bluuebunny/crossref_metadata_2025 huggingface dataset:
Water intakes from food, beverage, and drinking water of US older adults
Authors: Zizza Claire Ann , Wernette Catherine
Published: June 2021

Age‐related changes in behavior and health status predispose all older adults to dehydration. If left unchecked dehydration can cause changes in body chemistry, kidney failure, and death. Despite the dire consequences of improper hydration, very little attention has been devoted to the measurement of their total daily water intake. Our objective was to examine the water intake from beverage and food sources of independent, community‐living older adults. Data for 2,015 older adults (≥ 65 y) from the 1999–2002 National Health and Nutrition Examination Surveys were used for this study. Dietary data were collected through a 24‐h dietary recall method and after the recall, respondents were asked to estimate their intake of plain drinking water in the previous 24‐h period. Linear regression models controlling for age, income, sex, education and marital status were used to estimate total water intake from drinking water, beverages and food. Adjusted total water intakes were 2,877, 2557, and 2304 g/d for those aged 65–74 y, 75–84 y, and over 85 y respectively. On average food sources contributed 23, 24, and 26% of the total water intake for those same age groups. Presently no single measure completely discriminates between dehydrated and non‐dehydrated individuals. This study provides important data regarding the fluid intake and thus hydration status of older adults. Partially funded by Alabama Agricultural Experiment Station (ALA013‐020).
Basal and amino acid stimulated fractional synthesis rates of calf muscle protein subfractions in peripheral arterial disease
Authors: Tuvdendorj Demidmaa , Wolfe Robert R , Killewich Lois A
Published: June 2021

Basal fractional synthesis rate (FSR) and the effect of essential amino acid (EAA) supplementation on protein synthesis of specific subfractions of calf muscle in peripheral arterial disease (PAD) is presented. Six patients with PAD and 6 healthy, age‐ and sex‐matched controls were studied. FSR was measured in the basal (post‐absorptive, PA) state and following administration of a single oral bolus of 15g of EAA. Sarcoplasmic, mitochondrial and myofibrillar protein FSRs were calculated from gas chromatography/mass spectroscopy measurements of the incorporation of the stable isotope L‐[ring‐2H5]‐phenylalanine into muscle proteins. Data are expressed as mean±SE; statistical analyses were performed using two‐way repeated ANOVA. Results: In both groups, in comparison with PA, EAA stimulated mitochondrial (PAOD: 0.055±0.011 vs 0.110±0.017 %/h; control: 0.063±0.019 vs 0.130±0.027 %/h; PA vs EAA, P<0.05) and sarcoplasmic (PAOD: 0.065±0.011 vs 0.123±0.008 %/h; control: 0.075±0.012 vs 0.110±0.013 %/hr; P < 0.05) protein FSR, but not myofibrillar (PAOD: 0.070±0.020 vs 0.100±0.011 %/h; control: 0.070±0.022 vs 0.070±0.017 %/h; P= NS). Conclusion: Basal and EAA stimulated protein FSR subfractions of gastrocnemius muscle are not different in PAD compared to healthy control subjects.
Substituting Almonds for Saturated Fatty Acid Sources and Other Foods in the Typical American Diet Can Achieve the Dietary Guidelines for Americans’ Nutrient Recommendations
Authors: DeAngelis Michael A , Irish‐Hauser Sonya , Shiery Lesley , Lapsley Karen
Published: June 2021

The 2005 Dietary Guidelines for Americans recommend reducing consumption of certain nutrients such as saturated fat and cholesterol and increasing consumption of some nutrients such as vitamin E, magnesium and dietary fiber. Almonds are a low‐saturated fat source of dietary fiber, vitamin E and magnesium. This analysis was conducted to demonstrate the effects on nutrient intake of substituting an equicaloric load of almonds for primary saturated fat sources in a typical American dietary pattern. A 2000‐calorie meal pattern from the MyPyramid dietary guidance was altered to reflect typical consumption patterns without almonds and then substituting 220 calories of almonds for an equicaloric amount of other foods including saturated fat sources. The control and almond menus were analyzed using FoodWorks 7.0 (The Nutrition Company, Long Valley, NJ). Percent change in nutrients was calculated for each of the seven daily sets of dietary patterns and averaged for the seven days. The almond substitution reduced saturated fat (‐30%) and cholesterol (‐26%) and increased levels of vitamin E (296%), magnesium (265%) and dietary fiber (42%). The improvements in nutrient intake support a recommendation for consuming almonds in place of saturated fat sources in future dietary guidance. Further analyses will include serving‐size based substitutions and other nuts. Support: Almond Board of California
Relationships between fecal phytate and mineral excretions depend on the dietary phytate and age
Authors: Kim Jihye , Woodhouse Leslie , King Janet , Welch Ross , Paik Hee Young , Joung Hyojee
Published: June 2021

The study was done to determine the effect of dietary phytate and age on the relationships between fecal phytate and mineral excretions. Fourteen young and 14 elderly women were fed two experimental diets, high phytate (HP) and low phytate (LP), for 10 days each with a washout period. Duplicate diet samples from two different menus and complete fecal samples were collected for 5 days during each diet period to analyze phytate and mineral contents. Fecal phytate excretion (319 vs. 177 mg/d, p<0.05) and degradation rate of dietary phytate (80 vs. 74%, p = 0.002) increased significantly during the HP diet compared to the LP diet in young women. There was a positive relationship between fecal phytate and mineral excretions such as calcium, copper, iron and zinc in both HP and LP diet in young women (p<0.05). The linear relationship was greater during LP diet compared to the HP diet. However, no association was found in elderly women. Fecal phytate had a positive relationship with mineral excretion suggesting the negative effect of un‐degraded phytate on mineral absorption. The inhibitory effect of phytate on mineral absorption was affected by dietary phytate and age. Supported by Korea Research Foundation
Effects of vitamin E on serum cytokine levels in healthy adults
Authors: Sundaresan Sinju , Malone Brenda , Radcliffe John , Imrhan Victorine
Published: June 2021

Effects of Vitamin E on serum cytokine levels in healthy adults

Purpose: The study determined the effects of increased dietary intake of Vitamin E (~15g per day) on serum levels of vascular endothelial growth factor (VEGF), interleukins (IL)‐1α, 1β, IL 2, IL 4, IL 6, and tumor necrosis factor (TNF‐α) in healthy adults. Methods: Healthy volunteers were randomly assigned to Control (C) and Intervention (I) groups (n=15) and allowed to consume normal diets for one week. Group C continued their normal diets for 4 weeks and group I was given vitamin E‐rich foods (muffins with cottonseed oil, almonds, broccoli, wheat germ) for 4 weeks. Weekly 3‐day diet diaries were analyzed for nutrients and energy using NDSR program. Blood samples were assayed for VEGF, interleukins and TNF‐α at baseline and week 5. Results: At the end of week 5, Group I had 2.5‐fold higher IL‐α, and 2.2‐fold higher IL‐1β production than Group C (P<0.05) respectively. This group also had 2.0‐fold higher IL‐2, IL‐4, and 1.8‐fold higher TNF‐α production than Group C (P<0.05) respectively. However, IL‐6 production was not significantly enhanced in Group I at the end of week 5. VEGF levels in Group I was 0.8‐fold lower (NS; P=0.059) than Group C at the end of week 5. Conclusion: Serum pro‐inflammatory IL 1‐α, 1‐β, IL 2, IL 4, and TNF‐α levels were significantly improved by including vitamin E‐rich foods in the diet without affecting energy intake, with no changes in IL‐6 and VEGF levels in these healthy adults.
Production of distinct carotenoid mixtures from tomato cell suspension cultures varies by herbicide treatment and variety used.
Authors: Engelmann Nancy J , Rogers Randy B. , Lila Mary Ann , Erdman John W.
Published: June 2021

Tomato product consumption has been correlated with decreased chronic disease risk. Radiolabeled forms of tomato carotenoids are essential for tracking bioavailability, biodistribution, and metabolism in human and animal models; however, many of these compounds are not commercially available. In order to target specific compounds and efficiently produce mixtures of tomato carotenoids [phytoene (PE), phytofluene (PF), and lycopene (LYC)] for future radiolabeling, different herbicide treatments and tomato cell lines were investigated. Three herbicide treatments [norflurazon (NORF), 2‐(4‐chlorophenyl‐thio) triethylamine (CPTA), or NORF & CPTA (COMBO)] were investigated with ‘VFNT Cherry’. CPTA treatment yielded primarily LYC (2.49 mg/L culture) followed by PE & PF (0.30, 0.32 mg/L). NORF treatment yielded the greatest PE (1.00 mg/L) followed by PF (0.10 mg/L) and traces of LYC. COMBO treatment led to the best‐mixed yields of PE, PF, and LYC (1.09, 0.28, 1.45 mg/L). Five different cell lines (‘VFNT Cherry’, ‘Ailsa Craig’ hp‐1, ‘Ailsa Craig’ WT, ‘Manapal’ hp‐2, and ‘Manapal’ WT) were treated with CPTA and evaluated, and ‘VFNT Cherry’ and the ‘Ailsa Craig’ hp‐1 mutant accumulated the greatest LYC. Selected varieties and herbicide treatments will be applied for radiolabeled carotenoid generation for use in future animal and human trials. (Supported by NIH/NCI CA 112649‐01A1)
Stimulatory effect of dietary red ginseng on epidermal levels of hydration and ceramides in ultraviolet radiated hairless mice
Authors: Kim Hyunae , Oh Inngyung , Park Kyung‐Ho , Kim Na‐Mi , Do Jae‐Ho , Cho Yunhi
Published: June 2021

Ultraviolet (UV) radiation induces skin dryness, which largely depends upon disruption of epidermal barrier. As searching dietary and plant sources of protecting from skin dryness, hairless mice were fed diets of either 0.5% or 1.0% red ginseng extract (groups H0.5 and H1.0) for 5 wks in parallel with UV radiation. As controls, two groups were fed a control diet with (group C) or without UV radiation (group UV) for 5 wks. With no differences in food intake and weight gains among all groups, epidermal levels of hydration and ceramides, the major lipid maintaining epidermal barrier, in group H0.5 were similar to those in the non‐UV radiated, normal control group (group C). In addition, protein expression of serine palmitoyl transferase (SPT), an enzyme of de novo ceramide synthesis, was increased. However, epidermal levels of hydration and ceramides in group H1.0 did not differ from in the UV radiated control group (group UV), in which ceramidase, an enzyme of ceramide degradation, is highly expressed. Together, we demonstrate that dietary supplementation of 0.5% red ginseng protects skin from UV induced dryness with the accumulation of ceramides coupled with a high expression of SPT protein.
Purple carrot (Daucus carota L.) polyacetylenes decrease LPS induced expression of inflammatory proteins in macrophage and endothelial cells
Authors: Metzger Brandon Troy , Barnes David M. , Reed Jess D.
Published: June 2021

Carrots (Daucus carota L.) contain phytochemicals with health‐promoting properties including carotenoids, phenolics, polyacetylenes, isocoumarins, terpenes, and sesquiterpenes. Purple carrots also contain anthocyanins, a class of compounds with reported anti‐inflammatory activity. Purple carrot was investigated in macrophages and endothelial cells as a novel food capable of modulating inflammatory response to lipopolysaccharide (LPS). A bioactive methanol extract prepared by chromatography reduced inflammatory response in macrophages and endothelial cells as a model of vascular inflammation. Nitric oxide was reduced dose responsively, in addition to mRNA of several pro‐inflammatory cytokines (IL‐6, IL‐1β, TNF‐α), and iNOS in macrophage cells. Protein secretion of IL‐6 and TNF‐α were reduced 77 and 66% in porcine aortic endothelial cells with 6.6 and 13.3 μg/mL of purple carrot from LH‐20 respectively. Isolation of active phytonutrients in purple carrot resulted in a bioactive sub‐fraction enriched in polyacetylene compounds falcarindiol, falcarindiol 3‐acetate, and falcarinol. Isolated polyacetylenes reduced nitric oxide production in macrophage cells by as much as 65% without cytotoxicity. Falcarindiol was the most bioactive polyacteylene. Our results suggest that polyacetylenes, not anthocyanins, in purple carrots are responsible for anti‐inflammatory bioactivity.
Modular architecture and human disease alleles of the mitochondrial replicative DNA helicase
Authors: Kaguni Laurie S. , Ziebarth Tawn D. , Matsushima Yuichi , Farr Carol L.
Published: June 2021

The human mtDNA helicase shares amino acid sequence and functional similarities with bacteriophage T7 primase‐helicase. mtDNA helicase exists as a hexamer that is destabilized under low salt conditions, but can be maintained by inclusion of Mg2+ and ATP, and by temperatures above 20 °C. Limited proteolysis reveals distinct N‐ and C‐terminal polypeptides that represent minimal structural domains, and yields several stable fragments that differ in the presence of nucleoside di‐ and triphosphate and DNA. Truncations of the N‐ and C‐termini affect differentially DNA‐dependent ATPase activity, and whereas a C‐terminal domain polypeptide is functional, an N‐terminal domain polypeptide lacks ATPase activity.

Molecular analysis of the Drosophila homolog was used to evaluate the physiological consequences of active site and human disease alleles. RNA interference reduces expression of d‐mtDNA helicase to an undetectable level in Schneider cells, causing an ∼5‐fold decrease in mtDNA copy number, whereas overexpression increases mtDNA levels 1.4 fold. Overexpression of helicase active site mutants results in a severe depletion of mtDNA, and a dominant‐negative lethal phenotype. Interestingly, overexpression of mutants analogous to human disease alleles show differential effects, ranging from dominant negative effects to increases in mtDNA copy number. This research was supported by NIH grant GM45295.
A preliminary evaluation of the efficacy and safety of medicinally ingested vinegar in individuals with type 2 diabetes
Authors: Johnston Carol S. , White Andrea M. , Kent Shannon M.
Published: June 2021

Vinegar ingestion at mealtime reduces postprandial glycemia; however, it is unknown whether chronic vinegar use is associated with reductions in hemoglobin A1c. This 12‐wk pilot study examined the efficacy and safety of medicinally ingested vinegar in type 2 diabetics. Participants (n = 27) were randomized into three groups: vinegar pills (REF, 30 mg acetic acid daily; the reference treatment), pickles (PCK, ~1400 mg acetic acid daily), or vinegar (VIN, 2800 mg acetic acid daily). Hemoglobin A1c fell 2.4% in VIN participants over the 12‐wk trial but rose 1.1% and 3.7% in REF and PCK participants respectively (p = 0.068). Fasting glucose and insulin did not vary by treatment. One‐half of PCK and VIN participants reported at least one treatment‐emergent adverse event (e.g., burping, flatulence, acid reflux) at week 6 as compared to 11% of REF participants (p = 0.110). Urinary pH was reduced in VIN participants at week 12 as compared to the other groups (−9% versus +3% and +2% for the PCK and REF groups respectively, p = 0.023). At week 6, plasma aspartate aminotransferase tended to increase in the VIN group as compared to the other groups (+17% versus +8% and −8% for VIN, PCK, and REF respectively; p = 0.090). These data indicate that chronic vinegar ingestion may favorably influence hemoglobin A1c in individuals with type 2 diabetes but may also impact hepatic function and metabolic pathways aside from glucose metabolism.
Low level of Lemon Balm (Melissa officinalis) essential oils showed hypoglycemic effects by altering the expression of glucose metabolism genes in db/db mice
Authors: Chung Mi Ja , Cho Sung‐Yun , Lee Sung‐Joon
Published: June 2021

The effects of the lemon balm essential oils (LBEO) in the db/db mice were examined. Mice were divided into four groups and the LBEO was orally administered for 6 weeks: saline, control; 0.15mg/d, LBEO1; 0.05mg/d, LBEO2; 0.0125mg/d, LBEO3. The LBEO showed biphasic effects on plasma glucose levels; the low level feeding was hypoglycemic but the medium and the high levels exerted hyperglycemic effects. The LBEO3 group showed reduced fasting glucose (65%, P<0.05) and triglyceride concentrations compared with the controls. Glucose tolerance, assessed by oral glucose tolerance test, was improved in this group accordingly. However, in the LBEO1 and the LBEO2 groups, glucose tolerance was worsened according to the elevated fasting glucose levels compared with the controls (33% and 27% elevation in the LBEO1 and the LBEO2, respectively P<0.05). Plasma GOT and GPT levels were elevated in these groups as well. The hypoglycemic mechanism of low level feeding of the LBEO was further investigated with quantifying the expression of the glucose metabolism genes by real time PCR. The glucokinase and the GLUT 4 gene expressions were significantly upregulated, however, the expression of glucose‐6‐phosphase and phosphoenolpyruvate carboxykinase were downregulated in the mouse livers of the LBEO3 group. The results suggest that the LBEO may be hypoglycemic when administered at low concentration. These effects may be due to the enhanced glucose uptake and the inhibition of gluconeogenesis in the livers.
Dietary supplementation with cholesterol (Chol) and docosahexaenoic acid (DHA) Increases the activity of the arginine‐nitric oxide pathway in tissues of neonatal pigs
Authors: Li Peng , Kim Sung Woo , Datta Sujay , Pond Wilson G. , Wu Guoyao
Published: June 2021

Nitric oxide (NO) plays an important role in the development and functions of brain and other organs. The synthesis of NO from L‐Arg is catalyzed by tetrahydrobiopterin (BH4)‐dependent NO synthase. Sixteen newborn pigs were nursed by sows for 24 h and then assigned randomly to one of 4 treatment groups, representing supplementation with 0.0%, 0.2% Chol, 0.2% DHA, and Chol plus DHA to the basal formula consisting of coconut oil, soybean oil, soybean flour, whey, whey protein concentrate, minerals and vitamins. All piglets were euthanized at day 49 of life. Brain, liver and gastrocnemius muscle were analyzed for BH4 and arginine, as well as the activities of GTP cyclohydrolase‐I (the first and rate‐limiting enzyme for de novo BH4 synthesis) and NO synthase, using HPLC and radiochemical methods. NOS activity was below the detection limit in the livers of all pigs. DHA supplementation (P<0.01) increased GTP‐CH activities and BH4 concentrations in the brain, liver, and muscle by 24–46%, while enhancing (P<0.05) NOS activities by 45–48% in brain and muscle. Dietary Chol supplementation increased (P<0.05) NOS and GTP‐CH activities by 17–26% in the brain but had no effect in liver or muscle. DHA and Chol increased concentrations of arginine in the brain (35–42%), but not in the other two tissues. The tissue‐specific effects of cholesterol and DHA on the arginine‐NO pathway may play an important role in postnatal growth and development. Supported by TAES and NCSU.
Identification of genes associated with neural development in puppies as indicated by MRI
Authors: Frantz Nolan Z. , Zicker Steve C. , Yamka Ryan M. , Friesen Kim G. , Gao Xiangming , Al‐Murrani Samer
Published: June 2021

Microscopic resonance imaging (MRI) was performed on 78 (Age = 1 year) beagle puppies as a non‐invasive means to determine relative amounts of grey and white brain matter. The fifteen puppies with the greatest amount of total grey and white matter were compared to the fifteen puppies with the least amount of total grey and white matter from the original 78. Puppies were fed commercially available foods for one year and then a blood sample was drawn into a PAXgene Blood RNA tube. RNA was extracted according to the procedures provided in the PAXgene Blood RNA Kit Handbook (Qiagen, Valencia, CA). RNA was hybridized to an Affymetrix GeneChip Canine‐2 Genome Arrays and normalized using Robust Multi‐Array Average. Transcripts having a P < 0.01 (following a false discovery rate adjustment value of 0.1) and a fold change range of at least 1.3 were considered different among the two groups. Analysis of the RNA found differences in 107 genes between the fifteen puppies with greatest amount of grey and white matter and puppies with least amount of grey and white matter. Of the genes identified, 43 were up‐regulated and 64 down‐regulated in puppies with the greatest amount of grey and white matter when compared to puppies with least grey and white matter. Genes associated with growth hormone signaling and neuron development were increased, while inflammation and fatty acid biosynthesis were decreased in puppies with greatest amount of grey and white matter compared to puppies with the least amount of grey and white matter.
Neutralization of Unfractionated Heparin and Low Molecular Weight Heparin by Novel Salicylamide Derivatives
Authors: Jeske Walter , Brubaker Aleah , Liu Dahui , Young Trevor , Hoppensteadt Debra , Iqbal Omer , Fareed Jawed , Walenga Jeanine , Bakhos Mamdouh
Published: June 2021

Heparin (UFH) neutralization is critical post‐ bypass surgery to avoid excessive blood loss. Protamine use can be associated with severe side‐effects and less effectively neutralizes low molecular weight heparin (LMWH). We studied the ability of polycationic salicylamide derivatives (PolyMedix, Radnor, PA) to neutralize UFH and LMWH. Plasma was supplemented with UFH or enoxaparin (Sanofi‐Aventis, Paris, France) and protamine or one of six salicylamide derivatives and then analyzed using clotting (aPTT, Heptest, thrombin time) and amidolytic (anti‐Xa, anti‐IIa) assays. Protamine neutralized UFH in all assays. Two salicylamide derivatives produced a comparable effect, while three derivatives exhibited a stronger neutralization of UFH. While residual anti‐Xa and anti‐IIa activity (20% and 10%, respectively) was observed with a 5‐fold protamine excess, complete neutralization was observed with the salicylamide derivatives. Enoxaparin's anticoagulant activity was neutralized by 50% at a 5:1 protamine:enoxaparin ratio and completely by the salicylamide derivatives. 5 of 6 salicylamide derivatives inhibited anti‐Xa activity. The results show that salicylamide derivatives neutralize the anticoagulant and anti‐protease actions of UFH and may be more effective at neutralizing LMWHs. Future studies will characterize the compounds’ PK/PD profile in animal models of bleeding and thrombosis.
The characterization of the morphology and intrinsic oscillatory contractions in pudendal arteries of aging normotensive rats
Authors: Hannan Johanna , Blaser Mark , Pang Judy , Bennett Brian , Pang Stephen , Adams Michael
Published: June 2021

Aging increases the risk of erectile dysfunction (ED) and cardiovascular diseases. We examined the effects of age on erectile function and vascular structure and function, particularly with respect to intrinsic oscillatory properties (vasomotion) of the primary penile resistance artery (pudendal).

Methods: Erectile responses were assessed in 15 and 50 week Sprague‐Dawley rats. After sacrifice, myograph‐mounted pudendal arteries were pre‐contracted and responses to acetylcholine and L‐NAME assessed. Oscillations were analysed via FFT and the signals separated by power density spectra; very low (VLF) 0.002‐0.05 Hz, low (LF) 0.05‐0.2 Hz, high (HF) >0.2 Hz.

Results: Aged rats had severe ED that corresponded with a disproportionately thickened wall of the pudendal (~2‐fold). In both young and old rats, pudendal vasomotion was dominated by VLF oscillations, whereas aortic waves were mainly LF and HF. Aging increased VLF activity in the pudendal (4‐fold), but not aorta. L‐NAME abolished aortic VLF activity, but did not alter pudendal oscillations.

Conclusion: The preponderance of VLF oscillations in the pudendal arteries suggests there is a novel regulation for controlling penile inflow that is independent of nitric oxide. The aging associated morphological and vasomotion changes in the pudendal suggests a causative role for pathogenic vascular remodelling of pre‐penile vessels in aging‐induced ED.
No change in endothelium‐dependent relaxation or morphology of peripheral arteries following 30 days of aortic coarctation.
Authors: Newcomer Sean , Hsu Charlie , Caron Normand , Ingram David , Roseguini Bruno , Laughlin Harold
Published: June 2021

We designed this study to evaluate the effects of thoracic aortic coarctation (AC) on peripheral endothelial function and development of atherosclerosis. Endothelial function and development of atherosclerosis were assessed in the brachial (BR) and femoral (FEM) arteries of control (CON; n=6) and coarctated (COA; n=7) swine 30 days after AC. In vitro assessments of vasorelaxation to increasing doses of endothelium‐dependent and –independent vasodilators were performed in arterial rings harvested from the BR and FEM arteries. Intimal medial thickness (IMT) and sudan IV staining were performed in the BR and FEM arteries. AC produced a 30 day average blood pressure gradient of 56 ± 5mmHg across the coarctation. Elevated blood pressure proximal to the coarctation produced a significantly greater heart to body weight ratio in the COA (5.51 ± 0.06g/kg) vs. CON (4.66 ± 0.15g/kg) swine. Endothelium‐dependent and ‐independent relaxation was not significantly different in the BR or FEM arteries of COA and CON swine. In addition, there were no significant differences in either IMT or sudan IV staining of the BR or FEM arteries from COA and CON swine. These findings suggest that the stimulus and/or duration of 30 days AC were insufficient to produce endothelial dysfunction and atherosclerosis in the peripheral vasculatures of these swine.

Supported by NIH HL083597, HL36088 and HL52490
Luminal glucose protects against ischemia/reperfusion‐induced intestinal epithelial barrier defects in rats
Authors: Huang Ching‐Ying , Hsiao Jong‐Kai , Lu Yen‐Zhen , Yu Linda C.H.
Published: June 2021

Intestinal ischemia/reperfusion (I/R) results in mucosal barrier disruption and enteric bacterial translocation (BT). Previous study showed that enhanced sodium‐dependent glucose transport inhibits the LPS‐induced increase of epithelial permeability. The aim was to evaluate whether luminal glucose protects against I/R‐induced intestinal epithelial barrier defects in rats. A 10 cm jejunal loops was created containing Krebs buffer without or with 25 mM of sugar (glucose or mannitol). Rats then received sham operation or I/R challenge by occlusion of superior mesenteric artery for 20 min and reperfusion for 1 hr. Histological evidence exhibited shortened villi and denuded tips in the jejunum, and heightened bacterial colony forming units were seen in liver and spleen of I/R rats. A novel technique utilizing magnetic resonance imaging (MRI) was developed for the detection of real‐time in vivo intestinal permeability changes. A contrast probe gadodiamide was injected into the jejunal loop, and the increase of MRI signals in the liver, kidney and plasma were five fold higher in I/R rats compared to sham controls. The I/R‐induced jejunal mucosal damages, increased epithelial permeability and BT were reduced by administration of luminal glucose, but not of mannitol. These findings suggest that glucose plays a protective role against the intestinal barrier dysfunction induced by I/R.
Early intestinal tight junction loss after hemorrhagic shock
Authors: Thuijls Geertje , de Haan Jacco‐Juri , Derikx Joep P , Bamburg Jim R , Heineman Erik , Buurman Wim A
Published: June 2021

The intestinal barrier is based on tight junctions (TJ) between intact epithelial cells. These TJ are anchored via the filamentous (F‐)actin cytoskeleton. We hypothesize that hemorrhagic shock (HS) leading to intestinal hypoperfusion, causes loss of F‐actin, via activation of Actin Depolymerizing Factor/Cofilin (AC), and consequently results in TJ loss. This study investigates intestinal AC activation and cytoskeleton severing following HS.

Rats were subjected to HS and sacrificed, along with controls, at 15, 30, 60 and 90 min after induction of shock. Intestinal hypoperfusion, activation of AC, actin cytoskeleton severing (degradation of F‐ into globular actin), and TJ integrity loss (Claudin‐3, ZO‐1) were assessed.

HS caused intestinal hypoperfusion (−71.7% bloodflow) and activation of AC from 15 min after HS (by 21%, P<0.05 vs controls, student's t‐test (stt)). This was accompanied by actin cytoskeleton severing, followed by intestinal TJ loss from 30 min after HS (Claudin‐3: 100% vs 71%; ZO‐1: 100% vs 41%, p<0.05 vs controls, stt).

We show for the first time that HS results rapidly in intestinal hypoperfusion, AC activation, actin depolymerization and subsequent TJ loss. Elucidation of the etiology of barrier failure is a first step in understanding the disease entity, which will be followed by the development of therapeutic interventions to reduce the development of barrier failure.
Normalization of proliferation and paracellular permeability of bladder epithelial cells from interstitial cystitis patients by a synthetic inhibitor of antiproliferative factor
Authors: Keay Susan , Kaczmarek Piotr , Zhang Chen‐Ou , Michejda Christopher
Published: June 2021

Objectives: Interstitial cystitis (IC) is a chronic disorder with bladder epithelial thinning or ulceration, pain, urinary frequency and urgency. Bladder epithelial cells from IC patients make an antiproliferative factor (APF) that inhibits cell growth, decreases tight junctions, and increases paracellular permeability. We screened inactive synthetic APF derivatives for their ability to inhibit APF, then determined the ability of an inhibitory derivative to normalize proliferation and permeability of IC cells.

Methods: Normal bladder cells were pretreated with inactive APF derivatives, then incubated with active synthetic APF. IC cells were incubated with varying concentrations of D‐proline APF. Cell proliferation was determined by 3H‐thymidine incorporation. Gene expression was determined by quantitative RT‐PCR. Paracellular permeability was determined by 14C mannitol and 3H‐inulin flux on Transwell plates.

Results: One of 28 inactive derivatives (D‐proline APF) blocked APF activity in normal bladder cells. D‐proline APF also significantly increased cell proliferation, increased zonula occludens‐1 and claudin 1, 4 and 8 expression, and decreased permeability of IC cells.

Conclusions: D‐proline APF can normalize IC cell proliferation and paracellular permeability in vitro, making it a potential therapy for IC. Supported by NIH R01 DK52596 and the Intramural Research Program of the NCI, NIH
The involvement of Gab1 and PI 3‐kinase in β1 integrin signaling in keratinocytes
Authors: Kuwano Yoshihiro , Fujimoto Manabu , Watanabe Rei , Ishiura Nobuko , Nakashima Hiroko , Okochi Hitoshi , Tamaki Kunihiko
Published: June 2021

The control of the stem cell compartment in epidermis are closely linked to the regulation of keratinocyte proliferation and differentiation. β1 integrins are expressed 2‐fold higher by stem cells than transit‐amplifying cells and are considered as an epidermal stem cell marker. Signaling from these β1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of β1 integrins, we established HaCaT cells expressing high β1 integrins by repeated flow cytometric sorting of the cells with high β1 integrin expression. In these obtained cells expressing β1 integrins by 5‐fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high β1 integrin expression, while She phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3‐kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3‐kinase play pivotal roles in the β1 integrin‐mediated regulation of the epidermal stem cell compartment.
Redox state and PKC signalling in neuronal differentiation and sensitivity to glycoxidative stress
Authors: Nitti Mariapaola , Furfaro AnnaLisa , Cevasco Claudia , Traverso Nicola , Marinari Umberto Maria , Pronzato Maria Adelaide , Domenicotti Cinzia
Published: June 2021

Intracellular redox state modulation can influence cell proliferation, differentiation or death. Here we show that Retinoic Acid (RA)‐induced neuroblastoma cell differentiation is accompanied by increased expression of anion superoxide generating enzyme NADPH oxidase, decreased SOD1 expression and increased catalase expression. Moreover, cell differentiation induced PKC alfa inactivation and PKC delta activation. We believe these alterations responsible for the observed cell sensitivity to glycoxidative stress: indeed, only the RA‐differentiated cell viability decreased after exposure to Advanced Glycation End Products (AGEs), via intracellular ROS overproduction, while undifferentiated cells proved insensitive. AGE‐induced effects on differentiated cells were prevented by pre‐treating cells with NADPH oxidase or PKC delta inhibitors. These observations may be useful in order to identify redox sensitive molecular targets responsible for cell growing rate modifications.

Grants from PRIN n.2006065711_002
Effect of hypoxia‐regulated CD151 expression on cancer cell adhesion
Authors: Chien Chun‐Wei , Lee Jenq‐Chang , Lin Shih‐Chieh , Lin Po‐Wen , Lai Yen‐Yu , Tsai Shaw‐Jenq
Published: June 2021

CD151 is a member of tetraspanin family that plays important roles in cell adhesion and motility. However, the regulation of CD151 expression in normal and cancerous cells has not yet revealed. In cancer tissue, it is suggested that CD151 acts as a metastasis‐suppressor while hypoxia has been shown to be a factor of causing metastasis. We hypothesize that hypoxia‐induced cancer cell metastasis may be mediated via deregulation of CD151. Indeed, colon cancer cells expressed less CD151 than its normal counterpart, which is inversely correlated with the expression of hypoxia‐inducible factor‐1α (HIF‐1α). By using bioinformatic approach, we identified a putative hypoxia response element (HRE) in the promoter region of human CD151 gene. Treatment of colon cancer cells with hypoxia or chemicals that are known to increase accumulation of HIF‐1α resulted in decreased CD151 expression. Promoter activity assay further demonstrated that the predicted putative HRE was pivotal for HIF‐1α‐dependent downregulation of CD151 expression. Furthermore, inhibition of CD151 expression by hypoxia resulted in decreased cancer cell adhesion to laminin‐coated matrix, a process that enables cancer cell to detach from its surrounding matrix and be ready for metastasis. In conclusion, we demonstrated that hypoxia downregulates the expression of CD151 thus enhances the detachment of cancer cells from its basement membrane protein, an important first step for metastasis.
Recombinant Strongylocentrotus purpuratus hyalin binds to proteins isolated by disaggregating sea urchin embryos
Authors: Metzenberg Stan , Gilani Lila , Badali Oliver , Willis Billie‐Jean , Hutchins‐Carroll Virginia , Carroll Edward J. , Oppenheimer Steven B.
Published: June 2021

Hyalin is an extracellular matrix protein of sea urchins that is involved in embryonic development and in embryonic adhesive interactions. The protein consists primarily of tandemly repeated HYR modules, which are representative of an evolutionarily conserved family of domains thought to carry an immunoglobulin‐like fold structure. We have set out to characterize the biological and biochemical activities of an E. coli‐expressed recombinant HYR module, prepared as a fusion protein using the vector pGEX‐5X. The HYR module was cloned using a set of overlapping synthetic oligonucleotides, and placed under the control of the inducible tac promoter. Glutathione S‐transferase (GST)‐tagged HYR was purified from induced bacteria by affinity chromatography using glutathione sepharose. In a pull‐down assay with immobilized GST‐HYR module, or unmodified GST alone as a control, we have determined that specific S. purpuratus proteins, obtained by disaggregating sea urchin blastula‐gastrula stage embryos in calcium‐magnesium free sea water, bind to the recombinant HYR module. We are characterizing these specific binding targets of the HYR domain, and applying site‐directed mutagenesis to identify the sequences responsible for binding (Supported by NIH NIGMS SCORE (S0648680), NIH NIGMS RISE, NIH NIGMS MARC, and the Joseph Drown Foundation).
Inihbition of Protein Glycation with Varying Concentrations of Lysine
Authors: Sims Heather M. , Birdwell Angela L. , O'Reilly Kelley E. , Bwashi Adonis , Wing Barbara D.
Published: June 2021

The causes of the complications of diabetes mellitus have been explored in ongoing studies in our laboratory using an In Vitro system. This system is used to screen compounds for their ability to inhibit the glycation of proteins by sugars. Vascular damage seen in diabetic patients is impacted by such protein sugar interaction. During this study it was found that lysine is an effective inhibitor of glycation. The inhibition of glycation was studied using a spectrophotometric assay for glycoproteins. Studies indicated lysine as most effective as an inhibitor of glycation at a concentration of 100 μg/ml. Lower levels of inhibition were observed when a concentration of 500 μg/ml was used. When the concentration was lowered to 50 μg/ml and 200 μg/ml the results showed the inhibition of glycation was roughly equal at each respective concentration. Further research is being performed to find the actual concentration at which the lysine inhibition no longer performs efficiently. Preliminary results indicated that further investigation is needed to determine the mechanism of inhibition and the possible use of lysine in the treatment of diabetic patients.

This research project was funded by the Smith‐Glynn‐Callaway Medical Foundation.
In vivo Experimental Restorative Dentistry Model. Preliminary Study.
Authors: Farinha Rodrigo , Moreno M. Helena , Carrilho Eunice
Published: June 2021

An experimental model should comprise all aspects that can influence the pathology or the treatment tested. For restorative dentistry treatments, although all the recent achievements related mainly with the increasing use of adhesive techniques, the cavity design and the sound tissue preservation are crucial to the treatment's outcome.

The advantage of using an in vivo experimental model is that we can reproduce all variables affecting the restoration, carrying to the laboratory the problems of the clinic for a better understanding and new solutions. To collect data that can be comparable and directly applicable to humans, we must create a situation as quite as similar to the clinical approach. Although there are too many different caries experimental models drawings to test the mechanism of action of fluoride and dentifrice formulations, or the carcinogenicity of foods or those which can be protective, none has been made to test the materials used on restorative dentistry.

We propose an experimental in vivo model, which aims to be used to test caries cavities restorations. We propose two different cavity drawings to be used in dog's teeth.
Time‐course of corneal neovascularization and ocular surface squamous neoplasia development in bi‐transgenic Krt12‐rtTA/TetO‐FGF7 mice, depends on age of induction
Authors: Meij Johanna Theodora Antoinette , Liu Chia‐Yang , Zhang Yujin , Kao Candace W.C. , Kao Winston W.‐Y.
Published: June 2021

To investigate the role of the cytokine FGF7 (KGF) in corneal development and maintenance, we generated Krt12‐rtTA/TetO‐FGF7mice that overexpress FGF7 in the corneal epithelium after doxycycline (Dox) treatment. Previous findings showed that 1 mo induction in the adult results in reversible epithelial hyperplasia, whereas induction throughout embryonic development causes severe anomalies resembling ocular surface squamous neoplasia (OSSN). In the present study, a wider range of embryonic (E) and postnatal (P) starting ages and periods of induction were applied by treating Krt12‐rtTA/TetO‐FGF7 dams with 80 μ g/kg Dox ip, followed by Dox chow for different periods of time. Eyes were examined by in vivo confocal microscopy (HRT II‐cornea module) and immunohistochemistry. Embryonic induction up to E20 resulted in an OSSN‐like phenotype that did not fully reverse even after 2 mo. Interestingly, Dox up to E17 caused no surface anomalies, but blood vessels were present in the anterior stroma and persisted up to 5 mo. Reinduction of these adult mice led to OSSN within 1 mo. Postnatal induction for 2 mo starting within 7 d after birth caused OSSN, at P10 led to corneal hyperplasia and neovascularization, and at P15 or later, to hyperplasia. A novel finding was that after long‐term (4 mo) Dox, corneal neovascularization and OSSN ensued also in adult Krt12‐rtTA/TetO‐FGF7 mice, suggesting decreasing susceptibility with maturation. Supported by PHS EY013755, Research to Prevent Blindness, Ohio Lions Research Foundation
Cytochrome p450 2C Contributes to Peri‐Transplant Ischemic Injury and Cardiac Allograft Vasculopathy Following Heterotopic Heart Transplantation in Rats
Authors: Hunter Arwen L , Kerjner Alexandra , Mueller Katelyn J , McManus Bruce M , Granville David J
Published: June 2021

Peritransplant ischemia and reperfusion (I/R) injury contributes to post‐transplant vascular dysfunction and cardiac allograft vasculopathy (CAV). We have shown that cytochrome p450 (CYP) 2C inhibition significantly reduces I/R‐induced myocardial infarction and post‐ischemic vascular dysfunction. The objective of this study was to assess the contribution of CYP2C to CAV. Rat heterotopic heart transplants were performed between Lewis donors and Fisher recipients. Rats received CYP2C inhibition by sulfaphenazole (SP) or vehicle control 1hr prior to surgery. Organs were harvested 4, 7 and 30 days post‐transplant. Luminal narrowing was measured using morphometric analysis and immune infiltration was scored from absent to severe in a blinded manner. Smooth muscle cell (SMC) proliferation and apoptotic cells were detected by Ki‐67 and TUNEL staining, respectively. SP did not affect post‐transplant morbidity, mortality or weight gain. Coronary blood vessels from rats treated with SP showed significantly reduced luminal narrowing compared to control (12.07±4.09% vs 66.21±13.61%, p<0.05) and demonstrated a reduced SMC proliferation (3.32±3.27% vs. 7.20±2.32%). SP did not alter immune infiltration (p>0.1) nor did it significantly alter TUNEL positivity in myocardial, endothelial or SMC populations. In conclusion, CYP2C contributes to SMC proliferation CAV without affecting general immune infiltration.
Nodal and Lefty signaling regulates the growth of pancreatic cells
Authors: Zhang You‐Qing , Sarvetnick Nora
Published: June 2021

Nodal and its antagonist, Lefty, are important mediators specifying the laterality of the organs during embryogenesis. Nodal signals through activin receptors in the presence of its co‐receptor, Cripto. In the present study, we investigated the possible roles of Nodal and Lefty signaling during islet development and islet regeneration. By using immunohistochemistry, RT‐PCR and Western blotting methods, we found that both Nodal and Lefty are expressed in the pancreas during embryogenesis and islet regeneration. Exposure to recombinant mouse Nodal (rmNodal) inhibited the proliferation of a pancreatic progenitor cell line, AR42J cells, and conversely, recombinant mouse Lefty‐1 (rmLefty‐1) stimulated the proliferation of these cells. We also present evidence that the proliferation of the pancreatic progenitor cell line induced by Lefty‐1 involves phosphorylation of MAPK, activation of Akt and Bcl‐2 upregulation. In vivo blockade of endogenous Lefty using neutralizing a Lefty‐1 monoclonal antibody results in a significantly decreased proliferation of duct epithelial cells suggesting that Lefty‐1 is necessary for epithelial cell expansion during islet regeneration. Together, our data provide evidence for a novel role of Nodal‐Lefty signaling during pancreatic development and islet regeneration.
Genetic variations of the carbonic anhydrase gene were associated with hepatocellular carcinoma in Korean population
Authors: Lee Seung Ku , Yi Cheong‐Ho , Kim Min‐Ho , Cheong Jae Youn , Cho Sung Won , Kang Jason Jongho , Yang Song‐Ju , Kwack KyuBum
Published: June 2021

Carbonic anhydrase (CA) gene family has been identified at least 15 different isoforms in mammalian. Previous studies have shown that its expression is induced in the endothelium of neovessels in cancer. Altered CA genes expression was associated with hepatocellular carcinoma (HCC). Genetic variations can be analysed to assess the risk of an individual for a particular diseases. To genetic association test, we investigated that CA genes contains variants are associated with chronic liver diseases including chronic hepatitis (CH), liver cirrhosis, and HCC, we genotyped CAs SNPs markers on 117 patients with CH, 112 HCC and 117 spontaneous recovery (SR) controls. In this study, we found that CA genes SNPs, haplotypes, and diplotypes were associated with HCC. The results of the present study suggest that CA genes SNPs and haplotype might be associated with the susceptibility of HCC.
D1 versus D2 dopamine receptor selectivity is determined by intramolecular hydrogen bonding patterns in catechol‐containing novel dopamine analogues
Authors: Chemel Benjamin R , Bonner Lisa A , Watts Val J , Nichols David E
Published: June 2021

In 1991, DeNinno and colleagues described a novel class of isochroman dopamine analogues. Many of these ligands possessed greater than 100‐fold selectivity for dopamine D1 vs. D2 receptors. We studied a parallel series of chroman compounds. Surprisingly, we demonstrated that repositioning the oxygen atom in the heterocyclic ring conferred D2 receptor selectivity to the chroman compounds. In silico modeling led to the hypothesis that the reversed selectivity profile for the chroman compounds was due to a unique potential for intramolecular hydrogen bonding between the oxygen in the chroman ring and the meta‐hydroxyl of the catechol moiety. This has the potential to realign the catechol hydroxyl groups, thus disrupting proposed hydrogen bond interactions between these ligands and critical serine residues in TM5 of the D1 receptor. This hypothesis was tested by the pharmacological evaluation of a parallel series of carbocyclic compounds. These results suggested that when the potential for intramolecular hydrogen bonding is disrupted, D1 receptor selectivity is restored. We have completed the creation and characterization of HEK hD1 wt, S197A, S198A, S199A, and S202A stable cell lines. Currently, we are using these cell lines to evaluate the effects of each mutation on the pharmacological profile of analogous chroman, isochroman, and carbocyclic compounds. This work is supported by NIH grant DA02189 and MH60397.
Specificity of 5‐HT2A Receptor Ligands in Mice
Authors: Silva Uade Olaghere da , Sanders‐Bush Elaine
Published: June 2021

In‐Vitro studies of RNA editing of the serotonin type 2C receptor (5‐HT2CR) have shown a pronounced loss of function in the fully edited isoform (VGV). Our goal is to evaluate functional coupling of the edited 5‐HT2CR in‐vivo using transgenic mice that solely express the VGV isoform. In order to accomplish this goal, we must be able to establish selective activation of the receptor both in‐vitro and in‐vivo. This makes ligand specificity of the utmost importance. Since pharmacological studies are not available for mice, we compared the relative affinity of antagonists for 5‐HT2A versus 5‐HT2C receptors, confirming that M100907 and SB206553 were relatively selective. In order to examine receptor specificity in‐vivo, N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ), an irreversible alkylating agent, was injected i.p. to inactivate sites in frontal cortex. We then tested the ability of pretreatment with antagonists to block EEDQ. While 0.25 mg/kg of the 5‐HT2A antagonist M100907 prevented 5‐HT2A inactivation, it did not protect 5HT2C sites. In contrast, the 5HT 2C antagonist SB206553 did not protect 5‐HT2A receptors. (Supported by NIH grants T32MH065782 and R01MH34007)