bluuebunny/crossref_metadata_2025 huggingface dataset:
Supernumerary lumbar rib in human prehistory
Authors: Eaves‐Johnson K Lindsay
Published: June 2021

Unlike their cervical counterparts, supernumerary lumbar ribs are generally considered to be of little clinical significance, though they do constitute a topic of anthropological interest. The two most complete Neandertal lumbar regions (Kebara 2 and Shanidar 3) both show evidence of articular facets for bilateral lumbar ribs and a high incidence of the condition has been reported in the prehistoric Sadlermiut Eskimo. This pilot study tests the association of bi‐iliac breadth with the presence of supernumerary lumbar rib in two wide‐bodied prehistoric populations: Pt. Hope Inupiat (n=25), Tierra del Fuegan/Aonikenk (n=25), and one narrow‐bodied prehistoric population: South African Zulu (n=25). Moreover, as mouse models have shown a correlation between 12th rib length and lumbar rib presence, this was also investigated. Of the populations studied, only the Pt. Hope Inupiat evinced lumbar ribs (16%) even though their bi‐iliac breadth was not significantly greater than that seen in the Tierra del Fuegan/Aonikenk. Additionally, significantly longer ribs were observed for the Pt. Hope Inupiats in agreement with the predicted model. These results suggest that the presence of supernumerary lumbar rib in human prehistory is indicative of other basic alterations of the axial skeleton and is most likely related to population history.

Grant Funding Source: National Science Foundation Doctoral Dissertation Improvement Grant #0752723 and the Wenner‐Gren Foundation for Anthropological Research.
Effect of Low Doses of Nicotine on Early Avian Development
Authors: Brauer Philip R , Olley John T , Pham Evelyn Q , Reedy Mark V
Published: June 2021

Previous nicotine teratogenic studies in animals used nicotine levels exceeding those measured in human amniotic fluid of smoking mothers and were conducted at relatively late stages of embryonic development. Here, we injected a single dose of physiologically relevant amount of nicotine into chicken eggs after 24 hr of development to determine its teratogenic effect in younger embryos. After 4 additional days of incubation, embryos exposed to 100 or 1,000 nM nicotine were significantly smaller as evident by a decrease in wet weight and crown rump length and exhibited a higher frequency of gross morphological abnormalities. 1,000 nM nicotine also caused delayed developmental progression. Embryos treated with 10 nM nicotine were unaffected. Immunohistochemistry to analyze the expression of nicotinic acetylcholine receptors (nAChRs) in very early embryos revealed the presence of alpha1/5 nAChR subunit expression in the developing myotome as early as 3 days and beta2 nAChR subunit expression in pharyngeal arch tissue at 2 days and in neural tissue by 3 days. Our data shows very young embryos express nAChRs and that a single exposure to a low dose of nicotine increases the risk of congenital defects and causes a significant decrease in embryonic growth rate. Supported by Nebraska Health and Human Services (LB506 & LB595) and NIH (#P20‐RR0169).
Regulator of G protein Signaling 3 Modulates Wnt5b Calcium Dynamics and Somite Patterning
Authors: Freisinger Christina M , Slusarski Diane C
Published: June 2021

We have identified Ca2+ release as an output of Wnt signaling network and are investigating the mechanisms and context by which this Wnt signal mediates its biological effect on the developing embryo. We have determined that the early Ca2+transients are, in part, modulated by Wnt‐5 and require G‐protein signaling. To identify potential intracellular regulators of calcium release dynamics, we are characterizing Rgs3, a member of the Regulators of G‐protein Signaling (RGS) protein family.

This work integrates molecular, genetic and physiological approaches to investigate the mechanism and context mediating calcium release dynamics and the biological effect on developing zebrafish embryos in vivo. Using loss of function analysis, we demonstrate that Rgs3 is necessary for proper gastrulation and somite patterning during zebrafish development. These actions of Rgs3 require its ability to interact with and accelerate the rate of GTP hydrolysis by G proteins, as revealed by studies employing an Rgs3 mutant defective in these activities. This study identifies a previously unknown genetic interaction between RGS and the non‐canonical Wnt signaling network.

Grant Funding Source: March of Dimes (5‐FY99‐806); NIH (RO3 HD045488‐01), RO1 (GM067881‐01); and American Heart Association Predoctoral Fellowship
Discovering Interactions Present During the Growth and Development of a Craniofacial Bone
Authors: Duench Kellie Lynn , Franz‐Odendaal Tamara
Published: June 2021

The study of skeletal (bone and cartilage) development includes the investigation of intramembranous bones of the head and facial skeleton as well as endochondral bones (e.g. long bones) of the body. The development of skeletal tissues often relies on an interaction between the mesenchyme and the epithelium. Bone is formed by signalling interactions between these two tissues. Often these signals experience cross‐talk, where one signal is inducing or inhibiting another signal. When it comes to scleral ossicles (an example of intramembranous bone), it has recently been shown by our lab that the Hedgehog gene family is involved in its development. Using whole mount in situ hybridization further investigation was performed to discover if multiple members of the Hedgehog family are involved, and also to determine the location and distribution of the Patched (Ptc) receptor. Bead implantations using a common Bone morphogenetic protein inhibitor, noggin, were performed to determine the involvement of the BMPs during ossicle development. The relationship and interactions between these two genes families (BMP, and Hedgehog) during scleral ossicle development will be discussed. These investigations have allowed for further understanding of the mechanism of development for the ocular skeleton and intramembranous bones, in general.

Grant Funding Source: NSERC
Multiphoton Microscopy for Detection of GFP labeled Mesenchymal Stem Cells in Porcine AAA Model
Authors: Shah Tejas Rajendra , Shin Hyun Joo , Han Daniel K , Galende Eliza Yaniz , Malik Rajesh K , Salloum Alexander , Faries Peter L
Published: June 2021

Introduction

Multiphoton microscopy has transcended from a picture‐taking novelty to a powerful tool used to detect excitation fluorescence. This abstract aims to assess the ability to detect green fluorescence protein (GFP) labeled porcine mesenchymal stem cells (MSC) that were infused into aneurismal aortic tissue.

Methods/Results

MSCs were isolated from Yorkshire swine bone marrow and infected with GFP + lentivirus. An aortic aneurysm was created in the pig (by mechanical and chemical degradation) and GFP labeled MSC were introduced into the aneurismal aorta. 24 hours after implantation, the pig was sacrificed and the aorta was removed. The tissue was secured in agar and sent for multiphoton imaging. Imaging was performed at 0, 24, 48, 72, and 96 hrs after harvest. Multiphoton imaging detected GFP labeled cells with high fluorescence excitability in the aortic tissue up to 72hrs after tissue harvest. With second harmonic generation, collagen molecules were imaged and were distinctly different from the GFP excited cells. Presence of GFP positive cells was confirmed with subsequent immunohistochemistry and western blot.

Conclusions

Multiphoton microscopy is useful in tracking GFP labeled MSCs infused into the aneurismal aorta of swine. These cells can be tracked up to 72hrs after tissue harvest with presence of GFP by western blot. This powerful tool can be applied to in vitro tracking of in vivo placed MSCs.

Grant Funding Source: National Institute of Health K08HL073313
Magnetic Resonance Imaging in Evaluation of Tumoral Lesions of the Heart
Authors: Miclaus Gratian Dragoslav , Catau Gabriela , Zahoi Delia Elena , Ples Horia
Published: June 2021

Most frequent tumor lesions of the heart are represented by benign tumors and metastatic tumors. A primary tumor lesion of the heart represents a very rare pathology; with an incidence 20–40 times lower than metastatic tumors of the heart.

Magnetic resonance imaging is the method of choice used to confirm the diagnostic suspicion raised by other imaging techniques, to have additive morphologic and information on the signal behavior of the mass, and to exclude/confirm the neoplastic nature of a mass.

We examined a female patient with symptoms of fatigue, dispnoea and loss of weight. She was examined before with transthoracic and transesofageal ultrasonography with diagnostic presumption of myxoma.

We found a mass that involved left atrium, lateral wall of the left ventricle and invasion thru the cardiac sinus in the right atrium. After the administration of gadolinium behavior of the tumor was highly suggestive for malignant lesion (lymphoma or angiosarcoma).

In conclusion, magnetic resonance imaging of the heart represents a powerful tool in noninvasive evaluation of the cardiac tumor pathology.
Uncovering a conserved role for JMJD2 histone tri‐demethylases from worm to human
Authors: Whetstine Johnathan R , Black Josh C , Allen Andrew , Forbes Emily , Smallwood Andrea , Longworth Michelle , Tschoep Katrin , Ulmschneider Bryne , Walsh Ryan , Ren Bing , Dyson Nick
Published: June 2021

Histone 3 lysine 9 tri‐methylation (H3K9me3) is a hallmark of heterochromatin, a repressive structure found at repetitive and telomeric regions in the genome. This histone modification plays a critical role in the temporal control of heterochromatic replication and the regulation of euchromatic gene expression. Alterations in H3K9me3 and heterochromatin are directly associated with the onset of genomic instability and cancer. The lack of H3K9me3 has also been shown to be a contributor to the premature aging disease Hutchinson‐Gilford Progeria Syndrome. These few examples highlight an important link between the levels of H3K9me3, cancer, and premature aging. Identifying factors that influence H3K9me3 and heterochromatin dynamics will have profound implications in pathophysiology. We recently identified the first histone lysine‐specific tri‐demethylase family (JMJD2A‐D) in human and C. elegans (JMJD‐2) that regulate H3K9me3 and H3K36me3 levels. We uncovered a novel and highly conserved role for the JMJD2 proteins in multiple DNA‐templated processes. We have also demonstrated that the phenotypes are a reflection of altered chromatin structure. Our observations suggest that JMJD2 proteins are important regulators of the chromatin state from worm to human. We will present these findings as well as our most recent advances into understanding the molecular basis of the JMJD‐2‐related phenotypes.
Inhibiting the 26S Proteasome Expands Glucocorticoid Receptor Transcriptional Networks via Chromatin Modifications
Authors: Kinyamu Harriet Karimi , Yang Jun , Archer Trevor
Published: June 2021

Our goal is to understand the mechanisms by which the 26S proteasome regulates glucocorticoid receptor (GR)‐mediated gene transcription. The 26S proteasome, a multienzyme complex, known for its role in protein degradation, is now implicated in all stages of the transcription process. Biochemical and expression profiling experiments suggest a complex role for the 26S proteasome in GR‐mediated transcriptional regulation. Inhibiting 26S proteolytic activity results in differential expression of GR target genes, despite elevated levels of GR and RNA Pol II, indicating that GR/RNA Pol II turnover cannot explain the diververgent expression of GR targets. Chromatin modifications, such as posttranslational modifications of histones, are a dynamic way to expand transcriptional networks. In MCF‐7 breast cancer cells, transcript levels encoding several histone H3‐lysine‐methyltransferases and demethylases are changed significantly by proteasome inhibition. Furthermore, inhibiting proteasome activity leads to an increase in key histone H3‐lysine methyl marks. We stably integrated FLAG‐ tagged histone H3‐N‐terminus tail in MCF‐7 cells to query candidate effector proteins globally associated with these modifications. Mass‐Spectrometric analysis identified multiple proteins involved in mRNA processing, implicating the 26S proteasome in this process. We are currently examining the functional implications of these interactions.
Phosphorylation of histone H1 at specific sites during gene transcription
Authors: Mizzen Craig Andrew , Zheng Yupeng , Pesavento James , Schultz‐Norton Jennifer , John Sam , Nardulli Ann , Kelleher Neil , Hager Gordon
Published: June 2021

Histone H1 affects DNA‐templated processes by promoting chromatin folding. Phosphorylation of H1 has been implicated in chromatin decondensation to facilitate transcription and in mitotic chromosome condensation, but little is known about either process. We used novel chromatographic and proteomic approaches to identify sites in individual H1 variants that are phosphorylated exclusively in mitosis (M sites) and sites that are phosphorylated in both interphase and mitosis (I sites) in synchronized HeLa cells. Chromatin immunoprecipitation and other analyses reveal that I site phosphorylation is preferentially associated with chromatin during transcription by RNA pol I and RNA pol II. It colocalizes with RNA pol I and nucleolar sites of Br‐UTP incorporation. It is enriched at rDNA promoters in growing cells, and this association decreases when rRNA transcription is attenuated by actinomycin D. It is rapidly enriched at hormone response elements after cells are stimulated with dexamethasone or estradiol. I sites are only partially conserved among the human H1 variants, our data suggests that this may contribute to their functional differences.
The histone H2A N‐terminal tail regulates H2B monoubiquitylation and H3 K4 methylation via a novel trans tail pathway
Authors: Zheng Suting , Reese Joseph C
Published: June 2021

The roles of the histone H3 and H4 N‐terminal tails in regulating chromatin structure and gene transcription have been extensively studied. However, very little is known about the functions of the H2A N‐terminal tail in transcription regulation. We analyzed multiple histone modifications associated with active transcription in the absence of the histone H2A tail. Our results reveal a novel trans‐tail regulation between the H2A tail and H2BK123 monoubiquitylation (H2BK123ub). Deletion of the H2A tail, or as little as four amino acids (a.a. 16–20), leads to a significant decrease in H2BK123ub, followed by a global reduction in H3K4 methylation by COMPASS. COMPASS complex purified from a H2A tail deletion strain lacks the Cps35 subunit, similar to the complex purified from a RAD6 deletion mutant. Rad6, which is responsible for ubiquitylating H2B K123, is still capable of being targeted to genes in the absence of the H2A tail, suggesting a type of post‐recruitment regulation. Deletion of the H2A tail causes a lag in the induction time of the GAL1 gene compared to wild type. Additionally, we show that deleting the Ubp8 and Ubp10 deubiquitylases not only restores the normal rate of induction of GAL1, but also restores H2BK123ub and H3K4me3 levels within the cell. Our analysis uncovers a role for the H2A tail in regulating transcription by maintaining a proper level of H2BK123 ubiquitylation and H3K4 methylation.

This research was supported by funds from National Institutes of Health (GM58672) to J.C.R.
Identification and maturation of newly synthesized fungal histone H3
Authors: Waterborg Jakob Harm
Published: June 2021

Reversed‐phase hplc of histone H3 in Ustilago maydis, Saccharomyces cerevisiae and Schizosaccharomyces pombe revealed the existence of a distinct form, ~5–20% of total histone H3 depending on strains and culture conditions, with delayed column elution, i.e. a more hydrophobic character. This form was more highly labeled by methionine than bulk H3, consistent with increased levels of lysine methylation and protein hydrophobicity. It does not contain non‐acetylated histone H3 and, in U.maydis, and has higher steady‐state acetylation levels than bulk H3. In asynchronous cultures, acetylation of replacement and of replication‐dependent H3 variants was more than 3‐, and 2‐fold higher, respectively, than bulk H3. Lysine pulse label incorporated exclusively into this form, identifying it as newly synthesized histone H3. Consistent with this, its abundance decreased in cycloheximide. Cell fractionation demonstrated that it does not represent a cytoplasmic pool. Lysine pulse‐chase labeling in U.maydis demonstrated a discrete chase into bulk, mature histone H3 with a half‐life of 30 min, complete within the 150 min cell doubling time. Western blotting and mass spectrometry are used to identify sites of acetylation and methylation in new H3 and changes during histone H3 maturation.

Research supported by Missouri Life Sciences Research Board #13254
Non‐canonical inteins: Alternate mechanisms for protein splicing
Authors: Mills Kenneth V. , Reitter Julie N. , Duffee Lauren R. , York Daniel J. , Dorval Deirdre M. , Lewandowski Katherine T.
Published: June 2021

Protein splicing involves the post‐translational excision of an intervening polypeptide (the intein) from flanking domains (the exteins), as well as ligation of the exteins. The first step of splicing is an amide‐ester rearrangement of the peptide bond that links the N‐terminal extein and intein. However, the intein that interrupts a putative phage terminase in C. thermocellum lacks an N‐terminal nucleophile and should not be able to promote this step. We show that this intein can splice, and have examined the influence of conserved residues on efficient splicing. The third step of splicing is cyclization of Asn coupled to peptide bond cleavage. However, the PolII intein from P. abyssi has a C‐terminal Gln and can still promote splicing. We compare the effects of C‐terminal Asn and Gln on splicing efficiency, as well as the influence of conserved residues on the catalysis of splicing.

This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.
Site‐directed mutagenesis of a novel secondary alcohol dehydrogenase from M. luteus WIUJH20
Authors: Shoger Nicholas , Huang Jenq‐Kuen , Wen Lisa
Published: June 2021

A novel secondary alcohol dehydrogenase (2o‐ADH) has been characterized in this lab previously. This 2o‐ADH is categorized as secondary alcohol dehydrogenase based on its biochemical reactions, but it belongs to NAD+‐dependent short‐chain L‐3‐hydroxyacyl‐CoA dehydrogenase (SCADH) based on amino acid sequence homology. In literatures, 2o‐ADH and SCADHs are distinguished in term of their primary structure and substrate specificities. SCADH has tight substrate specificity; it only catalyzes L‐3‐hydroxyacyl‐CoAs with various hydrocarbon chain‐length. In comparison the 2o‐ADH has low substrate specificity; it can catalyze hydroxy fatty acids with hydroxyl group in either D or L form on a number of carbon positions. However, the enzyme can not use hydroxyacyl‐CoA as a substrate. This 2o‐ADH is an excellent model system to study structure‐function relationship and determine what distinguishes their substrate specificity. We report here generation of single and double amino acid substitution mutants (D60S, A67K, and D60S + A67K) of 2o‐ADH in order to probe substrate functional group specificity.

The research was supported in part by Western Illinois University Foundation.
Sulforaphane Enhances Proteasome Activity through Upregulation of Hsp27
Authors: Mi Lixin , Gan Nanqin , Chung Fung‐Lung
Published: June 2021

Stimulating proteasome activity for rapid removal of misfolded and oxidized proteins is a promising strategy to prevent and alleviate aging‐related diseases. Sulforaphane (SFN), an effective cancer prevention agent derived from cruciferous vegetables, has been shown to enhance proteasome activities in mammalian cells and to reduce the level of oxidized proteins and amyloid β‐induced cytotoxicity. Here we report that SFN induces proteasome activities through stimulating the expression of Heat shock protein 27 (Hsp27). Our results show that SFN‐induced proteasome activity correlated well with the upregulation of Hsp27. SFN‐induced proteasome activation was significantly reinforced in cells overexpressed with Hsp27 while nullified in cells with Hsp27 knocked down by si‐RNA. The role of Hsp27 in regulating proteasome activity was further confirmed in a pair of isogenic REG cells, in which SFN‐induced proteasome activation was only observed in cells stably transfected with Hsp27, but not in the parental cells that is Hsp27‐free. Finally, we demonstrated that phosphorylation of Hsp27 is irrelevant to SFN‐induced proteasome activation. This study provides a novel mechanism underlying SFN‐induced proteasome activity. This is the first report that heat shock response, in addition to antioxidant response through Keap1‐Nrf2 pathway, may contribute to SFN‐caused cytoprotection.
Nuclear Magenetic Resonance Evidence for the Role of the Flexible Regions of the E1 Component of the Pyruvate Dehydrogenase Complex from Gram Negative Bacteria
Authors: Song Jaeyoung , Nemeria Natalia S. , Kale Sachin , Kakalis Lazaros
Published: June 2021

Sequence‐specific NMR assignments were made for six residues in the N‐terminal 1–55 region, and one in each of the inner (401–413) and outer (541–557) active center loops of the E1 component of E. coli pyruvate dehydrogenase complex (E1ec, 2x100 kDa), flexible regions not seen in the x‐ray structure. The 2D HSQC spectrum revealed 103 resonances of the 109 expected in the three regions. Site‐specific substitutions and appropriate labeling patterns assisted the assignments. Resonances assigned to the N‐terminal region diminished in size/were absent in the spectrum in the presence of E2ec1‐190 comprising the lipoyl domain, peripheral subunit binding domain and hinge regions, indicating that the N‐terminal region became organized. Assignment of backbone NH resonances for residues G402 and G542 revealed dynamic features of the loops. These resonances, but not those assigned to the N‐terminal region, were no longer seen on saturation of the enzyme with a stable pre‐decarboxylation analogue C2alpha‐phosphonolactylThDP (PLThDP), indicating that the dynamic loop regions became ordered. The results provide the first structural support to the notion that the N‐terminal region of the E1ec of this entire class of bacterial pyruvate dehydrogenase complexes is responsible for binding the E2 component specifically, and dynamic motions of loops of E1ec are restricted by PLThDP. Supported by NIH‐GM‐050380.
A possible mechanism for the regulation of IRES‐mediated expression by eIF2A
Authors: Merrick William C. , Reineke Lucas C. , Cao Yu , Baus Diane
Published: June 2021

We have shown that eIF2A has the capability of repressing expression from the URE2 IRES in vivo.1 Current studies are addressing how this down regulation might be achieved. There are now five pieces of evidence that allow for the postulation of a model for this regulation:

the eIF2A mRNA disappears within 2–3 minutes with essentially any stress

eIF2A protein may or may not show enhanced degradation depending on the stress

eEF1A binds to the URE2 IRES element

by pull down experiments, eEF1A and eIF2A reciprocally bind each other

it appears that the interaction between eEF1A and eIF2A is through the C‐terminal regions of each protein

In the model to be presented, it is anticipated that the binding of eEF1A to the URE2 IRES‐element facilitates the binding of eIF2A (and Met‐tRNA) and that the resulting complex at the level of the 80S ribosome is slow to convert to an elongating ribosome. With the down‐regulation of eIF2A protein and/or activity, eIF2 is presumed to direct Met‐tRNA binding in a more efficient manner allowing for a 10‐fold increase in expression.

Preliminary evidence indicates that the regulation of IRES‐mediated expression is not exclusive to the URE2 IRES element but that eIF2A down‐regulates expression from several other IRES elements as well (identified by Gilbert et al.2).
A Proteomic Investigation of Arsenic and Antimony Stress on Sulfolobus solfataricus
Authors: Rastedt Danielle Elizabeth , Grypiuk Nicole , Selvig Kyla , Maaty Walid , Bothner Brian
Published: June 2021

Members of Archaea, the third domain of life, have adapted to some of the most severe environments known to support life. One in particular, Sulfolobus solfataricus, is thermoacidophilic, therefore, it can be found in extreme environments such as the hot springs in Yellowstone National Park. This archeaon can endure extreme temperatures, high acidity, and highly enriched metal environments. Understanding how these organisms survive and what particular mechanisms are used in these harsh environments have become of intrinsic interest. The aim of this study was to quantitatively define the regulation of proteins upon treatment with arsenic and antimony species in different time periods and concentrations. Proteomic analyses using two‐dimensional differential gel electrophoresis (2D‐DIGE) was used to determine proteins of interest after exposure to 50μM antimony. Furthermore, SDS‐PAGE analysis was used to determine insoluble proteins after being stressed with 240μM and 1mM arsenic. Regulated proteins lists were created, which can be further used to identify the mechanisms of proteomic response. Together these experiments begin to identify important proteins, which play a role in the environmental stresses of arsenic and antimony.
Structure of the First Fungal Enzyme Form of Aspartate β‐Semialdehyde Dehydrogenase
Authors: Arachea Buenafe T , Liu Xuying , Pavlovsky Alexander , Viola Ronald E
Published: June 2021

The enzyme aspartate semialdehyde dehydrogenase (ASADH) catalyzes the conversion of β‐aspartyl phosphate to aspartate β‐semialdehyde, a critical transformation step that is essential in microbial amino acid biosynthesis. This enzyme is found exclusively in bacteria, fungi and plants thus making it a potential target for drug development. Representative structures of bacterial and archael organisms have been previously determined; however no structural information on any fungal ASADH has been available. Here we report the first structure of ASADH purified from yeast Candida albicans and crystallized in the presence of its nucleotide cofactor. The fungal enzyme is a functional dimer with similar overall fold and domain organization with its bacterial counterparts. More detailed structural comparison between the fungal and bacterial ASADHs revealed differences in secondary structural elements and in the nucleotide cofactor binding that may explain the lower catalytic activity observed for the fungal enzyme. Moreover, alterations in the dimer interface through the deletion of a helical subdomain and the replacement of amino acids involved in critical hydrogen bonding network results in the disruption of intersubunit communication channels required to support an alternating site catalytic mechanism. Elucidation of the structural details of this fungal enzyme allows assessment of ASADH as a possible target for antifungal drug development. Crystallization studies of fungal ASADH‐inhibitor complexes are currently underway to provide additional information for inhibitor design.
Lipid rafts determine association of Orai1, STIM1 and the TRPC1 and TRPC6 proteins
Authors: ROSADO JUAN A , GALAN CARMEN , SALIDO GINES M
Published: June 2021

Different laboratories have shown that proteins involved in Ca2+ entry are localized in discrete plasma membrane lipid rafts, cholesterol and sphingolipid‐enriched membrane domains that scaffold certain signaling molecules while excluding others. Lipid rafts have been shown to determine targeting of STIM1 clusters to endoplasmic reticulum‐plasma membrane junctions upon store depletion, facilitating the interaction of STIM1 with TRPC1 and the activation of store‐operated Ca2+ entry (SOCE). The aim of the study was to investigate the role of lipid rafts in SOCE and the association between Orai1, STIM1 and TRPCs. Treatment of HEK293 cells with thapsigargin (TG; 1 μM) results in association of Orai1 with STIM1, TRPC1 and TRPC6. TG‐induced association of these proteins was significantly attenuated by preincubation for 30 min with 10 mM methyl‐β‐cyclodextrin (CD; p<0.05; Student's t‐test), which depletes cells of cholesterol and disrupts lipid rafts domains, without altering the association of Orai1 with STIM1 or the TRPCs in non‐stimulated cells. Preincubation with CD significantly reduced TG‐induced Ca2+ entry by 20% (p<0.05) without having any effect on Ca2+ release. In conclusion, here we show that lipid raft domains are necessary for the formation of Ca2+ signalling complexes, involving Orai1, TRPCs and STIM1, which are important for SOCE.

Supported by MEC grant BFI2007‐60104 and Junta de Extremadura.
MITOCHONDRIAL DYSFUNCTIONS INDUCED BY OXIDATIVE STRESS IN CACO‐2CELLS
Authors: Rame Taha , Seidman Ernest , Beaulieu Jean François , Boudreau François , Gendron Fernand Pierre , Delvin Edgard , Devendra Amre , Levy Emile
Published: June 2021

Despite the fact that oxidative stress (OS) has been implicated in IBD pathogenesis and mitochondria are the substantial source of ROS, little attention has been paid to these organelles. We hypothesize that OS can lead to mitochondrial dysfunctions recapitulating various IBD abnormalities. The present study will to investigate the impact of OS on mitochondrial antioxidant content, energy production, calcium homeostasis, cell death, transcription factors status and DNA integrity. We used FE/ASC to generate OS in Caco‐2 cells. Our results show that the administration of FE/ASC (0.2 mM/2 mM) (1) increased MDA levels; (2) reduced ATP production; (3) led to alterations in the gene expression and protein mass of mitochondrial transcription factors (mtTFA, mTFB1, mTFB2) without any effects on POLRMT; (4) provoked dysregulation of mitochondrial calcium homeostasis; (5) elicited DNA lesions as illustrated by the raised levels of 8‐OHdG; (6) lowered, OGG1, one of the first lines of defence against 8‐oxodG mutagenicity; and (7) upregulated cytochrome C and AIF protein expressions, indicating exaggerated apoptosis. The presence of the powerful antioxidant BHT prevented the occurrence of OS and most of the mitochondrial abnormalities. In conclusion, our results suggest that OS causes mitochondrial dysfunctions and modulates mitochondrial transcription factors. We believe that the identification of OS‐mediated mitochondrion disorders adds new information regarding their implication in IBD pathogenesis.
AHR (Acute hypoxia responsive element) As a New Tool Detecting Acute Hypoxia
Authors: CILEK Mehmet Zeynel , HIROHATA Satoshi , HATIPOGLU Omer Faruk , NINOMIYA Yoshifumi
Published: June 2021

Objective

Previously we reported an interesting gene that transiently induced in the endothelium in myocardial infarction which may be a hypoxia‐inducible gene expressed by endothelial cells. Its expression was limited to a few hours’ hypoxia. We tested if this switch element (we named this sequence as AHR (Acute hypoxia responsive element) for this induction can be used for detecting acute hypoxia.

Methods

We prepared several different constructs with different length of AHR promoter then performed luciferase assay in hypoxic cells. We identified the responsible AHR activated by acute hypoxia. We made green fluorescence protein (GFP) expressing construct (AHR‐GFP) under the control of AHR. We transfected AHR‐GFP construct into human umbilical vein endothelial cells (HUVECs), and examined GFP production under 30 minutes, 1, 3, 6, 12 or 24 hours of hypoxia respectively. After incubation, cells were fixed with 4% paraformaldehyde (PFA) and observed under the fluorescence microscope.

Results

When AHR‐GFP –transfected HUVEC was cultured in normoxic condition, GFP was very slightly observed. In contrast to this, the number of GFP‐positive HUVEC increased at 3h (~10‐fold compared to normoxia) under hypoxic stimulation. The induction of AHR under hypoxia returned to the normoxic level at 24h of hypoxia.

Conclusions

AHR works under acute hypoxia and may be used as a specific tool for detecting acute hypoxia.
Urokinase plasminogen activator receptor activation of invasion and migration in H460 cells requires Na+/H+ transporter activity and expression
Authors: Kutz Craig , Haak Andrew , Rasted Danielle , Provost Joseph J , Wallert Mark A
Published: June 2021

Non‐small cell lung cancer (NSCLC) is an aggressive cancer where 10% of patients survive beyond five years of initial diagnosis. One of the reasons for the poor survival rate is the robust invasion and metastatic properties of NSCLC. Urokinase plasminogen activator (uPA) and its receptor are directly associated with a poor prognosis in NSCLC. Na+/H+ transporter (NHE1) has been implicated in regulation of cell motility in a number of cancer cells. We wished to identify the role NHE1 plays in uPA activation of tumor progression in NSCLC. We used both uPA and the inactive‐receptor binding amino terminal fragment of uPA (ATF) to stimulate human lung H460 carcinoma cells. 10 nM uPA or ATF stimulated proliferation 1.4 ‐ 1.2 fold over non‐treated cells. Inhibition of NHE1 with 10 μM EIPA abrogated agonist‐stimulated proliferation. Tumor formation size and number was significantly increased by addition of either uPA or ATF, while NHE1 inhibition blocked the ability of either agonist to induce tumors in soft agar assays. Migration of H460 cells was inhibited in the presence of EIPA. Both uPA and ATF enhanced MMP9 activity but not in cells treated with EIPA. Only ATF increased MMP9 expression. We also show the effect of uPA in NHE1 and uPAR knockdown cell lines where we investigated MMP9 activity, and the location of NHE1 and uPAR in lipid rafts. Our results indicate that uPAR activation of cancer cell properties requires NHE1. Support from NIH‐1‐R15‐CA135616‐01
Tissue‐specific regulation of calpain and calpastatin activities by PIN1 in mice
Authors: Schneider Ryan A. , Liu Tongzheng , Hoyt Dale G.
Published: June 2021

The peptidyl‐proline isomerase, Protein Interacting with Never In Mitosis Gene A‐1 (PIN1), facilitates cis‐trans isomerization of phospho‐serine/threonine‐proline motifs in proteins, thereby modulating their synthesis, degradation and function. We previously found that depletion of PIN1 in endothelial cells (EC) reduced calpain activity. PIN1 may regulate calpains or the endogenous inhibitor, calpastatin. Here, we hypothesized that genetic deletion of PIN1 would reduce calpain activity and increase calpain inhibitory activity in mice. Calpain activity in different tissue extracts was measured with a fluorescently quenched peptide substrate. Calpain inhibitory activity was determined by first heat‐inactivating endogenous calpain, and then assessing the ability of extracts to inhibit cleavage of the peptide substrate by exogenous porcine erythrocyte μ‐calpain. As in EC, endogenous calpain activity was reduced in extracts of lung and kidney from PIN1 knockout mice. In addition, calpain inhibitory activity was increased in these knockout tissues. Surprisingly, the opposite phenotype was observed in knockout brain extracts: endogenous calpain activity was increased, and calpain inhibitory activity was reduced. These results indicate that PIN1 regulates calpain or calpastatin in vivo, but in a tissue‐specific manner.
Caspofungin induces programmed cell death in Saccharomyces cerevisiae
Authors: Chin Christopher , Helming Katherine , Austriaco Nicanor
Published: June 2021

Caspofungin is the first drug of a new class of drugs called echinocandins. It inhibits the enzyme β(1,3)‐D‐glucanase, disturbing the cell wall of target cells. However, it is not clear how the drug actually kills yeast cells. We now show that culturing the budding yeast, Saccharomyces cerevisiae, in media containing caspofungin induces several known hallmarks of programmed cell death, including mitochondrial fragmentation, reactive oxygen species (ROS) production, and DNA degradation. This suggests that caspofungin kills by inducing apoptosis. Thus, mutations that prevent programmed cell death may also lead to drug resistance. Experiments are underway to determine if a similar process occurs with the human fungal pathogen, Candida albicans. (Supported by RI‐INBRE Grant# P20RR016457 from NCRR, NIH.)
Antioxidant and cytoprotective effects of ajoene as a consequence of Nrf2‐mediated glutamate‐cysteine ligase induction
Authors: Kay Hee Yeon , Yang Jin Won , Ryu Jae Ha , Jeon Ra Ok , Kim Sang Geon
Published: June 2021

The cytoprotective effects of chemopreventive agents may be attributed to the induction of antioxidant enzymes. One of the key antioxidant enzymes is glutamate‐cysteine ligase (GCL), which enables cells to protect from oxidative injury by increasing cellular GSH synthesis. Nrf2 as an essential transcription factor regulates the expression of genes encoding for GCL and associated enzymes. The effects of organosulfurs derived from garlic on inflammation, tumor proliferation and cholesterol synthesis have been widely studied. This study investigated whether ajoene, a garlic component, has the ability to activate Nrf2 and induce GCL and, if so, the role of activating Nrf2 in cytoprotection against oxidative stress. Ajoene promoted Nrf2 activation, as evidenced by enhanced nuclear accumulation as well as decreases in Keap1 and Nrf2 interaction and Nrf2 ubiquitination. Consistently, ajoene increased antioxidant response element‐mediated reporter gene activity, the expression levels of GCL and its transcript. Among the upstream kinases of Nrf2, only protein kinase C δ (PKCδ) was activated by ajoene. Likewise, PKCδ inhibitor prevented Nrf2 activation and GCL induction by ajoene, suggesting that ajoene promotes antioxidant defense system by Nrf2 activation via PKCδ. Ajoene was effective in preventing GSH depletion, ROS production and cell death elicited by tert‐butylhydroperoxide treatment. The important role of activated Nrf2 in the cytoprotective effect of ajoene was verified by the reversal of ajoene's ability to protect hepatocytes in the Nrf2 gene knockout experiment. The results of this study demonstrate that ajoene promotes PKCδ ‐dependent Nrf2 activation and Nrf2‐mediated GCL induction, and which may contribute to protecting cells against oxidative stress.
Preliminary Screening of Capsaicin‐based Compounds as Potential Therapeutic Targets for Human Non‐small Cell Lung Cancer
Authors: Weidman Cory , Wallert Mark A , Provost Joseph J
Published: June 2021

Lung cancer is the second most common cancer and accounts for approximately 15% of all new cancers. During 2009, there will be an estimated 219,440 new cases of lung cancer. Lung cancer can be divided into two distinct types; small cell and non‐small cell cancer. Non–small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. The five‐year survival rate for non‐small cell lung cancer ranges from 11% to 17%. Current modalities treating the disease are focused on growth factor receptor therapy. Capsaicin is the active component of chili peppers plants belonging to the genus Capsaicinoids. The Vanilloids, a family of molecules to which capsaicin belongs, bind to targets in mitochondria of cancer cells to trigger apoptosis, without harming non‐transformed cells. Our goal was to determine the ability of a family of Vanilloid based compounds on three different NSCLC cell lines NCI‐H460, NCI‐H358, and NCI‐H1299. We found that compound LB‐24c12a caused a significant drop in proliferation in all three types of NSCLC cell lines, while SM‐1 was found to cause a slight rise in proliferation. A dose response to LB‐24c12a was determined in the micromolar range in H460 cells. The compounds were further analyzed for their effects on cytotoxicity, apoptosis and cell motility. These results shed light on possible effects on NSCLC by variations of capsaicin molecule.
Targeting NF‐κB in macrophages alleviates skin inflammation in a mouse model of psoriasis
Authors: Syrovets Tatiana , Wang Honglin , Buechele Berthold , Lunov Oleg , Scharffetter‐Kochanek Karin , Simmet Thomas
Published: June 2021

The pathogenesis and signaling in psoriasis, a chronic inflammatory skin disease, is not well‐understood. We found that, in contrast to skin from healthy subjects or from patients with atopic dermatitis, psoriatic skin exhibits strong activation of transcription factor NF‐κB mainly confined to dermal macrophages, whereas lymphocytes did not show activated NF‐κB. NF‐κB is required for the induction and/or function of many cytokines and aberrant cytokine expression has been proposed as an underlying cause of psoriasis. Therefore, we investigated whether NF‐κB targeting would affect the disease in the transgenic CD18 hypomorphic (CD18hypo) mouse model of psoriasis. When mice with severe psoriasiform lesions were treated systemically or locally with the natural IκB kinase inhibitor acetyl‐11‐keto‐β‐boswellic acid (AKβBA), NF‐κB signaling and the subsequent NF‐κB‐dependent cytokine production (TNF‐α, MCP‐1, IL‐12, and IL‐23) was profoundly suppressed. Additionally, application of the compound led to resolution of the abundant immune cell infiltrates, and reduced the increased proliferation of the keratinocytes. Overall, the AKκBA treatment reconstituted a nearly normal skin phenotype. Thus, NF‐κB signaling is pivotal for the pathogenesis in the CD18hypo mouse model of psoriasis and targeting of NF‐κB in macrophages might provide an effective therapeutic strategy.
The effect of bee venom against glutamate toxicity in microglial cells
Authors: Lee Sang Min , Yang Eun Jin
Published: June 2021

Bee venom (BV) extracted from honey bee has been used in a traditional Korean medical therapy. Several research groups demonstrated the anti‐inflammation effects of BV in immune‐related diseases in vivo and in vitro.

Glutamate is predominant excitatory neurotransmitter in the central nervous system (CNS). Prolonged exposure of glutamate receptors to high or persistently increased concentrations of glutamate leads the cell death. In addition, changes in glutamate release, uptake and activity of glutamate transporters have been reported in many neurodegenerative diseases such as Huntington¡s disease, Parkinsons¡s disease, Alzheimer¡s disease, and amyotrophic lateral sclerosis (ALS). However, BV is not clear whether BV has a protective effect against glutamate‐mediated cell death. To assess whether BV affords protection against glutamate mediated cell death, we examined the cell viability and signal transduction regarding cell survival with glutamate‐treated microglial cells in either the presence or absence of BV. First of all, we confirmed glutamatergic toxicity on microglial cells. Then, we found that BV had an inhibitory effect against the glutamatergic toxicity. As a result of it, our results showed that BV inhibited significantly the cell toxicity against glutamate and activation of proteins such as Akt, JNK, Erk and p38 were altered by exposure of glutamate time dependent manner.
The Involvement of WNT Signaling Pathway in Human Colon Cancer Metastasis
Authors: Li Qian , Chen Hong
Published: June 2021

Colon cancer is one of the most common cancers. Mortality from colon cancer mainly derives from its metastases. The mechanisms for colon metastasis are not clear. Aberrant WNT signals have been commonly observed in human colon cancer. Therefore, we hypothesize that WNT signaling pathway participates in the metastasis of colon cancer. To test this hypothesis, we used the cell line models SW480 and SW620, which are poorly and highly metastatic colon cancer cell lines, respectively. SW480 was established from a primary human colon cancer, while SW620 was from a lymph node metastasis in the same patient a year later. mRNA expression of genes involved in WNT pathway were detected in SW480 and SW620 by real‐time PCR. WNT5a decreased 1000 times in SW620 compared to SW480. N‐Myc downstream‐regulated gene 1 (NDRG1), which was reported to inhibit WNT pathway by interacting with WNT co‐receptors, decreased 10 times. On the other hand, secreted frizzled‐related protein 1(sFRP1), another WNT signaling antagonist, increased 3 times in SW620. The significant difference of WNT signaling observed between SW480 and SW620 showed strong correlation between the WNT signaling pathway and the invasive ability of colon cancer cells. Mechanisms of this interaction between WNT signaling pathway and colon cancer metastasis are under future investigation. This research was supported by the University of Illinois at Urbana‐Champaign.
Herpes Simplex Virus type 1 infection disrupts the ATR‐mediated damage response
Authors: Mohni Kareem N , Weller Sandra K
Published: June 2021

Herpes Simplex Virus type 1 (HSV‐1) is a large DNA virus that replicates in the nucleus of the host cell. HSV‐1 infection results in the activation of the ATM mediated damage response but not ATR. Since activation of ATM in uninfected cells generally results in the activation of ATR, we undertook the current study to determine how HSV‐1 infection disrupts the ATR mediated damage response. Preliminary data using phospho‐specific antibodies indicate that RPA and Chk1, which usually are targets of ATR, are not phosphorylated in infected cells. Using immunofluorescence we have shown that RPA and ATRIP are localized to the sites of viral DNA replication and that ATR and ATRIP do not colocalize in infected cells like they do during a normal damage response. We next wanted to see if we could activate the ATR pathway in infected cells by using hydroxyurea (HU). HU is a potent inhibitor of both cellular and viral DNA replication and will result in stalled replication forks and phosphorylation of the ATR substrate Chk1 in uninfected cells. Consistent with our previous data, Chk1 is not phosphorylated in response to HU in HSV‐1 infected cells. Taken together these results indicate that components of the ATR signaling pathway are present in viral replication compartments but that ATR signaling itself is disabled, perhaps by uncoupling ATR from ATRIP. This work was supported by Public Health Service grants AI21747 and AI69136.
Translesion synthesis of abasic sites by yeast DNA polymerase epsilon
Authors: Johansson Erik , Sabouri Nasim
Published: June 2021

Studies of replicative DNA polymerases have led to the generalization that abasic sites are strong blocks to DNA replication. Here we show that yeast replicative DNA polymerase ε bypasses a model abasic site with comparable efficiency to Pol η and Dpo4, two translesion polymerases. DNA polymerase ε also exhibited high bypass efficiency with a natural abasic site on the template. Translesion synthesis primarily resulted in deletions. In cases where only a single nucleotide was inserted, dATP was the preferred nucleotide opposite the natural abasic site. In contrast to translesion polymerases, DNA polymerase ε with 3′–5′ proofreading exonuclease activity bypasses only the model abasic site during processive synthesis and cannot reinitiate DNA synthesis. This characteristic may allow other pathways to rescue leading strand synthesis when stalled at an abasic site.