bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the chemical synthesis of N‐acylphosphatidylethanolamine
Authors: Son Jennifer , Garrett Teresa A.
Published: June 2021

N‐acylPE has been identified in lipid extracts from Escherichia coli. N‐acylPE levels are four‐fold increased in E. coli cells lacking phosphatidylglycerol phosphate synthase (pgsA) as compared to wild‐type E. coli(MG1655). N‐acylPE was originally identified in neutral, chloroform‐based lipid extracts of ΔpgsA and MG1655 but we have observed that extraction conditions alter N‐acylPE levels. ΔpgsA and MG1655 cells were extracted with neutral or acidic Bligh‐Dyer extraction containing chloroform or methylene chloride. N‐acylPE levels were analyzed using negative‐ion electrospray mass spectrometry. When methylene chloride replaced chloroform N‐acylPE persisted in lipid extracts from ΔpgsA but not in lipid extracts from MG1655. At acidic pH, N‐acylPE persisted in lipid extracts prepared in the presence of chloroform or methylene chloride from ΔpgsA but not MG1655. This suggests N‐acylPE maybe an in vivo metabolite of ΔpgsA but an artifact of extraction in MG1655. To understand the origin of N‐acylPE, we have investigated whether phosgene, a degradation product of chloroform, is responsible for chemical formation of N‐acylPE during extraction. Using 32P‐PE we studied the phosgene mediated synthesis of N‐acylPE from PE and free fatty acids (FFA) in a single‐phase neutral Bligh‐Dyer mixture. 32P‐PE, chloroform, methanol, buffer, palmitate and phosgene was incubated at 22°C for 10 minutes and visualized using thin layer chromatography. A metabolite that comigrates with N‐acylPE is formed in the presence of phosgene and FFA but not in the absence of phosgene. Thus phosgene may promote formation of N‐acylPE during lipid extraction. Future work is required to establish that this phosgene and fatty acid dependent metabolite is indeed N‐acylPE and lead us to firmly establish that N‐acylPE is an in vivo metabolite of E. coli.
Investigation of Lipid‐Based Assembly of Viral Particles
Authors: Zofkie Amanda , Cossell Christina M , Adu‐Gyamfi Emmanuel , Stahelin Robert V
Published: June 2021

Sustainment of viral infections from HIV, Ebola and the Human T‐Cell Leukemia Virus (HTLV) requires assembly at the host cell membrane to replicate via budding into new infectious viral particles. The assembly process requires lipid binding by viral matrix proteins as well as hijacking of host cell machinery to complete the budding and replication process. Currently, there is a paucity of information on the molecular architecture of this process including how lipids in the plasma membrane regulate the budding process. To analyze the lipids present when a virus buds from the plasma membrane we have begun a comprehensive and interdisciplinary investigation of the budding process. We have investigated the lipid binding and cellular targeting mechanisms of viral matrix proteins to understand their role in targeting the plasma membrane bud site. Next, we purified virus like particles (VLPs) from a number of infected human host cells to assess their lipid composition. The lipid compositions of these strains were then compared to the lipid composition of the plasma membrane of host cells via lipid mass spectrometry. Lastly, we performed a variety of mutagenic and lipid signaling assays to assess the role of different cofactors in VLP formation. Results elucidate the role of matrix proteins in viral budding and host cell lipid changes responsible for sustaining different types of viral infections.
Differential Regulation of IL‐27 Expression during Inflammatory Stress in Adipocytes
Authors: Nam Heesun , Ferguson Bradley S , Rainey Claire F , Hopkins Robin G , Stephens Jacqueline M , Morrison Ron F
Published: June 2021

Obesity can lead to insulin resistance by mechanisms involving macrophage infiltration of adipose tissue and adipocyte secretion of inflammatory adipokines that mediate systemic inflammation and disease. Interleukin‐27 is a heterodimeric cytokine encoded by independent p28 and EBI3 genes. While recent reports have demonstrated regulation of IL‐27 in antigen presenting cells, little information regarding IL‐27 in other cell types has emerged. Using 3T3‐L1 adipocytes stimulated with proinflammatory stimuli as a model of inflammatory stress, we demonstrate similar basal expression of p28 and EBI3 in undifferentiated preadipocytes (PAs) relative to mature adipocytes (ADs). Exposure of PAs to 100 pM tumor necrosis factor α (TNFα) resulted in marked, transient induction of p28 and EBI3 gene expression peaking at 4 hrs and 18 hrs, respectively. Interestingly, neither gene was induced equivalently in ADs following TNFα stimulation. Using specific inhibitors of pertinent signaling pathways, we determined that p28 and EBI3 were predominantly downstream of NFκB. However, the refractory nature of both genes in ADs was not likely due to differences in upstream signaling pathways that were similarly activated in PAs and ADs. These findings demonstrate that IL‐27 gene expression is regulated in PAs in response to inflammatory stress and that both p28 and EBI3 become refractory to such stress as PAs mature into ADs.
Three‐Tier Model for Inflammasome Expression and a New Concept of Inflammation Privilege
Authors: Yang Xiao‐Feng , Yin Ying , Mai Jietang , Liu Fang , Jiang Xiaohua , Wang Hong
Published: June 2021

We examined a new hypothesis that the tissue expressions of TLRs/NLRs/inflammasome/caspase‐1/IL‐1β pathway are differentially regulated. We made several findings: (1) Vascular tissues and heart express fewer types of TLRs and NLRs than immune tissues; and (2) Based on the expression data of inflammasomes (NALP1, NALP3 and IPAF), the tissues can be classified into three tiers: the first tier tissues including blood and thymus express inflammasome(s) in constitutive status; the second tier tissues have inflammasome(s) in nearly‐ready expression status (with the requirement of upregulation of one component); the third tier tissues, like heart, require upregulation of at least two components to assemble functional inflammasomes. We examined the mRNA levels of inflammasome components in human aortic endothelial cells after stimulated with lipopolysaccharide (LPS). In the presence of LPS stimulation, the mRNA levels of the main inflammasome component including ASC, caspase‐1, NALP‐1 and NALP‐3 are up‐regulated, which supports our three‐tier model. Our original model of three‐tier expression of inflammasomes would further suggest a new concept of tissues’ inflammation privilege, which provides an important insight to the differences among tissues in initiating acute inflammation and upregulation of inflammasomes in inflammation‐privileged tissues in response to chronic metabolic stimuli.
HIV Protease Inhibitors Differentially Regulate PPARγ expression in Adipocytes
Authors: Pecora Beth S , Zha Weibin , Hylemon Phillip B , Wabitsch Martin , Zhou Huiping
Published: June 2021

HIV protease inhibitor (PI)‐induced dysregulation of lipid metabolism is a major concern in the clinic. The peroxisome proliferator‐activated receptor γ (PPARγ) plays a pivotal role in regulating the expression of key genes involved in lipid metabolism and activating adipocyte differentiation. We have previously shown that individual HIV PIs had different effects on PPARγ expression and adipocyte differentiation. The aim of this study was to examine the potential cellular mechanisms by which HIV PIs regulate PPARγ expression in adipocytes.

Methods

Mouse 3T3‐L1 and human SGBS adipocytes were used in this study. The interaction between RNA binding proteins, HuR and CUGBP1, and PPARγ mRNA was identified by pull‐down assay and immunoprecipitation followed by real‐time RT‐PCR. The intracellular protein levels of HuR and CUGBP1 were measured via Western Blot analysis. Luciferase reporters containing PPARγ promoter, coding region, or 3’untranslated region were utilized to identify the potential mechanisms by which HIV PIs regulate PPARγ expression.

Results

HIV PIs differentially regulate the expression of PPARγ in adipocytes.

Conclusions

HIV PI‐mediated dysregulation of PPARγ expression may represent a critical mechanism by which HIV PIs induce dyslipidemia, lipodystrophy and metabolic syndrome.

This work was supported by NIH R01 and F30 grants.
Mutant FAR protein construction and their potential as vaccine candidates
Authors: Scheidt Michael , Bath Jennifer , Bakken Amanda , Clark Colin , Eneh Peace , Knox Megan
Published: June 2021

Fatty acid and retinol binding proteins are a family of proteins found in free living and parasitic nematodes. It is our hypothesis that parasitic nematodes secrete FAR into host tissues, resulting in localized immunosuppression, and hence chronic parsitic infection. Heligmosomoides polygyrus is a mouse model for hookworm that is commonly used in the laboratory for the investigation of the cellular responses to chronic helminth infections. Our laboratory is investigating a protein retrieved from the L4 larval stage of this parasite, a protein believed to be involved in assisting the parasite to form and maintain chronic infections. We have termed this protein Hp‐FAR‐1, for H. polygyrus Fatty Acid and Retinol Binding Protein 1. Creation of mutant FAR proteins that do not bind retinol are of interest for the development of potential vaccine candidates. The purpose of this study was to compare Ce‐FAR‐4, a non‐functional FAR protein from C. elegans, with parasitic FAR proteins. The primary amino acid structures of both parasitic and Ce‐FAR‐4 were analyzed for regions of conservation. Specifically, we target regions that were conserved among the parasitic proteins, including Hp‐FAR‐1, but not in Ce‐FAR‐4; hypothesizing that mutating the conserved regions of Hp‐FAR‐1to the amino acids found in Ce‐FAR‐4 would inhibit retinol binding.
Molecular Architecture of Viral Assembly and Bud Site Formation
Authors: Stahelin Robert V , Adu‐Gyamfi Emmanuel , Yu Hui , Cossell Christina M , Zofkie Amanda
Published: June 2021

Viruses cause an array of diseases, some fatal and others a minor nuisance, some transmitted through the air and others through bodily fluids. Strides have been made in eradicating some viruses from the planet through vaccination but others such as HIV, Ebola or the Human T‐cell Leukemia virus (HTLV) lack significant treatment options or preventive vaccination. The pathogenesis of these viruses depends on infection, replication and generation of new virions. This process requires concerted action of newly synthesized viral proteins and hijacking of mammalian machinery. The generation of new virions requires budding from the host cell membrane into a new particle with the ability to infect more cells. Over the last several years the matrix proteins (MA) and capsid proteins (CA) have been implicated in association with lipid membranes and budding from the host cell to create new virions but mechanistic insight is lacking. To address basic questions underlying virion formation, we have used biochemical, biophysical, structural, cellular, and single molecule analysis to investigate how MAs bind lipid membranes and change their shape. We then map these interactions in human cells and in the formation of new virions.
Kinetics of yeast dissociation from lectin beads: II. alpha methyl glucose
Authors: Zem Gregory C. , Dreyfuss J , Allen J , Kawashima R , Daco E , Kanda Y , Yaghoobian J , Cochrane A , Sobhani J , Pouromid K , Jahanian S , Solati A , Balazadeh H , Hernandez D , Cortes M , Kermani K , Oppenheimer S. B.
Published: June 2021

As concanavalin A (Con A) has preferential binding specificity for mannose and glucose, here we test the ability of alpha methyl glucose to dissociate yeast (Saccharomyces cerevisiae) from Con A beads. We chose only one concentration of each of these two sugars (mannose sugar in Part I of this series) to study so that we could do hundreds of replicate trials to statistically evaluate the reliability of this new model system. Future dose‐response experiments will be performed to precisely determine the effectiveness of the reagents at the lowest concentrations. In 247 repeated trials using 1.5 M alpha methyl glucose versus controls in the absence of sugar, with tiny Standard Errors, the sugar substantially increased dissociation of yeast that were bound to Con A derivatized beads over the 60 min time course compared with sugar‐free controls (2‐sample‐t‐test results: 20 min p less than 0.0001; 40 min p less than 0.0001; 60 min p less than 0.0001). Repeated measures ANOVA indicated a significant effect of the treatment on the number of bound yeast (F= 333, p less than 0.0001), as well as a significant interaction between treatment and amount of time elapsed (F=152, p less than 0.0001). The results are expected but the model is new. We believe that for the first time the kinetics of yeast elution from lectin beads is precisely measured in an easily visualized, simple and inexpensive model system to test elution reagents in glycan purification protocols and to test the efficacy of potential carbohydrate‐based drugs in dissociating pathogens from human cells (Supported by NIH NIGMS SCORE S0648680, MARC, RISE, the Joseph Drown Foundation and the Sidney Stern Memorial Trust).
Kinetics of yeast dissociation from lectin beads: III. potassium chloride
Authors: Zem Gregory C. , Dreyfuss J , Allen J , Kawashima R , Daco E , Kanda Y , Koleilat T , Khurrum M , Yaghoobian J , Danialian S , Marroquin M , Wolfenden L , Oppenheimer S. B.
Published: June 2021

Here we quantitatively examine the kinetics of salt‐induced dissociation of mannose‐rich yeast (Saccharomyces cerevisiae) from concanavalin A (Con A) derivatized agarose beads. This model can be used to test a reagent's ability to elute glycan‐containing molecules from lectin beads in purification protocols and to remove pathogens from human cell surfaces in drug development studies. We chose one salt concentration (4.0 M potassium chloride) in this study in order to do many replicate experiments to statistically evaluate the reliability of this model system. Future work will test many reagent concentrations in dose‐response experiments to determine reagents that are most effective at the lowest concentrations. In 65 repeated trials, the kinetics of dissociation of yeast previously bound to Con A derivatized agarose beads in the presence and absence of the salt over a 60 min time course was measured. This was done by simply counting the yeast remaining on the beads at each time point. A repeated measures‐ANOVA indicated a significant effect of the salt on the number of bound yeast (F=70, p less than 0.0001) as well as a significant interaction between the treatment and time elapsed (F=25, p less than 0.0001). Two‐sample t‐tests comparing salt treated samples with controls indicated substantially increased dissociation in the salt treated samples (20 min p less than 0.0001, 40 min p less than 0.0001, 60 min p less than 0.0001). What is new here is the model. We know of no past work that measures dissociation kinetics in so simple, inexpensive and easily visualized a manner (Supported by NIH NIGMS SCORE S0648680, MARC, RISE, the Joseph Drown Foundation and the Sidney Stern Memorial Trust).
Effect of Chronic Estradiol‐17β (E2) exposure on the expression of genes related to inflammation in the Arcuate Nucleus of female rats: Effects of aging.
Authors: Jagannathan Lakshmikripa , MohanKumar Sheba , MohanKumar Puliyur S
Published: June 2021

The use of estrogen is widespread among young women as a contraceptive and in older women for hormone replacement therapy. Although estrogen is known to have several beneficial effects on the brain, these effects are limited to non‐hypothalamic regions. Earlier studies have demonstrated inflammatory changes in the arcuate nucleus (AN) of the hypothalamus after E2 treatment. We hypothesized that chronic E2 exposure would increase the expression of cytokine and chemokine genes in the AN. To test this, young (Y) and middle aged (MA) female rats were sham implanted or implanted with slow release E2 pellets for 90 days. Ovariectomized Y and MA rats were treated the same way. At the end of treatment, animals were sacrificed and the AN was microdissected from brain sections. RNA extracted from the AN was subjected to a qPCR array. The array consisted of genes related to the inflammatory response like IL‐1β, TNF‐α, IL‐6, CCl2 and iNOS. In Y animals, E2 treatment significantly upregulated the following genes: IL‐6 by 3.73 fold, CCL2 by 4.76 fold, and iNOS by 9.42 fold compared to control (p<0.05). There was no change in the expression of these genes in MA animals after E2 treatment. These results indicate that chronic E2 exposure affects the expression of pro‐inflammatory genes in an age‐associated fashion. Supported by NIH AG027697.
Changes in BACE1 and BACE2 Expresssion in Alzheimer's Disease and Down Syndrome
Authors: Laux Ashley , Webb Robin , Holler Chris , Dowling Amy , Beckett Tina , Murphy M. Paul
Published: June 2021

Progressive cognitive decline is seen in Alzheimer's disease (AD) and Down syndrome (DS). In both, the amyloid precursor protein (APP) is believed to cause the development of amyloid pathology in the brain with the β‐site APP cleaving enzymes 1 and 2 (BACE1 and BACE2) as rate limiting enzymes. Individuals with DS have an extra copy of both APP and BACE2 on chromosome 21. Despite well studied mechanisms behind the common pathology, not much is known about regulatory processes. Evaluation of gene and protein expression levels in disease and control samples were studied to identify key regulation factors. Quantitative RT‐PCR and Western blot were used to measure the relative mRNA and protein expression levels of APP, BACE1 and BACE2 in DS, Mild Cognitive Impairment (MCI), presymptomatic AD (PCAD), frontotemporal dementia (FTD) and AD. In DS; APP, BACE1 and BACE2 mRNA increased while GAPDH decreased. APP expression increased, BACE1 expression decreased, and no change was found in BACE2 expression. In AD, MCI, PCAD and FTD, BACE1 and BACE2 increased. Regulation of APP and BACE2 are likely due to gene dosage effect while GAPDH and BACE1 are transcriptionally controlled in DS. Upregulation of BACE1, BACE2 and APP are likely early effects of MCI and AD and may be driven by general neurodegenerative processes. Thus the pathology seen in DS is regulated by different mechanisms than those seen in MCI and AD. Supported by NIH NS058382.
Autophagy as a survival response to sigma receptor ligand‐induced endoplasmic reticulum stress
Authors: Kim Felix J. , Stabler Stacy M. , Schrock Joel M. , Pasternak Gavril W.
Published: June 2021

The sigma1 receptor is a novel endoplasmic reticulum (ER) molecular chaperone that is highly expressed in certain tumor cell types. Small molecule ligands targeting sigma1 inhibit cell proliferation and induce apoptosis in vitro and inhibit tumor growth in vivo. However, the cellular pathways engaged by sigma1 receptor‐ligand interaction are not well defined. We previously observed that treatment with sigma1 antagonists activates autophagy. Here, we elucidate the events leading to this activation. Autophagy can function as a death or survival pathway in response to cell stress. As sigma1 is enriched in the ER, we asked if sigma1 ligand treatment induces ER stress. We found that sigma antagonists induce ER stress and activate the unfolded protein response (UPR) in a dose and time responsive manner. We then asked if autophagy functions as a death or survival pathway. We found that UPR activation precedes autophagosome formation and autophagy precedes apoptosis in antagonist‐treated cells. Furthermore, inhibition of autophagosome formation and degradation accelerates antagonist‐mediated apoptosis. Together, these data suggest that autophagy is a survival response to sigma antagonist‐induced ER stress.
Cysteine rich protein 1 (CRP1) in UV radiation‐induced stress responses
Authors: Latonen Leena , Jarvinen Paivi M , Saarialho‐Kere Ulpu , Laiho Marikki
Published: June 2021

UV radiation is the most prominent environmental carcinogen. We have assessed transcriptional differences between cell cycle arrest and apoptosis induced by UV radiation, and identified CSRP1 as a previously uncharacterized stress response gene. We have now studied the protein coded by this gene, named Cysteine rich protein 1 (CRP1), in UV radiation‐induced stress responses. CRP1 is an actin cytoskeleton‐interacting LIM domain protein, which has growth suppressive properties in human diploid fibroblasts. Yet, following severe UV‐ and staurosporine‐induced stresses inducing apoptosis, expression of CRP1 provides a survival advantage. CRP1 is induced in primary human keratinocytes by physiologically relevant wavelengths and doses of UV radiation. During UV‐induced keratinocyte apoptosis, CRP1 is cleaved in a caspase‐dependent manner. In human skin specimens, CRP1 is expressed in smooth muscle cells, vasculature, sweat glands, sebaceous glands, and hair root sheath, but very little or no CRP1 is present in keratinocytes of the normal skin. In contrast, CRP1 expression is elevated in basal keratinocytes of actinic keratosis (AK) representing UV‐damaged skin. However, in squamous cell carcinoma (SCC), a UV‐provoked skin cancer, no elevated CRP1 expression was found. Our results show that CRP1 is regulated by UV radiation, and suggest a role for CRP1 in phototoxic responses of human skin.
Nutlin‐3, a MDM2 antagonist, induces Erk1/2 activation in U2OS cells
Authors: Lee Sun‐Young , Ko Kyoung‐Won , Choe Yun‐Jung , Jeong Seong‐Whan , Kim Ho‐Shik
Published: June 2021

Nutlin‐3 is a small molecular antagonist of MDM2 which is a critical negative regulator of p53. Activation of p53 by nutlin‐3 has been reported to trigger cell cycle arrest and apoptosis in cancer cells. Here, we present that nutlin‐3 activates anti‐apoptotic pathway as well as apoptosis in human osteosarcoma U2OS cells.

Nutlin‐3 inhibited growth of U2OS cells, which was associated with apoptosis. Concomitantly, Erk1/2 was phosphorylated by nutlin‐3 treatment. Pretreatment of MEK inhibitors such as U0126 and PD98059 enhanced cell death by nutlin‐3, suggesting that activated Erk1/2 protects U2OS cells from apoptosis. In addition, nutlin‐3 induced transcription of EGFR ligands including HB‐EGF and Erk1/2 phosphorylation by nutlin‐3 was prevented by cycloheximide, implying that Erk1/2 activation might be resulted from autostimulation of EGFR by upregulated EGFR ligands.

Taken together, p53 activation by nutlin‐3 may induce both apoptotic and survival pathways consisted of Erk1/2. Therefore, combined treatment of nutlin‐3 and Erk1/2 inhibitors may enhance therapeutic efficacy of p53 activation to induce cancer cell apoptosis. This research was supported by MRC from cell death disease research center funded by KOSEF.
Cloning and expression of a type I metacaspase from Schizophyllum commune
Authors: Zheng Huiqing , Fox Kristin M.
Published: June 2021

Apoptosis, or programmed cell death, is an intracellular mechanism that induces ‘cell suicide’ and plays a very critical role in the development and homeostasis of all multi‐cellular organisms. Apoptosis is carried out by caspases, a family of cysteine proteases. At least fourteen distinct caspases have been identified in mammals, but only a few caspases have been identified in other less complex organisms. Recently, Madeo and his colleagues have investigated a protein, YCA1, in Saccharomyces cerevisiae, which demonstrates caspase‐like processing and is homologous to mammalian caspases. This finding is a fundamental starting point for the investigation of other caspases in non‐mammalian organisms, especially fungi. One type I metacaspase gene (scp1) has been successfully cloned from S. commune. We have amplified the open reading from of a second type I metacaspase gene (scp2) from an S. commune cDNA library via polymerase chain reaction (PCR). This PCR product was cloned and then subcloned into the expression vector, pQE‐80 (Qiagen). Expression and purification of the protein product of scp2 are underway.
Molecular Pathways Involved in RAGE Mediated Attenuation of Arteriogenesis
Authors: Rogers Jeffrey Keith , Jones Joshua , Waikel Rebekah , Anaya Paul
Published: June 2021

The receptor for advanced glycation endproducts (RAGE) has been implicated in promoting vascular dysfunction through the activation of proinflammatory cascades and the induction of oxidative stress. Chronic activation of RAGE is associated with diabetes‐related vascular complications including coronary and peripheral vascular atherosclerosis and diabetic retinopathy. We have demonstrated that RAGE activation in monocytes and macrophages also regulates vascular remodeling in response to acute ischemia in a mouse model of hind‐limb ischemia. Lentiviral‐directed overexpression of RAGE in monocytes prevents the development of collateral vessels (arteriogenesis) in response to hind‐limb ischemia. To better understand the molecular mechanisms responsible for these observations, we have undertaken a cell culture approach to address the effect of monocyte ‐specific RAGE activation on the production of proarteriogenic and proangiogenic factors under hypoxic conditions and determine how chronic RAGE activation influences receptor recycling. We have generated soluble RAGE using a baculovirus system, which will provide a valuable reagent to evaluate RAGE signaling in response to hypoxic conditions. In addition, we are generating N‐terminal GFP‐tagged versions of the RAGE receptor that will allow us to evaluate RAGE receptor recycling following activation in a macrophage cell line.
Non‐Structural (NS) proteins of Respiratory Syncytial Virus (RSV) target multiple components of the interferon induction and response pathways of innate immunity
Authors: Swedan Samer Fuad , Barik Sailen
Published: June 2021

RSV is a Pneumovirus of the family Paramyxoviridae. RSV is a leading cause of respiratory disease in the very young and the elderly. Our research focuses on the ability of RSV to inhibit innate immunity. We hypothesize that the two Non‐Structural (NS1 and NS2) proteins of RSV interfere with key signaling components in the interferon (IFN) induction and response cascades, thus inhibiting innate immunity and leading to disease. Our research goals are: 1) To identify key host targets of NS proteins and 2) To identify the IFN‐suppressor domains of the NS proteins. Our preliminary data reveal that NS1 is required for decreasing levels of TRAF3 and IKKε, which are key components for the IFN induction cascade, and that NS2 is required for the degradation of STAT2, a key component for the IFN response cascade. The C‐terminal 20 amino acids (AA) of NS1 were not required for its effect on TRAF3, whereas the C‐terminal 10 AAs were required for the reduction of IKKε. The C‐terminal 4 AAs of NS2 were required for its ability to degrade STAT2. Finally, we discovered that NS1 and NS2 can interact in vitro and can individually interact with the host MAP1B protein.

Sponsored by Alabama EPSCoR and NIH grant AI059267
The cytotoxic A1 domain of shiga‐like toxin 1 interacts with two distinct sites on the ribosomal stalk
Authors: McCluskey Andrew J , Gariepy Jean
Published: June 2021

Ribosome‐inactivating proteins (RIPs) such as Shiga‐like toxin 1 (SLT‐1) halt protein synthesis in eukaryotic cells by depurinating a single adenine base in the sarcin‐ricin loop of 28S rRNA. The molecular details leading up to the site‐specific depurination are lacking, despite a general understanding of the biochemical basis of SLT‐1 toxicity. Here we present evidence that the mechanism by which the catalytic domain of RIPs cleaves its substrate involves initial docking interactions with the ribosomal stalk by virtue of a conserved C‐terminal domain common to all three stalk proteins P0, P1, and P2. The A1 chain of SLT‐1 transiently binds to this peptide with a modest binding constant and rapid on and off rates. Mutagenesis of charged residues within the A1 chain identified a cationic surface that interacts with the peptide motif. In addition, peptide phage‐display revealed a complementary charged surface and an additional hydrophobic motif which may be involved in the interaction. Moreover, deletion mutagenesis performed on the ribosomal protein P0 revealed that the A1 chain binds to an alternate site on P0 in proximity to the contact sites for P1/P2 heterodimers. These results demonstrate that the catalytic chain of RIPs such as SLT‐1 dock on ribosomes using two classes of binding sites located within the ribosomal stalk which may aid in orienting their catalytic domain in close proximity to the depurination site.
Inhibition of nucleosome assembly by the mismatch repair system
Authors: Takahashi Tatsuro , Taki Kanae , Higashi Torahiko , Nakagawa Takuro , Masukata Hisao
Published: June 2021

The mismatch repair (MMR) system post‐replicatively corrects replication errors such as base‐base mismatches. The MMR reaction begins with recognition of the errors by the MutS complex. MutS and other factors then identify the erroneous newly synthesized strand. The nature of strand discrimination signals in eukaryotes remains elusive, but a gap or 3′‐end of DNA can work as the signal in vitro. Concurrent with the MMR reaction, nucleosomes re‐assemble on the replicated DNA strands. Nucleosome assembly might impede the MMR reaction, for example by obscuring strand discrimination signals. However, how the MMR system deals with nucleosome assembly remains highly unclear.

Using Xenopus egg extracts as a vertebrate model system, we investigated functional interaction between the MMR reaction and nucleosome assembly. In the nucleoplasmic extracts of Xenopus eggs, nucleosomes rapidly assemble on naked DNA. Surprisingly, nucleosome assembly was severely inhibited when a base‐base mismatch was present on the DNA molecule. The MutS complex specifically bound to the mismatch‐containing DNA, and this complex was essential for both repair of the mismatch and inhibition of nucleosome assembly. These results suggest that nucleosome assembly at replication error loci is temporally inhibited by a MutS‐dependent mechanism during the MMR reaction. We propose that this mechanism is important for eukaryotic MMR in the context of chromatin.
Acetylation of Dna2 and Fen1 by P300 Promotes Long Flaps Favoring DNA Stability
Authors: Balakrishnan Lata , Stewart Jason A , Polaczek Piotr , Campbell Judith L , Bambara Robert A
Published: June 2021

Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki fragments and repair of DNA damage. FEN1 can be post‐translationally modified by the transcriptional coactivator, p300. Acetylation of FEN1 by p300 inhibits its endonuclease activity impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2. However, in contrast to FEN1, acetylation of Dna2 stimulates its 5′–3′ endonuclease, the 5′–3′ helicase and DNA‐dependent ATPase activities, allowing more efficient processing of long flaps. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Dna2 and FEN1 form a complex with p300 both in vitro and in vivo. Differential regulation of the activities of the two endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as insuring correct processing. Such intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the re‐synthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair.

This work was funded by the National Institute of Health grant GM024441 to R.A.B.
Subunit exchange and DNA‐binding dynamics of Escherichia coli single‐stranded DNA binding protein (SSB)
Authors: Mason Claire Elise , Jergic Slobodan , Dixon Nicholas , Beck Jennifer
Published: June 2021

Single‐stranded DNA binding proteins (SSBs) are ubiquitous in all organisms, and are responsible for binding to and maintaining the structural integrity of single‐stranded DNA (ssDNA) produced during DNA replication, recombination and repair processes. Escherichia coli SSB is a homotetrameric protein, and its interaction with ssDNA has been well‐characterised in vitro, but the mechanism by which it is able to move rapidly upon DNA during dynamic processes such as replication is largely unclear. In this work, nanoelectrospray mass spectrometry was used to investigate aspects of the E. coli SSB‐ssDNA interaction. By examining subunit exchange between unlabeled SSB and uniformly labeled 15N‐SSB, it was found that ssDNA binding has a strongly inhibitory effect on subunit exchange, which occurs slowly but extensively in the absence of DNA. Furthermore, using mass spectrometry and surface plasmon resonance, intact SSB tetramers, under certain conditions, were found to be able to transfer directly between discrete ssDNA strands. Whether or not transfer occurs is dependent on both oligonucleotide length and salt concentration. This work shows that direct transfer is a possible mechanism of SSB mobility on DNA.

This work was funded by an Australian Research Council Discovery Grant
SV40 Large T Antigen Helicase/Initiator Structure and Function
Authors: Chen Xiaojiang , Gai Dahai
Published: June 2021

SV40 replication system serves as a model system for studying DNA replication in eukaryotic cells. SV40 large T antigen (LTag) is a replicative helicase and a “pleiotropic” replication initiator that recognizes replication origin, and initiates origin DNA melting and unwinding. To understand how LTag assembles on origin DNA to initiate DNA melting/unwinding, we determined the structure of the LTag‐dsDNA‐ADP complex, which represents the first structure for a hexameric helicase/initiator bound to dsDNA in the central channel. This structure captures a distinctive intermediate complex corresponding to an early stage of SV40 origin unwinding. The LTag‐DNA‐ADP complex structure reveals several unexpected features regarding: i) how a smaller hexameric channel accommodate a larger duplex DNA, ii) how the six subunits of LTag in the channel interact with dsDNA, and iii) how LTag distorts and melts the origin DNA using the β‐hairpin fingers and a nearby loop. I will present these new structural data and the implications for a plausible molecular mechanism for origin DNA melting and unwinding by this multi‐functional initiator and replicative helicase in eukaryotic cells.
MiR181 regulates Mcl‐1 expression in Lyn‐dependent imatinib‐resistant CML
Authors: Zimmerman Eric , Dollins Claudia M. , Hammond Scott M. , Graves Lee M.
Published: June 2021

Imatinib, a Bcr‐Abl inhibitor, is a successful front‐line treatment for chronic myelogenous leukemia (CML). Resistance to imatinib treatment has been shown to involve over‐expression of Lyn kinase (Lyn) and Bcl‐2 family proteins. MicroRNAs (miRNAs) are small ~22 nucleotide regulators of protein expression whose expression correlates with the development of drug resistance in cancer. However, the importance of miRNA regulation in imatinib resistance has not been investigated. MYL‐R cells are a recently‐established model of Lyn‐mediated imatinib‐resistant CML. Profiling miRNA expression in MYL‐R and drug‐sensitive MYL cells identified approximately 30 miRNAs whose expression differed >2‐fold. Of these miRNA the miR181 class of miRNAs were dramatically reduced in MYL‐R cells. Chemical (PP2, desatinib) and genetic (siRNA) inhibition of Lyn rescued miR181b and miR181d expression in MYL‐R cells. Futhermore, over‐expression of Lyn in MYL cells decreased miR181 expression. To determine the consequence of Lyn‐mediated miR181 regulation, we sought to discover protein targets of miR181. Mcl‐1 is a Bcl‐2 family member and pro‐survival protein associated with drug resistance in leukemia. Rescue of miR181b expression in MYL‐R cells reduced Mcl‐1 expression. In addition, using a luciferase construct containing the Mcl‐1 3′ UTR we observed a decrease in luciferase expression upon miR181b over‐expression, suggesting that miR181 directly targets Mcl‐1 mRNA. In summary our results have identified a novel pathway by which Lyn expression promotes cell survival through the up‐regulation of Mcl‐1.
Post‐transcriptional Control of Human Interleukin‐3 Mediated by its 3′‐UTR
Authors: Hernández‐Pérez Marimar , Almodov Natalia , Gonzalez‐Feliciano Jose A , Gonzalez Carlos
Published: June 2021

Human interleukin‐3 (IL‐3) is a cytokine that stimulates the growth and differentiation of early lymphoid stem cells and has been implicated in certain types of cancer. IL‐3 is a member of a class of transiently expressed mRNAs that harbor Adenosine/Uridine‐Rich Elements (ARE) in their 3′‐UTRs. The 3′‐UTRs of many cytokines play a role in post‐transcriptional control by altering mRNA stability and/or translation. Transfection assays using HeLa and Jurkat cells harboring a luciferase‐IL‐3 3′‐UTR reporter showed a significant reduction in luciferase activity. The mRNA levels of these luciferase chimera do not show any significant differences in HeLa and T‐cells, suggesting that the hIL‐3 3′‐UTR mediates post‐transcriptional control. To understand how the AREs from the hIL‐3 3′‐UTR are involved in controlling translation, we conducted site‐directed mutagenesis of four ARE clusters that were identified in the hIL‐3 3′‐UTR. Firefly luciferase reporters harboring ARE mutations were transfected into HeLa and T cells. The luciferase chimeras lacking the hIL‐3 ARE showed an increase in luciferase activity. Electrophoretic mobility shift assays (EMSA) demonstrated that TIA‐1 and HuR, two ARE‐BP previously associated with translational control, bind to the hIL‐3 ARE. These studies will help delineate the functionality of AREs in the translational control of human IL‐3 and its relationship with oncogenesis.

Supported by grants from NIH to C.I.G. (KO1 HL‐04355‐05, U54 CA96297, P20 RR 016174). M.H. is supported by the RISE Program, R25GM061838.
Non‐Coding ncRNA Modified by Environmental Factors and the Epigenetic Code: Translating the Entangled Cancer‐Angiogenesis‐Tolerance Epigenome
Authors: Wissler Josef H.
Published: June 2021

OBJECTIVE

From the genome of same genotype, genetic [Mendelian] and epigenetic [non‐Mendelian] information may be translated into different organized or mess‐chaotic [tumor] phenotype variations. ncRNA functions of cells activated by extrinsic [environmental] factors were investigated whether chance or necessity for structural codes govern genomic episcription [Ann. N.Y. Acad. Sci. 1022: 163–184, 2004; 1137: 316–342, 2008].

RESULTS

Nascently formed modified, edited, redox‐ and metalloregulated functional small hairpin ncRNA [<200n] as bioaptamers in RNP complexes were sequenced. Some address conserved homologous helix‐nucleating domains shared in epigenetic regulator proteins entangled in tolerated growth, vascularization, metabolic syndromes, cancer and epigenome indexing. At variance to usual interpretation by genetic complementary and triplet base codes, ncRNA are not “non‐ coding”, if read in another “language alphabeth” [complementary biomolecular imprint interaction]. This epigenetic code consists of different associated intrinsic and extrinsic interactive imprints and factors: [1] Non‐Mendelian nucleic acid 3D‐episcripts in helix‐ nucleating complementary interaction with [2]: Conserved homologous Mendelian domains in epigenetic regulator protein and nucleic acid matrices. [3] Extrinsic and intrinsic factors upon which formation of [1] and interaction with [2] depends [e.g. redox‐and metalloregulation].

CONCLUSIONS

The epigenetic code has more diversity, complexity and plasticity repertoires than genetic codes. It implies some chance and heuristic reactions in algorithmic [necessity] processes and Darwin‐Mendel's genetic principles in synergism with some environmental [Lamarck's] influences for epigenetic imprinting and inheritance.
A MicroRNA Upregulates Neuron‐Specific Transcripts through the NMD Pathway
Authors: Wilkinson Miles Frome , Huang Lulu , Bhalla Angela D , Bhardwaj Anjana , Song Hye‐Won , Karam Rachid , Bruno Ivone G
Published: June 2021

Cells are equipped with multiple RNA regulatory mechanisms that control the expression of specific proteins. Here we report that an miRNA expressed selectively in the brain, miR‐128, regulates another RNA‐repressing mechanism: nonsense‐mediated decay (NMD). miR‐128 directly regulates two NMD factors: the RNA helicase UPF1 and the exon junction complex core component MLN51. Gain‐of‐function and loss‐of‐function experiments demonstrated that miR‐128 downregulates the magnitude of NMD, and rescue experiments provided evidence that miR‐128 has this effect by acting through both UPF1 and MLN51. The level of miR‐ 128 dramatically increases in precursor neural cells induced to differentiate in vitro and during embryonic brain development in vivo. Because miR‐128 inhibits NMD, which itself inhibits RNA expression, we hypothesized that the rise in miR‐128 levels during brain development serves to upregulate transcripts with features recognized by the NMD pathway. Supporting this hypothesis, we found that miR‐128 increased the levels of many neuronal transcripts regulated by NMD, including those encoding the neuron‐specific RNA splicing regulator nPTP and the synaptic regulator ARC. Forced miR‐128 expression in either a stem cell line or primary neuronal stem cells upregulated several neuronal differentiation markers, suggesting that miR‐128 promotes neuron differentiation. Together, our results suggest the existence of interconnected RNA circuitry between the miRNA and NMD pathways, which provides a novel means to induce cell type‐ specific gene expression during development.
Biophysical Characterization of Effect of Zinc on Eukaryotic Translation Initiation Factor 4B
Authors: Sultana Shemaila , Gallie Daniel R , Goss Dixie J
Published: June 2021

Eukaryotic translation initiation factor 4B promotes RNA dependent ATP hydrolysis and ATP dependent RNA helicase activity of eIF4A and eIF4F. It has recently been reported that eIF4B also organizes assembly of the RNA, eIFiso4G, eIF4A, and poly‐(A) binding protein and zinc enhances the RNA binding and interaction with PABP. By the use of fluorescence spectroscopy, native protein tryptophan fluorescence, and fluorescence labeled RNA as internal molecular probes we are characterizing these interactions. We previously reported that zinc binds tightly with eIF4B, Kd of 19.7 nM (J Biol Chem. 2008 Dec 26; 283(52):36140–53). The Kd of binding of eIF4B with 20mer poly (A) RNA is 77 ± 7 nM and in presence of zinc it lowers down to 45 ± 3 nM. There was no significant difference between the binding of eIF4B full length and eIF4B (320–527) deletion mutant with 20mer poly(A) RNA. Circular dichroism showed that addition of zinc resulted in a more than 50% decrease in alpha content of the eIF4B protein. Biophysical characterization of the effect of eIF4B versus eIF4B‐Zn and its interactions with other (eIF) factors is also carried out. Supported by National Science Foundation grant MCB 0814051
Conversion of Adenines to Inosines in RNA During Virus Infections Activates Innate Immune Responses
Authors: Liao Jie‐Ying , Prater Chrissy , Thakur Sheetal , Imani Farhad
Published: June 2021

Innate immune responses generated during viral infections are critical for protective anti‐viral immunity. The exact viral‐derived structures (also known as pathogen‐associated molecular patterns, PAMPs) that activate uninfected cells to induce activation signals are not completely characterized. To date, the viral PAMPs that are identified include dsRNA and 5′ triphosphate ssRNA, which are recognized by several cellular pathways such as SR‐A, PKR, RIG‐I, TLR‐3 and MAVS. During viral infections, the cellular enzyme ADAR‐1 (adenosine deaminase acting on RNA‐1) catalyzes conversion of adenines to inosines (A‐I editing), which is a pathway for blocking viral replication. ADAR‐1 activation in the infected cells leads to the presence of inosine‐containing viral and cellular RNA (Ino‐RNA). Here, we provide in vitro and in vivo data to affirm our hypothesis that the release of Ino‐RNA from infected cells and its interaction with surrounding uninfected cells is a potent stimulus for inflammatory innate immune responses. The innate response was confirmes by rapidly and potently induction of IFN‐b, TNF‐a, IL‐6 and RANTES. The A‐I editing increased secondary structures of RNA which resulted in activation of the dsRNA‐specific protein kinase (PKR). Our data uncover a novel strategy for uninfected host cells to detect viral infections in the surrounding tissue.
LC‐FTMS‐based metabolomics reveals age‐ and gender‐related differences in marmosets
Authors: Soltow Quinlyn A. , Promislow Daniel E.L. , Mansfield Keith G. , Wachtman Lynn M. , Jones Dean P.
Published: June 2021

Metabolic profiling provides a global assessment of metabolites and evaluates the impact of diet, environment, and behavior on whole‐body metabolism. The purpose of this study was to use liquid chromatography‐Fourier transform mass spectrometry (LC‐FTMS) to identify distinct metabolic profiles and metabolites that differ with age in male and female marmosets. After protein extraction, 10 μl of marmoset plasma was analyzed by LC‐FTMS. The LC consisted of a C18 precolumn and anion exchange/C18 tandem column switching. Detection of 3 million ions at a maximum of 500 msec provided 2 scans/sec over 10 min for the m/z decade of 85–850. Features were defined as having m/z ± 0.002 amu and the same elution time. Results revealed more than 2000 m/z features from 25 male and 37 female healthy marmosets ages 2–13 yrs old. Unsupervised principal component analysis separated young and old and also male and female marmosets. Cluster analysis revealed that metabolomic networks in young marmosets maintained greater integrity than the old, and the networks between sexes demonstrated no overlap. Predicted elemental formulas of metabolites differing with age and sex provide putative biomarkers of aging and identify specific modules disrupted with age. In conclusion, LC‐FTMS‐based metabolomics provides a means to evaluate age‐ and gender‐related metabolic changes in detail. Funded by NIEHS and NCRR.
Metabolomic evaluation of Masseter muscle in an experimental malocclusion model
Authors: Farinha Rodrigo , Gomes Marcolino , Tavares Ludgero , Carvalho Rui , Silvério Cabrita A. M.
Published: June 2021

Occlusal disarrangements are very often in the basis of the sintomatology related with temporomandibular disfunction, which courses with muscular and articular involvement.

The aim of this study is to give a contribution to the understanding of metabolic changes in the masseter muscle in an experimental model of malocclusion.

20 Wistar male rats, 12 weeks old were distributed in two groups: the control group, kept in the standard conditions and the test group. In the test group we created a change of the normal occlusal contacts by adding fotopolimerized resin on the left superior teeth. After two weeks, all animals were killed and the complete necropsy was performed. Fractions of masseter muscle were collected for histopathological analysis and for NMR analysis. The fragments for NMR analysis were snap‐frozen in liquid nitrogen and stored at −80ºC until the spectroscopy was performed.

After the spectra and statistical analysis we observed that Taurine (3.261 PPM); β‐glucose (3.42 PPM); Creatin (3.93 PPM) and a metabolite at 4.334 PPM were significantly changed between the control and test groups on left masseter. Taurine (3.261 PPM), β‐glucose (3.422 PPM), and two metabolites on 4.964 and 1.482 PPM were significantly changed between the control and test groups on right masseter. We didn't observe any statistically significant changes between left and right masseter metabolic profile on control group

The results of this study suggest that occlusal interferences can produce metabolic changes on masseter muscle on the working and non‐working sides. Taurine and β‐glucose suffered statistically significant changes on both left and right masseter muscles.