bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum mineral levels according to iron nutrition state and physical activity in young women
Authors: Lee Okhee , Moon Jongwha , Chung Yongsam
Published: June 2021

The iron nutrition status influences other micro‐mineral status through interacting in the intestinal absorption or in the uptake into tissue. The iron‐deficiencies of women are usually not a kind of an anemia, but a state of only iron storage depletion without anemia. Extracellular mineral concentrations can be changed by exercise through water and electrolyte loss of sweat. We compared the minerals levels in serum according to body iron stores and physical activity in young women. We collected bloods of young collegiate sedentary women and physically active athletic women, and serum concentrations of Na, Cl, Ca, Fe, Zn, Se were measured by neutron activation analysis method. The proportion of females with iron depletion(ID, serum ferritin <20μg/L) were 50% in sedentary and athletic women, respectively. The subjects were divided into 4 groups according to physical activity and iron storage. The serum Na, Cl and Ca level showed increasing tendencies in sedentary with normal iron status compared to the athletes with ID, although no significant differences among 4 groups. The serum Fe levels did not show significant differences among 4 groups, although lower values in the athletes than sedentary women. The serum Zn and Se concentrations were the highest in athletes with normal iron status and the lowest in sedentary with ID. In conclusion, this study suggests that the serum Se and Zn concentrations in young women might be influenced by the chronic physical activity and iron status. This study was supported by the MOST of Korea
Effect of Undaria pinnatifida extract on glucose tolerance and insulin secretion in the neonatal Streptozotocin‐induced diabetic rats fed a high‐fat diet
Authors: Choi Hyunju , Nam Jeongsu , Lee Wonjoon , Yoon Imsil , Kim Youngsik , Lee Dongseok , Kim Yongho
Published: June 2021

The purpose of this study was to determine anti‐diabetic activities of Undaria pinnatifida extract. Neonatal diabetes was induced by i.p. injection of Streptozotocin (80mg/kg body weight) to the male Sprague‐Dawley rats at the age of 1 day. When the rats were grown to the adult (about 300g body weights), a high‐fat diet (40 % fat by kcal) was fed for two weeks. Then, rats were divided into two diet groups; one group fed a high‐fat diet with 5% of Undaria pinnatifida extract (UPEX) for the next four weeks, and the other group fed only a high‐fat diet as a control. The glucose tolerance was evaluated using an oral glucose tolerance test, and the glucose curves were compared among the diabetic rats which have similar fasting blood glucose levels. Insulin secretion was also measured using an in vitro pancreas perfusion. In results, rats in the UPEX group showed the lower glucose curves compared to the rats in the control group. Moreover, the amount of insulin secreted from the pancreas stimulated with glucose (200mg/dL) was greater in the UPEX group than those in the control group (P<0.05). These results are suggesting that Undaria pinnatifida extract has a beneficial effect on the glucose control and insulin secretion. (This work was supported by Korea Research Foundation Grant KRF‐F00014, Brain Korea 21, NURI, and FIRST of the Inje Research and Scholarship Foundation.)
Dietary fructose during pregnancy and lactation causes enlarged livers in rat dams and impairs growth of offspring
Authors: Zou Mi , Koser Stephanie L. , Teegarden Dorothy , Donkin Shawn S.
Published: June 2021

Maternal malnutrition is a predisposing factor for development of metabolic syndrome. Dietary fructose is linked to several metabolic disorders. We hypothesized that maternal fructose consumption would impact fetal growth and postweaning fructose intake would magnify this effect. Pregnant Sprague Dawley rats (n=27) were fed for ad‐libitum intake (AD), 30% of ad libitum intake (UN), or fed a diet containing 63% fructose (FR). Diets for AD and UN groups contained 60% carbohydrate from sucrose, corn starch and maltodextrin. Pups from UN dams weighed less (P < 0.05) at birth than AD or FR offspring (3.87 vs. 6.55 ± 0.25 g). Pups (n=107) were cross‐fostered onto dams across maternal diet groups. During lactation dams were retained on their respective gestation diets but were fed ad libitum. Pups nursed by FR dams gained 12.4% less than pups nursed by AD fed dams. At weaning male pups were fed either control diet or a diet containing 63% fructose. Liver for FR dams were 207 % of AD or UN dams (27.5 vs 13.3 g; P < 0.05). UN pups weighed less (P < 0.05) than AD or FR pups at harvest (129 d )(365.1 vs. 424.8 or 437.1 ± 8.5 g) but had greater (P < 0.05) proportion of epidydimal tissue (1.15% vs. 0.95%). In utero and suckling nutrition alter body weight, adiposity and liver mass in offspring. Fructose enlarges liver in pregnant and lactating dams. Effects of postweaning fructose are independent of maternal and suckling nutrition. NIH DK077581.
Oat reduces serum total cholesterol in healthy adult dogs
Authors: Yu Shiguang
Published: June 2021

Hypercholesterolemia occurs in various diseases in dogs, such as chronic kidney disease, hypothyroidism, diabetes mellitus, cholestasis, hyperadrenocorticoidism, idiopathic hypercholesterolemia, and lipoproteinlipase deficiency. The objective of this study is to investigate whether dietary oat reduces serum cholesterol in dogs. Forty‐eight healthy adult beagles were fed a basal dry dog food for 4 weeks. The basal food is nutritionally complete and balanced for an adult dog. Afterwards, dogs were randomly divided into 4 groups (week 0 of the study), 12 dogs per group. Each group was given either the basal food (control), basal food with 5%, 10%, or 20% of oat groat for additional 8 weeks. Oat groat was added to the basal food at the expense of rice and corn so that proximate analysis was similar among the foods. Serum total cholesterol was measured in weeks 0 and 8 of the study. Comparing to week 0, serum total cholesterol change was 1%, 2.1%, −5.6%, and −14.7% after dogs were fed the food containing 0%, 5%, 10%, or 20% of oat groat for 8 weeks, respectively. There was a significant difference in serum total cholesterol change between the control food and the food containing 20% oat groat (p < 0.05). Food intake was similar among dietary groups. Body weight was not affected by dietary oat groat during the study. The results of this study show that dietary oat at 20% may be beneficial to dogs with hypercholesterolemia.
Effect of total dietary fiber on satiety in cats.
Authors: Yamka Ryan Michael , Friesen Kim
Published: June 2021

The objective of this study was to investigate the effects of feeding two foods with elevated levels of total dietary fiber on satiety in cats. Both foods were formulated to contain similar levels of total dietary fiber (TDF; > 11.5% dry matter basis). The foods were fed to two groups of 10 cats (average age = 5.48 ± 1.54 years; average weight = 3.21 ± 0.53 kg) for a period of 16 weeks in order to determine the effect of total dietary fiber on satiety. During the study cats were given ad libitum access to the foods throughout the day. Food intake was recorded daily and body weight was recorded weekly to determine the effects of fiber on satiety and weight maintenance. During the 16 week study, body weight and intake did not change for both treatments. There were no differences between the two treatments for intake or body weight at week 1 or 16 indicating that the level of TDF in both foods was efficacious in promoting satiety in cats. The results of this study demonstrate that satiety and body weight can be controlled in cats fed ad libitum with foods containing > 11.5% total dietary fiber.
Potency Adjusted Generic Versions of Argatroban can be Differentiated from Branded Argatroban in Thrombin Generation and Platelet Activation Assays.
Authors: Adiguzel Cafer , Iqbal Omer , Sammikannu Michael , Cunanan Josephine , Hoppensteadt Debra , Fareed Jawed
Published: June 2021

Argatroban (ARG) is used to anticoagulate patients with heparin‐induced thrombocytopenia. Generic ARGs are available in Japan. Despite similar antithrombin potencies, pharmacodynamic differences have been noted. To study their bioequivalence, generic ARGs were supplemented in whole blood, platelet rich plasma, platelet poor plasma and biochemical systems. Anticoagulant activity was measured using activated clotting time (ACT), PT, APTT, Heptest, prothrombinase activated clotting time (PICT) and thrombin time assays. The effect on tissue factor mediated platelet activation was studied using flow cytometry. Thrombin generation markers were measured. Anticoagulant effects of branded and generic ARGs were the same. Upon addition of tissue factor to the ACT assay, relative anticoagulant effects of these agents differed. All ARGs blocked microparticle generation and P‐selectin expression, though with varying potencies. Differences in thrombin and Xa generation assays and in the ability to inhibit thrombin activatable fibrinolysis inhibitor (TAFI) activation were noted. Despite similar effects in antithrombin titration and global anticoagulant assays, differences between the generic and branded ARG were noted in other assays. Such differences, which may be due to the solution matrix or the relative proportion of different ARG isomers, suggest the need for additional pharmacoequivalent studies.
Neutralization of the Anticoagulant and Anti‐Xa Effects of Fondaparinux and Idraparinux by a Novel Synthetic Antagonist. Pharmacologic Implications
Authors: Fareed Jawed , Jeske Walter , Cunanan Josephine , Adiguzel Cafer , Iqbal Omer , Hoppensteadt Debra
Published: June 2021

Synthetic analogues of the oligosaccharide sequence mimicking the antithrombin binding sites of heparin (UFH) have been developed as antithrombotic agents. Arixtra (Fondaparinux) (F) is currently approved for the management of post surgical DVT and ACS, while its methylated derivative, namely Idraparinux (I) is in clinical development for different clinical indications. Despite their clinical efficacy, bleeding complications have been reported with the use of these agents. Currently, there is no pharmacologic antagonist available to neutralize these agents. Recently, a series of synthetic salicylamide derived antagonists for (UFH) and low molecular weight heparins are developed (Polymedix, Radnor, PA). To further investigate the relative neutralization of F and I, each of these agents were added to citrated whole blood and citrated plasma at a concentration range of 0–10 ug/ml. PS and PMX 60056 were added at fixed concentrations of 6.2, 12.5 and 25 ug/ml. Heptest was used to measure the anticoagulant effects of these agents. In addition, FPA and thrombin generation was also measured. PMX 60056 effectively neutralized the heptest prolongation by both the F and I. However, PS did not produce any antagonism of the anticoagulant effects of these two agents. In the anti‐Xa assay as measured by using the amidolytic assays, neither the PMX 60056 nor the PS produced any neutralization of these effects at any concentrations. In the whole blood assays, PMX 60056 was effective in the neutralization of the fibrinopeptide A generation inhibition, however PS did not produce any inhibition. These results suggest that PMX 60056 is effective in neutralizing the anticoagulant effects of Fand I and warrant additional in vivo validation of these studies.
CXCR2 may be critical in recruiting neutrophils (PMN) into the inflamed colon in DSS‐induced colitis
Authors: Shukkur Farooq Muhammed , Stillie RoseMarie , Rostom Reem , Svensson Majlis , Svanborg Catharina , Streiter Robert M. , Stadnyk Andrew W.
Published: June 2021

Dextran sodium sulphate (DSS)‐induced colitis in mice is characterized by PMN infiltration into the colonic mucosa and lumen. The mechanism by which this occurs is unclear. To address this, we determined the role of CXCR2 in DSS‐induced colitis by neutralizing CXCR2 or using CXCR2 KO (knockout) mice. DSS (5% w/v) was administered through drinking water to CXCR2 KO and Balb/c mice (WT) for 5 days followed by tap water for 1 day. In the neutralization study, DSS‐treated or control WT mice were injected i.p with goat serum or goat anti‐CXCR2 serum and disease severity was assessed. DSS‐treated CXCR2 KO mice failed to recruit PMN into the mucosa and had less severe crypt damage with no ulceration compared with WT mice. In the neutralization experiments, goat serum‐injected mice receiving DSS lost weight and showed severe clinical illness. Histological observation revealed edema, PMN infiltration into the submucosa and mucosa, extensive crypt damage with abscesses, and ulceration. In contrast, the anti‐CXCR2 serum‐treated mice showed less clinical illness with no weight loss. This was associated with less histological damage and a lack of PMN infiltration. Our experiments suggest a role for CXCR2 in DSS‐induced colitis and that it may be critical in recruiting PMN into the inflamed mucosa. Funded by the CCFC and the Swedish Medical Research Council, and an IWK Health Centre Fellowship to SMF.
Ligand specific integrins may be involved in the endothelial cell (EC) apoptosis pathway suppressed by cyclic strain (CS)
Authors: Hirayama Yo , Sumpio Bauer E.
Published: June 2021

The precise mechanism of how the EC surface receptor mediates a CS signal and regulates the apoptosis pathway remains to be elucidated. We hypothesized that ligand specific integrins (LSI) play a pivotal role in this mechanism. Thus, the current study was designed to investigate whether LSI are involved in the CS regulated apoptosis pathway or not. ECs were cultured on collagen‐I (a ligand specific for α2β1 integrin but not for α5 and β4) coated flexible plate. Then, ECs were exposed to CS (10% average strain, 60 cpm) for 24h with or without 10μg/ml of α2‐integrin blocking Ab (anti‐α2), anti‐α5, anti‐β1 and anti‐β4. The apoptosis group was kept in serum‐free medium without strain for the same time period. After that, apoptotic cells were detected by TUNEL assay. The apoptotic cell ratio of EC with CS was 5.4±2.5%, and without CS was 16.2±4.9%, suggesting CS suppressed serum withdrawal induced EC apoptosis (p<0.05). Meanwhile, the apoptotic cell ratio of EC with anti‐α2, anti‐α5, anti‐β1 and anti‐β4 was 14.9±2.9, 3.8±1.6, 17.6±1.3, 6.9±1.1 % respectively. There are significant differences between the LSI groups and the non‐LSI groups (α2 vs α5; p<0.01, β1 vs β4; p<0.01). Our preliminary study also showed that anti‐α2 and β1 inhibited the phosphorylation of AKT. These results indicate that CS suppresses EC apoptosis, and LSI may be implicated in mediating CS and regulating the EC apoptosis signaling pathway.
Characterizing membrane clustering of the β2 integrin CR3 using fluorescence resonance energy transfer (FRET)
Authors: Heflin Katie Elizabeth , O’Brien Xian M. , Hyun YoungMin , Kim Minsoo , Albina Jorge E. , Reichner Jonathan S.
Published: June 2021

β‐glucan, a molecule found in yeast cell walls, has been implicated in priming the immune system without causing an increase in inflammatory cytokines. Our previous data indicates that in neutrophils the primary receptor for β‐glucan is CR3 (αMβ2), a β2 integrin crucial for leukocyte recruitment to areas of inflammation. We undertook a fluorescence resonance energy transfer (FRET) approach to investigate the mechanism of CR3 priming. CR3 activation, like that of the related integrin αLβ2, may be regulated through the affinity of a single molecule for its ligand and/or the clustering of many receptors. Our lab had previously generated constructs of αM and β2 with a C‐terminal fusions of mCFP and mYFP respectively for FRET investigations of affinity activation. Now we have created a new construct of αM with a C‐terminal fusion of mYFP. Both αM constructs, along with a wild‐type β2, were transfected into K562 and HL‐60 cells. All constructs were validated for function and are currently being used to investigate the clustering patterns of CR3 under previously characterized priming conditions. Research supported by NIHGM016694.
Choline deficiency alters angiogenesis in the fetal brain
Authors: Mehedint Mihai George , Craciunescu Corneliu , Niculescu Mihai , Sin Kam May , Zeisel Steven
Published: June 2021

Maternal dietary choline intake during pregnancy alters normal fetal neurogenesis and associates with lifelong changes in memory function in the offspring. Mitosis is decreased, while apoptosis is increased, in neural progenitor cells of the hippocampus in the fetuses of rodent dams fed choline deficient (CD) diets. We now report that maternal choline deficiency increases angiogenesis in the fetal hippocampus. Timed pregnant C57BL/6 mice were fed a CD or control (CT) AIN‐76 diet from day 12 to day 17 (E17) of gestation. Fetal brains were studied at day E17. We found increased numbers of endothelial cell‐clusters expressing factor VIII in the fetal hippocampal area in the CD group (68±3 in CD vs. 54±3 in CT, p <0.01). In other experiments, neural progenitor cells from E14 fetal mouse brains were grown in low choline (5 μM) or control (70 μM) for 72 h. In the low choline group we observed increased relative gene expression for vascular endothelial factor (VEGF) (5.03±2.24 in CD vs. 0.52± 0.19 in CT, p<0.001) and angiopoietin 2 (ANGPT2) (4.92±1.7 in CD vs. 1.43±0.6 in CT, p<0.05). We conclude that the changes in neurogenesis are the trigger for altered angiogenesis, probably by increasing VEGF and ANGPT2 signaling. This phenomenon contributes to the effects that maternal intake of choline has on fetal brain development. This research is supported by the grant from NIH‐ AG09525.
Progressive heart block and ventricular hypertrophy in mice lacking urea transporter UT‐B
Authors: Meng Yan , Zhou Lei , Zhao Dan , Guo Lirong , Zhao Xuejian , Yang Baoxue
Published: June 2021

Urea transporters B(UT‐B) is membrane proteins that facilitate urea movement across biological membranes, expressed in kidney, erythrocytes, heart, etc. The aim of this study was to determine the phenotype of UT‐B deletion in heart. UT‐B expression in heart was studied by RT‐PCR and Western blot. Surface electrocardiogram recording (lead II) was measured in wildtype mice and UT‐B null mice. Cardiac function was evaluated by echocardiography, left ventricle internal diameters and wall thicknesses were measured at end systole and end diastole (LVPW(d) and LVPW(s)). RT‐PCR and Western blot analysis showed that UT‐B was expressed in heart in wild‐type mice. No UT‐B transcript and protein was found in UT‐B null mice. ECG recording showed that P‐R interval was significantly delayed in UT‐B null mice vs wildtype mice (p < 0.05). Atrial ventricular heart block type and only appeared in 52 weeks old UT‐B null mice. Echocardiography revealed that the LVPW(d) and LVPW(s) in 52 weeks old UT‐B null mice (1.1128 ± 0.0981 and 1.6157 ± 0.1322 mm) were greatly thicker than age matched wild‐type mice (0.8914 ± 0.1433 and 1.2500 ± 0.1786, P < 0.05 respectively). These results provide the first evidence that UT‐B deletion cause progressive heart block in mice and ventricular hypertrophy is involved in the pathogenesis of heart block. *This study was supported by grant 30670852 from the Natural Science Foundation of China.
Elimination of Heparan Sulfate Glycosaminoglycans from the Glomerular Basement Membrane has Profound Effects on Podocyte Ultrastructure and Function
Authors: McCarthy Kevin John , McCarthy Deborah Jane , Chen Shoujun , Holzman Lawrence , Yamaguchi Yu , Woods Anne , van Kuppevelt Toin
Published: June 2021

Heparan sulfate (HS) proteoglycans (PG) are considered important in GBM ultrafiltration and are synthesized primarily by podocytes. The EXT1 gene encodes for the glycosyltransferase that is the limiting step in HS‐GAG polymerization on PG core proteins. PEXTKO (podocyte EXT1 knockout) mice were developed by breeding EXT1fl/fl with mice transgenic for CRE‐recombinase expression driven by the podocin promoter. Podocytes from PEXTKO mice lack HS‐GAG, yet renal development and maturation occurs. H&E stained sections from kidneys of PEXTKO animals were similar to control animals. Electron microscopy (EM) studies showed podocyte foot process effacement occurred PEXTKO mice at all ages. GBM ultrastructure was compromised in 1 month old animals, with GBM splitting in some areas. In adult animals there was evidence of enhanced GBM synthesis yet overall GBM thickness appeared normal. Immunostaining kidney sections from PEXTKO mice with core protein specific antibodies against perlecan showed no difference in the pattern of staining between control animals and PEXTKO animals, however agrin core protein staining was increased. Albuminuria was not seen in 3 month old PEXTKO animals but mild microalbuminuria was seen in 8 month old animals. The data indicate that the presence of HS chains on GBM proteoglycan core proteins may not exclusively function as a passive sieve, but rather modulate podocyte behavior.
Differences in longevity between Old Master sculptors and painters
Authors: Greenspan Phillip , Heinz Grete , Hargrove James L.
Published: June 2021

The art of sculpting in stone expends more energy than painting on canvas or wood. To investigate whether European Old Master sculptors outlived painters, a database was established based on two art historical reference texts. Artists living in the 20th century were excluded from the database. Analysis of 406 artists from sixteen countries over five centuries revealed that sculptors lived significantly longer than painters (67.4 ± 1.1 vs. 63.6 ± 0.9 years, P < 0.01). This finding correlates with the work of Paffenbarger and colleagues who documented that longshoremen with a heavy workload live significantly longer that men assigned to light work activity. It is estimated that the caloric expenditure of sculpting is approximately 2.5 times greater than painting and approximates the caloric expenditure of cargo handling longshoremen. Since the major cause of death prior to the 20th century was infectious disease, the increased longevity of Old Master sculptors may reflect the effect of moderate exercise on the immune system.
Susceptibility of the aging Brown Norway rat to carbaryl, an anticholinesterase‐based insecticide: Thermoregulatory and cardiovascular responses.
Authors: Gordon Christopher John
Published: June 2021

The proportion of aged in the United States is projected to expand markedly for the next several decades. Hence, the U.S.EPA is assessing if the aged are more susceptible to environmental toxicants. The thermoregulatory and cardiovascular responses of young adult, mature adult, and aged Brown Norway (BN) rats were used to assess susceptibility to carbaryl, an anticholinesterase insecticide. Male BN rats acquired from the National Institutes of Aging at 4, 12, and 24 m of age were implanted with transmitters to monitor heart rate (HR), core temperature (Tc), and motor activity (MA). Baseline data were monitored in undisturbed animals for 7 d. Age had little effect on HR or MA, but there was a significant increase in daytime Tc in the 24 m rats. Rats were dosed by oral gavage with corn oil or 25 mg/kg CAR. Exposure to CAR led to reductions in Tc and HR; however, the hypothermic and bradycardic effects were attenuated in aged rats. In another study, Tc and MA were followed for 22 m in BN rats beginning at 3 m of age. Mean 24 hr Tc decreased from 3 to 6 m, then increased gradually from 6 to 22 m. MA was unaffected by age. Overall, aging is associated with attenuation in the toxicological response to carbaryl. The subtle elevation in Tc with aging in the BN rat may be an important physiological biomarker for aging studies in rodent models. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.
Short‐term(S‐T) dietary magnesium(Mg) deficiency(MD) results in generation of cardiovascular risk factors, free radicals & apoptosis, which are inhibited by water‐borne Mg
Authors: Shah Nilank C. , Liu Jian‐ping , Sica Anthony L. , Hussain Mahmood , Altura Bella T. , Altura Burton M.
Published: June 2021

It has been suggested that low dietary intake of Mg is an unappreciated risk factor for cardiovascular diseases(CVD), and leads to generation of free radicals and programmed cell death. Can water‐borne Mg overcome these pathological alterations? Using S‐T( 21 days) dietary deficiency of Mg( 10 % normal intake) in Wistar rats, we determined whether S‐T MD would lead to lowering of serum (s)‐Mg,s‐HDL‐cholesterol(HDL‐C),elevation of s‐LDL‐cholesterol ( LDL –C),and s‐calcium(Ca),activation of nitric oxide synthase ( n‐NOS),lipid peroxidation (LP),DNA fragmentations (DNA‐fr.),and activation of caspase‐3( CAS‐3) apoptotic pathways in cardiac ( CA) and aortic smooth muscle ( AOSM) in‐vivo. The MD‐animals were allowed to drink triply‐distilled water containing one of five different levels of Mg while control animals received normal Mg‐containing diets and given triply‐distilled water . On the 22nd day, sera and tissues ( left & right ventricles,atria & abdominal aorta) were analyzed. The data indicate that S‐T MD resulted in reductions in s‐Mg ( ~ 75%),and s‐HDL‐C(~50%) and elevation in s‐Ca and s‐LDL‐C( 3 to 5 times normal),activation of n‐NOS,LP,DNA‐fr and activation of CAS‐3 in CA and AOSM. Intake of water‐borne Mg attenuated, or inhibited the alterations observed in the serum analytes and molecular CVD risk factors. In conclusion, we demonstrate , that S‐T MD results in generation of multiple cardiovascular risk factors and programmed cell death in several CV tissues in rats.
Neurogenesis from Neuroglia: Curcumin's potential role in transforming neuroglia into neurons
Authors: Gouw Arvin , Panchal Heena , Ho Jacqueline , Ghazi Kamelia , Chang Tinny , Singh Satbir , Timiras Paola S.
Published: June 2021

The anti‐oxidative, anti‐inflammatory and anti‐proliferative actions of Curcumin, the active ingredient in turmeric are well known. Previous studies on Rat C6 Glioma cells show that curcumin stimulates neuroglial cell maturation and differentiation. Neuroglial cells, astrocytes, and oligodendrocytes are closely related to neurons developmentally, and support neuronal function. DNA microarrays reveal that neurofilament M, involved in neurogenesis, is upregulated by curcumin in cultured neuroglioma. These studies investigate curcumin's possible role in transdifferentiation of these cells into precursor cells. Cultured Rat C6 Glioma cells were cultured with 1μM and 5 μM curcumin for 2 and 7 days. They were then stained for glial fibrillary acid proteins (GFAP), a neuroglial marker, and with Nestin, a neuronal marker, at the end of each trial. Our immunofluorescent images showed loss of GFAP neuroglial expression and a progressive increase in expresssion of Nestin in cells treated with curcumin for 7 days. Thus curcumin may also play a role in neurogenesis from neuroglia. Supported by NIH Grant AG 19145‐05.
An intrasubunit salt‐bridge linking Loop A and Loop 2 is critical for GABAA receptor activation
Authors: Venkatachalan Srinivasan P , Thao Say , Czajkowski Cynthia
Published: June 2021

Current models for cys‐loop receptors propose that ligand binding triggers channel gating via a conformational wave involving sequential movements of protein blocks from the binding site to the channel. How movements are relayed from one block to another is unknown. In GABAA receptor (GABAAR) models of the beta (β) subunit, a pair of oppositely charged residues in Loop 2 (D56) and Loop A (K102) are in close proximity and potentially link the GABA binding site (Loop A) to the gating interface (Loop 2) via a salt bridge. We reversed and swapped these charges and expressed wild‐type (WT) and mutant GABAARs in Xenopus oocytes and examined using voltage clamp. Charge reversals (D56K, K102D) significantly decreased GABA efficacy and increased GABA EC50 ∼1000‐fold as compared to WT (13.3 ± 1.5μM). The charge swap (D56K‐K102D) restored GABA efficacy to near WT values and increased GABA EC50 (900‐fold), much less than the shift predicted (∼106‐fold) in the absence of any interaction. Mutant cycle analysis yielded a ∼4.0 kcal/mol of coupling free energy between the two residues. Notably interaction between these residues is also important for pentobarbital activation of GABAAR. In summary, the data indicate that D56‐K102 form a salt‐bridge that links the GABA binding site to Loop 2. Breaking this salt bridge hinders receptor activation, identifying a potentially new structural path that links GABA binding to channel gating.
Evidence of calpain and PKC cosignaling in diabetic endothelial dysfunction
Authors: Smolock Amanda R , Scalia Rosario
Published: June 2021

Vascular complications are a well‐known cause of morbidity and mortality in diabetes. Endothelial dysfunction is thought to be a primary event in vascular inflammation. Both protein kinase C (PKC) and, more recently, calpain have been implicated in this process. Calpains are a family of calcium‐dependent cysteine proteases that are involved in many cellular processes including the activation of PKC. We hypothesized that calpain and PKC cosignal in the endothelial dysfunction of hyperglycemia. Studies were performed in streptozotocin‐induced diabetic rats before and after calpain inhibition. Biochemical analysis of mesenteric tissue extracts demonstrated a 1.5‐fold increase in calpain activity and a 1.3‐fold increase in PKC activity in diabetic rats. Intravital microscopy of mesenteric post‐capillary venules demonstrated a calpain‐dependent increase in leukocyte adhesion in diabetic rats (adherent leukocytes/100 μm vessel length were 2.8±0.5, 6.9±0.4; and 3.5±1.2 in control rats, diabetic rats and diabetic rats treated with the calpain inhibitor ZLLal, respectively; p<0.05). Taken together, these data indicate a novel relationship between calpain and PKC in the endothelial dysfunction of hyperglycemia.
An ethanol extract Gastrodia elata inhibits TNF‐α‐induced cell adhesion in human umbilical vein endothelial cells
Authors: Hwang Sun Mi , Kang Dae Gill , Lee Yun Jung , Kim Jin Sook , Lee Ho Sub
Published: June 2021

Gastrodia elata is Oriental herbs that have been widely used for the treatment of inflammatory diseases. Endothelial dysfunction and associated vascular inflammatory processes play pivotal roles in the development of atherosclerosis. The present study was designed to examine the effect of ethanol extract of Gastrodia elata (EGE) on vascular inflammation response in primary cultured human umbilical vein endothelial cells (HUVEC). EGE significantly inhibited tumor necrosis factor‐α (TNF‐α)‐induced increase in monocyte adhesion to HUVEC in dose‐dependent manner. In addition, pretreatment with EGE attenuated the expression levels of cell adhesion molecules such as intercellular cell adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), and endothelial selectin (E‐selectin) induced by TNF‐α in HUVEC. Real time RT‐PCR also showed that EGE decreased the expression levels of ICAM‐1, VCAM‐1, E‐selectin, MCP‐1, and IL‐8 mRNA. Furthermore, EGE significantly inhibited the TNF‐α‐induced intracellular reactive oxygen species (ROS) production and NF‐κB activation by preventing IκB phophorylation. In conclusion, EGE has an anti‐inflammatory activity, which is at least in part the result of it reducing the cytokine‐induced HUVEC adhesion to monocytes through the inhibition of intracellular ROS production, NF‐κB activation, and cell adhesion molecules expression in HUVEC.
Modulation of endothelium‐dependent contractions by chronic inhibition of nitric oxide synthase in the rat aorta
Authors: Qu Chen , Leung Susan W.S. , Vanhoutte Paul M. , Man Ricky Y.K.
Published: June 2021

Acute inhibition of nitric oxide (NO) synthase (NOS) augments the release of endothelium‐derived contracting factor (EDCF). The present study investigates whether or not chronic inhibition of NOS modulates endothelium‐dependent contractions (EDC). Eighteen weeks old male Sprague Dawley rats were treated for 6 weeks with vehicle or the NOS inhibitor, N‐nitro‐L‐arginine methyl ester (L‐NAME). Aortic rings with or without endothelium were used for isometric tension recording. Chronic NOS inhibition increased systolic blood pressure and reduced acetylcholine‐induced relaxations in rings with endothelium. This reduction was reversed by L‐arginine (1 mM). In quiescent aortic rings, A23187 caused contractions, which were greater in the presence of the endothelium and after L‐NAME treatment. These contractions were abolished by the cyclooxygenase (COX)‐2 (NS‐398) but not the COX‐1 (SC‐560) inhibitor, and by the selective thromboxane (TP)‐receptor antagonist (terutroban). In the treated group, COX‐1 and COX‐2 expressions, as determined by Western blotting, were up‐regulated while the TP receptor expression remained unchanged, and eNOS expression and NO level became lower. These experiments demonstrate that chronic NOS inhibition in the rat enhances EDC by inducing COX‐2 expression and augmenting the production of EDCF. (Supported by the Research Grant Council of Hong Kong)
In vitro assessment of oxidative stress in the thoracic aorta of a mouse model of Marfan syndrome
Authors: Yang Huei‐Hsin Clarice , Chung Ada WY. , van Breemen Cornelis
Published: June 2021

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder, and thoracic aortic aneurysm is a fatal complication. We have shown that MFS compromised aortic vasomotor functions. Endothelial dysfunction (ED) may be caused by oxidative stress where excessive reactive oxygen species interacts and degrades nitric oxide, reducing its bioavailability. Hypothesis: We hypothesized that oxidative stress contributed to ED in the thoracic aorta (TA) of Marfan syndrome. Thoracic aorta from mice (n = 20, 6 months) heterozygous for FBN1 allele (Fbn1C1039G/+, the most common class of MFS) were compared with those from control littermates. In the isometric force measurement, Marfan aorta demonstrated 40% reduction in phenylephrine (PE)‐induced contraction and 30% acetylcholine (ACh)‐mediated relaxation compared with the control. Preincubation with superoxide dismutase (SOD, 150 Units/ml), an enzyme converting superoxide to H2O2, and apocynin (100 μM), an NAD(P)H oxidase inhibitor, suppressed PE response but improved ACh‐relaxation to a greater extent in the Marfan TA. In conclusion, oxidative stress is elevated in the Marfan aorta, and the excess level of superoxide may cause ED. Project was funded by CIHR.
ATP Release via Connexin Hemichannels from Rat Ventricular Fibroblasts
Authors: Soleymani Sahar , Aroonsakool Nakon , Landeen Lee , Yokoyama Utako , Giles Wayne R. , Insel Paul A.
Published: June 2021

ATP can be released by and regulate the function of many cell types via the activation of membrane receptors. However, the release of ATP from cardiac fibroblasts (CF) has not been studied. We hypothesized that CF release ATP in response to physical perturbation, and that this release may be mediated by connexin (Cx) hemichannels. We isolated ventricular CF from adult Sprague Dawley rats, exposed them to hypotonic challenge or cyclic mechanical stress, and assayed extracellular media for ATP by a luciferin‐luciferase method. We found that hypotonic challenge and cyclic mechanical stretch increased ATP release from CF. Decreasing extracellular calcium from 1 mM to 0 mM, a maneuver that opens Cx hemichannel, increased ATP release, while carbenoxolone, a Cx blocker, markedly decreased ATP release. Real‐time PCR revealed that CF express multiple Cx proteins: Cx 43 > Cx 45 > Cx 40 and that TGF‐β treatment decreased expression of Cx 43 by 40%. We conclude that ATP release occurs from CF in response to hypotonic conditions and mechanical stretch and that this release likely occurs through Cx hemichannels, whose expression can be regulated by TGF‐β, a profibrogenic stimulus. Since CF and myocytes express receptors for ATP and its metabolites, release of ATP by CF likely contributes to autocrine/paracrine regulation of both fibroblasts and myocytes in the heart. (Supported by grants from NIH, AHA, and Ellison Medical Foundation)
Enzymes as Escorts in Organometallic Cofactor Trafficking
Authors: Banerjee Ruma
Published: June 2021

Many coenzymes are vitamins that are assimilated in mammals into their active form from precursors obtained from the diet. They are often both rare and reactive rendering the likelihood that the cell uses a collision‐based strategy for their delivery to dependent enzymes, low. The problem is especially acute for B12, an organometallic cofactor that is reactive in all three of its biologically relevant oxidation states, is usually present at very low concentrations, and is only used by a limited group of enzymes. To avert problems of cofactor dilution and inactivation in an intracellular milieu, we have proposed that enzymes that catalyze the ultimate step in cofactor assimilation pathways may also function as escorts for targeted delivery of their products. We will discuss recent insights into this problem is handled for coenzyme B12, which is synthesized and delivered to its target enzyme by adenosyltransferase.
Effect of endothelin receptor antagonists on gap junction channels in post‐infarcted rats
Authors: Lee Tsung‐Ming , Chen Chien‐Chang , Chang Nen‐Chung
Published: June 2021

Myocardial infarction leads to derangements in cellular electrical stability and the generation of lethal arrhythmias. Endothelin (ET) receptor blockers (ERAs) have been shown to contribute to the antiarrhythmic effect. We investigated whether ERAs exert antiarrhythmic effects through increased expression of connexin43 (Cx43) after infarction. Twenty‐four hours after ligation of the left anterior descending artery, male Wistar rats received either vehicle, ABT‐627 (selective ETA receptor antagonist), bosentan (nonselective ETA/ETB receptor antagonist), or hydralazine for 4 weeks. Measurement of myocardial ET‐1 levels at the border zone revealed a significant increase in vehicle‐treated rats compared with sham‐operated rats, consistent with increased ET‐1 levels after infarction. Immunoblotting revealed that at the border zone the vehicle group showed a marked reduction in the amount of phosphorylated Cx43, whereas either ABT‐627‐ or bosentan‐treated groups showed only a slight reduction compared with sham. Immunohistochemistry and real‐time RT‐PCR confirmed that the Cx43 attenuation was prevented by ERA administration. Arrhythmic scores during programmed stimulation in ERA‐treated rats were significantly lower than those treated with vehicle. The effects of ERAs on Cx43 and arrhythmais were dissociated from their blood pressure‐lowering effect because ERAs and hydralazine reduced arterial pressure similarly. Our data indicate that the ET system plays an important role in the attenuated expression of Cx43 after infarction. Chronic inhibition of either ETA or ETA/ETB receptors restores the decreased expression of phosphorylated Cx43 and improves electrical instability.
Augmented Myocyte Protection And Proliferation In The Infarcted Heart Following Treatment With Cell Lysate From Pharmacologically Preconditioned Mesenchymal Stem Cells
Authors: Afzal Muhammad Rizwan , Haider Husnain Kh , Idris Niagara Muhammad , Jiang Shujia , Rahman Shahid , Ashraf Muhammad
Published: June 2021

We previously showed that preconditioning of mesenchymal stem cells (MSCs) with diazoxide upregulates expression of multiple trophic factors. We hypothesize that intramyocardial injection of cell lysate from these MSCs (pcMSCs) will prevent myocyte apoptosis, stimulate proliferation and attenuate infarct size. Real time PCR showed higher expression of a series of trophic factors in pcMSCs. Cardiomyocytes pretreated with pcMSC lysate were more resistant to oxidant stress and showed higher phosphorylation of PKC å and PKC ä compared to cardiomyocytes treated with lysate from non preconditioned cells. This effect was blocked by Chelerythrin. For in vivo studies, 50 μl of pcMSC lysate (group 1) or non‐pcMSC lysate (Group 2) or DMEM (group 3) was injected into the acutely infarcted wall. Echocardiography showed improved LV function in group 1 at 6 weeks. Hearts were harvested at day 7 and 6 weeks for Masson Trichrome staining that showed significantly attenuated infarct size, reduced area of fibrosis and well preserved LV wall thickness in group 1 compared to other groups. Immunostaing for Ki67 and á‐actinin showed higher number of proliferating cardiomyocytes in group 1. Capillary density was also highest in the peri‐infarct region in group 1.

Conclusion: The trophic factors rich lysate from pcMSC triggers proliferation of cardiomyocytes and preserves LV wall thickness and function.
Intracellular redox status and NAD(P)H:Quinone Oxidoreductase 1 (NQO1) activity in pulmonary arterial endothelial cells (PAEC)
Authors: Bongard Robert , Lindemer Brian , Merker Marilyn
Published: June 2021

NQO1 is a phase II antioxidant enzyme induced in PAEC by oxidative stress. Since isolated NQO1 utilizes either NADH or NADPH as electron donors, we asked whether either donor was preferentially utilized by NQO1 in intact calf PAEC. Intact PAEC NQO1 activity was measured using duroquinone (DQ) as the electron acceptor and cell pyridine nucleotide levels were measured by HPLC. Glycolysis was inhibited with 2‐deoxyglucose (2‐DG) or iodoacetate (IOA) and the pentose phosphate pathway by epiandrosterone (EPI). 2‐DG decreased NADH/NAD+ and NADPH/NADP+ ratios, by 70% and 52%, respectively, and intact cell DQ reduction capacity by 70%. For 2‐DG, lactate restored NADH/NAD+ but not NADPH/NADP+ or DQ reduction capacity. IOA decreased NADH/NAD+, had relatively little effect on NADPH/NADP+ and did not decrease DQ reduction capacity. While EPI alone had no effect on redox status, DQ reduction decreased by 64%, and, when DQ was also present, NADPH/NADP+ decreased by 87% with no decrease in NADH/NAD+. DQ alone also depressed NADPH/NADP+ but not NADH/NAD+. Thus, intact PAEC DQ reduction capacity is more highly correlated with NADPH/NADP+ than NADH/NAD+, implying NADPH as the preferred intracellular electron donor for NQO1. PAEC H2O2 induced oxidative stress was also increased by EPI, suggesting that NADPH dependent NQO1 activity might be compromised under such conditions. Support: HL‐65537 and Dept. Veterans Affairs.
Potentiation of the hepatotoxicity of carbon disulfide by chlordane‐induced cytochrome P450 enzymes
Authors: Dalvi Ramesh R. , Dalvi Prasad S. , Billups Leonard H.
Published: June 2021

Dithiocarbamate drugs such as disulfiram are extensively metabolized in the body to carbon disulfide (CS2), which can produce a variety of toxic effects. When metabolized by phenobarbital‐induced cytochrome P450s (CYPs), CS2 produces hepatic necrosis in rats. In this study we investigated whether CYPs induced by chlordane (CLD), a paradigm of chlorinated hydrocarbon pesticides, potentiate hepatotoxicity of CS2. Male Sprague‐Dawley rats (n=3) were treated with CLD (25 mg/kg, ip) daily for 4 days and 24 h after the final injection, the rats were injected with CS2 (380 mg/kg, ip) in oil while controls received vehicle alone. The rats were then sacrificed at 3, 6 and 24 h. At 3 h post‐treatment, total hepatic GSH decreased modestly but lipid peroxidation increased markedly while all CLD‐inducible CYPs (1A1, 2B1, 2E1 and 3A2) were inhibited by CS2 significantly and variably. At 24 h there was a significant increase in GSH, lipid peroxidation, and ALT activity. Activity of the CYPs was also increased but still remained significantly depressed, especially that of CYP2B1. Livers taken at 3 and 6 h showed subtle to distinct apoptotic changes and at 24 h a severe lesion of hydropic degeneration of the centrilobular cells with apoptosis was observed. These results indicate that the metabolism of CS2 by CLD‐induced CYPs and the generation of lipid peroxides may have contributed to the enhanced hepatocellular damage.
Morphological actions of isoprostanes on bovine retina, ex vivo
Authors: Zhao Min , Zhan Guilin , Zhang Yan , Ohia Sunny E , Opere Catherine A
Published: June 2021

Isoprostanes (IsoPs) can regulate the release of excitatory amino acid from isolated bovine retina (Opere et al., Neurochem Res 30: 2005). The effect of IsoPs on morphology of mammalian retinal tissues is unknown. We examined the effects of intravitreally injected 8‐isoPGE2 on retinal tissue integrity and viability, ex vivo. Using retina obtained at the beginning (time=0) and 30 mins after intravitreal injection of 8‐isoPGE2 or vehicle, tissue viability was examined by TdT‐mediated dUTP nick‐end labeling (TUNEL) assay for apoptosis while tissue integrity was examined by hematoxylin and eosin (H&E) staining assay for detection of necrosis. In positive controls (pretreated with DNase I), TUNEL stained retinal nuclei green while DAPI stained retinal nuclei blue. There was no apoptosis (green) detected in TUNEL‐ stained nuclei in negative controls (pretreated with reaction mix lacking rTdT Enzyme) while DAPI still stained retinal nuclei blue. Compared with the untreated eyes at the beginning of the experiment, retina isolated from the eyeballs exposed to 8‐isoPGE2 (1μM and 100 μM) or vehicle (time = 30 mins) exhibited no apoptosis in TUNEL‐stain. H&E ‐ staining assay revealed no necrosis and no changes in retinal structures between untreated eyes (time =0) and after 30 mins of treatment with 8‐isoPge2 (1μM and 100 μM) or vehicle. In conclusion, IsoPs had no significant action on retinal tissue integrity and viability.
Novel free radical scavengers protect retinal cells and decrease elevated intraocular pressure in a rat ocular hypertension model
Authors: Hosseini Alireza , Lattanzio Frank A. , Schellenberg Karl A. , Samudre Sandeep S , Shaeffer James , Williams Patricia B.
Published: June 2021

Glaucoma is a neurodegenerative disease with damage retinal cells (RC) often associated with elevated intraocular pressure (IOP). Novel free radical scavengers (FRS) with esterified ion chelator and reducing side groups on a methoxypolyethylene glycol backbone (MPs) were evaluated as anti–glaucoma agents capable of both IOP reduction and RC protection.

The operated eye of surgical rat glaucoma model received topical MPs or amifostine, a clinical FRS (all agents 20 ml, 0.3 – 100 mM). IOP was measured via Goldmann tonometer. Slit lamp, confocal microscopy and chronic toxicity studies were performed on normal rats (10 or 87 mM MPs, 3x/day for 30 days). 14C MPs were used for pharmacokinetic studies. Intravitreal injection with NMDA (2μL, 10–40mM) to naïve rats induces RC ERG and histological losses in 2 weeks. This damage was challenged with co–administered intravitreal MPs (n=5–6).

Topical MPs (3–100 mM), but not amifostine, decreased IOP up to 29.6 % for T1/2s of 1–2 hrs. MPs also caused no acute or chronic ocular/systemic toxicity. 14C topical MPs showed ocular penetration with elimination via urine. MPs also reduced NMDA induced RC loss and A and B waves decreases. In vitro isolated rat and bovine RC assays are currently evaluating the role of free radical generation, elevated intracellular calcium and mitochondrial depolarization in the MPs mechanism of action in vivo.

Supported in part by CHRB and AHAF.