bluuebunny/crossref_metadata_2025 huggingface dataset:
Roles of proline‐rich regions of the actin patch protein App1p in its phosphatidate phosphatase activity and membrane localization
Authors: FAKAS STYLIANOS , CARMAN GEORGE
Published: June 2021

Actin patch protein App1p is a component of cortical actin patches. It has been recently identified as a Mg2+‐dependent phosphatidate phosphatase (PAP) that catalyzes the conversion of phosphatidate (PA) to diacylglycerol (DAG) and Pi. The protein contains a canonical DXDX(T/V) catalytic motif that is typical of Mg2+‐dependent phosphatase enzymes and several proline‐rich regions (PXXP motifs). These proline‐rich regions are known to interact with Src homology 3 (SH3) domains that are characteristic of actin patch proteins (e.g., Abp1p and Rvs167p) involved in endocytosis. In fact, App1p has been shown to physically interact with several actin patch proteins. The fact that App1p possesses PAP activity indicates that it may regulate the local concentrations of PA and DAG found in cortical actin patches. PA and DAG are known to facilitate membrane fission/fusion events in model systems, and they are also known to interact with and regulate enzymes that play important roles in vesicular trafficking. In this work, the proline residues in the PXXP motifs in App1p were mutated to glycine by site‐specific mutagenesis. The mutant App1p enzymes were expressed in app1Δ and app1Δ pah1Δ dpp1Δ lpp1Δ quadruple mutant cells to assess the roles of PXXP motifs in the interaction with actin patch proteins, PAP activity, and the association of App1p with membranes. Supported by NIH grant GM‐28140.
Characterization of the cytosolic pool of perilipin 5
Authors: Brackbill James A , Moeller Katherine , Beussman Douglas J , Tansey John T.
Published: June 2021

The PAT family of lipid storage droplet (LD) associated proteins consists of five members, each an established regulator of neutral lipid metabolism. The most recently discovered family member, perilipin 5, has been shown to localize to two distinct intracellular pools: the lipid storage droplet and a cytosolic fraction, the nature of which is less understood. Characterization of the cytosolic fraction by way of sucrose gradient ultracentrifugation and non‐denaturing gel electrophoresis has indicated a density of 1.15 g/mL and a mass of 575 kDa for the particles on which LD‐dissociated perilipin 5 resides. The observed density and susceptibility of these structures to lipolytic degradation suggest that their core is rich in neutral lipid, much like the larger LD's. Based on the current understanding of these complexes, our lab is using a combination of immunoprecipitation and crosslinking techniques to identify other proteins that localize to these particles and interact with perilipin 5. Characterization of these candidate proteins holds the promise of yielding new insights into the regulation and function of the perilipin 5 protein, and to expand our understanding of lipid metabolism as a whole.
Sialic Acids Attached To Nav1.4 DIS5‐S6 N‐ And O‐glycans Contribute To Channel Gating
Authors: Ednie Andrew R , Harper Jean M , Bennett Eric S
Published: June 2021

Sialic acid residues attached to the Nav1.4 Domain I S5‐S6 extracellular loop (DIS5‐S6) contribute significantly to channel gating. Previous studies focused on the role of N‐glycans in channel gating. Here we questioned whether sialic acids attached to Nav1.4 D1S5‐S6 O‐glycans also contribute to modulation of channel gating. Gating of wild type Nav1.4 and an Nav1.4 chimera containing the Nav1.5 D1S5‐S6 loop was compared among three CHO cell types with varying abilities to glycosylate proteins. Nav1.4 but not the chimera gated at 15 mV (in Lec2 cells, reduced N‐ and O‐linked sialic acids) and 8 mV (in Lec1 cells, truncated N‐glycans) greater depolarizations compared to fully glycosylating control conditions (in Pro5 cells). This suggests N‐linked sialic acids account for ~1/2 of the impact of sialic acids on Nav1.4 gating. The converse experiment was performed using the ldl‐D cell system to express glycoproteins under varying conditions of N‐ and O‐glycans. Under conditions of full N‐ but no O‐glycosylation, gating of Nav1.4 but not the chimera was shifted by a depolarizing 9 mV and by 17 mV when both N‐ and O‐glycans were truncated. Further, all sialic acid effects were shown to be localized to the Nav1.4 D1S5‐S6 loop and could be described by an electrostatic mechanism. The data suggest that the Nav1.4 D1S5‐S6 loop is N‐ and O‐glycosylated and that the full impact of sialic acids on Nav1.4 gating can be accounted for by sialic acids attached to D1S5‐S6 N‐ and O‐glycans that modulate channel gating through electrostatic mechanisms.
Novel role of cytohesin‐2 in regulation of macropinocytosis pathway and cell proliferation
Authors: Marshansky Vladimir , Hosokawa Hiroyuki , Zhuang Zhenjie , Randazzo Paul A. , Grüber Gerhard , Ausiello Dennis A.
Published: June 2021

Recently, we uncovered four V‐ATPase a‐subunit isoforms derived peptides a1N1–17, a2N1–17, a3N1–17 and a4N1–17 as a potent inhibitors of the GDP/GTP‐exchange activity of cytohesin‐2 in vitro. Here, we used these peptides to study the role of cytohesin‐2 in regulation of macropinocytosis pathway and cell proliferation in vivo. Treatment of HeLa and MTC cells by cell‐permeable and fluorescently labeled peptides (FITC‐a2N1–17‐TAT or CMTR‐a2N1–17‐TAT) give rise to cell shape remodeling and activation of the macropinocytosis pathway. Uptake of RITC‐dextran but not albumin‐Alexa555 by this pathway was insensitive to inhibitors of NHE‐exchanger (amiloride, DMA, EIPA) and V‐ATPase (bafilomycin A1). Pulse‐chase and confocal time‐lapse imaging revealed that anti‐cytohesin peptides are targeted to endosomal/lysosomal compartments and slow down vesicular trafficking. We also showed that all four anti‐cytohesin peptides are potent inhibitors of the EGFR/cytohesin‐2 dependent proliferation of human lung cancer A549 cells. In summary, here we demonstrated an important role of signaling between V‐ATPase and cytohesin‐2 in regulation of macropinocytosis pathway and cell proliferation. Importantly, these anti‐cytohesin peptides could be used to treat EGFR/cytohesin‐2 dependent cancers and to prevent development of diabetic nephropathy. Supported by grants NIH DK038452 and BADERC DK05752–08 to VM.
Immune‐enhancing effect of high molecular weight portion of dextran glycated alpha‐lactalbumin
Authors: Hong Seung‐Taek , Koo Yun‐chang , Chun Su‐Hyun , Son Da‐Hee , Nam Mi‐Hyun , Oh Jun‐Gu , Lee Kwang‐Won
Published: June 2021

Proteins and reducing sugars can be conjugated by glycation reaction which is Maillard reaction. We have tried to find a new functional material through this reaction. In the previous study, we found that there was a potency of immune‐modulating effect on alpha‐lactalbumin (LA) and dextran (D) conjugate (LADC). There was no a significant difference between LA and LADC in terms of immune response in RAW 264.7 macrophage cell line. However, in this study, the immune‐enhancing effects of high molecular weight portion of LADC were shown DLA in RAW 264.7 was shown. LA and D were dry‐heated at 60¡É and 79% relative humidity for 1 and 15 days. SDS‐PAGE analysis was used to confirm the occurrence of the conjugated formation. 100 kDa membrane cut‐off was carried out to gain a high molecular weight portion of LADC. Immune‐enhancing effects were screened by nitric oxide (NO) production. TNF‐α, IL‐1β and IL‐6 cytokines were estimated in vitro. Data showed that the high molecular weight cutoff portion of LADC (LADCCO) showed dramatically increase of NO production. Also, LADCCO treated group presented high expression of immune‐related cytokines, TNF‐α, IL‐1β, and IL‐6, in RAW 264.7 macrophages. Therefore, our study suggests that high molecular weight cutoff portion of dextran glycated with alpha‐lactalbumin might be a new useful material for enhancing the immune responses.
Identification of Divergent Regulatory Mechanisms across the RGK Family of Small GTPases
Authors: Kaminski Catherine , Manning Janet R. , Magyar Janos , Shi Geng‐Xian , Sievert Gail , Satin Jonathan , Andres Doug
Published: June 2021

The small GTPases Rem, Rem2, Rad, and Gem/Kir (RGK) potently block L‐type calcium current (ICa,L) when overexpressed. Multiple RGK proteins are expressed in excitable tissues such as the heart. This study aims to understand how RGK proteins are regulated and how they function physiologically. Analysis of cardiac hypertrophy and ICa,L in Rem and Rad knockout mice suggests separable functions for these closely related G‐proteins. As a potential regulatory mechanism, Rem phosphorylation by protein kinase D (PKD) after α‐adrenergic stimulation was reported to relieve Rem‐mediated ICa,L inhibition (Jhun BS et al. Circulation Research 2012; 110:59–70). Using phospho‐Rem and phospho‐Gem specific antibodies, we also observed PKD‐dependent phosphorylation of Rem downstream of α‐adrenergic stimuli. Interestingly, while α‐adrenergic stimulation had no effect on the phosphorylation state of Gem, robust phosphorylation of Gem was achieved downstream of β‐adrenergic stimuli and was dependent on protein kinase A. Together, these studies suggest that individual RGK proteins have divergent roles in ICa,L modulation and that distinct kinase cascades may allow differential regulation of RGK proteins for fine tuning of ICa,L control.

This work was supported by the National Institutes of Health R01 HL072936 (to DAA and JS) and T32 HL072743 (to CNK).
Gα12 regulation of polycystin‐1 proteolysis and nuclear localization
Authors: Cavagnini Kyle S , Snelson Mark T , Denker Bradley M , Meigs Thomas E
Published: June 2021

Autosomal dominant polycystic kidney disease frequently leads to end‐stage renal failure, and mutations in the cell surface glycoprotein polycystin‐1 (PC1) have been implicated in 85% of disease cases. Previous studies have shown the cytoplasmic tail of PC1 to undergo proteolytic cleavage and subsequent nuclear localization, where it regulates several key genes related to cell proliferation, differentiation, and apoptosis. We previously characterized the interaction of PC1 with the trimeric G protein Gα12 as requiring a PC1 region encompassing a G protein binding sequence (GPS) located on the cytoplasmic tail. In separate studies, a region closely overlapping the GPS was reported as a nuclear localization signal for the cleaved PC1 tail. To test the hypothesis that binding of Gα12 regulates proteolysis of the PC1 tail or its subsequent trafficking, we engineered a PC1 construct HA‐tagged at the C‐terminus, to allow monitoring of proteolysis and trafficking. Here, we describe effects of constitutively activated Gα12 on generation of the PC1 cytoplasmic tail, and are examining whether Gα12 expression regulates its nuclear localization. In addition, we have engineered a panel of charge‐reversal mutations within the GPS/nuclear import signal of PC1, and are utilizing these to examine whether disruption of Gα12 binding affects PC1‐mediated activation of the transcription factor AP‐1. We acknowledge support from the UNC‐Asheville Undergraduate Research Program (KSC and MTS), Harvard Polycystic Kidney Center (BMD), and Lineberger Comprehensive Cancer Center (TEM).
eYFP reporter system for pyroglutamate incorporation
Authors: Benhaim Mark , Sheppard Kelly
Published: June 2021

Pyroglutamate (pGlu) is an amino acid formed from modification of glutamine in proteins like β‐amyloid peptides associated with Alzheimer's disease and the anti‐cancer agent, onconase. To allow for in‐depth studies into the role pGlu plays in proteins and its relationship to diseases like Alzheimer's, it would be beneficial to synthesize polypeptides with pGlu incorporated during translation (i.e., expand the genetic code to include pGlu). We have developed a means to synthesize pGlu on transfer RNA by modifying the archaeal system for tRNA‐dependent glutamine formation and placed the system into E. coli to decode amber stop codons (UAG) with pGlu. To determine if there is site‐specific incorporation of pGlu at amber stop codons, we have selected the enhanced yellow fluorescent protein (eYFP) as our reporter protein and codon 14 for pGlu incorporation. Position 14 in the protein is normally proline, a chemical analog of pGlu. We have mutated efyp to have an amber, opal, or ochre stop codon at codon 14. After optimizing eYFP overproduction, we will determine if our modified archaeal system in E. coli allows for specific decoding of the amber stop codon in eyfp with pGlu.
Effect of omega‐3 fatty acid intake on homocysteine in African and Haitian Americans with type 2 diabetes
Authors: Vaccaro Joan A , Exebio Joel C , Ajabshir Sahar , Zarini Gustavo G , Huffman Fatma G
Published: June 2021

Objective

Homocysteine (HCY) has been considered an independent risk factor for cardiovascular disease. The aim of the study was to assess the relationships among ethnicity, diabetes, omega‐3 fatty acid (n‐3) intake and HCY.

Methods

This was a cross‐sectional study of N=410 African (AA) (n=172) and Haitian (HA) (n=238) Americans with and without type 2 diabetes recruited from the area of Miami‐Dade and Broward counties in Florida, USA using mailing lists and community methods. Sociodemographics, anthropometrics and blood samples were collected after participants signed informed consent. All dietary variables were collected using a semi‐quantitative food frequency questionnaire.

Results

Ethnicity by n‐3 intake was associated with HCY (p=.004) in an adjusted model [χ2 (10) = 30.9 (N=406) p = 0.001]. The odds of having high HCY for AA with low n‐3 intake (≥ 1.0 g/day) was [OR = 4.36 (1.59, 12.0) p = .004]. Ethnicity (p = .155), diabetes (p = .770) and diabetes by n‐3 intake (p = .965) were not significantly associated with HCY.

Conclusion

Omega‐3 fatty acid intake and HCY should be investigated within ethnicities to determine beneficial levels. Funding was by an NIH/NIDDK grant.
Elevated plasma C‐Reactive Protein (CRP) and low carotenoid levels are associated with increased mortality in colorectal cancer patients
Authors: Cooney Robert Vincent , Chai Weiwen , Franke Adrian A. , Caberto Christian , Wilkens Lynne R. , Kolonel Laurence N. , Custer Laurie , Morrison Cynthia , Le Marchand Loic
Published: June 2021

Objective

To determine the associations of plasma CRP and carotenoids with survival in colorectal cancer patients.

Methods

Using stored patient plasma samples from a multiethnic population‐based case‐control study of colorectal cancer, we determined associations of post‐treatment blood levels of CRP and carotenoids with survival. Patients (n = 455) were followed (mean of 8 years) after phlebotomy, during which time 20% died of colorectal cancer and 24% from other causes. Hazard ratios were estimated by Cox proportional hazards regression with adjustment for stage and age at diagnosis, ethnicity, sex, and smoking status.

Results

A strong positive association with all‐cause mortality was observed for plasma CRP (Ptrend < 0.0001). Inverse associations were seen for α‐carotene (Ptrend = 0.02), α‐cryptoxanthin (Ptrend = 0.002), lycopene (Ptrend = 0.006), and lutein/zeaxanthin (Ptrend = 0.07), whereas no significant associations were observed for the pro‐vitamin A carotenoids, β‐carotene and β‐cryptoxanthin.

Conclusions

Results are consistent with a direct relationship for increased inflammation with poor survival among colorectal cancer patients. Future studies of nutritional approaches to reduce inflammation and CRP levels and increase specific carotenoids in deficient patients may be warranted as a complementary treatment strategy for colorectal cancer.
Food group consumption and its association with BMI z‐score and socioeconomic characteristics in rural school‐aged children
Authors: Choumenkovitch Silvina Furlong , McKeown Nicola M , Hyatt Raymond R , Kraak Vivica I , Cohen Juliana F W , Economos Christina D
Published: June 2021

U.S. children's compliance with dietary recommendations varies by age, gender, and socioeconomic and weight status. We analyzed data from 1273 children (grades 1–6) participating in the Creating Healthy, Active and Nourishing Growing‐up Environments (CHANGE) study, an obesity‐prevention intervention in eight rural US communities. Children's intakes of fruits, vegetables, whole grains, protein foods and dairy were assessed using the Block Food Screener for ages 2–17 y. General linear models were used to calculate adjusted prevalence of meeting 50% of recommended whole‐grain servings and 100% of recommended servings for the other groups and to study the association between covering recommendations and weight. Prevalence of meeting dietary recommendations decreased across age groups for all food groups except fruits. Prevalence of meeting the fruit recommendations was lower in boys than girls. Hispanic children were more likely to consume recommended fruit and dairy servings, compared to black and white children, and whole grains compared to white children. BMI z‐score and prevalence of overweight and obesity was lower in children meeting recommendations for all food groups compared to children who met no recommendations (1.13 vs.0.77, P=0.02 and 39 vs. 61%, P < 0.001, respectively). Adhering to dietary recommendations varied by age, gender, race and weight status in this population of rural children.

Support was provided by Save the Children's U.S. Programs.
World Health Organization infant and young child feeding indicators and their associations with child growth: a synthesis of recent findings
Authors: Jones Andrew David , Ickes Scott B , Smith Laura E , Mduduzi Mdu NN , Chasekwa Bernard , Heidkamp Rebecca A , Menon Purnima , Zongrone Amanda A , Stoltzfus Rebecca J
Published: June 2021

This study aims to determine cross‐country patterns of associations of each of the World Health Organization (WHO) infant and young child feeding (IYCF) indicators with child stunting, wasting, height‐for‐age Z‐score (HAZ) and weight‐for‐height Z‐score (WHZ) in order to assess the constructs measured by the indicators across contexts. Seven studies using recent Demographic and Health Surveys data from a total of eight countries in sub‐Saharan Africa (5), Asia (2), and the Caribbean (1) were identified. The WHO indicators showed mixed associations with child growth across regions. Exclusive breastfeeding (EBF) under six months and continued breastfeeding demonstrated negative associations with HAZ while introduction of complementary foods (CF) and dietary diversity were positively associated (P<0.01 and P<0.05, respectively) with HAZ in Bangladesh and Zambia. EBF was also associated with higher WHZ in Bangladesh (P<0.05) and Zambia (P<0.001). The “minimum acceptable diet” (MAD) indicator was positively associated with HAZ in Bangladesh, Zambia and Ethiopia (P<0.05). Child stunting was associated only with introduction of CF in Bangladesh (P<0.01) and the MAD indicator in Zimbabwe (P<0.05). These indicators are useful tools for monitoring trends in breastfeeding and complementary feeding practices. However, additional measures of dietary quality that demonstrate consistent associations with child growth may be needed for research purposes.
Low retinol levels virtually undetected in coastal Guatemala: Findings from a convenience sample of women and schoolchildren
Authors: Soto‐Méndez María José , Campos Raquel , Martínez Ana Carolina , Detlefsen Michelle , Solomons Noel W
Published: June 2021

Background

Guatemala was the first nation ever to mandate fortification of granulated table sugar with vitamin A. Norms specify a concentration of from 10 to 20 mg of retinol as retinyl palmitate per kg of sugar. The program has been in continuous operation since 1987.

Objective

To assess vitamin A status with circulating retinol concentration in a convenience sample of women and children from coastal plain of Guatemala.

Methods

Plasma samples were collected from 157 adult women and 134 schoolchildren from among 12 rural communities in the sugarcane‐growing Pacific lowlands of Guatemala. Retinol concentration was determined by high precision liquid chomatography at INCAP, and values were expressed in μg/dL.

Results

The global median retinol concentration was 52 μg/dL. The mean retinol concentration of 63±23 μg/dL for women was significantly higher than the 46±14 μg/dL in children (p<0.001). The overall distribution of retinol concentration values was: <20 μg/dL, 0%; 20–29 μg/dL, 3%; 30–49 μg/dL, 44%; 50–69 μg/dL, 37%; 70–89 μg/dL, 11%; >;90 μg/dL, 5%.

Conclusion

Retinol concentration in the deficient (<20 μg/dL) and low (20–29 μg/dL) ranges were virtually undetected in coastal Guatemala, related, in part, we propose to the national sugar fortification program.

Funded by Cabcorp, Guatemala and the Hildegard Grunow Foundation, Munich
Factors Associated with Low Water Intake among Korean Adolescents ‐ Korea National Health and Nutrition Examination Survey, 2007–2010
Authors: Kim Mi‐Hyun , Park Sohyun , Lee Haeng‐Shin
Published: June 2021

Drinking water is essential for life and consuming plain water instead of calorically sweetened beverages is one approach for decreasing energy intake. Due to limited information on characteristics associated with water intake among Korean adolescents, we examined water intake and associations of demographic characteristics and behavioral factors with water intake among a nationally representative sample of Korean adolescents. We used the 2007–2010 Korea National Health and Nutrition Examination Survey data for 1,288 adolescents (15–18 y). Multivariable logistic regression was used to calculate adjusted odds ratios (OR) for factors associated with low water intake (<4 cups/d). Nationwide, mean water intake was 5.7 cups/d for males, 4.1 cups/d for females, and 38.4% of adolescents reported drinking water <4 cups/d. Statistically significant factors associated with greater odds for low water intake were being female (OR=2.8) and not meeting physical active recommendation (≥20 min/d on <3 d/wk) (OR=1.6). Being underweight, overweight, and obese were significantly associated with reduced odds for low water intake (OR=0.7, 0.4 and 0.5, respectively). However, intake of milk, soda, coffee, fruits, vegetables, and sodium and eating out were not significantly associated with low water intake. Our findings may be used to tailor intervention efforts to increase plain water intake as part of a healthy lifestyle.
Breastfeeding improves biochemical control in children with phenylketonuria
Authors: O'sullivan Elizabeth , Boyle Fiona , Mehta Saurabh , Kearney John
Published: June 2021

Objective

This research sought to analyze blood levels of phenylalanine (Phe) in children with phenylketonuria (PKU) by breastfeeding status and duration.

Methods

173 patients with severe and moderately severe PKU born between January 1st 1994 and December 31st 2008 were retrospectively identified from the patient database of the NCIMD, Ireland. Data on duration of breastfeeding were collected from medical charts and all recorded Phe blood levels from 3 weeks to 5 years of age were accessed from patients’ digital and paper records. Data was analyzed using SAS 9.3; repeated measures modeling was used to examine the impact of breastfeeding on blood Phe levels.

Results

All models were fitted controlling for severity of disease (moderate/severe). A significant main effect of breastfeeding was found, suggesting that infants ever breastfed had lower Phe levels than those never breastfed. Breastfeeding for a longer duration reduced Phe levels significantly more than breastfeeding for shorter durations.

Conclusions

Preliminary analysis of this data indicates that breastfeeding may lower blood Phe levels in children with PKU. Additionally, there may be a dose‐response effect with longer durations of breastfeeding leading to better biochemical control.
Breastmilk and the lactating breast as a tool to elucidate breast cancer
Authors: Hassiotou Foteini , Hartmann Peter E. , Blancafort Pilar
Published: June 2021

Mammary stem cells (MaSCs) are responsible for the normal expansion of the breast during pregnancy/lactation. MaSCs are also primary targets of malignant transformation, giving rise to invasive breast carcinomas, a subset of which display lactating features. We have recently reported expression of OCT4 and associated embryonic stem cell (ESC) genes in the normal lactating breast and breastmilk stem cells (hBSCs). This prompted us to examine OCT4 expression in breast cancers with lactating features and to compare it with that observed during normal lactation. Minimal OCT4 expression was found in the normal resting breast, whereas this significantly increased in the normal lactating breast. Further increase was found in breast tumours with lactating features. Comparison of normal hBSCs with OCT4‐overexpressing tumorigenic breast cell lines (OTBCs) showed that OTBCs had unbalanced expression of ESC genes. Similar to hBSCs, OTBCs differentiated into functional lactocytes synthesising milk proteins both in vitro and in vivo. These findings highlight ESC genes as potential regulators of the normal remodelling of the breast towards a milk‐secretory organ and of aberrant breast cell proliferation upon deregulation of their expression, suggesting a role in both normal lactation and breast oncogenesis.

Grant Funding Source: Women and Infants Research Foundation; and Medela AG (Switzerland)
Plasma glutathione resettlement by oral glutamine supplementation is associated with glycine normalization in HIV+ treated patients
Authors: Burini Roberto Carlos , Borges‐Santos Maria Doroteia , Moreto Fernando , Ming‐Yu Yong
Published: June 2021

HIV+ patients present low concentrations of reduced GSH and its precursors. To investigate the responses of the GSH pathway to Gln dietary supplements in 12 HIV+ treated patients (6 men and 6 women, 22 – 45 years old) and 20 healthy controls (Co, 10 men and 10 women, 20 – 59 years old) were studied at their usual diet (Do) and after 7‐day dietary‐supplements containing glutamine (Gln, 20g/d). Blood samples were drawn after overnight fast and HPLC plasma analysis of amino acids, total (GSH) and oxidized glutathione (GSSG) were carried out at moments before (M0) and after the intervention period (M1). Statistical comparisons were made between groups (Co X HIV+) with p<0.05. Under Do the HIV+ group showed higher GSSG along with lower concentrations of GSH and all amino acids except Hcy. Met, Gly and GSH were normalized by the dietary Gln. The 71.4% increase of GSH in Gln group at M1 was followed by the increase of its precursors markedly Gly (27.2%) and slightly by Glu (13.7%) and Cys (10.8%), nevertheless without detectable reduction in the GSSG/GSH ratio. Moreover the intervention led to an increased Tau/Cys (18.9%) and decrease of Glu/Gln (6.6%) ratios relatively to Co. In conclusion, the restoration of GSH normal levels by Gln was markedly supported by Gly replenishment. The restricted contribution of Cys and Glu to GSH generation could be due to either their limited formation (Gln→Glu) and/or higher utilization (Cys→Tau).

Supported by FAPESP, CAPES, CNPq
Estimation of Dietary Vitamin K Intakes and Food Sources: Using the Korean National Health and Nutrition Examination Survey
Authors: Sohn Cheongmin , Kim Eunsu , Kim Misung , Na Woori
Published: June 2021

It is well known that vitamin K works for blood clotting reaction and helps forming bones by boosting formation of osteoblast. Recently it has been reported that it also prevents various illnesses like type 2 diabetes, obesity and cardiovascular disorders. However, there has been insufficient development of nutrition intake tools for estimating vitamin K intakes and analyzing the amount of vitamin K intakes for Korean people. Therefore, this study aims to provide the basic data for developing tools to analyze vitamin K intakes by analyzing Koreans’ vitamin K intakes and its food sources. This study used the primitive data of the 5th the Korean National Health and Nutrition Examination Survey, and analyzed data from 8,027 subjects who participated in the survey among total 9,691 subjects of the nutrition examination survey.

The vitamin K intakes were shown as 248.03±4.34 μg/day for men and 199.25±3.54 μg/day for women(p<0.001). In case of intakes for each age group, this study found 127.64±3.09 μg/day for teens, 208.10±8.67 μg/day for 20s, 264.88±7.08 μg/day for 30s, 271.63±7.68 μg/day for 40s, 294.17 ± 10.49 μg/day for 50s, 256.20±11.28 μg/day for 60s and 220.05±6.55 μg/day for 70s(p<0.001). For the contribution of vitamin K to each food group, vegetable was the highest(84.3%) and then respectively seaweed(5.5%), fruits(2.9%), seasoning(2.8%), fat and oil(2.2%), beans(1.2%). As dish sources for vitamin K, we found respectively cabbage kimchi(14.9%), seasoned spinach(9.5%), soybean past soup with dried radish greens(4.8%), rich soybean paste stew(3.9%) and gimbap(2.9%) in the order.

This study found that Koreans’ average vitamin K intakes is 233 μg/day, which means that Koreans’ vitamin K intakes is relatively higher than Westerners. It is considered that such results reflect Koreans’ dietary composition which helps to frequently intake food that contains a lot of vitamin K like cabbage, spinach and radish greens as side dishes.
Metabolism of ginger component [6]‐shogaol in liver microsomes from mouse, rat, dog, monkey, and human
Authors: Soroka Dominique Nicole , Chen Huadong , Zhu Yingdong , Sang Shengmin
Published: June 2021

[6]‐Shogaol, a major bioactive component of ginger, has been reported to have potent anti‐cancer activity. The in vitro metabolism of [6]‐shogaol was compared among five species with respect to metabolic profiles using liver microsomes from mouse (MLM), rat (RLM), dog (DLM), monkey (CyLM), and human (HLM). Following incubations with [6]‐shogaol, major reductive metabolites 1‐(4′‐hydroxy‐3′‐methoxyphenyl)‐4‐decen‐3‐ol (M6), 1‐(4′‐hydroxy‐3′‐methoxyphenyl)‐decan‐3‐ol (M9), and 1‐(4′‐ hydroxy‐3′‐methoxyphenyl)‐decan‐3‐one (M11) were found in all species. Two new oxidized metabolites of 6‐shogaol, M14 and M15, were identified as (1E,4E)‐1‐(4‐Hydroxy‐3‐ methoxyphenyl)deca‐1,4‐dien‐3‐one and (E)‐1‐(4‐Hydroxy‐3‐ methoxyphenyl)dec‐1‐en‐3‐one, respectively. The kinetic parameters of M6 in liver microsomes from each respective species were quantified using Michaelis‐Menton theory. A broad CYP450 inhibitor, 1‐aminobenzotriazole (ABT), precluded formation of oxidative metabolites M14 and M15 in HLM, and diminished oxidative metabolism in MLM, RLM, DLM, and CyLM. Additionally, 18β‐glycyrrhetinic acid, an aldo‐keto reductase (AKR) inhibitor, eradicated the formation of the reductive metabolites M6, M9, and M11 in all species. Metabolites M14 and M15 were tested for cancer cell growth inhibition and induction of apoptosis and both showed substantial activity, with M14 displaying greater potency than [6]‐shogaol.

We conclude that [6]‐shogaol is metabolized extensively in mammalian species mouse, rat, dog, monkey, and human, and that there are significant interspecies differences to consider when planning pre‐clinical trials towards [6]‐shogaol chemoprevention.

This work was supported by NIH grants CA138277 and CA138277S1 to S. Sang.
Leucine modulation of AMPK and mitochondrial biogenesis in C2C12 myotubes is Sirt1 dependent
Authors: Liang Chunzi , Zemel Michael B
Published: June 2021

We previously showed leucine and its metabolites to stimulate Sirt1 expression and activity, fat oxidation and mitochondrial biogenesis, in both adipocytes and myotubes. However, the mechanism of these effects is poorly understood. Here we show that leucine increased cellular NAD+ levels 5‐fold (p=0.025) and that there were subsequent increases [150%] in NAD+‐dependent Sirt1 activity (p=0.001). Further, leucine and its metabolites (KIC and HMB) significantly increased fatty acid oxidation [350%] (p=0.014) and these effects were inhibited by the SIRT1 inhibitor (EX527) (p=0.0001). Similarly, leucine stimulated mitochondrial biogenesis (200%, p=0.001), with associated increases in expression of PGC‐1a (268%, p=0.0001) and SIRT3 (227%, p=0.009), while EX527 inhibited this effect (p=0.013). Finally leucine activated AMPK (310%, p=0.025), while this effect was also blocked by EX527. These data indicate that leucine stimulates of AMPK and mitochondrial biogenesis is mediated by Sirt1, possibly due to increase in NAD+ levels.
Increasing Tomato Intake from Current to MyPlate Recommended Amount: Effect on Serum Lycopene
Authors: Reimers Kristin , Andon Mark , Jacobson Mary , Nguyen Von , Cooper Lisa , Rippe James
Published: June 2021

To encourage greater vegetable intake, USDA's MyPlate food guidance recommends 0.8 cup equivalents of tomatoes/d, 0.5 cups more than the 2005 recommendation. Tomatoes are the primary dietary source of lycopene, and both tomato intake and serum lycopene are inversely correlated with chronic disease risk. To determine the effect of consuming an amount of tomatoes similar to MyPlate recommendation on serum lycopene, participants (n = 58, ages 35 – 70, BMI 27 – 37) with a baseline tomato intake of 0.15 cups/d were asked to consume 1.0 cup equivalent/d of canned tomatoes, sauce, or paste as part of their usual diet for six weeks. Compared to baseline, median lycopene increased 32% from 0.40 ± 0.18 to 0.53 ± 0.21 μg/ml. Lycopene values by quartile were:

Median (sd) Serum Lycopene, μg/ml

Baseline
Six Weeks
% Change

Q1
0.22 (0.05)
0.32 (0.06)
45

Q2
0.33 (0.03)
0.49 (0.04)
48

Q3
0.47 (0.04)
0.62 (0.04)
32

Q4
0.68 (0.14)
0.88 (0.13)
29

These data show that increasing tomato intake from usual amounts to an amount consistent with MyPlate recommendations causes an upward shift in serum lycopene that is equivalent to approximately one quartile across the concentration spectrum.
Lentinula edodes Consumption: Proliferation, Activation and Modification of Memory and Naive Innate Immune Cell Populations
Authors: Stanilka Joy M , Rowe Cheryl A , Creasy Rebecca A , Dai Xiaoshuang , Percival Susan S
Published: June 2021

A human nutrition intervention was conducted to evaluate the effect of dried Lentinula edodes (Shiitake) mushrooms on immunological changes in healthy subjects. 51 subjects were randomized to 1(50g) or 2 (100g) servings/d for 28d. Peripheral blood mononuclear cells were isolated and cultured for 24hr or 6d with PHA‐L, before and after mushroom consumption. Proliferation and activation of gamma delta T (γδTCR+/CD3+), NK (CD56+/CD3−) and NKT (CD56+/CD3+) cells were determined by flow cytometry. In addition, naïve (CD45RA+) and memory (CD45RO+) γδT cell populations were determined. After mushroom consumption, significant increases in proliferation were observed after 24hr for NK and NKT cells and after 6d for all three cell types. An increase in the ability to proliferate ex vivo suggests a more robust response may occur in vivo. Changes in the distribution of CD45RO+ and CD45RA+ on γδ TCR+ cells were observed suggesting mushroom consumption influences maturation of γδ‐T cells in vivo. Expression of CD69 on γδ T cells and CD314 on NK and NKT cells was significantly increased during 24hr of culture, after 28d of mushroom consumption. Increased ability to express these activation markers suggests the cells may be primed. Overall, changes observed in these innate immune cells suggest positive benefits of regular Shiitake mushroom consumption. Support from US Mushroom Council & Australian Mushroom Growers Assn.
Agonist‐induced conformational change of the Formyl Peptide Receptors
Authors: Cooray Sadani Nandima , Clark Adrian , Flower Roderick , Perretti Mauro
Published: June 2021

The human Formyl Peptide Receptor family (FPR1, FPR2/ALX and FPR3) belongs to the superfamily of class A GPCRs and is responsive to a variety of agonists. ALX is the first GPCR known to be able to bind to both peptides and lipids and transduce both pro and anti‐inflammatory cellular responses. To investigate the versatility and potential ligand biased agonism of ALX, we explored the possibility that FPRs may exist as dimeric structures and tested whether these interactions may be modulated by different ligands. We used a combination of the co‐immunoprecipitation (co‐IP) technique and Bioluminescence Resonance Energy Transfer (BRET) in HEK293 cells transfected with epitope tagged receptors. We show that FPRs are constitutively dimerised and reveal opposing effects on the BRET signal between the ALX homodimers in response to the anti‐inflammatory (AnxA1 and LXA4) and proinflammatory (SAA) agonists. The pan agonist Ac2–26 enhanced the BRET signal only when ALX was expressed, alone or together with FPR1. We conclude that ALX can adopt a ligand‐directed conformation upon application of pro‐resolving agonists leading to specific intracellular signalling. Our findings indicate that agonist binding, activation of signalling molecules as well as the dimerisation state of the receptors may contribute to the conformational landscape of the FPRs.

Support: Wellcome Trust (Programme 08667/Z/08/Z)
Differential Trafficking of the FPR2/ALX receptor in response to endogenous or synthetic ligands
Authors: Thompson Dawn , Perretti Mauro
Published: June 2021

Following agonist activation G protein‐coupled receptors (GPCR) undergo rapid endocytosis and are recycled back to the cell surface to once again bind agonist (resensitisation), or targeted to the lysosomes (downregulation). The formyl peptide family (comprising FPR1, FPR2/ALX and FPR3) regulate both pro and anti‐inflammatory effectors in the immune response. Despite several articles identifying important phosphorylation residues, little is known about the endocytosis and subsequent post‐endocytic sorting of these receptors. Here we compare the trafficking and signalling of FPR2 in response to synthetic or endogenous agonists. The synthetic peptide (500nM WKYMWm) induced rapid endocytosis of FPR2 followed by extensive recycling after agonist washout. Furthermore, FPR2 was stable following prolonged agonist treatment (180mins). Consistent with recycling, ERK phosphorylation was desensitised after 30mins agonist treatment but recovered after agonist washout. In contrast, neither the endogenous anti‐inflammatory Annexin A1 nor the pro‐inflammatory Serum Amyloid A (SAA) induced endocytosis of the receptor, despite robust ERK phosphorylation and calcium release. These findings have profound implications for the design of new ligands to treat inflammatory disorders, since use of synthetic ligands promoting endocytosis may help reset the system returning signalling to a physiological level.
Heparanase upregulation in the inflammatory airway facilitates syndecan shedding and sustained inflammation
Authors: Wu Lap Kei Kenneth , Chan Stanley CH , Ip Mary SM , Shum Daisy KY
Published: June 2021

Shed syndecan‐1 and neutrophil elastase forms anti‐elastase resistant complexes in the airway fluids. This is thought to be the cause of airway damage and deteriorating lung function in COPD and bronchiectasis. We aim to develop strategies to decrease syndecan‐1 shedding for the treatment of COPD and bronchiectasis.

Elevated levels of heparanse but not matrix metalloproteinase‐7 (MMP‐7) in sputum samples of such patients suggested that the action of the GAG digesting enzyme heparanase modifies syndecan‐1 to become more susceptible to shedding by MMP‐7 cleavage.

We show in vitro that heparanase induced syndecan‐1 shedding in cultured human epithelial cells, and that heparanase‐treated recombinant syndecan‐1 was more readily cleaved by MMP‐7 than control treatment of the recombinant syndecan‐1. Surface plasmon resonance analysis demonstrated an increase in affinity of syndecan‐1 for MMP‐7 upon heparanse treatment. Co‐immunoprecipitation study targeting syndecan‐1 found associated MMP‐7, significantly higher in the sputum samples of bronchiectasis and COPD patients than in induced sputum of healthy individuals. The results support that heparanase activity in the inflammatory airway environment enhances shedding of syndecan‐1 and thus a target for the treatment of bronchiectasis and COPD. This work is supported by the HKRGC.
Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemia–reperfusion: Therapeutic potential of mitochondrially targeted antioxidants
Authors: PACHER PAL , Mukhopadhyay Partha , Horvath Bela , Zsengeller Zsuzsanna , Batkai Sandor , Cao Zongxian , Kechrid Malek , Holovac Eileen , Erdelyi Katalin , Liaudet Lucas , Stillman Isaac E , Joseph Joy , Kalyanaraman Balaraman
Published: June 2021

Mitochondrial reactive oxygen species generation has been implicated in the pathophysiology of ischemia‐reperfusion (I/R) injury; however, its exact role with inflammation are elusive. Herein we explore the spatial‐temporal relationship of oxidative/nitrative stress and inflammatory response during the course of hepatic I/R and the possible therapeutic potential of mitochondrial‐targeted antioxidants, using a mouse model of segmental hepatic ischemia‐reperfusion injury. Hepatic I/R was characterized by early mitochondrial injury, decreased complex I activity, increased oxidant generation in the liver or liver mitochondria, and profound hepatocellular injury/dysfunction with acute proinflammatory response without inflammatory cell infiltration, followed by marked neutrophil infiltration and a more pronounced secondary wave of oxidative stress in the liver. Mitochondrially targeted antioxidants, MitoQ or Mito‐CP, dose‐dependently attenuated I/R‐induced liver dysfunction, the early and delayed oxidative stress response, and mitochondrial and histopathological injury/dysfunction, as well as delayed inflammatory cell infiltration and cell death. Mitochondrially generated oxidants play a central role in triggering the deleterious cascade of events associated with hepatic I/R, which may be targeted by novel antioxidants for therapeutic advantage
Cytotoxicity effects of tert‐butyl hydroperoxide‐induced p53 ‐ mediated cell‐cycle arrest, apoptosis and aging in rat aortic endotheial cells and aorta: inhibition by Angelica sinesis extracts
Authors: Yeh Yueh‐Chiao , Liu Tsun‐Jui , Shin Tzu‐Hui , Wang Li‐Chuan , Lai Hui‐Chin
Published: June 2021

Endotheial dysfunction is one of the underlying causes for vascular diseases. tert‐butyl hydroperoxide (t‐BHP), a short‐chain lipid hydroperoxide analog, has been referred to cause cellular senescence in different systems. This study aims to investigate whether the potent antioxidant Angelica sinesis (AS) extracts, often used in traditional Chinese medicine, could protect against the t‐BHP induced toxicity in rat aortic endothelium. Primary cultured endothelial cells (ECs) were treated with vehicle or t‐BHP. Exposure of ECs to lower doses t‐BHP caused cellular senescence and apoptosis, and higher doses initiated cell‐cycle arrest, telomerase acitivty diminished, and cellular inflammation. However, these adverse effects could be antagonized by AS extracts pretreatment via p53‐dependent mitochondrial signaling pathways. In terms of in vivo study, male rats at 6‐or 24‐week of age were administrated with vehicle or t‐BHP. Significanty increased TUNEL‐ and SA‐beta‐gal‐positive cells, provoked caspase‐3 activities, and inhibition of telomerase activities in aorta from t‐BHP‐treated groups. These results suggest t‐BHP impaired vascular cell survival at least partially by activating the p53‐mediated signaling pathway and AS can counteract these actions of t‐BHP to protect the aortic endotheium from t‐BHP induced dysfunction.(Supported by research grants from NSC992313B343001‐MY3, Taiwan)
Increased plasma ammonia concentration contributes to methamphetamine‐induced blood‐brain barrier damage
Authors: Northrop Nicole Alia , Halpin Laura E , Yamamoto Bryan K
Published: June 2021

Recently our lab has shown that the psychostimulant, methamphetamine (Meth), produces liver damage. In models of acute liver failure, increases in plasma ammonia mediate brain edema and disruption of tight junction proteins of the blood‐brain barrier (BBB). Neurotoxic doses of Meth that produce liver damage also disrupt the structure and function of tight junction proteins, occludin and claudin‐5. Therefore, we hypothesized that Meth‐induced increases in plasma ammonia will disrupt BBB structure and function. A neurotoxic Meth regimen (10 mg/kg, ip, q 2hr, ×4) produced an 86±12% increase in plasma ammonia 2 hr after the last Meth injection, compared to saline treated controls. Pretreatment with lactulose (5.3 g/kg, po, q 12hr), a drug that increases the excretion of ammonia, prevented these increases. Meth significantly decreased expression of occludin by 51±8% and claudin‐5 by 51±7%, and increased BBB permeability as measured by FITC‐dextran extravasation, compared to saline treated controls 24 hr after treatment. Meth‐induced BBB disruption was prevented by lactulose pretreatment. These data indicate that Meth‐induced increases in plasma ammonia contribute to the disruption of BBB structure and function. These findings identify a novel mechanism through which Meth disrupts the BBB and a novel consequence of Meth‐induced liver damage.

Supported by NIH DA07606.
Selectively silencing Nitric Oxide Synthase 1 Adaptor Protein in the Nucleus Tractus Solitarius leads to QT Interval Prolongation via Glutamate‐mediated neurotoxicity
Authors: SINGH NEHA , ZHONG TIECHENG , O'ROURKE STEPHEN T. , SUN CHENGWEN
Published: June 2021

Variants of the nitric oxide synthase 1 adaptor protein (NOS1AP) locus are strongly related to QT interval prolongation and sudden cardiac death (SCD) in human. Our aim was to study the action of NOS1AP in the NTS by knockdown of NOS1AP using lentiviral vector‐mediated shRNA (Lv‐NOS1AP‐shRNA). qPCR data showed NOS1AP mRNA levels were expressed in NTS 3‐fold higher than organs such as kidney and heart in SD rats. Microinjection of Lv‐NOS1AP‐shRNA in NTS caused significant reduction in NOS1AP expression in SD rats. NOS1AP knockdown in NTS did not alter blood pressure (BP), heart rate (HR) recorded by radiotelemetry. ECG analysis revealed heart rate variability (HRV) was significantly reduced (SDNN, 51.2±5.6 vs 5.0±1.3 ms, P< .001) and QTc interval was markedly prolonged (72.4±4 vs 105±11 ms, P< .05) in NOS1AP knockdown rats. To study the cellular mechanism underlying NOS1AP action, we studied the effect of NOS1AP knockdown on glutamate‐induced neurotoxicity in Cath‐a cells. Treatment of cells with Lv‐NOS1AP‐shRNA significantly reduced NOS1AP expression, and was associated with increased glutamate‐induced neurotoxicity, suggesting a protective effect of NOS1AP. In summary, all results indicate that NOS1AP dysfunction in NTS might play an important role in the pathogenesis of cardiac arrhythmia which, in part, could be due to increased sensitivity to glutamate induced neurotoxicity.

Research Support: NDSU‐COBRE
Preserved Left Ventricular Performance In Spontaneously Hypertensive Rats Following Preload And Afterload Challenges
Authors: Fan Ming , El‐Mas Mahmoud M , Abdel‐Rahman Abdel A
Published: June 2021

Hypertension‐induced left ventricular (LV) hypertrophy is an independent risk factor for heart failure. We studied LV performance and myocardial nitric oxide (NO) signaling and reactive oxygen species (ROS) in 6‐month‐old SHRs and age‐matched Wistar‐Kyoto rats (WKY) subjected to preload and afterload challenges. Under basal conditions, LV nNOS, but not eNOS, expression and nitrite/nitrate (NOx) were higher in SHR than in WKY rats. SHRs also exhibited higher LV weight, LV developed pressure (LVDP), LV end‐systolic pressure (LVESP), rate of rise (LV dp/dtmax) and fall (LV dp/dtmin) in LV pressure. In the acute volume overload model (dextrose infusion), SHRs showed significantly smaller reductions in LV dp/dtmax and LVDP and greater increases in LVEDP and isovolumic relaxation constant (Tau, τ), compared with WKY rats, suggesting enhanced systolic and depressed diastolic functions in SHRs. We also report improved LV performance in SHRs during pressure overload (phenylephrine infusion) as reflected by the increases in LVDP, LVESP, LV dp/dtmin, and LV dp/dtmax. LV ROS was increased by the two overload challenges in WKY, but not SHR, rats. The results suggest that despite evidence of early LV hypertrophy, the ability of the SHR to curtail myocardial oxidative stress may accounts, at least partly, for the preservation of the left ventricular performance during pressure or volume overload challenge in SHRs.