bluuebunny/crossref_metadata_2025 huggingface dataset:
A chemical‐genetic screen to unravel the genetic network of CDC28/CDK1 links ubiquitin and Rad6–Bre1 to cell cycle progression
Authors: Zimmermann Christine , Chymkowitch Pierre , Eldholm Vegard , Putnam Christopher D. , Lindvall Jessica M. , Omerzu Manja , Bjørås Magnar , Kolodner Richard D. , Enserink Jorrit M.
Published: June 2021

Cyclin‐dependent kinases (CDKs) control the eukaryotic cell cycle, and a single CDK, Cdk1 (also known as Cdc28), is necessary and sufficient for cell cycle regulation in the budding yeast Saccharomyces cerevisiae. Cdk1 regulates cell cycle‐dependent processes such as transcription, DNA replication and repair, and chromosome segregation. To gain further insight into the functions of Cdk1, we performed a high‐throughput chemical‐genetic array (CGA) screen aimed at unraveling the genetic network of CDK1. We identified 107 genes that strongly genetically interact with CDK1. Although these genes serve multiple cellular functions, genes involved in cell cycle regulation, transcription, and chromosome metabolism were overrepresented. DOA1, which is involved in maintaining free ubiquitin levels, as well as the RAD6–BRE1 pathway, which is involved in transcription, displayed particularly strong genetic interactions with CDK1. We discovered that DOA1 is important for cell cycle entry by supplying ubiquitin. Furthermore, we found that the RAD6–BRE1 pathway functions downstream of DOA1/ubiquitin but upstream of CDK1, by promoting transcription of cyclins. These results link cellular ubiquitin levels and the Rad6–Bre1 pathway to cell cycle progression.
Betulinic acid decreases Sp1 level via increasing the sumoylation of Sp1 to inhibit lung cancer growth
Authors: Hung Jan-Jong , Chang Wen-Chang
Published: June 2021

Previous study has known that Sp1 inhibited by a natural product, betulinic acid (BA), involves in the cancer progression, but the mechanism still need to delineate. In this study, we found that BA treatment increases the sumoylation of Sp1 by inhibiting the SUMO1/sentrin specific peptidase 1 (SENP1), increasing the recruitment of E3 ligase, RNF4, and then results in ubiquitination and degradation in a 26S‐proteosome dependent pathway. Gene expression profile of the MMA‐treated KRAS‐induced lung cancer transgenic mice studied by the cDNA array found that 542 of genes were affected by repressing Sp1 activity. One of these important genes, cyclin A2, addressed deeply indicates that BA and MMA affect cyclin A2 expression by decreasing Sp1 stability and DNA binding affinity, respectively. Downregulation of cyclin A2 led to decrease pRb phosphorylation and cause cell cycle G2‐arrest. Finally, Sp1 level studied during in Kras‐induced lung cancer transgenic mice and clinical lung cancer specimen found that Sp1 was accumulated significantly. Taken together, this study clearly clarifies the mechanism of BA‐mediate Sp1 degradation: BA modulates the posttranslational modification, such as increased sumoylation and ubiquitination, of Sp1, to destabilize Sp1 to affect its target genes such as cyclin A2, and then repress lung cancer growth.
Swe1‐dependent and Swe1‐independent pathways controlling Hydroxyurea resistance in Saccharomyces cerevisiae
Authors: tripathi kaushlendra , Matmati Nabil , Qin Tingting , Zheng W. Jim , mohanty Bidyt K , Hannun Yusuf A
Published: June 2021

Exposure to Hydroxyurea (HU) slows down or stalls DNA replication fork and activates the replication checkpoint. In Saccharomyces cerevisiae the replication fork component proteins Tof1, Csm3, Ctf4 and Mrc1 as well as its regulators such as Dcc1, Ctf18 and Ctf8 confer HU resistance. HU treatment of yeast cells causes actin depolarization in which several DNA integrity checkpoint proteins as well as morphogenesis controller Swe1 have been implicated. Recently we have shown that a sphingolipid gene ISC1 also controls cell morphology under HU stress by monitoring Swe1 and Cdk1 activity.

Chromatin remodeling protein complexes such as Ino80 and Swr1 that function to reorganize histones during transcription and DNA repair, have recently been implicated in genotoxic tolerance in HU and methyl methanesulfonate (MMS). We have observed that although replication checkpoint proteins Mrc1, Tof1, Csm3 and Ctf4 and certain chromatin remodeling complex proteins such as Arp6 and Arp8 are involved in replication fork stalling, genotoxic tolerance and recovery from HU and MMS, their modes of action may be different. We find that they cooperate in HU/MMS sensitivity and recovery from HU/MMS.

We have also observed that HU treatment causes morphological aberrations in strains lacking replication associated genes such as CTF4 and DCC1. Interestingly, although HU causes morphological aberrations in the absence of ARP8, an INO80 complex gene, it does cause any aberrations in the absence of ARP6, a component of SWR1 complex gene. First time we are showing the functional interactions among replication checkpoint proteins, nuclear actin‐related proteins of chromatin remodelers, and actin regulators in the genotoxic tolerance and genotoxin‐induced cell morphology.
Insulin disregulation and neurodegeneration: a possible role for CDK5
Authors: Khost Daniel Emerson , Cotton Renita , Aguanno Ann
Published: June 2021

Cyclin dependent kinase 5 (CDK5) is a member of the cyclin dependent kinase family that, unlike its fellow CDKs, is not involved in regulation of the cell cycle, but is instead predominantly active in terminally differentiated neurons. CDK5's role in the developing nervous system in regards to dendrite extension, synapse formation, axonal guidance, as well as an array of other functions has been well studied over the years. It has also been implicated in Alzheimer's Disease (AD), where disregulation of CDK5 via the cleavage of its activating partner p35 to p25 contributes to the formation of neurofibrillary tangles. CDK5 activity is also found in other tissues, including the pancreas, where it is believed to play a role in insulin exocytosis. A correlation between Type II diabetes and AD has led to the hypothesis that a connection exists between insulin disregulation and neurodegeneration, and as CDK5 is active in both tissues it represents a possible common feature in the two pathologies. To study this, our lab models the developing and mature nervous and endocrine system using PC12 and AR42J cell lines, respectively. We induce transdifferentiation of both of these cells lines with various growth factors and investigate the impact of CDK5's chemical inhibition. Our results suggest a role for CDK5 in the link between insulin disregulation and neurodegenerative disease.

Funding from the US Dept. of Education and the Rose M Badgely Charitable Trust
Role of Hepatic Monounsaturated Fatty Acid Synthesis in Metabolic Regulation
Authors: Strable Maggie , Flowers Matthew , Liu Xueqing , Ntambi James M
Published: June 2021

Stearoyl‐CoA desaturase (SCD) catalyzes the de novo synthesis of monounsaturated fatty acids (MUFA) from saturated fatty acids. Past work demonstrated SCD1 deficiency impairs hepatic lipogenesis and protects against diet‐induced obesity. Our objective was to determine if hepatic MUFA synthesis is sufficient to restore the impaired lipogenic program in SCD1 global knockout mice (GKO). To address this, we produced liver‐specific transgenic mice expressing either human SCD5, which preferentially synthesizes oleate (18:1n‐9), or mouse SCD3, which preferentially synthesizes palmitoleate (16:1n‐7), and introduced these transgenes into GKO mice. Hepatic oleate synthesis largely prevented very‐low‐fat diet‐induced weight loss and increased white adipose tissue weight to a greater extent than hepatic palmitoleate. In females, hepatic SREBP‐1 maturation and lipogenic gene expression increased in hSCD5/GKO while expression of these genes remained low in mSCD3/GKO mice. Hepatic oleate increased plasma glucose levels greater than hepatic palmitoleate. This work suggests that hepatic MUFA are involved in regulation of lipogenesis and gluconeogenesis with oleate being more potent than palmitoleate. Supported by NIH.
Potato consumption among adults in the U.S.
Authors: Storey Maureen L , Anderson Patricia A
Published: June 2021

According to CDC, 26.3 percent of U.S. adults consumed the recommended 3 servings of vegetables per day in 2009. Potatoes, one of the most commonly consumed vegetables, are an important and affordable source of potassium (K) and dietary fiber (DF). We examined potato consumption using data from the 2005–2006 and 2007–2008 National Health and Nutrition Examination Survey for males and females aged 19–30, 31–50, 51–70, and 71+ y. Appropriate survey weights were used to calculate average consumption of white potatoes (WP) on the day of the survey, including baked, boiled, roasted, stewed, stuffed, mashed, French fried (FF), potatoes with sauce, hash browns, homefries, potato skins and potato salad. Males 19–30, 31–50, 51–70, and 71+y consumed an average of 92.7, 64.1, 67.1, and 62.2 calories (kcal) a day from WP, respectively. Females 19–30, 31–50, 51–70, and 71+ consumed a daily average of 61.4, 51.5, 36.3, and 35.7 kcal from WP, respectively. Average kcal from FF potatoes declined from 78.5 and 39.4 calories among men and women 19–30 to 9.7 and 4.6 calories among men and women 71+. Among adults 19–99, 50 percent of adults did not eat any WP on the day of the survey, and the 90th percentile of WP consumption was 103 grams, or less than one medium potato. Seventy‐five percent of adults did not eat FF potatoes on the day of the survey. Encouraging potato consumption may be an effective way to increase vegetable consumption among adults.
Selective Activation of Human Pregnane X Receptor, Glucocorticoid Receptor, and Constitutive Androstane Receptor by Individual Ginkgolides
Authors: Lau Aik Jiang , Yang Guixiang , Chang Thomas K. H.
Published: June 2021

Pregnane X receptor (PXR), glucocorticoid receptor (GR), and constitutive androstane receptor (CAR) regulate the expression of genes involved in diverse biological functions. Ginkgolide A, ginkgolide B, ginkgolide C, and ginkgolide J are structurally‐related terpene trilactones present in Ginkgo biloba extract. In the present study, we compared the effect of individual ginkgolides on the function of human PXR (hPXR), human GR (hGR), and human CAR (hCAR). As assessed in a cell‐based luciferase reporter gene assay, none of the ginkgolides activated hGR or hCAR (wild‐type and its SV23, SV24, and SV25 splice variants). Whereas ginkgolide A and ginkgolide B activated hPXR to a greater extent than ginkgolide C, ginkgolide J had no effect. Among the ginkgolides investigated, only ginkgolide A and ginkgolide B bound to the ligand‐binding domain of hPXR, recruited steroid receptor coactivator‐1, and increased hPXR target gene (CYP3A4) expression. In conclusion, the individual ginkgolides regulate the function of nuclear receptors in a chemical‐dependent and receptor‐selective manner. This study identifies ginkgolide A and ginkgolide B as naturally‐occurring agonists of hPXR, provides mechanistic insight into the structure‐activity relationship of ligand‐activation of hPXR, and suggests that specific ginkgolides may modulate the biological actions mediated by this receptor. [Supported by CIHR and MSFHR]
Disruption of soluble epoxide hydrolase modulates adenosine‐induced response: role of adenosine A2A receptor and cyp‐epoxygenases
Authors: Nayeem Mohammed A. , Zeldin Darryl C. , Pradhan Isha , Mustafa S Jamal , Falck John R. , Morisseau Christophe , Marowsky Anne
Published: June 2021

We hypothesize the deletion of sEH, adenosine induces relaxation through A2A AR via cyp‐epoxygenases. Using sEH−/− & sEH+/+ mouse aorta, response for acetylcholine (ACh), NECA & CGS 21680 were obtained with L‐NAME, ZM‐241385, SCH‐58261, 14,15‐EEZE, AUDA, t‐AUCB. In sEH−/−, ACh‐response was not different from sEH+/+ (p>0.05), and L‐NAME blocked ACh‐responses in sEH−/− & sEH+/+ (p>0.05). NECA @ 10‐6 M relaxes in sEH−/− (+12.94 ± 3.2%) vs. contraction (sEH+/+; −5.35 ± 5.2%), ZM‐241385, SCH‐58261 blocked relaxation in sEH−/−. In sEH−/− @10‐6 M CGS‐21680 relaxes (+25.90 ± 5.1%) vs. contraction (sEH+/+; −10.56 ± 2.9%), L‐NAME did not block CGS‐21680 (sEH−/−, p>0.05) response, whereas 14,15‐EEZE did (p<0.05). The CGS‐21680‐induced response was not increased by AUDA & t‐AUCB in treated and non‐treated sEH−/− (p>0.05), whereas AUDA & t‐AUCB have reversed in sEH+/+ (from −10.56 ± 2.9 to +40.83±4.1 with AUDA, to +50.25±6.0 with t‐AUCB). Systolic (21 mmHg) & diastolic (19 mmHg) pressure were down in sEH−/− vs. sEH+/+. Compared to sEH+/+, in sEH−/−, no sEH protein was there, whereas increased in mEH, A2A AR, Cyp2c29 & Cyp2j5 expressions (37%, 31%, 24% and 46%, respectively), but decreased in A1 AR (30%), & Cyp2j2 not changed. Data suggest that adenosine induces relaxation, and lowers the BP with up‐regulation of A2A AR, Cyp2c29 & Cyp2j5 in sEH−/− vs. contraction in sEH+/+ mice. NIH: HL 027339 & HL 094447, GM 31278 & NIEHS: z01 ES025034
NADPH oxidase activation in equine palmar digital arteries and veins
Authors: Schwartz Dean David , Wooldridge Anne A , Waguespack R Wayne
Published: June 2021

A common factor in equine laminitis, metabolic syndrome and obesity is oxidative stress due to the accumulation of reactive oxygen species (ROS). Several enzymes contribute to the production of ROS including NADPH oxidase (NOX). In the present study, we examined superoxide generation by NOX in equine palmar digital arteries and veins. Palmar digital arteries and veins were isolated from clinically healthy horses euthanized for other reasons. Gene expression of NOX 1, 2 and 4 was detected in both arteries and veins using PCR primers designed from predicted sequences in the Equus caballus (domestic horse) genome. Western blot analysis confirmed protein expression of these isoforms. NOX activity was measured in freshly isolated rings using the superoxide detector lucigenin. The NOX inhibitor diphenylene iodonium (1 μM), as well as the superoxide dismutase mimetic, EUK‐134 (1 μM) significantly inhibited superoxide generation in both arteries and veins suggesting equine digital vessels generate superoxide through NOX. The nitric oxide synthase inhibitor, L‐NAME (10 μM), the mitochondrial inhibitor rotenone (1 μM) and the cyclooxygenase inhibitor indomethacin (1 μM) had no effect on NOX activity. These data suggest that equine palmar digital arteries and veins can generate superoxide through activation of NADPH oxidase. Funded by Auburn University Animal Health & Disease Research Funds.
Sca‐1 positive cells are more sensitive than adult rat cardiomyocytes to doxorubicin‐induced changes
Authors: Ludke Ana , Sharma Anita K , Bagchi Ashim K , Singal Pawan K
Published: June 2021

Cardiac progenitor cells in the adult heart have recently been implicated in Doxorubicin (Dox)‐induced cardiomyopathy. This study compared the Dox‐induced changes, mediated by oxidative stress, in cardiac stem cells expressing antigen‐1 (Sca‐1 pos) and in cardiomyocytes isolated from adult Sprague‐Dawley rat hearts. Sca‐1 pos cells as well as cardiomyocytes were incubated for 24h with different concentrations of Dox (0.01–10 μM), with and without Vitamin C (25 μM) ‐ a potent antioxidant. Using the same number of cells, an analysis of cell viability (MTT assay), cell injury (AK release), oxidative stress (DCFDA), and ATP levels was performed. Sca‐1 pos cells showed significant decrease in viability and ATP levels and increase in oxidative stress and cell injury at all doses of Dox. Cardiomyocytes were affected only at doses higher than 0.1 μM and significantly less change in these parameters were seen as compared to the Sca‐1 pos cells. Vitamin C was able to mitigate Dox‐induced changes in both cell types. It is suggested that cardiac progenitor cells are more susceptible to Dox‐induced damage in comparison to cardiomyocytes which appear to be mediated by oxidative stress. These findings contribute to a better understanding of the delayed vulnerability of the heart after a prolonged Dox administration. (Supported by Manitoba Health Research Council).
Klotho protects against oxidative damage via amelioration of phosphotoxicity
Authors: Ravikumar Priya , Flaherty Kellie L. , Esser Victoria , Hsia Connie C.W. , Moe Orson W.
Published: June 2021

Klotho mitigates aging in various organs via a host of mechanisms including anti‐oxidation and anti‐phosphotoxicity. Organ damage in Klotho‐deficient animals can be rescued by dietary phosphate (Pi) restriction. The pulmonary epithelium is exposed to constant environmental insults. Klotho‐deficient animals exhibit alveolar airspace enlargement suggesting accelerated aging. To test the direct effects of Klotho against O2 and Pi toxicity in vitro, we exposed A549 lung epithelial cells to hyperoxia (95‐5% O2‐CO2) and increasing Pi (1, 3, 5 mM). Soluble (truncated) or transmembrane (full length) Klotho was over‐expressed by transfection. Apoptosis was measured from caspase‐8 activity (Table, nmol.[hr.104 cells]−1, mean±SD).

Both hyperoxia and Pi induce apoptosis in A549 cells; the effects are synergistic (p<0.05, repeated measures ANOVA with respect to O2 and Pi). Both transmembrane and soluble Klotho overexpression markedly reduced cell apoptosis induced by Pi loading especially in hyperoxia. We conclude that Klotho exerts direct cytoprotective effects on pulmonary epithelium via anti‐oxidative and anti‐phosphotoxic effects.

Support: F32 HL103043, RO1 DK091392, DK092461
Sympathovagal imbalance in type 2 diabetic (T2D) Goto‐Kakizaki (GK) rats associates with abnormal hypothalamic and brainstem gene expressions relevant to food intake
Authors: Yang Hong , Zhao Ke-Wei , Chen Ai
Published: June 2021

Background

Brainstem thyrotropin‐releasing hormone (TRH) resides in nerve fibers innervating sympathetic and vagal motor neurons and regulates food intake through vagal stimulation of ghrelin release (Endocrinology, 2006). TRH induced sympathetic activation is potentiated and vagal activation is impaired in GK rats, resulting in profound visceral dysfunctions (Endocrinology, 2005, Neuroscience, 2010, Hypertension Res 2011), including reduced food intake (Endo meeting 2010).

Method

Hypothalamic and brainstem gene expression relevant to food intake was measured by qPCR in Wistar and GK rats normally fed, fasted for 48 h and fasted for 48 h + refed 2 h.

Results

Fasting increases TRH/TRH receptor mRNA in brainstem and ghrelin in serum in Wistar rats but not in GK rats. Fasting also increased hypothalamic gene expression of NPY, AgRP, ghrelin receptor, and leptin receptor, and decreased that of POMC in Wistar rats. These changes were not found in GK rats. Differences in gene expression of brainstem NPY, POMC, leptin receptor, MC4R, and 5‐HT receptor3a, and hypothalamic TRH, Y2 receptor and MC4R were found between fasted or refed Wistar and GK rats.

Conclusion

sympathovagal imbalance resulting from brainstem TRH dysfunction associates with disabled hypothalamic and brainstem responses to energy demand in T2D GK rats. Supported by VA Merit Ward (Yang).
Calcium permeability in NMDA receptors in NTS baroreceptive neurons is not decreased at low temperature in Syrian hamsters, a hibernating species
Authors: Sekizawa Shin-ichi , Horowitz John , Horwitz Barbara , Chen Chao
Published: June 2021

Neurons in the nucleus tractus solitarius (NTS) receiving cardio‐respiratory afferent information continue to process signals at 15°C in the Syrian hamster. It is not known if intrinsic adaptations in NMDA receptors limit Ca2+ influx and hence augment neuroprotection in this hibernating species. As an initial step in identifying potential intrinsic adaptations, we tested the hypothesis that the Ca2+/Cs+ permeability ratio of NMDA receptors is decreased in hamsters compared to rats at 15°C. Using fluorescent tracer techniques, we identified 2nd‐order NTS baroreceptive neurons and recorded whole‐cell currents with Cs+ replacement of K+ in the pipette solution. From I‐V plots before and after bath perfusion of NMDA (0.3mM) in the Na+ and K+ free extracellular solution containing 2 mM Ca2+, we determined reversal potentials. Calculated Ca2+/Cs+ permeability ratios were 6.6±0.6 (n = 8 neurons) in euthermic hamsters, 7.1±1.0 (n = 13 neurons) in rats, and 7.7±1.0 (n = 9 neurons) in hibernating hamster (p = 0.782). These data negate our hypothesis and suggest that intrinsic NMDA adaptations do not reduce Ca2+ conductance relative to K+ conductance. Barring other adaptations in the NMDA receptor itself, adaptations of other membrane properties that affect activation of NMDA receptors may take on a major protective role in hamster NTS neurons. (support: R01 HL091763)
A cafeteria‐style diet induces obesity and increases lumbar sympathetic nerve activity in rats
Authors: Muntzel Martin S. , Al-Naimi Omar Ali
Published: June 2021

Obesity is a primary cause of essential hypertension. Despite the importance of this link, little is known about how weight gain causes increases in arterial blood pressure (BP). A leading hypothesis suggests that obesity may activate long‐term elevations in sympathetic nerve activity (SNA) that increase peripheral resistance. We therefore predicted that a cafeteria‐style diet would chronically increase body weight and lumbar SNA in rats. To test this, we implanted Telemetry Research (Auckland, New Zealand) radio‐telemetry devices into female Wistar rats. Starting at 5 days following surgery, rats were divided into a Control group (n = 4) that received rat chow and water for 16 days and a Cafeteria group (n = 3) that received crackers, vanilla wafers, buttered popcorn, cheetos, chocolate, salami, smoked fish, condensed milk, and soda pop also for 16 days. At study completion, body weight gains were lower in Controls (from 257±21g to 284±16 g) than in Cafeteria (from 254±21 g to 306±28 g) rats. Whereas lumbar SNA remained at baseline levels in Control rats after 16 days, it increased by 20% in Cafeteria‐fed rats. A testing of lumbar SNA quality revealed that the nerves responded appropriately to sodium nitroprusside (from 100% baseline to 128±14%) and to hexamethonium bromide (100% to 16±6%). These data show, for the first time, that the high‐fat cafeteria‐style diet stimulates chronic increases in lumbar SNA in rats.
Sex‐specific pattern of respiratory control persist in adult mice KO for nuclear progesterone receptors.
Authors: Joseph Vincent , Dallongeville Arnaud , Doan Van Diep
Published: June 2021

We used adult, male and female mice knock out for the nuclear progesterone receptors (PR−/−) to measure respiratory frequency (fR), tidal volume (Vt), minute ventilation (Ve), and CO2 production (VCO2) under normoxia and hypoxia (10% O2 – 30 minutes) using whole body plethysmography. In normoxia, male and female PR−/− mice had lower Ve, and a tendency towards lower Vt (p=0.058) than wild type (WT). VCO2 tended to be lower in PR−/− compared to WT (p=0.075), resulting in similar Ve/VCO2 between PR−/− and WT. Females (WT and PR−/−) had higher Ve, Vt, VCO2 and Ve/VCO2 compared to males, indicating effective hyperventilation, not dependent on PR expression. After 30 minutes of hypoxia, a similar pattern of difference was observed. We conclude that nuclear PR plays an important modulatory role on ventilatory control in normoxia and hypoxia in male and female adult mice. However, nuclear PR does not account for sex‐specific differences in respiratory control. Founded by CIHR.
Cervical spinal demyelination with ethidium bromide reversibly impairs phrenic nerve activity in adult rats
Authors: Johnson Rebecca A. , Nichols Nicole L. , Duncan Ian D. , Punzo Antonio M. , Mitchell Gordon S.
Published: June 2021

Respiratory complications are a major cause of morbidity/mortality in many diseases associated with demyelination. Little is known concerning mechanisms whereby lack of myelin impairs ventilation, or how patients compensate for such changes (compensatory plasticity). We tested the hypothesis that respiratory function is diminished with focal spinal demyelination using unilateral ethidium bromide (EB) injections into C2 bulbospinal tracts. Ventilation (plethysmography) and phrenic nerve activity (neurophysiological recordings) were made 7–21 days post‐EB at baseline and in hypoxia±hypercapnia. EB produced maximal demyelination at day 7 with spontaneous remyelination in 14–21 days (all P<0.05). Ventilation did not differ between groups (all P>0.05). Ipsilateral phrenic nerve voltage was lower than the contralateral voltage 7 days post‐EB during hypoxia and hypoxia/hypercapnia (both P<0.05), as well as the ipsi‐ and contralateral voltage of SHAM and 14 day rats in all conditions (all P<0.05). Average voltage ratios (ipsi:contra) correlated to lesion size at baseline (P<0.05) and were signficantly reduced in 7 day rats (all P<0.05). Our reversible demyelination model provides a powerful tool to study respiratory motor impairment, mechanisms underlying compensatory plasticity/spontaneous recovery, and therapies to reduce demyelination and its associated respiratory motor dysfunction. Support: Supported by grant 1UL1RR025011 from the Clinical and Translational Science Award (CTSA) program of the National Center for Research Resources (NCRR), National Institutes of Health (NIH).
The Binding of G‐Quadruplex DNA to Insulin: From Single Molecule to Bulk‐Methods
Authors: Sinniah Kumar , Lynch Susanna , Timmer Christine , Witte Amanda
Published: June 2021

The formation of guanine (G)‐quadruplex structures in the guanine‐rich tandem repeats of the insulin‐linked polymorphic region (ILPR) is linked to transcriptional effects on the insulin gene. Recent studies demonstrate that the ILPR G‐quadruplexes can bind to insulin, and while this may impact the transcription of insulin, little is known about the binding mechanism. We have performed single molecule atomic force microscopy (AFM), differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and fluorescence quenching spectroscopy to characterize the binding interaction between G‐quadruplex DNA and insulin. In this presentation, I will describe the bulk thermodynamic measurements performed at various temperatures from 10 to 37 °C. The experimental results based on KD demonstrate a moderate affinity by the ILPR consensus sequence to insulin. In addition to thermodynamic measurements, kinetic off‐rates were measured by single molecule force‐pulling measurements and these results will be discussed in relation to kinetic off‐rates measured from the unfolding of G‐quadruplex DNA.
Structural requirements for N‐terminus‐mediated basolateral targeting (BLT) in multipass membrane transporters
Authors: Kuo Shiu-Ming , Yu Siyuan , Wang Li-Yuan , Campbell Christine E.
Published: June 2021

Using the homologous chimera approach, we have previously identified a BLT sequence in the N‐terminus of sodium‐dependent vitamin C transporter 2 (SVCT2) including 5 critical amino acids ELMAI (N55–59). The C‐terminal sequence of SVCT is required for the membrane incorporation of both apical and basolateral SVCTs. Here, the minimal sufficient peptide for BLT was determined. N50–72 and N45–72 but not N50–64 and N45–64 showed BLT activity as determined by confocal and Transwell studies. N50–80 had more exclusive basolateral presence and higher BL uptake. Replacing Y60, L72, L76 by A did not affect BLT previously but amino acid deletion and rearrangement in this region led to the partial loss of BLT in the N50–80 construct. Two other published basolateral transporters, AQP3 and SLC13a3, have BLT activity in their respective N‐terminus in homologous chimera experiments. We used the N‐termini of these two transporters to test whether N‐terminus can independently mediate BLT. The resultant SVCT heterologous chimeras had little plasma membrane localization and showed novel intracellular distribution. Heterologous chimera between N50–64 of SVCT2 and apical SLC11a2v2 also had a similar phenotype. In conclusion, N‐terminal sequence downstream of the critical ELMAI likely plays a role in stabilizing BLT structure and BLT may depend on a bipartite mode of action between N‐terminus and a partner sequence within the gene.
TAK1 kinase signaling regulates Nrf2 and intestinal homeostasis
Authors: Simmons Alicia Nicole , Ninomiya-Tsuji Jun
Published: June 2021

Inflammatory bowel disease is a chronic disease that causes inflammation of the intestine, and is one of the major health problems in the US. However, effective treatments remain to be explored. Understanding the mechanism of intestinal inflammation and damage will aid in effective treatments. TGF‐β activated kinase 1 (TAK1), which is a member of the MAPKKK and is known to be activated by many different stressors and cytokines. We have previously shown that TAK1 deficiency in the intestinal epithelium causes oxidative damage including cell death and loss of paneth cells, which resemble IBD pathology. However, it was not known the mechanism of how reactive oxygen species (ROS) are accumulated and paneth cells are depleted. We tested a hypothesis that TAK1 signaling prevents commensal bacteria‐induced ROS. We found that loss of bacteria or ablation of TLR signaling by MyD88 knockout could rescue the ROS accumulation in TAK1 deficient intestinal epithelium, while either of them could not restore the loss of paneth cells. We also identified that TAK1 modulates the stability of an antioxidant transcription factor Nrf2. These results suggest that TAK1 regulation of Nrf2 is important for elimination of bacteria‐induced ROS in the intestine. Activation of TAK1 may be a possible new approach to reduce oxidative damage in the intestine. We are currently investigating the molecular mechanism of how TAK1 regulates paneth cells.
Nanoanatomy Museum: Creating a Protein Family ProfileGrid Database
Authors: Roca Alberto
Published: June 2021

ProfileGrids allow the easy visualization of very large protein multiple sequence alignments (MSAs). Conserved and especially variable regions are represented as a matrix color‐coded according to the residue frequency occurring at each column position. While databases of protein families exist (such as Pfam), there are few curated repositories of user‐generated MSAs possibly due to the lack of paradigms for visualizing large MSAs. Here we present progress toward building a database of protein family ProfileGrids that we call the Nanoanatomy Museum. Our initial dataset was the pre‐calculated MSAs of the largest protein families from the Pfam database (ranging up to 160,000+ homologs). We describe our high‐throughput method for calculating ProfileGrids with the new JProfileGrid v2.0 software. It allows rapid and automated generation of ProfileGrids due to algorithm optimization, a command‐line interface, and a new PNG image file output format. The final database will be a proof of principle for how established databases can incorporate ProfileGrids in the standard description about protein families thus replacing other visualizations such as sequence logos.
Venous malformations: from causes to pathogenic mechanisms and murine models
Authors: Vikkula Miikka S.
Published: June 2021

Vascular malformations are localized errors of vascular development. They are often identified on the skin as “birthmarks” of various sizes and shapes but may be encountered in other organs, such as the liver, intestine and the brain. Venous malformations are the most commonly seen vascular malformations in specialized multidisciplinary centers, due to associated morbidity. Most are sporadic, but familial forms exist: VMCM, for mucocutaneous venous malformation, and GVM, for glomuvenous malformation. We unravelled that these hereditary forms are basically caused by a dominant activating TIE2 mutation (VMCM) or a loss‐of‐function glomulin mutation (GVM). The large clinical variability regarding the location, number and size of lesions, made us hypothesize that a somatic second‐hit may be needed, as suggested by Knudson for retinoblastoma. Proof for this has started to pile up. Moreover, our tissular screens have also identified local, somatic genetic defects that cause the most common sporadic form of venous malformations. These mutations differ from the inherited ones in regard to hyperphosphorylating effects and downstream transcriptomic profiles when expressed in HUVECs. These data highlight the importance of assessing for tissue‐based genetic changes, especially acquired genetic changes, as possible pathophysiological causes, which have been largely overlooked in developmental disorders.
Transcription factor Reb1p regulates DGK1‐encoded diacylglycerol kinase in Saccharomyces cerevisiae
Authors: Qiu Yixuan , Fakas Stylianos , Carman George
Published: June 2021

In the yeast Saccharomyces cerevisiae, DGK1‐encoded diacylglycerol (DAG) kinase catalyzes the CTP‐dependent phosphorylation of DAG to form phosphatidate. This activity is required for phospholipid synthesis during growth resumption from the stationary phase when de novo fatty acid synthesis is inhibited. Growth resumption was accompanied by the induction of DAG kinase activity and protein amount. The DGK1 promoter contains the sequence TTACCCG that is a binding site for the transcription factor Reb1p. This sequence in the DGK1 promoter was mutated to TTCACCG, and the DGK1 gene was expressed in dgk1Δ mutant cells. Cells expressing the mutant gene showed a defect in growth resumption from stationary phase, and a low constitutive level of DAG kinase activity and protein amount. Lipid analysis data showed that cells expressing the DGK1 gene with the mutation in the Reb1p binding site failed to mobilize triacylglycerol for membrane phospholipid synthesis as shown for cells that expressed the wild type DGK1 gene. The Reb1p mutation in a PDGK1‐lacZ reporter gene caused a decrease in β‐galactosidase activity when compared with the activity directed by the control wild type reporter gene. These results indicated that DGK1 expression was regulated by Reb1p, and that this regulation was important for growth resumption from stationary phase. Supported by NIH grant GM‐28140.
Clinical significance of organs vascularization: The role of vascular grafts for revascularization surgeries
Authors: Cabral Richard Halti , de Prates Nadir Eunice Valverde Barbato
Published: June 2021

The use of vascular grafts to bypass occluded arteries is still an important therapeutic option for revascularization surgeries. However, it is still not clear which factors may play positively for the longer durability of the grafts. The objectives of the present study is to identify morphological and structural parameters which may explain the different behavior between the most frequently used vascular grafts and also help choosing the best conduit for myocardial revascularization.

We have studied the morphology and the constitution of the extracellular matrix of the wall from the following vessels: coronary, internal thoracic, radial and gastromental arteries and saphena magna vein. Light microscopy, scanning electron microscopy and confocal microscopy were used for histological and morphometric study. The analyses of the vessel wall under different staining added to the information provided by the immunohistochemistry, allowed us to evaluate the extracellular matrix constitution of the arterial and venous grafts.

The three arterial grafts presented the same architectural pattern except for differences in layer thickness and also in the extracellular matrix distribution pattern. The proximal segment of the internal thoracic artery showed a double elastic layer in the adventicia, and the distal segment showed multiple elastic membranes in the media. Radial artery presented a collagen distribution pattern similar to the saphenous vein. No outer elastic limitant was seen in the venous conduit; however it presented lots of elastic fibers and two layers of smooth muscle cells in the media. It is worthy saying that the saphenous vein presented a wavy intima which could enhance its contact with platelets, facilitating thrombosis. In none of the of the studied vessel it was possible to identify vasa vasorum in the media layer.

We can conclude that there is not a major single factor that makes a vessel become the perfect graft. A combination of morphological features may help in the understanding of grafts behavior and give some clues of how we can deal with them to achieve better results.
Anthropometry in achondroplasia adults
Authors: Henry Bobbie , Koerner Celide , Alade Yewande , Schulze Kerry , Hoover-Fong Julie
Published: June 2021

Background

Achondroplasia is the most common short stature skeletal dysplasia with macrocephaly, lordosis, and rhizomelia. Elevated hypertension prevalence and earlier median cardiovascular disease death are prompting research to elucidate the cause in this population. Standardized anthropometry methodology, for correlation with health outcomes, is needed for accurate assessment of body measurements in achondroplasia.

Methods

20 adults with achondroplasia (11 M,9 F) had extensive anthropometric evaluation.

Results

Aberrant skinfolds, displaced anatomic landmarks, and excessive body fat necessitated modification of most measures and dismissal of triceps and subscapular skinfolds and iliac crest waist circumference. Reliable measurements requiring minimal or no modifications included maximal hip, umbilical and neck circumference, height and weight. Stratified by gender, only hip circumference (male 91.9±10.9 cm, female 109.6±9.3 cm) was statistically significantly different (p = 0.001).

Discussion

These measures were readily accessible without specialized equipment. Novel standardized methodology are now established for the clinical setting and future health outcomes research in achondroplasia.

Research support from the Alan and Kathryn Greenberg Center for Skeletal Dysplasias, McKusick‐Nathans Institute of Genetic Medicine, Johns Hopkins University
The effect of naringenin on the phosphorylation of AMPK in diet‐induced obese mice
Authors: Ke Jia-Yu , Tian Min , Kliewer Kara , Belury Martha A
Published: June 2021

Previously, we reported that naringenin, the predominant flavanone in grapefruit, acted like metformin to decrease glucose output from hepatocytes. The objective of this study is to investigate whether dietary naringenin modulates energy balance and metabolism through p‐AMPK, a central regulator of glucose and lipid metabolism in several tissues. Male FVB mice were fed a high fat diet for 19 days, and then switched to AIN‐93G diet with (N= 10) or without (N=8) naringenin for 13 days. Body weight and food intake of mice fed naringenin were not significantly different than values in mice fed diet without naringenin. Mice were fasted for 12 hr then necropsy performed. There was a trend of enhanced p‐AMPK:AMPK in quadriceps muscle in mice fed diet with naringenin but p‐AMPK:AMPK was not significantly different in liver or inguinal fat pad. The induction of p‐AMPK in skeletal muscle suggests that naringenin exerts effects on energy pathways regulated by AMPK. Currently, we are conducting a new study to investigate the effects of various doses of naringenin on p‐AMPK activity and energy balance and metabolism in a mouse model for diet‐induced obesity.
Ascorbic acid‐induced reduction in adiponectin is not improved by alpha tocopherol and is accompanied by a small reduction in protein levels of Peroxisome Proliferator‐ Activated Receptor Gamma in 3T3‐L1 adipocytes
Authors: Narayanan Vijaya , David Leslie , Kane Christopher
Published: June 2021

Adipocytes play a role in metabolism and regulation of homeostasis through the release of cytokines such as adiponectin. Adiponectin is an anti‐inflammatory adipokine and has been shown to improve insulin sensitivity and enhance beta‐oxidation of fatty acids. A reduction in adiponectin has been associated with increased inflammation, metabolic syndrome and chronic diseases. Since oxidative stress has been known to regulate metabolic disease, we previously investigated the role of ascorbic acid and found that it abolished adiponectin expression in adipocytes. We hypothesized that treatment with ascorbic acid may cause a reduction in PPARgamma protein levels and that alpha tocopherol may mitigate the effect of ascorbic acid on adiponectin synthesis. We used 3T3‐L1 cells to monitor adiponectin protein levels. Differentiated 3T3‐L1 (Day 8), cells were treated with ascorbic acid, with and without alpha tocopherol. We found that the reduction of adiponectin in cell lysates was not overcome with the addition of alpha tocopherol. Further, we also observed a small reduction in PPARgamma in adipocytes treated with ascorbic acid. These results suggest that adipocyte metabolism may be influenced by dietary antioxidants. This research was funded by Florida International University.
A moderate carbohydrate‐restricted diet results in weight loss and improves clinical parameters of metabolic syndrome in adult men and women and addition of egg yolk further improves inflammation
Authors: Blesso Christopher N , Andersen Catherine J , Barona Jacqueline , Volk Brittanie , Volek Jeff S , Fernandez Maria Luz
Published: June 2021

Men and women (n = 18) with metabolic syndrome (MetS) were instructed to follow a carbohydrate (CHO)‐restricted diet (<30% energy) and randomly assigned to consume either 3 whole eggs (EGG, n = 9) or egg substitute (SUB, n = 9) per day for 12 wk. Dietary intake, parameters of MetS, inflammatory markers and body composition by DXA were assessed at baseline, wk 6 and wk 12. Total calories (p<0.0001), total CHO (p<0.0001), % CHO energy (p<0.001), and glycemic index (p<0.0001) decreased in all subjects over time. Reductions in weight (p<0.0001), body mass index (p<0.0001), fat mass (p<0.0001), and trunk fat (p<0.0001) were also observed. MetS parameters were improved with increases in plasma HDL‐cholesterol (p<0.01) and decreases in waist circumference (p<0.01), plasma glucose (p<0.05) and plasma triglycerides (TG) (p<0.01) with no differences between groups. Reductions in total CHO correlated with reductions in weight (0.484, p<0.05) and plasma TG (0.483, p<0.05). The EGG group consumed more cholesterol (p<0.0001) and choline (p<0.0001) than the SUB group. There was a greater reduction in plasma tumor necrosis factor‐α (TNFα) in EGG vs. SUB groups (p< 0.05) and increases in adiponectin (0.419 vs 0.014 mg/L). These results suggest that the addition of egg yolk does not suppress the beneficial effects of carbohydrate‐restriction in MetS and may modulate plasma TNFα and adiponectin. [Supported by the American Egg Board].

Grant Funding Source: American Egg Board/Egg Nutrition Center
Exploratory analysis of facilitators and barriers to weight loss maintenance in premenopausal women
Authors: Metzgar Catherine J , Nelson Sarah A , Preston Amy G , Miller Debra L , Nickols-Richardson Sharon M
Published: June 2021

Weight loss maintenance (WLM) is critical for long‐term health, yet difficult to sustain. Methods to qualitatively explore facilitators and barriers to WLM in premenopausal women with overweight/obesity will be described. Women will be recruited from an original, 18‐wk randomized clinical trial that promoted WL via an energy‐restricted diet that included one sugar‐free cocoa beverage and two dark chocolate snacks/d (n= 30) or one sugar‐free non‐cocoa beverage and two licorice snacks/d (n=30). In this follow‐up study, success with WLM will be explored. Three focus groups of 3–5 women/group will be conducted. Focus groups will be guided by a facilitator using open‐ended questions and probes. Topics for questions will include perceptions of snacking, WL and WLM success, self‐efficacy, stages of change, and facilitators/barriers to WLM. Focus groups will be held in a private room and tape‐recorded. Phenomenologic analysis will be used to evaluate transcripts and will be independently coded by two investigators. Common themes will be identified. Women will complete questionnaires to gather demographic data. Descriptive statistics will be used to characterize women. Findings are expected to provide insight into development of future interventions that facilitate WLM in premenopausal women. Funded by The Hershey Center for Health and Nutrition, The Hershey Company, Hershey, PA.
Body weight reduction by exercise is more effective than by dietary restriction even when dietary condition is equal except for energy
Authors: Yamada Tetsuo , Kurasawa Shin-ichi , Matsuzaki Masami , Tanaka Akira
Published: June 2021

Composition of reducing diet during weight‐loss programs has not been examined in detail. We investigated the effects of reducing body weight by exercise and by dietary restriction on lipid metabolism and nutritional status.

Methods

In crossover trial 1, six adult male volunteers participated in two experiments over ten days. The first five days were an adjustment period (energy intake, 2,656 kcal/day; mean). During the second five days, the participants either exercised on a bicycle ergometer to consume 886 kcal/day or reduced their energy intake to 1,770 kcal/day by reducing all foods in proportion to the energy intake. In crossover trial 2, similar experiments were carried out reducing only carbohydrate and lipid for dietary restriction.

Results

In trial 1, serum lipid levels were significantly improved more after exercise (Ex) than after dietary restriction (DR), but total protein and albumin levels unchanged. Whereas nitrogen and potassium balances were significantly more positive during Ex than during DR in only trial 1, urinary 3‐methylhistidine, adrenaline and nor‐adrenaline excretion levels were larger during Ex than during DR in both trials.

Conclusions

Body weight reduction by exercise brings larger muscle protein turnover and more vigorousness than by dietary restriction even when dietary condition is equal except for energy. This study was supported by a grant from Kanto Gakuin University.
Anti‐hyperglycemic Effect of 2 Amadori Rearrangement Compounds, Arginyl‐fructose and Arginyl‐fructosyl‐glucose
Authors: Kwon Young-In , Ha Kyoung Soo , Jo Sung-Hoon , Apostolidis Emmanouil , Jang Hae-dong , Lee Mee sook
Published: June 2021

During the heat processing of raw ginseng to produce red ginseng, amino acid derivatives such as arginyl‐fructose (AF) and arginyl‐fructosyl‐glucose (AFG) are formed at high levels, through Amadori rearrangement, the early step of Maillard reaction, from arginine and glucose or maltose, respectively. However, very limited information is available about the effect of structural difference between AF and AFG on various biological activities. This is the first report of the mode of action and effect of AF and AFG on the type 2 diabetes management related inhibition of postprandial hyperglycemia in vitro and in animal model. In our previous study, standards AF and AFG were chemically synthesized and in this study their inhibitory activities against rat intestinal α‐glucosidases and porcine pancreatic α‐amylase were investigated in vitro. The effect of AF and AFG on postprandial blood glucose increase after meal was investigated in Sprague Dawley (SD) rats fed on starch or sucrose meals. Both Amadori compounds significantly reduced the postprandial blood glucose levels after starch or sucrose loading. These results indicate that AF and AFG, Maillard reaction products, may have anti‐diabetic effect by suppressing carbohydrate absorption in the gastrointestinal level, and thereby reducing the postprandial increase of blood glucose.

Grant Funding Source: National Research Foundation of Korea (NRF)