bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular translocation in Aktion: small molecule inhibitor effects on AKT localization
Authors: Strelko Cheryl L. , Roberts Mary F.
Published: June 2021

Upregulation of the AKT pathway leads to a variety of human cancers. Phosphatidylinositol ether analogs, or PIAs, are small molecules designed to inhibit the Ser/Thr kinase AKT, by binding to its pleckstrin homology (PH) domain and preventing translocation from the cytoplasm to the nucleus. D‐3′‐deoxy‐diC8‐phosphatidylinositol is a soluble PIA that has an IC50 of 40 µM in U937 human lymphoma cells, which predominantly express Akt1. This compound (and not related further deoxygenated inositol lipids or L‐inositol species) does not appear to affect activation and growth of cells that have other Akt isoforms as well as Akt1 (e.g., B cells). We have shown that D‐3′‐deoxy‐diC8PI inhibits phosphorylation of proteins downstream of Akt1. However, near normal levels of phosphorylation of Akt1 strongly indicate that plasma membrane translocation of Akt1 has not been significantly inhibited. A GFP‐Akt1 fusion protein was expressed in U937 and MCF‐7 cell lines, and the movement of Akt1 when treated with D‐3′‐deoxy‐diC8PI was tracked using confocal microscopy. Treatment with D‐3′‐deoxy‐diC8PI did not inhibit plasma membrane or nuclear translocation, indicating cytotoxicity occurs by a different mechanism. Binding of the Akt1 PH domain in the absence and presence of the short‐chain PIA has also been examined to confirm the in vivo results.

(This work is supported by N.I.H. GM60418).
Dolichol‐linked oligosaccharide synthesis inhibitor reduces the survival potential of capillary endothelial cells
Authors: Banerjee Aditi , Miller Meriandrys , Baksi Krishna , Banerjee Dipak K
Published: June 2021

Proliferation and differentiation of capillary endothelial cells (i.e., angiogenesis) are essential for breast tumor growth and metastasis. We have recently shown that dolichol‐linked oligosaccharide (i.e., LLO) biosynthesis and turnover are required for angiogenesis. Its inhibition by a glycotherapeutic, tunicamycin resulted in cell cycle arrest in G1 and induction of apoptosis mediated by unfolded protein response (upr) signaling. The objective of our study was to evaluate that tunicamycin reduces the survival potential of capillary endothelial cells. We have used vascular endothelial growth factor (VEGF) and insulin‐like growth factor‐1 (IGF‐1) to answer this question. VEGF165 (10ng/ml) stimulated cellular proliferation but it could not protect the cells from tunicamycin. A similar result was also obtained when the cells were treated with IGF‐1 (10ng/ml). There was however upregulation of p85PI3K(Tyr), Akt(S473) and p53(S15) phosphorylation in tunicamycin treated cells following IGF‐1 treatment for 10 min. The phosphorylation of NFκB (p65 S536) on the other hand remained silent under the experimental condition. These results together with the activation of caspase‐9, p21 and down‐regulation of GSK3‐β following tunicamycin treatment in the absence of IGF‐1 suggest shifting of the survival potential of VEGF165/IGF‐1 to apoptosis. Supported in part by grants U54‐CA096297 and Susan G. Komen for Cure BCTR0600582 (DKB), and G12‐RR03035 (KB).
Estradiol regulation of thimet oligopeptidase in the hypothalamus of female mice
Authors: Dai Yi , Cyr Nicole E. , Tetel Marc J. , Kua Lindsay H. , Chadwick Joe G. , Bruce Lisa A. , Wolfson Adele J.
Published: June 2021

Thimet oligopeptidase (TOP) is a metallopeptidase that degrades neuroendocrine peptides such as gonadotropin‐releasing hormone (GnRH), a key player in the hypothalamic‐pituitary‐gonadal axis. In females, GnRH levels affect the synthesis and release of the ovarian steroid hormone estradiol (E2). Changes in E2 levels, in turn, alter GnRH concentrations via positive and negative feedback loops at the level of the hypothalamus and pituitary. Some previous studies in rats and ewes have shown that increased E2 levels decrease hypothalamic TOP levels. To test whether E2 regulates hypothalamic TOP levels in female mice, and whether specific regions are involved in regulation, we collected samples of whole hypothalamus and hypothalamic slices from ovariectomized mice treated with estradiol or an oil control. Brain slices immunolabeled for TOP showed no difference in TOP concentrations in the medial preoptic, arcate nucleus, or median eminence where GnRH is produced and released. However, the ventral lateral portion of the ventral medial nucleus (VMNvl) showed lower levels of TOP in E2 treated animals. Using a co‐expression assay, we demonstrated that TOP and estrogen receptor αare co‐localized in all hypothalamic regions investigated including the ventral medial nucleus. Our data suggest that TOP is regulated by E2 in the hypothalamus of female mice, but only in the VMNvl.
Evolution of PKA Signaling: Structure of Yeast Regulatory Subunit
Authors: Rinaldi Jimena , Wu Jian , Yang Jie , Rossi Silvia , Moreno Silvia , Taylor Susan S.
Published: June 2021

cAMP is known to mediate, in both prokaryotes and eukaryotes, a wide variety of cellular responses to external stimuli. In eukaryotes, the major receptor for cAMP signaling is the regulatory subunit (R) of cAMP‐dependent protein kinase (PKA). In mammals there are two major classes of R subunits (I and II), however, Saccharomyces cerevisiae has only one R gene (Bcy1). As the yeast is far from mammals in the phylogenetic tree the study of this protein is important from the point of view of molecular evolution. Sequence analysis of the cyclic‐AMP binding domain (CNB) has shown that the fungal R subunits belong to a distinct group, yet exhibits some similarity to both mammalian RI and RII. In order to a molecular understanding of the mechanism of activation of PKA by cAMP in yeast and to provide insight into the evolution of cAMP receptor, we purified and crystalized a deletion mutant of Bcy1, Bcy1(168‐416) bound to cAMP and then compared it with the previous structures of mammalian RIα and RII ? cAMP‐bound isoforms. Surprisingly the relative orientation of the two CNB domains in Bcy1 is very different from RIα and RIIβ, although each domain superimposes very well. This structure serves as a hallmark for the yeast PKA. Closer examination of the structural differences between mammalian R subunits and Bcy1 identifies a hinge point in the kink between the B and C helices of the A domain that accounts for the major deviation in tertiary structure of the different R subunits. The similarity and differences of the interface between the two CNB domains in Bcy1 was also described and compared with the mammalian isoforms. (Supported in part by NIH GM34921 to SST)
Investigating PEA‐15's ability to regulate ERK1/2 MAP Kinase activation
Authors: Haling Jacob , Ginsberg Mark
Published: June 2021

PEA‐15 is a highly‐conserved protein expressed in many vascular cells. Increased PEA‐15 expression leads to dramatically increased activation of ERK1/2 MAP kinase, a key regulator of cell proliferation and gene expression, due to activation of Ras and MEK, the upstream components of the MAP kinase pathway. Here we show that PEA‐15 mediates activation of this pathway by interrupting a key negative feedback loop in the MAP kinase pathway. FRS2 is a central adaptor for receptor tyrosine kinase activation of Ras signaling; ERK1/2 phosphorylates FRS2 to inhibit Ras activation. We report that PEA‐15 binds to ERK1/2, thereby preventing their membrane recruitment. In consequence, ERK1/2 are unable to phosphorylate FRS2, thereby prolonging its capacity to recruit Grb2‐Sos and thereby activate Ras. Genetic deletion of FRS2 blocks the capacity of PEA‐15 to activate the MAP kinase pathway and reconstitution with phosphorylation mutants of FRS2 abrogates the ability of PEA‐15 to regulate ERK1/2 activation. Thus, PEA‐15 serves as a modulator of the classical MAP kinase pathway by preventing the ERK1/2‐dependent phosphorylation of FRS2.
Gossypol suppresses invasive behavior of human prostate cancer cells by modulating AP‐1 and NF‐kappaB signaling pathways
Authors: Jiang Jiahua , Slivova Veronika , Jedinak Andrej , Sliva Daniel
Published: June 2021

Gossypol (GP) is a naturally occurring yellow pigment present in cottonseed that was originally identified as a potent anti‐fertility agent in both men and women. As previously demonstrated, GP suppresses proliferation of a variety of cancer cells in vitro and in vivo through the mechanism including the induction of apoptosis. However, the molecular mechanism(s) responsible for the inhibitory effects of GP on the metastatic behavior of human prostate cancer cells remain to be elucidated. In the present study, we used DNA‐microarray, reporter gene assay, Western blot analysis and ELISA to investigate the effects of GP on the invasiveness of human prostate cancer cells PC3. Here, we show that GP inhibits the cell adhesion, cell migration, and cell invasion of PC3 cells. Moreover, GP suppresses the NF‐kB activity in PC3 cells by the down‐regulation of the expression of p65/p50, while AP‐1 activity is inhibited by the down‐regulation of the expression of c‐fos. The inhibition of AP‐1 and NF‐kB activity results in the modulation of cell cycle regulatory proteins and inhibition of the secretion of vascular endothelial growth factor (VEGF). Collectively, our findings suggest that GP exerts its effects on cancer cells by multiple mechanisms and may have potential therapeutic use for the treatment of prostate cancer.
Effect of Pepsin Hydrolysis on Amylase Activity and Lack of Amylase Inhibitory Activity of Sweet Potato Protein
Authors: Patterson Katherine W. , Truong Van den , Clare Debra A. , Allen Jonathan C.
Published: June 2021

The objectives were to determine the effect of pepsin hydrolysis on amylase activity and test for amylase inhibitory activity of Beauregard sweet potato protein. Protein was extracted from sweet potato flesh, peel, and leaf. Samples with or without pepsin digestion at pH 2 were run on SDS PAGE. Proteins resistant to pepsin digestion were found in the flesh, peel, and leaf. Amylase activity and amylase inhibitory activity were determined using starch as substrate, sugar reducing ends as product, and dinitrosalicylic acid as the detection medium. Dinitrosalicylic acid reacts with sugar reducing ends to absorb at 540 nm. No amylase activity was found in the leaf extract. Digestion with pepsin eliminated amylase activity in the flesh and peel extracts. Amylase inhibitors were not apparent in any of the fractions before or after pepsin hydrolysis. Amylase inhibition by sweet potato protein does not appear to be the mechanism for the low glycemic response to Beauregard sweet potatoes. Gastric digestion is likely to eliminate native amylase activity in the sweet potato thereby requiring pancreatic amylase to digest carbohydrate in vivo.

Support: N.C. Sweet Potato Commission.

Grant Funding Source

N.C. SWEET POTATO COMMISSION
Effects of Medicinal plant extracts on Blood pressure in Spontaneously Hypertensive Rats
Authors: HAHM TAE‐SHIK , PARK SUNG‐JIN , LO Y. MARTIN
Published: June 2021

This study was carried out to investigate the effects of medicinal plant extracts on angiotensin converting enzyme (ACE) activities. Among the fifty medicinal plant extracts, Morus alba Linne, Prunella vulgaris var. lilacina Nakai and Scutellaria baicalensis Georgi extracts showed strong ACE inhibitory activities. The inhibitory activities of extracts which Morus alba Linne, Prunella vulgaris var. lilacina Nakai and Scutellaria baicalensis Georgi were 50.3%, 56.9%, 48.1%, respectively. Systolic blood pressure was significantly decreased in the group fed Morus alba Linne extracts (21.1 mmHg), Prunella vulgaris var. lilacina Nakai extracts (20.4 mmHg), Scutellaria baicalensis Georgi extracts (26.3 mmHg) compared to control group. Diastolic blood pressure was significantly decreased in the group fed Morus alba Linne extracts (11.7 mmHg), Scutellaria baicalensis Georgi extracts (14.1 mmHg) compared to control group (P<0.05). it is expected that follow up study of medicinal plant through developing processed food and evaluation of their functional properties would provide useful information as a source of medicinal foods.
Validation of dual stable isotope method for measuring cholesterol absorption in hamsters
Authors: Ghanam Khadija , Wang Yanwen
Published: June 2021

Although dual stable isotope method has been used to measuring cholesterol absorption rate in hamsters, this method is adopted from humans and has not been well validated in hamster model. Thus, we have conducted two studies in hamsters to validate this method in terms of optimum time points and effective dose of two cholesterol tracers. After 2 wk of adaptation, male Golden Syrian hamsters were administered orally with 18O‐cholesterol (0.45 mg in study 1 and 0.6 mg in study 2) and intravenously injected with 13C‐cholesterol (0.18 mg in study 1 and 0.24 mg in study 2). Blood samples were collected at 0, 6, 24, 48, 72, 96, and 120 h, respectively following the injection. The enrichment of 18O and 13C in free cholesterol of red blood cells was analyzed using isotope ratio mass spectrometry (IRMS) and used to calculate cholesterol absorption rate. The results showed that both tracers increased gradually in the first 6 or 24h and gradually declined over next 4 days. The tracers at low and high doses behaved similarly over the time. The ratio of 18O/13C reached a plateau at 48 h and stayed constant until 96 h. The percent cholesterol absorption rate did not differ among 48, 72 and 96 h. These results suggest that cholesterol absorption rate can be measured precisely using the dual stable isotope method at 48 or 72h with the injection of lower dose of stable isotope.

Supported by Atlantic Innovation Fund, Canada
Maternal MTHFR C677T Genotype & Depressed Mood during Pregnancy Affects SLC6A4 Methylation in Infants at Birth
Authors: Wade Rachel E , Oberlander Tim F , Brain Ursula , Austin Jehannine , Devlin Angela M
Published: June 2021

In utero and early postnatal exposure to depressed maternal mood may program childhood behaviour via epigenetic processes. Methylenetetrahydrofolate reductase (MTHFR) is an enzyme important for methyl metabolism. A common variant in the MTHFR gene, C677T, is associated with depression. We investigated the effect of maternal MTHFR C677T genotype on maternal mood (Edinburgh Postnatal Depression Scale, EPDS), during pregnancy (n=82 women, all receiving folate supplements) and on promoter methylation of two genes implicated in depression, brain derived neurotrophic factor, BDNF, and sodium‐dependent serotonin transporter, SLC6A4, in the women and their infants at birth. Women with the MTHFR 677TT genotype had greater (P<0.05) mid gestational depressed mood (mean age 24.98±0.51 weeks). Maternal MTHFR 677TT genotype was also associated with lower (P<0.05) maternal and infant SLC6A4 promoter methylation but not BDNF methylation. We also found a negative relationship (P<0.01) between maternal depressed mood (EPDS scores) and maternal SLC6A4 promoter methylation. These findings show that maternal MTHFR C677T genotype and mood influence maternal and infant SLC6A4 promoter methylation and suggests that disrupted maternal methyl metabolism and depression during pregnancy affects gene‐specific DNA methylation patterns, which may have long‐term consequences for childhood behaviour.

Grant Funding Source

CIHR
Student Involvement in Local Wellness Policies of Local Education Agencies in Pennsylvania
Authors: Jomaa Lamis Hassan , McDonnell Elaine , Hartman Terryl , Weirich J. Elaine , Orlofsky Charles , Probart Claudia
Published: June 2021

The purpose of this study is to investigate Student Involvement (SI) in Local Wellness Policies (LWPs) of Pennsylvania Local Education Agencies (LEAs) and to explore predictors of SI. According to the 2004 federal mandate, each LEA participating in a school meal program must develop a LWP and involve a number of stakeholders in the development process. Research supports SI in school‐based interventions to promote healthy eating and physical activity. LWPs from Pennsylvania LEAs (n=721; including 539 public and 182 private LEAs) were abstracted and analyzed using SPSS 15. Logistic regressions were run to examine predictors for involving students on wellness committees, in annual reviews, in school menu planning, as well as assessing student needs and promoting peer‐education. LEAs with comprehensive and rigorous policies were more willing to engage students in LWPs, regardless of the sponsor type (public or private), enrollment, location, or socioeconomic status. LEAs that included students in policy development were more likely to involve students on on‐going wellness committees. In conclusion, LEAs that wrote comprehensive and rigorous policies were more likely to have SI. Demographic characteristics did not appear to be barriers to SI. Involvement of students in drafting wellness policies increases the likelihood of on‐going SI, which may lead to greater student buy‐in for wellness initiatives.

Grant Funding Source

Robert Wood Johnson Foundation
A Culturally tailored Nutrition Intervention to Promote Omega 3 fatty acid Consumption from fish Among African‐American Women of Childbearing age: A Pilot Study
Authors: Jones Samira Soyini , Greenwood MRC , Zidenberg‐Cherr Sheri
Published: June 2021

The prevalence of obesity, type 2 diabetes and cardiovascular disease continues to rise among African‐American women. Consumption of high caloric, non‐nutrient dense food choices and lack of adequate physical activity may increase their risk for these detrimental outcomes. Additionally, studies have shown the importance of omega 3 fatty acids, specifically docosahexanoic acid (DHA) and eicosapentanoic acid (EPA), on reducing the risk of cardiovascular disease, insulin resistance, and improved neurocognitive functioning in adult populations. The aim of this project is to assess the effectiveness of a four week nutrition intervention tailored to the needs of African‐American women on increasing nutrition knowledge and improving dietary behaviors. Preliminary data on fish consumption prior to intervention indicate most women, on average, consumed fish 1‐2 times per month and improved to at least 4 times per month. Increases in knowledge of the health benefits of fish consumption and the risks associated with consumption of mercury‐contaminated fish were observed. Dietary behaviors such as 'limiting food portions' were found to be consistently correlated at baseline to post‐intervention (r =0.368, p=0.027) indicating improvements in this area. Intervention strategies have included use of evidence‐based nutrition education curricula that are community‐based participatory approaches for adults.

Grant Funding Source

UC Davis Department of Nutrition
A Pilot Study to Examine the Impact of the Farmers' Market Nutrition Program
Authors: Racine Elizabeth Fussell , Vaughn Ashley
Published: June 2021

This pilot study examined WIC participants' use of farmers' markets. As well as evaluating the impact of the Farmers' Market Nutrition Program (FMNP) on the consumption of fruits and vegetables.

Participants were American born, Black, pregnant women in Washington, DC (DC) and Charlotte, NC who enrolled in WIC. The sample included women who completed the pre and post survey at their WIC offices in DC (n=44) and Charlotte (n=39) during the spring/summer 2007 and fall/winter 2007. In addition to receiving the standard WIC vouchers, women in DC also received $30 in FMNP vouchers while women in Charlotte did not.

Paired t test was used to examine differences in intake from spring to fall. A two sample independent t test was used to compare the difference in intake and sociodemographic characteristics between women in Charlotte and DC.

Women in DC were more likely to ever use farmers' markets; 56.8% in DC vs. 33.3% in Charlotte (p= 0.008). Two thirds (66%) of women in DC used their FMNP vouchers; these women were older than those who did not use their vouchers. Fruit and vegetable intake decreased from pre to post test among both groups, there was no difference in this decrease between groups.

This study demonstrated that women in a city with the FMNP were more likely to use farmers' markets. It also found that nutrition education to encourage fruit and vegetable consumption in the fall/winter is warranted.
Complementary feeding education to mothers in health facilities of Manya‐Krobo district of Ghana
Authors: Robinson Jasna Lin , Marquis Grace S , Lartey Anna
Published: June 2021

In Ghana, the prevalence of young child undernutrition is highest between 9 and 21 mo of age; inadequate complementary feeding (CF) is one of its major causes. This study examined health service education focused on CF and its relationship with mothers' knowledge and reported practices. Two focus groups were conducted with mothers of young children (n=14). Semi‐structured interviews were conducted with Ministry of Health (MOH) personnel (n=3), doctors (n=3), nurses (n=13), health assistants (n=5), Queen Mothers (n=6), and fathers (n=8). All focus group discussions and interviews were tape‐recorded and translated for coding and analysis. Messages on CF were provided via 10‐min health talks, posters, and private counseling. While all health workers recommended starting CF at 6 mo, messages for older infants did not consistently follow MOH feeding guidelines. Reported barriers to CF education included lack of audience‐appropriate teaching aids and limited in‐service training. Like health workers, mothers and fathers' knowledge of the recommended timing for CF initiation was good but, in practice, progression to a family diet was problematic. Practical CF advice is needed and should be integrated and reinforced throughout the health system.
Type 2 diabetes peer counseling intervention improves knowledge and self‐management skills
Authors: Cruz Roberto C , Segura‐Pérez Sofia , Vega‐Lopez Sonia , Chhabra Jyoti , Damio Grace , Pérez‐Escamilla Rafael
Published: June 2021

The objectives of this abstract are to: 1) show pre‐post test preliminary findings of 10 peer counseling educational sessions and 2) report participants' (N=30) level of satisfaction in the intervention arm of a parallel randomized‐controlled peer counseling trial. The DIALBEST 17 session peer counseling interventions included nutrition, diabetes and medication management. Participants mean age was 58 ± 8.8 years and 73% were women. 53.3% had less than an 8th grade education and 93.3% of participants were unemployed and preferred the intervention in Spanish. Preliminary findings show that 60% of those who could not identify carbohydrates sources at baseline were able to do so after the intervention. Likewise, 100% of those who could not identify at baseline which foods were likely to increase blood sugar answered correctly after being exposed to the peer counselors. The corresponding improvement for food label understanding was 80%. 73% of participants reported that 9 of the peer counseling sessions assessed were very good or excellent. All participants reported feeling better prepared to take care of their diabetes needs after being exposed to the peer counselor interventions. Funded by The Connecticut Center for Eliminating Health Disparities among Latinos (NIH‐NCMHD grant # P20MD001765).
Toddler‐feeding practices in Latinos: an early start in obesity prevention
Authors: Chaidez Virginia , Kaiser Lucia , Vosti Stephen
Published: June 2021

The goal of this research is to examine the relationships between child‐feeding practices, dietary intake, and growth status in Latino toddlers. Data collection consisted of in‐depth interviews with Latino mothers with toddlers (n=94) to collect information on demographics, child‐feeding practices, toddler's dietary intake and anthropometrics. Current feeding practices were assessed using a newly developed Toddler Feeding Questionnaire (TFQ) which contains two subscales; the TFQ Indulgent Subscale (TFQIS) and the TFQ Authoritative Subscale (TFQAS). Only the TFQIS revealed a positive correlation with toddler dietary variables, while neither subscale was associated with any of the toddler anthropometrics. Results of stepwise regressions indicated that only the TFQIS remained significantly associated with total energy of toddlers (β=8.885, P=0.020, Adjusted R2=0.0477). Maternal birthplace (β= ‐9.707, P=0.0111) and the TFQIS (β=0.495, P=0.0046) were both associated with total fat (grams), (Adjusted R2=0.1149, overall model P=0.0015). Maternal birthplace (β= ‐5.840, P=0.0019) and the TFQIS (β=0.176, P=0.0335) were both associated with percentage of calories from fat, (Adjusted R2=0.1131, overall model P=0.0018). Findings suggest indulgent practices are occurring early in Latino toddlers and may contribute to obesogenic eating patterns.

Grant Funding Source

National Research Initiative of the Cooperative State Research, Education and Extension Service, USDA, Grant # 2006‐55215‐16720; the University of California Institute for Mexico and the United States (UCMEXUS); the Western Center for Agricultural Health and Safety; the Food Stamp and Nutrition Education Program (FSNEP); and the Gustavus and Louise Pfeiffer Research Foundation
The addition of cysteine to the total sulphur amino acid requirement as methionine does not increase erythrocytes glutathione synthesis in the TPN fed human neonate
Authors: Courtney‐Martin Glenda , Moore Aideen M. , Ball Ronald O. , Pencharz Paul B.
Published: June 2021

Controversy exists as to whether the total parenteral nutrition (TPN) fed human neonate is capable of synthesizing adequate cysteine from methionine when the total sulphur amino acid (TSAA) is provided as methionine only. Our recent study, in which we determined the TSAA requirement as methionine only, provided evidence that the TPN fed human neonate is capable of synthesizing adequate cysteine from methionine; at least for protein synthesis. However, there is evidence that protein synthesis takes precedence over glutathione (GSH) synthesis when sulphur amino acid supply is low. The goal of this study was to determine whether GSH synthesis is maximized at a methionine intake adequate for protein synthesis in the TPN fed human neonate. We measured GSH concentration, fractional and absolute synthesis rate in 5 TPN fed post‐surgical human neonates in a repeated measures design. Each neonate received a cysteine free methionine adequate TPN for 3 d followed by a methionine adequate cysteine supplemented TPN for another 3 d and GSH kinetics measured on the 3rd d of each study period. The isotope used was [U‐13C2 ‐15N]glycine. Cysteine supplementation did not increase glutathione synthesis. Further studies in neonates on TPN for prolonged periods with evidence of TPN associated liver disease are required to determine if cysteine supplementation positively affects GSH synthesis under such conditions.
Effects of amino acids on phosphorylation of S6 ribosomal protein in mammary epithelial cells
Authors: APPUHAMY JAYASOORIYA RANGA , Hanigan Mark D , Escobar Jeffery
Published: June 2021

Protein synthesis is a highly regulated process responding to various signals including hormones and supply of energy and amino acids. The interactions of these signals have not been explored in bovine mammary epithelial (BME) cells. The objective of this study was to investigate the effects of individual amino acid deprivation on phosphorylation of ribosomal protein S6 which regulates protein synthesis. An established BME cell line (MAC‐T) was used as a model. Cells were grown to 85% confluence, serum starved for 12 h, and treated with serum‐free media containing all amino acids (Ctrl) or media singly deprived of Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, or Val for one hour. Then, cells were lysed, and cell lysates were subjected to Western immunoblotting using an antibody against phosphorylated S6 (rpS6235/236). Membranes were subsequently stripped and reprobed with an antibody against total S6. Results were expressed as ratios of pS6235/236: total S6 which is indicative of the S6 phosphorylation state. Deprivation of Arg, Val, and Ile reduced (P < 0.05) S6 phosphorylation by 96%, 90%, and 85%, respectively, compared to Ctrl. Deprivation of Phe and Leu reduced (P < 0.10) S6 phosphorylation by 45% and 30%, respectively. Branched chain amino acids, Arg, and Phe appeared to have key roles in regulation of protein translation initiation in BME cells.
Leucine supplemented meals improve muscle protein synthesis in older adults
Authors: Casperson Shanon , Sheffield‐Moore Melinda , Paddon‐Jones Douglas
Published: June 2021

Protein‐energy supplementation is routinely employed to combat the sarcopenia of aging. However, many regimens are impractical or ineffective due to issues including increased satiety, poor palatability, cost and compliance. To this end, we conducted a 14 day study in which we supplemented regular daily meals of community dwelling elders (n=8, 63‐76 yrs) with a small amount of the key regulatory amino acid leucine (4 g per meal; 3 meals per day; days 2‐13). Stable isotope studies consisting of a primed continuous L‐[ring‐13C6] phenylalanine infusion, arterialized blood samples and vastus lateralis muscle biopsies were performed on days 1 and 14. Mixed muscle fractional synthesis rate (FSR) was measured during the post‐absorptive state and following ingestion of a standardized test meal containing 10 g of carbohydrate and 7 g of mixed essential amino acids. Leucine supplementation significantly increased postabsorptive FSR (Day 1: 0.061 + 0.005 vs. Day 14: 0.074 + 0.006 %/hr; p=0.003) and the response to the standardized meal (Day 1: 0.073 + 0.006 vs. Day 14: 0.100 + 0.007 %/hr; p=0.02). These data suggest that a low volume leucine supplement may provide a minimally disruptive and efficient means of chronically stimulating muscle protein synthesis and improving the anabolic response to meals ingested by older adults.

Grant Funding Source

Funded by the UTMB Claude D. Pepper Older Americans Independence Center # P30 AG024832. Studies conducted on the GCRC at UTMB funded by grant M01 RR 00073 from the National Center for Research Resources, NIH, USPHS.
Influence of monosodium glutamate supplementation on plasma glutamine and on enzymes related to glutamine metabolism in rats
Authors: Boutry Claire , Bos Cecile , Matsumoto Hideki , Azzout‐Marniche Dalila , Tome Daniel , Blachier Francois
Published: June 2021

Free glutamate could exert signal function and/or metabolic effects due to the presence of receptors in the digestive tract and/or the adjustment of enzymes related to glutamine metabolism. We assessed the physiological and metabolic effects of monosodium glutamate (MSG) in rats at supplemental doses. Seventy four rats received either a MSG‐supplemented diet chronically for 15 days (MSG‐C) or acutely on the day of sacrifice (MSG‐A). A control group (C) received NaCl instead of MSG. The rats were sacrificed after ingestion of a 15N‐labeled meal. The 15‐d MSG supplementation had no effect on food intake, weight gain, body composition and leptinemia. Glutamine concentrations were significantly higher in plasma (+23%) and in urine (+309%) in MSG‐A vs. C. This was associated with an enhanced glutamine synthetase (GS) activity in muscle (+52%) and a decreased glutaminase activity (‐20%) in intestinal mucosa. The dietary N metabolic fate was not influenced by MSG supplementation, except for a higher protein accretion in intestinal mucosa (+13%) in MSG‐C vs. C. In conclusion, an acute MSG supplementation elicited increased glutamine concentration and consistent changes to enzymes activities related to glutamine metabolism. This effect was not maintained when MSG was supplemented chronically for 15 days.
Ultrastructure of mitochondria in various human pituitary tumor types
Authors: Sharma Soniya , Horvath Eva , Kovacs Kalman , Rotondo Fabio
Published: June 2021

The mitochondria, the major powerhouse for cellular functions, are predominantly ovoid or rod‐shaped cytoplasmic organelles enclosed by double outer membrane. The cristae, are formed by the infoldings of the inner membranes. Morphologic variations in size, shape, number of cristae exist, attuned to the function of the resident organ. Specific ultrastructural features characteristic of certain tumor types may occur. Such mitochondrial alterations were recorded by EM in more than 5000 various types of surgically removed pituitary adenomas. Changes are chiefly quantitative: A) size and density of internal structures (large/small; active/functionally suppressed prolactinoma); B) increase in number, i.e. oncocytic change. Generally occurring within the endocrine system owing to functional decline. Most prevalent in gonadotroph adenomas lineage. Specific alterations: A) giant mitochondria: specific marker of acidophil stem cell adenoma. With preservation of the double membranes, progressive enlargement and disappearance of the internal compartment and occurrence of unidentified tubular structures take place; B) Mitochondrial enlargement and homogenization of the entire internal compartment evident only in gonadotroph adenoma. C) Close spatial association of mitochondria and RER. It occurs only in silent adenoma subtype 3. The specific alterations may be used as ultrastructural markers.
Estimate of fibrosis in carbon tetrachloride induced chronic liver injury in rat by breath test with L‐[1‐13C] phenylalanine
Authors: Yan Weili , Qu Xinhua , Huang Xiaolu , Huang Gang , Xiong Ping
Published: June 2021

Liver malfunction is a major problem in the world. Traditional diagnoses which are based on indices of blood biochemistry and liver biopsy have limitations and disadvantages. Developing new non‐invasive methods to accurately assess liver function is always an interesting topic. The objective of this study is to investigate the relationship between the parameters of breath test and the histological grading, and provide valid parameters for the evaluation of liver function. We injected L‐[1‐13C]phenylalaline (13C‐Phe) into rats in the control group (n=8) and carbon tetrachloride (CCl4 ) induced chronic injury group (n=21) through tail vein, quantified the 13CO2 using air isotope ratio mass spectrometry, and examined histological changes of rat livers. We tested 12 parameters and found that only unit‐liver‐weight (LW) parameters, including maximum unit abundance (13Cmax/LW), unit abundance at 2 min and 7 min (13C2/LW and 13C7/LW), cumulative unit 13C excretion in 10 min and 30 min (AUC10/LW and AUC30/LW) and unit 13C excretion rate constant (PheBT‐k/LW) were significantly affected in the chronic injury group. Our findings indicate that the breath test using 13C‐Phe (PheBT) together with the unit‐liver‐weight (LW) parameters can quantitatively reflect the degree of fibrosis in the livers of chronically injured rats.
Insulin regulation of dopamine transporter activity.
Authors: Speed Nicole K , Owens William A , Kennedy Jack Philip , Vaughan Roxanne , Lindsley Craig , Niswender Kevin D , Daws Lynette C , Galli Aurelio
Published: June 2021

A growing body of literature points to insulin as an important regulator of dopamine transporter (DAT) function. Importantly, in vitro studies suggest that insulin regulation of DAT function might be mediated by the downstream effectors phosphotidylinositol 3‐kinase (PI3K), and its downstream effector protein kinase B (PKB, or Akt), which fine tunes DAT cell surface expression. Insulin status in rats was modified by administration of the diabetogenic agent streptozotocin (STZ), food restriction and high‐fat feeding (diet induced obesity (DIO)). A battery of biochemical and molecular approaches were used to measure DAT cell surface expression and activity of insulin downstream effectors. We found that Akt activity, as well as DAT cell surface expression, was significantly reduced in hypoinsulinemic rats and DIO rats. In addition, we have looked at the specific isoform of Akt involved in this pathway. We have available to us highly specific inhibitors of Akt1 (Akti‐1), Akt2 (Akti‐2) and of both (Akti‐1/2), allowing us to determine which Akt isoform regulates DA efflux and DAT trafficking. This work looks to further define the hypothesis that insulin signaling regulates DA clearance and AMPH actions.
Identification, Ki determination and CoMFA analysis of nuclear receptor ligands as competitive inhibitors of OATP1B1‐mediated estradiol‐17β‐glucuronide transport
Authors: Gui Chunshan , Wahlgren Brett , Lushington Gerald H. , Hagenbuch Bruno
Published: June 2021

Evidence shows that drug‐drug interactions can occur at the level of drug transporters such as the organic anion transporting polypeptide (OATP) 1B1, which mediates uptake of amphipathic organic compounds from blood into hepatocytes. Nuclear receptors are ligand‐activated transcription factors that play an important role in xenobiotic disposition and human diseases. Several of these nuclear receptor ligands have been shown to interact with OATP1B1. To understand the mechanisms of the polyspecificity of OATP1B1 and to predict and prevent the adverse drug‐drug interactions knowledge of a high‐resolution structure is critical. Because there is no crystal structure of Oatps/OATPs available to date, we attempted to elucidate the characteristics of the substrate binding site of OATP1B1 based on interactions with small molecules. First, we identified inhibitors of the model substrate estradiol‐17β‐glucuronide of OATP1B1 from about 40 nuclear receptor ligands. Then, we performed inhibition kinetic studies to identify competitive inhibitors and determine their Ki values which ranged from submicromolar to submillimolar. Finally, we performed CoMFA analysis on the identified competitive inhibitors. The CoMFA results indicate that the substrate binding site of OATP1B1 consists of a large hydrophobic middle part with basic residues at both ends that could be very important for substrate binding.
Interaction of Green Tea Catechins with Organic Anion Transporting Polypeptides
Authors: Roth Megan , Timmermann Barbara , Hagenbuch Bruno
Published: June 2021

Organic anion transporting polypeptides (OATPs) are multispecific transporters that mediate the uptake of numerous drugs and xenobiotics into cells. Here, we examined the effect of green tea (Camellia sinensis) flavanols on the function of three OATPs expressed on the basolateral membrane of human hepatocytes. Uptake of the model substrate estrone‐3‐sulfate was measured using CHO cells stably expressing OATP1B1, OATP1B3, or OATP2B1 in the absence and presence of the four most abundant flavanols found in green tea. Uptake by OATP1B1 and OATP2B1 was inhibited by epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) in a concentration dependent way. In contrast, OATP1B3‐mediated uptake of estrone‐3‐sulfate was strongly stimulated by EGCG (5‐fold increase, EC50 = 10.9 μM). The effect of EGCG on OATP1B3 was also studied with additional substrates: uptake of estradiol‐17β‐glucuronide was not affected, while uptake of Fluo‐3 was inhibited (IC50 of 8.3μM). These results indicate that two of the major flavanols found in green tea have a substantial effect upon the function of OATPs expressed in hepatocytes and can potentially alter the pharmacokinetics of drugs and other OATP substrates. These substrate dependent effects of EGCG on OATP1B3 seem to affect different transport/binding sites. In the future, we will use LC‐MS/MS analysis to identify whether these compounds are directly transported by OATPs.
Genetic variability of cytochrome b5 and NADH cytochrome b5 reductase: SNP discovery and characterization
Authors: Sacco James Carmel , Trepanier Lauren A
Published: June 2021

NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX‐HA) to the parent drug sulfamethoxazole. SMX‐HA and a spontaneous nitroso metabolite are thought to contribute to sulfonamide hypersensitivity. Variability in hydroxylamine reduction could therefore contribute to drug toxicity. The aim of this study was to characterize variability in SMX‐HA reduction in 111 human livers, and investigate the functional significance of non‐synonymous polymorphisms (SNPs) in b5R and b5 cDNA. Hepatic SMX‐HA reduction displayed a 19‐fold variability (0.09‐1.69 nmol/min/mg protein), with the Vmax for the reaction varying 5‐fold between low‐activity and high‐activity outliers. The expression of immunoreactive b5 and b5R were significantly correlated to each other (p<0.0001). SMX‐HA reduction activity increased with increasing b5 and b5R protein content (p=0.003, p=0.0239). A novel S5A SNP in b5 was associated with very low activity and protein expression. Two novel b5R SNPs (R59H, R297H) displayed biphasic SMX‐HA reduction kinetics, with the mutant enzyme contributing to the lower affinity component, and, in the case of the R59H SNP, contributing to a reduction in activity. The b5 S5A and b5R R297H SNPs are associated with reduced SMX‐HA reduction in human liver and work is ongoing to clone, express, and characterize these mutant b5 and b5R proteins.

Supported by National Institutes of Health grant GM61753
Dynamic patterns of histone methylation are associated with ontogenic expression of the Cyp3a genes during mouse liver maturation
Authors: Li Ye , Yue Cui , Hart Steven N , Klaassen Curtis D
Published: June 2021

Human cytochrome P450 3A members, CYP3A4 and CYP3A7, exhibit a developmental switch in gene expression during liver maturation. This developmental switch results in the variation of drug metabolism between children and adults. Mouse Cyp3a11 and Cyp3a16 are orthologous to CYP3A4 and CYP3A7 respectively. They also exhibit a similar developmental switch and may serve as a model to study human CYP3A switch. Since epigenetic mechanisms are implicated in gene expression and cellular differentiation during development, we hypothesized that they may be involved in controlling the developmental switch of Cyp3a genes. The current study determined the status of DNA methylation, histone 3 lysine 4 dimethylation (H3K4me2), and histone 3 lysine 27 trimethylation (H3K27me3) around the mouse Cyp3a locus by ChIP‐chip assays in mice livers at various developmental ages. DNA was not hypermethylated in the Cyp3a locus across from prenatal to adult stages. However, increases in Cyp3a11 expression in livers from perinatal to adult ages were associated with increases of H3K4me2 around Cyp3a11 gene. Suppression of Cyp3a16 expression in adult mice coincided with the decrease of H3K4me2 and increase of H3K27me3 around Cyp3a16. In conclusion, H3K4me2 and H3K27me3 are dynamically changed in a locus‐specific manner along the Cyp3a locus during liver maturation, and involved in the regulation of the developmental switch of Cyp3a genes.
TNFα enhances the expression and secretion of chemerin from adipocytes
Authors: Parlee Sebastian D , Ernst Matthew C , Sinal Christopher J , Goralski Kerry B
Published: June 2021

We recently identified a novel adipokine, chemerin that regulates adipogenesis and adipose metabolism via the chemokine‐like receptor 1 (CMKLR1). In humans, serum chemerin levels modestly correlate with measures of body fat and markers of metabolic syndrome suggesting a potential role for chemerin in obesity related diseases. The object of our study was to examine the regulation of chemerin secretion in models of obesity and inflammation. In 12 to 14‐week‐old ob/ob obese mice, serum chemerin levels but not adipocyte expression of chemerin were elevated 2‐fold. In comparison, CMKLR1 expression in adipocytes of ob/ob mice was reduced 70 % compared to controls. In 3T3‐L1 adipocytes, TNFα treatment produced a dose‐ and time‐dependent induction of chemerin and inhibition of CMKLR1 expression and stimulated the secretion of biologically active chemerin from 3T3‐L1 adipocytes. We conclude that serum chemerin levels are elevated in a mouse model of obesity, which may arise from TNFα‐mediated secretion of chemerin from adipocytes. However, chemerin/CMKLR1 signaling in adipocytes may be blunted in obesity due to reduced expression of CMKLR1. Funding: NSHRF, CIHR, DMRF, DPEF.
A probe for chaperone/multiprotein complexes and inhibitors in biological matrices
Authors: Moaddel Ruin , Sanghvi Mitesh , Marszall Michal , Kole Sutapa , Bernier Michel , Wainer Irving W.
Published: June 2021

Objective

Hsp90α was immobilized onto the surface of magnetic beads and used to extract small ligands and client proteins from chemical mixtures and biological matrices.

Methods

Hsp90α was immobilized through the N‐terminus, Hsp90α‐NT‐MB or C‐terminus, Hsp90α‐CT‐MB. Ligand fishing experiments were carried out using C‐terminus binders (CA1, NOVO), N‐terminus binders (17‐AAG, GM) and non‐binders (nicotine, propanolol). Protein fishing experiments were performed with recombinant eNOS, p60 Hop and Hsp70. The supernatants and eluates were analyzed using lc‐ms (small ligands) or Western blot analysis (proteins). The effect of ATP and Hsp90α thiol modifications on multiprotein complex formation was also studied.

Results

Data indicated that Hsp90α‐CT‐MB selectively bound N‐terminus ligands and Hsp90a‐NT‐MB selectively bound C‐terminus ligands. With isolated proteins, Hsp90α‐NT‐MB formed complexes with eNOS and p60 HOP but not Hsp70. All three proteins were extracted when Hsp90α‐NT‐MB was incubated with a mixture of proteins and p60 Hop was isolated from KU‐812 cellular extracts using Hsp90α‐NT‐MB. Hsp90α‐protein complex formation was reversibly reduced by ATP/MgCl2 and thiol modification.

Conclusions

The Hsp90α‐MBs can be used to isolate small molecule ligands and client proteins from complex chemical and biological matrices and to study Hsp90α multiple conformations.
Understanding ligand affinity and efficacy at mu and delta opioid receptors using cyclic pentapeptides
Authors: Purington Lauren , Mosberg Henry I. , Traynor John R.
Published: June 2021

Chronic use of mu opioid drugs such as morphine causes neurochemical adaptations leading to tolerance and dependence. While the effects of morphine are mediated by mu‐opioid receptors, several studies have shown that antagonism or knockdown of delta‐opioid receptors can modulate development of tolerance and dependence. To test the hypothesis that delta‐opioid receptors contribute to chronic effects of mu opioids, we developed cyclic pentapeptides displaying both mu agonism and delta antagonism and equivalent affinity as tools to gain a better understanding of the functional mu‐delta interaction(s). Based on in silico modeling data at putative active and inactive conformations of the mu‐ and delta‐opioid receptors, several peptides containing a napthylalanine substitution were synthesized. These compounds displayed variable mu agonist/delta antagonist profiles. One candidate (Peptide 8), displayed full agonism at the mu receptor (99% stimulation versus mu agonist DAMGO) and antagonism at the delta receptor (Ke = 20 nM versus delta agonist SNC80) in the GTPγ35S assay. Peptide 8 bound with equipotent affinity (ranging between 1.2‐1.4 nM) to all opioid receptors stably expressed in C6 rat glioma cells. In SH‐SY5Y cells endogenously expressing mu‐ and delta‐opioid receptors, Peptide 8 was a mu agonist with greater potency but lower efficacy than DAMGO. Supported by DA007281, DA007267, DA03910, and DA04087