bluuebunny/crossref_metadata_2025 huggingface dataset:
Kidney HIF‐1: Effects of altitude, exercise and cell‐free hemoglobin
Authors: van Patot Martha Tissot , White Molly P , Hamilton Karyn L , Gotshall Robert , Irwin David
Published: June 2021

Objective. To determine if hemoglobin‐based oxygen carriers (HBOC) improves kidney oxygenation during global hypoxia at rest and exercise. Methods. Relative tissue oxygenation was assessed by determining hypoxia‐inducible transcription factor 1 (HIF‐1) DNA‐binding (ELISA) and metabolic profiles (magnetic resonance spectroscopy) in Sprague Dawley rat kidneys. Animals were exposed to 1600 (NX) or 5500 (HX) m for 4 h, after treatment (3 ml) with either lactated Ringer's solution or hemoglobin‐based oxygen carrier (HBOC, 1300 mg/kg), during rest or after run to exhaustion. Results. At rest, HX‐induced HIF‐1 DNA‐binding was significantly reduced with HBOC treatment. In accordance, kidneys of animals at rest during HX showed evidence of oxidative stress, high hydroxybutyerate and low phosphotidyl choline. Oxidative stress was not apparent in the kidneys of HBOC‐treated animals during HX. HBOC had no effect on HIF‐1 DNA‐binding during NX. Exercise during NX increased HIF‐1 DNA binding, but lowered it during HX. HBOC‐treatment during exercise had no effect on kidney HIF activity. Conclusion. Data indicate that HBOC lowered HIF‐1 activity in the kidneys of rats exposed to 5500 m, as did exercise, and may provide a therapeutic measure by which to improve tissue oxygenation during global hypoxia. Funded by the USA Defense Advanced Research Project Agency (DARPA) #BAA 06‐19‐F64.
Quantification of physiological adaptations during altered loading states in rodents
Authors: Bederman Ilya , Silva Fatima , Camesasca Marco , Shuster Jeffrey , Ewart Steve , Cabrera Marco
Published: June 2021

Muscle unloading due to space travel, bed rest, or immobilization leads to many physiological adaptations at the whole‐body, tissue, and cellular levels. Specifically, hindlimb suspension unloading (HSU) has been devised as a highly effective technique to simulate the effects of microgravity that lead to muscle atrophy, as well as metabolic and endocrine changes. To quantify metabolic adaptations at various levels, we administered a single bolus of “doubly‐labeled” water (Bederman et al, 2006) to determine: (a) energy expenditure and body composition (whole‐body level); (b) turnover rates of tissue‐specific lipids and proteins from the labeling patterns of protein‐bound alanine and triglyceride‐bound glycerol, rates of de novo lipogenesis, (tissue and cellular level); and (c) labeling patterns of pathway specific metabolites (cellular level). Here, we present the application of this novel methodology in a model of muscle disuse. After three weeks of HSU, rats demonstrated changes at the whole body level (elevated VO2 and VCO2 (lower RQ) and leaner body composition). We report on the changes in fuel selection and rates of tissue remodeling using biochemical fluxes of proteins and lipids. Our work provides the experimental basis for multiscale modeling of the metabolic adaptations to altered loading states.
Activation of the hexosamine biosynthesis pathway alters the balance between hypertrophy and apoptosis in cardiomyocytes from diabetic mice
Authors: Shan Dan , Marchase Richard B , Dell'Italia Louis J , Chatham John
Published: June 2021

Studies have shown that hyperglycemia blunts Angiotensin II (ANG) and phenylephrine (PE) induced cardiomyocyte hypertrophy by increased hexosamine biosynthesis pathway (HBP) flux. Therefore, we tested the hypothesis that diabetes attenuates ANG and PE induced hypertrophy and increases apoptosis due to HBP activation. Cardiomyocytes isolated from type 2 diabetic db/db mice and non‐diabetic controls were treated with 1μM ANG and 10μM PE for 24h. Both ANG and PE significantly increased expression of hypertrophy markers, atrial natriuretic peptide and alpha‐sarcomeric actin in the non‐diabetic but not the diabetic group. Apoptosis assessed by annexin‐V was significantly increased in db/db group (ANG: 30±0.5%; PE: 29±1%) compared to control group (ANG: 20±1.2%; PE: 21±0.6%). Treatment of db/db cardiomyocytes with HBP inhibitors azaserine (5μM) or Don (20μM), partially restored the hypertrophic response to ANG and PE and decreased apoptosis. Non‐diabetic cardiomyocytes incubated with high glucose medium (25mM) or glucosamine (GlcN, 5mM), to activate the HBP exhibited a decreased hypertrophic response to ANG or PE and increased apoptosis compared to normal glucose (5mM). These results suggest HBP pathway may play an important role in mediating the balance between cardiomyocyte hypertrophy and apoptosis in type 2 diabetes.
Coronary and Mesenteric Vascular Dysfunction in High Fat Fed Rats
Authors: Oltman Christine L. , Kleinschmidt Travis L. , Coppey Lawrence J. , Davidson Eric P. , Yorek Mark A.
Published: June 2021

The purpose of this study was to examine microvascular complication in rats fed a high fat (HF) diet. Sprague‐Dawley rats 12 weeks of age were fed a diet containing 45% kcal as fat. Reactivity of small (75 – 150 μm intraluminal diameter) coronary and mesenteric vessels was evaluated. After 8 weeks, vascular relaxations were not altered. After 16 weeks, acetylcholine (ACh)‐mediated relaxation of coronary arteries from HF fed rats was impaired (relaxation to 10 μM Ach: control=47 ± 5, high fat fed=27 ± 8%, p≤0.05). After 24 weeks, reactivity of coronary arteries to ACh was further impaired in rats fed the HF diet (relaxation to 10 μM Ach: control=47 ± 9, HF fed=14 ± 13%, p≤0.05). After 32 weeks on the diet, 10 μM ACh induced 42 ± 28 and 23 ± 8% relaxation in coronary arteries from control and HF fed rats, respectively. Hydroethidine staining suggests increased superoxide formation in coronary arteries from HF fed rats. ACh‐mediated responses were not altered in mesenteric arteries from HF fed rats for the first 24 weeks. After 32 weeks on the diets, 10 μM Ach induced 89 ± 4 and 54 ± 13% relaxation in mesenteric arteries from control and HF fed rats, respectively, p≤0.05. Reversal of diet for 8 weeks, after 24 weeks on HF diet, did not improve vascular function. These studies suggest that a HF diet causes a progressive impairment of endothelium‐dependent vascular function in small arteries. Supported by VA Merits to Drs. Oltman and Yorek.
Alterations in mitochondrial function and cytosolic calcium transients induced by hyperglycemia are restored by over‐expression of mitochondrial transcription factor A (TFAM) in cardiomyocytes
Authors: Suarez Jorge , Hu Yong , Makino Ayako , Fricovsky Eduardo , Dillmann Wolfgang H.
Published: June 2021

TFAM is essential for mitochondrial DNA transcription and replication. TFAM expression is decreased in diabetic cardiomyopathy; however, the functional implications are unknown. We hypothesized that a reduced TFAM activity may be responsible for some of the alterations caused by hyperglycemia. Therefore, we investigated the effect of TFAM over‐expression on hyperglycemia‐induced cytosolic calcium handling and mitochondrial abnormalities. Neonatal rat cardiomyocytes were exposed to high glucose (30mM) for 48 h and examined whether TFAM over‐expression, by protecting mtDNA, could reestablish calcium fluxes and mitochondrial alterations towards normal. Our results show that TFAM over‐expression increased two fold mitochondrial DNA copy number in cells treated with high glucose. High glucose induced a reduction in ATP content and mitochondrial [Ca2+] (30% and 40% respectively) and TFAM over‐expression returned these parameters to control values. Calcium transients were prolonged by 70 % after high glucose treatment which was associated with diminished SERCA2a expression. These parameters were improved by TFAM over‐expression. In addition, we found that TFAM activity can be modulated by O‐linked β‐N‐acetylglucosamine glycosylation. In conclusion, TFAM over‐expression protected cell function against the damage induced by hyperglycemia in cardiomyocytes.
The effects of antioxidants on insulin signaling in spontaneously hypertensive rat vascular smooth muscle cells
Authors: Gardner Carla , Motley Evangeline , Washington Benny
Published: June 2021

The objective of this study was to investigate the possible role of reactive oxygen species on insulin signaling in a hypertensive rat model. Because Protein kinase B/Akt serves as a link between upstream insulin signals, such as insulin receptor substrate‐1 and their cellular consequences, we sought to determine the phosphorylative characteristics of Akt in the insulin signaling pathway. To elucidate the effects of reactive oxygen species, spontaneously hypertensive vascular smooth muscle cells were treated with 6.25–50 μM hydrogen peroxide (H2O2). Our results indicated that 6.25 μM H2O2 enhanced Akt phosphorylation, while 25 μM decreased insulin‐induced Akt phosphorylation. We also demonstrated that H2O2 decreased insulin receptor substrate‐1 phosphorylation. Further, H2O2‐induced inhibition of insulin signaling was inhibited by the antioxidant, pyrrolidine dithiocarbamate. These studies provide more evidence that reactive oxygen species inhibit insulin signal transduction thereby causing insulin resistance leading to cardiovascular disease.

This work was supported by a grant from the Faculty Research Award program of the Division of Research and Sponsored Programs, Tennessee State University.
ATTENUATED EXPRESSIONS OF SOD‐1 IN THE MESENTERIC ARTERIAL TISSUE OF STREPTOZOTOCIN (STZ)‐INDUCED DIABETIC RATS
Authors: Odetunde Juliana O. , Adeagbo Ayotunde , Bratcher Adrienne , Joshua Irving G.
Published: June 2021

Various studies have found that oxidative stress, which can be due to an increase in the production of reactive oxygen species (ROS) and/or a decrease in the elimination of ROS by enzymatic antioxidants, plays a critical role in vascular dysfunction that is associated with diabetic mellitus. Our study performed Western blot analysis of superoxide dismutase (SOD), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) of mesenteric arterial (MA) tissues of control, 6‐ and 12‐week STZ‐induced diabetic Sprague‐Dawley rats with blood glucose levels of 113 ± 4.24, 598 ± 4.47, and > 600 mg/dl, respectively. Our results showed an increase in eNOS (>1 fold), iNOS (>4 fold), and SOD (>8 fold) expressions in the 6‐wk STZ MA tissues (n=5) versus control tissues (n=5). The results also showed relatively unchanged levels of eNOS, increased iNOS (>1 fold), and increased SOD (>5 fold) expressions in 12‐wk STZ MA tissues (n=3) versus control tissues. Past studies have shown an increase in the production of ROS at 6 weeks of STZ diabetes, which is sustained through 12 weeks of the disease in the rat aortic tissue. Our study suggests that diabetes initially stimulates a compensatory increase in SOD expression, which attenuates as the disease progresses to 12 weeks. Supported by grant #T35‐HL071486 from the NIH.
Reduction in Mn‐superoxide dismutase (SOD2) corrects insulin resistance due to high fat feeding in oxidative tissues of mice
Authors: Conniff Mary Elizabeth , James Freyja D , Huang Ting‐Ting , Wasserman David H
Published: June 2021

SOD2 is a mitochondrial antioxidant enzyme responsible for converting superoxide radical to H2O2. Certain levels of H2O2 are essential while excessive levels are harmful to normal metabolic function. We hypothesized that reducing the formation of H2O2 by decreasing SOD2 expression by 50% would improve metabolic function. SOD2+/−mice and wild type (WT) littermates (n=14/13) were chow or high fat fed (HF) for 3 months. Catheters were placed in the jugular vein and carotid artery. 5 days later a hyperinsulinemic glucose clamp was performed. 2‐deoxyglucose was given for analysis of tissue glucose uptake. Body weight, 5 h fasted blood glucose or insulin levels were not different between SOD2+/− and WT on either diet. Glucose infusion rate (mg/kg/min) to maintain euglycemia was similar in SOD2+/− and WT on chow (49±4 vs 49±6) and HF (32±3 vs 35±4). The rates of glucose uptake (Rg; μmol/100gtissue/min) in vastus lateralis muscle were not different between SOD2+/−and WT on either diet. Heart Rg was significantly higher in SOD2+/−compared to WT (174±9 vs 130±13) but there was no difference in diaphragm Rg (46±19 vs 46±8) on chow. WT mice on HF had impaired insulin stimulated glucose uptake in heart (71±9) and diaphragm (22 ± 3). Heart Rg (167±26) and diaphragm Rg (157±12) were not impaired by HF in SOD2+/−. A 50% reduction in SOD2 activity corrects the HF induced insulin resistance that occurs in highly oxidative tissues, such as diaphragm and heart. Supported by DK 54902 and DK 59637
Reduced cytokine production by glycogen‐elicited peritoneal cells from diabetic rats.
Authors: Alba‐Loureiro Tatiana Carolina , Pithon‐Curi Tânia Cristina , Curi Rui
Published: June 2021

Studies with diabetic rats and mice showed important alterations in neutrophil function such as decreased cell migration, phagocytosis capacity and release of TNF‐α, IL‐1β and PGE2 upon exposure to LPS. Elicited peritoneal cells (mainly neutrophils) obtained from STZ‐induced diabetic male Wistar rats (65mg/Kg b.w, 2 and 6 weeks) were investigated. Diabetic (D) and control (C) rats received an intraperitoneal injection of glycogen solution (1%). Four hours afterwards, the peritoneal lavage was performed and the following parameters were: total cell counts in peritoneal lavage and the measurement of the cytokines (IL‐1β, CINC‐2α/β, TNF‐α and IL‐10) was performed in samples of the peritoneal cavity fluid by ELISA. A similar number of cells migrated to the peritoneal cavity in all groups. The diabetic state caused a significant decrease of IL‐1β (45%), TNF‐α (47%), IL‐10 (50%) and CINC‐2α/β (55%) levels in the intraperitoneal cavity fluid. However, the amount of cytokine produced by elicited peritoneal cells from insulin treated diabetic rats was the same observed in control rats. Therefore, the impairment in the production/liberation of cytokines by elicited peritoneal cells might contribute to an impaired inflammatory response and high susceptibilityto infections observed in diabetic state.

Research supported by: FAPESP and CNPq
Hyperglycemia compromises mitochondrial function
Authors: Edwards John G. , Medikayala Sushma , Zhao Xiangmin
Published: June 2021

Mitochondrial dysfunction has a significant role in the development and complications of diabetic cardiomyopathy. Hyperglycemia‐induced mitochondrial oxidative stress has been suggested as one contributing factor. The impact of hyperglycemia was studied using H9c2 myotubes (a cardiac‐derived cell line) and Cyt C‐ cells (an embryonic cell line having reduced levels of cytochrome C). Cells were maintained in the presence of 5.5, 16.5, or 35.0 mM glucose for up to 13 days. Hyperglycemia significantly increased protein levels in the Cyt C‐ cells but not the H9c2 cells. Functional tests of mitochondria (succinate dehydrogenase and ATP generation) suggested that although the H9c2 myotubes were sensitive to hyperglycemia, the Cyt C‐ cells were not. Hyperglycemia did not significantly alter generation of extracellular H2O2 in either cell line. However, intramitochondrial superoxide (mtSO) levels (detected using MitoSox) was increased in the H9c2 cells following 13 days of hyperglycemia. Conversely, up to13 days of hyperglycemia did not significantly altered mtSO levels in the Cyt C‐ cells. Hyperglycemia increased H9c2 caspase 3/7 activity that appeared to track mtSO levels. These results suggest that mitochondrial function may be compromised by hyperglycemia as a consequence of altered intramitochondrial oxidative stress. Supported in part by the New York Medical College Research Endowment Fund.
Endothelial‐specific BH4 expression accelerates refractory wound healing by enhancing eNOS activity and suppressing oxidative stress in type 1 diabetes
Authors: Tie Lu , Marrotte Eric J. , Channon Keith M. , Chen Alex F.
Published: June 2021

Background: Refractory wound is a severe complication in diabetes that leads to limb amputation, but the mechanisms are poorly understood. eNOS plays a key role in normal wound repair, but is uncoupled in streptozotocin (STZ)‐induced type 1 diabetes due to reduced cofactor BH4. We tested the hypothesis that GTP cyclohydrolase I (GTPCH), the rate‐limiting enzyme of BH4 synthesis, retards eNOS uncoupling with accelerated wound healing in STZ mice.

Methods and Results: Blood glucose levels were significantly increased in both male GTPCH transgenic mice (Tg‐GCH) and the wild‐type (WT) littermates 5 days after STZ regimen. A full‐thickness excisional wound was created on mouse dorsal skin by a 4‐mm punch biopsy. The rate of wound closure in STZ mice was delayed by 18.48% compared to normal WT mice, which was rescued in STZ Tg‐GCH mice by an average of 8.48% (n=3–5, p<0.05). HPLC analysis showed that cutaneous BH4 level was significantly reduced in STZ mice vs. WT mice (n=4–5, p<0.05), which was maintained in STZ Tg‐GCH mice. In STZ mice, cutaneous eNOS activity and nitrite level were decreased compared to WT mice, paralleled by increased superoxide (O2−) level. In STZ Tg‐GCH mice, nitrite level was potentiated and O2− level was suppressed compared to STZ mice.

Conclusion: Endothelial‐specific BH4 overexpression accelerates wound healing in STZ type 1 diabetes mice by enhancing eNOS activity and suppressing oxidative stress.
Dose‐dependent mechanisms mediate lipopolysaccharide (LPS) hypotension in male rats
Authors: Marlow Joshua , Yilmaz Mustafa S , Millington William R , Feleder Carlos
Published: June 2021

Previous data from our laboratory indicate that lipopolysaccharide (LPS) initially lowers arterial pressure through a central mechanism that involves the preoptic anterior hypothalamic area (POA). In this study, we investigated how the inflammatory signal induced by peripherally injected LPS reaches the POA. Indeed, it is well known that LPS raises body temperature through two different mechanisms, depending on the dose. Low doses of LPS cause fever by activating vagal nerve afferents; high doses cause fever by activating receptors in the organum vasculosum of the lamina terminalis (OVLT). In the present study we found that the effect of a relatively low hypotensive dose of LPS (1 mg/kg iv) was blocked by subdiaphragmatic vagotomy or lidocaine injection into the nucleus tractus solitarius (NTS), but not by lidocaine injection into the OVLT or the area postrema in isoflurane anesthetized rats, suggesting that initiation of LPS hypotension is mediated by vagal nerve afferents. Neither vagotomy nor NTS lidocaine injection prevented the hypotension evoked by high dose LPS (15 mg/kg), however. Interestingly, lidocaine injection into the OVLT prevented the hypotension evoked by high dose LPS. These data indicate that LPS lowers arterial pressure initially by two different pathways. Low doses lower arterial pressure by activating vagus nerve afferents; high doses act through the OVLT.
Remodeling of the guinea pig intracardiac plexus following pressure overload‐induced cardiac hypertrophy
Authors: Baran Caitlin N , Southerland E. Marie , Ardell Jeffery L , Hardwick Jean C
Published: June 2021

Chronic pressure overload (PO) of guinea pig hearts was induced by a banding of the descending dorsal aorta (10–15% constriction). Animals were allowed to recover for 2 months, at which point the heart and intracardiac nerve plexus were isolated for electrophysiological and immunohistochemical analysis. Sham surgeries, in which the aorta was surgically visualized but undisturbed, were run in parallel. PO animals showed a significant increase in the size of the heart and lungs as a percent of body weight versus sham surgeries. Intracellular voltage recordings from parasympathetic postganglionic neurons showed no change in resting membrane properties in the sham or PO tissues versus age‐matched controls. However, neurons from PO hearts showed a significant increase in the magnitude of the histamine‐induced increase in neuronal excitability (as determined by the frequency of action potentials produced with depolarizing stimuli) as compared to both controls and sham surgeries. PO hearts also showed a three‐fold increase in the number of neuronal nitric oxide (nNOS) immunoreactive neurons (PO 15.2 + 1.8% versus controls 5.4 + 2.9%, p<0.02). These results indicate that chronic pressure overload induces a differential remodeling of intrinsic cardiac cellular phenotypes and functional up‐regulation of local network excitability. Supported by HL71830 (JLA) and R15 HL060619‐02 (JCH).
Delta‐opioid receptor (DOR) activation is neuroprotective during oxygen‐glucose deprivation (OGD) in neonatal rat medullary slices in vitro
Authors: Majewski David J. , Johnson Stephen M.
Published: June 2021

OGD can cause long‐lasting damage to the CNS and cell death. DOR activation prior to OGD is neuroprotective in cardiac tissue, but it is not known whether DOR activation protects oscillatory motor networks, such as the respiratory rhythm generator. To address this question, respiratory‐related hypoglossal (XII) motor bursts were recorded with suction electrodes from rhythmically active neonatal rat (P0–P3) medullary slices. Application of OGD solution (oxygen‐ and glucose‐free) decreased spontaneous respiratory burst amplitude by 53 ± 5% within 10 min and bursts were abolished within 21.6 ± 1.5 min (n = 5). In contrast, application of DPDPE (1.0 μM; DOR agonist) for 10 min prior to and during OGD solution application caused XII burst amplitude to decrease by only 24 ± 5% at 10 min and bursts were abolished at 31.4 ± 3.9 min (n = 5; p < 0.01 for drug effect). OGD‐induced changes in burst frequency were nearly identical during OGD solution application alone or with DPDPE co‐application. These data suggest that XII motoneurons are more vulnerable to OGD than rhythm‐generating neurons, and that DOR activation is neuroprotective during OGD in oscillatory motor networks.
Relationships between inflammatory markers and regional fat and muscle volumes in morbidly obese women
Authors: Vendela Mandolyn Jade , Nicklas Barbara J. , Fernandez Adolfo , You Tongjian , Miller Gary D.
Published: June 2021

Obesity is considered a chronic inflammatory state as adipokines are released from fat depots and increase circulating levels of cytokines. The relationships between regional fat and muscle depots and circulating levels of inflammatory markers are not well described in morbid obesity (BMI ¡Ý 40.0 kg/m2). The study' s purpose was to examine associations of pro‐ and anti‐inflammatory markers and regional body composition in morbidly obese women. Volumes of fat (intermuscular (IM) thigh, visceral, IM abdominal) and thigh muscle were measured by computer tomography in obese women (n=16; 45.9 + 9.6 yrs; BMI = 54.0 + 7.9 kg/m2). Pearson correlations were performed between weight, BMI, fat depot and thigh muscle volumes and fasting plasma levels of interleukin‐6 (IL‐6), tumor necrosis factor alpha (TNFa), TNFa soluble receptor 1 (TNFsR1), C reactive protein (CRP) and adiponectin. Significant relationships (p<0.05) were observed with body weight and CRP (r=0.586) and TNFsR1 (r=0.523); BMI and TNFsR1 (r=0.548); intermuscular thigh fat and TNFsR1 (r=0.516); and adiponectin and intermuscular abdominal fat (r=0.581). Thigh muscle was correlated with TNFa (r=0.641) and adiponectin (r=−0.605). Circulating levels of pro‐ and anti‐inflammatory markers are related to body weight, BMI, and levels of fat and muscle in several regional depots fat, but not to visceral fat.
Exercise‐induced muscle damage and oxidative stress in untrained and moderately trained subjects
Authors: Park Kyung‐Shin , Sedlock Darlene A. , Navalta James W.
Published: June 2021

Muscle damage, which is greater following an unaccustomed downhill run (eccentrically‐biased exercise) compared to a level grade run or a second downhill run, increases intracellular free radicals which reduce oxygen radical absorbance capacity (ORAC).

PURPOSE

to investigate differences in markers of muscle damage and oxidative stress between moderately trained (MT) and untrained (UT) college students.

METHODS:

Nine MT and nine UT [VO2max (ml.kg−1.min−1) = 54.9±1.1(SE) vs. 45.7±1.8] performed three 40 min treadmill runs at ~70% VO2max: a level run (L) and two downhill (−10%) runs (DH1 and DH2). Blood was sampled before and immediately (POST), 2h, 24h, and 48h postexercise, and analyzed for creatine kinase activity (CK) and ORAC.

RESULTS

Running speed was faster for MT than UT in L (p<.05) and DH (p<.01). Following DH1, CK activity was significantly elevated at 24h in both groups (p<.01), and significantly higher at 2h in MT than UT (p<.01). ORAC of MT was significantly lower at 24h in DH1 vs L and DH2 (p<.01). ORAC did not significantly change in UT.

CONCLUSIONS

Muscle damage and oxidative stress were greater in MT than UT following unaccustomed downhill running at the same relative metabolic intensity. Therefore, factors other than relative metabolic rate, e.g., muscle activation (running speed, stride length), or other exercise training‐related mechanisms, may be mediating this response.
In vitro effect of fatty acids on human neutrophil function and death
Authors: Pithon‐Curi Tania Cristina , Levada‐Pires Adriana Cristina , de Lima Thais Martins , Hatanaka Elaine , Curi Rui
Published: June 2021

The highest concentration of fatty acids (FA) that are nontoxic to human neutrophils was determined. The effect of nontoxic fatty acid concentrations on neutrophil phagocytic capacity and ROS production was also investigated. The cells were treated with various concentrations (50 until 600 μM) of docosahexaenoic (DHA); eicosapentaenoic (EPA); arachidonic (AA); γ‐linolenic; stearic (SA); linoleic (LA); palmitic (PA); palmitoleic; myristic(MA); lauric; oleic (OA); elaidic; capric; caprylic and caproic acids for 3 h. The % of cells with intact membrane and fragmented DNA varied depending on the type of fatty acid and the concentrations used. The toxicity was as follows: DHA=LA=OA>EPA=AA=palmitoleic>capric>γ‐linolenic=lauric>SA=PA=myristic=elaidic= caprylic= caproic acids. The % of human neutrophils DNA fragmentated was increased as follows: DHA= AA= LIN= PA= palmitoleic= OA= elaidic= capric> EPA= γ‐linolenic= SA= myristic>lauric>caprylic=caproic acids. Therefore, unsaturated fatty acids that caused low neutrophil toxicity were: γ‐linolenic and EPA acids. Caprylic and caproic acids caused the low toxic effects on neutrophils. All FA tested had no effect on neutrophil phagocytosis at non toxic concentration. FA can regulate neutrophil function by modulating this cell death. Financial Support: FAPESP/CNPq
DAPK mediates TNF‐alpha induced apoptosis by TNF‐receptor 1 association and JNK activation through MKK7
Authors: Gentry Patrick Lee , Zhang Liguo , Duan Rui , Gallagher Patricia Jane
Published: June 2021

Death Associated Protein Kinase (DAPK) is a calcium calmodulin (CaM) regulated serine/threonine kinase, which has a pivotal role in regulation of apoptosis and survival in cells. The cellular activities of DAPK are tightly regulated by intracellular calcium levels, an inhibitory autophosphorylation site (S308) within the CaM‐binding domain, and by proteasomal degradation. Preliminary studies from this laboratory have suggested that DAPK has a major role in signaling by the inflammatory cytokine, TNF and becomes associated with TNFR1 in response to TNF. To better understand the mechanism by which DAPK regulates apoptotic responses to the cytokine TNF, we have examined the downstream arms of TNFR1 signaling cascade. These studies have revealed that maximal activation of c‐Jun N‐terminal kinase (JNK) in response to TNF is dependent upon the activities of DAPK. Data shows that upon siRNA mediated depletion of DAPK, JNK 1 activation is significantly attenuated. However, there is no detectable effect on NFkB, p38, or ERK 1/2 activation. This data has identified a novel‐signaling pathway for DAPK, in the regulation of survival, inflammation, and proliferation signals through modulation of MAPK/JNK activation. Ongoing studies are focused on elucidating the mechanism of DAPK activation of the MAPK/JNK pathway and the physiological role of this interaction. Supported by HL54118 and DK062810 to PJG
Necroptosis is involved in cardiac cell death induced by chemical ischemia
Authors: Chua Chu Chang , Chua Kao‐Wei , Gao Jinping , Hamdy Ronald C , Chua Balvin HL
Published: June 2021

Necrosis is generally believed to be a passive cellular response to external damage. To date, it has been difficult to investigate the potential mechanisms of necrosis. Recently, a new pathway to necrosis has been described by Yuan and colleagues. This pathway, called necroptosis, is a form of cell death that leads to necrosis. A chemical compound, necrostatin‐1 (Nec‐1), specifically inhibits this non‐apoptotic cell death. Iodoacetate (IAA) is an irreversible inhibitor of glycolysis and has been used as a tool for inducing chemical ischemia. We hypothesize that Nec‐1 can confer protection in this model. In this study, we explored the protective effects of Nec‐1 on IAA‐induced ischemia in rat myoblastic H9c2 cells. These cells were exposed to DMEM containing 7.5% dialyzed fetal bovine serum in the presence of 10–100 uM IAA for 2 h followed by incubation with regular medium overnight. Cell proliferation was measured by MTS assays. IAA was found to induce cell death in a dose‐dependent manner. At 50 uM IAA, cell proliferation was 30% as the control level. The addition of 25 uM Nec‐1 significantly attenuated IAA‐induced cell death. The protective effect of Nec‐1 was also dose‐dependent. These results demonstrate for the first time that necroptosis is involved in IAA‐induced cell death (Supported by American Heart Association Southeast Affiliate, Department of Veterans Affairs Merit Review and NIH HL‐087271).
Inhibition of glycogen synthase kinase‐3β protected human oral gingival fibroblasts from etoposide‐induced apoptosis
Authors: Reddix Rhoda Ann , Vastardis Sotirios , Lucchesi Pamela A
Published: June 2021

Background: A key feature of gingival enlargement is hyperproliferation and prolonged survival of human oral gingival fibroblasts (HOGF). Several studies have demonstrated that glycogen synthase kinase(GSK)‐3β inhibitors are cytoprotective in neurons. Their protective effects may be mediated by β‐catenin, which has also been shown to promote the survival of various cell types. Aim: To determine whether inhibition of GSK‐3β promotes the survival of HOGF cells. Methods: HOGF cells were isolated from healthy human gingival tissues obtained from two patients during periodontal surgery. The cells were grown in 24 well plates and examined for cell viability using the MTT assay. Initially, the cells were serum starved for 24 hrs, pretreated with vehicle or 10mM LiCl, a GSK‐3β inhibitor and then exposed to the pro‐apoptotic agent, etoposide (2.5 mM, ETOP). We also examined relative changes in the phosphorylation of Akt using immunoflourescence analysis in HOGF cells exposed to LiCl for 30min and 24 hrs. Results: ETOP reduced HOGF cell viability. Pretreatment with LiCl prevented the ETOP‐induced apoptosis in these cells. (p<0.01, n=6 exp). In addition, LiCl increased phosphorylated Akt levels within 30 min and it remained elevated for 24 hrs. Conclusion: The results suggest that inhibition of GSK‐3β promotes the survival of HOGF cells by activating the prosurvival protein, phosphorylated Akt. Supported by HL056046.
Leptin protects rat cardiomyocytes from H2O2‐ and hypoxia‐induced apoptosis
Authors: Shin Eyun‐Jung , Eguchi Megumi , Liu Yuantao , Sweeney Gary
Published: June 2021

Obesity increases the risk of heart failure, yet there is also compelling evidence suggesting a protective effect of obesity on subsequent events post‐myocardial infarction. Leptin, the product of the obese (ob) gene, circulates at higher concentrations in obese individuals. The objective of this work was to investigate the effect of leptin on cardiomyocyte apoptosis; a well known component of cardiac remodeling that can contribute to heart failure in obesity. We used H2O2 and hypoxia to mimic myocardial ischemia in vitro and first demonstrated that the decreased viability of H9c2 rat cardiomyocytes exposed to H2O2 was attenuated by pretreating cells with leptin (6nM) for 1hr. Similarly, exposure to H2O2 or hypoxia increased phosphatidylserine exposure as measured via annexin V binding, decreased mitochondrial membrane potential, increased cytochrome c release from mitochondria and elevated activity of caspase‐3 measured by cleavage and enzymatic assay. Importantly, all of the above were prevented when cells were pretreated with leptin for 1hr. In conclusion, we have demonstrated that leptin has anti‐apoptotic effects in rat cardiomyocytes which may be of significance in regulating the development of heart failure in obesity.
Negative pericardial pressure can impede ventricular systole in elasmobranch fishes
Authors: Lai N. Chin , Fang HongFei , Machaj Laura , Tang Tong
Published: June 2021

The duration of ventricular contraction in elasmobranch fishes (sharks and rays) has been thought to greatly exceed ventricular filling, and was posited that this long systolic event is required for developing negative extracardiac (pericardial) pressure for atrial filling. Conversely, this temporal extension could be due in part to the negative force – that can be artificially induced in the pericardium ‐ impeding contraction or to an absence of key protein(s) affecting the contractile rate. This study evaluates if ventricular systole is indeed intrinsically prolonged in the absence of a negative pericardial pressure and examines whether cAMP activity, a key element of the contractile process, was attenuated in anesthetized shortfin mako (Isurus oxyrinchus) and blue (Prionace glauca) sharks. Ventricular pressures were obtained with the pericardium exposed by a pressure transducer, and ECG signals were used to discern the systolic and diastolic durations. Systolic and diastolic durations were similar for both species with systole accounting for about a third of the cardiac cycle, which differs from classical observations (Table). The activities of cAMP performed on the shark's ventricular tissues were comparable to mammals, implying that the initial phase of the contractile process was not hindered (Table). These findings suggest that the prolonged systolic event observed previously could be the manifestation of a strong negative pericardial pressure, however, similar chronic measurements on conscious sharks are needed. Data expressed as mean±sem (n). Supported by an unrestricted grant from IGAM
Identification of an NHE8 ortholog in the gills of the anadromous sea lamprey Petromyzon marinus
Authors: Edwards Susan L , Fortier Jennifer , Hyndman Kelly A , Evans David H , Diamanduros Andrew , Claiborne James B
Published: June 2021

The maintenance of internal pH homeostasis is critical to the survival of all organisms. The transmembrane exchange of hydrogen ions for sodium ions is seen across all phyla. Na+/H+exchangers (NHEs) are a family of transmembrane proteins that facilitate this exchange. To date, 10 NHE isoforms (NHE1–10) have been characterized in mammals. Epithelial isoforms NHE2 and NHE3 have been previously identified in the gills of fishes from a wide diversity of classes, where they are thought to play a role in the maintenance of systemic pH. A recent study has described the localization of NHE8 to both the organelles and apical membrane of mouse proximal tubule epithelial cells. These studies suggest that similar to the epithelial isoform NHE3, NHE8 may have the ability to recycle to the plasma membrane. The NHE8 clade appears to have originated in slime mold suggesting that NHE8 isoform could be the precursor to the development of the plasma membrane recycling NHE isoform NHE3. Single orthologs of NHE8 have been identified in the genomes of invertebrates, fish and mammals. However, the NHE8 group is not as well characterized. In this study we have identified an NHE8 ortholog in the gills of the primitive vertebrate, the anadromous sea lamprey, the NHE8 cDNA shares a high homology to other known NHE8 sequences. Tissue distribution PCR suggests that the transcripts of the lamprey NHE8 are present in multiple tissues: gill, heart, stomach, liver, muscle and intestine. The functional role of NHE8 in the lamprey is yet to be determined.
Apical Na+/K+‐ATPase and strong alkalinization in the anterior stomach of larval yellow fever mosquitoes (Aedes aegypti)
Authors: Onken Horst , Patel Malay , Javoroncov Margarita , Moffett Stacia B , Moffett David F
Published: June 2021

Anterior stomachs of larval mosquitoes maintain luminal pH values of 10–12. Recently, Na+/K+‐ATPase was localized in apical membranes of this tissue in larval Aedes aegypti (Patrick, ML et al. 2006: JEB 209, 4638). We tested the hypothesis that this apical ATPase acts as a primary active Na+/H+‐exchanger, contributing to luminal alkalinization by active H+ absorption. m‐Cresol Purple (mCP, 0.04 %) in the water was used to visualize alkalinization in the anterior stomach of the transparent larvae. In the presence (N = 10) and absence (N = 12) of 5 mM ouabain the anterior stomach was found to be purple, indicating an alkaline content. Isolated anterior stomachs were bathed in mosquito saline and perfused with 100 mM NaCl and mCP. The transepithelial voltage (Vte) was measured and luminal alkalinization was determined by the color change of mCP after luminal perfusion stop. Luminal ouabain (5 mM) did not affect Vte or the alkalinization of the lumen (N = 7). Ouabain‐sensitive ATPase activity was measured in homogenates of single stomachs in the presence (86 ± 3 nmol Pi h−1 gut−1 or 10.6 ± 2.2 ìmol Pi h−1 mg−1, N = 3) and the absence of K+ (0.1 ± 0.1 ìmol Pi h−1 mg−1, N = 3). Omission of K+ abolished the ouabain‐sensitive ATPase activity. The results seem to rule out that apical Na+/K+‐ATPase contributes to luminal alkalinization by generating ATP driven Na+/H+‐exchange across the apical membrane. Supported by NIH (1R01AI063463‐01A2).
The Use of OsiriX & Adobe Flash to Deliver Case‐Based Radiology to the Classroom & through the Internet
Authors: Foreman K. Bo , Morton David A. , Albertine Kurt H.
Published: June 2021

The cadaver is the primary teaching tool for the study of medical gross anatomy. However, clinical evaluation of internal anatomy is frequently accomplished using various imaging techniques (e.g. MRI and CT scans). Therefore, we created an Internet‐based tutorial as an ancillary teaching tool for students taking gross anatomy. Using a HIPPA‐approved protocol, we obtained permission to access DICOM files from the hospital medical server (PACS). Using PACS, DICOM files were sent directly to OsiriX. From OsiriX all personal identifying information was removed (anonymized) and the files were exported as a JPEG image set and QuickTime Virtual Reality (QTVR) movie. The exported material was used to populate the Internet‐based interactive tutorial that was created using Adobe Flash. Tools within the tutorial enable the user to scroll through the image set and select an individual image that can be panned, zoomed, rotated, and reset. Links from the Internet‐based tutorial to QTVR movies allow rotation of objects in three dimensions. In addition, the interactive application displays self‐learning text related to the displayed material. We conclude that the Internet‐based tutorial aids in the education, review, and understanding of normal human anatomy.
Cytosolic Triglyceride Storage in Mouse Enterocytes during Dietary Fat Absorption Visualized by Coherent Anti‐Stokes Raman Scattering Microscopy
Authors: Cheng Ji‐Xin , Zhu Jiabin , Lee Bong Gi , Buhman Kimberly K.
Published: June 2021

Although triglyceride (TG) storage in cytoplasmic lipid droplets (CLDs) in hepatocytes is well accepted, it remains controversial whether enterocytes store TG in CLDs during normal dietary fat absorption. Herein coherent anti‐Stokes Raman scattering (CARS) microscopy was applied to directly visualize TG accumulation in enterocytes based on the resonant CARS signal from symmetric CH2 stretch vibration. Small intestinal tissues from C57BL/6J mice fed 0, 50, or 300 μL olive oil were examined. CLDs were observed in CARS images. TG storage as a function of digestion time and location were quantified by image‐J analysis and confirmed with a biochemical assay. Combined CARS and two‐photon‐excited fluorescence imaging of individual isolated enterocytes revealed that CLDs are located adjacent to the Golgi complex towards the apical side. In addition, in two mouse models with perturbation of chylomicron secretion, diacylglycerol acyltransferase 1 deficient and PL81‐treated mice, CARS images of enterocytes confirmed increases in CLDs in enterocytes. Taken together, these results demonstrate storage of TGs in CLDs within enterocytes during dietary fat absorption. Further study of the formation, trafficking, and lipolysis of CLDs in enterocytes would deepen the current understanding of dietary fat absorption and facilitate the development of new therapeutic methods for fighting obesity.
A‐002, a secretory phospholipase A2 inhibitor, reduces cholesterol accumulation in aorta and aortic cytokines in guinea pigs
Authors: Leite Jose Oyama , Vaishnav Ushma , Puglisi Michael , Trias Joaquim , Fraser Heather , Fernandez Maria Luz
Published: June 2021

Secretory phospholipase A2 enzymes (sPLA2) are involved with the generation of proinflammatory molecules such as leukotrienes and prostaglandins and are associated with atherosclerotic plaques. In this study we tested the effects of an sPLA2 inhibitor (A‐002) on aortic inflammatory cytokines and aortic lipid accumulation in a guinea pig atherosclerosis model. For this purpose, 24 male Hartley guinea pigs (12 per group) were fed a diet containing 0.25% dietary cholesterol to induce atherosclerosis, for 3 months. Animals were randomized into placebo (PL) or treated (A‐002) group, and gavaged daily either with vehicle (10% acacia, PL) or A‐002 (150 mg/kg/d, A‐002). At the end of 12 weeks, guinea pigs were sacrificed and blood was collected to measure plasma lipids and lipoprotein subfractions. The aorta was harvested for histological evaluation, measurement of inflammatory cytokines and lipid accumulation. Aortic cytokines including Interleukin (IL)‐2, IL‐10, IL‐12 and GMSCF measured by use of antibodies and Luminex technique were significantly lower (p < 0.05) in the A‐002 group. In addition, the A‐002 group had 26.5% lower cholesterol accumulation in the aorta (p < 0.05), but not in plasma, in association with a reduction in atherosclerotic lesion content in the aortic sinus. From these studies we conclude that A‐002 was effective in reducing atherosclerosis, as indicated by reductions in aortic cholesterol content, inflammatory cytokines and lesion content. [Study supported by Anthera Pharmaceuticals, Inc., San Mateo, CA].
Rhodiola extract inhibits adipocyte differentiation in 3T3‐L1 cells
Authors: Lee OK‐Hwan , Kwon Young‐In , Apostolidis Emmanouil , Shetty Kalidas , Lee Boo‐Yong , Kim Young‐Cheul
Published: June 2021

Adipocyte differentiation is a key process in the development of obesity as well as in normal development and function of adipose tissue. We have previously shown that Rhodiola extract high in phenolics dose‐dependently inhibited the enzyme activity of both glucose‐6‐phosphate dehydrogenase (G6PDH), the rate‐limiting enzyme of the pentose phosphate pathway (PPP), and proline dehydrogenase (PDH) in differentiating 3T3‐L1 preadipocytes. The present study is designed to investigate whether the inhibitory effects of Rhodiola extract on enzyme activities correlate with gene expression pattern and further PPARγ mRNA and protein expression, a key adipogenic transcription factor, by semi‐quantitative RT‐PCR and Western blot analysis. 3T3‐L1 preadipocytes were treated with various concentrations of Rhodiola extract for up to 8 days following standard induction of differentiation. Rhodiola treatment significantly inhibited expression of G6PDH and PDH mRNAs as well as mRNA and protein expression in a dose‐dependent manner. These results indicate that Rhodiola extract can inhibit adipogenesis through a mechanism(s) that involves direct downregulation of PPARγ gene expression or via modulation of pentose phosphate pathway linked to PDH, which may be important for cellular energy and redox environment in adipocytes.
Identification of a novel phytoestrogen in coffee beans
Authors: Allred Kimberly , Vanamala Jairam , Murano Peter , Allred Clinton
Published: June 2021

Trigonelline (TRG), a niacin related compound, is a natural constituent of coffee accounting for approximately 1% dry matter in roasted beans. Studies exploring the effects of this bioactive compound on mammalian cells are limited. However, treatment of neuroblastoma cells with TRG resulted in increased formation of neurites and TRG inhibits invasion of hepatoma cells. The purpose of these studies was to determine if TRG also has endocrine actions in mammalian cells. Specifically, we sought to determine if TRG exhibits estrogenic properties by activating the estrogen receptor (ER). Proliferation of ER‐dependent breast cancer (MCF‐7) cells was used to screen for potential estrogenic actions of the compound. TRG stimulated MCF‐7 cell proliferation in a dose responsive manner and significantly enhanced cell growth at concentrations as low as 100pM. Co‐treatment of MCF‐7 cells with TRG and ICI, an ER antagonist, inhibited TRG induced cell proliferation. In addition, when cells were co‐treated with suboptimal doses of estradiol and TRG, an additive enhancement of MCF‐7 growth was observed. Estrogen response element (ERE)‐reporter assays and modulation of ER target genes (pS2, progesterone receptor, and cyclin D1) were also analyzed. These data demonstrate that TRG at low concentrations stimulates MCF‐7 cell growth and that this effect is mediated through ER. Thus, we have identified TRG as a novel phytoestrogen.
Mechanisms of impaired eosinophil recruitment and humoral response induced by endotoxin tolerance in murine asthma
Authors: Natarajan Sudha , Kim Jiyoun , Remick Daniel G.
Published: June 2021

Previous data suggest that persistent endotoxin (ENDO) exposure protects against inflammation induced by allergen challenge. We hypothesized that persistent exposure to ENDO results in ENDO tolerance (ET) and a specific reduction in both cellular and humoral immunity. ET was induced in female BALB/c mice by direct intratracheal installation of 1ug of ENDO for 4 consecutive days. On day 5, mice were immunized with 2ug cockroach allergen, challenged on days 14 & 21 and sacrificed at subsequent timepoints. Airways hyperresponsiveness to methacholine was significantly increased in ET (350% above baseline) vs. non ET (144%) mice. Numbers of eosinophils recruited to the lung (ET vs. non‐ET, 14,486vs.74,636), eosinophil specific peroxidase activity and pulmonary levels of eotaxin (67pg/ml vs. 48) and eotaxin‐2 (2349 vs. 249) were significantly decreased in ET mice. Differences were not seen in other eosinophil chemoattractants IL‐5 or RANTES. Neutrophil numbers, CXC chemokines, and pulmonary expression of Th1 (IFN‐g;, IL‐12) and Th2 (IL‐4, 5, 13) cytokines were not affected by ET, although plasma IgE (141ng vs. 45) and IgM (11ug vs. 7) were significantly decreased in ET mice. Our data indicate ET specifically blunts eosinophilic inflammation and the humoral response to allergen challenge, providing mechanistic insight into protection from asthmatic inflammation.