bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of PI4,5P2 by septin complexes is required for their essential function in cytokinesis in budding yeast
Authors: Votin Violet , Allyn Theresa , Thorner Jeremy
Published: June 2021

Septins comprise a conserved family of proteins that are involved in processes of membrane remodeling, such as cytokinesis. Cytokinesis in budding yeast (Saccharomyces cerevisiae) requires assembly of septins adjacent to the plasma membrane of the mother‐bud neck, where they serve as a protein scaffold and diffusion barrier. The aim of our studies is to determine how septins are recruited to and anchored at the plasma membrane. Among plasma membrane constituents, phosphatidylinositol‐4,5‐bisphosphate (PI4,5P2) is a potential candidate for recruiting septins, because at the site of cytokinesis, the plasma membrane in mammalian cells is enriched in PI4,5P2, which is required for cytokinesis [Emoto et al. (2005) J. Biol. Chem. 280: 37901]. PI4,5P2 is also required for proper septin localization in yeast [Rodríguez‐Escudero et al. (2005) Biochem. J. 390: 613]. Our work indicates that a PI4,5P2‐binding reporter localizes preferentially to the yeast bud neck. In addition, liposome flotation assays show that the yeast septins Cdc3, Cdc11 and Cdc12 bind to PI4,5P2‐containing vesicles in vitro. Furthermore, complexes of Cdc3, Cdc10, Cdc11 and Cdc12 also bind PI4,5P2. Mutation of basic residues in Cdc3 decreases PI4,5P2 binding in vitro and causes defects in cytokinesis in vivo, supporting the view that septin‐PI4,5P2 interaction is required for the septins to execute their function in cytokinesis. NIH R01 GM21841
Functional architecture of the retromer cargo‐recognition complex
Authors: Rojas Adriana Lucely , Hierro Aitor , Rojas Raul , Murthy Namita , Effantin Gregory , Kajava Andrey , Steven Alasdair , Bonifacino Juan , Hurley James
Published: June 2021

The retromer complex is required for the sorting of acid hydrolases to lysosomes, transcytosis of the polymeric immunoglobulin receptor, Wnt gradient formation, iron transporter recycling and processing of the amyloid precursor protein. Human retromer consists of two smaller complexes: the cargo recognition VPS26‐VPS29‐VPS35 heterotrimer and a membrane‐targeting heterodimer or homodimer of SNX1 and/or SNX2. Here we report the crystal structure of a VPS29‐VPS35 subcomplex showing how the metallophosphoesterase‐fold subunit VPS29 acts as a scaffold for the carboxyterminal half of VPS35. VPS35 forms a horseshoe‐shaped, righthanded, alpha‐helical solenoid, the concave face of which completely covers the metal‐binding site of VPS29, whereas the convex face exposes a series of hydrophobic interhelical grooves. Electron microscopy shows that the intact VPS26‐VPS29‐VPS35 complex is a stick‐shaped, flexible structure, approximately 21nm long. A hybrid structural model derived from crystal structures, electron microscopy, interaction studies and bioinformatics shows that the a‐solenoid fold extends the full length of VPS35, and that VPS26 is bound at the opposite end from VPS29. This extended structure presents multiple binding sites for the SNX complex and receptor cargo, and appears capable of flexing to conform to curved vesicular membranes.
Cell surface expression of system xc− is upregulated in non‐confluent cell cultures
Authors: Sherburn Stephanie , Condelee Mazi , Goltz Abby , Chase Leah A.
Published: June 2021

System xc− is a transport system that exchanges extracellular cystine for intracellular glutamate. The transporter plays an important role in providing cysteine as a precursor for glutathione synthesis and in maintaining the redox balance within the cell. We recently reported that oxidants regulate the activity of System xc− by altering the rate of trafficking of the transporter to the plasma membrane. Additionally, we have demonstrated that cell surface expression of System xc− is influence by cell culture density. Using cell surface biotinylation assays, we showed that only 35‐45% of the transporter is localized to the plasma membrane in confluent cultures of human glioma cells, while 90% of the transporter is localized to the membrane in cultures which are only 50% confluent. In this study, we tested the hypothesis that the difference in membrane localization of System xc− is a result of a greater glutathione demand in actively dividing non‐confluent cells compared to confluent cells which are no longer actively dividing. We used enzymatic assays and immunotcytochemical methods to measure the glutathione levels in confluent and non‐confluent cultures of human glioma cells. These studies reported here combined with our previous studies suggest that the localization of System xc− is influenced by the glutathione levels in the cells independent of the mechanism by which gluathione levels are modulated.
Using Diffusion Tensor Imaging (DTI) to Study Neuroanatomical Structures
Authors: Wisco Jonathan J.
Published: June 2021

Diffusion Tensor Imaging (DTI) is becoming the preferred radiological method to assess and quantify white matter fiber tract integrity in the human brain. Its applications have been widespread in studies of normal aging, Alzheimer's Disease, schizophrenia and stroke, and in clinical improvements of pre‐surgical planning and post‐surgical assessment. DTI is also becoming the method of choice for studying functionally connected areas of cerebral cortex through the reconstruction of white matter fiber tracts. Anatomists have an important role of providing expert validation of DTI methodological development and its application to basic science and clinical problems. This talk will focus on the methodological basics of DTI and present examples of its applicability to the study of the neuroanatomy. Future directions for the role of anatomists in this field of research will be proposed.
Novel Phosphatase SMP3 Controls Outputs of BMP Signal Transduction
Authors: Shen Tao
Published: June 2021

Bone Morphogenetic Proteins (BMPs) and other members of the TGFb superfamily regulate cell growth and differentiation. Abrogation of BMP/TGFb signaling is associated with cancer, cardiovascular, autoimmune and skeletal diseases. The key step of BMP signal transduction is the phosphorylation of transcription factor Smad1, 5 and 8 by BMP receptors. While phosphorylated Smad1/5/8 (P‐Smad1/5/8) activates BMP‐induced gene transcription in the nucleus, protein phosphatases dephosphorylate P‐Smad1/5/8 and consequently terminate BMP signaling. The identity of the Smad phosphatase has been one of the major unanswered questions in BMP/TGFb biology.

To identify Smad phosphatases, we generated cDNA encoding all the protein phosphatases in human genome and undertook a genome‐wide screen. Using combined methods of functional genomics and biochemistry, a novel cytoplasm‐localized Smad1/5/8‐specific phosphatase, SMP3 was discovered. The effects of SMP3 on controlling the level of P‐Smad1/5/8 in vivo and in vitro were analyzed by immunofluorescence and western blotting, using a phospho‐specific antibody against P‐Smad1/5/8. In vitro binding, immunoprecipitation, and bimolecular fluorescence complementation assays demonstrated that SMP3 and Smad1 directly interact in the cytoplasm. Consequently, ectopic expression of SMP3 in cells abolished BMP‐induced gene expression as measured by real‐time PCR and transcriptional reporter assays, and loss of SMP3 activity (site‐directed mutagenesis) or expression (RNA interference) greatly enhanced BMP signaling.

In conclusion, SMP3 acts as a gatekeeper to prevent excessive BMP signaling through dephosphorylation and subsequent nuclear exclusion of Smad1/5/8. Our results not only gain insights into the important role of BMP signaling in human growth and development, but also are pertinent towards novel therapeutic approaches for the prevention and treatment of human diseases.
Role of JNK and p38 MAPK in Taiwanin A induced cell death
Authors: yeh sheau farn
Published: June 2021

Taiwanin A, a lignan isolated from Taiwania cryptomerioides Hayata, has been reported to have cytotoxicity against human cancer cells. It was shown that Taiwanin A damages microtubule, induces mitotic arrest and apoptosis. Taiwanin A induced apoptosis in HepG2 cells was mediated through activating the tumor suppressor protein p53. However, whether there are other intracellular signaling pathways also involved in Taiwanin A mediated apoptosis is not known. In this study, we examined the activation of three MAPKs (mitogen activated protein kinase): ERK, p38 and JNK in HepG2 cells upon Taiwanin A treatment and the role of MAPK activation in Taiwanin A induced apoptosis.

We found that Taiwanin A activated all three MAPKs. Taiwanin A induced cytotoxicity could be partially blocked by either the p38 inhibitor SB203580 or JNK inhibitor SP600125, but not by ERK inhibitor PD98059. Combined treatment of SB203580 and SP600125 showed additive effects on the inhibition of Taiwanin A's cytotoxicity suggested that both JNK and p38 may play role in Taiwanin A induced apoptosis. Inhibition of p38 activity reduced Taiwanin A‐induced p53 phosphorylation on serine 15 residue. Direct interaction of Taiwanin A activated p38 and p53 was demonstrated by immuno‐precipitation. On the other hand, inhibition of JNK activity by either SP600125 or by silencing the JNK scaffold protein JIP2 was also shown to reduce phosphorylation of Bcl‐2.

Therefore, we identified two Taiwanin A induced signaling pathways; p38‐p53 and JNK‐Bcl‐2; that are involved in Taiwanin A induced apoptosis in HepG2 cells.
Effect of dietary phytochemicals on peroxide induced oxidative stress in Lumbricus terrestris
Authors: Hutton Trey S , DeLeon Robert Corey , Lester Christen , Maitin Vatsala , Crixell Sylvia , Vattem Dhiraj A
Published: June 2021

Oxidative stress has been linked to several chronic diseases, including heart disease, cancer and inflammation. Phenolic phytochemicals abundant in natural products have high antioxidant activity and can potentially reduce the effects of oxidative stress. However, their antioxidant response mechanism in vivo is not very well understood. Our objective was to investigate the effect of several natural products on modulating the antioxidant response in vivo in hydrogen peroxide (HP) stressed Lumbricus terrestris. Sexually mature L. terrestris were randomly divided into 4 treatment groups and were allowed to feed ad libitum on 1.25% Gerber‐oatmeal agar (GA). The animals in control, peroxide, treatment and treatment‐peroxide groups, fed on GA, GA + 0.13% HP, GA+ 10% extract and GA+0.13% HP+ 10% extract respectively. Two animals from each group were euthanized after 2d, 4d and 6d and 1 inch of circular muscle posterior to the clitellum was dissected and extracted. Malondialdehyde (MDA) levels were determined to assess peroxide induced lipid oxidation. The antioxidant response was determined by measuring the radical quenching activity of muscle, the levels of superoxide dismutase (SOD) and catalase (CAT) using standard methods. Our results indicated that compared to controls, animals on grapefruit, grape, ginger, and cranberry diet had the lowest MDA levels and high radical quenching activity. Animals on grapefruit, ginger, raspberry, and cranberry diet had 3‐7 fold more muscle CAT levels. SOD levels in animals on turmeric, mace, blackberry, and oregano diet was almost 100‐200 fold higher. Our results suggest that dietary phytochemicals can manage acute oxidative stress in vivo by inducing antioxidant enzyme response mediated by SOD and CAT.
Acute consumption of very low carbohydrate meals lowers postprandial concentrations of insulin, leptin, and total ghrelin
Authors: Kumagai Melissa , Bergman Sarah , Smith Julie , Jordan Julia , Clemons Aaron , Gillingham Melanie , Duell Bart , Stadler Diane
Published: June 2021

People following low carbohydrate (LC) diets report "food disinterest" that may lead to greater weight loss than other diets. Changes in weight regulation hormones that affect hunger and satiety may contribute to this heightened response. A random‐order, crossover design was used to test the hypothesis that acute consumption of LC meals reduces postprandial insulin, leptin, and ghrelin concentrations compared to high complex carbohydrate (HC) meals.

Seven normal‐weight adults completed two 4‐day controlled dietary phases. Subjects consumed a standard diet for 3 days and LC (4% CHO) or HC (58% CHO) meals providing 10 kcal/kg on day 4. Insulin, leptin, and ghrelin were measured in blood samples hourly for 9.5 h after breakfast. Area under the curve (AUC) and patterns of change for each analyte were determined with differences between diets compared using paired t‐tests and Wilcoxon Signed‐Rank tests.

AUC's for insulin (91±13 vs 259±30 μIU ·h/ml, p=0.0002) and leptin (4±0.7 vs 5±1.0 ng·h/ml·kg fat mass, p=0.03) were significantly lower after the LC than the HC diets. The linear term was significant for leptin, p=0.03. The linear (LC), quadratic and quartic (HC) terms were significant for ghrelin, p=0.02.

Consumption of LC and HC meals resulted in different patterns of change for all analytes, and lower AUCs for insulin and leptin. These differences may explain the food disinterest and heightened weight loss associated with LC diets.
Dietary monounsaturated fatty acids, genetic polymorphism of nucleotide binding oligomerization domain 1 (NOD1) and serum HDL concentrations
Authors: Cuda Cristina , Cahill Leah , El‐Sohemy Ahmed
Published: June 2021

Dietary fatty acids have been associated with cardiovascular disease, in part, by influencing risk factors such as serum lipids and inflammation. Nucleotide Binding Oligomerization Domain 1 (NOD1) is an intracellular protein of the innate immune system, and genetic polymorphisms of NOD1 have been associated with inflammatory conditions. The objective was to determine whether a polymorphism in the NOD1 gene (G796A) modifies the association between dietary fats and serum HDL. Men and women (n= 961) aged 20‐29 years completed a 196‐item food frequency questionnaire and provided a fasting blood sample for determining serum cholesterol concentrations and genotyping. No significant diet‐gene interactions were observed for total (p=0.16), saturated (p=0.19), monounsaturated (MUFA) (p=0.19) or polyunsaturated fat (p=0.60) on serum HDL concentrations. However, a significant association was observed between MUFA and HDL cholesterol (r=0.13, p<0.0001). The Spearman correlation between dietary MUFA and serum HDL was 0.15 (p=0.001) for those with the GG genotype, 0.18 (p=0.0003) for the GA genotype and ‐0.01 for the AA genotype (p=0.9), after adjusting for energy intake, sex, ethnicity and BMI. These results indicate that NOD1 genotypes modify the association between dietary MUFA intake and HDL cholesterol concentrations.

Grant Funding Source

Advanced Food Materials Network
Anti‐obesity effect of green tea catechin in the uncoupling protein‐2 transgenic mouse
Authors: Lee Mak‐Soon , Kim Yangha
Published: June 2021

In this study, we was evaluated the effects of green tea catechin EGCG (epigallocatechin gallate) in the transgenic mouse model with uncoupling protein‐2 (UCP‐2)‐Luciferase. UCP‐2 is a mitochondrial membrane transporter, controlling energy expenditure and thermogenesis. UCP‐2 promoter (‐1830/+30 bp) was subcloned into the pGL3‐Basic vector that includes luciferase as a reporter gene. Biologycal activities of dietary polyphenol EGCG were carried out in UCP‐2 transgenic mice. Transgenic mice were fed on a high‐fat diet for 8 weeks to induce obesity. Subsequently they were maintained on a high‐fat control diet, high‐fat diets supplemented with 0.2% and 0.5% EGCG (w/w) respectively for a further 8 weeks. EGCG diets reduced the body weight and also considerably lowered the levels of plasma triglyceride and leptin. Luciferase activity was significantly increased in the liver, white adipose tissue, brown adipose tissue and muscle of UCP‐2 transgenic mice. These results suggest that anti‐obesity effects of green tea catechin EGCG are associated with an increase in the UCP‐2 gene expression, regulating thermogenesis. "This study was supported by the Project of Bio‐Food Research from the Korea Science and Engineering Foundation (KOSEF, M10510130005‐08N1013‐00510)".
Applying the Theory of Reasoned Action to evaluate soy consumption intention and influential factors
Authors: li suyun , Camp Shirley , Finck Jananne , Winter Martha , Chapman‐Novakofski Karen
Published: June 2021

The objective was to determine variables associated with intention to consume soy products, using a pre‐ (N=166) and post‐(N=141) survey based on the Theory of Reasoned Action in a 3‐session diabetes program. At pre‐ subjective norm (SN), behavior attitude (BA), behavior control (BC) were correlated with intention(R=0.302,0.330,0.493, respectively, P<0.01); at post‐ SN, BA, BC were correlated with intention (R=0.588,0.383,0.615, respectively, P<0.01). Multiple regression analysis at post‐ revealed that 50% of variance in intention to consume soy was explained by two models: "‐2.007+0.787BC +0.210 BA" and "‐1.788+0.939 BC +0.432 texture"(P<0.01). Differences between pre‐/post‐ were significant for BA, BC and intention, but not for SN (P<0.01). The percentage regarding the cost and health benefit impact on purchasing/ consuming soy was lower in pre‐ vs post‐, but at both times, health benefits had a larger impact than cost.(26%/49% for cost, 46%/71% for health benefits, pre‐/post‐ respectively). BC includes issues of cost, availability, and ability to prepare, and seems to be most influential in intention to consume soy. In conclusion, having soy foods available and knowing how to prepare and knowing the health benefits of soy are important determinants in intention to consume soy. The project was supported by Illinois Soy Association and University of Illinois Extension.
The relationship of dietary habits with TV watching and internet usage in part of Gwangju and Jeonnam elementary school children
Authors: Khil Jinmo , Cho A Ra
Published: June 2021

The purpose of this study was to examine the dietary habits according to TV watching and internet usage and to establish the desirable extracurricular activity and eating habits in elementary school children. A questionnaire was filled out by children in 4th, 5th and 6th grades in 6 elementary schools within Gwangju and Jeonnam region and 712 cases were analyzed by SPSS 12.0. TV watching time was related with dietary habit score in subjects. The children who watch TV "=1 hour/day" in weekday had a better dietary habit score than the children who watch TV "=1 hour (p<0.0001). The children who watch TV "=2 hours/day in weekend had a better dietary habit than the children who watch TV "=2 hours" (p<0.0001). The internet usage was related with dietary habit score in children. The children who use internet "=1 hour/day" in weekdays had a better dietary habit score than the children who use internet "=2 hours/day" (p<0.0001). The children who use internet "=2 hours/day" in weekend had a better dietary habit than the children who use internet "=2 hours/day" (p<0.0001). These results indicate that the children who have more appropriate daily activity pattern showed better dietary habit score and TV watching and internet usage time occupy much of the elementary school children's extracurricular activities. Therefore, it is essential that the program development of both educational and social levels for active and outdoor extracurricular activity of school children.
Kinetics of myogenic progenitor cell (MPC) expansion and bone marrow‐derived cell recruitment in skeletal muscle regeneration
Authors: Martinez Carlo Obet , McManus Linda M , Shireman Paula K
Published: June 2021

Muscle regeneration requires myogenic progenitor cells (MPC) and recruitment of bone marrow (BM)‐derived cells. MPC (CD34+/CD45−/Sca‐1−) and BM‐derived progenitor cells (CD45+/Sca‐1+) isolated from injured muscle undergo myogenic differentiation in vitro. However, the kinetics of MPC expansion and BM‐derived cell recruitment to injured muscle are poorly understood and were quantitated in tibialis anterior muscle using flow cytometry after cardiotoxin (CTX) injury in wild type (WT, C57Bl/6J) male mice. Maximal MPC expansion occurred 3 days post‐CTX and BM‐derived progenitor and immune cell recruitment exhibited similar kinetics; few cells present at baseline with maximal accumulation 3 days post‐CTX.

MPC expansion and BM‐derived cell recruitment occurred within days of injury and demonstrated similar accumulation kinetics suggesting interactions between MPC and BM‐derived cells may be crucial to skeletal muscle regeneration. A better understanding of interactions between different cell types could result in improved strategies for muscle regeneration and tissue engineering.

Supported by Veterans Administration and HL074236 and HL090196.
Aromatic Hydrocarbon Receptor Down‐regulates MYCN Expression and Promotes Neural Differentiation of Neuroblastoma
Authors: Wu Pei‐yi , Hsu Wen‐Ming , Lee Hsinyu
Published: June 2021

Neuroblastoma (NB) is a heterogeneous neoplasm derived from sympathoadreanal lineage of the neural crest progenitor cells, and is the most common malignant disease of infancy. MYCN amplification is a prognostic factor of NB. It indicates a highly malignant disease and poor patient prognosis. However, how MYCN affects the NB cell behavior remains unclear. By DNA arrays and pathway analysis, aromatic hydrocarbon receptor (AHR) was found to reversely correlate with the MYCN expression in NB tumors. In addition, positive AHR expression by immunostaining of NB tumors predicted a favorable prognosis. AHR was known as the mediator of the toxicity of dioxin‐like compounds. However, recent studies demonstrated that AHR is important in regulating cell death, proliferation, and differentiation. We hypothesize that AHR could regulate MYCN expression in NB. Over‐expressed AHR promotes SK‐N‐DZ cell neuronal differentiation and down‐regulate the mRNA expression of MYCN. Our results suggested that AHR not only relates to the MYCN expression but also plays an important role in regulating tumorigenesis of NB.
The Effects of Acute Injections of Selective Dopamine Receptor Agonists and Antagonists on Three Models of Impulsivity in the Rat
Authors: Koffarnus Mikhail , Woods James
Published: June 2021

A role for dopaminergic transmission in the expression of impulsivity in animal models has been demonstrated. The role of individual dopamine receptors in these actions is not entirely clear, however. Sprague Dawley rats were trained to respond on one of three purported models of impulsivity: the delay discounting task, a paced fixed consecutive number schedule, or an uncertain visual discrimination task. Agonists and antagonists selective for individual dopamine receptor subtypes or compounds acting at the dopamine transporter were administered and changes in behavior were recorded. Results indicate that dopaminergic transmission is important for each of the impulsivity models examined. Furthermore, the effects of specific compounds across models are sometimes in concordance and sometimes in discordance, indicating these models are not measuring the same aspect of behavior. This research was supported by USPHS/NIDA grants R01 DA020669 and T32 DA007267.
Novel non‐nucleoside HCV NS5B inhibitors exhibit low potential for CYP‐mediated drug‐drug interactions
Authors: Sergeeva Maria , LeBrun Laurie A , Hou Huiying , Kirkovsky Leo , Zhou Yuefen
Published: June 2021

INTRODUCTION

HCV RNA‐dependent RNA polymerase (NS5B), essential for viral replication, is an attractive target for HCV drug discovery. We previously disclosed a novel series of potent non‐nucleoside NS5B inhibitors which bind to the "palm" site of the NS5B protein. Here we report the results of in vitro ADME studies with representative NS5B inhibitors aimed at evaluation of their drug‐drug interaction potential.

METHODS

Test articles were incubated in the presence of human or animal liver microsomes or Cytochrome P450 SupersomesTM (1A2, 1A6, 2C8, 2C9, 2C19, 2D6 and 3A4) and NADPH+ regeneration system and the remaining parent molecules were quantified by LC/MS/MS. CYP inhibition was assessed using Vivid® Cytochrome P450 Screening Kits (1A2, 2C9, 2C19, 2D6 and 3A4) or, for selected compounds, in human liver microsomes.

RESULTS

Test article stability was assessed in liver microsomes of male and female mice, rats, rabbits, dogs, monkeys and humans. The compounds showed good metabolic stability (t½ > 60 minutes) in liver microsomes of all species tested with the exception of rabbit (both genders) and male rat microsomes. 2C8, 2C9 and 3A4 were identified as CYP isoforms involved in metabolism. CYP inhibition was insignificant for all isoforms tested.

CONCLUSIONS

The results suggest that CYP related drug‐drug interaction potential of the compounds evaluated in the current study is minimal.
Age‐related increases in microsomal quercetin glucuronidation in rat small intestine.
Authors: Bolling Bradley W , Court Michael H , Blumberg Jeffrey B , Chen C‐Y. Oliver
Published: June 2021

It is not known whether age modifies the metabolism of quercetin. Pooled microsomal fractions of 3 equidistant small intestine (SI) segments from 4, 12, 18, and 28 mo male F344 rats (n=8/age) were employed to model the kinetics of UDP‐glucuronosyltransferase (UGT) activity toward quercetin. Intrinsic clearance (CLint=Vmax/Km) values in the upper, middle and lower SI increased with age. At 18 and 28 mo, CLint was 2‐fold of 4 mo at 0.0665 mL/min/mg. Within individual microsomes at 30 and 300 µM quercetin, age‐related increases were significant in the upper and lower SI. In the upper SI at 30 µM quercetin, UGT activity was 2‐ and 3‐fold at 18 and 28 mo, respectively, of 0.348 nmol/min/mg at 4 mo. Lower SI UGT activity of 12 and 28 mo rats increased 70% from 1.44 nmol/min/mg at 4 mo. The lower SI preferentially catalyzed glucuronidation at the 7‐ position with 51% of total metabolites. The upper SI produced the greatest proportion of 4′‐ and 3′‐ conjugates at 26 and 33% respectively. Quercetin UGT regioselectivity did not change with age. Thus, age‐related increases in quercetin glucuronidation depend upon intestinal segment and are more pronounced in the upper and lower sections.
Characterization of mutation in the 395DQxD398 motif of the glucuronic acid binding site in human UGT1A6: Comparison to UGT1A10
Authors: Brezden Anna , Mazur Anna , Xiong Yan , Miller Grover P. , Finel Moshe , Radominska‐Pandya Anna
Published: June 2021

Human UGTs catalyze the glucuronidation of a variety of endogenous and exogenous compounds, and require the common co‐substrate, UDP‐glucuronic acid (UDP‐GlcUA). Due to exon sharing, the primary structure of the C‐terminal half of all the UGTs of subfamily 1A is identical. Therefore, we hypothesize that these isoforms will also share a common binding site for the GlcUA moiety. To test this hypothesis, three mutants of UGT1A6 (D395A, Q396A, and D398A) have been generated and characterized by Western blot and glucuronidation assays. While D395A showed no activity, Q396A and D398A glucuronidated p‐nitrophenol (pNP), albeit with altered kinetic parameters. Q396A did not affect Vmax but did decrease the Km value ~5‐fold. For D398A, the Km was similar to that of Q396A, and there was a 7‐fold decrease in the Vmax value. These findings are consonant with our prior studies using the corresponding UGT1A10 mutants and thus support the hypothesis. We compared these results to the binding residues identified for UGT2B7 modeling of the crystallized C‐terminal domain. UGT2B7 and the UGT1A family possess a number of distinctive UDP‐glucuronic acid contact amino acids. Differences between the tertiary structure and function of these two isoforms could facilitate the design of affective activators and/or inhibitors of glucuronidation specific for UGT1A and 2B isoforms. NIH‐DK60109, GM075893 (AR‐P)
Copy number of human SULT1A1 gene among newly menopausal Caucasion women
Authors: Moyer Ann , Weinshilboum Richard M , Miller Virginia M
Published: June 2021

There is large variation in response to menopausal hormone treatments among women and it remains unclear as to what genetic factors might increase risk of breast cancer in women using such treatments to relieve symptoms of menopause. SULT1A1 is a ubiquitous enzyme involved in the metabolism of steroid hormones including estrogen. A number of polymorphisms in the gene for this enzyme are associated with various cancer risks and the activity level of the enzyme is correlated closely with variation in copy number of the gene. This pilot study evaluated SNP variation and copy number of SULT1A1 in a subset of women (n=156) being screened for the Kronos Early Estrogen Prevention Study (KEEPS). All participants gave written informed consent. DNA prepared from blood was genotyped for SULT1A1 copy number, rs3760091 and rs750155 from the promoter region of SULT1A1, and rs9282861 from the coding region (SULT1A1*2 variant). In this cohort, 5.2% had 1 copy of SULT1A1, 65.2% had 2 copies, 23.9% had 3 copies, and 5.8% had 4 copies. Previously reports of the allele frequency for the three SNPs did not take into account gene copy number. When copy number was taken into account, allele frequency was significantly different, a fact that will have to be taken into account in future genotype‐phenotype correlations. (Supported by grants from the Kronos Longevity Research Institute, NIH HL90639, RR24150 and GM61388.)
Measurement of Membrane‐Bound Human Heme Oxygenase‐1 Activity Using a Chemically Defined Assay System
Authors: Huber Warren , Marohnic Christopher , Peters Michelle , Alam Jawed , Masters Bettie Sue S , Backes Wayne L
Published: June 2021

Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH‐cytochrome P450 reductase (CPR). Although most studies with HO used a 30‐kDa form, lacking the C‐terminal membrane binding region, the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity being elevated 5‐fold, and the Km for CPR decreased approximately 50‐fold. The goal of these studies was to accurately measure membrane‐bound heme oxygenase‐1 (HO‐1) activity using a coupled assay containing purified biliverdin reductase (BVR). When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2‐3 min; however, the reaction was only linear for 10‐30 sec when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO‐1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ml) and hBVR (0.025‐0.05 µM), but neither supplement increased the time that the reaction remained linear. Superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4‐5 minutes, even at high CPR levels. These results not only demonstrate that hydrogen peroxide leads to a decrease in HO‐1 activity, but also provide a chemically defined system to further examine the function of full‐length HO‐1 in a membrane environment.
Preventive effects of quercetin against pulmonary injury Induced by paraquat in rats
Authors: Kim Young C , Park Hee K , Kwon Do Y , Kim Sun J
Published: June 2021

We examined the antioxidant and anti‐inflammatory components isolated from Moutan radicus cortex. The antioxidant activity toward peroxyl radical, hydroxyl radical, and peroxynitrite was quantified using the Total Oxyradical Scavenging Capacity (TOSC) assay. Among the components examined, the highest TOSC toward peroxyl radical was shown by (+)‐catechin (CT) followed by quercetin (QC), methyl gallate (MG), and gallic acid (GA). The TOSC toward hydroxyl radical was MG > QC > CT > GA. The TOSC toward peroxynitrite was GA > CT > MG > QC. All of the four components decreased the NO release in lipopolysaccharide‐stimulated RAW264.7 cells, but only QC and GA depressed iNOS expression. The protective effects of QC against paraquat (PQ)‐induced pulmonary injury were measured. Rats received an intratracheal dose of PQ and were treated with QC for 2 weeks. The TOSC and GSH levels in lung were reduced and malondialdehyde in lung and NO concentrations in plasma were increased by the PQ challenge, but these changes were inhibited by QC treatment. The elevation of 4‐hydroxyproline in lung tissues was also decreased in the rats treated with QC. Histopathological examination of lung tissues showed similar results. The results suggest that the protective effects of QC against PQ‐induced lung injury are probably associated with its antioxidant activity.
RENAL FUNCTION EVALUATION INDUCED BY HIGH SALT INTAKE IN PERFUSED KIDNEYS.
Authors: Fiorino Patricia , Farah Vera , Yokota Rodrigo , Longo Vitoria Moreau , Leite Ana Paula , Fonteles Manassés Claudino
Published: June 2021

There is a significant association between salt intake and high blood pressure that could be related to alterations in the ability of the kidneys to excrete sodium chloride. Preliminary data from our group showed an increase in blood pressure of 11% in rats with salt added to the drinking water (1%) during 10 days; therefore the aim of this study was to evaluate renal function changes induced in rats by high salt intake. Male Wistar rats (n=5/group) were divided in Control group (CG) and Salt group (SG), (1%, drinking water, 10 days). Isolated kidneys were perfused with Krebs‐Henseleit solution containing 6% (g weight) of previously dialyzed bovine serum albumin. Glomerular filtration rate (GFR), renal vascular resistance (RVR), urinary flow (UF), fractional sodium and potassium tubular absortion (%TNa+ and %TK+) and Clearance osmolar (Cosm) were determined during 120 min. In SG group we observed an increased in GFR, (SG: 1.1 ± 0.04 vs CG: 0.4±0.01 mL/g/min), associated with markedly augments in UF (SG: 0.2±0.05 vs CG: 0.1±0.01 mL/g/min). There were no changes in RVR (SG: 6±1 vs CG: 4±0.4 mmHg/mL/g/min). The %TNa+ (SG: 73±3 vs CG: 74±6, %) was similar between the groups. However, salt intake increased the %TK+ (SG: 75±5 vs CG: 58±4 %) and Cosm (SG: 0.2±0.06 vs CG: 0.1±0.01, mL/g/min). These findings demonstrate that renal alterations observed herein are not related to blood pressure increased.
A novel Embryonic Stem Cell differentiation model for High‐Throughput Antivasculogenesis Screening
Authors: Piecewicz Stephanie Marie , Harfouche Rania , Sengupta Shiladitya
Published: June 2021

Recent studies have implicated the contribution of stem cells towards tumor neovascularization through the process of de‐novo differentiation or vasculogenesis, thus establishing the need for an effective assay to screen antivasculogenesis agents. The goal of this work was to establish an in vitro system for the efficient differentiation of embryonic stem cells (ES) into endothelial cells (EC) with the aim of creating an effective model for high‐throughput vasculogenesis inhibitor screening. We and others have shown that ES cells associate into embryoid bodies, which can differentiate into EC when exposed to extracellular matrix and/or growth factors. Here we describe an effective model for ES cell differentiation into EC as an assay system for screening drugs. We established that matrix composition is critical for the differentiation of embryoid bodies into EC, as confirmed by immunohistochemistry and imaging for selective expression markers, and EB plated with no matrix exhibited no vascular phenotype. The addition of growth factors increased vasculogenic sprouting by up to 16.5 times as compared to control (P<0.01). This model was used to test various permutations of signal transduction inhibitors to explore downstream pathways. In conclusion, we have established a novel in vitro model for screening antivasculogenic agents for potential cancer therapies in preclinical trials.
APS Actives AMPK and Alleviates Glucose Toxicity in High‐glucose Treated C2C12 cells
Authors: Zou Feng , Liu Jian , Jin Huan , Ouyang Jingping
Published: June 2021

To investigate the effects of Astragalus polysaccharide(APS) on the activity of AMPK on normal or high‐glucose treated mouse C2C12 cells. The C2C12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum , supplemented with 100 U/mL penicillin,100 μg/mL streptomycin, and 2 mmol/L l‐glutamine in a humidified atmosphere with 5% CO2 at 37°C. The cells were plated at a density of 3×104 cells/cm2 and maintained in culture medium for 24h before treatment. The C2C12 cells were pretreated with APS for 24h, AICAR for 30 min, Compound.C for 15min and insulin for 24h.High glucose (30 mmol/L) is suitable to induce significant insulin resistance. The activity of AMPK, the activity of ACC, and the expression of total AMPK were measured by Western blotting. Glucose uptake were measured by GOD‐POD method. The transduction of GLUT4 were measured by immunofluorescence. In this research, the activity of AMPK and ACC were increased by the raise of concentration of APS(50‐150 ug/ml).However, the activity decreased with the concentration of APS(200ug/ml). APS enhanced glucose uptake and stimulate GLUT4 translocation in C2C12 cells via AMPK pathway. APS can also increase AMPK activity in high glucose‐treated cell group and this effect can block by the AMPK inhibitor: Compound.C. The results showed that APS can actives AMPK in normal C2C12 cells and alleviates glucose toxicity in high glucose‐treated C2C12 cells. This subject has a financial assistance by NSFC(30370673).
LC‐MS‐based metabolomics of acetaminophen‐induced acute toxicity
Authors: Chen Chi , Krausz Kristopher W , Shah Yatrik M , Idle Jeffrey R , Gonzalez Frank J
Published: June 2021

Acetaminophen (APAP)‐induced acute toxicity is a clinically important model for studying drug‐induced liver injury. In order to discover potential biomarkers of APAP‐induced toxicity, a LC‐MS‐based metabolomic study was conducted to investigate the differential response from wildtype and cyp2e1‐null mice to APAP overdose. Serum samples collected during APAP treatment were analyzed by an UPLC‐QTOF LC‐MS system. Chromatograms and mass spectra were deconvoluted into a data matrix for multivariate data analysis. Results from these analyses revealed the correlation between hepatotoxicity and metabolomic profiles of serum samples since APAP treatment led to a far greater changes in the metabolomic profile of wild‐type mice than that of cyp2e1‐null mice in the multivariate model. Among the group of serum ions contributing this observation, several acylcarnitines including pamitoylcarnitine and myristoylcarnitine are very prominent because of their parallel increase with transaminase activity and their role in fatty acid beta‐oxidation. Overall, the combination of genetically‐modified animal models and metabolomics provide a powerful tool to characterize APAP‐induced liver toxicity and undercover novel mechanism.
Novel antiangiogenic peptides inhibit tumor growth in breast cancer xenografts
Authors: Koskimaki Jacob E , Rosca Elena , Raman Venu , Bhujwalla Zaver , Popel Aleksander
Published: June 2021

Breast cancer is the most commonly diagnosed female malignancy in the United States, proving fatal to nearly 40,000 people in 2007. Angiogenesis or neovascularization, the process of new blood vessel formation from pre‐existing microvasculature, is necessary for homeostasis in tissue, and tumor progression. We previously introduced a novel systematic methodology to identify putative endogenous antiangiogenic peptides and validated these predictions in vitro in human umbilical vein endothelial cell (HUVEC) proliferation and migration assays, and have evaluated the ability of several peptides to suppress angiogenesis in breast cancer orthotopic xenograft models in severe combined immunodeficient (SCID) mice using MDA‐MB‐231 human breast cancer cells. Peptides were administered intraperitoneally, once per day, and tested at several concentrations. After demonstrating significant suppression of tumor growth in vivo with unmodified sequences, we have modified peptides using synthetic amino acids to replace cysteines with {alpha}‐amino‐n‐butyric acid (Abu). Using these peptide modifications, we have shown peptide bioactivity in vitro in cell proliferation assays and significant suppression of tumor growth in vivo. These modified sequences are stable under oxidizing conditions, and represent significant steps towards translational clinical research.
IP3 receptors, but not ryanodine receptors mediate subsarcolemmal Ca2+ oscillations in arteriolar smooth muscle cells
Authors: Boerman Erika M. , Jackson William F.
Published: June 2021

The role of ryanodine receptors (RYR) and IP3 receptors (IP3R) in Ca2+ signalling in arteriolar smooth muscle cells (ASMC) has not been established. Therefore, we tested the hypothesis that RYR and IP3R mediate subsarcolemmal Ca2+ oscillations (OSC) in ASMC. Second or 3rd order hamster cremaster arterioles were cannulated, loaded with 5 μM Fluo‐4 AM for confocal measurement of OSC and pressurized to 80 cm H2O at 34 °C. In 6 vessels with myogenic tone, OSC were observed in 47 ± 9% of ASMC along vessels, with amplitude (AMP) = 1.42 ± 0.09 (Fluo‐4 intensity relative to baseline, F/Fo) and frequency (FRE) = 0.33 ± 0.06 s−1. The RYR antagonist, ryanodine (RYD; 10 μM) had no effect on OSC. With RYD, OSC were observed in 48 ± 9% of ASMC with AMP = 1.46 ± 0.10 F/Fo and FRE = 0.34 ± 0.06 s−1 (n = 6, p > 0.05), although 10 μM RYD abolished Ca2+ signals induced by the RYR agonist, caffeine (10 mM; p < 0.05), demonstrating the efficacy of RYD. In contrast, the IP3R antagonist, 2‐aminoethoxydiphenyl borate (2‐APB;100 μM) significantly decreased OSC occurrence (from 48 ± 5% to 11 ± 6%), AMP (from 1.67 ± 0.06 to 1.04 ± 0.05 F/Fo), and FRE (from 0.21 ± 0.06 to 0.04 ± 0.01 s−1) (n = 6, p < 0.05). Similarly, OSC were inhibited by xestospongin D (5 μM; n = 3, p < 0.05), another IP3R antagonist. Thus, IP3R, but not RYR, mediate subsarcolemmal Ca2+ oscillations in arteriolar smooth muscle cells. Supported by PHS grants HL 32469 & HL 086483 to WFJ and AHA Fellowship 0815778G to EMB.
Resveratrol reverses both thromboxane A2 receptor and non‐receptor (K+) mediated pulmonary vasoconstriction
Authors: Natale Elizabeth Kathleen , Slutzky Jessica L , Chicoine Louis G
Published: June 2021

Resveratrol (3,5,4′‐trihydroxystilbene) is an antioxidant found in red wine. Recent studies have demonstrated that resveratrol attenuates platelet activation/aggregation via increased NO/cyclic GMP formation. We tested the hypothesis that resveratrol reverses Thomboxane A2 receptor mediated pulmonary vasoconstriction. The lungs of adult male Spague‐Dawley rats (weight 225‐300 g) were isolated, ventilated (5% CO2, balance RA) and perfused with a physiological salt solution (PSS) at constant flow rate (30 mL/kg/min). U46619, a thromboxane A2 mimetic, was added to the perfusate to induce a stable pulmonary arterial pressure increase of 8‐10 mmHg (n=3). Increasing doses of resveratrol (3‐300 ug/mL) were added to the perfusate. The addition of resveratrol resulted in a dose dependent decrease in pulmonary arterial pressures. We then tested the hypothesis that resveratrol reverses non‐recptor mediated pulmonary vasoconstriction using KCl. We preconstricted the lungs with K+ (20‐30 mM), which resulted in an arterial pressure increase of 8‐10 mmHg (n=3). Increasing doses of resveratrol (3‐300 ug/mL) were added to the perfusate. The addition of resveratrol resulted in a dose dependent decrease in pulmonary arterial pressure, such that resveratrol reversed K+ constriction at concentrations >100 ug/mL. These results demonstrate that resveratrol reverses both, receptor‐mediated as well as non‐receptor mediated pulmonary vasoconstriction.
Rosuvastatin Attenuates Pulmonary Arterial Hypertension in the Transgenic (mREN2)27 (Ren2) Rat
Authors: DeMarco Vincent Gerard , Habibi Javad , Whaley‐Connell Adam T , Schneider Rebecca I , Andresen Bradley T , Ferrario Carlos M , Sowers James R , Dellsperger Kevin C
Published: June 2021

Activation of the intrapulmonary renin‐angiotensin system in male transgenic Ren2 rats is associated with increases in lung oxidative stress, medial thickening of pulmonary arterioles, and pulmonary hypertension (PH). Statins are potent antioxidants and are vasculoprotective. We hypothesized that rosuvastatin (RSV) ameliorates PH and pulmonary vascular remodeling in Ren2 rats, in part, by reducing oxidative stress. Ren2 and Sprague‐Dawley (SD) rats were injected daily with RSV (10 mg/kg) for 3 wk, after which we measured right ventricular systolic pressure (RVSP) and mean arterial pressure (MAP). To evaluate remodeling, we measured the thickness of pulmonary arterioles. To evaluate oxidative stress in the lung we measured 3‐nitrotyrosine, NADPH oxidase activity, NOX2 and Rac1 expression, and ROS. Ren2 rats had elevations in MAP, RVSP, thickness of pulmonary arterioles, NADPH oxidase activity, Nox2 and Rac1, ROS, and 3‐nitrotyrosine compared to SD rats. RSV blunted the increase in RVSP, but not MAP, in the Ren2. RSV attenuated elevations in surface area of small pulmonary arteries, reduced NADPH oxidase activity and ROS, and 3‐nitrotyrosine, but did not blunt elevated levels of Nox2 and Rac1 protein expression in the Ren2. In summary, RSV reduces oxidative stress, pulmonary vascular remodeling and PH in male Ren2 rats. NIH R01‐HL‐63904 and VA Merit (JRS), and MU Research Board (VD).
Intermittent hypoxia increases PKCα/β‐ and reactive oxygen species‐dependent myofilament Ca2+ sensitization in small pulmonary arteries
Authors: Snow Jessica , Kanagy Nancy , Walker Benjimen , Resta Thomas
Published: June 2021

Intermittent hypoxia (IH) elicits pulmonary hypertension, increases circulating levels of the vasoconstrictor peptide, endothelin‐1 (ET‐1), and increases ET‐1‐mediated Ca2+ sensitization in pulmonary vascular smooth muscle (VSM). We hypothesized that, similar to effects of IH in systemic arteries, IH enhances ET‐1‐induced Ca2+ sensitization in pulmonary.VSM through stimulation of PKCδ. To test this hypothesis, we assessed vasoconstrictor and VSM Ca2+ responses to ET‐1 in the presence of the PKCδ inhibitor, rottlerin, or the PKCα/β inhibitor, myr‐PKC(20‐28), in endothelium‐disrupted, pressurized pulmonary arteries (~160 µm inner diameter) from IH or sham rats, as previously reported. In contrast to our hypothesis, rottlerin did not attenuate ET‐1‐mediated vasoconstriction in either group. However, myr‐PKC normalized reactivity between IH and sham groups without altering the associated Ca2+ responses. Because reactive oxygen species (ROS) contribute to ET‐1‐induced Ca2+ sensitization after IH, and ROS can activate PKC, we additionally assessed responses to ET‐1 in the presence of both myr‐PKC and the ROS scavenger, tiron. This drug combination inhibited ET‐1‐induced Ca2+ sensitization in vessels from IH but not sham rats to a similar degree as myr‐PKC or tiron alone. We conclude that IH augments ET‐1‐induced Ca2+ sensitization via PKCα/β and ROS, which may signal through a common pathway.
CD40 activation is angioproliferative in pulmonary microvascular endothelial cells
Authors: Baker Jeffrey T , Moore Timothy M
Published: June 2021

Introduction

Endothelial CD40 is important during inflammation as an angiogenic stimulus. We tested whether CD40 expression in PMVEC is associated with angioproliferative potential.

Methods

Western analysis for CD40 expression was performed. Growth curves, barrier measurements, and scratch‐wound assays were carried out using an ECIS apparatus (Applied Biophysics) and a digital image system composed of an inverted microscope.

Results

Basal CD40 expression was high in PMVEC, whereas expression was absent in rat pulmonary arterial‐derived EC (PAEC). CD40 expression could be up‐regulated in PAEC following over‐expression of NAP‐1, an endothelial progenitor cell‐associated protein. Exposure of PMVEC to sCD40L (1 μg/mL) for 1 ‐ 48 hours increased proliferation and improved barrier integrity. Exposure to sCD40L also produced an accelerated PMVEC migration response with decreased wound closure times. These effects were inhibited by pre‐treatment with anti‐CD40 antibodies.

Conclusions

Our data indicate activation of PMVEC‐expressed CD40 results in angioproliferative responses. These responses may be inherent to cell phenotypes expressing NAP‐1. Identification of PMVEC expressing CD40 in situ may localize pulmonary resident endothelial progenitor cells. Support: Parker B. Francis Foundation and AHA (0765421B) to T.M.M.