bluuebunny/crossref_metadata_2025 huggingface dataset:
Host microenvironment dictates tumor growth, angiogenesis and vascular permeability
Authors: Oh Phil , Borgstrom Per , Czarny Malgorzata , Schnitzer Jan
Published: June 2021

With new potential therapies for the treatment of human solid tumors constantly arising, the need for a suitable animal model which is clinically relevant becomes critical. An important criterion in the treatment of solid tumors is the understanding of the molecular and microenvironmental events that influence tumor formation, growth, metastasis and vascularization. Here we develop new tumor models that appear to more accurately depict human tumors in many ways beyond the standard subcutaneous models used predominately in the past. When we tested orthotopic, subcutaneous as well as non‐orthotopic engrafted tissues it was still clear that the orthotopic models provided a microenvironment more suitable for tumor growth and angiogenesis as well as being more restrictive. It may not be surprising with this pervasive vascular leakiness in the subcutaneous tumor models enable many treatments to work preclinically. But unfortunately this does not translate in clinical trials in humans. We are in the process of testing clinical drugs in our model. Here we show evidence that the host microenvironment plays an important role in tumor growth, angiogenesis and vascular permeability. Therefore, we feel this "pseudo‐ectopic/orthotopic" method may be more clinically relevant for testing potential therapies for the treatment of both primary and metastatic human solid tumors.
The role of the plasminogen activator system in the cellular physiology of MDA‐MB‐231 breast cancer cells
Authors: Gramling Mark , Church Frank C.
Published: June 2021

The purpose of this study is to examine the contribution of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI‐1) to invasion, proliferation, and survival of MDA‐MB‐231 breast cancer cells and how their expression is controlled. Inhibition of uPA activity inhibited invasion 30%, while inhibition of PAI‐1 increased invasion 50%. Inhibition of uPA, PAI‐1, and their cell surface receptors (urokinase receptor and low density lipoprotein‐related protein 1) inhibited proliferation and survival in response to tumor necrosis factor. Growth of cells in three dimensional matrices appeared to be inhibited by the presence of a PAI‐1 blocking antibody. Immunoblot and PCR indicate inhibition of either uPA or PAI‐1 activity led to decreased uPA expression and increased PAI‐1 expression. Inhibition of either uPA or PAI‐1 activity led to decreased phosphatidyl inositol‐3 kinase (PI3K) activity whereas incubation with pre‐formed uPA‐PAI‐1 complexes led to increased PI3K activity. Inhibition of PI3K led to decreased uPA protein expression with concomitant increased PAI‐1 expression. These results indicate uPA and PAI‐1 contribute to MDA‐MB‐231 invasiveness and activity of both proteins promotes cell proliferation and survival. Furthermore, PI3K positively regulates uPA, negatively regulates PAI‐1, and PI3K may be activated by recognition of uPA‐PAI‐1 complexes at the cell surface.
Suppression of autophagy enhanced proteasome inhibitor‐induced cell death selectively in cancer cells
Authors: Ding Wen‐Xing , Ni Hong‐Min , Gao Wentao , Chen Xiaoyun , Kang Jeong Han , Yin Xiao‐Ming
Published: June 2021

Autophagy is a lysosomal degradation pathway that has been involved in many chemotherapy drugs and radiation‐treated tumor cells. Autophagy principally serves as a protective mechanism to protect organisms against diverse pathologies but paradoxically can also leads to cell death, a controversy that has not been elucidate yet. In this study we found that suppression of proteasomal degradation induces compensatory activation of autophagy. When autophagy is suppressed by a pharmacological agent, such as 3‐MA or chloroquine, or via a genetic approach using siRNA‐mediated knockdown of specific autophagy genes, proteasome‐induced cell death was significantly enhanced only in cancer cell lines but not in non‐transformed normal cells. Therefore it seems that autophagy plays a protective role in cancer cells but has less effect on non‐transformed normal cells. Thus suppression of autophagy will enhance the chemotherapeutic effects of proteasome inhibitor via selectively killing of cancer cells. Indeed, results form the in vivo xenograft tumor studies indicate that the combined use of proteasome inhibitors and an autophagy inhibitor, such as chloroquine, can suppress tumor growth more significantly than the use of either agent alone. In summary these findings indicate that blocking both autophagic and proteasomal degradation could be an effective strategy for cancer therapy.
RNA interference techniques as an approach to elucidating ranavirus gene function.
Authors: Sample Robert Cressten , Whitley Dexter , Henegar Jeffrey , Sullivan Donna C , Chinchar Greg
Published: June 2021

Viruses of the genus Ranavirus, family Iridoviridae, are a group of large, icosahedral dsDNA viruses which have emerged as new pathogens of poikilothermic vertebrates. To confront their spread, it is vital to understand the mechanisms which control virus replication, host‐range, and pathogenesis. Therefore, Frog virus 3 (FV3), the ranavirus type species, was chosen as the model system. The FV3 genome has been completely sequenced and ~25% of the genes have recognized functions whereas the remaining ~75% have no known function. An antisense approach using RNA interference (RNAi) and antisense morpholinos (asMOs) was chosen to identify gene function and determine the requirements for replication in vitro and pathogenesis. The major capsid protein (MCP), the viral homologue of RNA Polymerase II (vPol‐IIα}), myristolated membrane protein (MMP), ICP‐46 (46K), and the cytosine DNA‐methyltransferase (DMT) were targeted using gene specific siRNAs or asMOs. Reductions of >80% in gene expression and >90% in viral titers were observed along with reductions in cytopathic effect. Transmission electron microscopy revealed reductions in virion assembly and production of atypical elements. These results support antisense techniques as a way of identifying and elucidating the function of genes involved in viral replication and pathogenesis.
Flagellin positively and negatively regulates IL‐1β expression via Ipaf and TLR5
Authors: Vijay‐Kumar Matam , Sitaraman Shanthi S. , Gewirtz Andrew
Published: June 2021

IL‐1β plays a pivotal role in health/disease. Consequently, regulation of IL‐1β bioactivity is multi‐faceted including inflammasome‐mediated processing of pro‐IL‐1β to mature IL‐1β and production of secretory IL‐1 receptor antagonist (sIL‐1Ra), which blocks IL‐1β activity. We sought to define how IL‐1β bioactivity is regulated in response to bacterial flagellin. We examined production of IL‐1β and sIL‐1Ra in vitro, in intestinal epithelial cells (IEC), macrophages, and mice treated with flagellin. Exposure of macrophages to 10μg/ml flagellin did not induce typical activation markers (e.g. IL‐6) but resulted in internalization of flagellin and production of Il‐1β that was dependent on both Ipaf and caspace‐1, but not TLR5. Conversely, although quite responsive to flagellin, IEC neither internalized flagellin nor produced IL‐1β suggesting that, in vivo, flagellin‐induced IL‐1β production is mediated by macrophage Ipaf. In contrast, production of sIL‐Ra, in vivo, was mediated by TLR5 suggesting loss of TLR5 might result in excessive flagellin‐induced IL‐1β activity. In accordance, while administration of flagellin to mice lacks toxicity in WT mice, flagellin‐treated TLR5−/− developed lasting inflammatory pathology. Thus, dual opposing regulation of flagellin‐induced Il‐1β activity by Ipaf and TLR5 mediates host defense while protecting against excessive inflammation.
Problem‐based teaching in a large classroom setting for on‐campus and off‐campus students
Authors: Scofield Margaret Ann , Bockman Charles S. , Huggett Kathryn D. , Abel Peter W.
Published: June 2021

Previous studies emphasize that large group settings hinder successful learning, particularly for problem‐based discussions. Our on‐campus pharmacy students have previously used small group sessions to discuss problem‐based cases that reinforce didactic lectures in a year‐long ten credit course in pharmacology. However, instructor led, small group sessions are not practical for problem‐based discussions by off‐campus students. Our objective was to develop, implement and evaluate a problem‐based discussion in a large group environment and capture the visual and auditory portions of the discussion for off‐campus students. On‐ and off‐campus students evaluated the new (large group) and past (small group) environments through standard course evaluations and specific questionnaires. The retention of information in both learning environments for both on‐ and off‐campus students was assessed using exam and quiz grades. We found an increase in on‐campus student participation in the new (large group) environment and favorable comments by both on‐ and off‐campus students. Knowledge retention was enhanced in the new (large group) environment as evidenced by an increase in quiz and exam median scores for both sets of students. This methodology may prove valuable and reassuring where on‐ or off‐campus resources may not be available to implement numerous small group sessions for problem‐based learning.
CEA‐CAM‐1 positive stem cells from the brain of the adult pig
Authors: Katner T L , Fenley C L , Fenley J L , Long G F , Alena C , Lochner F , McCommon G , Samples O , Collins J A , Hixson D , Bowyer F P , Black A C , Young H E
Published: June 2021

Carcinoembryonic antigen‐cell adhesion molecule‐1 (CEA‐CAM‐1) positive stem cells have been visualized in the white matter and gray matter in the cerebral cortex of adult rats. Similar cells have also been isolated and characterized from the skeletal muscle of and peripheral vasculature of adult mammals, including humans. The current study was undertaken to determine the location of these cells with respect to the brains of the adult pig. Adult pigs were euthanized following the guidelines of Fort Valley State University's IACUC. The heads were fixed by immersion fixation, the brains isolated, cryosectioned and stained with an antibody diagnostic for adult pluripotential stem cells, i.e. CEA‐CAM‐1. The current study will discuss the location with respect to the white and gray natter in the adult pig. Studies are ongoing to address their functional significance in neuronal injury and repair.
Curcumin and Ginger Extract Improves Abrasion Wound Healing in Damaged Skin
Authors: Warner Roscoe L. , Bhagavathula Narasimharao , Dasilva Marissa , McClintock Shannon D. , Barron Adam , Aslam Muhammad N. , Johnson Kent J. , Varani James
Published: June 2021

Topical treatment of hairless rats with curcumin and a ginger extract led to increased collagen production and decreased expression of MMP‐9 in treated skin. Rats were pre‐treated topically (21 days) with the botanical preparation followed by a combination of botanicals and Temovate (corticosteroid) for an additional 15 days. Superficial abrasion wounds healed more slowly in the skin of Temovate‐treated rats than in skin of control animals. Healing was more rapid in skin of rats that had been pre‐treated with the curcumin ‐ ginger extract (plus Temovate) than in the skin of rats treated with Temovate and vehicle alone. Each of the botanicals alone also improved wound healing but the individual agents were not as effective as the combined preparation. Dermal punch biopsies were obtained at the time of wound closure. Collagen production was increased and MMP‐9 production was decreased in the recently‐healed skin from rats treated with the botanical preparation relative to rats treated with Temovate plus vehicle. Neither of the botanical treatments alone or in combination caused skin irritation during the treatment phase or during wounding and repair. Taken together, these data suggest that a combination of curcumin and ginger extracts might provide a novel approach to improving structure and dermal function often seen in non‐healing wounds. Work supported by NIH HL07097
The effects of replicative senescence and telomerase on contraction and motility of fibroblast cells
Authors: Peterson Joanne Lykins , Vaughan Melville
Published: June 2021

Fibroblast cells are important in wound healing as they differentiate into myofibroblasts that can migrate into a wound and create tension and aging skin seems to take longer to repair a wound. This study was designed to look at the migration and contraction ability of early (EP) and late (LP) passage cells as compared to cells with telomerase (hTERT), an enzyme that lengthens telomeres delaying senescence. To assess contraction, all cells were grown on coverslips with and without transforming growth factor beta, a wound healing agent, and stained for alpha smooth muscle actin (α‐sma) a myofibroblast marker. Collagen lattice assays were used to assess contraction by measuring lattice diameter as it contracted over time then stained for the presence of α‐sma. Finally, a scratch assay was used to measure the ability for each type of cell to migrate into a "wounded" area. Contraction by EP cells was greater than LP cells in a five day collagen lattice. Results on migration assays and α‐sma in coverslips and collagen lattice are still being analyzed; however expected results would show that EP and hTERT cells should migrate faster and have more α‐sma than LP cells. If these results are shown, they would suggest that the formation of α‐sma in EP and hTERT cells allows faster migration and better contraction than in LP cells. Funding provided by the University of Central Oklahoma.

Grant Funding Source

UCOIRB
Classroom Clickers Provide Reliable Formative Feedback: Three years experience in gross and microscopic anatomy curriculum
Authors: Alexander Cara Jae , Crescini Weronika , Lachman Nirusha , Pawlina Wojciech
Published: June 2021

The goal of our study was to evaluate the effectiveness of the audience response system (ARS) as a tool for providing formative feedback to first year medical students. Over a three year period, we collected data from anatomy and histology courses in which students answered clinically oriented multiple choice questions using the ARS. We compared each student's cumulative ARS and final exam scores and we calculated a correlation coefficient between these two values. In addition, we conducted a questionnaire to evaluate the students' perception on ARS. According to our results, students saw ARS as useful for providing feedback on performance with a mean of 3.61 (1‐5 scale). There was a strong positive correlation between ARS and final exam scores. R values for the ARS and NBME Anatomy subject exam was R=.9556; and for the Anatomy/Radiology written exams, R=.9882. Students believed that ARS improved their performance with a mean of 3.94 (1‐5 scale). Students also preferred more frequent ARS quizzes with fewer questions to less frequent sessions with more questions (mean=3.74). In conclusion, students use ARS as a feedback tool and can rely on it as a good predictor of final course performance. We have found that ARS feedback motivates students to seek help from faculty when appropriate. It is user friendly, interacts well with PowerPoint, and is not time consuming for faculty.

Grant Funding Source

AAA
Interactive group learning in a virtual microscopy world
Authors: Goldman Haviva M , Smith Janet D , Churchill Judy R , Smolen Arnold J , DePace Dennis M
Published: June 2021

Virtual microscope technology presents new opportunities and challenges for curricular development in medical education. In our Problem Based Learning (PBL) curriculum at Drexel University College of Medicine, we chose to replace traditional microanatomy laboratory sessions with web‐based annotated virtual slide based assignments, self‐study guides, and self‐assessments for students to complete at home. We then used the laboratory time for group‐based review sessions that were comprised of structure scavenger hunts with student presentations and faculty facilitation. Following the first year of this format change, no significant difference in student practical exam performance was evident. However, performance on microanatomy multiple‐choice questions increased by 13%, suggesting enhanced understanding of microanatomy concepts. Since other curricular changes were also implemented this year, it is not possible to determine the degree to which the lab format change contributed to this increase. Student evaluations suggest the new format was preferred, and was an effective use of time. Laboratory participation averaged 99%, and 89% of students reported completing most at‐home assignments in advance of in‐lab sessions. We conclude that the new lab format encourages both independent and interactive‐group learning, and is well suited to the learning style of our PBL students.

Grant Funding Source

Drexel University Professional Enrichment and Growth Grant
An analysis of the effectiveness of team roles during anatomy dissections
Authors: Elzie Carrie A. , Herring Nicole , Petrusa Emil , Pettepher Cathy , Dalley Arthur F.
Published: June 2021

Vanderbilt University recently adopted an integrated medical curriculum which, like many schools, resulted in a reduction in the number of teaching hours designated for gross anatomy. The reduction in hours primarily affected the lecture portion of the class, while preserving as much time as possible for laboratory dissection. The active components of laboratory dissection are deemed essential to the learning process of the students at Vanderbilt University. Thus, to maximum the time spent in the laboratory students at each dissecting table have access to a large touch‐screen PC that provides the students with daily laboratory goals, dissection instructions and several electronic textbooks and videos that provide resources to aid in their dissection. Typically each table is shared by five students ‐ one of which is assigned to operate the computer, two serve as primary dissectors and two as assistant dissectors. The roles rotate every lab session to ensure that all students are exposed to each responsibility. Thus, this study seeks to investigate if there is a correlation between the student's assigned role and their performance on that particular portion of the practical exam. For instance, on the day of the heart dissection, will the primary dissector perform better on the practical questions specific to that lab than the assisting dissector or the computer operator? Data collected from this study will allow us to evaluate the effectiveness of our team roles during gross anatomy as well as assess the impact of the computers during dissection.

Grant Funding Source

IRACDA NIH 5K12 GM068543‐04
A NEW COMPLEMENTARY ACTIVITY TO HISTOLOGY TEACHING
Authors: Monte‐Alto‐Costa Andréa , Romana‐Souza Bruna
Published: June 2021

Objective

To test a new complementary activity to histology teaching aiming to improving histology knowledge of undergraduated nursing students.

Methods

Students were divided into groups (6 per group). Each group received a file containing introduction, objectives, methodology and figures (such as graphics, tables and photomicrographs) of a scientific article focused in histological parameters (pathology of an organ). Students should analyze the material received and complete the article inserting title and conclusion, describing results and proposing clinical application. Furthermore, groups prepared an oral presentation and wrote an abstract about complete article.

Results

All groups showed a good presentation and abstract describing results (mainly photomicrographs and graphics), connecting these results with its clinical relevance and presenting a creative title and a final conclusion. At the end, the students evaluated this activity and described three main positive points about it: it permitted to improve their histology knowledge, to associate theoretical concepts of histology with practical ones and to deepen theoretical concepts of histology.

Conclusions

This complementary activity to histology teaching improved histology knowledge of undergraduate nursing students, because these students deepened their theoretical concepts of histology, associated theoretical and practical concepts of histology and recognized the importance of histology in their professional life.

This work was supported by Department of Histology and Embryology, State University of Rio de Janeiro (DHE‐UREJ).
Thyroid hormone receptor recruits histone methyltransferase PRMT1 to regulate transcription and developmental rate
Authors: Shi Yun‐Bo
Published: June 2021

Protein arginine methyltransferase 1 (PRMT1) acts as a coactivator for nuclear receptors through histone H4 R3 methylation. Here we investigated the role of PRMT1 in thyroid hormone (T3) receptor (TR)‐mediated transcription in vivo during vertebrate development by using T3‐dependent Xenopus laevis metamorphosis as a model. We first showed that PRMT1 expression was upregulated during metamorphosis when both TR and T3 were present. We then demonstrated a role of PRMT1 in TR‐mediated transcription by showing that PRMT1 enhanced transcriptional activation by liganded TR in the frog oocyte transcription system and was recruited to the T3 response element (TRE) of the target promoter in chromatin in the oocyte as well as to endogenous TREs during frog metamorphosis. Surprisingly, we found that PRMT1 was only transiently recruited to the TREs in the target during metamorphosis. Mechanistically, we showed that overexpression of PRMT1 enhanced TR binding to TREs in vivo, leading to increased histone modifications and recruitment of other coactivators. Importantly, transgenic overexpression of PRMT1 enhanced gene activation in vivo and accelerated both natural and T3‐induced metamorphosis. These results indicate that PRMT1 functions transiently as a coactivator in TR‐mediated transcription by enhancing TR‐TRE binding to regulate the rate of metamorphosis.
Yeast High Mobility Group Protein HMO1 Negatively Regulates Its Own Expression
Authors: Xiao Lijuan , Grove Anne
Published: June 2021

The Saccharomyces cerevisiae high mobility group protein HMO1 is composed of two DNA binding domains, box A and box B, and a lysine‐rich C‐terminal tail. HMO1 plays various crucial functions in chromosomal dynamics processes, and it has been proposed to coordinate rRNA and ribosomal protein (RP) gene transcription. Strains bearing hmo1 mutant alleles are viable, but have growth defects. For recovery of the growth phenotype, we found that excess HMO1 is deleterious to cell growth, while expression of truncated HMO1 lacking the C‐terminal tail can recover the growth phenotype, suggesting that expression of HMO1 is strictly regulated. A β‐galactosidase reporter construct driven by the Hmo1 promoter was created, and the β‐galactosidase activity assay showed that HMO1 negatively regulates its own expression, and that the C‐terminal tail is unnecessary for this regulation. The hmo1Δ strain is less sensitive to rapamycin, which inhibits the Target of Rapamycin (TOR) kinase. The β‐galactosidase activity assay showed that rapamycin represses HMO1 gene expression, but only when HMO1 is present, which is very similar to what was reported for RP gene expression, where the TOR‐dependent repression of RP genes is alleviated in the absence of HMO1. We therefore propose that HMO1 mediates a similar regulation of its own expression and that of RP genes in response to TOR signaling.
Nascent peptide‐dependent regulation of protein synthesis
Authors: Vazquez‐Laslop Nora , Martinez‐Garriga Blanca , Kannan Krishna , Klepacki Dorota , Mankin Alexander S.
Published: June 2021

The ribosome has the intrinsic capacity to monitor the structure of the nascent peptide. Sensing of the nascent peptide structure takes place in the exit tunnel of the large ribosomal subunit and often leads to ribosome stalling. This property of the ribosome is used to regulate gene expression. However, the nature of the sensory elements, the molecular bases involved in ribosome response and the spectrum of genes regulated by this mechanism are unknown. We are using genetic and biochemical analyses to address these questions. Mutational analysis reveals that specific 23S rRNA residues forming the walls of the exit tunnel participate in sensing the nature of the nascent peptide. Alterations at position A2062 have been shown to alleviate drug‐dependent ribosome stalling. The effect of mutations at other rRNA positions in the tunnel, including A2058, A2503, U2586 and U2609 are being analyzed. The expression profile of proteins in cells carrying A2058G mutant ribosomes was compared to that of wild type cells. Several proteins were found to be differentially expressed, suggesting that nascent peptide‐ribosome interactions might be involved in regulation of a variety of cellular genes.
Characterization of Zeaxanthin glucosylase (CrtX) from Paracoccus haeundaensis
Authors: Kim Young Tae , Ji Keunho , Seo Yong Bae
Published: June 2021

Zeaxanthin, one of the carotenoids, is found most abundantly in dark, leafy green vegetables and crucial to the good health of the eyes. We have previously reported the isolation of a new marine bacterium, Paracoccus haeundaensis, which produces carotenoids, mainly astaxanthin. The astaxanthin biosynthesis gene cluster from the P. haeundaensis, consisting of six carotenogenic genes, was cloned and their molecular characteristics were previously reported. Zeaxanthin glucosyltransferase (CrtX) mediates the formation of zeaxanthin to zeaxanthin diglucoside. In the present study, zeaxanthin glucosyltransferase gene (crtX) was isolated from the marine bacterium, Paracoccus haeundaensis, and consisted of 1248 bp encoding 415 amino acids residues. The nucleotide sequence of crtX was analyzed and turned out to be well conserved during evolution. The expression plasmid containing crtX gene (pSTCRT‐X) was constructed and E. coli cells containing this plasmid produced the recombinant protein of approximately 46 kDa, which corresponds to zeaxanthin glucosyltransferase. The biosynthesis of zeaxanthin diglucoside was obtained when pSTCRT‐X plasmid was co‐transformed into the E. coli containing the plasmid pET‐44a(+)‐CrtEBIYZ carrying the astaxanthin biosynthesis genes (crtE, crtB, crtI, crtY, and crtZ) from the P. haeundaensis required for zeaxanthin β‐D‐diglucoside biosynthesis.
Characterization of lycopene cyclase (CrtY) from marine bacterium, Paracoccus haeundaensis
Authors: Kim Young Tae , Jeong Tae Hyug , Seo Yong Bae
Published: June 2021

Lycopene cyclase (CrtY) converts lycopene into β‐carotene by catalyzing the formation of two beta‐rings at each end of the linear carotene. This reaction takes place as a two step reaction which both sides of the lycopene molecule are cyclized into β‐carotene rings via the monocyclic γ‐carotene as an intermediate. Lycopene cyclase gene (crtY) involved in the carotenoid biosynthetic pathway of the Paracoccus haeundaensis was subcloned into the expression vector, pET‐44a(+), and expressed in E. coli BL21(DE3) codon plus cell. The resulting protein was expressed as a fusion protein containing an N‐terminal six‐histidine extension which derived from the expression vector. The expressed protein was purified to homogeneity by affinity chromatography and incubated with the substrate mixture. The products of the enzymatic reaction were separated with HPLC and analyzed by spectroscopic method. The results showed that lycopene was converted into γ‐carotene and β‐carotene, suggesting that the purified enzyme (CrtY) is enzymatically active. Furthermore, substrate specificity and cofactor requirements of the purified protein were determined. The enzymatic reaction was stimulated by NADH or NADPH.
Regulation of stress granule dynamics by Grb7 and FAK signalling pathway
Authors: Tsai Nien‐Pei , Ho Ping‐Chih , Wei Li‐Na
Published: June 2021

Cells form stress granules (SGs) in response to environmental stresses, which constitute cytoplasmic domains where mRNAs are stored and translation is halted. Although several components are found in SGs, it is poorly understood as to how SGs are formed and dissolved. We identified growth factor receptor‐bound protein 7 (Grb7), an RNA‐binding, translational regulator, as an integral component of SGs, which directly interacts with Hu antigen R (HuR) and is required for cells to form SGs. When stress is terminated, Grb7 is hyperphosphorylated by focal adhesion kinase (FAK), loses its ability to directly interact with HuR and is dissociated from SG components, thereby disrupting SGs in recovering cells. Consistently, dominant‐negative hypophosphorylated mutants of FAK and Grb7 significantly attenuate SG disassembly during recovery. FAK activation followed by its phosphorylating Grb7 constitutes a cell‐autonomous signalling pathway that regulates the disassembly of SGs and translational stimulation during recovery. This is the first reported pathway actively regulating the dynamics of SGs. This work was supported by DA11190, DA11806, DK54733, DK60521, K02‐DA13926 to L.‐N. W.
Characterization of AS Regulatory Protein, a Novel Protein Implicated in Endothelial Stress Response
Authors: Pendleton Laura C. , Corbin Karen D. , Shriver Sandra K. , Solomonson Larry P. , Eichler Duane C.
Published: June 2021

The citrulline‐nitric oxide cycle is a tightly regulated system for the production of nitric oxide (NO) in endothelial cells. The cycle consists of the proteins endothelial nitric oxide synthase (eNOS), argininosuccinate synthase (AS), and argininosuccinate lyase (AL). AS and AL serve to recycle citrulline, a product of the production of NO, to arginine, the principle substrate for eNOS. AS regulatory protein (ARP) is a small protein encoded by an upstream open reading frame in minor expressed AS mRNAs. Overexpression of ARP suppressed AS expression and NO production in endothelial cells while selective knockdown of the longer AS transcripts that produce ARP resulted in increased AS protein expression and NO production. To investigate the subcellular localization of ARP, a custom polyclonal antibody was generated. Fluorescence microscopy studies demonstrated that ARP is expressed throughout the cytoplasm but that expression seems predominant in the perinuclear endoplasmic reticulum and golgi, co‐localizing with both eNOS and AS. Co‐immunostaining for ARP and TIA‐1, a marker for stress granule formation, demonstrated that ARP moves to stress granules when stress is induced artificially by sodium arsenite in endothelial cells. This data demonstrates that ARP may play a role in the sequestration of mRNAs under conditions of stress. (Supported by the James & Esther King Biomedical Research Program Grant).
NMR dynamics investigation of ligand induced changes of main and side chain arginine N‐H's in human phosphomevalonate kinase
Authors: Olson Andrew L , Yao Huili , Herdendorf Timothy , Miziorko Henry , Cai Sheng , Sem Dan S
Published: June 2021

Human phosphomevalonate kinase (PMK) is involved in phosphoryl transfer from mevalonate 5‐phosphate (M5P) to mevalonate 5‐diphosphate. There are 17 arginines in PMK with the most important being R18, which is part of the "Walker A" domain, a highly conserved motif for binding ATP. Other important arginines are R42, R73, R110, and R142, as these are in the binding pocket and are potentially used to stabilize charge of interacting ligands. In order to characterize the effect/importance of these arginines in ligand binding, PMK was overexpressed with selectively labeled 15N‐arginines and monitored by 2D‐NMR. This is the first report of monitoring ligand induced side chain mobility and dynamics by modelfree analysis. Apo‐PMK side chains are relatively mobile (S2= 0.44 ± 0.04) and binding of M5P causes nearly all of the labeled side chains to get more ordered (S2= 0.71 ± 0.05), indicating ligand‐side chain interaction. It's curious that more side chains, than are in the binding site, get less mobile and to assess this we are attempting to assign the side chains to main chain residues that are assigned. We are also investigating arginine dynamics in ATP binding as well as the ternary complex (PMK/ATP/M5P). (AHA 05303072 to D.S.S.)
Cardioprotection increases phosphorylation of the mitochondrial electron transport chain and promotes supercomplex formation
Authors: Wong Renee , Steenbergen Charles , Murphy Elizabeth
Published: June 2021

It is proposed that preconditioning (PC) signaling converges on mitochondria and results in cardioprotection. How this signaling affects mitochondrial organization is yet to be understood. We used proteomics to test whether PC and pharmacological PC similarly alter mitochondrial proteins. Langendorff murine hearts were treated with a GSK‐3β inhibitor (GSK Inhib) for 10 min or preconditioned for 4 cycles of 5‐min I/R, prior to 20‐min ischemia and 120‐min reflow. PC and GSK Inhib improved LVDP recovery, decreased infarct size, and reduced ischemic lactate production compared to their time‐matched controls. We used proteomics to examine mitochondrial protein levels/phosphorylation changes that were present only in PC and GSK Inhib, but not in control. Levels of cytochrome c oxidase (COX) subunits 5A and 6B, ATP synthase coupling factor 6, and cytochrome b‐c1 complex (complex III) subunit 6 were increased while cytochrome c was decreased in PC and GSK Inhib. Changes in COX and complex III levels led us to investigate the formation of electron transport chain (ETC) supercomplexes, which consist of complexes I, III, and COX and reportedly play a role in e− transport and ROS production. PC and GSK cardioprotection increased the amount of COX in the supercomplex. Thus, the ability of PC and GSK inhibition to alter ETC complexes and supercomplex composition will have important implications for mitochondrial function.
Regulation of p21 Expression by Sphingosine Kinase 2
Authors: Hait Nitai C , Milstien Sheldon , Spiegel Sarah
Published: June 2021

Sphingosine‐1‐phosphate (S1P) is a potent sphingolipid mediator formed by phosphorylation of sphingosine catalyzed by two sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2. Whereas SphK1 is overexpressed in breast cancers and has been linked to cancer progression, not much is known about SphK2. Expression of SphK2, which is enriched in the nucleus of MCF7 human breast cancer cells, increased expression of the cyclin‐dependent kinase inhibitor p21 but had no effect on p53 or its phosphorylation. The phorbol ester PMA is known to induce growth arrest of MCF7 cells accompanied by induction of p21. Indeed, overexpression of SphK2, but not catalytically inactive SphK2, enhanced PMA‐mediated increases in p21 mRNA and protein expression in MCF7 cells. Conversely, downregulation of SphK2 with specific siRNA decreased basal p21 levels as well as its elevation mediated by PMA. Moreover, overexpression of SphK2, but not the inactive mutant, in HeLa cells, where they are also localized to the nucleus, enhanced basal as well as PMA‐stimulated p21‐luciferase promoter activity. Our data support the notion that SphK2 and formation of S1P in the nucleus might be important regulators of p21 transcription. Supported by R01CA61774 to SS.
Regulation of Autophagy by Sphingosine‐1‐Phosphate Phosphohydrolase 1
Authors: Lepine Sandrine , Schramm Danielle , Milstien Sheldon , Spiegel Sarah
Published: June 2021

Sphingolipid metabolites have recently been implicated in autophagy. In this work, we showed that downregulation of sphingosine‐1‐phosphate phosphohydrolase 1 (SPP1), an enzyme that dephosphorylates S1P induced autophagy, as determined by increased formation of LC3‐positive autophagosomes and immunoblotting for LC3II. Downregulation of PERK, IRE1_, or ATF6, three initiators of ER stress signaling, suppressed autophagy mediated by SPP1 depletion. Moreover, CHOP, Bip and phospho‐eif2, and cleavage of pro‐caspase 4 and pro‐caspase 2, downstream targets of ER and Golgi stress, was increased after downregulation of SPP1. However, silencing SPP1 did not affect either beclin levels or mTor downstream targets, suggesting that these pathways are not involved. Interestingly, downregulation of SPP1 did not lead to apoptosis. Exogenous S1P or dihydro‐S1P, which activate S1P receptors, in contrast to increased intracellular S1P, do not induce autophagy. Thus, SPP1 may regulate protective autophagy through ER stress signaling by regulation of intracellular S1P levels. Supported by R37GM043880 to SS.
Investigating the anionic phospholipid regulation of diacylglycerol kinase epsilon
Authors: Lung Michael , Topham Matthew K. , Epand Richard M.
Published: June 2021

Mammalian diacylglycerol kinases (DGKs) form critical links in cell signaling pathways by catalyzing the phosphorylation of diacylglycerol (DAG) to phosphatidic acid (PA); regulating the levels of these two essential second messengers in the cell. The epsilon isoform of DGK (DGKε), possesses the simplest primary structure of any DGK isoform, yet is unique in its inhibition by anionic phospholipids such as PA. The molecular mechanisms underlying this unique feature of DGKε are unknown. Here we show that there is a combined acyl chain specificity in the PA inhibition of DGKε, favouring a combination of an arachidonyl sn‐2 and a stearoyl sn‐1 acyl chain. These results contrast to those of previous reports in which an arachidonyl‐DAG specific isoform of DGK did not exhibit PA acyl chain specific inhibition. Also, deletion of DGKε's cationic cluster retains its PA acyl chain specific inhibition, a region previously hypothesized as a candidate for PA binding. Overall, these results suggest that the PA acyl chain specific inhibition of DGKε occurs through a binding site within DGKε with a definite shape that is not found in DGKε's cationic cluster. This work is funded by NSERC and the NIH.
AMP‐activated protein kinase suppresses LXR‐dependent sterol regulatory element‐binding protein‐1c transcription in rat hepatoma McA‐RH7777 cells
Authors: Yang Jian , Craddock Lauren , Hong Sharon , Liu Zhi‐Mei
Published: June 2021

AMP‐activated protein kinase (AMPK) is an intracellular fuel sensor that plays a key role in regulating fatty acid synthesis in liver. Sterol regulatory element‐binding protein (SREBP)‐1c is a master regulator of hepatic lipogenic gene expression. It has long been documented that AMPK activation suppresses hepatic SREBP‐1 mRNA and nuclear SREBP‐1 protein. But the mechanism remains undefined. In this study we investigated the molecular mechanisms by which AMPK downregulates hepatic SREBP‐1c mRNA using a novel model cell line McA‐RH7777. We found that AMPK is robustly activated in rat hepatoma McA‐RH7777 cells treated with two widely used AMPK activators, AICAR and metformin, and AMPK activation sharply suppresses SREBP‐1c mRNA and nuclear SREBP‐1c protein, but not SREBP‐1a mRNA derived from the same gene. These inhibitory effects are reversed by the AMPK inhibitor Compound C or 8‐BrAMP, demonstrating the requirement of AMPK in the suppression of SREBP‐1c mRNA and nuclear SREBP‐1c protein by AICAR and metformin. AMPK does not enhance SREBP‐1c mRNA degradation in the presence of the general transcription inhibitor actinomycin D; instead it inhibits SREBP‐1c promoter activity in a luciferase reporter assay. AMPK‐mediated inhibition of SREBP‐1c promoter activity can also be abrogated by the AMPK inhibitor Compound C. Furthermore AMPK activation significantly attenuates the synthetic liver X receptor (LXR) ligand T0901317‐induced SREBP‐1c promoter activity. AMPK also inhibits cleavage of LXR ligand‐induced SREBP‐1c precursor. We conclude that AMPK suppresses hepatic SREBP‐1c mRNA expression by inhibiting LXR‐dependent SREBP‐1c transcription via inhibition of endogenous LXR ligand production and by inhibiting SREBP‐1c processing.
Triphenylphosphine promotes production of 12‐HETE during COX‐1‐dependent metabolism of arachidonic acid
Authors: Pernecky Steven John , Sengupta Aditi , Ailaboina Lalini , Sidhu Rohini , Basu Hemendra
Published: June 2021

Lipid hydroperoxides (LOOH) play a role in reactions catalyzed by PGH2 synthase (COX). Commercial preparations of arachidonic acid (AA) contain LOOH as judged by TLC, and these hydroperoxides are reduced to alcohols by the reducing agent triphenylphosphine (TPP). PGE2 formed by ovine COX‐1 via PGH2 is produced during AA metabolism as judged by GC‐MS following formation of the methyl ester and methoxime/silylated derivatives regardless of whether AA, AA treated with TPP, or AA isolated from TLC following TPP treatment are used as substrates. When AA pretreated with TPP was incubated with COX‐1 that was also treated with TPP, there was a marked decrease in the amount of PGE2 produced and the appearance of 12‐HETE. No 12‐HETE was produced when COX‐1 was heat inactivated prior to the reaction with TPP‐treated AA. When the same experiment was conducted with tributylphosphine (TBP), PGE2 production was not compromised and no 12‐HETE was produced. These results suggest that TPP stimulates a lipoxygenase‐type reaction with ovine COX‐1 while suppressing COX‐1 activity that does not involve the reductive function since an alternate reductant, TBP, was incapable of supporting 12‐HETE production.
Role of Obesity and Aging on Intestinal Stem Cell Function
Authors: Graham Suzanne , Choi Joan , Birse Ryan , Oldham Sean
Published: June 2021

Changes of somatic stem cell (SSC) functionality has been proposed to contribute to tissue and organismal aging. Because the effects of diet and aging on SSC function can impact organ senescence and longevity, the need to determine the differences between young and old SSC in vivo and elucidating general principles of how SSC respond to diet and aging signals are critically important for many aspects of aging and regenerative biology. However, we have a limited understanding of how SSCs change with age and under different diet conditions as well as how modifying aged SSCs affects organ function. Given the important contributions of the intestine to metabolism, growth, and immunity, we used Drosophila intestinal stem cells (ISCs) as a model system to understand these questions. We show that there is an age‐dependent increase in ISC numbers. Furthermore, using a novel High Fat Diet‐induced obesity model, we also see a similar increase in ISC numbers. Both of these age and High Fat Diet‐induced ISC phenotypes can be prevented by decreasing the insulin‐nutrient sensing Target of Rapamycin (TOR) signaling pathways. Thus, these results show that reduction of insulin‐metabolic pathways provides protection against age‐ and high fat diet‐dependent changes in the ISCs and intestine. Future work will look at how other dietary conditions like Dietary Restriction can also modify the ISC aging phenotypes. These results are highly relevant to metabolic diseases like obesity and diabetes as well as colorectal cancer. In many species, reduction of insulin‐nutrient sensing pathways promotes longevity and these results suggest longevity may be linked to stem cell aging via alterations of these pathways.
Melatonin is up‐regulator on small G‐protein mediated apoptotic cell death in cervical and endometrial cancer cells of human
Authors: Hong Yonggeun , Won Jinyoung , Park Kanghee
Published: June 2021

Many gynecologic tumors are associated with infertility and spontaneous abortion that constitute a public problem. Recent studies have shown the main hormone of the pineal gland inhibit directly many different types of human tumor cells and naturally secreted cytotoxin, which induces tumor cell death(apoptosis). Additionally, activated Ras proteins have important roles on the regulation of apoptosis depending on cell type and other factors. Ras protein, a small GTP binding protein, send growth stimulating message to the nucleus through downstream small G protein such as Rac1 and Cdc42. Ras protein is an initiator of many tumors, and melatonin shows oncostatic action in various tumors cells. Whether melatonin can counteract the Ras‐involved functions remains unknown and is worth exploring. Thus, our overall hypothesis is that natural hormone melatonin regulates small GTP binding protein mediated apoptosis and we would demonstrate anti‐tumor function of melatonin in primary cultured human uterus cervix and endometrial cancer cells. What Ras gene transfected into cells were treated with melatonin, then observed increasing caspase‐3 expression suggesting turn of the apoptotic signal machinery. At physiological concentration of melatonin (1 nM), cell proliferation was decreased, suggesting cell cycle arrest through increasing p21 expression, which is mediated by p53(tumor suppressor gene). These observations indicate that melatonin, an endogenous hormone, inhibits the tumor cell proliferation suggesting melatonin should be developed as a therapeutic reagent.

Supported by BioGreen21 Program (#20070401034006) of Rural Development Administration, South Korea
ERK Inhibition by SU1498 Alters Expression of Bcl‐2 Family Proteins in Breast Cancer Cells
Authors: Kovala Tom , Poulin Josee
Published: June 2021

The extracellular signal‐regulated kinase (ERK) pathway is vital to the cell survival. We have demonstrated the ability of SU1498, an inhibitor of ERK kinase activity, to sensitize breast cancer cells with constitutively active ERK to apoptosis. To identify the mechanisms involve, the expression of the antiapoptotic Bcl‐2 and Bcl‐xL and the proapoptotic Bad, Bax, Bik and Bak were examined under apoptotic conditions. One breast cell line (MCF‐10A) and three breast cancer cell lines (MCF 7, MDA‐MB‐231 and MDA‐MB‐468) were used to examine the effects of SU1498 and serum starvation on the expression of the Bcl‐2 family proteins. The MCF‐10A and MCF7 cells exhibit normal ERK regulation while the MDA‐MB‐231 and MDA‐MB‐468 cells lines have a constitutively activated ERK. Across all the cell lines, the combined treatments resulted in either decreased expression, or no changes in the expression, of the antiapoptotic Bcl2 proteins. The expression of the proapoptotic proteins Bad or Bak were dramatically upregulated only in those cells with constitutive ERK activation, while they tended to demonstrate reduced expression in the cells lines with normal ERK regulation. These cell lines with upregulate Bad and/or Bak expression were also those most sensitive to SU1498 induced cell death. The ability of SU1498 to sensitize breast cancer cells to apoptosis by inhibition of ERK may have therapeutic uses.

Supported by NSERC
The macronutrient composition of the evening meal before glycemic index testing has no effect on glycemic response
Authors: Brown Rachel Clare , Ning Boris , Williams Sheila , Venn Bernard , Green Tim John
Published: June 2021

Background

A criticism of glycemic index (GI) is the large variation in glycemic response, both between and within individuals. To reduce this variation it is recommended that GI testing of a food be conducted under standardized conditions. One recommendation is that each subject consumes the same evening meal before each test day. Given the demands placed on participants it is important to assess whether such a restriction is necessary.

Aim

To determine whether manipulating the fat and carbohydrate content of the evening meal preceding GI testing has an effect on glycemic response.

Methods

Using a randomized crossover design, 20 participants completed a series of 8 tests. Test foods consisted of 2 glucose beverages and 2 bread samples, containing 50g of available carbohydrate. Each test food was consumed following a high carbohydrate or a high fat meal the night preceding testing. Blood glucose concentration was measured at regular intervals over 2 hours and area under the curve (AUC) determined.

Results

There were no significant differences in the AUC for the glucose drink [ratio (95% CI); 0.91 (0.80, 1.03)] or bread [1.15; (0.97, 1.36)] with the high carbohydrate and the high fat evening meal.

Conclusion

The fat and carbohydrate content of the evening meal prior to GI testing does not influence blood glucose response, thus participants should be allowed some flexibility in evening meal selection.