to external stimuli. Recently, browning of white adipocyte upon stimulation has been considered as a key aspect in energy metabolism due to its energy expenditure characteristics. The current study aims to investigate the direct role of Ganoderma extracts on adipocyte formation and lipid metabolism as well as regulation of energy homeostasis. To examine the relevance of these effects mediated by Ganoderma, in vitro and in vivo assays were performed after treatment with Ganoderma extracts. Here, we demonstrate that a quality assured extract of Ganoderma tsugae (GTE), a popular species of Lingzhi locally cultivated in Taiwan, had no obviously cytotoxic effect but contributed to adipogenesis accompanied with the changes of morphological features (lipid droplet and mitochondria) in 3T3‐L1 adipocytes. Moreover, the histological analysis of white fat‐pad tissues from mice during the development of obesity showed browning characteristics after treatment with GTE. Subsequently, western blotting further confirmed the browning of white adipocyte formation, including up‐regulation of adipogenic‐, mitochondrial‐ and energy‐related proteins induced by GTE. In addition, GTE treatment not only reduced lipogenesis and lipid droplet fusion but also promoted lipid mobilization. Taken together, our findings reveal for the first time that GTE promotes metabolic health benefit and regulates the vital energy, as mentioned in the traditional Chinese Materia Medica, by modulating intracellular lipid homeostasis and induction of browning of adipocyte formation. This study provides new perspectives to elucidate the role and physiological effects of Ganoderma via current biotechnological techniques.

Support or Funding Information

This study was supported by the Ministry of Science and Technology (MOST 104‐2320‐B‐039‐024), R.O.C.
Increased Endurance Exercise Capacity Secondary to Liver Fatty Acid‐Binding Protein Ablation
Authors: Xu Heli , Gajda Angela , Zhou Yin Xiu , Fatima Anam , Agellon Luis B. , Storch Judith
Published: November 2023

Two fatty acid‐binding proteins (FABPs) are abundantly expressed in proximal enterocytes, intestinal FABP (IFABP, FABP2) and liver FABP (LFABP, FABP2), both with high affinity for binding long chain fatty acids. We previously showed that LFABP and IFABP knockout mice have divergent phenotypes in response to high‐fat feeding, with LFABP−/− mice gaining more weight and body fat, and IFABP−/− mice remaining lean relative to wildtype (WT) mice. Interestingly, despite their obese phenotype, LFABP−/− mice showed signs of being metabolically healthy, with a similar glucose tolerance to WT mice regardless of their markedly greater fat mass and an increased endurance exercise performance. To explore this surprising exercise phenotype, here we focused on possible metabolic alterations in the skeletal muscle secondary to LFABP ablation. The results showed higher mitochondrial enzyme activities in LFABP−/− mouse muscle, along with an increased triglyceride level and decreased AMPK activity, indicating that the mice have abundant energy availability at rest, compared with WT mice. Upregulation of PGC1α and PPARα mRNA levels was also found, suggesting an induction of mitochondrial biogenesis and an adjustment in lipid utilization. These results suggest that although LFABP is not normally expressed in muscle, its ablation in liver and intestine appears to contribute to a higher mitochondrial content and efficiency in energy utilization in the skeletal muscle, leading to the higher endurance exercise capacity in the LFABP−/− mice.

Support or Funding Information

This work was supported by National Institutes of Health Grant DK‐38389 from NIDDK, and by funds from the New Jersey Agricultural Experiment Station (to J. S.).
Sirtuin 3 is a Novel Regulator of Mitochondrial Ceramide Metabolism in Stroke
Authors: Gudz Tatyana I. , Riley Christopher , Keffler Jarryd , Yu Jin , Kindy Mark , Macklin Wendy , Lombard David , Novgorodov Sergei
Published: November 2023

Experimental evidence supports the role of mitochondrial ceramide accumulation as a cause of mitochondrial dysfunction and brain injury after stroke. Herein, we report that SIRT3 regulates mitochondrial ceramide biosynthesis via deacetylation of ceramide synthase (CerS) 1, 2 and 6. Reciprocal immunoprecipitation experiments revealed that CerS1, CerS2 and CerS6, but not CerS4, are associated with SIRT3 in cerebral mitochondria. Furthermore, CerS1, 2 and 6 are hyper‐acetylated in the mitochondria of SIRT3‐null mice and SIRT3 directly deacetylates the ceramide synthases in a NAD+‐dependent manner that increases enzyme activity. Investigation of SIRT3 role in mitochondrial response to brain ischemia/reperfusion (IR) showed that SIRT3‐mediated deacetylation of ceramide synthases increased enzyme activity and ceramide accumulation after IR. Functional studies demonstrated that absence of SIRT3 rescued the IR‐induced blockade of the electron‐transport chain at the level of Complex III, decreased ROS generation and protein carbonyls in mitochondria. Importantly, Sirt3 gene ablation protected the brain from IR injury. These data support the hypothesis that IR activates SIRT3, resulting in the deacetylation of ceramide synthases and the elevation of ceramide which could inhibit Complex III, leading to increased ROS generation and brain injury. The results of these studies highlight a novel mechanism of SIRT3 involvement in modulating mitochondrial ceramide biosynthesis and suggest a critical role of SIRT3 in promoting mitochondrial dysfunction and brain injury after stroke.

Support or Funding Information

NIH grant NS083544
Alterations in Acyl Phosphatidylglycerol Levels in ΔpagP, ΔpldB, and Δcls Escherichia coli leads to Phenotypic Variance
Authors: Pourmaleki Maryam , Garrett Teresa A
Published: November 2023

Headgroup‐acylated glycerophospholipids (GPLs) are a low abundance class of lipids involved in cell membrane function during cell division in Escherichia coli. Acyl phosphatidylglycerol (acyl PG) is a minor headgroup‐acylated GPL found in the cell membrane. Three enzymes, PagP, PldB, and At1g78690 (AT), play roles in the synthesis of acyl PG in E. coli. PagP synthesizes acyl PG in the outer membrane from phosphatidylglycerol (PG) and PG or phosphatidylethanolamine (PE). PldB synthesizes acyl PG in the inner membrane from PG and 2‐acyl lyso PE. AT, a lysoglycerophospholipid acetyltransferase, through an unknown mechanism, synthesizes GPLs by acylating lyso GPLs, which leads to an accumulation of acyl PG when overexpressed in E. coli. This study aims to characterize the cellular phenotype of E. coli with altered levels of acyl PG. Strains of wild‐type (WT) MG1655 E. coli with pagP and pldB deletions, ΔpagP and ΔpldB, were transformed with an arabinose induced empty pBAD33.1 plasmid, or pBAD33.1 containing PagP, AT, or PldB. A triple cardiolipin synthase (cls) knockout mutant, BKT12, and its WT control, W3110, were transformed similarly. Cellular morphology, temperature sensitivity, lipid composition, cellular permeability, and minimum inhibitory concentration to a variety of antibiotics was determined for each strain. Compared to the WT controls, overexpression of PldB leads to an elongation of cell length in BKT12, MG1655, ΔpagP, and ΔpldB. No strains showed altered temperature sensitivity. Overexpression of PagP leads to an altered growth rate and the presence of an additional unidentified lipid. Additionally, PagP overexpression leads to an increase in membrane permeability in all strains. There was no change in the minimum inhibitory concentrations upon expression of PagP, AT, or PldB. In the future, a ΔpldBΔpagP strain will be constructed and similarly analyzed to help us better understand the role of headgroup‐acylated GPLs in the cell.

Support or Funding Information

This work was supported by National Science Foundation Research at Undergraduate Institutions grant #1152463 to T. A. G.
Toward a Functional, ChemoProteomic Interrogation of Kinome and Nucleotide Binding Space
Authors: Kozarich John W
Published: November 2023

Protein kinases represent the single largest mammalian enzyme family with ~ 518 members in the human proteome and have been implicated in a wide array of complex cellular functions and pathways, ranging from metabolic regulation to tumorigenesis. The physiological functions of the majority these enzymes remain unknown. The central role of protein kinases in signal transduction has generated interest in targeting these enzymes for a wide range of therapeutic indications.

We have reported a method for identifying and quantifying protein kinases in any biological sample from any species. The ActivX Activity Based Proteomics technology for kinase relies on acylphosphate nucleotide probes, prepared from biotin and ATP or ADP that react covalently at the ATP binding sites of virtually all known protein kinases. Biotinylated peptide fragments from labeled proteomes are captured, sequenced and identified using a mass‐spectrometry based analysis platform to determine the kinases present and their relative levels. Direct competition between the probes and inhibitors can be measured to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling.

In this presentation, the development of the acylphosphate nucleotide probes and the scope of the reaction with the kinome and other nucleotide binding proteins will be discussed. In addition, other issues that speak to the breadth and versatility of this method will be addressed. The proteome‐specific kinase selectivity by drugs will also be demonstrated. Finally, the potential of the approach in other areas of nucleotide binding space, such as the GTP‐dependent enzymes, will be demonstrated.
Structural basis of distinct salicylic acid glucosylation in Arabidopsis thaliana by two homologous enzymes: implications for plant stress response
Authors: Thompson Alayna Michelle George , Iancu Cristina V , Dean John , Choe Jun‐yong
Published: November 2023

The objective of this work is to understand SA‐glucose conjugate formation by two enzymes from Arabidopsis thaliana, UGT74F1 and UGT74F2. Salicylic acid (SA) is utilized as a signaling molecule by plants in response to various stresses. To store SA in an inactive form, conjugates are formed with small organic molecules such as glucose, and inactive SA can then be transported and stored in plant vacuoles. In the model organism Arabidopsis thaliana SA‐glucose conjugates are formed by two homologous enzymes (UGT74F1 and UGT74F2) that transfer glucose from UDP‐glucose to SA. Despite being 77% identical with conserved active site residues, these enzymes catalyze the formation of different products: UGT74F1 forms salicylic acid glucoside (SAG), while UGT74F2 forms both SAG and salicylic acid glucose ester (SGE). SAG and SGE serve different cellular functions in terms of storage, so understanding their creation is important. As UGT74F1 and UGT74F1 are very similar, there are likely very subtle differences responsible for the observed final products. To investigate the difference between these enzymes, we began with crystallizing purified proteins. We solved the crystal structure of UGT74F2 complexed with its substrates UDP and SA, and this structure allowed the identification the residues that coordinate SA – H18, Y180, and M274. These residues are conserved between UGT74F1 and UGT74F2, but the loops containing these residues have several amino acid substitutions. In silico docking of SA to UGT74F1 (homology model) and UGT74F2 suggests that SA binding could be more constrained in UGT74F1. From our crystal structure and in silico modeling, we theorize that amino acid substitutions around the binding site are responsible for the observed activity differences between these enzymes. Some substitutions could affect hydrogen bonding: position 15 is threonine in UGT74F2, but serine in UGT74F1, while position 112 is alanine in UGT74F2 but a serine in UGT74F1. Other substitutions could drastically change the geometry of loops near the binding pocket, such as position 178 which is either a leucine (UGT74F1) or a proline (UGT74F2). For our future directions, we are investigating the involvement of these substitutions in product formation. We have produced mutant proteins and are investigating their products by enzymatic assays. Additionally, we are using in silico ligand docking, x‐ray scattering on solution phase protein, and x‐ray diffraction crystallography to investigate the subtle differences between UGT74F1 and UGT74F2.

Support or Funding Information

Supported by RFUMS‐DePaul Pilot Grant Award
Seasonal Variation in Urinary Aflatoxin (AFM1) of Lactating Women from Rural Households in Southern Ethiopia
Authors: Gebreegziabher Tafere , Tsegaye Workneh , Stoecker Barbara J.
Published: November 2023

Aflatoxins (AFB1, AFB2, AFG1 and AFG2) are highly toxic compounds produced by Aspergillus species. These contaminants can be present in a wide range of food stuffs especially in humid and hot climates. AFM1 is a metabolic byproduct of the potent carcinogen AFB1. As well as being carcinogenic, aflatoxins have been associated with impaired growth in infants and children. Despite the high likelihood of exposure to these contaminants, there have been only a few studies done in Ethiopia. Urine samples were collected from 101 lactating women during 2013 and short‐term aflatoxin exposures were assessed. Samples were collected in a dry season (January to February) and a wet season (June to August). Mean ± SD age of mothers was 23 ± 4 y. Mean maternal BMI was 21.3 ± 2.5 and mid‐upper arm circumference (MUAC) was 23.1 ± 1.9 cm. Corn and enset are the major staple foods of the study population. Corn would have been recently harvested in January to February and would have been stored for several months by June. Urine samples were analyzed for AFM1 using ELISA kits from Helica Biosystems Inc. AFM1 was detected in 58% and 68% of urine samples in the dry and wet seasons respectively. Among those who were exposed, the median (IQR) AFM1 was 75 (42, 165) pg/mL in the dry season and 152 (77, 352) pg/mL in the wet season. The difference of AFM1 between the two seasons was significant (p < 0.001). High urinary AFM1 in the dry season was correlated with high urinary AFM1 in the wet season (r = 0.381, p = 0.009) and this association was increased when urinary AFM1 was corrected for creatinine (r = 0.418, p = 0.001). Because of the humid and hot climatic conditions of the study area and poor quality of food storage the women are at high risk of dietary exposure of aflatoxin. Further studies are recommended to assess the prevalence of the problem and its related health implications.

Support or Funding Information

Supported by Oklahoma State University