bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxygen Sensing by the HIF Pathway
Authors: Simon M Celeste
Published: June 2021

Prolyl hydroxylation of hypoxible‐inducible factor alpha (HIF‐α) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O2)‐replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O2 deprivation (hypoxia). Using a fusion protein containing the human HIF‐1α O2‐dependent degradation domain (ODD), we monitored PHD activity both in vivo and in cell‐free systems. This novel assay allows the simultaneous detection of both hydroxylated and nonhydroxylated PHD substrates in cells and during in vitro reactions. Importantly, the ODD fusion protein is regulated with kinetics identical to endogenous HIF‐1α during cellular hypoxia and reoxygenation. Using in vitro assays, we demonstrated that the levels of iron (Fe), ascorbate, and various tricarboxylic acid (TCA) cycle intermediates affect PHD activity. The intracellular levels of these factors also modulate PHD function and HIF‐1α accumulation in vivo. Furthermore, cells treated with mitochondrial inhibitors, such as rotenone and myxothiazol, provided direct evidence that PHDs remain active in hypoxic cells lacking functional mitochondria. Our results suggest that multiple mitochondrial products, including TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, and cellular responses to O2 depletion.
Kinetic and thermodynamic assessment of binding of serotonin transporter (SERT) inhibitors
Authors: Martin Renee , Henningsen R , Suen A , Stockdale D , Apparsundaram S , Milla M E
Published: June 2021

Considering their shared mechanism of action, there is surprising diversity in the clinical outcomes of SERT inhibitors. Such behavior may arise from the binding modes of those inhibitors with SERT. We report here on the thermodynamics of ligand‐SERT interactions determined from equilibrium and kinetic binding analyses. All drugs excepting fluvoxamine displayed remarkably similar binding thermodynamics with relatively equal contributions of entropy and enthalpy to free energy of binding. Binding enthalpies correlate with ligand polar surface areas suggesting an allowance for charged/polar interactions without enormous energetic penalties for dehydration. In the case of fluvoxamine, which possesses the greatest conformational flexibility yet binds entropically, flexible interaction is implied such that the entropic penalty expected from conformational restriction is limited. Rapid association was observed for all ligands, reflecting low energy barriers and diffusion‐limited binding reactions. The inhibitors bind in a mutually‐exclusive manner with each other as well as with 5‐HT. Taken together, the antidepressant binding site is probably solvent exposed, possibly along the substrate transport pore as predicted from structural modeling based on the leucine transporter.
Structural insights into metabolite‐sensing mRNAs
Authors: Batey Robert T.
Published: June 2021

Riboswitches are structured elements found in the 5′‐untranslated regions of mRNAs that specifically bind small molecule metabolites to regulate gene expression in a cis‐fashion. These riboregulatory elements typically contain two domains: an aptamer domain that specifically recognizes and binds a ligand and an expression platform that directs expression of the mRNA at the transcriptional or translational level. Our laboratory has solved the X‐ray crystal structures of the aptamer domains of the purine and two S‐adenosylmethionine responsive riboswitches in complex with their cognate ligands, revealing complex tertiary architectures that scaffold the ligand‐binding pocket. In each case, almost all of the functional groups of the ligand are directly or indirectly read by the RNA resulting in their extremely high specificity. To complement these bounds structures, we have used biochemical and structural methods to reveal the nature of the unligand form of these aptamer domains, illuminating aspects of their ligand‐dependent folding. Regulation is achieved through a series of ligand‐induced tertiary structural changes in the RNA that serve to stabilize a helix that forms part of a secondary structural switch with the expression platform.
Influence of dietary availability of protein and carbohydrate on ERK1/2 and Akt signaling in rat liver
Authors: Zhang Zhi‐Ying , Devkota Suzanne , Walker Denise , Layman Donald Keith , Pan Yuan‐Xiang
Published: June 2021

The dietary reference intakes (DRIs) established an acceptable macronutrient distribution range (AMDR). However, the ideal ratio of dietary protein to carbohydrate for adult health and its nutritional regulation remains unknown. The present study examined the regulation of p44/42 mitogen activated protein kinases (MAPK) pathway and AKT pathway in rats fed with different levels of dietary proteins. Male rats (n=36,~200 g) were trained to meal feed on a Baseline diet (20% of energy as protein, 50% carbohydrates, 30% fat) for 6 days and sacrificed (day 0) at fasted or 90 minutes postprandial (pp), to a 4g breakfast meal. The remaining rats were randomly assigned to either a low‐protein diet (LP group: 15% protein, 55% carbohydrates, 30% fat) or a high‐protein diet (HP group: 30% protein, 40% carbohydrates, 30% fat) for 21 days and sacrificed at fasted or 90 min pp. Protein and RNA samples were extracted from rat liver tissue and western blot were used to detect the protein expression. Phosphorylation of ERK1/2 was significantly highest in LP diet group, followed by HP group and Baseline diet group. In contrast, phospho‐AKT(Ser 473) was lowest in LP group, followed by Baseline diet group and HP diet group. No significant postprandial changes in p‐ERK1/2 and p‐AKT were observed. For phospho‐ERK1/2, three bands were seen in 30% HP diet 90min pp group, indicating high protein diet may induce additional modification in phospho‐ERK1/2. This study demonstrates that changes in protein and carbohydrate intakes within the AMDR results in different nutritional regulation of MAPK pathway and AKT pathway, which may play an important role in adult health.
Nutritional and hormonal regulation of angiotensinogen in human adipose tissue
Authors: Fletcher Sarah , Lee Mi‐Jeong , Heo Young‐Ran , Kalupahana Nishan , Hall Lorin , de Barros‐Mazon Silvia , Glynn Nicole M , Voy Brynn , Fried Susan K , Moustaid‐Moussa Naima
Published: June 2021

White adipose tissue (WAT) is a dynamic endocrine tissue that secretes several adipokines important in whole body homeostasis. Angiotensinogen (AGT), the only known precursor for the hypertensive adipokine angiotensin II (Ang II), is highly expressed in WAT. The purpose of this work is to investigate nutritional (fatty acids) and hormonal (insulin and glucocorticoid) regulation of AGT expression and Ang II secretion in human WAT. AGT protein level and Ang II synthesis were higher in omental vs. subcutaneous WAT; results were consistent for both freshly isolated samples and for WAT maintained in culture. Ang II secretion positively correlated with BMI and adipocyte size. Although the level of secretion and induction were quantitatively different among patients, all fatty acids regardless of the length or degree of unsaturation exerted positive, dose‐ and time‐ dependent effects on Ang II secretion, compared with vehicle‐treated tissues. Further, AGT protein levels were increased by insulin alone and further enhanced by combined insulin and dexamethasone treatment. In conclusion, human adipose AGT and Ang II are regulated in a depot‐specific manner and induced by fatty acids, insulin and glucocorticoids, confirming the importance of this gene in metabolic and nutritional disorders associated with obesity.
Dietary vitamin E is critical for T cell trafficking to the brain during infection
Authors: Sheridan Patricia Ann , Beck Melinda A
Published: June 2021

Previous studies from our laboratory have shown that dietary a‐tocopherol (VE) is critical to regulating the cytokine and chemokine response in the brain to herpes simplex virus (HSV) infection. In addition to the cytokine response, the timing of T cell infiltration is critical to the resolution of central nervous system HSV‐1 infections. Specifically, the appearance of “neuroprotective” IFN‐g‐producing CD8+ T cells is crucial. During CNS infection CD8+ T cell priming and expansion occurs in the draining lymph node, followed by recruitment and expansion occurs in the spleen with subsequent accumulation in the brain. In order to determine how VE deficiency and VE supplementation would alter T cell trafficking during HSV‐1 infection of the brain, we placed weanling male BALB/cByJ mice on VE deficient (Def), adequate (Adq) or 10X supplemented (Sup) diets for 4 weeks and then infected the mice intranasally with HSV‐1. VE Def mice had suppressed antigen‐specific T cell trafficking to the brain. In contrast, VE supplementation had no effect on T cell trafficking. In contrast to both VE Def and VE Adq mice, VE Sup mice did not increase splenic regulatory T cells in response to infection. Our results demonstrate that adequate levels of VE are important for trafficking antigen‐specific T cells to the brain and dietary VE levels modulate T regulatory and dendritic cells in the periphery. Support:P30ES10126 and DK56350.
Rapid synthesis and degradation of the Regulated in Development and DNA damage (REDD1) protein in response to IGF‐I in C2C12 myotubes
Authors: Frost Robert A , Lang Charles
Published: June 2021

REDD1 is a stress response gene whose protein product negatively regulates signaling through the mammalian target of rapamycin (mTOR) while desensitizing the response of cells to apoptosis. Given the anabolic and protective affect of insulin‐like growth factor‐I (IGF‐I) on skeletal muscle we examined the regulation of REDD1 by IGF‐I in C2C12 myotubes. IGF‐I rapidly increased the expression of REDD1 mRNA 3‐fold. IGF‐I‐induced REDD1 expression was blocked by inhibitors of transcription and translation. IGF‐I withdrawal resulted in a rapid 70% loss of REDD1 protein within 2h. Although the loss of REDD1 was blocked by inhibition of the proteasome this was negated in the presence of cycloheximide. Consistent with this finding, MG132 increased REDD1 expression. Both IGF‐I and insulin stimulated REDD1 and they had an additive affect with dexamethasone another inducer of REDD1. IGF‐I‐stimulated REDD1 was mTOR‐dependent based on its inhibition by rapamycin. Although IGF‐I and MG132 induced REDD1 these agents had little affect on signaling downstream of mTOR. In vivo both IGF‐I and insulin stimulated REDD1 expression in the gastrocnemius. Our results suggest the REDD1 gene is positively regulated by IGF‐I and that endogenous REDD1 expression has little affect on mTOR signaling under these conditions. We speculate that increased REDD1 may function to protect muscle during stress conditions.
Retinoid storage in hepatic stellate cells
Authors: Libien Jenny , Oxberry William C. , O'Byrne Sheila , Blaner William S.
Published: June 2021

The liver is the main site of retinoid storage in the body. Within the liver, retinyl esters are hydrolyzed to retinol within hepatocytes and then may be secreted from the liver into the circulation bound to retinol‐binding protein (RBP) or may be re‐esterified by lecithin:retinol acyl transferase (LRAT) to form retinyl ester. Retinyl esters are then stored within the lipid droplets of hepatic stellate cells. We have previously reported that LRAT −/− mice have impaired absorption and storage of retinyl esters and have hepatic stellate cells devoid of their characteristic lipid droplets. Immunohistochemical studies of mouse liver using a monoclonal antibody against LRAT show expression primarily in hepatic stellate cells. We propose that LRAT is necessary for the formation of the lipid droplet by the stellate cell. To determine how retinoids are released from the lipid droplet, histologic examination of livers of RBP−/− mice was performed. In 18 month‐old RBP−/− mice, hepatic stellate cell lipid droplets were markedly enlarged. In 31 month‐old RBP−/− mice, numerous lipogranulomas, composed of lipid laden macrophages, were identified. EPON‐embedded thin sections and electron microscopy showed that the lipogranulomas were centered around hepatic stellate cells. Minimal steatosis of the RBP−/− livers was present. We propose that in the absence of RBP, hepatic stellate cell lipid droplets become distended and leak lipid eliciting a granulomatous reaction. The findings suggest that LRAT is necessary for lipid droplet formation and RBP is involved in mobilization of retinyl esters from lipid droplets in hepatic stellate cells. This work was supported by grants R01 DK061310, DK068436 and DK079221.
Relative bone mass decreased in mice fed high dietary fat despite an increase in body mass and bone formation markers
Authors: Cao Jay
Published: June 2021

Osteoporosis and obesity are interrelated health disorders. Osteoblasts and adipocytes are derived from common mesenchymal stem cells and age‐related osteoporosis is associated with increased bone marrow adipogenesis. To determine whether bone mass and osteoblast number and activity are affected by dietary fat intake, six‐week‐old male C57BL/6 mice were randomly assigned to two groups (n=10–11/group) and fed either a high fat diet (HFD, 45% kcal as fat) or a low fat control diet (LFD, 10%) for 14 weeks. Bone marrow stromal cells (BMSCs) were cultured and bone architecture was evaluated by micro computed tomography. Mice fed HFD were 31% heavier (P<0.01) than those fed LFD, 41±6 vs. 31±2g, respectively. Higher numbers of alkaline phosphatase (ALP) positive colonies and calcium nodules were formed by BMSCs from HFD than from LFD mice (P<0.01). At d 14 of culture, ALP mRNA levels in BMSCs from HFD were higher (P<0.05) than from LFD mice. There were no significant differences in tibial fat‐free weight, length, and mid‐shaft diameter between the two groups (P>0.05). Although HFD increased total tibial volume by 14% as compared with LFD (2.05±0.15 vs. 1.80±0.29 mm3, respectively, P<0.05), bone volume relative to body mass decreased (8.5±1.8 vs. 11.2±3.4 mm3/kg, respectively). Our data suggest that feeding HFD results in bone resorption that exceeds any positive effect that increased body weight has on bone formation.
Wnt / RANKL‐mediated bone growth promoting effects of blueberries in weanling rats
Authors: Chen Jin‐Ran , Lazarenko Oxana P. , Badger Thomas M. , Ronis Martin J.J.
Published: June 2021

We studied the effects of dietary blueberry supplementation on bone growth in weanling rats. Weanling male and female rats were fed AIN‐93G semi‐purified diets supplemented with 10% whole blueberry powder for 14 and 30 days beginning on PND 21. In both sexes tibial bone mineral density and content and cortical bone thickness were all increased dramatically in blueberry‐fed rats as compared to control casein AIN‐93G fed animals (p < 0.05). The serum bone formation marker alkaline phosphatase (ALP) increased but the resorption marker RatLaps decreased in blueberry‐fed rats. Ex‐vivo osteoclastogenesis and osteoblastogenesis assays and bone histomorphometric studies revealed decreased osteoclastogenesis and increased osteoblastogenesis in blueberry‐fed rats compared to controls (p < 0.05). In contrast to control diets, gene expression from both bone and bone marrow of blueberry‐fed rats showed dramatic decreases in RANKL and tartrate resistant acid phosphate (TRAP) mRNAs but increases in bone formation markers ALP, osteocalcin, RUNX2 and the Wnt signaling related components Wnt5a, LRP5, LRP6 mRNAs. Beta‐catenin protein level was increased in blueberry‐fed rats in bone tissue relative to control rats (p < 0.05). These results indicate significant stimulation of bone growth associated with blueberry consumption in early development. Supported in part by ARS CRIS #6251‐51000‐005‐03S.
Effect of green tea polyphenols on chronic inflammation‐induced bone loss in female rats
Authors: Shen Chwan‐Li , Yeh James K. , Liu Xiao‐Qing , Dunn Dale M , Stoecker Barbara J , Wang Piwen , Tang Yuntian , Wang Jia‐Sheng
Published: June 2021

Our recent study demonstrated a bone‐protective role of green tea polyphenols (GTPs), extracted from green tea, in estrogen‐deficiency‐induced bone loss of middle‐aged female rats through an increase of antioxidant capacity and/or a decrease of oxidative stress damage. This study further examines effects of GTPs on chronic‐inflammation‐induced bone loss of female rats. A 2 (placebo vs. lipopolysaccharide, LPS) × 2 (no GTPs vs. 0.5% GTPs in drinking water) factorial design using 40 female rats (3‐month‐old) assigned to 4 groups (n=10/group): placebo (P, placebo implantation), LPS implantation (L), P+0.5% GTP (PG), and LPS+0.5% GTP (LG) for 12 wks. After 12 weeks, LPS resulted in a significant increase in inflammation index based on white blood cell count (WBC), accompanied by a significant decrease in bone mineral density (BMD). Supplementation of GTP significantly lowered the WBC and increased BMD. Neither LPS nor GTP affected cortical bone parameters measured by microcomputed tomography at femur midshaft. These results demonstrated that GTPs supplementation mitigates LPS‐induced bone mineral density loss in female rats. Such a protective role of GTPs may be, in part, attributed to decreased inflammation. This study suggests a potentially significant prophylactic role of green tea in bone health of women with chronic inflammation‐induced bone loss.
Compound A from Plantago asiatica essential oils reduced plasma triglyceride and cholesterol levels and suppressed the expression of 3‐hydroxy‐3‐methyl‐glutaryl‐Coenzyme A reductase in the liver of diet‐induced obese mice
Authors: cho sungyun , Chung Mija , Lee Sung‐joon
Published: June 2021

Plantago asiatica is a commonly used folk medicine in Eastern Asia and its essential oils have various bioactive compounds. Previously, we have showed that Plantago asiatica essential oils (PAEO) have hypolipidemic effects. Since compound A(82.6%) was a major component in the PAEO, we further examined hypolipidemic effects of compound A as well as the PAEO in diet‐induced obese mice. Mice (C57BL/6CrSlc) were fed a high fat diet for 5 weeks and then compound A and the PAEO were orally administered for 6 weeks under high fat diet. The PAEO and the compound A feeding significantly reduced plasma total cholesterol and triglyceride levels after 3 and 6 weeks, respectively, compared with the controls. Compound A reduced plasma cholesterol concentrations by approximately 20%. We investigated the effects of the PAEO and compound A on the expression of cholesterol metabolism genes. The expression of the HMG‐CoA reductase, assessed by RT‐PCR, was significantly reduced by 50% in the livers of compound A‐fed mice compared with the control livers after 6 weeks. Our data suggest that compound A, a major compound in the PAEO, may exert hypocholesterolaemic effects by downregulation of the HMG‐CoA reductase expression in diet‐induced obese mouse.
Induction of immune tolerance through an IL‐10 dependent mechanism allows Entamoeba histolytica successful cololization in the colon
Authors: Rybicka Joanna Malgorzata , Chadee Kris
Published: June 2021

Entamoeba histolytica infects 10% of the world's population, which can lead to amoebic dysentery and/or liver abscess. The majority of infected individuals are completely asymptomatic. We hypothesized that E. histolytica is capable of inducing immune tolerance in antigen presenting cells thus promoting parasite survival in the colon in the absence of an overt inflammatory or immune response. In support of this, we show that dendritic cells (DC's) and macrophages (mφ) exposed to amoeba secreted proteins can induce a significantly up‐regulation of IL‐10 mRNA expression and increase phosphorylation of ILT2. Furthermore, low concentrations (10μg/mL) of amoebic proteins caused rapid 3–8‐fold up‐regulation of ILT2 inhibitory receptor on CD4+ T cells in the presence of mφ or DCs 36 h following exposure. However, there was no significant up‐regulation of ILT2 if T cells were deficient in IL‐10. Moreover, amoebic proteins caused a significantly increased in apoptosis in IL‐10 deficient T cells after 48 or 72 hr as compared to wild type controls. We conclude that E. histolytica is capable of inducing IL‐10 production in antigen presenting cells, which is required for up‐regulating ILT2 on CD4+ T cells and inhibition of T cells apoptosis. This work was supported by NSERC. JMR is a recepient of the QE II Graduate Scholarship from the Province of Alberta and CAG/CIHR studentship.
Helicobacter pylori activates μ‐calpain to cleave E‐cadherin in epithelial monolayers
Authors: O'Connor Pamela M , Lapointe Tamia K. , Buret Andre G.
Published: June 2021

H.pylori (Hp) is a risk factor for gastritis, gastric ulcers, and adenocarcinoma. The ability of Hp to disrupt epithelial intercellular adhesion may influence disease severity. Disruption of adherens junction (AJ) protein E‐cadherin has a well‐established role in carcinogenesis. Calpain family proteases are upregulated in several types of adenocarcinoma and can cleave E‐cadherin to a functionally‐impaired 100kDa form. AIMS: 1) to determine the effects of Hp on calpains, 2) to assess the role of calpains in Hp‐induced AJ alterations. RESULTS: Epithelial (SCBN) monolayers challenged with Hp strain SS1 or 60190 for 24h had increased calpain activity and elevated levels of the cleaved form of the calpain substrate α‐spectrin, as determined by activity assay and immunoblotting. Immunoblotting for the μ and m calpain isoforms revealed that μ, but not m, calpain is activated in response to Hp. Immunostaining and western blotting of subcellular fractions revealed loss of membrane‐associated E‐cadherin and increased cytosolic E‐cadherin. Western blotting of whole cell lysates demonstrated that Hp challenge induced cleavage of the 120kDa full‐length E‐cadherin to a 100kDa form. Calpain inhibition prevented these junctional defects. CONCLUSION: Carcinogenic Hp induces isoform‐specific calpain activation which results in E‐cadherin truncation in non‐tumorigenic epithelial cells. This work is funded by CIHR.
Blockade of host CD147 by small interfering RNAs suppresses growth of human colon cancer xenografts
Authors: Abraham Dietmar , Zins Karin , Sioud Mouldy , Lucas Trevor , Aharinejad Seyedhossein
Published: June 2021

Tumor cells stimulate matrix metalloproteinase (MMP) production by stromal cells through cell–cell interactions mediated by adhesion molecules such as the extracellular MMP inducer (human CD147/EMMPRIN, mouse basigin). This study investigated whether tumor‐stromal cell interactions mediated by CD147 promote colon cancer growth by utilizing small interfering (si)RNAs directed against EMMPRIN or basigin in co‐cultures of cancer cells with macrophages and fibroblasts and human SW620 colon cancer xenografts in immune deficient mice. Blocking host basigin but not cancer cell‐derived EMMPRIN expression suppressed tumor growth in colon cancer xenografts illustrating the complex role of CD147 in tumor growth. Experiments in vitro showed CD147 mediated SW620‐stromal cell interactions led to increased MMP‐2 expression in fibroblasts but not macrophages. Expression of host vascular endothelial growth factor‐A in both fibroblasts and macrophages was independent of CD147 in vitro and in vivo. However, inhibition of cancer cell‐derived EMMPRIN leads to increased MMP‐9 levels in vivo. Our findings provide new insights into CD147‐mediated tumor‐host interactions regulating colon cancer growth and may lead to the development of novel siRNA‐based therapeutic strategies to treat patients with colon cancer.
Pegylated‐Interferon 2α (Peg‐IFN) suppresses β‐catenin signaling in liver
Authors: Thompson Michael David , Monga Satdarshan
Published: June 2021

Peg‐IFN is an effective first line therapy in treatment of chronic Hepatitis C virus (HCV) infection. Interestingly, HCV patients treated with Peg‐IFN are less likely to develop hepatocellular carcinoma (HCC). HCC remains the most common form of liver cancer and activation of the Wnt pathway is observed in a significant subset. We explored the possibility that Peg‐IFN may act through inhibition of Wnt signaling. Treatment of 2 human hepatoma cell lines (HepG2 & Hep3B) with various concentrations of Peg‐IFN led to a decrease in the active form of β‐catenin. Likewise, β‐catenin dependent transcriptional activity was decreased by up to 50% as measured by the TOPFLASH luciferase reporter assay. To examine Peg‐IFN.s effects in vivo, transgenic mice overexpressing a stabilized, mutant form of β‐catenin were exposed to DEN and treated with Peg‐IFN (7,000 or 70,000 U) for six weeks. Treatment with Peg‐IFN decreased nuclear β‐catenin compared to control. This was associated with a decrease in Ki‐67 positive hepatocytes. No change in the transcription of β‐catenin was evident suggesting the effect to be through a post‐ translational mechanism. Thus Peg‐IFN effectively downregulates Wnt/β‐catenin signaling and might have important treatment implications in HCC.
Signaling pathways regulating the airway phenotype of mice exhibiting airway surface dehydration
Authors: Livraghi Alessandra , Grubb Barbara R. , O’Neal Wanda K. , Mall Marcus A. , Boucher Richard C. , Randell Scott H.
Published: June 2021

Overexpression of the epithelial Na+ channel βsubunit (gene Scnn1b) in mouse airways results in airway surface dehydration, neonatal mortality, mucous cell metaplasia and airway inflammation. Crossbreeding Scnn1b‐Tg mice with gene‐deficient mice enables quick and efficient determination of specific pathways relevant to the development of lung pathology. We bred Scnn1b‐Tg mice with mice deficient in: 1) IL‐4 receptor alpha chain (IL‐4Rα), shared by IL‐4 and IL‐13 receptors; 2) TNFα; 3) TNFα receptor 1 (TNFαR1), and evaluated inflammatory infiltrate, cytokine profile, lung pathology, and airway bioelectrics. In Scnn1b‐Tg mice, absence of IL‐4Rα improved neonatal survival and was associated with decreased mucous cells and eosinophilic infiltrate at day 10. However, absence of IL‐4Rα did not affect the chronic inflammatory infiltrate and lung pathology in adult Scnn1b‐Tg mice. Although TNFα was increased in Scnn1b airways, absence of TNFα or TNFαR1 did not ameliorate the lung phenotype of Scnn1b‐Tg mice nor modify airways bioelectrics. To assess the contribution of IL‐1β and TLR pathways, we are currently breeding the Scnn1b‐Tg mice with mice deficient in the adaptor molecule MyD88. These results indicate that multiple pathways contribute to airway inflammation and remodeling in the setting of surface dehydration and mucus stasis.

Funded by CFF grants LIVRAG04I0 and RANDEL07P0, and NIH P50 HL060280 to R.C.B
Nanostructured substrates reveal myosin‐mediated mechanotransmission in endothelium
Authors: Helmke Brian P. , Zawislak Jennifer D. , Mott Rosalind E. , Kolasinski Kurt W.
Published: June 2021

Intracellular mechanical strain focusing implicates the cytoskeleton in transmission of external force to intercellular junctions, but whether mechanotransmission within the endothelium requires active contractility remains undetermined. Here, endothelial cells were grown on nanostructured substrates consisting of pillars with height 10 μm, pitch 6 μm, and tip diameter 0.1 μm. Cells formed nascent focal adhesions and fibronectin matrix fragments, but focal contact maturation and matrix fibrillogenesis were inhibited. In subconfluent layers, pillars impinged significantly on both the cell cytoplasm and nucleus, suggesting that cells relaxed mechanically around the 3D topography. In confluent monolayers, actin stress fiber and VE‐cadherin distributions were indistinguishable from those in confluent monolayers on coverslips, and pillars were no longer detectable. Inhibition of myosin IIA with blebbistatin caused cells in a confluent layer to relax around the pillars. Thus, myosin‐mediated internal tension was required for monolayer stabilization above the pillars, suggesting that active tension among adjacent endothelial cells in a monolayer dominates mechanotransmission. Supported by NIH grant HL071958.
Control of microvascular tube assembly by endothelial cell‐pericyte interactions
Authors: Davis George E , Stratman Amber N. , Koh Wonshill , Sacharidou Anastasia , Davis Michael J.
Published: June 2021

Endothelial cell (EC) tube morphogenesis is controlled by downstream signals derived from a cell surface metalloproteinase, MT1‐MMP, the Rho GTPases, Cdc42 and Rac1, and the α2β1 integrin. These signals promote lumen formation and interconnection of neighboring cells to form multicellular tube networks in 3D collagen matrices. This process depends on Cdc42‐mediated signaling and involves the downstream effectors, Pak2, Pak4, the cell polarity protein, Par3, and the protein kinase C isoforms, epsilon and zeta. Also, during tube formation, ECs generate MT1‐MMP‐dependent proteolyzed spaces, termed vascular guidance tunnels, which represent physical imprints in the matrix allowing for rapid cellular movement and tube remodeling. In EC‐pericyte cocultures, marked recruitment of pericytes occurs into these vascular guidance tunnels and they are distributed in a polarized fashion along the ablumenal surface of EC tubes. These tunnels facilitate dynamic migration of ECs and pericytes, leading to deposition of basement membrane matrix components (only occurs with pericytes) and EC tube stabilization. Thus, EC microvascular tube assembly is a function of initial EC signaling and morphogenic events leading to vascular guidance tunnel formation and downstream stabilization signals catalyzed by EC‐pericyte interactions within these tunnel spaces. Supported by NIH grants HL59373 and HL79460.
Nuclear mRNA export requires coordinated FG‐Nup binding and DEAD‐box protein‐mediated remodeling events
Authors: Wente Susan R. , Terry Laura J. , Alcazar‐Roman Abel R. , Tran Elizabeth J.
Published: June 2021

Nucleocytoplasmic transport of proteins and nucleic acids is essential for proper gene expression. Using the Saccharomyces cerevisiae model, we discovered key events for directional export of messenger (m) RNA. Distinct steps were pinpointed at nuclear, central and cytoplasmic nuclear pore complex (NPC) structures. Nearly half of the ∼30 NPC proteins harbor Phe‐Gly (FG) repeat domains that potentially serve as binding sites for the shuttling mRNA export receptor Mex67. A large‐scale deletion strategy was employed to generate mutants lacking discrete combinations of FG‐domains, and specific subsets of FG domains were required for mRNA export. The FG mutants also revealed multiple functionally independent translocation routes through NPCs. mRNA export required FG domains on the nuclear NPC face (presumably for initial docking at the NPC) and in the NPC central core. At the cytoplasmic NPC face, we found that the RNA‐dependent ATPase activity of the DEAD‐box protein Dbp5 was stimulated by inositol hexakisphosphate‐bound Gle1. The ADP‐bound form of Dbp5 triggered release of the RNA‐binding protein Nab2 from RNA. This novel nucleotide‐dependent switch mechanism remodeled mRNA‐protein complexes for directional translocation. We predict that these FG‐binding and mRNP remodeling steps are coupled for spatial regulation of mRNA export through and subsequent release from the NPC.

Supported by grants from the NIGMS.
Eggs modulate the inflammatory response to carbohydrate restricted diets in overweight men.
Authors: Ratliff Joseph C , Mutungi Gisella , Puglisi Michael , Volek Jeff S , Fernandez Maria Luz
Published: June 2021

Carbohydrate restricted diets (CRD) lower glucose and insulin levels and improve atherogenic dyslipidemia. Dietary cholesterol has been shown to increase plasma HDL‐C independent of macronutrient composition. Twenty eight overweight men [body mass index (BMI) 26–37 kg/m²] aged 40–70 y consumed an ad libitum CRD (% CHO:fat:protein = 17:57:26) for 12 weeks. Subjects were matched by age and BMI and randomly assigned to consume eggs (EGG, n=15) (640 mg cholesterol/day provided by eggs) or placebo (SUB, n=13) (no added cholesterol). Fasting blood samples were drawn before and after the intervention to assess plasma lipids, insulin, adiponectin and markers of inflammation including C‐reactive protein (CRP), tumor necrosis factor‐alpha (TNF‐α), interleukin‐8 (IL‐8), monocyte chemoattractant protein‐1 (MCP‐1), intercellular adhesion molecule‐1 (ICAM‐1), and vascular cell adhesion molecule‐1(VCAM‐1). Body weight, percent total body fat and trunk fat were reduced for all subjects after 12 wk (P < 0.0001). Increases in adiponectin were also observed (P < 0.01). Subjects in the EGG group had a 21% increase in this adipokine compared to a 7% increase in the SUB group (P < 0.05). Plasma CRP was significantly decreased only in the EGG group (P < 0.05). MCP‐1 levels were decreased for all subjects but they were lower in the SUB compared to the EGG group (P< 0.05). VCAM‐1, ICAM‐1, TNF‐α, and IL‐8 were not modified by CRD or eggs. A CRD with daily intake of eggs decreased plasma CRP and increased plasma adiponectin compared to a CRD without eggs. These findings indicate that eggs are an important part of a CRD, possibly due to the presence of cholesterol and antioxidants such as lutein and vitamin E that modulate inflammatory responses.
Effect of dietary protein level on whole body protein turnover in the adult cat (Felis catus)
Authors: Wester Timothy J , Weidgraaf Karin , Ugarte Claudia E
Published: June 2021

The domestic cat is an obligate carnivore and as such has a high protein requirement. However, little is known about how protein turnover is affected by dietary protein intake in the cat. In the current study, kinetics of [1‐13C]Leu metabolism were used to quantify rates of whole body protein synthesis, degradation, and oxidation in adult cats (n = 6) fed diets containing 15, 40, or 65% of the ME intake as CP. Cats were fed the test diets to maintain BW for a minimum of 3 wk before the day of [1‐13C]Leu infusion. They were fed hourly on day of infusion. Values for Leu flux, non‐oxidative Leu disposal (NOLD, an indication of protein synthesis), Leu rate of appearance into the plasma (Ra, an indication of protein degradation), and Leu oxidation on different diets were as follows:

As indicated by rate of whole body Leu flux, cats fed a diet that supplied 65% of ME intake as CP had elevated rates of whole body protein turnover via chiefly increased rates of amino acid oxidation. This study showed that during a period of marginal protein intake, cats modulated protein turnover to conserve body protein. Research supported by the Massey University Research Fund.
Oral‐iron‐associated depletion of the fecal antioxidant capacity state is improved by concurrent dietary antioxidants
Authors: Orozco Monica N , Friel James K , Schuemann Klaus , Solomons Noel W
Published: June 2021

Background:

Iron's oxidative nature and ease to participate in redox reactions confer it the ability to produce reactive oxygen species (ROS) that may alter the normal antioxidant capacity state of feces, leading to oxidative damage in the colon.

Objectives:

To evaluate dietary antioxidants’ effect on the fecal antioxidant capacity state depleted by oral Fe supplementation.

Methods:

12 adult males received 3 7‐d treatments of: A=120 mg Fe (FeSO4), B=120 mg Fe + 30 mg carotenoids (CT) + 36.8 mg tocopherols and tocotrienols (TT), and C=120 mg Fe + 60 mg CT + 73.6 mg TT. Stool samples from baseline, active supplementation and washout phases were assayed for their capacity to blunt the production of indicator hydroxylated salicylic acid derivatives resulting from in vitro free‐radical attack by Fe3+ exposure.

Results:

A significant reduction on the production of hydroxylated compounds with respect to regimen A was seen for treatments B and C. Baseline antioxidant capacity state was virtually restored with dietary CT + TT antioxidants.

Conclusions:

Dietary antioxidants may improve the depleted fecal antioxidant capacity state of persons taking Fe supplementation.

Sponsored in part by the Malaysian Palm Oil Board and INF‐EMF.
Green tea increases insulin sensitivity and decreases brain oxidative stress in fructose‐fed rats
Authors: Roussel Anne Marie , Hininger Isabelle , Coves Sara , Cao Heping , Anderson Richard A.
Published: June 2021

Hyperglycemia and insulin resistance are leading causes of early brain alterations. Our objective was to investigate the in vivo effects of green tea extract on insulin sensitivity, insulin signaling, and brain oxidative stress using an experimental rodent model of diet‐induced insulin resistance, the fructose‐fed rat. Wistar rats, 10 per group, received a fructose rich diet (FD) or FD plus 1g green tea extract/kg diet. After consuming tea extracts, plasma insulin decreased from 412 ± 100 to 13 ± 28 pmol/L. There were also associated decreases in glucose and triglycerides. Real‐time PCR measurements showed that increases in insulin sensitivity were associated with an up‐regulation of GLUT4, insulin receptor, IRS‐2, glycogen synthetase kinase, p‐110 phosphoinositol kinase (PI3‐K), and protein kinase B in the liver. Addition of green tea extract to the diet resulted in significant decreases in lipid oxidation and DNA oxidative damage in brain tissue while sulfhydryl groups and glutathione remained unchanged. Ferric‐reducing/antioxidant power (FRAP) in brain was also enhanced. This study shows that green tea extract protected against brain oxidative damage and suggests possible neuroprotective effects against insulin resistance‐induced, early oxidative brain alterations. (Funded by USDA/ARS and Unilever, France).
Antioxidant Properties of Human Milk Fractions
Authors: Friel James Kenneth , Tsopmo Apollo , Diehl‐Jones Bill , Aluko Rotimi
Published: June 2021

Human milk (HM) contains antioxidants such as vitamins (A, C, E) and enzymes (SOD, GPx, CAT) which can reduce radical‐mediated diseases among infants. The current study is aimed at identifying novel antioxidants that might help to reduce some diseases of prematurity and to enhance infant formulas for babies who cannot be breastfed. To achieve our goal, pooled HM samples were freeze‐dried and initially separated into lipid, whey and casein proteins. The antioxidant power of each fraction was measured using the oxygen radical absorbance capacity (ORAC) assay: results were 67.2 μmol Trolox/g for whole milk; 7.1, 13.1, and 205.6 μmol Trolox/g for lipid, whey and casein, respectively. The HM sample was subsequently digested with pepsin, pancreatin and bile salt to mimic the infant digestion system. ORAC (μmol Trolox/g) values were 172.4 for the whole pepsin‐pancreatin digested milk, 234.3 for peptides above 3000 Da, and 146.9 for peptides below 3000 Da. The resulting fractions were separated by preparative HPLC and the fractions dried. Antioxidant capacity of each fraction was obtained and the identity of four antioxidant peptides confirmed by LC‐MS/MS. This work is supported by the Advanced Food and Material Network (AFMNet), the Manitoba Institute of Child Health and Canadian Institutes of Health Research (CIHR).
Manipulation of lipid bioaccessibility of almonds influences postprandial lipemia in healthy human subjects
Authors: Berry Sarah Elizabeth Emma , Ellis Peter , Tydeman Elizabeth , Lewis Hannah , Picout David , Phalora Ravneet , Rosborough Jennfier
Published: June 2021

An exaggerated postprandial lipemic response is associated with increased risk of coronary heart disease, due to acute effects on lipids, oxidative stress, hemostatic factors and vascular function. Lipid released slowly from a complex food matrix during digestion may attenuate postprandial lipemia1. The present study therefore investigates the effects of lipid release (lipid bioaccessibility (LB)) on postprandial changes (over 8h) in plasma triacylglycerol (TAG) concentration, oxidative stress (measured by 8‐isoprostane F2α) and vascular function (measured by peripheral augmentation index (PAIx)). Subjects (n = 20, aged 18–40y) were fed test meals containing 50g fat of low LB (intact whole almonds; WA), high LB (almond oil + defatted almond flour; AO) or as a control meal (CO) in a randomised cross‐over design, with 1‐wk washout. The postprandial increase in plasma TAG was significantly lower (74% & 58% lower incremental area under curve) following the WA vs. the AO and CO meals, respectively (P<0.001). The peak reduction in PAIx (by −9.5%, −10.1% & −12.6% at 2h following WA, AO and CO meals respectively, time effect; P<0.001) was not significantly different between meals. There were no changes in plasma 8‐isoprostane F2α concentrations. Reducing LB of almonds beneficially influences postprandial lipemia.

This study was funded by the Almond Board of California
The vitamin A contribution of high β‐carotene maize ingested by women as measured with electrochemical detection
Authors: White Wendy S , Li Shanshan , Shaik Parvin , Nugroho Angela , Rocheford Torbert
Published: June 2021

High β‐carotene maize is being developed through plant breeding as a sustainable agronomic approach to alleviate vitamin A deficiency in Africa. Our objective was to quantify the vitamin A equivalence of high β‐carotene maize based on ingestion of a single realistic serving of maize porridge. Women (n = 6) each consumed in random order 3 maize porridges (250 g each) containing 62.5 g maize flour: 1) high β‐carotene maize porridge (526.8 μg β‐carotene); 2) white maize porridge with β‐carotene reference dose (535.7 μg added β‐carotene); 3) white maize porridge with vitamin A reference dose (285.6 μg added retinol). Blood samples were collected over 9 h. Retinyl palmitate was analyzed in triacylglycerol‐rich lipoprotein fractions by HPLC with coulometric array electrochemical detection. Areas under the curve (AUC) for retinyl palmitate were 9.46 ± 1.41 nmol·h/L, 34.06 ± 4.31 nmol·h/L, and 32.32 ± 5.05 nmol·h/L after ingestion of high β‐carotene maize porridge, white maize porridge with β‐carotene reference dose, and white maize porridge with vitamin A reference dose, respectively. The vitamin A equivalence of the β‐carotene in high β‐carotene maize was 6.90 ± 1.53 to 1 (by wt); the vitamin A equivalence of the β‐carotene reference dose (2.11 ± 0.59 to 1) approximated that established by the U.S. Institute of Medicine. High β‐carotene maize is a bioavailable source of vitamin A. Supported by USAID/CIAT and HarvestPlus.
Mammalian Scribble regulates the assembly of epithelial tight junctions
Authors: Young Cheryl , Ivanov Andrei , Nusrat Asma
Published: June 2021

Scribble, Disc‐large (DLG), and Lethal giant larvae (LGL) tumor suppressor proteins have been implicated in the regulation of epithelial cell polarity. However, the mechanisms by which these proteins contribute to epithelial polarity are not well understood. Since tight junctions (TJs) play an important role in regulating intestinal epithelial cell (IEC) polarity and barrier function, we examined if these proteins participate in TJ dynamics and in the establishment of the IEC barrier function. Model IECs, SK‐CO15 and T84 cells were used for these studies, and TJ assembly/disassembly was analyzed using the calcium switch assay. Fluorescence labeling/confocal microscopy demonstrated that Scribble and LGL1 but not DLG1 co‐localized with TJ proteins, occludin and zonula occludens‐1. Additionally, Scribble and LGL1 co‐internalized with the TJ proteins during TJ disassembly induced by calcium depletion. Furthermore, siRNA‐mediated downregulation of Scribble, but not LGL1 or DLG1 significantly attenuated the functional and morphologic assembly of TJs during calcium repletion. Downregulation of Scribble did not influence expression of DLG1 or LGL1 and vise versa, and these proteins did not co‐associate as determined by immunoprecipitation. In conclusion, we demonstrate a unique role of mammalian Scribble in regulation of IEC TJ assembly and in the development of epithelial barrier function.

Supported by CCFA Career Development Award (AII), NIH DK‐59888 (AN), and NIH DK‐55567 (AN)
MnSOD and ALDH‐2 deficiency potentiate aging‐associated vascular dysfunction, mitochondrial oxidative stress and mtDNA strand breaks
Authors: Daiber Andreas , Wenzel Philip , Schuhmacher Swenja , Kienhöfer Joachim , Oelze Matthias , Schulz Eberhard , Münzel Thomas , Bürkle Alexander , Bachschmid Markus
Published: June 2021

Vascular aging is associated with progressive deterioration of vascular homeostasis and reduced relaxation. These effects can be explained by a reduction in free bioavailable NO that is inactivated by an age‐dependent increase in superoxide formation. In the present study mitochondria as a source of reactive oxygen species (ROS) and the contribution of manganese superoxide dismutase (MnSOD) and aldehyde dehydrogenase (ALDH‐2) were investigated. Age‐dependent effects on vascular function were determined in aortae of these mice by isometric tension measurements. Mitochondrial ROS formation was measured by enhanced chemiluminescence in isolated heart mitochondria. ROS‐mediated mitochondrial DNA (mtDNA) damages were detected by the fluorescent‐detection alkaline DNA unwinding (FADU)‐assay. Expression of antioxidant proteins were assayed by quantitative RT‐PCR. Endothelial dysfunction was observed in aged C57/Bl6 WT mice in parallel to increased mitochondrial ROS formation and mtDNA damage. In contrast, middle‐aged ALDH‐2−/− mice showed a marked vascular dysfunction that was similar in old ALDH‐2−/− mice. Aged MnSOD+/− mice showed the most pronounced phenotype such as severely impaired vasorelaxation, highest levels of mitochondrial ROS formation and mtDNA damage. Mitochondrial radical formation significantly contributes to age‐dependent endothelial‐dysfunction.
Electronegative LDL disrupts mitochondrial homeostasis: a novel mechanism for cigarette smoking‐associated endothelial dysfunction
Authors: Tang Daming , Burns Alan R , Lu Jonathan , Chen Hsin‐Hung , Wu Huanzhu , Engler David A , Sawamura Tatsuya , Yang Chao‐Yuh , Chen Chu‐Huang
Published: June 2021

Background and objectives: We reported that cigarette smoking provokes formation of L5, an electronegative low‐density lipoprotein (LDL), which inhibits maturation of endothelial progenitor cells. Here we investigated whether L5 disrupts mitochondrial homeostasis essential for EC survival.

Methods and results: Using ion‐exchange chromatography, L5, an electronegative LDL, was detected in smoker's plasma but nonsmokers. L5 dose‐dependently decreased mitochondrial membrane potential in cultured human umbilical venous ECs which was fluorescently exhibited by leakage of JC‐1 into the cytoplasm and flow cytometry assay. Trans‐electron microscopy revealed crista disappearance and membrane disintegration of the mitochondria, accompanied by chromatin fragmentation in the nucleus. These changes were associated with activation of caspase‐3 and mitochondrial release of cytochrome c and Smac. L5 markedly reduced expression of antiapoptotic proteins Bcl‐2 and c‐IAP. Addition of L1 in physiological ratios to the culture media did not compromise L5's bioactivity. Blockade of LOX‐1 with its neutralizing antibody or silencing it with siRNAs protected mitochondrial integrity and prevented apoptosis induced by L5.

Conclusions: cigarette smoking is associated with the formation of L5, which promotes HUVEC apoptosis by impairing mitochondrial integrity via the LOX‐1 receptor.