bluuebunny/crossref_metadata_2025 huggingface dataset:
Hydrogen sulfide protects against hepatocyte cell death and mitochondrial reactive oxygen during hypoxia
Authors: Norris Eric , Schleser Franziska , Culberson Cathy , Clemens Mark G
Published: June 2021

Hydrogen sulfide (H2S) is an endogenous gas with important physiological functions. The role of H2S in liver injury remains controversial. Exogenous H2S is protective in ischemia/reperfusion and burns, whereas the inhibition of endogenous H2S is protective in sepsis models. We have demonstrated increased oxygen consumption in the liver with low levels of exogenous H2S (<200ìM) via mitochondrial sulfide oxidation but inhibition at higher levels. In vivo H2S infused into the portal vein decreased hepatic tissue PO2. Thus, we hypothesized that H2S contributes to liver injury via exacerbation of cellular hypoxia. To test this, primary rat hepatocytes were subjected to one hour of hypoxia (PO2 < 25 mmHg) in the presence of the slow releasing H2S donor, GYY 4137 (GYY). Hypoxia caused significant cell death in hepatocytes plated at >75% confluence. Contrary to our hypothesis, GYY significantly improved cell viability following hypoxia (84% survival vs. 30% in control, P= 0.0017). Since mitochondrial stress contributes to cell injury, we tested whether GYY decreased mitochondrial reactive oxygen during hypoxia. GYY significantly reduced mitochondrial ROS in hypoxia as visualized by the mitochondrial ROS specific dye Mitosox (TM) (50.3 vs 31.3 units, P=0.006). Thus, H2S protects against hepatocellular injury through a reduction in mitochondrial‐derived reactive oxygen production. Supported by DK38201
TMEM16A and NHERF1 Regulate Ca2+ and cAMP Stimulated Cl‐ Secretion in Murine Colon
Authors: Kazi Mirajul Hoque , Ali Irshad , Sarker Rafiquel , Cha Boyoung , Zachos Nicholas C , Harfe Brain D , Donowitz Mark , Tse Chung Ming
Published: June 2021

TMEM16A is a calcium activated chloride channel (CaCC). However, its role in colonic Cl secretion is unknown. By qPCR, we identified TMEM16A as the predominant CaCC in mouse colon and hypothesized that it was involved in basal and Ca2+‐ stimulated colonic Cl secretion. Because the NHERF family of PDZ domain containing proteins has been shown to regulate intestinal ion transporters, we tested whether NHERF1 and NHERF2 are involved in TMEM16A regulation. Colonic Cl secretion was measured as the change in short circuit current (ΔIsc) by Ussing chamber. Digallic acid, a CaCC inhibitor inhibited basal Isc by 50% (p<0.05) and completely abolished carbachol (CCH) stimulated Isc in the wild type (WT) mouse colon. In TMEM16A+/− colon, basal, CCH and forskolin (FSK) stimulated Isc were reduced by 47% (p<0.04), 67% (p<0.001) and 75% (p<0.001), respectively. In NHERF1−/− mice, basal and CCH stimulated Isc were reduced by 60% (P<0.01) and 92% (P<0.001), while there was no significant change in the FSK stimulated Isc. Basal, CCH and FSK stimulated Isc in NHERF2−/− colonic tissues were not different from WT. Our results suggest that in mouse colon (1) TMEM16A is a major contributor of basal, CCH and unexpectedly also cAMP stimulated Cl secretion; (2) NHERF1, but not NHERF2 plays an important role in basal and CCH stimulated Cl secretion. In conclusion, NHERF1 affects colonic Cl secretion via effects on TMEM16A.
3H‐D‐glucose transport is both Na+‐ and K+‐dependent in the marine shrimp, Litopenaeus setiferus
Authors: Ahearn Gregory A. , Obi Ijeoma , Sterling Kennth M.
Published: June 2021

This study reports the occurrence of a brush border 3H‐D‐glucose transporter in the digestive tract of a marine shrimp that is similarly stimulated by both Na+ and K+. Hepatopancreatic brush border membrane vesicles loaded with 300 mM mannitol exhibited 0.1 mM 3H‐glucose uptake overshoots, that were membrane‐potential‐ sensitive, when incubated in media containing either 150 mM NaCl or KCl. Sugar influxes (15 sec uptakes) in both NaCl or KCl were significantly (p < 0.05) inhibited by 5 mM phloridzin. 3H‐glucose (0.1 mM) influxes in media containing concentrations of NaCl or KCl from 0 to 400 mM were hyperbolic functions of the salt concentrations with both Km‐ and Jmax –values generally higher for KCl than for NaCl. Significant increases (p < 0.05) in Km‐ and Jmax‐values occurred in both media in the presence of a membrane potential difference (inside negative). 3H‐glucose influx was a hyperbolic function of [glucose] from 0.1 to 5.0 mM in the presence of either 150 mM NaCl or KCl. Influx kinetic constants (Km and Jmax) were higher in NaCl than in KCl, and imposed inside‐negative membrane potentials decreased the kinetic constants in both instances. Results suggest that sugar absorption in shrimp may be largely K+‐dependent during juvenile herbivory and Na+‐dependent in adult omnivory/carnivory. Supported by USDA agriculture and food initiative competitive grant no. 2010‐65206‐20617.
Functional and structural regulation of urate handling by human SLC2A9 isoforms in the Xenopus laevis oocyte expression system
Authors: Cheeseman Chris I , Witkowska Kate , Long Toto
Published: June 2021

Human GLUT9 (SLC2A9) is a high capacity urate transporter, backed by genome‐wide association scans. This gene's SNPs coincide with urate pathology1. The protein is predominant in liver and kidney, main organs in human urate homeostasis. Two splice variants of the carrier, SLC2A9a (A) and SLC2A9b (B), which differ in their residue composition in the N‐terminus, target differently to cell membranes of polarized epithelium2. Hydrophobic residues in this family of transporters were shown to play a role in hexose selectivity3.

OBJECTIVES

To investigate differences in urate transport function and selectivity by SLC2A9 variants, for uptake and efflux modes.

METHOD

hSLC2A9 isoforms and appropriate mutants were overexpressed in X. laevis oocytes. Radiolabelled urate uptake and efflux was measured under varied conditions.

PKC agonist affects (A)‐ but not (B)‐mediated urate uptake and efflux. Kinetic experiments reveal that both affinity and capacity of the transporter are altered, without changes in funcional surface expression of the protein. Elevation of intracellular cAMP altered (B)‐ but not (A)‐mediated urate uptake under specific conditions. These observations suggest that urate reabsorption (A) and urate secretion (B) may be differentially regulated in polarized epithelia. Furthermore, hydrophobic residues around exofacial vestibule may act as hexose and urate selectivity determinants.
Generation of Gαi2G184S conditional mutant mice to study regulator of G protein signaling (RGS) proteins
Authors: Parra Sergio , Huang Xinyan , Wade Susan , Kaur Kuljeet , Charberneau Raelene , Neubig Richard R
Published: June 2021

GTPase activity of G inhibitory proteins (Gαi) is strongly accelerated by RGS proteins. A mutant mouse (Gαi2 G184S) in which RGS‐mediated negative regulation is lost shows protection against ex vivo cardiac ischemia‐reperfusion injury, and had worsened outcomes in two in vivo models of heart failure. However, such mice have a complex phenotype and decreased viability. In order to better define the mechanisms mediating these cardiovascular effects, we have developed a novel Gαi2 G184S conditional knock‐in mouse model with a loxP‐flanked minigene encoding wild‐type exons 5–9 in front of the mutant exon 5. We show that floxed‐allele‐ carrying mice appear normal and fertile. Upon crossing the Gαi2 G184S conditional to the Cre‐ERT2 transgenic mice, the wildtype sequence is efficiently deleted by Cre‐mediated excision in vivo after tamoxifen treatment and Gαi2 mRNA contains mostly mutant sequence. Levels of Gαi2protein were somewhat reduced in several tissues, but responses to the stimulation of α2 and muscarinic M2 receptors were similar between Cre‐ERT2/Gαi2 +/+ and Cre‐ERT2/Gαi2 G184Scond/G184Scond mice before tamoxifen treatment. After tamoxifen treatment, Gαi2 mediated responses are enhanced in Cre‐ERT2/Gαi2 G184Scond/G184Scond mice. This mouse model will be a valuable tool to dissect temporal and tissue‐specific roles of GPCRs, Gαi2, and RGS proteins. (Supported by NIH R01 GM039561‐23)
Electrophysiological properties of YM‐244769, a new and selective NCX blocker, in heart
Authors: Watanabe Yasuhide , Kita Satomi , Iwamoto Takahiro
Published: June 2021

A selective Na+/Ca2+ exchange (NCX) inhibitor would be very useful for studying both the physiological roles of NCX and a new drug therapy for cardiovascular diseases. In the present study, we examined electrophysiological properties of YM‐244769, a novel benzyloxyphenyl NCX inhibitor, in mammalian heart. NCX currents (INCX) were measured in single cardiac ventricular myocytes of guinea pig by using the whole‐cell voltage‐clamp technique. YM‐244769 suppressed the bi‐directional INCX in a concentration‐dependent manner (0.01–3uM). The IC50 values of YM‐244769 for the bi‐directional outward and inward INCX were both about 0.1uM. YM‐244769 suppressed the uni‐directional outward INCX (Ca2+ entry mode) with the IC50 value of 0.05 uM. In contrast, 10 uM YM‐244769 inhibited it only by about 50%. At 5 mM intracellular Na+ concentration, YM‐244769 suppressed INCX more potentially than it did at 0 mM [Na+]i. Intracellular application of trypsin via the pipette solution did not change the blocking effect of YM‐244769. On the other hand, YM‐244769 (1–10uM) did not affect the other membrane currents; INa, ICa, IK1, ISS, Ito and IOAG. YM‐244769 (0.3–3uM) suppressed hypoxia/reoxygeneration‐induced Ca2+ overload.

YM‐244769 inhibits the Ca2+ entry mode of NCX more potently than the Ca2+ exit mode in cardiac myocytes. YM‐244769 is a representative NCX inhibitor against the Ca2+ entry mode.
RhoA, phospholipase Cε and PKD signaling mediate S1P induced cardioprotection against ischemia/reperfusion
Authors: Xiang Sunny Yang , Ouyang Kunfu , Chen Ju , Smrcka Alan V. , Brown Joan Heller
Published: June 2021

The lysophospholipid sphingosine‐1‐phosphate (S1P) has established roles in protecting the heart against ischemia/reperfusion (I/R) injury. The mechanism by which this occurs is not fully understood. We previously reported that S1P activates the small GTPase RhoA, and more recently demonstrated that RhoA activation in adult mouse hearts protects against I/R injury. Here we tested the hypothesis that RhoA signaling mediates S1P induced cardioprotection. In cultured neonatal rat cardiomyocytes, S1P induces a 4–5‐fold increase in active RhoA. Inhibiting RhoA with C3 significantly attenuates PI hydrolysis induced by S1P and, using RNA interference, we demonstrate that phospholipase C epsilon (PLCε) is the S1P/RhoA target. To determine whether PLCε mediates S1P induced cardioprotection, PLCε knockout (KO) mouse hearts were isolated and challenged by global I/R. There is no difference in infarction in PLCε KOs versus wild‐type (WT) controls. However, S1P pretreatment reduces infarct size by 50% in WTs but has no effect on PLCε KOs, demonstrating that PLCε is critical for S1P protection. We also find that protein kinase D (PKD) is activated by S1P through RhoA and PLCε signaling and that PKD1 gene deletion completely prevents S1P induced cardioprotection against I/R. Together, we revealed a novel S1P signaling pathway mediated through RhoA, PLCε and PKD which protects the heart against I/R damage. (AHA#11POST7580130, HL28143)
An apoptosis signal‐regulating kinase 1 inhibitor reduces cardiomyocyte apoptosis and infarct size in a rat ischemia‐reperfusion model
Authors: Gerczuk Paul Z. , Breckenridge David G. , Liles John T. , Shryock John C. , Belardinelli Luiz , Kloner Robert A. , Dai Wangde
Published: June 2021

Apoptosis Signal‐Regulating Kinase 1 (ASK1) is a critical mediator of oxidative stress‐induced apoptosis. We hypothesized that a small molecule inhibitor of ASK1 (ASK1‐i; Gilead Sciences, Palo Alto, CA) would reduce myocardial infarct size in a rat ischemia/reperfusion model. Forty‐seven SD rats were randomized to 3 groups: ASK1‐i infusion (n=16), vehicle infusion (n=16), or ischemic preconditioning (IPC; n=15). Infusion of ASK1‐i (10 mg/kg) or vehicle commenced 45 minutes prior to myocardial ischemia. IPC consisted of 3 cycles of 3 minutes coronary occlusion and 5 minutes reperfusion immediately prior to index myocardial ischemia. Rats were then subjected to 30 minutes of coronary occlusion followed by 180 minutes of reperfusion. Pathologic analysis revealed no significant difference in the size of the ischemic risk zone between the 3 groups. Infarct size, expressed as a percentage of the risk zone averaged 27.7 +/− 3.3%, 16.5 +/− 3.4%, and 41.5 +/− 4.8% in the ASK1‐i group, the IPC group, and the vehicle group, respectively (p < 0.05 for ASK1‐i and IPC versus vehicle). The percentage of apoptotic nuclei as assessed by TUNEL positivity averaged 12.4 +/− 1.1%, 11.1 +/− 1.9%, and 19.2 +/− 2.2% in the ASK1‐i group, IPC group, and vehicle group respectively (p < 0.05 for ASK1‐i and IPC versus vehicle). Inhibition of the ASK1 was shown to reduce apoptosis and myocardial infarct size in a rat model of ischemia/reperfusion.
Angiopoietin‐1 reduces myocardial apoptosis and promotes cardiac repair via recruitment of hematopoietic progenitor cells in diabetic db/db mice
Authors: Chen Jian-Xiong , Zeng Heng
Published: June 2021

Hematopoietic progenitor CD133+ cells have been shown to be involved in myocardial healing following myocardial infarction (MI). Previously we demonstrated that angiopoietin‐1(Ang‐1) is beneficial in the repair of diabetic infarcted hearts. We now investigate whether Ang‐1 affects CD133+ cell recruitment to the infarcted myocardium thereby mediating cardiac repair in type II (db/db) diabetic mice. db/db mice were administered either adenovirus Ang‐1 (Ad‐Ang‐1) or Ad‐â‐gal systemically followed by ligation of the left anterior descending coronary artery (LAD). Overexpression of Ang‐1 resulted in a significant increase in CXCR‐4/SDF‐1 expression and promoted CD133+/CXCR‐4+ and CD133+/SDF‐1+ cell recruitment into ischemic hearts. Overexpression of Ang‐1 led to significant increases in number of BM‐derived CD31+ and smooth muscle‐like cells and VEGF expression in bone marrow (BM). This was accompanied by significant decreases in cardiac apoptosis, MI size and fibrosis and an increase in myocardial capillary density. Ang‐1 also upregulated Jagged‐1 and Notch3 expression followed by increases in VSMC recruitment and newly formed arterioles in the infarcted myocardium. Our data strongly suggest that Ang‐1 attenuates cardiac apoptosis and promotes cardiac repair by promoting recruitment of hematopoietic progenitor CD133+ cells and angiogenesis via upregulation of CXCR‐4/SDF‐1.
Physalin promotes positive cardiac inotropism by a PKARYR2‐ dependent pathway
Authors: Gomes Victor Martins , Pessoa Otília Deusdênia Loiola , Fonteles Manassés Claudino , Santos Cláudia Ferreira , Sudo Roberto Takashi , Nascimento Nilberto Robson Falcão
Published: June 2021

Long‐term positive cardiac inotropic therapy that increases both life quality and expectancy is still lacking. A phytosteroid isolated from Physalis angulata (Solonacea), and denominated F9, was probed in guinea‐pig cardiac tissues in vitro. Concentration‐response curves (0.2 to 200 μM) were performed in both spontaneous beating right atria and eletrically driven left atria. The effects of F9 in the atria were evaluated either in the absence or presence of propranolol (1 μM), verapamil (0.1 μM), staurosporine (0.1μM ) and H89 (10μM). Saponin‐skinned fibers were studied in order to further study its mechanism of action. F9 increased left atria tension to 377.57±24.73 % without any remarkable arrhythmogenic, chronotropic or tonotropic effect. This positive inotropic effect was not affected by pretreating tissues with propranolol, staurosporine or verapamil. Nevertheless, this effect was blunted by 91.8% by H89 (n=5) a PKA inhibitor. F9 (10μM) evoked a contraction of trabecular skinned fiber that attained 53% of the maximal contraction achieved with 0.25 μM Ca2+. This compound did neither affect calcium loading nor shifted the concentration‐response curve to pCa (7.0– 4.8). The increase in tension promoted by F9 in skinned fibers was not blocked by xestopongin C (3μM) or herbymicin A (1μM) but was blunted by either ruthenium red (30μM) or genistein (100μM). F9 does neither affect NKA enzymatic activity nor cAMP levels. The positive cardiac inotropic effect of F9 seems to be dependent of activation of PKA and RYR2 phosphorylation. This hypothesis is being currently tested.
Neuropeptide Y antagonist improves bone density in ovariectomized female rats
Authors: Tucci Michelle A , Benghuzzi Hamed A , McGuire Robert
Published: June 2021

Neuropeptide Y plays a key role in the physiological control of energy homeostasis. The role of neuropeptide Y in ovariectomized animals has not been fully established. Multiple neuropeptide Y receptor subtypes are thought to mediate neuropeptide Y activity in both energy regulation and bone metabolism. We examined the role of neuropeptide Y(1) on bone density and strength in adult ovariectomized female rats compared with estrogen administration. A sustained‐dose of a neuropeptide Y(1) receptor antagonist (0.1 μg/day) reduced body weight similar to animals receiving sustained physiologic estrogen levels. In addition to body weight reductions, bone densities were increased and bone strength was improved over ovariectomized animals that were untreated. The histological and biomechanical evaluations showed Y(1) receptor antagonist had similar improvements compared with estrogen treated animals. These findings suggest neuropeptide Y(1) may play a role at the central nervous system and ultimately regulate energy homeostasis and bone metabolism.
Targeting Potassium Channels On Fibroblast‐like Synoviocytes For The Treatment Of Pristane Induced Arthritis In A Rat Model
Authors: Hu Xueyou , Tajhya Rajeev , Laragione Teresin , Huq Redwan , Koshy Shyny , Khan Fatima , Gulko Percio , Beeton Christine
Published: June 2021

Targeting Potassium Channels On Fibroblast‐like Synoviocytes For The Treatment Of Pristane Induced Arthritis In A Rat Model Rheumatoid arthritis (RA) is a chronic disease that leads to inflammation of the joints and other organs. Fibroblast‐like synoviocytes (FLS) play an important role in the pathogenesis of RA. We have previously identified and defined the roles of the major functional K+ channels KCa1.1 channels (BK, Maxi‐K, slo1) expressed at the plasma membrane of FLS isolated from patients with RA (RA‐FLS). In the present study, we used RT‐PCR, western blotting, immunocytochemistry, and patch‐clamp electrophysiology to identify KCa1.1 channels as the major functional K+ channels expressed by FLS isolated from rats with pristane‐induced arthritis (PIA), an animal model for RA. Blocking KCa1.1 channels inhibited the proliferation of these cells and their ability to produce MMP‐2 without inducing cytotoxicity. In keeping with these results, blocking KCa1.1 channels also inhibited the invasion of RA‐FLS in Matrigel assays. Furthermore, a KCa1.1 blocker, injected every other day after onset of PIA clinical signs, stopped disease progression. Our findings demonstrated that KCa1.1 channels on fibroblast‐like synoviocytes are promising therapeutic targets for the treatment of RA.

This study is supported by funding from Baylor College of Medicine and the NIH (AI084981).
Effects of maternal milk lactoferrin supplementation on neurodevelopment and neuroprotection
Authors: Wang Bing , Larvaron Pierre , Somm Emmanuel , Sizonenko Stéphane
Published: June 2021

Background

Lactoferrin (Lf), an iron‐binding glycoprotein containing terminal sialic acid residues, is secreted in milk. Lf is synthesized in brains of both humans suffering from Alzheimer's dementia, and in the mouse model of Parkinson's disease, suggesting that Lf may play a role in neurodegenerative diseases.

Objective

To investigate the neuroprotective effects of Lf in rats following prenatal exposure to dexamethasone (DEX) by 1H‐MRS and hippocampal gene expression profiling.

Method

Intrauterine growth restriction (IUGR) in rat pups was induced by maternal exposure to DEX supplemented with Lf (1g/kg b.w./day) during gestation and lactation. Neurochemical profiles of hippocampus (HC) and cortex (C) were determined in vivo by 1H‐MRS at 9.4 T, and selected gene expression in hippocampus at postnatal day 7.

Results

DEX in the rat model showed a significant decrease in the concentration of NAA‐NAAG and Glu‐Gln, markers of neuronal integrity and function, in the HC and C. This confirmed the altered brain development after DEX exposure and revealed the potential protective effect of Lf given to gestational and lactating dams in the rat model of prenatal exposure to adverse conditions. DEX‐induced transcriptional changes in BDNF and DMT1, normalized with maternal bLf supplementation.

Conclusion

: Maternal milk Lf supplementation has a protective effect for neurodevelopment delayed IUGR in the rat model.
Wnt Signaling Mediates De‐differentiation of Endothelial Cells during Neovascularization
Authors: Kohler Erin , Baruah Jugajyoti , Azar Dimitry T , Chang Robert , Malik Asrar B , Wary Kishore K
Published: June 2021

RATIONALE

The ability to induce de‐differentiation of resident cells in situ at the site of injury or ischemic tissues may be a useful approach for reparative and regenerative medicine. To address this possibility, here we test the capacity of 6‐bromoindirubin‐3′‐oxime (BIO) to induce neovascularization by inducing a transcriptional program centering the NANOG gene networks.

RESULTS

BIO not only induced interaction of β‐catenin with NANOG, but also increased the expression of mesenchymal and hemangioblastic cell markers NANOG, VEGFR2, BRACHYURY, and CD133, while decreasing mature EC markers CD31 (PECAM‐1) and von Willibrand Factor (vWF). Increased expression of these molecules induced mesenchymal/stem‐like cellular phenotypes and increased neovascularization in Matrigel and in Corneal neovascularization assays. Furthermore, BIO induced asymmetric cell division in these cells, a hallmark of stem cell selfrenewal. In contrast, NANOG knockdown inhibited the ability of BIO treated ECs to divide asymmetrically, to form cellular aggregates, and to undergo angiogenesis.

CONCLUSION

These findings, for the first time, demonstrate the ability of BIO to induce dedifferentiation of ECs into primitive cells in relation to neovascularization by up‐regulating the transcriptional network centering the NANOG gene.

Supported by NIH (R01HL079356), AHA, and T32HL072742 NIH training grants.
Taste and smell receptors depend upon growth factor secretion to maintain normal taste and smell function
Authors: Henkin Robert I
Published: June 2021

Taste and smell receptors are unique; they do not exhibit mitosis and have no endogenous blood supply or lymphatics. Receptors in taste buds depend upon growth factors secreted in saliva to maintain normal taste. Any process inhibiting secretion, caused by many diseases, inhibits receptor activation causing taste dysfunction. Receptors in olfactory epithelium depend upon growth factors secreted into nasal mucus to maintain normal smell. Any process inhibiting secretion, caused by many diseases, inhibits receptor activation causing smell dysfunction. Taste and smell are chemical senses. They depend upon salivary and nasal mucus growth factors to maintain normal taste and smell function. Many chemical moieties secreted from multiple organ systems (e.g., endocrine, metabolic, hematological), act as these growth factors comprising hormones (e.g., thyroid), vitamins (e.g., B12, A), trace metals (e.g., copper, zinc), adenylyl cyclases (e.g., cAMP, cGMP) and other moieties. These growth factors act on stem cells in taste buds and olfactory epithelium to induce receptor maturation. Decreased secretion of these growth factors cause receptor loss through apoptosis with subsequent taste and smell dysfunction. Treatment of these pathological conditions restores normal growth factor secretion initiating receptor regeneration, inhibiting apoptosis and restoring normal taste and smell function.
Site‐specific arginine dicarbonyl modifications of serum albumin are reduced in metformin‐controlled T2DM patients
Authors: Kinsky Owen Robert , Kimzey Michael J , Allred Serena F , Yassine Hussein N , Tsaprailis George , Stump Craig , Billheimer Dean , Monks Terrence J , Lau Serrine S
Published: June 2021

Advanced glycation end products (AGE) are a possible link between hyperglycemia and the development of diabetic complications. Careful control of AGE could be vital to improve diabetic complications. Biomarkers to evaluate control of AGE are needed to guide development of emerging therapies. Using an unbiased proteomics approach, dicarbonyl (methylglyoxal, glucosone, and 3‐deoxyglucosone) on arginine (R) and oxidative markers on methionine residues on serum albumin were studied in 120 subjects with varying degrees of type‐2 diabetes mellitus (T2DM) with or without metformin treatment. Trypsin digested delipidated serum samples were analyzed by multiple reaction monitoring on a QTRAP mass spectrometer, based on transitions that were validated in vitro. Strong signals for dicarbonyl modification were observed at R186, R257, and R428. Statistical analyses of the dicarbonyl‐R in relation to known T2DM clinical markers including HbA1c, fasting glucose, and waist circumference were performed between groups. Dicarbonyl markers were significantly higher in T2DM (n=47) compared to non‐T2DM (n=46), and certain markers were significantly decreased in T2DM/metformin compared to T2DM (n=26; p<0.001 [post hoc]). The study reveals that dicarbonyl adducts may be a useful biomarker for monitoring the progression of T2DM and metformin treatment efficacy. (P30ES006694, R24DK083948, T32ES016652, T32ES007091).
Interrogating Therapeutic Manipulation of the Endocannabinoid System in Human Colon
Authors: Vase Hollie Francesca , Drew Janice Elizabeth , Ross Ruth , Carey Frank , Steele Robert , Bunton David , Farquharson Andrew
Published: June 2021

The endocannabinoid system (ECS) plays a role in colon inflammation and cancer. Human colon explants and a novel gene expression profiling technology, the GenomeLab System (Beckman) have been developed to investigate ECS regulation in human colon. A custom designed GeXP (Beckman) multiplex gene expression assay, the hECSplex, was developed. hECSplex gene expression signatures of EC receptors (CNR1, CNR2, GPR55 and TRPV1), ECS enzymes (NAPE‐PLD, GDE1, DAGLA, DAGLB, FAAH, FAAH2 and PTGS2), inflammatory (IL1B, IL10, IL6, LEP, TNF and SOCS3), signalling pathway (ID1, BCL2, CFL1, BIRC5, TP53, MYC and KRAS), lipid production (SREBF1, ACACA), and plasma‐membrane (OCLN) markers were assayed using 50ng of total RNA from human colon explants treated with either cannabidiol (CBD) or tetrahydrocannabinol (THC). Preliminary results indicate that both therapeutics regulate EC receptor expression, but notably regulate diverse expression of ECS enzymes, signalling targets and inflammatory markers. The GenomeLab System presents new opportunities to interrogate multiple components of the ECS in small colon explant tissue samples in response to novel therapeutics.
Investigation of hypoglycemic activity of four extracts of Petiveria alliacea (Guinea hen weed) in normoglycemic experimental rat models
Authors: Christie Sara-Lou Fredricka , A Levy Arkene S.
Published: June 2021

Petiveria alliacea has ethno‐traditional use as a hypoglycemic in Jamaica. These anecdotal claims however, are yet to be scientifically validated. In this study, we evaluated the hypoglycemic activity of four extracts from P. alliacea in normoglycemic experimental rat models. Crude extracts were prepared from the aerial parts of P. alliacea and single oral doses (200 and 400 mg/kg body weight) were assessed for hypoglycemic activity in overnight fasted normoglycemic rats and glucose loaded normoglycemic rats. Blood glucose level was estimated at 30 minute intervals for 3 h using Accu‐chek Active glucometer and test strips. The aqueous, ethanol, ethyl acetate and hexane extracts produced no significant decline in fasting blood glucose (FBG). No significant improvement in glucose tolerance was observed following treatment with the extracts. However, the aqueous extract at 400 mg/kg increased FBG from 4.75±0.69 mmol/L to 5.88±1.14 after 3 h when compared to the vehicle treated group (P=<0.00001). Glybenclamide decreased FBG from 4.25±0.24 to 2.52±0.46 (P=0.0007) and improved glucose tolerance (P=<0.001). These results indicate that Petiveria alliacea demonstrates no significant hypoglycemic activity in normoglycemic rats. By contrast, the aqueous extract at high doses appears to possess a hyperglycemic effect. This will be evaluated in further studies. Funding: UWI, Office of Graduate Studies & Research
Flavonoids and Fibrate modulate APOE4‐induced processing of Amyloid precursor protein in rat neuronal cells
Authors: Davra Viralkumar R. , Benzeroual Kenza E.
Published: June 2021

Several studies reported a role of Apolipoprotein E4 (ApoE4) in processing Amyloid precursor protein (APP) to Amyloid β (Aβ) peptide, thus playing a major role in Alzheimer's disease. This study, investigated the effect of flavonoids and fenofibrate, a hypolipidemic agent, on ApoE4‐induced APP processing in B103 cells stably transfected with human wild‐type APP695. Cells were pretreated with varying doses of flavonoids and fenofibrate for 1 hour, prior to ApoE4 exposure for 24 hours. Results showed that pretreatment with fisetin and diosmetin significantly reduced, whereas naringenin and fenofibrate had no effect on APOE4‐increased expression of APP. In addition, the expression of carboxy terminal fragments‐α and ‐β generated by the α‐ or β‐secretase cleavage of APP increased significantly in cells pretreated with fisetin as compared to disometin, naringenin, and fenofibrate. Conditioned medium collected from treated B103‐APP cells were used to treat Rat pheochoromocytoma (PC12) cells and cell viability assay showed more pronounced effect of flavonoids on reversing ApoE4 induced cell death as compared to fenofibrate. β‐secretase activity showed that flavonoids may not have a direct effect on the enzyme but may increase APP processing and prevent cell death, most likely through a decrease in Aβ production as reported by the conditioned media experiment. This work was supported by Long Island University, NY.
Mechanical Circulatory Unloading Promotes Proteins Synthesis and Maintains Leucine Oxidation
Authors: Kajimoto Masaki , O'Kelly Priddy Colleen M. , Ledee Dolena R. , Bouchard Bertrand , Isern Nancy , Olson Aaron K. , Des Rosiers Christine , Portman Michael A.
Published: June 2021

Mechanical ventricular unloading by arterial‐venous extracorporeal membrane oxygenation (ECMO) serves as a bridge to recovery in infants with severe acute heart failure. The influence of mechanical circulatory support on protein synthesis and degradation is unknown. We tested the hypothesis: ECMO induces reductions in myocardial protein synthesis and promotes increased amino acid oxidation. Anesthetized immature swine (n=20) received intracoronary infusion of 13C6, 15N L‐leucine alone or with 2‐13C pyruvate after 8 hours of loading (LOAD) or ECMO. Leucine incorporation into protein and contribution to the citric acid cycle (CAC) relative to pyruvate was determined using 13C‐magnetic resonance spectroscopy and GCMS. ECMO increased the protein fractional synthesis rate (0.91%±0.04% vs. 0.63%±0.10%, p<0.06), but did not alter leucine oxidation relative to pyruvate. ECMO also increased mTOR phosphorylation (by immunoblot) and decreased eukaryotic‐Elongation factor‐2 phosphorylation.

Conclusions

Mechanical unloading in the form of ECMO enhances protein synthesis without reducing amino acid contribution of acetyl‐CoA to the CAC. The protein synthesis occurs with activation of the mTOR pathway. These data suggest that atrophy induced by clinical mechanical circulatory support and ventricular unloading is caused by accelerated protein degradation.
Overexpression of phospholipid hydroperoxide glutathione peroxidase (MPHGPx) attenuates cardiac mitochondrial proteomic loss and reverses protein import detriments observed with type 1 diabetes mellitus
Authors: Baseler Walter A. , Dabkowski Erinne R. , Jagannathan Rajaganapathi , Thapa Dharendra , Nichols Cody E. , Shepherd Danielle L. , Croston Tara L. , Schnell David M. , Hollander John M.
Published: June 2021

Mitochondrial dysfunction is a contributor to diabetic cardiomyopathy. Previously, we observed proteomic decrements within the mitochondrial inner membrane (IMM) and matrix of diabetic cardiac interfibrillar mitochondria (IFM) correlating with dysfunctional mitochondrial import. The goal of this study was to determine whether overexpression of MPHGPx, an antioxidant capable of scavenging membrane‐associated lipid peroxides, could preserve the mitochondrial proteome and import process. MPHGPx transgenic mice and controls were made diabetic by multiple low‐dose streptozotocin. Proteomic analyses revealed MPHGPx overexpression preserved proteins decreased within the diabetic IFM. Ingenuity Pathway Analyses indicated that OXPHOS, TCA, and FAO processes most influenced in diabetic IFM were preserved by MPHGPx overexpression. Further, IMM proteins were the most highly preserved (63%) followed by matrix proteins (34%). Mitochondrial protein import decrements were also preserved in the MPHGPx diabetic IFM (P<0.05) which correlated with increases in protein import constituents. The results indicate overexpression of MPHGPx can protect essential mitochondrial complexes in both IMM and matrix and may provide cardioprotective benefits to the diabetic heart. (Support: NIH DP2DK083095, NIH T32HL090610, AHA 10PRE3420006)
Systemic Leptin Alters Response of Nucleus Tractus Solitarius Neurons That Innervate Rostral Ventrolateral Medulla to Peripheral Chemoreceptors
Authors: Ciriello John , Moreau Jason M. , Messenger Scott A. , Iqbal Waseem , Migchels Megan J.
Published: June 2021

Experiments were done in anesthetized rats to investigate the effects of systemic injection of leptin on the response of neurons within nucleus tractus solitarius (NTS) that innervate the rostral ventrolateral medulla (RVLM) during activation of carotid chemoreceptors. The NTS region containing neurons expressing the leptin receptor Ob‐Rb was explored for single units antidromically activated by stimulation of pressor sites in RVLM. Antidromically activated units were tested for their response to injection of leptin (50 ng/0.1 ml) and KCN (80 μg/0.1 ml). Of 64 units tested in NTS, 28 units were antidromically activated by RVLM and of these units, 17 (~61%) were excited by intracarotid injections of KCN. Additionally, 11 (~65%) of these KCN responsive units were also excited by leptin injections. Furthermore, the response of these single units to KCN was potentiated (83%) immediately following the leptin injection. Control intracarotid injection of saline (vehicle for all injections) neither altered the activity of the NTS neurons nor the response of NTS units to KCN. Taken together, these data suggest that increases in circulatory levels of leptin can alter the sympathoexcitatory responses to peripheral chemoreceptor reflex activation by modulating the activity of neurons within the NTS. Supported by HSFO
Increased fat and simple carbohydrate consumption results in obese prepubertal pigs without an increase in body weight
Authors: Reeves H.F. , van Eyk G.R. , Seelenbinder K.M. , Price K.L. , Scheffler T.L. , Gerrard D.E. , Scheffler J.M. , Escobar J.
Published: June 2021

The objective was to determine how carbohydrate (CHO) source affects body adiposity, intravenous glucose tolerance (IVGT) and insulin levels in female prepubertal pigs. Pigs (28‐d‐old) were used in an 8‐wk trial. Diets were Control or high‐fat (HF), which was further divided into containing complex (Corn) or simple CHO (SC, Glucose or Fructose). Control pigs were pair‐fed to HF pigs resulting in similar weight gain and final body weight across treatments (P=0.81). Compared to Controls, metabolizable energy intakes were higher for HF (37%, P<0.001) and SC (14%, P<0.03) diets. Plasma cholesterol was higher (P<0.02) in pigs fed Corn and Fructose compared to those fed Glucose or Control diets. Ultrasound backfat depth, an indicator of body adiposity, was 84% higher (P<0.001) in HF, and SC tended to increase fat (P=0.11). HF intake increased (P<0.001) fasting blood glucose levels but lowered insulin, HOMA‐B, and HOMA‐IR (P<0.01). SC intake tended to reduce (P=0.08) fasting insulin and lowered HOMA‐B and HOMA‐IR (P<0.05). Glucose area under the curve (AUC) was higher (P<0.05) in HF and SC pigs compared to Control, but insulin AUC was not different (P=0.14) across all treatment combinations. No differences between Glucose and Fructose were found for any measured parameter. Consumption of SC increases body adiposity and alters glucose homeostasis without changes in weight gain.

Grant Funding Source : College of Agriculture and Life Sciences
Dietary fat and sugar induce obesity and impair glucose tolerance in prepubertal pigs
Authors: van Eyk G.R. , Fisher K.D. , Kasten S.C. , Scheffler T.L. , Price K.L. , Reeves H.F. , Gerrard D.E. , Scheffler J.M. , Escobar J.
Published: June 2021

A translational swine model of childhood obesity was used to determine the interaction between dietary energy source, body adiposity, and oral glucose tolerance (OGT). Prepubertal female pigs weaned at 21 d of age were fed control (C), refined sugar (S), fat (F), and sugar‐fat (HE) diets in a 2×2 factorial arrangement for 16 wk. Calories from fat were 8.9% for C, 5.6% for S, 35.5% for F and 32.3% for HE. Calories from sugar were 36.0% for S and 30.7% for HE. Adding fat or sugar increased (P<0.003) calorie intake indicating impaired ability to regulate energy consumption. Daily fat accretion and percent body fat were higher (P<0.03) in all treatments compared to C, and in HE compared to S. Ultrasound backfat depth was associated (P<0.001) positively with percent body fat (r2 = 0.909) and negatively with percent body protein (r2 = −0.912). Fat and sugar intake raised (P<0.02) plasma cholesterol levels. Glucose area under the curve (AUC) in response to OGT at 14 wk was higher (P<0.03) in F (+15%) and HE (+32%) pigs compared to C. Glucose AUC from S‐fed pigs was not different (P=0.2) from C‐ or F‐fed pigs. Adding sugar, fat, or their combination to diets increased (P<0.008) glucose AUC. Inclusion of fat in the diet reduced (P<0.001) insulin AUC during OGT, while sugar had no effect. Inclusion of fat and refined sugar in pig diets increases body adiposity and impairs glucose homeostasis. College of Agriculture and Life Sciences
Flow‐Mediated Dilation is Attenuated in Young Patients with Cystic Fibrosis
Authors: Harris Ryan A , Poore Spencer , Berry Breana , Eidson Dabney , Pollock Jennifer S , McKie Kathleen T
Published: June 2021

Cystic fibrosis (CF) is a fatal genetic disorder that affects the lungs as well as multiple biological systems. This study sought to determine if endothelial function, measured by the flow‐mediated dilation (FMD) test, was lower in patients with CF compared to controls. 15 patients with CF and 15 demographically matched controls participated in this study. Overnight fasting blood samples were taken and FMD and spirometry were performed. No differences in age, height, or BMI were observed between patients with CF and controls (all p>0.05). Although lung function was in the normal range, patients with CF had lower FEV1 when compared to healthy controls (92.8±15 vs. 104.5±11 % predicted). In addition, brachial artery FMD was lower in patients with CF compared to controls (5.0±2.7 vs. 7.6±3.1%; p=0.02) which was accompanied by a slightly higher plasma concentration of 8‐ isoprostane (16±2 vs. 12±1 ng/ml; p=0.20) and NOx (48±5 vs. 35±4 uM; p=0.06) and a lower total antioxidant capacity (1.29±0.11 vs. 1.42±0.08 mM; p=0.38) compared to controls. These data indicate that young patients with cystic fibrosis have impaired endothelial function that precedes the decline in pulmonary function. Systemic oxidative stress and inflammation may be a contributor; however, more subjects are needed to elucidate this mechanism.

Supported in part by the American Heart Association (R.A.H) and Child Health Discovery Institute (R.A.H).
The Influence Of PKCδActivity On RhoA Interaction With Its Effector Proteins
Authors: Chichger Havovi , Duong Huetran , Harrington Elizabeth O
Published: June 2021

The balance of RhoA activation is vital in maintaining endothelial function in part due to its role in cell contraction, a function which regulates vascular permeability. Previous studies have implicated PKCδ activity in the regulation of endothelial barrier function via modulation of RhoA activity. To understand the relationship between PKCδ and RhoA in the pulmonary endothelium, we used co‐precipitation experiments to identify Rho effectors that bind RhoA in the presence and absence of the PKCδ inhibitor, rottlerin.

We noted PKCδ binding with both constitutively inactive, RhoAG17A, as well as the constitutively active RhoAQ63L, forms of RhoA. Chemical inhibition of PKCδ resulted in increased PKCδ binding to RhoAG17A but decreased PKCδ binding to RhoAQ63L. Altered binding of RhoAQ63L and RhoAG17A with p190RhoGAP and p115RhoGEF, respectively, was observed upon PKCδ inhibition. Using electric cell‐substrate impedance sensing (ECIS) system, we noted that similar to PKCδ inhibition, Rho kinase inhibition with Y27632 promoted endothelial barrier dysfunction.

The data demonstrates that PKCδ influences RhoA activity in pulmonary endothelial cells and the decline in PKCδ or Rho kinase signaling correlated with dysfunction of the endothelial barrier. We propose that PKCδ functions as a regulator of vascular permeability and thus presents an interesting target in the regulation of the pulmonary endothelium.
Acute inhibition but not chronic deficiency of G protein‐coupled estrogen receptor reduces bioactivity of nitric oxide
Authors: Meyer Matthias R. , Field Angela S. , Barton Matthias , Prossnitz Eric R.
Published: June 2021

The transmembrane G protein‐coupled estrogen receptor (GPER) has been implicated in rapid vascular signaling. Whether endogenous GPER affects bioactivity of nitric oxide (NO) is unknown. We studied gene expression of endothelial NO synthase (eNOS), basal NO bioactivity, and stimulated NO release in the aorta of GPER+/+ and GPER−/− mice (3 mo) isolated for recording of isometric tension. We also determined acute effects of the GPER‐selective antagonist G15 on NO bioactivity. Vascular eNOS gene expression was analyzed by qPCR. Basal NO bioactivity was calculated as the difference in contraction to phenylephrine (PE, 300 nmol/L) in the absence and presence of the NOS inhibitor L‐NAME (300 μmol/L), and receptor‐mediated NO release to acetylcholine (ACh, 0.1 nmol/L – 0.1 μmol/L) was induced in PE‐precontracted rings. Chronic GPER deficiency had no effect on eNOS gene expression, basal NO bioactivity, or ACh‐stimulated, NO‐mediated relaxation. In contrast, acute inhibition of GPER by G15 reduced basal NO bioactivity by 34% (38.3±5.7% vs. 57.7±5.2%, n=4–6, P<0.05 vs. vehicle) and inhibited relaxation to ACh‐stimulated NO release by 18% (−60.7±3.5% vs. −74.4±2.5%; AUC 90±11 vs. 133±9 units; n=5–6, P<0.05 vs. vehicle) in GPER+/+ mice. These data demonstrate that acute inhibition of GPER reduces basal NO bioactivity and inhibits stimulated NO release. In contrast, the NO pathway appears to be unaffected by chronic GPER deficiency.

Supported by Swiss National Science Foundation (SNSF) grant PBZHP3‐135874.
Determination of actively circulating volume (ACV) by injection of normal saline
Authors: Nunez Fiesky A. , Kislukin V. , Thurmalla N. , Krivitski N. , Smith T. L. , Callahan M. F.
Published: June 2021

Examination of the indicator concentration curves after rapid injection of normal saline reveals the existence of transient damped oscillations followed by a new, relatively stable level of arterial blood density. We hypothesized that the variation in density could be used to estimate ACV. To address this issue we analyzed the variation of arterial hematocrit (Ht1) during the injection/infusion of 0.9% NaCl; and performed experiments to answer the question: what is the rate of the restoration of blood density, in other words, what is the rate of leakage of injected normal saline from active circulation if (a) the blood volume is elevated up to 50% of expected and (b) if blood is withdrawn up to 30% of expected BV.

Male SD rats (n=15) were anesthetized and received an extracorporeal A‐V loop connecting the carotid artery and the jugular vein with a controlled flow rate (8ml/min). Transit time clamp‐on sensors, TS450 flowmeter and an automatic algorithm (Transonic Systems Inc.) were used to calculate the cardiac output and central and actively circulating blood volume following injection of isotonic saline (0.3ml) into the venous limb of the AV loop. Hemorrhage was done by controlled blood withdrawals(~30 ml/kg in 3–4 steps). Increase of ACV was performed by series of 15–20 injections of 0.5–1 ml of normal saline.

Results

(1) Analysis of Ht1 variations reveals if Htb/Ht1 is constant then 0.9% NaCl is an indicator for ACV determination; (2) The rate of leakage of injected normal saline from active circulation is ~ 1–3%/min in normal or hypovolemic states and is up to 10%/min if ACV is elevated up to 30% of expected baseline.
Angiotensin 1–7 Contributes to Disrupted Nitric Oxide control of Vasopressin Response to Hemorrhage during Acute Alcohol Intoxication
Authors: Whitaker Annie M. , Molina Patricia E.
Published: June 2021

Acute alcohol intoxication (AAI) impairs hemodynamic counterregulation and blunts the arginine vasopressin (AVP) response to hemorrhagic shock (HS). We have demonstrated that AAI disrupts signaling mechanisms controlling AVP release through upregulation of paraventricular (PVN) nitric oxide (NO) and attenuated angiotensin (ANG) II response to HS. We hypothesized that AAI may also enhance ANG Converting Enzyme (ACE) 2 activity promoting the conversion of ANG II to the NO‐stimulating peptide ANG (1–7) further contributing to AAI‐induced suppression of AVP release. Male Sprague‐Dawley rats (250–300g) received a primed (2.5g/kg + 0.3g/kg/h) 15h intragastric infusion of alcohol or dextrose (DEX) prior to fixed‐pressure (40 mmHg) HS. AAI increased PVN ACE2 activity (41%) compared to DEX+HS. The contribution of ANG (1–7) to the AAI‐induced increase in PVN NO, was demonstrated by central administration of the Mas receptor antagonist A‐779 prior to HS. AAI increased NOS activity (30%) and NO levels (40%) in the PVN compared to DEX controls. Administration of A‐779 decreased NOS activity (66%), NO levels (27%; p=NS) and partially restored AVP levels (20%) in AAI‐treated animals. These results suggest that increased hypothalamic Mas receptor activation partially contributes to the alcohol‐induced disruption of NO control of AVP release during HS. DOD PR‐054196, NIAAAAA7577, APS Porter Fellowship.