bluuebunny/crossref_metadata_2025 huggingface dataset:
A functional role of superoxide dismutase 3 in nitric oxide‐mediated protection against catabolic wasting in skeletal muscle
Authors: Okutsu Mitsuharu , Lira Vitor A , Zhang Mei , Yan Zhen
Published: June 2021

Oxidative myofibers are resistant to catabolic wasting caused by many chronic diseases, such as congestive heart failure and sepsis. We have recently identified a nitric oxide‐dependent antioxidant defense mechanism in this oxidative phenotype‐associated protection. Here, we show that systemic administration of NO donor GSNO (4 mg/kg/day, i.p.) significantly suppressed glucocorticoid‐induced (dexamethasone, 25 mg/kg/day, i.p.) loss of muscle mass and increases in E3 ligases MAFbx/atrogin‐1 mRNA expression in glyocolytic muscles. GSNO also prevented dexamethasone‐induced reduction of superoxide dismutase 3 (SOD3) protein expression. Importantly, somatic gene transfer of SOD3 in glycolytic tibialis anterior muscle significantly attenuated dexamethasone‐induced muscle atrophy. These findings suggest the functional importance of SOD3 in NO‐mediated protection against catabolic wasting in skeletal muscle.
Factor binding sites within the antisilencer/enhancer region of the myelin proteolipid protein gene are important for transcriptional activity in cultured oligodendroglial cells
Authors: Greuel Brian T. , Cooper Phoebe , Lehn John , Betz Adam , Stevenson Andrew
Published: June 2021

The first intron of the myelin proteolipid protein (Plp) gene contains a positive regulatory region known as the antisilencer/enhancer (ASE) that is located between nucleotides 1083 and 1177 of the intron and is sufficient to restrict high‐level expression of the gene to oligodendrocytes in mice. To identify critical transcriptional control elements within the ASE, we have created a series of twelve non‐overlapping 8 bp linker scan mutations across the ASE region. These mutant ASE fragments were inserted in both orientations into a Plp‐lacZ fusion gene containing a shortened form of intron 1 that lacked the ASE. Relative transcriptional activities of these PLP‐lacZ constructs were assessed indirectly by measuring the β‐galactosidase activities in extracts of transiently transfected oligodendroglial (N20.1) cells. Five of the mutants with linkers inserted near the center of the ASE demonstrated reduced activity (10–50% of wild‐type levels). This effect was relatively independent of the orientation of the ASE inserted into the Plp‐lacZ constructs, consistent with the function of this region as a transcriptional enhancer. LS mutations at these same positions were previously shown to interfere with the binding of factors present in nuclear extracts of N20.1 cells, suggesting that these factors may be important in the oligodendrocyte‐specific regulation of the Plp gene. Supported by NIH grant P20 RR‐16460.
Differential effects of two different beta‐adrenergic agonists, clenbuterol and ractopamine, on muscle growth in rats
Authors: Talmadge Robert J , DeGuzman Cathryn , Reecy James M , Gabler Nicholas K
Published: June 2021

This study compared the muscle growth promoting effects of two beta‐adrenergic agonists: clenbuterol (CLN) and ractopamine (RCT). Three groups of rats (10/group) were treated for a period of 6 days with either CLN (3 mg/kg body weight/day, 1 ml subcutaneous), RCT (4 mg/kg body weight/day, 1 ml intraperitoneal), or sterile saline (1 ml subcutaneous/day). The saline group served as the control (CON). The rats were then euthanized and muscles were dissected, weighed, snap frozen and stored at ‐70oC. Soleus (SOL), plantaris (PLN), and tibialis anterior (TA) muscle masses were significantly increased in CLN rats by 14, 26, and 17%, respectively. RCT rats displayed no change in absolute SOL mass, but elevations in MG and TA mass by 12, and 8%, respectively. The CLN rats had elevated phosphorylation of 4EBP‐1 and Akt in the PLN muscle, suggesting an elevation in activation of the PI3K‐Akt‐mTor growth‐related pathway. In contrast, RCT showed an elevation in Akt, but not 4EBP‐1 phosphorylation. The data suggest that differences in the effects of CLN and RCT on muscle mass may exist. Future studies will elucidate the physiological and cellular mechanisms that account for these differences.
Student Group Seminars‐ An Useful Tool for Active Learning of Physiology in Medical Schools
Authors: Ghosh Sarmishtha
Published: June 2021

Medical students need to keep abreast of available medical information, organizing and processing them properly in these days of exponential increase in medical information and technology. A need was felt to develop a teaching – learning module which would not only introduce a different approach of active learning of basic sciences but also train the students in gathering, organizing, processing and presenting information. Five common clinical problems associated with the central nervous system, not taught in a didactic lecture class were chosen for seminars. Three week were allotted for search of relevant materials from books, journals and online sources and the students were encouraged to construct a presentation out of that and present a seminar using varied audiovisual aids in front of their peers group and faculty. A structured feedback from the students using a 5 point Likert scale showed 80% considered the method to be very useful in improving both written and oral communication skills. Library records showed 70% increase in usage of the Library for both printed versions and online searches. 60% reported that it motivated them towards self directed learning and improved overall confidence in them. 77% rated group seminars as an important teaching‐learning component, useful for understanding basic science in clinical context. It is hence concluded that student group seminars can be used as an additional tool for effective learning of physiological concepts, improving skills of information retrieval and management together with increased overall confidence to help them handle problem based learning sessions in later days of their clinical training. The work was conducted as part of FAIMER Fellowship by the author.
Using team‐based learning (TBL) approach in teaching cardiovascular physiology to first‐year osteopathic medical students
Authors: Ioudina Marina V , Guadagnoli Jutta , Miller Terrence
Published: June 2021

Background

TUNCOM physiology is a lecture based course augmented by small group exercises and class presentations. Group exercises were deemed ineffective: they did not lend themselves to objective student assessment, and did not develop integration and application skills.

Purpose

TBL was implemented to address group exercise shortcomings.

Methods

The cardiovascular (CV) system for the past 2 years consisted of lectures, self‐ and group‐ study of objectives, and a TBL session utilizing traditional tools: individual and group readiness assurance tests (IRAT, GRAT); group application problems (GAP); and peer evaluation. Multiple faculty members developed each TBL tool. The system concluded with a multiple choice exam.

Results

GRAT (88%) and GAP (86%) scores were significantly higher than IRAT (68%), P<0.001. The mean system exam score did not differ from that of the exam given in the year preceding TBL (P= 0.611).

Conclusion

Students learned objectives equally well before and after TBL implementation as evidenced by system exam results, but GRAT and GAP performance indicated that TBL improved integration and application skills. TBL offered objective, measurable outcomes for student assessment.
Differential regulatory response for the Δ9 desaturase in Saccharomyces cerevisiae based on fatty acid species and intracellular amount (1000.3)
Authors: Schriemer Clara , Willey Marshall , Ochs Matthew , Hillers Lauren , McDonough Virginia
Published: June 2021

The OLE1 gene in Saccharomyces cerevisiae encodes the ∆9 desaturase, which inserts a double bond in saturated fatty acids to create unsaturated fatty acids (UFAs). OLE1 expression is controlled in part through the transcriptional regulators Mga2p and Spt23p in response to supply of UFAs. We investigated whether the regulation was uniform in response to different UFAs and at different concentrations. We found that in wild type cells, reporter gene assays show a stronger decrease in expression of OLE1 when fed 16:1∆9 or 18:2∆9, 12 as opposed to 18:1∆9 or 17:1∆10. Concentration of the fed fatty acid also impacted the regulation of OLE1 with higher levels of each UFA impacting expression to a greater degree. Fatty acid profiles of wild type cells show cells accumulate a higher concentration of 16:1∆9 and 18:2∆9, 12 than fed 18:1∆9 or 17:1∆10. This leads to the conclusion that the expression of OLE1 is dependent both on properties of fed fatty acids and the amount in the cell. While our initial hypothesis was that OLE1 is regulated in response to membrane fluidity, subsequent work does not support that idea. We have found that conditions that would affect membrane fluidity (besides UFA species and amount), such as growth temperature and saturated or trans fatty acid supplementation do not regulate OLE1 in the direction predicted by fluidity changes. Recently our lab has isolated a mutant that is deficient in regulation of OLE1, called nro1 (no regulation of OLE1). The signaling mechanism for the NRO1 protein’s action is unknown. Tests using the OLE1 promoter‐reporter gene constructs suggest that Nro1p responds more strongly to the fatty acids 16:1∆9 and 18:2∆9, 12, than 18:1∆9 and 17:1∆10. Characterization of NRO1 is discussed.

Grant Funding Source: Supported by NSF‐REU DBI‐0754293
Palmitate‐induced alterations in phospholipid composition promote ER stress and cellular dysfunction in hepatic lipotoxicity (1001.2)
Authors: Leamy Alexandra , Egnatchik Robert , Shiota Masakazu , Jacobson David , Young Jamey
Published: June 2021

Lipotoxicity induced by saturated fatty acid (SFA) accumulation has been postulated as a contributing factor in the pathogenesis of diseases such as non‐alcoholic fatty liver disease (NAFLD) and type II diabetes. Our central hypothesis is that overexposure to SFAs results in increased saturation of ER membrane phospholipids, which induces ER stress marked by calcium efflux and CHOP overexpression, ultimately culminating in apoptosis. Our objective is to determine how perturbations of pathways involved in lipid metabolism modulate SFA‐induced lipotoxicity, as assessed by measurements of phospholipid (PL) composition, ER stress, reactive oxygen species (ROS) accumulation and apoptotic cell death. Using the calcium chelator BAPTA, we suppressed downstream markers of lipotoxicity such as ROS accumulation and caspase activation, but were unable to prevent ER dysfunction. Interventions designed to reduce PA incorporation into PLs by increasing triglyceride synthesis resulted in elevated levels of lipid accumulation but significantly reversed PA‐induced increases in PL saturation, abnormalities in ER membrane structure and homeostasis, and all other phenotypic markers of hepatic lipotoxicity. Our data indicate that by modulating alternative pathways of hepatic lipid metabolism upstream of PL incorporation, the lipotoxic effects of SFAs can be significantly reduced.

Grant Funding Source: Supported by: National Science Foundation Career Award 0955251
Regulation of CCN2 and CCN3 in bone marrow through myloid zinc finger‐1 and its medical implication in hematopoiesis (1005.4)
Authors: Rozado André , Piszczatowski Richard , Rafferty Brian , Lents Nathan
Published: June 2021

CCN2, also known as CTGF, the connective tissue growth factor, has recently been found to be in high abundance in platelets and released upon activation, an effect inhibited by aspirin, suggesting additional role in blood clotting and/or wound healing. It has been demonstrated that another member of the CCN family, the Nephroblastoma overexpressed gene (NOV, CCN3) functions in hematopoietic potential, blood cell formation, and helps to maintain a competent immune system. Taken together, these two CCN family members represent interesting new areas of research in blood cell biology, with dramatic clinical potential.

Recent research in our lab discovered that the transcription factor MZF‐1 regulates both the CCN2 and CCN3 genes. Specifically, we found that MZF‐1 binds to the promoter of both CTGF (CCN2) and NOV (CCN3). It is known that both vitamin D (calcitriol) and vitamin A (all‐trans retinoic acid) stimulate transcription of the MZF1 gene. We have thus discovered a potential new pathway in which Vitamins A and D induce MZF‐1 to upregulate CCN3 and CCN2 resulting in increased hematopoietic potential and increased differentiation of HSCs and their progeny. It is known that the presence of CTGF in platelets is the result of endocytosis from extracellular environment. However, we have confirmed that treatments with either vitamin A or D activate the MZF‐1 pathway and results in increased production of CCN2 in stromal fibroblasts, which in turn results in enhanced loading of CCN2 into developing platelets in vitro.

Furthermore, the regulation of CCN3 by MZF‐1 represents a testable new clinical treatment. We have observed that both vitamin A and vitamin D induce CCN3 expression, through MZF‐1, in multiple blood cell types. We are currently testing if this will lead to enhanced hematopoietic potential. This work could impact the efficacy of blood donation and transfusion, bone marrow transplants, and the treatment of bleeding and clotting diseases as well as lymphomas and leukemias.
Pulsed EPR distance measurements resolve the impact of site‐specific calmodulin methionine oxidation (1009.4)
Authors: Klein Jennifer , McCarthy Megan , Reuter Mitch , Olenek Michael , Moen Rebecca , Thomas David
Published: June 2021

We have examined the structural impact of oxidizing specific protein methionine (Met) residues in the C‐ter lobe of calmodulin (CaM); these oxidation sites are known to abolish CaM regulation of the major calcium release channel, the ryanodine receptor complex (RyR). We have linked oxidation‐induced changes in RyR regulation to changes in CaM‐RyR structure using (1) protein mutagenesis to mimic oxidation at specific sites and (2) spectroscopy to resolve oxidation‐induced changes in protein structural dynamics.

Protein oxidation by reactive oxygen species (ROS) is strongly associated with loss of strength in skeletal muscle and is proposed to play major roles in aging and degenerative muscle disease. Targeted interventions that block or repair specific oxidative damage, while supporting normal levels of oxidation, could decrease morbidity associated with muscle dysfunction_this level of discrimination demands molecular‐level insights into the mechanisms that underlie the decline in muscle force associated with oxidative stress.

Pulsed EPR distance measurements across CaM’s lobes were sensitive to large‐scale conformational changes that accompany both calcium binding and RyR peptide binding. In the absence of calcium, CaM was highly disordered, populating a broad distribution of conformations. Calcium binding strongly stabilized an elongated conformation, while RyR peptide binding to calcium‐loaded CaM strongly stabilized the compact conformation. CaM conformation, particularly the distribution over structural states, was sensitive to Met to Gln substitutions (M109Q and M124Q) designed to mimic CaM Met oxidation. Structural sensitivity to M‐to‐Q mutations was observed in both the presence and absence of calcium, and in complex with RyR peptide. We conclude that Met oxidation alters CaM’s functional interaction with RyR through changes in the orientation and flexibility of CaM’s lobes.

Grant Funding Source: This work is supported by a University of Wisconsin‐La Crosse Faculty Research Grant to JC Klein, NI
Leucyl‐tRNA synthetase is an amino acid sensor for the activation of Vps34 upstream of mTORC1 (1012.5)
Authors: Yoon Mee Sup , Chen Jie
Published: June 2021

Amino acids availability regulates the mammalian target of rapamycin (mTOR) complex, mTORC1, a master regulator of cell growth. We previously reported that a pathway consisting of the class III PI‐3‐kinase Vps34 and the phospholipase PLD1 mediates amino acid activation of mTORC1, via PLD1 activation and translocation to the lysosome. The mechanism by which the Vps34‐PLD1 pathway senses amino acids is not known. In the current study, we identify leucyl‐tRNA synthetase (LRS) as the amino acid sensor for the activation of Vps34‐PLD1. LRS knockdown impaired amino acid‐induced Vps34 activation, cellular PI3P level increase, PLD1 activation, and PLD1 lysosomal translocation. Conversely, overexpression of LRS enhances activation of Vps34 and PLD by amino acids. Results with LRS mutants suggest that this function of LRS is dependent on its ability to bind leucine but independent of its tRNA charging activity. In addition, we find that LRS physically interacts with Vps34 through Vps15 and this interaction may be directly responsible for the activation of Vps34 in response to amino acids. Taken together, these observations strongly suggest that LRS mediates amino acid activation of Vps34‐PLD1 upstream of mTORC1. Our findings reveal a new regulator for Vps34 and fill a previous gap in the amino acid‐sensing signaling network.

Grant Funding Source: NIH GM089771 and AR048914
NAADP evokes calcium signals and plays a critical role in downstream physiological responses in naïve T cells (1013.5)
Authors: Ali Ramadan , Camick Christina , Wiles Katherine , Slama James , Giovannucci David , Wall Katherine
Published: June 2021

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing second messenger discovered to date and has been implicated in Ca2+ signaling in human Jurkat lymphoma T cells via ryanodine receptor activation. In contrast the role of NAADP in Ca2+ signaling or the identity of the Ca2+ stores targeted by NAADP in conventional naïve T cells has not been investigated. In the current study, we demonstrated for the first time to our knowledge the importance of NAADP in the generation of Ca2+ signals in murine conventional naïve T cells. Combining live‐cell imaging methods and a pharmacological approach using the NAADP antagonist Ned‐19, we addressed the impact of NAADP on the generation of Ca2+ signals evoked by TCR stimulation and the role of this signal in downstream physiological endpoints such as proliferation, cytokine production, and other responses to stimulation of murine naïve T cells. Moreover, we demonstrated that acidic stores in addition to the endoplasmic reticulum are the relevant Ca2+ stores that were sensitive to NAADP in naïve T cells.

Grant Funding Source: Supported by NIH GM100444
Microarray analysis of the effects of elevated cAMP in differentiating osteoblasts (1013.6)
Authors: Ghanayem Kathryn , Cuppini Candice , Rylaarsdam Robin
Published: June 2021

McCune‐Albright Syndrome (MAS) is an acquired genetic disorder caused by a mutation in Gs alpha at Arg201 that inhibits GTP hydrolysis, constitutively activating the protein and resulting in elevated levels of basal cAMP in the affected cells. A major pathological feature of MAS is polyostotic fibrous dysplasia of bone (FD), a finding for which very little effective therapy exists. In an effort to identify novel potential drug targets to treat FD, we utilized a human fetal osteoblast cell line, hFOB1.19 (ATCC) that proliferates at 33C and differentiates into mature osteoblasts at 39C. We treated cells with the fungal metabolite forskolin at its EC50 concentration for elevating cAMP in these cells, and then either maintained the cells at 33C or shifted them to the differentiation conditions for 24 hours. Total RNA was collected and analyzed for levels of gene expression using an Illumina HT12 bead chip. Pairwise analysis of changes in gene expression identified 370 genes that were aberrantly expressed during differentiation in the presence of elevated cAMP. Twenty‐eight genes with known functions in osteoblast biology and/or differentiation were further analyzed by RT‐PCR to confirm the expression changes seen in the microarray assay.

Grant Funding Source: Supported by NIH grant 1R15ED020190‐01
Cultural capital associated with children’s food consumption and physical activity among parents of first and second grade children in urban, suburban, and rural Japan (1014.1)
Authors: Goto Keiko , Fukushima Naho , Wolff Cindy , Giovanni Maria , Murayama Nobuko
Published: June 2021

This study examined children’s food consumption and physical activity and their parents’ cultural capital levels in three Japanese communities. A total of 729 parents of first and second grade children enrolled in nine elementary schools in Chiba (urban), Niigata (suburban), and Miyazaki (rural) participated in a survey. Factor analysis resulted in three cultural capital variables: preservation of traditional Japanese food culture, parents’ aversion to Western foods, and parents’ openness to foods from other cultural groups. Correlations and ANOVA were used for statistical analysis. Children in Chiba consumed significantly higher amounts of store bought vegetables, fruits, and Western franchise fast foods compared to children in Miyazaki. Consumption of local vegetables was the highest in Miyazaki. Preservation of traditional Japanese food culture was associated with a higher level of physical activity among children. Western food aversion was associated with consumption of fresh vegetables and local/seasonal foods. Openness to trying foods from other cultures was associated with consumption of sweetened beverages. Our findings underline the significance of identifying cultural capitals for child health. These may ultimately contribute to developing policies and programs for healthier feeding practices in countries that are undergoing nutrition transition.

Grant Funding Source: CSU Research grant
Global trends in breastfeeding duration (1015.1)
Authors: Kay Melissa , Bentley Margaret , Adair Linda
Published: June 2021

Breastfeeding is an important determinant of child survival and strategy for improving child health in developing countries. The World Health Organization recommends exclusive breastfeeding up to 6 months, with continued breastfeeding along with appropriate complementary foods up to 2 years or beyond.

We analyzed nationally representative Demographic and Health Survey (DHS) data from 21 countries between 1990 and 2011. Countries had at least 2 years of data, including one from 2008 or later for a total of 83 surveys. Our sample includes singleton, last‐born infants 0‐36 months living with their mother (n=185,469). We compared median breastfeeding duration calculated from current status data.

Trends within countries did not exhibit meaningful patterns. Median duration changed by <1 month in 43% of intervals. Significant differences are seen between countries; 14% of country‐years had medians 2 years.

Despite progress promoting awareness of breastfeeding, adherence to recommendations is not common. Analyzing DHS data is a unique opportunity to assess trends and explore noncompliance. These observations can be used to guide promotion efforts. Future studies should exam the influence of social, economic and demographic characteristics on breastfeeding duration.

Grant Funding Source: Supported by an educational grant from Wyeth Nutrition
Adults’ attitudes toward and purchasing intentions for child‐friendly shaped healthy fruit and vegetable snacks (1019.23)
Authors: Baker Selena , McCabe Sean , Swithers Susan , Payne Collin , Kranz Sibylle
Published: June 2021

Fruit and vegetable intake in children falls short of meeting national recommendations. Children’s liking of fruits and vegetables (FV) and availability and accessibility are important determinants of intake. We hypothesized that parents/caregivers would recognize fun‐shaped FV as being child‐friendly (CF) and would be willing to pay slightly more for CF‐shaped FV. A convenience sample of adults was recruited for a consumer survey and optional taste‐test of regular‐shaped (chunks/slices) or CF‐shaped (butterfly, chick, flower, teddy bear) FV. Participants (n=298) were 34% more likely to select CF vs. regular‐shaped FV for taste‐test (p=0.033). The majority of adults (68 ‐ 96.3%) reported high sensory ratings (蠅6 on a 7‐point scale) for both CF‐ and regular‐shaped FV, and 100% of parents/caregivers recognized the shaped FV as CF. Only 33.3% who tasted the CF FV reported high willingness to “pay a little extra” for the product. Therefore, while participants recognized the shaped FV as child‐friendly and were more likely to taste‐test them, product price appears to play an important role in adults' decision to purchase FV, impacting availability of FV for child consumption.
Consumption of low‐fat dairy foods for 6 months improves insulin resistance without adversely affecting lipids or bodyweight in healthy adults (1025.2)
Authors: Rideout Todd , Marinangeli Christopher , Rempel Curtis
Published: June 2021

Given the highly debated role of dairy food consumption in modulating biomarkers of metabolic syndrome, this study was conducted to examine the influence of long‐term (6 month) dairy consumption on metabolic parameters in healthy volunteers under free‐living conditions without energy restriction. Twenty‐three healthy subjects completed a randomized, crossover trial of 12 months. Participants consumed their habitual diets and were randomly assigned to one of two treatment groups: a high dairy supplemented group instructed to consume 4 servings of dairy per day (HD); or a low dairy supplemented group limited to no more than 2 servings of dairy per day (LD). Baseline, midpoint, and endpoint metabolic responses were examined. Endpoint measurements of body weight and composition, energy expenditure, blood pressure, blood glucose, and blood lipid and lipoprotein responses did not differ (p>0.05) between the LD and HD groups. HD consumption improved (p<0.05) plasma insulin (‐9%) and insulin resistance (‐11%, p=0.03) as estimated by HOMA‐IR compared with the LD group. Study results suggest that high dairy consumption (4 servings/d) may improve insulin resistance without negatively impacting bodyweight or lipid status under free‐living conditions.

Grant Funding Source: Supported by the Government of Manitoba Department of Industry, Energy and Mines
Soda consumption and the risk of hip fractures in postmenopausal women (1027.6)
Authors: Fung Teresa , Arasaratnam Meredith , Grodstein Francine , Katz Jeffrey , Rosner Bernard , Willett Walter , Feskanich Diane
Published: June 2021

Background: The frequency of soda consumption remains high in the U.S. Soda consumption has been associated with poor bone health in children, but few studies have examined this relationship among adults, and none have examined the relation of soda consumption to risk of hip fractures.

Objective: We examined the association of soda, including specific types of soda, and risk of hip fracture in postmenopausal women.

Design: Analysis was conducted using data from the ongoing Nurses’ Health Study cohort. Diet was assessed at baseline using a semi‐quantitative food frequency questionnaire, and updated approximately every four years. In up to 30 years of follow‐up, we identified 1,968 incident hip fractures in postmenopausal women (N=76,123). We computed relative risks (RR) for hip fracture by amount of soda consumption using Cox proportional hazards models, adjusting for potential confounders.

Results: In multivariable models, each additional serving of total soda per day was associated with a significant 14% increased risk of hip fracture (RR=1.14, 95% confidence interval (CI) 1.07‐1.23). Risk was significantly elevated among consumers of both regular (RR=1.19, 95% CI= 1.03‐1.38) and diet soda (RR=1.12, 95% CI= 1.04‐1.21) and also did not significantly differ between colas and non‐colas or sodas with or without caffeine. Association between soda and hip fractures did not differ by body mass index or by diagnosis of diabetes.

Conclusion: Increasing soda consumption of all types may be associated with increasing risk of hip fracture in postmenopausal women.

Grant Funding Source: Supported by: NIAMS T32 AR 055885; NIAMS P60 AR 47782, NIH CA87969
Expression of insulin‐like growth factor family members in myostatin null mice (1032.3)
Authors: Clark Daniel , Clark Diana , Hogan Elizabeth , Kroscher Kellie , Dilger Anna
Published: June 2021

Myostatin inhibits while insulin‐like growth factors (IGF) 1 and 2 increase pre‐ and postnatal skeletal muscle growth. However, the contribution of altered IGF expression to the hypermuscular phenotype of myostatin null (MN) animals is unclear. Wild type (WT) and MN mice were euthanized at approximately 14 days of gestation (‐7d), birth (0d), 7 days (7d), weaning (21d), sexual maturity (42d), and mature size (70d). From fetal mice, RNA was extracted from the entire body. For the neonatal periods, 0d and 7d, all muscles from the hind limbs were compiled for RNA extraction. At 21d, 42d, and 70d, biceps femoris, tibialis anterior, triceps brachii, and gastrocnemious‐soleus complex were collected. As expected, muscle weights increased up to 90% in MN mice compared with WT mice at 21d, 42d and 70d. However, IGF1 expression tended to be decreased by 25 ‐ 50% (P < 0.10) in MN mice compared to WT mice. Additionally, IGF2 expression in MN fetuses tended to be decreased by 30% (P < 0.10) in MN mice compared to WT mice; however IGF2 expression did not differ between genotypes at 0d and 7d. At 21d in biceps femoris muscles, IGF2 expression was increased more than 2 fold (P < 0.01) in MN compared to WT mice. Similarly, at 42d and 70d in biceps femoris muscle, IGF2 expression was increased over 4 fold (P < 0.01) in MN compared to WT mice. Therefore, increased postnatal IGF2 expression may contribute to increased muscle hypertrophy observed in MN mice.
Interaction of the protease resistant proteins with the intestinal mucosa membrane proteins (1036.3)
Authors: Yum Youjin , Lee Juyoung , Lim Jinkyu
Published: June 2021

Many studies have shown that some substances in soy, such as digested peptides or isoflavones from soy have effects on satiety control and nutrient absorption functions. However, it is still clearly not known how soy can control those functions. We have shown that some of the soluble soy proteins were resistant to proteases, such as pepsin and pancreatine, in the in vitro digestion analysis. We named these proteins as PRFPs (protease‐resistant food proteins), and postulated that PRFPs can interact with the mucosa membrane on the villi or microvilli in the small intestine to trigger the biological activities mentioned above. The interactor proteins on the intestinal membrane and soy proteins were identified by crosslinking and mass spectrometric analysis. The identified intestinal membrane proteins were mostly channel proteins and galectins. The soy partners to the membrane proteins were found to be glycinin and beta‐conglycinin, which are the majority proteins of soy. Phosphoproteomic changes in the intestinal cell line upon soy protein treatment were tested. After the soy protein treatment, phosphoproteins enriched from the cell extract by IMAC column were separated on 2D gels and compared for the up and down regulations. Interestingly, many of the proteins involved in protein folding or energy metabolism were significantly phosphorylated by soy protein treatment to the cell line.

For further studies, detailed cell signaling pathway/mechanism by soy PRFPs treatment to the cells are under investigation in view of revealing the function of soy proteins in satiety, nutrient absorption, and others.
γ‐Conglutin, the lupin seed glucose‐lowering glycoprotein, interferes on insulin pathway in Hep G2 cells (1045.16)
Authors: Parolari Anna , Manzoni Cristina , Faoro Franco , Duranti Marcello , Lovati Maria
Published: June 2021

Gamma‐conglutin (γ‐C), a Lupinus albus, seed glycoprotein, has been shown to lower blood glucose in hyperglycaemic rats and increase glucose consumption in HepG2 cells. Aim of the present work was to assess the modalities of interaction of this protein with the target cells, so as to contribute unveiling its mechanism of action. γ‐Conglutin internalization: HepG2 cells, pretreated for 30’ w/wo inhibitors of caveolae mediated endocytosis [filipin (5 μg/ml) and genistein (200 μM)], clathrin‐mediated pathway [chlorpromazine (25 μM), methyl‐β‐cyclodextrin (5 mM)], and macropinocytosis [amiloride (5 mM)], were incubated with FICT‐γ‐conglutin (50 μg/ml) for 4 h at 37°C, followed by FACS analysis. The macropinocitosis pathway was the mechanism of γ‐conglutin internalization (69% inhibition). 2D‐electrophoresis of cell lysates and antibody reaction of the blotted maps showed that γ‐conglutin is internalized in intact form. Moreover, the protein is modified inside the cell by multiple phosphorylation. Insulin pathway: reduced effect on AMPK activity (‐50% vs controls) and increased expression of phosphorylated AKT form (+ 25% vs controls) were recorded in HepG2 cells, following western‐blot analysis, exposed to 10 mM γ‐conglutin and/or 100 nM insulin, or metformin 10 mM.

Grant Funding Source: MIUR of Italy
cDNA and lincRNA microarrays: identification of expression patterns related to metastatization in oral squamous cell carcinoma (1048.6)
Authors: Oliveira‐Costa Joao Paulo , Gigliola Mabel , Macedo Mariana , Carraro Dirce , Soares Fernando
Published: June 2021

Oral squamous cell carcinoma (OSCC) metastatic cascade still a poorly understood mechanism, in terms of gene expression differences between primary site and metastatic cells at lymph nodes. Using 20 matched samples of OSCC cases, being 10 from primary sites and 10 metastastic lymph nodes we performed lincRNA and cDNA microarrays analysis (Agilent`s Sureprint G3 Human Genome V2.0). Our results showed 617 genes and lincRNAs significantly differentially expressed between metastatic lymph nodes and primary tumors. Integrated pathway analysis showed alteration in canonical pathways such as Ras homolog gene family, member A signaling pathway (RhoA pathway; p=0.002) and tumor necrosis factor receptor 2 signaling pathway (TNFR2 pathway; p=0.009). The most up‐regulated genes included SNORA65 (9,045‐fold), ZMAT1 (4,776‐fold) HNRNPU‐AS1 (4,626‐fold), RNU12 (4,557‐fold), TLE4 (4,555‐fold), and the most down‐regulated included MYBPH (‐67,818‐fold), ACTA1 (‐58,551‐fold), TNNC1 (‐23,119‐fold), F2RL2 (‐17,431‐fold) and MYLPF (‐13,785‐fold). Regarding lincRNAs, MALAT1 was significantly overexpressed in metastatic lymph nodes, with a 4.2‐fold increase. Our study shows the existence of several genes and lincRNAs with different expression patterns between OSCC primary tumor and metastatic lymph nodes, and also points to novel genes, which has not yet been implicated in OSCC metastasis, providing new potential targets in oral cancer research.

Grant Funding Source: Supported by FAPESP grants 2011/18606‐7‐0 and 2010/08400‐0
Cooperative gating and sensitivity of TRPV4 channels are regulated by distinct factors (1057.8)
Authors: Sonkusare Swapnil , Villalba Nuria , Freeman Kalev , Bonev Adrian , Nelson Mark
Published: June 2021

TRPV4 (transient receptor vanilloid 4) sparklets represent Ca2+ influx through single TRPV4 channels (Sonkusare et.al., Science, 2012). In this study, we determined the differences in endothelial cell (EC) TRPV4 channel activity in mouse mesenteric (MA) and basilar (BA) arteries by recording TRPV4 sparklet activity in slit open, en face preparation, under physiological conditions of temperature and ionic solutions. TRPV4 channels in MAs and BAs exhibited a number of similarities, including a 4‐channel meta‐structure, the same quantal levels (0.19 ΔF/F0), and cooperative channel gating at myoendothelial projections (MEPs). However, an analysis of TRPV4 sparklets indicated that the channel open probability (Po) in response to TRPV4 agonist GSK1016790A (GSK101, 100 nM) is ~ 15‐fold lower in BAs than MAs. In agreement with the Ca2+ imaging results, the sensitivity of TRPV4 channels to the agonist GSK101, measured as currents in freshly isolated cells is shifted by ~10‐fold in cerebral arteries. The TRPV4 channel gating at MEPs was cooperative in both MAs and BAs, as indicated by similar coupling coefficients (~0.2). In MAs, the cooperative channel gating at the MEPs was dependent on a kinase anchoring protein 150 (AKAP150). Notably, AKAP150 exhibited similar clustering at MEPs in MAs and BAs. These results suggest that cooperativity and agonist sensitivity of TRPV4 channels are functionally separable.

Grant Funding Source: HL044455, 1P01HL095488, R37DK053832, R01HL098243 to MTN
Self‐assessment of research competencies during a summer undergraduate research fellowship in pharmacology and toxicology (1058.3)
Authors: Aleksunes Lauren , Caswell Erin , Gutierrez Jorge , Liang Laura , Laskin Debra
Published: June 2021

Rutgers University has developed a summer research fellowship program to promote pharmacology, toxicology and environmental health sciences as careers in biomedical research. The program consists of a 10‐week basic science and translational research experience for undergraduates and was also designed to include weekly events including laboratory safety and responsible conduct of research training, a field trip to a pharmaceutical company, career development and research seminars and student presentations. To assess the improvement of participants in key areas, students completed an adapted version of the Undergraduate Research Student Self‐Assessment survey from the University of Colorado at the beginning and the end of the 10‐week fellowship. The response rates for the pre‐ and post‐program surveys were 100% and 71%, respectively. The survey captured changes in three key areas: Application of Knowledge to Research (data analysis, problem solving), Scientific Abilities (daily independent and group activities), and Research Abilities (laboratory and presentation activities). A 7‐point scale was used by participants to assess their level of confidence on 27 competencies. Significant improvement was observed in 15 of 27 competencies (55%). The greatest advancements were made in the ability of students to identify experimental limitations (31%), to determine the next step in a research project (26%) and to understand the connections between scientific disciplines (25%). A summer research program engages undergraduate students in full‐time research experiences and provides unique opportunities to promote pharmacology and toxicology as research areas for the next generation of scientists and enhances career development skills. Supported by ES020721, ES005022, and ASPET SURF.
G protein‐coupled estrogen receptor mediates relaxation of coronary arteries via cAMP/PKA‐dependent activation of MLCP (1065.14)
Authors: Yu Xuan , Stallone John , Han Guichun
Published: June 2021

Activation of GPER exerts a protective effect in hypertension and ischemia‐reperfusion models and relaxes arteries in vitro. However, the mechanism of GPER‐mediated vascular regulation is far from complete. The objective of this study is to test the hypothesis that GPER induced porcine coronary arteries relaxation is via cAMP/PKA signaling. We used isometric tension study, Western blotting and kinase activity essays. Our findings revealed that coronary artery relaxation to GPER agonist G‐1 (0.3‐3 μM) was associated with increased cAMP production in a concentration‐dependent manner. Furthermore, inhibition of adenylyl cyclase with SQ‐22538 (100 μM) or inhibition of PKA with either Rp‐8‐CPT‐cAMPS (5 μM) or PKI (5 μM) attenuated G‐1‐induced relaxation of coronary arteries preconstricted with PGF2α (1 µM). G‐1 also increased PKA activity in coronary artery smooth muscle cells (CASMC). To determine downstream signals of cAMP/PKA cascade, we measured RhoA activity in CASMC and myosin light chain phosphatase (MLCP) activity in artery rings by detecting phosphorylation of its regulatory myosin‐targeting subunit (pMYPT1). G‐1 decreased PGF2α induced pMYPT1, while Rp‐8‐CPT‐cAMPS prevented this inhibition of G‐1. RhoA activity was down regulated by G‐1, whereas GPER antagonist G36 restored it. Lastly, an inhibitor of the interaction between PKA and A kinase anchoring protein, MFMP‐API‐1 (100 µM), attenuated G‐1‐mediated relaxation and pMYPT1 response. We conclude that localized cAMP/PKA signaling is involved in GPER‐mediated coronary vasodilation by activating MLCP via inhibition of RhoA pathway.
Palmitoylation is required for activated PAR1 ubiquitination and p38 MAPK signaling (1066.16)
Authors: Canto Isabel , Grimsey Neil , Trejo JoAnn
Published: June 2021

Palmitoylation has diverse functions in the regulation of G‐protein coupled receptor (GPCR) signaling and trafficking, yet its function in signal regulation of the protease‐activated receptor‐1 (PAR1), a GPCR for thrombin, is not known. PAR1 is a unique GPCR that couples to multiple G‐protein subtypes, but the mechanisms that regulate G‐protein selectivity are not well understood. We have previously established that PAR1 is basally palmitoylated, and regulates adaptor protein complex‐2 and ‐3 accessibility of tyrosine‐based sorting motifs within the C‐tail domain, but whether palmitoylation affects activated PAR1 G protein signaling is not known. We utilized a palmitoylation‐deficient mutant of PAR1 in which C‐tail cysteines (C387 and C388) were converted to alanines (A), to examine the role of palmitoylation on PAR1 signal regulation. In endothelial cells, palmitoylation is not critical for PAR1 recruitment to caveolar microdomains. Although the PAR1 CC/AA mutant is capable of signaling to Gq in HeLa cells, it is defective in thrombin‐induced p38 MAPK signaling. However, PAR1 CC/AA retains normal ERK1/2 signaling. Additionally, PAR1 CC/AA is defective in agonist‐induced ubiquitination, a modification that is critical for p38 MAPK signaling. Thus, palmitoylation is likely to stabilize the proper PAR1 C‐tail conformation important for regulation of receptor signaling.
Analysis of functional selectivity at the nociceptin opioid receptor (1066.2)
Authors: Chang Steven , Spangler Skylar , Zhang Nancy , Planer William , Carroll F , Bruchas Michael
Published: June 2021

The Nociceptin/Orphanin FQ Opioid Receptor (NOPR) is the most recently discovered and least characterized G‐protein coupled receptor in the opioid receptor family. The activity of NOPR has been thought to play a role in neuromodulation in the contexts of pain, opioid tolerance, and the modulation of stress and anxiety behaviors. It is thought that the NOP receptor may bind functionally selective ligands which can bias signal transduction, resulting in multiple behavioral manifestations. Functional selectivity at the NOPR has yet to be explored, but this information is integral to understanding the complex modulation of behaviors. To study the functional selectivity of NOPR, we used live‐cell cAMP assays, BRET, as well as MAP kinase activity assays to study the Gα‐protein mediated and arrestin‐mediated signaling profiles. We screened multiple known NOPR ligands and novel, small molecules aimed at elucidating structural significance. We have found that NOPR does exhibit different signaling profiles, depending on the interacting ligand. Specifically, we show that minute changes in the structure can illicit opposing signaling profiles ranging from agonism to inverse agonism. Additionally, we propose ligand‐receptor docking configurations that may help elucidate the conformational influence on functional selectivity. Additionally, we screened the same ligands in an internalization and arrestin‐recruitment deficient NOPR point mutant (Zhang et al., 2012). Interestingly, the mutation of the Serine 363 phosphorylation site shows altered signaling profiles for the same ligands in comparison to the wild‐type receptor. In addition, the signaling profiles of the novel NOPR ligands may provide new insight into the structural relationship between ligands and biased signal transduction. Taken together, these data provide novel insight into the functional selectivity of the NOP receptor, and how it affects behavioral manifestation. Supported by: NIH R00 DA025182 and T32 DA007261.

Grant Funding Source: T32 DA007261
GPCRs and heterotrimeric G proteins directly regulate membrane type‐1 matrix metalloprotease (1066.6)
Authors: Overland Aaron , Insel Paul
Published: June 2021

Matrix metalloproteases (MMPs) mediate the transactivation of epidermal growth factor receptor (EGFR)‐dependent signaling pathways upon stimulation of GPCRs, but mechanisms of GPCR‐stimulated EGFR transactivation are poorly understood. A direct, membrane‐delimited mechanism has not previously been investigated. Several MMPs localize to the plasma membrane, including membrane type‐1 matrix metalloprotease (MT1‐MMP, also known as MMP14). We hypothesized that heterotrimeric G proteins directly activate MT1‐MMP, initiating transactivation of EGFRs via release of heparin‐binding EGF (HB‐EGF). We found that MT1‐MMP is activated by GPCR agonists, GTP, or GTPγS in a concentration and time‐dependent manner in both fibroblast and cardiac myocyte membranes. MT1‐MMP activation does not occur with GMP or ATPγS and is not blunted by Src, PKC or PLC inhibitors. This activation is specific to MT1‐MMP, as shown using siRNA knockdown and a highly selective MT1‐MMP peptide inhibitor, and results in release of HB‐EGF and activation of EGFR. We found a role for G protein βγ subunits, based on use of the selective βγ inhibitor, gallein. MT1‐MMP is thus a previously unrecognized direct GPCR/G protein membrane effector that can produce EGFR transactivation. Further understanding of these mechanisms may define roles for this membrane‐delimited signaling module in pathophysiology and as a therapeutic target.
Calcium/calmodulin‐dependent protein kinase II modulates inositol‐3‐phosphate receptors in vascular endothelium from mouse mesenteric arteries (1075.1)
Authors: Toussaint Fanny , Charbel Chimène , Blanchette Alexandre , Comtois Philippe , Ledoux Jonathan
Published: June 2021

Endothelial Ca2+ pulsar consists in a spontaneous Ca2+ release from IP3R localized within myoendothelial projections (MEP). We recently showed CaMKII activation by Ca2+ pulsars, thus modulating endothelial function through NO production. Moreover, CaMKII is a known regulator of intracellular Ca2+ homeostasis in several cell types. Our hypothesis was that Ca2+ pulsar‐activated CaMKII modulates endothelial intracellular Ca2+ dynamics. Our investigation showed that preincubation with KN‐93 (10 µM), CaMKII inhibitor, significantly altered endoplasmic reticulum (ER) Ca2+ content (≍ ‐60%). A similar KN‐93‐induced alteration of ER Ca2+ levels (≍ ‐55%) was observed while SERCA pumps were inhibited (Thapsigargin; 1 µM). However, inhibition of IP3R (2APB; 100 µM) precluded KN‐93 effect on ER Ca2+ content. Colocalization (<40nm) of CaMKII and IP3R was observed by PLA in situ hybridization. In addition, an isoform‐specific distribution within MEP (IP3R2>IP3R1>IP3R3) has been found by in situ immunofluorescence. Finally, KN‐93 induced recruitment of new Ca2+ pulsars sites (1.8‐Fold). These results suggest that CaMKII modulates intracellular Ca2+ dynamics through modulation of IP3R activity. Thereby, CaMKII is involved in a negative feedback loop as an endothelial calcium‐sensor switch regulating Ca2+ homeostasis.

Grant Funding Source: Supported by FRQS, FICM, CFI, SQHA and HSFC