bluuebunny/crossref_metadata_2025 huggingface dataset:
The behavioral actions of creatine vary with duration of intake
Authors: D'Anci Kristen E. , Allen Patricia J. , Kanarek Robin B.
Published: June 2021

Dietary creatine has been promoted for its positive health effects, and is best known for its use by athletes to boost physical performance. Creatine also has protective effects on the brain in models of neuronal damage, and improves cognitive performance on stressful tasks. However, there is little research examining how soon behavioral effects are seen after supplementation with creatine. To further explore the behavioral actions of creatine in rats, we used the forced‐swim test (FST) as a stressor, and assessed anxiety‐like behavior using the Elevated Plus Maze (EPM), and spatial learning using the Morris Water Maze (MWM). Female rats were fed chow alone or chow mixed with 4% w/w creatine for 1, 3, or 5 wks prior to the FST. Rats were tested in the EPM 24h after the FST and in the MWM 48h after the FST. There were no differences in any measures after 1 wk of creatine. At 3 wks, rats given creatine showed improved performance on the MWM relative to controls, but tended to show more anxiety‐like behavior in the EPM. At 5 wks, rats given creatine showed worse performance on the MWM and more anxiety‐like behavior in the EPM relative to controls. These findings indicate that there are time‐related differences in the behavioral effects of creatine supplementation, and suggest that continuous chronic intake of creatine may not be the most advantageous use of the supplement. Supported by internal research funds from Tufts University.
Medicinal herbs inhibit castrate‐resistant prostate cancer via mechanisms involving AMPK
Authors: Zhao Yi , Whelan Jay
Published: June 2021

Prostate cancer (PCa) is the second most common type of cancer among males in the USA. Preliminary data has shown that Zyflamend®, a mixture containing extracts of 10 medicinal herbs, inhibited growth of CWR22Rv1 cells (a castrate‐resistant human PCa cell line), by down‐regulating the expression of key mediators of lipogenesis, including SREBP‐1c, fatty acid synthase and HMG CoA reductase. Down‐regulation of lipogenic genes is associated with tumor growth inhibition. These same pathways are negatively regulated by 5’adenosine monophosphate (AMP)‐activated protein kinase (AMPK), a potential tumor suppressor protein that functions as a central energy sensor within the cell. Proliferation of CWR22Rv1 cells was enhanced following chemical inhibition of AMPK by Compound C and inhibited with constitutive over‐expression of AMPK following adenoviral infection. These effects are believed to be mediated by AMPKs phosphorylation of mTOR (mammalian target of rapamycin), a known activator of cell proliferation and a downstream target of AMPK. Similarly, Zyflamend® up‐regulates AMPK activation, inhibits cell growth, and coordinately increases the phosphorylation of down‐stream targets of AMPK, mTOR and acetyl CoA carboxylase (negatively effecting fatty acid synthesis). Thus, our results suggest that the polyherbal mixture Zyflamend® inhibits castrate‐resistant PCa through mechanisms involving AMPK.
Effects of blackberry and blueberry polyphenol extracts on NO, TNF‐α, and COX‐2 production in LPS‐stimulated RAW264.7 macrophages
Authors: Feresin Rafaela G , Zhang Jiyao , Elam Marcus , Hooshmand Shirin , Kim Jeong-Su , Arjmandi Bahram H.
Published: June 2021

Epidemiological studies have shown that populations who consume plant foods rich in polyphenols have lower incidence of chronic inflammatory diseases. These polyphenols have been shown to modulate the inflammatory response by inhibiting NO, TNF‐α, and COX‐2. The objective of this study is to investigate the extent to which blackberry and blueberry polyphenols modulate the production of NO, TNF‐α, and expression of COX‐2 in LPS‐stimulated RAW 264.7 macrophages. Berry polyphenols were extracted using ethanol and total phenolics were quantified by Folin‐Ciocalteu method. Macrophages were treated with different doses of polyphenols (0, 10, 100, 1000 μg/ml) 1 hr prior to stimulation with 10 ng/ml LPS for 6 hrs. Supernatants were collected to measure NO and TNF‐α production and cells were harvested to assess COX‐2 via western blot. Blackberry and blueberry polyphenol extracts contained 55.56 and 79.06 mg/g expressed as gallic acid equivalents, respectively. Blackberry polyphenol extract strongly inhibited NO production at doses of 10 and 100 μg/ml by 56 and 59%, respectively, without cytotoxicity whereas blueberry polyphenol extract had no effect on NO production. Thus far, these cell culture results indicate that polyphenol extracts from blackberry possess anti‐inflammatory properties; however, these findings need to be confirmed in in vivo models.
Effects of blueberry extract supplementation on antioxidant activity in chronically stressed rats
Authors: Lee Bog-Hieu , Kim Mi Young , Kim Eun Jin , Choi Joo-Yeon , Yu Miri , Han Chan-Kyu
Published: June 2021

The study was conducted to elucidate the effects of blueberry(Vaccinium cyanococcus) supplementation on antioxidant enzyme activities in rat blood, skin and brain tissue. Forty‐eight 10‐week‐old male Sprague‐Dawley rats were assigned to four groups (n=12/group); no stress‐normal diet(−CON), stress‐normal diet(+CON), no stress‐normal diet + 5% blueberry water(−BB), and stress‐ normal diet + 5% blueberry water(+BB). The +CON and +BB groups were given the stress during all experimental period and stress scheme was followed the chronic mild stress model. Body weight gain and feed efficiency ratio were decreased in given stress groups compared with non stress groups (p<0.05). Antioxidant activity levels in blueberry supplemented rats tended to increase compared to control groups. The stressed rats groups tended to have low antioxidant levels compared with unstressed rats groups. The superoxide dismutase(SOD) activity in serum and brain was not different among the treatments. The serum glutathione peroxidase activity was the highest in −BB group, but +BB group was lower than +CON(p<0.05). The skin SOD activity was significantly higher in blueberry supplementation groups than that of −CON and +CON groups. The skin catalase activity was higher in blueberry groups(p<0.05). From the findings, blueberry supplementation might provide the protection against oxidative stress induced by chronic stress.
Usefulness of dietary flaxseed in abrogating lung damage associated with space exploration: antioxidant and anti‐inflammatory effects of flaxseed in a murine model of repeated double‐hit low‐level radiation and hyperoxia exposure
Authors: Pietrofesa Ralph A , Dukes Floyd , Tyagi Sonia , Arguiri Evguenia , Solomides Charalambos C , Christofidou-Solomidou Melpo
Published: June 2021

Crew members during spaceflight missions must conduct extra‐vehicular activities (EVA) which may expose them to lung damaging hyperoxia and low levels cosmic/galactic radiation. Flaxseed (FS) has lung protective properties in mouse models of acute and chronic lung injury. To test the usefulness of FS as a dietary supplement for such missions, we evaluated FS in a murine model of double‐hit hyperoxia and radiation. Mouse cohorts (n=5/group) were pre‐fed diets containing either 0% FS or 10% FS for 3 wks and exposed to: a) normoxia; b) >95% O2 (O2); c) 0.25Gy gamma radiation (RAD); or d) a combination of O2 and RAD (O2+RAD). Challenge (O2 or RAD) was given 3X weekly for 2 wks; 8‐hour hyperoxia was spanned by normoxic intervals. Lungs were evaluated for oxidative damage, inflammation, injury and fibrosis. 0% FS‐fed mice developed significant lung injury and inflammation across all challenges, as evidenced by bronchoalveolar lavage (BAL) neutrophils (p<0.003), BAL protein levels and increased oxidative tissue damage (p<0.008). Lung hydroxyproline content increased in 0% FS‐fed mice exposed to RAD and O2+RAD (p<0.001). All adverse effects due to challenge conditions were ameliorated in FS‐fed mice. In conclusion, dietary FS is protective in a murine model of repeated low‐level radiation and hyperoxia exposure. Funded by: NIH/NCI, NASA (MCS)

Grant Funding Source: NIH/NCI/NASA
Anti‐oxidant and Anti‐inflammatory Activity of Tocopherol Isomers Modulates the Nrf‐2 and NfκB Activation in Caco‐2 Human Intestinal Cell Line
Authors: elisia ingrid , Kitts David
Published: June 2021

The objective of this study was to determine if the antioxidant activity of tocopherol (Toc) isomers translated to a protection against inflammation through oxidative‐stress related pathways in the Caco‐2 intestinal cell line. Antioxidant activities of major Toc isomers, determined using Oxygen Radical Absorbance Capacity (ORAC) chemical assay, followed a relative order of δToc>γToc>αToc. The anti‐inflammatory activity of Toc isomers was determined using interferon γ/phorbol myristate acetate (IFNγ/PMA) cocktail to stimulate interleukin 8 (IL8) secretion from Caco‐2 cells. Both αToc and γToc concentration‐dependently suppressed IL8 production from IFNγ/PMA‐stimulated cells. Delta Toc on the other hand was more effective at a lower concentration to modulate IL8 production compared to αToc and γToc, while being the least effective high concentration. Exposure of Caco‐2 cells to IFNγ/PMA activated both stress‐responsive NfκB pro‐inflammatory and Nrf2 antioxidant enzyme pathways. Only αToc concentration‐dependently suppressed NfκB activation induced by IFNγ/PMA cocktail. There was also an isomer‐dependent difference in the NfκB modulating activity, with αToc being the greatest followed by γToc and δToc (P<0.05). On the other hand, δToc was shown to further promote Nrf2 nuclear translocation, while αToc had no effect. To conclude, the antioxidative pattern exhibited by Toc isomers does not correspond to the anti‐inflammatory activity in Caco‐2 cells; a finding that is attributed to either a non‐antioxidant function of Toc isomers, or the formation of pro‐oxidants at high concentrations of Tocs.

Grant Funding Source: UBC‐FNH vitamin research fund and NSERC
Renal Adaptation in Gestational Iron Deficiency
Authors: Sun Mary (Yue) , Woolley Joseph C , Drake Keri A , Siddappa Asha Jyothi M , Georgieff Michael K , Magness Ronald R , Kling Pamela J
Published: June 2021

Background

Severe gestational iron deficiency (ID) can impair fetal growth and renal development, ultimately causing adult hypertension. Nephrogenesis continues until P7 in rats. Our aim was to examine rat kidney adaptations during gestational ID.

Methods

From gestational day 2 to postnatal day (P)7, pregnant rats were fed IS iron sufficient (198 mg Fe/kg diet) or ID rat diet (<6 mg Fe/kg diet), with the biological lactating dam nursing the pups. At P7 all ID dams were fed IS diet. At P15 and P30, rats were weighed, blood was drawn, and tissues were harvested. Kidneys were weighed and fixed renal sections were blindly examined for radial glomerular counts (RGCs), glomerular size, cortical depth, glomeruli per cortical surface area and total glomerular planar surface area. P<0.05 was considered significant.

Results

At P15, ID pups were 20% smaller than IS pups, but only 8% smaller at P30. The ratio of kidney/rat weight was similar in all groups, except higher in P15 ID. At P15, hematocrit was lower in ID than IS, but not at P30. Glomerulogenesis was altered by gestational ID. Compared to P15 IS, RGCs in P15 ID were lower, mean glomerular size smaller, cortical depth shorter, glomerular density greater, and glomerular planar surface area lower. Microscopic renal morphometric parameters were normalized by P30.

Conclusion

Gestational ID caused newborn anemia, growth restriction, with smaller and more‐dense glomeruli. The kidneys adapted to equalize glomerular density and filtering area, in contrast to published work in adult offspring of gestation ID. Delayed cessation of nephrogenesis may hold potential clues to understanding fetal renal adaptation to tissue iron depletion.

UW Department of Pediatrics and UW Graduate School, Meriter Foundation, Minnesota Viking Children's Fund

Grant Funding Source : UW Department of Pediatrics and UW Graduate School, Meriter Foundation, Minnesota Viking Children's Fund
454 Pyrosequencing reveals a beneficial shift in fecal microbiota of healthy adult men consuming polydextrose or soluble corn fiber
Authors: Hooda Seema , Boler Brittany M Vester , Serao Mariana C Rossoni , Staeger Michael A , Boileau Thomas W , Dowd Scot E , Fahey George C , Swanson Kelly S
Published: June 2021

The objective of the study was to test the impact of polydextrose and soluble corn fiber on the composition of the human gut microbiota using 454 pyrosequencing. Healthy adult males (n = 20) were enrolled in a randomized, double‐blind, placebo‐controlled crossover study. Subjects consumed 3 treatments (snack bars) containing either no supplemental fiber (NFC), polydextrose (PDX; 21 g/d), or soluble corn fiber (SCF; 21 g/d) for 21 days. Fecal samples were collected on day 16–21; DNA was extracted, followed by amplification of the V4–V6 region of the 16S rRNA gene for 454 pyrosequencing. The consumption of PDX and SCF led to an increase (P<0.05) in the families of Clostridiaceae and Veillonellaceae, and a decrease in Eubacteriaceae. The abundance of Faecalibacterium, Phascolarctobacterium, and Dialister was increased (P<0.05) in response to PDX and SCF intake, whereas Lactobacillus was higher (P<0.05) only after SCF intake. In contrast, PDX and SCF intake led to a decrease (P<0.05) in Eubacterium, Ruminococcus, and Bifidobacterium. Faecalibacterium prausnitzii was increased (P<0.05) by consumption of both fibers. The principal component analysis clearly indicated distinct clustering of individuals consuming novel fibers as compared to those consuming NFC. In conclusion, data demonstrated a beneficial shift in the gut microbiome of adults consuming PDX and SCF.

Grant Funding Source: General Mills, Inc., Minneapolis, MN
Fatty Acid Binding Protein 4‐deficient Mice are Protected from Oxygen‐induced Retinal Neovascularization
Authors: Saint-Geniez Magali , Ghelfi Elisa , D'Amore Patricia , Hotamisligil Gökhan , Cataltepe Sule
Published: June 2021

Fatty acid binding protein 4 (FABP4) is a member of the FABP family, which consists of highly conserved proteins that function as chaperones for free fatty acids. FABP4 plays a critical role in maintenance of glucose and lipid homeostasis as well as in the regulation of inflammation. Initially, FABP4 was thought to be expressed primarily by adipocytes and macrophages. We recently determined that FABP4 is also expressed in certain endothelial cells (ECs) where it plays a pro‐angiogenic role. The goal of this study was to determine whether FABP4 plays a role in oxygen‐induced retinal neovascularization.

We induced oxygen‐induced retinopathy (OIR) by exposing pups to 75% oxygen between postnatal (P) days 7 and 12. FABP4 mRNA and protein levels were significantly increased during OIR. Immunostaining revealed that FABP4 was strongly expressed in pathological neovessels but absent in normal retinal vessels. In addition, both the neovascular and avascular areas of FABP4−/− retinas were significantly reduced at P17 compared to WT. The effect of FABP4 deficiency on OIR appears independent from macrophages recruitment.

The observations that FABP4‐deficient mice are significantly protected from OIR and that FABP4 expression is restricted to neovascular tufts suggests that FABP4 inhibition may be a potential therapeutic target for proliferative retinopathies.

This project is funded by the NEI grant 1R21EY021873‐01.
Mono‐ubiquitination of annexin A1, acetylation of PML and apoptosis of MCF7 cells treated by HDAC inhibitors
Authors: Hirata Aiko , Senanayake Thulani H , Woster Patrick M , Hirata Fusao
Published: June 2021

When MCF7 human breast adenocarcinoma cells were treated by HDAC inhibitors (HDACi), SAHA or THS79‐12, considerable portions of MCF7 cells became floated and underwent apoptosis. Annexin A1 in floated cells was mono‐ubiquitinated as determined by Western blots, suggesting that DNA was damaged. PML, a protein that plays an important role in DNA damage induced apoptosis was acetylated. Consequently, BclxL was induced, and caspase 3 was cleaved, demonstrating features of apoptosis. On the other hand, annexin A1 in attached cells was not ubiquitinated, and acetylation of PML did not occur. Cleavage of caspase 3 did not increase, showing no apoptotic changes. Annexin A1 was phosphorylated at tyrosine by EGF, and cell proliferation was reduced. MCF7 cells were morphologically differentiated. Contrary to the previously proposed hypothesis that induction of annexin A1 in cancer cells by HDACi causes apoptosis, our present observations suggest that two distinct responses to HDACi take place in attached and floating cells: in floating cells, deacetylation of PML is inhibited and oxidative stress is induced by HDACi, whereas in attached cells, annexin A1 induced by HDACi influences cell proliferation signaling by EGF. (Supported in parts by a grant from The Susan G. Komen Breast Cancer Foundation.)
Detection of nanoparticles in mice using an integrated photoacoustic micro‐ultrasound system
Authors: Sun John , Heinmiller Andrew , Bates Dave , Needles Andrew , Theodoropoulos Catherine
Published: June 2021

VisualSonics have recently developed a photoacoustic imaging system (VevoLAZR, Toronto, Canada) that combines the sensitivity of optical imaging modalities and the high resolution of micro‐ultrasound. The high sensitivity enables detection of nano‐scaled contrast agents. Due to their small size, nanoparticles likely can cross the leaky tumor microvasculature and deposit in the extra‐ and intracellular space. This presents countless potential benefits in cancer therapy. Using VevoLAZR we investigated the use of various gold nanoparticles and common optical fluorescent dyes as photoacoustic contrast agents in a vessel phantom study. We also injected gold nanorods intravenously in mice bearing tumors and investigated the preferential accumulation of these nanoparticles in the tumor tissue. Tumor perfusion was also quantified using ultrasound contrast imaging. Gold nanorods showed the greatest photoacoustic intensity amongst the nanoparticles tested. Optical fluorescent dyes used were also readily detectable in photoacoustic imaging. In the tumor study, preferential accumulation of gold nanorods was observed in the tumor relative to mammary tissue. Interestingly, tumor perfusion appeared to be inversely correlated with amount of gold nanorod accumulation. Collectively, we have demonstrated photoacoustics as a novel and powerful tool to detect and quantify nanoparticles in mice tumors.
Inhibition of Myeloperoxidase by a Carbon Monoxide‐Releasing Molecule, CORM‐3
Authors: Patterson Eric Kenneth , Serzawa Fukashi , Capretta Alfredo , Fraser Douglas D , Potter Richard F , Cepinskas Gediminas
Published: June 2021

Carbon monoxide (CO)‐releasing molecules (CORMs) have recently been shown to suppress inflammation by interfering with neutrophil function. However, the mechanisms are not completely understood. Neutrophilic myeloperoxidase (MPO) is known to induce tissue damage through: 1) the direct oxidation and/or 2) through the hypohalous acid‐dependent oxidation of targets.

We hypothesized that CORM‐released CO inhibits MPO activity and thereby reduces neutrophil‐associated injury.

In these studies, we used a water‐soluble, ruthenium‐based CORM (CORM‐3) that we synthesized and known to suppress inflammation. In vitro assays were used to study CO release under conditions relevant to inflammation. Further studies investigated CORM‐3's ability to inhibit total MPO activity as well as the activity of the peroxidation and halogenation components.

Results indicate that CORM‐3‐released CO inhibited total MPO activity in a dose‐dependent manner. Furthermore, both the peroxidation and halogenation activities of MPO were inhibited by CORM‐3 in a dose‐dependent manner. On the contrary, control molecules; inactive CORM‐3 (a CORM lacking CO), or RuCl3 were less effective.

In summary, CORM‐3‐derived CO inhibits MPO activity and therefore may suppress neutrophil‐induced tissue damage. (HSFO NA6914 and LHRI IRF‐025‐09)
Expression of pentraxin 3 in the lungs of human, horse, cattle and pig
Authors: Soron Michelle , Singh Baljit
Published: June 2021

Bacterial lung diseases cause significant mortality and morbidity, causing enormous economic damage to the animal industry and cost billions of dollars in human health care. The innate immune system plays a critical role in the initiation of immune response in the lung. Pentraxin 3 (PTX3) is an acute phase protein that is produced at sites of inflammation by many cell types upon stimulation by proinflammatory cytokines, and agonists such as endotoxins. PTX3 recognizes and binds to many pathogens, activates the complement cascade, and has a role in the clearance of apoptotic and necrotic cells. Because there are very few data on the expression of PTX3 in the lungs, we examined PTX3 expression in normal and inflamed lungs of the horse, pig, human, and cattle using light and electron microscopic immunochemistry. We show PTX3 staining in bronchial epithelial cells and vascular endothelium in normal and inflamed lungs. Alveolar macrophages and inflammatory cells recruited into the lungs of bacteria‐infected animals stain intensely for PTX3. Immuno‐gold electron microscopy on horse, and calf lungs showed PTX3 in the nuclei, cytoplasm, and vesicular organelles of alveolar macrophages, endothelial cells and pulmonary intravascular macrophages. These data show PTX3 is expressed in normal lungs and its expression is increased in inflamed lungs.

Grant Funding Source: Alberta Livestock and Meat Agency
Oxidative Stress Regulation of Regulator of G‐Protein Signaling 4 (RGS4)
Authors: Monroy Carlos Aaron , Roman David
Published: June 2021

Regulators of G‐protein signaling (RGS) proteins are a family of proteins that regulate G‐protein coupled receptors (GPCRs) by acting as GTPase accelerating proteins (GAPs). RGS proteins increase the rate of hydrolysis of GTP to GDP in select Gα subunits, including Gαo, Gαi, and Gαq but not Gαs. One of these proteins, RGS4, has been implicated in several neurological diseases. In this study, we analyze the effect of oxidative stress on RGS4 regulation in neuronal cells and the effect of lipid peroxidation products, such as 4‐hydroxy‐nonenal (4HNE), on RGS4 activity. Select RGS4 isoforms were induced during oxidative stress with H2O2. 4HNE forms covalent adducts on reactive amino acids such as cysteine. Previous research revealed that modification only occurs at select cysteine residues. Several mutants were made containing only one of the modified cysteines, either C71, C148, and, C183, and were treated with increasing amounts of 4HNE and then assayed for coupling to the native binding partner Gαo by ALPHA‐Screen. The wild type construct showed sensitivity to 4HNE but the cysteine null mutant did not. Of the single cysteine containing mutants, only C148 conferred sensitivity to RGS4. Research support provided by the Pharmacological Sciences Training Grant (NIH/NIGMST32GM067795).
Activation of PKCα or PKCε as an approach to increase morphine tolerance in respiratory depression and lethal overdose
Authors: Lin Hong-Yiou , Law Ping-Yee , Loh Horace H.
Published: June 2021

Identifying mechanisms that increase morphine respiratory depression and overdose tolerance could lead to the safer use of opioids. Since PKC activity mediates morphine antinociceptive tolerance, we hypothesized that activating PKCα or ε at the pre‐Bötzinger complex (preBötC) can increase morphine tolerance in respiration and overdose. Laser microdissection and qRT‐PCR were used to compare the relative mRNA abundances of PKCα, γ, and ε between vlPAG vs. preBötC. To test whether PKCα or ε could enhance tolerance in respiratory depression and overdose, lentivirus carrying the wild type (WT), constitutively activated mutants (CA), and siRNA against PKCα or ε were microinjected into the preBötC. Expressing CAPKCα or ε, but not WTPKCα or ε, at the preBötC allowed rats to develop tolerance to morphine respiratory depression. In terms of lethality, expressing either WTPKCε, CAPKCα, or CAPKCε at preBötC increased morphine tolerance to lethal overdose. CAPKCε expressing rats developed the highest level of respiratory depression tolerance. Furthermore, when CAPKCε lentivirus was injected into the vlPAG, rats were able to develop significant antinociceptive tolerance at low doses of morphine that normally do not cause tolerance. The approach of increasing morphine respiratory depression and lethality tolerance by increasing PKCα or ε activity at preBötC could make opioids safer for chronic use. This research was supported by NIH grant [DA000564].
Endothelial NOS‐independent release of nitric oxide in the aorta of the spontaneously hypertensive rat
Authors: Zhao Yingzi , Leung Susan WS , Vanhoutte Paul M
Published: June 2021

Preliminary experiments showed that in the aorta of male spontaneously hypertensive (SHR), but not in that of normotensive Wistar‐Kyoto (WKY) rats, contractions to phenylephrine (PE) are inhibited in preparations with endothelium despite the presence of indomethacin and L‐NAME. The present study aimed to identify the mechanism underlying this endothelium‐dependent inhibition in the SHR aorta. Aortic rings, with and without endothelium, of male SHR and WKY were suspended in organ chambers for the measurement of isometric tension. Contractions to PE were smaller in SHR aorta with than in those without endothelium. The inhibitory effect of the endothelium was larger in preparations from adult than in those from young SHR. The endothelium‐dependent difference in contraction to PE was abolished by the NO scavengers oxyhemoglobin and carboxy‐PTIO, by the inhibitor of soluble guanylyl cyclase ODQ, and the by inhibitor of cytochrome P450 reductase (CPR) diphenyleneiodonium. PE‐induced contractions in adult WKY aortic rings with endothelium were not affected by carboxy‐PTIO. Thus, in the SHR, there is an endothelium‐dependent release of NO which is not produced by NOS but by CPR. Nitrate is the likely source of this NOS‐independent endothelial NO. The NOS‐independent NO release is observed only in preparations from hypertensive animals and increases with age (supported by the Hong Kong Research Grant Council).
A role for PKCβ in altering calcium homeostasis under hyperglycemic conditions in human endothelial cells
Authors: Zhang Rui , Thor Der , Chaharmahali Pegah , Anderson Leigh , Rahimian Roshanak
Published: June 2021

Cardiovascular diseases (CVD) are leading causes of morbidity and mortality for diabetic patients. Endothelial cell dysfunction is a hallmark of CVD. Intracellular Ca2+ concentration ([Ca2+]i) may regulate endothelial cell function. Here, we investigated the effects of hyperglycemia on [Ca2+]i and a role for protein kinase C (PKC) in regulating [Ca2+]i in the human endothelial cell line, EA.hy926. Cells were cultured in normal (5.5 mM, NG) or high (25 mM, HG) glucose media then treated with either a) vehicle (0.1% DMSO), b) LY341684 (selective PKCβ inhibitor, 100 nM, 24h), or c) phorbol 12‐myristate 13‐acetate (PMA, PKC activator, 200 nM, 4h). Using a spectrofluorometer, [Ca2+]i in cells loaded with Fura 2‐AM was monitored in the absence of extracellular Ca2+. Thapsigargin (SERCA inhibitor, 1 μM,) induced a transient increase in [Ca2+]i which was similar among the experimental groups of cells. However, when 1.5 mM Ca2+ was added, Ca2+ entry was significantly increased in cells cultured in HG compared to NG. This elevated Ca2+ entry in HG‐treated cells was attenuated by treatment with LY341684. Cells treated with PMA in NG showed an increased Ca2+ entry similar to cells cultured in HG. Our results suggest that hyperglycemia enhanced Ca2+ entry in endothelial cells is PKCβ dependent. Thus, PKCβ inhibitors may restore normal endothelial cell function under hyperglycemic conditions. Supported by NIH/NIDCR.
Impaired K+ channel function leads to increased catecholamine secretion by adrenal chromaffin cells in DOCA‐salt hypertension
Authors: Fhaner Matthew J , Galligan James J , Swain Greg M
Published: June 2021

Sympathetic nerve activity is increased in deoxycorticosterone acetate (DOCA) salt model of hypertension and catecholamine secretion from adrenal chromaffin cells is increased in DOCA‐salt rats. This includes the number of vesicles released by stimulation (acetylcholine, 1 mM). We tested the hypothesis that individual chromaffin granule size was increased and that K+ channel function was impaired in chromaffin cells from DOCA‐salt rats. Using the charge value obtained from individual events a vesicle radius can be determined by taking the cubed root of the charge. These data showed a positive shift in the mean Q1/3 value for DOCA‐salt release events compared to sham events suggesting that the increase in charge observed is due in part to increased vesicle radii. To determine why DOCA‐salt cells have increased release frequency compared to sham cells, K+ channel function was investigated. We used the K+ channel blockers paxilline (BK), 4‐aminopyridine (Kv1), glibenclamide (ATP‐sensitive) and apamin (SK). Paxilline and glibenclamide (but not apamin or glibenclamide) increased the number of secretory events and total number of catecholamines oxidized from sham chromaffin cells, however did not effect release from DOCA‐salt cells. We conclude that increased vesicle radius and impaired potassium channel function contribute to increased secretion and quantal charge in DOCA‐salt chromaffin cells.
Cleavage of L‐glutamate decarboxylase 65 is associated with brain injury
Authors: WU JANG-YEN
Published: June 2021

Previously, we have shown that the GABA synthesizing enzyme, L‐glutamic acid decarboxylase 65 (GAD65) is cleaved to form its truncated form (tGAD65) which is 2–3 times more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiological stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this communication, we have showed that formation of tGAD65 progressively increases with increase of stimulus concentration both in rat brain synaptosomes and primary rat neuronal cultures. More importantly, direct cleavage of SV‐associated fGAD65 by calpain was demonstrated and the resulting tGAD65 bearing the active site of the enzyme detached from the SVs. Vesicular GABA transport of the newly synthesized GABA was found to be reduced in calpain treated SV. Furthermore, we also observed that the level of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to elevation of local glutamate as measured by microdialysis. Moreover, the level of tGAD65 is also proportional to the degree of cell death when the primary neuronal cultures were exposed to high KCl. Based on these observations, we conclude that calpain‐mediated cleavage of fGAD65 is pathological due to decrease of activity of SV‐associated GAD65 resulting in decrease of GABA synthesis/packaging coupling process leading to decrease in GABA transmission. Supported in part by James & Esther King Grant # 09KW‐11, DOH, State of Florida.
Dynamic Interaction of α‐synuclein and dopamine transporter at the plasma membrane
Authors: Butler Brittany Renee , Goodwin Shawn , Khoshbouei Habibeh
Published: June 2021

The dopamine transporter (DAT) regulates the dimension and duration of DAergic transmission in the brain. We and others have shown that overexpression of α‐synuclein alters DAT activity. Previous studies have shown that α‐synuclein interacts at the C‐terminus of DAT, but the functional consequences of this interaction are not well known. This study examines the hypotheses that α‐synuclein interacts with DAT at the plasma membrane and this interaction alters DAT trafficking. To address these hypotheses we used Immunocytochemistry, live cell imaging, and co‐patching. The immunocytochemistry experiments in CHO cells co‐expressing YFP‐DAT and α‐synuclein suggest these proteins co‐localize at the plasma membrane. We found that although α‐synuclein is distributed homogenously in cells not expressing DAT, the intensity profile of α‐synuclein peaks at the plasma membrane of DAT cells, overlapping with that of DAT at the plasma membrane. Co‐patching experiments in living cells expressing YFP‐HA‐DAT +/− CFP‐α‐synuclein confirm co‐localization of the proteins at the membrane. We found a dynamic interaction between DAT and α‐synuclein and the recruitment of α‐synuclein to the plasma membrane. Fluorescence Recovery After Photobleaching experiments show that DAT decreases the lateral mobility of α‐synuclein. These results suggest the interaction between DAT and α‐synuclein is transient and dynamic.
The Protective Effects of Nicotine in Cells Expressing Beta Amyloid and Presenilin: Implications for Alzheimer's Disease
Authors: BROWN DWAYNE , Ramlochansingh Carlana , Tizabi Yousef , Manaye Kebreten F , Drew Amy C.
Published: June 2021

Alzheimer's disease (AD) is a progressive age‐related neurodegenerative disease characterized by cognitive impairments and formation of plaques and tangles. In addition to Aβ, the major component of plaques, mutation in the protein presenilin has also been shown to contribute to AD's pathology. Since protective effects of nicotine against several neuronal toxins have been observed both in‐vivo and in‐vitro, we hypothesized that nicotine would also protect against cellular damage or death due to high expression of Aβ alone or Aβ and presenilin. We also sought to determine whether this effect by nicotine is mediated through nicotinic receptors. A wild type neuroblastoma (N2a) cell line, one transfected with APP695 (N2a‐695) and another with the combination of APP695 and presenilin (N2a‐695+P) were pretreated with various concentrations of nicotine 24 hours after plating. The survival of the cells was determined and nicotine treatment dose‐dependently provided protection against cellular loss. 0.01uM nicotine resulted in a 22% increase in protection in N2a cells, 33% in the N2a‐695 and 44% in the N2a‐695+P cells. These results suggest a differential sensitivity to the protective effects of nicotine in cells representative of AD pathology. Nicotine effects were blocked by mecamylamine pre‐treatment. Although the mechanism for the nicotine‐induced protection is not known, the data suggest that nicotinic agonists could be of therapeutic use in AD.
EFFECT OF TIBOLONE ON MILD COGNITIVE IMPAIRMENT IN POSTMENOPAUSAL PATIENTS
Authors: Fernández-Hernández Said , Olivares-Corichi Ivonne Maria , Jimenez-Zamarripa Carlos Alberto , Rivera-Garcia Carlos , Calzada-Mendoza Claudia Camelia
Published: June 2021

OBJECTIVE

To evaluate the effect of tibolone on mild cognitive impairment in postmenopausal patients, the concentration of malondialdehyde/4‐ hidroxinonenal and anthropometric parameters.

METHODS

Twenty menopausal patients (50–55 years old) were included. The determinations were clinical history, body mass index, waist‐hip ratio, percentage of body fat, concentration of malondialdehyde/4‐ hidroxinonenal, and the cognitive function; which were evaluated before and 3 months after of treatment with tibolone.

RESULTS

Tibolone improved cognitive function in 77% of patients who had mild cognitive impairment baseline. Also reduced damage to lipid marker for oxidative stress (17 vs 11 nM MDA‐HNE before and after treatment, respectively), but anthropometric parameters were unchanged.

The correlation analysis showed that at low lipid damage is a higher score in memory.

CONCLUSIONS

Tibolone decreased mild cognitive impairment and the concentration of markers of lipid damage by oxidative stress, but did not reduce body mass index, waist‐hip index and fat percentage.
Substituted phenyl groups improve the pharmacokinetic profile of urea‐based soluble epoxide hydrolase inhibitors
Authors: Liu Junyan , Lin Yan-Ping , Morisseau Christophe , Lee Kin Sing Stephen , Rose Tristan E. , Hwang Sung Hee , Hammock Bruce D
Published: June 2021

Soluble epoxide hydrolase inhibitors (sEHIs) are anti‐inflammatory, analgesic, anti‐hypertensive, cardio‐protective and renal‐protective in multiple animal models. However, the earlier adamantyl urea‐based inhibitors are metabolically susceptible which limits their pharmacological use. Therefore, novel potent inhibitors with the replacement of an adamantyl group by a substituted phenyl group are synthesized to presumptively offer better pharmacokinetic (PK) properties including metabolic stability. Here we present the PK profiles of new inhibitors in a murine model. The PK profiles of sEHIs were determined following p.o. administration and serial bleeding in mice. The blood levels of inhibitors were measured by a HPLC/MS/MS method. Compared with the earlier adamantyl urea‐based inhibitors, substituted phenyl urea‐based inhibitors afford more favorable PK properties, such as higher Cmaxs, higher AUCs and longer t1/2s, which, as expected, show more stable metabolic stability. The sEHIs with substituted phenyl group are more metabolically stable than those with adamantly group. This indicates a new strategy for development of sEHIs for further study toward clinical trials.
Metabolism of Cyclophosphamide by the Human Cytochrome P450 2B6 Polymorphic Variants
Authors: Calinski Diane , Zhang Haoming , Sridar Chitra , Hollenberg Paul F.
Published: June 2021

The alkylating agent, cyclophosphamide, is a common therapeutic agent used in the treatment of many cancers. Importantly, the efficacy and toxicity of cyclophosphamide varies greatly in patient populations. Cytochrome 2B6 is the primary P450 isoform involved in the activation and metabolism of cyclophosphamide and a potential explanation for the observed therapeutic discrepancies is believed to be the highly polymorphic nature of CYP2B6. Common CYP2B6 polymorphisms are known to cause alterations in the metabolism of other known substrates, such as bupropion and efavirenz. Using a reconstituted purified system, the kinetics of cyclophosphamide metabolism have been determined for wild type CYP2B6 as well as several common polymorphisms including CYP2B6.4–7 and CYP2B6.9. Interestingly, CYP2B6.4 shows only mild variations from CYP2B6.1 in the Km, Vmax, and kcat whereas CYP2B6.7 and CYP2B6.9 have significantly lower kcat values. Other mutants to be tested are CYP2B6.5 and CYP2B6.6. In addition the kinetics of cyclophosphamide metabolism by CYP2B6 WT and the other polymorphic variants will be investigated in the lipid bilayer system, the Nanodisc, to reveal the impact that the lipid bilayer has on P450 function. [This work is supported in part by CA16954]
Functional analysis of cytochrome P450 2B isoforms using ticlopidine as an active site probe
Authors: Jang Hyun-Hee , Wilderman P. Ross , Dalvie Deepak , Halpert James R.
Published: June 2021

A recent study of in vitro metabolism of ticlopidine by reconstituted human P450 2B6 and rabbit P450 2B4 indicated that both enzymes generated multiple and similar metabolites but in different ratios. In this study, we determined the metabolism of ticlopidine by additional cytochrome P450 2B enzymes, including rat P450 2B1 and dog P450 2B11. Based on the X‐ray crystal structure of the P450 2B4‐ticlopidine complex, two active site mutants, F206A and F297A were also created. All enzymes were expressed in E. coli as N‐terminal modified, and C‐terminal His‐tagged constructs termed 2B4dH. The separation and characterization of the different metabolites of ticlopidine were achieved using HPLC‐MS/MS. P450 2B1 and 2B11 produced ticlopidine N‐oxide as the major product, and the production of this metabolite by the P450 2B subfamily exhibited a rank order of 2B1 > 2B11 > 2B4 > 2B6. The pattern of the products observed for P450 2B1 and 2B11 was similar to that of P450 2B4. In contrast, the 2B4dH F206A and F297A mutants formed predominantly ticlopidine S‐oxide dimer previously seen with P450 2B6. The highest rate of the dimer formation was observed in F206A compared with other 2B isoforms including 2B4dH F297A. These results suggest that ticlopidine is a valuable probe for differentiating P450 2B subfamily members and for assessing the effect of site‐directed mutations.(Supported by NIH grant ES003619)
A selective reversible azapeptide inhibitor of human neutrophil proteinase 3 derived from a high affinity FRET substrate
Authors: Korkmaz Brice , Kellenberger Christine , Cadène Martine , Viaud-Massuard Marie-Claude , Gauthier Francis
Published: June 2021

The biological functions of human neutrophil proteinase 3 (PR3) remain unclear because of its close structural and functional resemblance to neutrophil elastase (NE). Thus, all natural inhibitors of PR3 preferentially target NE. We have designed a selective PR3 inhibitor based on the sequence of one of its specific, sensitive FRET substrates. This azapeptide inhibits free PR3 in solution, PR3 bound to neutrophil membranes, and the PR3 found in crude lung secretions from patients with chronic inflammatory pulmonary diseases. But it does not inhibit significantly NE or cathepsin G. Unlike most of azapeptides, this inhibitor does not form a stable acyl‐enzyme complex; it is a reversible competitive inhibitor with a Ki comparable to the Km of the parent substrate. Low concentrations (10 μM) of the azapeptide (azapro‐3) totally inhibited the PR3 secreted by triggered human neutrophils (200,000 cells/100 μL) and the PR3 in crude biological samples. Azapro‐3 resisted proteolysis by its target enzyme, by the proteases in neutrophils homogenates and by those in lung secretions of patients with neutrophilic lung inflammation for hours.
Human iPS‐derived hepatocyte‐like cells as a model for hepatitis C virus infection
Authors: Yamane Seiji , Yoshida Takeshi , Takayama Kazuo , Kondoh Masuo , Sakurai Fuminori , Mizuguchi Hiroyuki , Yagi Kiyohito
Published: June 2021

Approximately 130–200 million people are estimated to be infected with hepatitis C virus (HCV) worldwide, and over 350 thousand people die from HCV‐related disease every year worldwide. Therefore, overcoming HCV is a critical issue for the World Health Organization. The host tropism of HCV is limited to human and chimpanzee, and a convenient model for HCV infection has never been fully developed. The establishment of human induced pluripotent stem (iPS) cell technology has led to innovative iPS‐based models for drug discovery. Protocols for preparation of iPS‐derived hepatocyte‐like cells (iPS‐Hep) have been developed; however, the suitability of iPS‐Hep cells as a model for HCV infection remains unclear. In the present study, we investigated infection of HCV pseudotype virus (HCVpv) and replication of the HCV subgenome in iPS‐Hep cells. We demonstrated that HCVpv infected iPS‐Hep cells but not iPS cells, and the HCV subgenome replicated in iPS‐Hep cells but not in iPS cells. Inhibitors of HCV replication, interferon, and HCV entry, anti‐CD81 antibody, attenuated HCV replication within the iPS‐Hep cells, and the HCVpv entry into the iPS‐Hep cells. These findings suggest that iPS‐Hep cells are a potent model for HCV infection. As a promising new tool for HCV research, iPS‐Hep cells will facilitate our understanding of the biology of HCV and the development of anti‐HCV drugs.
Molecular Determinants of Reactivation Potency for Novel, Efficacious, Centrally Active Oxime Reactivators of Phosphylated Acetylcholinesterase
Authors: Radic Zoran , Sit Rakesh K , Kovarik Zrinka , Berend Suzana , Garcia Edzna , Zhang Limin , Amitai Gabriel , Fokin Valery V , Sharpless K Barry , Taylor Palmer
Published: June 2021

We present here systematic structural optimization of uncharged but protonated N‐substituted 2‐hydroxyiminoacetamide reactivators of phosphylated human acetylcholinesterase (hAChE) intended for reactivation of organophosphate (OP) inhibited hAChE in the CNS. Starting with library of 138 uncharged reactivators we identified the initial lead, RS41A and refined its structure culminating in the azepine analogue RS194B, several‐fold enhanced for reactivation of OP‐hAChE conjugates formed by sarin, cyclosarin, VX, paraoxon and tabun. Detailed in vitro reactivation kinetics of RS194B revealed superiority over standard, pyridinium, reactivator antidote 2PAM and approached the potency of bisquaternary, bipyridinium dioxime MMB4. The pH dependence studies of UV, 1H NMR and oximolysis properties of lead oximes revealed pKa values of their ionizable groups in the range from 8.1 (for 2PAM) to 8.9 (for RS41A and RS194B). The low toxicity of RS194B (LD50=500 mg/kg) in mouse animal model in combination with its enhanced intrinsic reactivity resulted in great improvement of its therapeutic properties compared to RS41A, and standard oxime 2PAM. The improvement was particularly noticeable when pretreatment of mice with RS194B before OP exposure was combined with RS194B reactivation therapy following the OP insult.

Supported by the CounterACT Program, NIH OD, and the NINDS, Grant Number U01 NS058046.
THE EFFECT OF NORMOBARIC OXYGEN ON THE INJURY INDUCED BY FOCAL CEREBRAL ISCHEMIA IN RATS VIA HIF‐1‐NOTCH SIGNAL PATHWAY
Authors: chen chunhua , Zhang Weiguang , Yang Lei , Wang Ke , zhou Changman
Published: June 2021

Objective

To investigate the effect of pretreatment of inhalation of normobaric oxygen(NBO) on the injury induced by focal cerebral ischemia via HIF‐notch pathway.

Methods

Male SD rats were randomly divided into four groups: Control (sham surgery), Middle Cerebral Artery Occlusion and Reperfusion(MCAO), MCAO pretreated with Normobaric Oxygen (NBO), MCAO with NBO injected with 2ME2. 2ME2 was injected intraperitoneally at 1h before the inhalation of NBO. NBO was inhaled for 4h a day and for 6d continuous. We observed the neurological deficits in different groups. Rats were sacrificed at 24h for TTC staining, western blot, nissl and immunohistochemical staining.

Results

NBO pretreatment improved neurological deficits, reduced the infarct volume (P<0.05), inhibited the neuronal deformation shown by nissl staining and increased the expression of HIF‐1¦Á and Notch1 detected by Western Blot and immunohistochemical staining. But all the improvement were lessened by the 2ME2 injection.

Conclusion

NBO pretreatment may protect brain from cerebral ischemic injury via HIF‐Notch1 signal pathway.
Effect of a diet rich in fat and/or fructose on potassium channel gene expression in rat small mesenteric artery
Authors: Jensen Lars Jørn , Sorensen Charlotte Mehlin , Plogmark Oscar , Holstein-Rathlou Niels-Henrik , Salomonsson Max
Published: June 2021

We tested if arterial K+ channel gene expression is reduced under dietary conditions mimicking the metabolic syndrome. Male Sprague‐Dawley rats were fed high‐fat + high‐fructose (HFF; N=4) or high‐fructose (HF; N=4) diet from 4 to 32 weeks (wks) of age. Age‐matched control rats (CTRL) were fed normal chow (N=4 in each group). Systolic blood pressure (SBP) was measured using the tail cuff method. mRNA expression of K+ channels in mesenteric arteries was measured using quantitative real‐time polymerase chain reaction (Q‐PCR). At 32 wks body weight (BW) was increased in HFF (639±37g; P<0.05) compared to CTRL (522±22g), whereas BW was similar in HF (574±35g) and CTRL (564±15g). Average SBP through wks 10–32 was higher in both HFF and HF rats compared to their respective CTRL (HFF: 134±4 mmHg vs. 121±2 mmHg, P<0.05; and HF: 131±5 mmHg vs. 118±2 mmHg, P<0.05). In small mesenteric artery of HFF rats, mRNA expression of SK3 was reduced to 59% of that in CTRL (P<0.05) and KV1.5 was reduced to 73% of that in CTRL (P<0.01), while Kir2.1, Kir6.1, BKCa, IKCa, KV1.2 and KV7.4 expression was unchanged. HF diet did not significantly change K+ channel expression. We conclude that reduced expression of SK3 and KV1.5 channels might partially explain the increased SBP in metabolically challenged rats fed a diet rich in fructose + fat but not in those fed high‐fructose only.

Grants: Danish National Research Foundation (F.); Danish Heart F.; Lundbeck F.