bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired coronary artery vasodilation during skin surface cooling in healthy older adults
Authors: Gao Zhaohui , Wilson Thad E. , Hess Kari L. , Monahan Kevin D.
Published: June 2021

Cardiovascular‐related mortality increases in the cold winter months, particularly in older adults. To determine whether coronary artery blood flow responses to skin surface cooling differ with age, we studied 9 young (25±1 yrs) and 10 older healthy adults (65±2 yrs). We hypothesized that left anterior descending coronary artery blood velocity (CBV) responses, measured using Doppler transthoracic echocardiography, to skin surface cooling (15–18° C water perfused suit for 20 min) would be impaired with age. We found that cooling increased left ventricular wall stress, a determinant of myocardial oxygen consumption, in young (185.6±6.5 to 202.9 ±10.4 mmHg, P < 0.05) and older adults (189.1±8.8 vs. 222.7±14.7 mmHg, P < 0.01) although the magnitude of this response was twofold greater in older adults (Δ9.13±3.48% vs. Δ17.62±3.24%, young and older, respectively; P=0.09). Despite increased left ventricular wall stress during cooling in young and older adults CBV increased only in the young (20.86±1.32 to 25.56±1.24 cm/s, p<0.01) and not older group (17.60±1.09 to 19.93 ±0.97 cm/s, p=0.11). These data suggest that an impaired coronary vasodilator response to cold stress may contribute to age‐dependent increases in cardiovascular‐related mortality during the winter months in healthy adults.

Support: NIH HL92309, AG24420 and M01 RR10732
Effect of heat stress and ergopeptine alkaloids on the immune system of rats
Authors: Kishore Deepan , Eichen Peggy Ann , Waldron Matthew R , Evans Tim J , Fritsche Kevin L , Spiers Donald E
Published: June 2021

Rats were fed diets containing alkaloids (E+), minus alkaloids (E−), or pair‐fed (PF to E+). Groups were further divided into temperature treatments of thermoneutrality (TN; 21°C) or heat stress (HS; 33°C). Rats received diet treatments at TN for 1 week followed by 3 days HS or TN treatment. Feed intake and body weight were reduced by alkaloids, heat stress, and combination of both stressors. Feed intake was reduced 82% from preheat level in E+HS rats compared to 75% in HS rats. Blood samples were collected at trial end and analyzed by flow cytometry. Natural killer (NK) cells increased with all stressors, such that E+HS level was twice that of E‐TN rats (4.2% vs. 2.1%; α=0.05). Caloric restriction (HS and TN) modestly increased NK cells. The combination of stressors dimished B cells by one‐half (α=0.05) compared to E‐TN. Treatment differences in T cell percentage, however, were not statistically significant. In summary, combination of alkaloid intake and heat stress significantly affected the proportion of circulating NK and B cells, with more modest changes in T cells. Caloric restriction (PF) increased NK cells, regardless of environmental conditions. (USDA Agreement No. 58‐6227‐3‐016)
Effects of XS® Energy Drink on aerobic exercise capacity
Authors: Sheehan Kevin Michael , Hartzler Lynn K
Published: June 2021

This project examines effects of taking XS® Energy Drink for the purpose of enhancing aerobic exercise capacity and performance. XS® Energy Drink contains B vitamins, caffeine, taurine, and an adaptogen blend and is primarily designed and marketed to stimulate alertness; however, it is also marketed to athletes claiming to enhance performance. Our hypothesis is that consuming the XS® Energy Drink before exercise increases aerobic capacity and the time to muscle fatigue when compared with a placebo. We also hypothesize that consuming XS® Energy Drink before exercise decreases recovery time. Twelve physically fit 18–24 year olds performed two VO2 max tests according to a modified Ellestad Treadmill Protocol using a randomized double‐blind cross over method. We found no significant differences with XS® Energy Drink: VO2 max (p = 0.99), time until muscle fatigue (p = 0.48), maximum heart rate (p = 0.66), VE max (p = 0.10), time at which R > 1 (p = 0.50), or recovery time to 50% VO2 max (p = 0.67). We found the second trial to be significantly longer than the first (p = 0.01) likely due to desire to improve exercise time or familiarization with testing procedures. Our results show no physiological effects of XS® Energy Drink; however, we have not eliminated a possibility of psychological advantage. Supported by an undergraduate research fellowship and University Honors Program grant from Wright State University (KMS).
Class I and II anabolic steroids produce opposite hedonic and rewarding effects in pubertal and adult mice
Authors: Brito Paul , Huertas Adhly , Villafañe Blanca , Ramos Keyla , Rosa Dariana , Barreto Jennifer
Published: June 2021

Anabolic androgenic steroids (AAS) are synthetic derivates of testosterone. There are approximately 60 different AAS compounds that can be classified in three classes based upon their chemical structure and metabolites. Even short exposure to AAS can produce mood and behavioral symptoms. In previous experiments, we found that class I and II AAS induce hedonic and rewarding effects in adult mice. Recently, NIDA reported that 1.4 – 2.2% of adolescents ever tried steroids. In this study, we aimed to determine if AAS have hedonic properties in adolescent male mice. Animals received alternate androgen injections in a conditioned place preference (CPP) for 10 days. In addition, anxiety behaviors were measured. Androgens tested were testosterone propionate (TP) and 17α‐methyltestosterone (17α‐meT), class I and class III AAS, respectively. Three doses were tested (0.075, 0.75 and 7.5 mg/kg) for each drug. We have found a shift in place preference in animals treated with 17α‐meT in all doses tested, while TP showed no effect. In exploratory‐based anxiety, using light‐dark transitions, we found an increase in this parameter only with 17α‐meT (7.5 mg/kg). Body and gonadal weight were not affected in any of the AAS treatments. Our results suggest that developmental age, hormonal environment and AAS metabolism are important modulators of hedonia and reward.
α1‐Adrenergic receptor (α1‐AR) subtypes: Involvement in regulation of supraoptic nucleus (SON) vasopressin (VP) and oxytocin (OT) neurons
Authors: Sladek Celia D. , Stevens Wanida , Song Zhilin
Published: June 2021

Noradrenergic pathways (that co‐release ATP) transmit cardiovascular and suckling information to stimulate VP and OT secretion. Both ATP and phenylephrine (PE, α1‐AR agonist) increase intracellular calcium ([Ca++]i) in SON neurons and induce transient VP/OT release from explants of the hypothalamo‐neurohypophyseal sysem (HNS). Simultaneous exposure to ATP+PE induces a synergistic increase in VP/OT release that is sustained for hours. SON neurons express several α1AR subtypes (α1A, α1B, and α1D). Antagonists of these subtypes were used to evaluate their respective roles in inducing Ca++ and VP/OT responses to PE and ATP+PE. Live cell Ca++ imaging studies were performed in Fura‐2 loaded HNS explants. VP/OT release from perifused HNS explants was measured using radioimmunoassay. Antagonists of α1A‐ARs [WB4101 (100 μM) and 5‐methylurapidil (1 μM)] prevented PE‐induced increases in [Ca++]i, and attenuated the response to ATP+PE. Neither α1B‐AR (AH11110A, 100 nM) nor α1D‐AR antagonists (BMY7378, 10 nM) affected the Ca++ response to PE. However, neither WB4101 nor AH11110A altered VP/OT release induced by exposure to ATP+PE. These findings demonstrate functional expression of α1A‐ARs in SON neurons, and indicate that PE‐induced increases in [Ca++]i are primarily mediated by α1A‐ARs. However, additional α1‐AR subtypes participate in the sustained response to ATP+PE. Supported by NIH RO1‐NS29759.
Electrical stimulation of the subfornical organ induces feeding and drinking in satiated rats
Authors: Smith Pauline M , Ferguson Alastair V
Published: June 2021

The subfornical organ (SFO), a circumventricular structure which lacks the normal blood brain barrier has been shown to play a key role in body fluid homeostasis. A role for the SFO in sensing circulating satiety signals has been suggested by electrophysiological studies demonstrating that anorexigenic (leptin and amylin) and orexigenic (ghrelin) satiety signals influence the excitability of separate populations of SFO neurons. The present study examined whether electrical stimulation of the SFO influenced feeding in satiated rats.

Male Sprague Dawley rats implanted with concentric bipolar SFO stimulating electrodes received 5 min electrical (100 or 200μA) or sham stimulation and food and water intake measured. Once animals had undergone both electrical and sham stimulation protocols the brain was removed to confirm the location of the stimulating electrode.

SFO stimulation (100μA) elicited drinking in 5/6 animals tested (latency to drink: 6.2±2.6 min) but did not elicit eating. Higher stimulation intensities (200μA) elicited eating in all animals tested (food consumption: 0.6 ± 0.12g/100g body weight, n=6) with a mean latency to eat of 8.0 ± 4.0 min and elicited drinking in 5/6 animals (latency to drink: 15.2±2.6 min.) Sham SFO stimulation or stimulation (200μA) in non‐SFO sites (n=6) did not elicit feeding or drinking.

The results of the present study showing that SFO stimulation induces feeding in satiated rats, lends support for a role for the SFO as an integrator of circulating peptides that regulate feeding. Supported by Canadian Institutes of Health Research
Humor‐associated laughter affects appetite hormones
Authors: Berk Lee , Prowse Michelle , Bains Gurinder , Batt Jennifer , Petrofsky Jerry , Daher Noha , Danner Harmony , Ludeman Laura , Lahman Michael , Tan Stanley , Berk Dottie
Published: June 2021

We have previously shown that repetitious “mirthful laughter” (Laughercise), like physical exercise (PE), decreases stress hormones (cortisol & catecholamines), enhances immune activity (NK cell cytotoxicity, B‐cells, activated T‐cells, immunoglobulins, CD4/CD8 ratio), and lowers cholesterol & systolic blood pressure (Berk, 2001, 2009). Additionally, PE can modulate the appetite hormones leptin (decrease) and ghrelin (increases) immediate post‐PE (Jurimae, 2007). Therefore, the objective was to compare the response of 14 healthy individuals who viewed a 20 min videos, humorous vs. distressing (cross‐over design 1 wk apart), and measure leptin and ghrelin levels 1 wk before study (base), pre‐ (PR‐V) and post‐viewing (PO‐V). Friedman's ANOVA and Wilcoxon Signed Ranks tests were used for statistical analysis. Leptin decreased from base to PR‐V −12% (p=0.02), base to PO‐V −15% (p=0.03), and PR‐V to PO‐V −4% (p=0.03) for the humor group. Ghrelin decreased from base to PR‐V −11% (p=0.05) (anticipatory effect), increased base to PO‐V 9.0% (p=0.03), and PR‐V to PO‐V 52% (p=0.02). There was no significant change with the distress video. Laughercise appears to modulate and may optimize the appetite hormones leptin and ghrelin similar to PE. This may have clinical relevance for those where PE is difficult, the handicap, depressed or elderly individual/patient. Further research is needed to elaborate these data.
Intramuscular testosterone and trenbolone enanthate elevates hemoglobin concentrations
Authors: McCoy Sean C , Yarrow Joshua F , Conover Christine F , Lipinska Judyta A , Santillana Cesar , Borst Stephen E
Published: June 2021

Testosterone enanthate (TE) increases serum testosterone (T), prostate mass, and hemoglobin (Hb) in orchidectomized (ORX) rodents. Trenbolone enanthate (TREN) is a synthetic analogue of T which does not undergo 5‐alpha reduction or aromatization; suggesting that it may increase Hb without inducing prostate enlargement. Our purpose was to determine whether TREN administration results in elevated circulating trenbolone (TR) and elevated Hb without prostate enlargement. Thirty‐five 10 month old male Brown Norway rats were randomized into Sham+vehicle (V), ORX+V, ORX+T, and ORX+TREN groups and received weekly drug injections for 42 days. Injections of TE, resulted in elevated serum concentrations of T compared to other treatments (p<0.05). Whereas, injections of TREN increased serum TR levels without increasing serum T (p<0.05). Prostate mass was 78% lower in ORX and 30% lower in TR groups vs. SHAM (p<0.05); while prostate mass in ORX+T was nearly double SHAM (p<0.05). Both T and TREN elevated Hb by 11% compared to ORX (p<0.05). Ultimately, ORX+TREN elevated Hb concentrations and reduced prostate mass; suggesting that TR may selectively activate androgen receptors. Supported by a VA Merit Award (SEB).
Disruption of the tight junction complex (TJ) by the cins‐mutation of NOD2
Authors: Poritz Lisa S , Harris Leonard R
Published: June 2021

A mutation in the NOD2 gene is associated with Crohn's disease (CD). Patients with CD also have increased intestinal permeability and a defect in the TJ. The hypothesis for this study is that mutations in NOD2 disrupt the TJ.

Methods

IEC‐18 cells (IEC‐18p) were stably transfected with human wild‐type NOD2 (IEC‐18w) or the c‐ins mutation (IEC‐18m). Monolayers were assayed for permeability and for TJ proteins (ZO‐1, 2, occludin, claudin‐1, 2, 4) by Western blot (WB), immunofluorescence, and qRT‐PCR.

Results

NOD2 was detected by WB only in IEC‐18w cells. By qRT‐PCR expression of NOD2 was < 1 (relative to GAPDH) in IEC‐18p, 10 in IEC‐18m, and >200 in IEC‐18w. The NOD2 protein was functional only in IEC‐18w and when stimulated upregulated NF‐κB. ZO‐1 was decreased in IEC‐18m and markedly disrupted (fig). In IEC‐18w cells ZO‐1 was unchanged from IEC‐18p. Claudin‐1 was significantly increased in IEC‐18w by WB and >600 fold by qRT‐PCR compared to IEC‐18p and IEC‐18m (p<0.01). ZO‐1, ZO‐2 and occludin were significantly decreased by qRT‐PCR in IEC‐18m compared to IEC‐18p and IEC‐18w, p<0.01. Permeability to RITC 70,000 was increased >150% in IEC18m cells compared to IEC‐18p.

Conclusions

Expression of mutant NOD2 significantly decreased protein expression and disrupted the TJ resulting in a functional increase in permeability. These changes may contribute to alterations in permeability seen in CD.

Support by NIH K08 GM081595
Identification of RGS4 structures involved in its plasma membrane localization
Authors: Bastin Guillaume , Lachhar Karanjit , Seong Alex , Heximer Scott
Published: June 2021

RGS4 is highly expressed in the sinoatrial node of the heart and plays an important role in the regulation of M2R‐mediated heart rate control. In order understand the intracellular mechanisms that may regulate RGS4 function in the heart and other tissues we set out to identify the key determinants of RGS4 function in cell culture models. Previous studies have shown that optimal function of R4 group proteins requires their efficient plasma membrane targeting. Based on their high degree of similarity, we set out to compare the localization and function of RGS4 and RGS5 in mammalian cells. Confocal microscopy shows that RGS4‐YFP is mainly localized to the plasma membrane of HEK cells whereas RGS5‐YFP, as reported previously, is found primarily in the cytosol. Comparison of the sequence differences between RGS4 and RGS4 has identified several regions in RGS4 that may promote its increased localization to the plasma membrane. Chimeric YFP‐tagged cDNA constructs for RGS4:RGS5 and RGS5:RGS4 indicate that critical residues in the carboxyl‐terminal domain of RGS4 (including the RGS box) may promote its specific targeting to the plasma membrane but that G‐protein binding is not required for this function. Current studies are aimed at defining the specific residues within the carboxyl terminal that promote its increased localization and function. This study was funded by the Heart and Stroke Foundation of Ontario Grant‐in‐Aid Program grants T6799
Estrogen receptors as regulators of syndecan‐4 expression in solid tumors
Authors: Karamanos Nikos K , Gialeli Chrisostomi , Tsonis Anastasios
Published: June 2021

Estrogens regulate several physiological processes and are implicated in the growth of solid tumors, such as breast and colon. 17â‐estradiol (E2), particularly influences estrogen receptor (ER) expression. ER is synthesized in many cell types as two protein forms, ERá and ERâ, which are products of separate genes. E2 signaling is supposed to be mediated via linear pathway involving insulin‐like growth factor receptor (IGF‐R) and epidermal growth factor receptor (EGFR). The aim of this study was therefore to examine whether estradiol affects the expression of ERs and proteoglycans (PGs) implicated in breast and colon cancers as well as whether this effect is associated with ER/IGF‐R/EGFR system. In vitro studies were performed on a panel of breast and colon cell lines in the presence or absence of E2 and specific IGF‐R, EGFR tyrosine kinase inhibitors. The results showed that E2 affected on the gene expression of syndecan‐4 and is closely related with the expression balance between ERá/ERâ and the crosstalk between crucial ER/IGF‐R/EGFR pathways.
A role of GTP‐restricted ARL1 in the dissociation of p230/golgin‐245 from trans‐Golgi network
Authors: Yu Chia‐Jung , Huang Lien‐Hung , Li Chun‐Chun , Hsu Chia‐Yi , Lin Yi‐Ling , Lee Fang‐Jen Scott
Published: June 2021

The small GTPase, ARL1, regulates the structure and function of the Golgi complex through mechanisms that have been partly elucidated, which include an ability to regulate the recruitment of two GRIP domain proteins, p230/golgin‐245 and golgin‐97, to the Golgi. However, the regulation of dissociation of golgins from the Golgi remains unknown. Here, we report a novel property of GTP‐restricted ARL1, specifically, the ability to mediate the dissociation of p230/golgin‐245, but not golgin‐97 or GCC185, from the Golgi complex. An in vitro binding assay disclosed higher binding affinity of GTP‐restricted ARL1 to golgin‐97 than to p230/golgin‐245 and GCC185. In addition, ARL1 depletion in cells led to weakened association of golgin‐97 with the TGN, but had a less effect on p230/golgin‐245 and GCC185. We infer that different golgins have distinct targeting and dissociation specificities on the Golgi, which are differentially dependent on GTP‐restricted ARL1.
Mitochondrial Dysfunction Impairs Tumor Suppressor P53‐Mediated Cell Death
Authors: Yadava Nagendra , Compton Shannon , Kim Chul , Patel Pinal , Jerry D. Joseph , Schneider Sallie
Published: June 2021

To explore the tumor suppressing function of mitochondria we have investigated the link between mitochondrial dysfunction and the tumor suppressor protein p53. Using a set of respiration deficient (Res−) cells with impaired respiratory chain (RC) assembly, we have determined the effects of mitochondrial dysfunction on p53‐mediated cell death. Our data show that normal mitochondrial function is required for γ‐irradiation induced cell death, which is mainly p53‐dependent. The Res− cells are protected against radiation induced death. Depending upon the type of RC deficiency, the p53 protein expression is differentially regulated. However, irrespective of the differences in p53 protein expression profile, the p53 transactivation activity is impaired in all Res− cells. As expected, the pharmacological inhibition of RC function also protects cells from radiation‐induced cell death. The negative effect of mitochondrial dysfunction on p53 expression/function is a reversible phenomenon. We believe that these findings provide a mechanistic basis for explaining the role of mitochondria as tumor suppressors, and also provide plausible link why age is the major risk factor for cancer considering mitochondrial function declines during ageing. Research support: Rays of Hope Foundation, startup funds, NIH grant to NY.
Genome‐wide Localization of SREBP‐2 in Mouse Liver‐Chromatin by ChIP‐seq
Authors: Seo Young‐kyo , Chong Hansook Kim , Biesinger Jacob , Xie Xiaohui , Jeon Tae‐Il , Osborne Timothy F.
Published: June 2021

Sterol regulatory element‐binding proteins (SREBPs) are a family of basic helix‐loop‐helix leucine zipper (bHLH‐LZ) transcription factors that regulate lipid homeostasis. SREBP‐1 and SREBP‐2 are mainly involved in fatty acid and cholesterol metabolism, respectively, but a comprehensive genome‐wide distribution of the two SREBPs has not been possible until now. We previously reported the location of SREBP‐1 binding sites in mouse liver. Here, we report global targets of SREBP‐2 in mouse liver using chromatin imunoprecipitation–high‐throughput DNA sequencing. Antisera to SREBP‐2 were used with liver chromatin from mice fed a chow diet supplemented with lovastatin plus ezitimibe for one week, which leads to a huge induction of hepatic SREBP‐2 nuclear protein levels. After massive parallel DNA sequencing, mapping these reads to the mouse reference genome identified about 1500 peaks of SREBP‐2 binding in FDR, 0.05 and p‐value 10‐7. These binding peaks show a striking enrichment in proximal promoter regions, with 45% located within 1 kb upstream of a transcription start site. Our preliminary analyses indicates SREBP‐2 bound not only to many well‐characterized SREBP‐2 target genes but to several other previously unknown targets in lipid and carbohydrate metabolism as well as many putative target genes in other diverse biological pathways. Our report should be useful for understanding the mechanistic flexibility that has evolved at the individual gene/promoter level to maintain metabolic homeostasis in response to shifting diet conditions.
Adipokine Orosomucoid Restores Adipose Tissue Inflammation and Energy Homeostasis in Obesity
Authors: Lee Yun Sok , Kim Jae Bum
Published: June 2021

Orosomucoid (ORM), also called α‐1 acid glycoprotein, is an abundant plasma protein that is an immunomodulator induced by stressful conditions such as infections. We observed that ORM is induced selectively in the adipose tissue of obese mice and highly expressed in adipocyte fraction. Adipose ORM levels were elevated by metabolic signals including insulin, high glucose, and free fatty acid, as well as proinflammatory cytokine such as Tumor Necrosis Factor (TNF)α. ORM suppressed proinflammatory gene expression in adipocytes and suppressed interaction between adipocyte and macrophage. In addition, ORM suppressed TNFα‐induced Nuclear Factor Kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and Mitogen‐Activated Protein Kinase(MAPK). Concomitantly, ORM relieved hyperglycemia‐induced insulin resistance as well as TNFα‐mediated lipolysis in adipocytes. Accordingly, ORM improved glucose and insulin tolerance in obese and diabetic db/db mice. Taken together, our results suggest that ORM integrates inflammatory and metabolic signals to modulate immune responses to protect adipose tissue from excessive inflammation and thereby from metabolic dysfunction.
Effects of Procedural Pain on ATP Metabolism and Oxidative Stress in Premature Neonates
Authors: Plank Megan Sue , Asmerome Yayesh , Slater Laurel , Ahmed Mohamed , Angeles Katherine , Boskovic Danilo , Angelea Danilyn
Published: June 2021

Infants in the neonatal intensive care unit routinely undergo painful and invasive procedures. This study examines the effects of procedural pain on ATP metabolism and oxidative stress in two groups of preterm neonates: neonates experiencing a tissue‐damaging procedure (TDP) and those not experiencing any TDP (control group). Procedural pain was measured before and during the TDP using the Premature Infant Pain Profile (PIPP). ATP metabolism and oxidative stress were quantified by measuring levels of hypoxanthine, xanthine, uric acid and malondialdehyde(MDA) in plasma obtained before and thirty minutes after a TDP. We found that pain scores and MDA were higher in neonates undergoing a TDP. Purine breakdown was also found to be significantly enhanced in neonates undergoing a TDP. There were significant correlations between the PIPP pain score and hypoxanthine, xanthine and uric acid. Our data demonstrate that commonly performed NICU procedures increase pain as well as ATP utilization and oxidative stress measured by significant changes in plasma levels of hypoxanthine, uric acid and MDA.
CYP24A1 and the Metabolism of Vitamin D
Authors: Bushong Wednesday , Hollman Cassie , Lee Sarah , Akers Karlene , Akers Landon , Bitterman Jamie , Stout C. David
Published: June 2021

CYP24A1 belongs to a class of enzymes called mitochondrial P450s, which catalyze specific reactions in the biosynthesis and degradation of hormones. CYP24A1 metabolizes vitamin D, which functions in calcium homeostasis and cellular growth processes. An overexpression of CYP24A1 in disease states may lead to bone disorders, kidney disease, and breast or prostate cancer. A potential treatment could be an inhibitor of CYP24A1. CYP24A1’s mechanism of action is poorly understood as it has been relatively difficult to crystallize. As a membrane protein, CYP24A1 has extensive hydrophobic regions, making this process more difficult. The Stout Lab recently overcame this challenge and solved the first crystal structure for CYP24A1, but without vitamin D bound. Now that a structure of CYP24A1 has been determined, further understanding of enzyme function can be gained by solving structures with vitamin D and designed inhibitors bound in the active site. To assist in this effort the Lutheran High School of San Diego SMART Team (Students Modeling A Research Topic) used data from the Stout Lab to create a physical model of CYP24A1 using 3D printing technology. The model will be very useful in understanding the conformational changes that occur in this large complex protein structure upon vitamin D binding. Supported by grants from the HHMI Pre‐College Program, and Cytochroma, Inc. for research at TSRI.
Characterization of Novel BBS Mutants in Chlamydomonas reinhardtii
Authors: Mckenzie Nerissa , Lechtreck Karl F. , Witman George B.
Published: June 2021

Defects of cilia and flagella contribute to a multitude of human diseases, including Bardet‐Biedl Syndrome (BBS), a rare genetically inherited disorder characterized by obesity, retinal degeneration, polydactyly, and mental retardation. Thus far, defects in 12 genes have been linked to BBS, classified as BBS 1–12. Chlamydomonas reinhardtii, a biflagellate unicellular alga, has eight well conserved BBS genes (BBS 1, 2, 3, 4, 5, 7, 8, and 9) suggesting it could serve as unicellular model for BBS. Seven of the eight (excluding BBS3) the gene products form a complex, the BBSome, whose functions are currently unknown. Here, we attempt to characterize 13 BBS mutants, seven containing normal amounts of BBS4 in the flagella but sharing a nonphototactic phenotype with established BBS mutants of Chlamydomonas, five lacking BBS4 in the flagella, and one harboring reduced amounts of BBS4 in the flagella. The goal was to identify the genetic defects in these uncharacterized mutants, and to assay their photo‐behavioral responses. The results suggest that assembly, stability, and/or transport of the BBSome can be impaired despite the presence of the genes encoding all seven BBSome proteins. These findings indicate that other genes are involved in the functionality of the BBSome pathway and that the identification of these genes could further studies of BBS in humans. Supported by NIH T34GM008411
Fatty acids induce a conformational change in UCP1 and UCP3 in brown adipose tissue mitochondria
Authors: Divakaruni Ajit , Humphrey Dickon M. , Brand Martin D.
Published: June 2021

In brown adipose tissue (BAT) mitochondria, uncoupling protein 1 (UCP1) dissipates the protonmotive force to generate heat. UCP1 is activated by fatty acids and inhibited by nucleotides such as GDP, but the precise mechanisms involved remain controversial. Even less is known about the physiological role and regulation of uncoupling protein 3 (UCP3) in BAT. Here we present the first demonstration of a conformational change induced by fatty acids for both UCP1 and UCP3 in rat BAT mitochondria. Conformational changes were inferred from the kinetics of proteolysis when isolated mitochondria were treated with exogenous trypsin. Palmitate increased the rate of proteolysis for both proteins, showing that palmitate binds and affects their conformation. Trypsinolysis of UCP1 could be fully rescued by GDP, consistent with its ability to compete functionally with fatty acids. However, UCP3 degradation was GDP‐independent, suggesting that GDP interacts differently (or not at all) with UCP3. Further evidence for an acute interaction between fatty acids and UCP1 was provided by altered binding of the fluorescently tagged nucleotide derivative mant‐GDP to UCP1 in the presence of palmitate. Supported by a British Marshall Scholarship and NSF Graduate Research Fellowship (ASD), the UK Medical Research Council, and the Buck Institute for Age Research.
Correlation in species, cofactor, oligomerization and substrate specificity in short‐chain oxidoreductase enzymes
Authors: Huether Robert , Duax William
Published: June 2021

The Rossmann nucleotide‐binding fold is an abundant, stable fold, which includes the classical short‐chain oxidoreductase (SCOR) sub‐family. SCOR proteins are vital to growth and development of all organisms. Using cofactor identifier sites at adjacent positions in 19,003 SCOR enzymes, we have correlated the divergent evolution of these positions to the oligomerization interfaces and substrate identification loops. Cofactor preferences of 86% of the SCORs are associated with combinations of xR, Dx, Yx or xT. 27 combinations of amino acids in these two positions account for 83% of all SCORs. There is structural information about 22 of these combinations. Proteins with Dx and Yx correlate with NAD binding and xR and xT correlate with NADP binding which are associated with oxidation and reduction reactions respectively. On the basis of structure, cofactor and substrate recognition, 16% of the SCOR proteins can be assigned to families of orthologs for which x‐ray crystal structures have been done. There are 249 groups of proteins where 10 or more proteins contain the same substrate fingerprint that account for 48% of all SCOR proteins.
Systematic analysis of essential genes reveals new regulators of G protein signaling
Authors: Cappell Steven , Skowyra Dorota , Dohlman Henrik
Published: June 2021

The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify new signaling components we systematically evaluated 870 essential genes using a library of repressible‐promoter strains. Quantitative transcription‐reporter and MAPK activity assays were used to identify strains that exhibit altered pheromone sensitivity. Of the 92 newly identified essential genes required for proper G protein signaling, those involved with protein degradation were most highly‐represented. Included in this group are members of the SCF (Skp‐Cullin‐F‐Box) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCFCdc4 acts together with the Cdc34 ubiquitin conjugating enzyme at the level of the G protein, promotes degradation of the G protein α subunit, Gpa1, in vivo and catalyzes Gpa1 ubiquitination in vitro. These new insights to the G protein signaling network reveal the essential‐genome as an untapped resource for identifying new components and regulators of signal transduction pathways.
A Breath of Fresh Air
Authors: Reyes Carolina , Debenham William , Martinez Antonio , McCraine Krysten , Thornton Mackenna , Walger Rebecca , Galea‐Martinez Sandra A. , Sok Devin
Published: June 2021

According to the Collaborative on Health and the Environment, asthma is estimated to affect 300 million people worldwide. Asthma is the leading cause of hospitalization in children. G‐protein coupled receptors (GPCR) are a large family of transmembrane proteins that cells use to communicate with the environment. Beta‐2‐adrenergic receptor (B2AR) is a member of this family and binds to the small hormone adrenaline. The G‐protein relays the message to relax the muscles in the lungs and to open airways. In the case of a person with asthma, this interaction causes the lungs to constrict and airways to close. Our hypothesis will model the interaction of B2AR and GalphaS. These components move inward surrounding the ligand binding site, forming a "toggle switch" for receptor activation. By getting a better understanding of the structural changes following G protein binding, improved therapeutics can be developed to target the active receptor. People afflicted with respiratory diseases will be able to take a breath of fresh air. The El Capitan High School SMART (Students Modeling a Research Topic) Team is a partnership between a researcher and a group of students who used current data to create a physical model of B2AR interacting with GalphaS using 3D printing technology. Supported by a grant from the HHMI Pre‐College Program.
Mechano‐regulation of contractile phenotype in myofibroblasts
Authors: Risinger George Mitchell , Haaksma Carol J , Tomasek James J
Published: June 2021

During wound healing, myofibroblast (MF) appearance correlates with increased wound tension leading to the hypothesis that the mechanics of the wound environment regulate MF formation and function. In this study we examined how the mechanics of a high‐tension environment regulate the MF contractile phenotype. To address this we cultured rat dermal fibroblasts in compliant (low‐tension) and stiff (high‐tension) collagen lattices. Stiff‐cultured cells acquired the MF contractile phenotype of increased focal adhesions and stress fiber bundling and highly expressed the contractile cytoskeletal genes smooth muscle (SM) α‐actin, SM γactin and SM 22‐α. In contrast, compliant‐cultured cells had a fibroblast phenotype and low expression of contractile genes. We next asked if this mechano‐regulation was associated with dynamic changes in the actin cytoskeleton. In support of this, we found induction of F‐actin formation increased contractile gene expression in compliant‐cultured cells, and destabilization of the F‐actin cytoskeleton down‐regulated contractile gene expression in stiff‐cultured cells. We next plan to address the role of the actin‐sensitive transcription factor, myocardin‐related transcription factor‐A, in this process. Our goal is to identify the combination of external and internal factors regulating MF formation, maintenance, and function. Supported by NIH grant R01 GM60651.
Isoform‐specific modulation of CYP450 omega hydroxylases by cyclosporine in glomerular mesangial cells
Authors: Sharma Mukut , Sharma Ram , Srivastava Tarak , Savin Virginia J. , McCarthy Ellen T.
Published: June 2021

Cyclosprine A (CSA), an immunosuppressive agent used in the treatment of many proteinuric conditions, protects the glomerular filtration barrier. We have shown that 20‐hydroxyeicosatetraenoic acid (20‐HETE), an eicosanoid generated by the CYP4a and 4f omega hydroxylase activities, maintains and protects the glomerular filtration barrier. We hypothesize that CSA induces expression of CYP4a and/or 4f in mesangial cells to protect the filtration barrier and mitigate proteinuria. We incubated murine mesangial cells in CSA (10 nM, 100 nM, 1 μM, 10 μM) or vehicle for 1, 8 or 24 hrs. We then isolated total RNA and performed real time RT‐PCR using specific primers to measure expression of CYP4f13, CYP4a12a and CYP4a12b. CYP4f13 expression showed the greatest response to CSA treatment. At 8 hr CSA 1 μM and 10 μM increased CYP4f13 by 91% and 74%, respectively. At 24 hr CSA 100 nM, 1 μM and 10 μM increased CYP4f13 by 36%, 268% and 200%, respectively. At 24 hr, CSA 10nM and 100 nM increased expression of CYP4a12a by 75% and 88%, respectively, while CSA 10 μM appeared to suppress expression to 48% of control at 24 hr. CSA 10 μM increased expression of CYP4a12b by 85% at 8 hr; this concentration appeared to suppress expression at 24 hr. Thus, mesangial cells express CYP4a and 4f, and CSA modulates the expression of these CYP isoforms in this cell type in a time and dose‐dependent manner.

Supported by NIH/NIDDK R01 DK064969.
A zinc binding site by negative selection induces metallodrug susceptibility in an essential chaperonin
Authors: Cun Shujian
Published: June 2021

GroES is an indispensable chaperonin virtually found throughout all life forms. Consequently, mutations of this protein must be critically scrutinized by natural selection. Nevertheless, the homolog from a potentially virulent gastric pathogen, Helicobacter pylori, strikingly features a histidine/cysteine‐rich C terminus that shares no significant homology with other family members. Additionally, three more (H45, C51 and C53) are uniquely present in its apical domain. The statistical analyses show that these residues have probably originated from negative selection, assumedly driven by either dependent or independent amino acid mutations. In the absence of the C‐terminal metal‐binding domain, the mutant protein still exhibits a substantial capacity for zinc binding in vivo. The biochemical properties of site‐directed mutants indicate that H45, C51 and C53 make up an oxidation‐sensitive zinc binding site that may donate the bound metal to a zinc acceptor. Of interest, bismuth antiulcer drugs strongly bind at this site (Kd of ~7 × 10−26 M), replacing the bound zinc and consequently inducing the disruption of the quaternary structure. Since biological features by negative selection are usually inert to change during evolution, this study sheds light on a promising field whereby medicines can be designed or improved to specifically target the residues that uniquely evolved in pathogenic proteins so as to retard the emergence of drug resistance.
Expression, Purification and Preliminary Activity of a Novel Dehydratase Domain from a Polyketide Synthase.
Authors: Oyola‐Robles Delise J , Bermúdez Mei‐Ling , Márquez Suheiry , Carballeira Néstor M , Baerga‐Ortiz Abel
Published: June 2021

Polyunsaturated fatty acids (PUFAs) are essential membrane components in higher eukaryotes and important components of human health. PUFAs from deep‐sea bacteria are synthesized by a modular polyketide synthase consisting of several domains including two dehydratase (DH) domains, DH1 and DH2, which are responsible for the introduction of cis or trans double bonds into the final product. In order to study the mechanism and substrate specificity of these DH domains, protein fragments were expressed containing one or both domains. Domain boundaries were chosen by bioinformatic analysis using the Udwary‐Merski Algorithm (UMA) for the prediction of protein linkers. Recombinant proteins were purified by nickel agarose and anion exchange chromatography. Enzyme assays were performed by monitoring the consumption of synthetic substrates by UV spectroscopy. Results from the UMA sequence analysis suggest the presence of two additional, unexpected domains preceding DH1 and DH2. Results suggest that these unexpected domains are important for protein structure and stability, since constructs that lack these domains are insoluble. Preliminary enzyme assays show that all soluble DH‐containing constructs were inactive towards the short‐chain thioester substrate mimic, suggesting that longer thioesters substrate mimics will be required in order to assay dehydratase activities.
TTK Kinase Inhibitor Development through Chemical Proteomics
Authors: Flick Jeff S. , Gassman Andrew , Bramhall Dave , Fleischer Tracey , McAlexander Ian , Willardsen J. Adam , Chan Ashley , Brown Brita , Woodland Ann‐Marie , Boniface J. Jay
Published: June 2021

Myriad Pharmaceuticals has developed a chemical proteomics process to identify cellular proteins that bind immobilized small molecules. This process was used to identify off‐targets of early stage TTK inhibitors and to guide development of TTK inhibitors for our oncology program. It is an unbiased method and reduces the need to generate and test a large number of kinases in vitro. MPI‐0471537 inhibits TTK kinase in vitro with an IC50 of 8 nM and is cytotoxic to HCT116 cells (IC50 = 1.2 μM). MPI‐0471537 coupled to beads was used to affinity purify proteins from cellular lysates, with subsequent LC‐MS/MS‐based protein identification. As expected, TTK was identified as a high affinity binder of the MPI‐0471537‐beads. Additional kinases identified included AurA, FER, JNK1 and JAK1. In vitro assay of these kinases revealed IC50 values of 420, 15, 57 and 320 nM, respectively. MPI‐0478574, a derivative of MPI‐0471537 with modifications designed to increase the selectivity for TTK kinase (IC50 = 3 nM) was subsequently analyzed. MPI‐0478574‐beads failed to bind AurA and showed reduced affinity for FER, JNK1 and JAK1, but maintained similar affinity for TTK. Biochemical assays confirmed the reduced activity of MPI‐0478574 against AurA, FER, JNK and JAK1 (IC50 values >5,000, 380, 120 and >5,000 nM, respectively). These results demonstrate the efficacy of chemical proteomics to assess and advance kinase inhibitor selectivity.
Innovations in Mentoring for Secondary and Higher Education through UAN Activities
Authors: McCormick Casey Jerome
Published: June 2021

A look into some of the activities that Undergraduate Affiliate Networks of the ASBMB can use to achieve high success in outreach to their members and K‐12 students. Topics such as STEM education development at the secondary level and undergraduate research interest will be discussed. Most importantly, the presentation will look at how regional resources can prove to be an asset in these types of plans.
Structure and Function of the G‐protein Coupled Receptor Family
Authors: Stevens Raymond
Published: June 2021

G protein‐coupled receptors comprise the largest family of human eukaryotic signal transduction proteins that communicate across the membrane. We have solved the crystal structures of the human β2‐adrenergic receptor bound to the partial inverse agonist carazolol and timolol at 2.4 Å and 2.8 resolution Å, respectively. More recently, we determined the structure of the human adenosine A2a receptor bound to the antagonists ZM281345 at 2.6 Å resolution and conducted a community wide assessment of the current status of GPCR modeling and docking. The structures provide a high‐resolution view of a human G protein‐coupled receptor bound to diffusible ligands. Ligand‐binding site accessibility is enabled by the extracellular loops which are held out of the binding cavity by a set of disulfide bridges and unique structural motifs. An exciting discovery is the role of cholesterol in receptor stability and potential function. Future studies include the determination of representative members from the different branches of the GPCR phylogenetic tree including class A, B, and C GPCR's, as well as the receptors bound to agonists and G‐proteins in an activated state. New structural data will be reported. This work was supported by the NIGMS Protein Structure Initiative (ATCG3D) U54 GM074961 and the NIH Roadmap Initiative (JCIMPT) P50 GM073197.
Effect of γ‐irradiation on the antioxidant acticity of hot‐air and freeze drying grapes pomace
Authors: YOON MI HYANG , JANG SOON AE , YOOK HONG SUN
Published: June 2021

In this study, antioxidant properties of gamma‐irradiatied hot‐air drying and freeze drying grape pomaces were evaluated. Samples were irradiated at doses ranging of 1, 3, 5, 10 and 20 kGy. They were then analyzed to investigate antioxidant properties, including DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) radical scavenging activity, total phenolic compound content and reducing power. The DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) radical scavenging activity of nonirradiatied hot‐air and freeze drying grapes pomaces were 84.98% and 88.86%, respectively. And 3 kGy gamma‐irradiated hot‐air drying(86.99%) and freeze drying(89.90%) grape pomaces showed the highest activity in all samples. Total polyphenol content of hot‐air drying grape pomace showed the highest activity at 3 kGy‐irradiated samples of 700.21 ppm but freeze drying grape pomace showed the highest activity at nonirradiated sample of 900.63 ppm. The result of reducing power, 3 kGy‐irradiated sample showed the highest activity for the both hot‐air drying and freeze drying grape pomaces. Accordingly, the 3 kGy gamma irradiation of hot‐air drying or freeze drying grape pomace showed that it could improving the antioxidative ability.
Effect of Aralia Contientalis extracts in rat primary cultured chondrocytes viability and mRNA expressions related cartilage metabolism under oxidative condition
Authors: Yang Jiwon , Jang Ja‐Young , Lee Jeongmin , Jun Woo‐Jin , Hwang Kwon‐Tack , Lee You‐Yeon
Published: June 2021

This study was researched on the effect of Aralia Contientalis(AC) extracts on rat chondrocytes viability and mRNA expression related to cartilage synthesis and erosion in oxidative stress. AC fractions were extracted from 80%, 50%, 5% ethanol and hot water. Chondrocytes were harvested 6~10 weeks Spraque‐Dawley rats and all experiments were fulfilled within the passage number 6. At first, cytotoxity of AC fractions was tested by MTT assay, 80% and 50% EtOH fractions were under the concentration of 200ug/ml, 5% EtOH and hot water fractions were under the concentration of 400ug/ml with no cytotoxity. Cell viability and mRNA expression related cartilage metabolism against the oxidative stress were measured after 300uM of hydrogen peroxide(H2O2) treated. All extracts of AC were effective on cell viability in oxidative condition in a dose‐dependant manner, among the AC extracts, 80% EtOH fraction of AC was most effective in cell viability. mRNA expression related to cartilage anabolism and catabolism in oxidative circumstance, genes of cartilage synthesis, aggrecan, collagen type I, collagen type II and procollagen X were most expressed in 80% EtOH AC fraction. mRNAs related cartilage catabolism, MMP‐3, MMP‐7 and MMP‐13 were also expressed under the H2O2 treatment. Consequently, AC 80% ethanol extracts were most effective in rat chondrocytes viability and mRNA expression related to cartilage synthesis in oxidative condition.
Effect of chitosan on the induction of DNA damage response by selenium compounds
Authors: Zhang Shu , Zeng Huawei , Luo Yangchao , Wang Qin , Cheng Wenhsing
Published: June 2021

Selenium (Se), a nutrient trace mineral, plays important roles in optimizing human health. Chitosan is an effective, natural‐oriented material for synthesizing nanopolymers, with preferable properties such as biocompatibility, biodegradation and resistance to certain enzymes. In this study, encapsulated sodium selenite (Na2SeO3) and methylseleninic acid (MSeA) with low and medium molecular weight chitosan were used to determine the effect of chitosan on Se‐induced DNA damage response. A range of Se compounds was applied to the HCT 116 colon cancer cell line and the MRC‐5 normal fibroblasts. Analysis of cellular Se content demonstrated that: 1) cellular Se retention was greater in HCT‐116 than in MRC‐5 cells, 2) chitosan encapsulation enhanced Se contents in both the cells. Cell survival analysis showed that chitosan encapsulation protected against Na2SeO3 or MSeA induced cell killing, and the extent of which was more pronounced in MRC‐5 than in HCT 116 cells. Chitosan encapsulation decreased Se‐induced ATM phosphorylation Ser‐1981 in both MRC‐5 and HCT‐116 cells. Taken together, the results suggest that, when encapsulated with chitosan, cells are less susceptible to Se treatment, possibly through a mechanism by which the presence of chitosan attenuates Se‐induced activation of ATM and corresponding DNA damage response pathway.