bluuebunny/crossref_metadata_2025 huggingface dataset:
Preventive Effects of Phyllanthus urinaria Extract on Paw Edema in Adjuvant‐Induced Arthritis Rats
Authors: Kang Myung Hwa , Kim Tae Soo , Park Chun Geon , Park Hee Woon , Ju Young Woon , Cho Young Min , Koh Sang Bum , Seong Ha Jeong
Published: June 2021

The aim of this study was to examine the preventive effects of Phyllanthus urinaria Extract (PUE) on paw edema in adjuvant‐induced arthritis (AA) rats. Arthritis was induced by the injection of 200 ul of a suspension of 10 mg/ml heat‐killed tuberculosis (Mycobacterium tuberculosis) into the plantar region of the right hind foot of rats. Paw edema of the right hind feet of AA rats were reduced by the administration of PUE for 14 days and 28 days after adjuvant injection in comparison with those of AA rats administered physiological saline. The beta‐sitosterol had similar effect as the PUE applied. Myeloperoxydase activity (MPO) was reduced by the PUE in AA rats. These results indicate that the PUE are effective as anti‐inflammatory agents in AA rats and their action is markedly influenced by the inhibition of neutrophil migration into inflamed tissue. Therefore, these findings may provide the pharmacological basis of the clinical usage of PUE in treatment of inflammation.
Evaluating kava extracts and kavalactones for mutagenicity and toxicity
Authors: Whittaker Paul , Clarke Jane J. , San Richard H.C. , Betz Joseph M. , Mazzola Eugene P. , Dunkel Virginia C.
Published: June 2021

Kava (Piper methysticum) has been cultivated by South Pacific islanders for centuries and used as a social and ceremonial drink. The active components of kava rootstock are contained primarily in the lipid‐soluble resin. The compounds of greatest pharmacological interest are the styryl α‐pyrones or kavalactones and represent 3–20% of the dried rhizome. At least 16 lactones have been isolated from kava and six compounds, namely, kawain, dihydrokawain, methysticin, dihydromethysticin, yangonin, and desmethoxyyangonin are the major kavalactones, and account for approximately 96% of the lipid resin. Kava beverages and other preparations are known to be anxiolytic and are used for anxiety disorders. Dietary supplements containing the root of the kava shrub have been implicated in several cases of liver injury in humans, including several who required liver transplants after using kava supplements. In order to study the cellular toxicity and mutagenicity, two commercial samples of kava and the eight pure kavalactones, were evaluated in L5178Y mouse lymphoma cells. Neither the kava samples nor the kavalactones induced a mutagenic response in the L5178Y mouse lymphoma mutation assay with the addition of human liver S9 activation, but there were significant differences in toxicity among the kavalactones.
TRAP is a more sensitive assay than FRAP for evaluation of human plasma antioxidant capacity in response to acute coffee intake
Authors: Perrone Daniel , Moura‐Nunes Nathália , Farah Adriana , Donangelo Carmen Marino
Published: June 2021

This study aimed to evaluate the plasma antioxidant capacity (AC) in response to acute coffee intake using two assays based on different mechanisms: single electron transfer (Ferric ion Reducing Antioxidant Parameter) and hydrogen atom transfer (Total Radical‐trapping Antioxidant Parameter). Nine healthy subjects drank 200 mL of instant coffee or water after overnight fast, with 7d interval between treatments. Blood was drawn before (baseline) and 90 min after treatment. Plasma AC was evaluated by FRAP and TRAP at both time points. Albumin, bilirubin and uric acid levels were also measured. At baseline, FRAP and TRAP were correlated (r = 0,86, p < 0.001), and both FRAP and TRAP were correlated with uric acid (r = 0.88 and r = 0.91, p < 0.001, respectively). After coffee ingestion, FRAP and TRAP values increased 1.7 and 8.7%, whereas after water ingestion, FRAP and TRAP decreased 1.3 and 3.0%, respectively. Net percent increase in response to coffee intake was significantly higher (p < 0.02) for TRAP (11.7%) than for FRAP (3.0%). Changes in FRAP and TRAP were not correlated with those of uric acid, bilirubin and albumin levels, indicating that these endogenous components do not contribute to the antioxidant response to coffee intake. Although FRAP and TRAP assays were both able to detect the increase in plasma AC after coffee intake, TRAP showed to be a more sensitive assay. Financial support: CBP&D Café, CNPq, FAPERJ (Brazil).
Anti‐inflammatory effects of cranberry juice in lipopolysaccharide (LPS)‐stimulated RAW 264.7 murine macrophage cells.
Authors: Beverly Kandice , Basu Arpita , Lucas Edralin A.
Published: June 2021

Regular consumption of fruits and vegetables may protect against many chronic diseases due to its anti‐inflammatory and anti‐oxidative flavonoids. Proanthocyanidins are a subclass of flavonoids with powerful anti‐oxidative and anti‐inflammatory effects and are found in Vaccinium berries such as cranberries. In this study, we have investigated the in vitro anti‐inflammatory effects of cranberry juice. RAW 264.7 murine macrophage cells were treated with different concentrations of cranberry juice (0, 2.5, 5, 10, and 20%, v/v) for 24 h and then stimulated with 500 ng/mL of E. coli lipopolysaccharide (LPS) for another 24 h to induce inflammation. Cell viability, nitric oxide (NO), cytokines, and protein levels of cyclooxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS) were assessed. Cranberry juice at concentration of 20% significantly reduced iNOS expression, NO, and prostaglandin E2 secretion (P<0.05). Western blot data showed that cranberry juice did not affect protein levels of COX‐2. Cranberry juice has no effect on interleukin‐6 (IL6) and tumor necrosis factor‐α (TNF‐α) concentrations. The results from this study indicate that cranberry juice effectively inhibits pro‐inflammatory markers such as NO and PGE2 but has little effect on TNF‐α and IL‐6, and warrants further investigations as a component of medical nutrition therapy for chronic diseases. (Supported by College of Human Environmental Sciences, Oklahoma State University)
Isoflavones intake of young Korean adult males and its relation to their bone status
Authors: Kim Mi‐hyun , Surh Jeonghee , Chung Yoon‐Sok
Published: June 2021

To examine the potential role of habitual isoflavones intakes in bone status of men, a total of 463 Korean healthy adult males aged 20–26 were observed for their diet intake and calcaneus bone density using 3 day 24‐hr recall method and quantitative ultrasound, respectively. Critical markers reflecting bone metabolism such as blood total alkaline phosphatase activity (ALP) and N‐mid osteocalcin (OC) concentrations were also analyzed. For quantitative data on the intake levels of isoflavones, previous study monitoring phytoestrogens in 220 Korean leguminous foodstuffs was employed and the median value for each food was adopted. Isoflavones intake of the subjects was estimated as 14.80 mg/day, which constituted 7.49 mg daidzein, 6.49 mg genistein, 0.82 mg glycetin, 0.00 mg formononetin, and 0.00 mg biochanin A. The intake level turned out to be lower than the average daily isoflavones intake of Korean (23.0 mg, Surh et al, 2006) and much lower than the amount reputed to prevent osteoporosis (40–50 mg/day, Anderson & Garner, 1997), which should lead to no appreciable correlations among isoflavones intakes, bone mineral density of calcaneus, and blood bone metabolism markers measured in the young Korean adult males.
Induction of experimental colitis by dextran sulphate sodium in a piglet model of irritable bowel syndrome
Authors: Lackeyram Dale , Kim Connie , Young Denise , Archbold Tania , Yang Chengbo , Mine Yoshinori , Fan Ming
Published: June 2021

This study was conducted for chemical induction of intestinal inflammation with dextran sodium sulphate (DSS) in piglets. A total of 11 Yorkshire piglets of 5 d of age, with an average body weight (BW) of 3 kg, were fitted with intra‐gastric catheters and divided into a control (CON, n=6) and treatment groups (DSS, n=5). Both groups were infused with equal volumes of either saline or 1.25 g of DSS/kg BW.d in saline, respectively, for 10 d. Histological grading of the colon indicated that DSS animals had severe, transmural inflammation. ELISA analysis indicated that DSS‐treated piglets had higher (P<0.05) levels of the pro‐inflammatory cytokines TNF‐α and IL6 in the colon (DSS, 476.64±62.91 vs. CON, 83.66±22.52 and DSS, 189.72±9.93 vs. CON, 64.91±5.84, pg/g tissue), respectively. Quantitative real time RT‐PCR confirmed higher (P<0.05) expression levels of TNF‐α mRNA in DSS‐treated pigs (DSS, 1.21±0.51 vs. CON, 0.011±0.002, arbitrary units) while the anti‐inflammatory cytokine IL10 was undetectable (DSS, ND vs. CON, 0.025±0.005, arbitrary units) in the colon of DSS‐treated piglets. Gut D‐mannitol permeability into blood circulation subsequent to an intra‐gastric bolus infusion of D‐mannitol (0.6 g/kg BW) after 75 min was higher (P<0.05) in the DSS group (DSS, y = 1.691x, r2= 0.97 vs. CON, y = 0.463x, r2= 0.93). We conclude that the dosage of 1.25 g DSS/kg BW.d is suitable to induce experimental colitis and IBS pathology in piglets. Supported by OMAFRA of Canada.
PPAR‐gamma ligands modulate growth factor and bacteria induced cellular responses in prostate carcinoma cells
Authors: Koenig Wolfgang , Allhoff Ernst Peter , Arnold Ralf , Koenig Brigitte , Ghebremedhin Beniam , Tammer Ina , Koenig Jens Ekkehard
Published: June 2021

Prostate carcinoma cells (PC3, LNCAP, DU145) as well as prostate hyperplasia (BPH cells) can be activated for proinflammatory mediator release and parameters of innate immunity by bacteria as well as by various growth factors. Peroxisome proliferator‐activated receptors (PPAR) are ligand activated transcription factors with antiinflammatory, antineoplastic effects on tumor cell proliferation and angiogenesis. The effect of 15d‐PGJ2, and glitazones was analyzed on various prostate carcinoma cells which were stimulated with either VEGF, EGF, HGF, IGF, TNF, SEB, C5a for 5‐ to 48 hours. As bacterial stimuli were chosen Staph. aureus (n=5) or E. coli (n=5).

The release of IL‐6, IL‐8, PGE2, TGFβ was studied by Elisa, by RT‐PCR and Taqman analysis the expression of p22‐, 47‐, 67‐phox, gp91‐phox, cysteinylleukotriene receptors 1, 2, trefoil peptides (TFF1‐3), protease activated receptors (PAR1‐4), toll‐like receptors (TLR2, 4, 9), cyclooxygenase 2. We show: PPARγ‐ligands 1.) interact differently with carcinoma cells for mediator release as well as cell surface receptor expression and 2.) differences in pro‐ and antiinflammatory responses depend on the individual cell as well as the stimulus via differences in signal transduction. The results suggest a potential use of PPARγ‐ligands as adjuvant therapy of prostate carcinoma and further one has to consider the individual cell type and mode of cellular activation.
Copyright Issues: A Publisher's Perspective
Authors: Hyde Madelene
Published: June 2021

The face of publishing has changed dramatically over the last few decades. We are no longer an industry that simply prints books and journals but have now moved into an era of global content dissemination. A simple book may now be accompanied by online resources including the e‐book, an image bank, PowerPoint slides and test bank; a large reference work may include weekly updates direct from the author ensuring the content is constantly up‐to‐date. Everyone knows that photocopying a book contravenes copyright but the vast array of digital content that is now offered to faculty and students today makes the question of copyright even more difficult to navigate. There is much confusion over what is appropriate to download or use in teaching. The issue of “Fair Use” has been established but the definition remains fuzzy. What is the publishers’ interpretation of “Fair Use” and what steps do we take to enforce that? How does infringement of copyright adversely affect future publications? These questions and other issues will be addressed by this discussion of copyright in a digital age from a publisher's perspective.
Thrombosis/activation (TA) score: An early predictor of atherothrombosis risk in heart transplantation
Authors: Labarrere Carlos A. , Woods John R. , Hardin James W. , Campana Gonzalo , Ortiz Miguel A. , Sosa Marcelo J. , Walker Candace L. , Pitts Douglas E. , O’Donnell Jacqueline A. , Hormuth David A.
Published: June 2021

The discovery of biomarkers that signal atherothrombosis in heart transplantation is vital for preventing subsequent disease. We created a thrombosis/activation (TA) score to quantify the risk relationship of thrombosis (fibrin deposits and loss of vascular antithrombin and tissue plasminogen activator) and endothelial activation (intercellular adhesion molecule‐1 expression) with patient outcomes. Serial biopsies obtained in 172 patients at 10 days, 1‐, 2‐ and 3‐months post‐transplant were analyzed immunohistochemically. Patients were followed annually with coronary angiograms. Logistic regression was used to develop scaled TA scores. Kaplan‐Meier analyses were used to validate the score's capacity to predict outcomes occurring over a 6‐year follow‐up. TA scores were significantly associated with development of cardiac allograft vasculopathy (CAV) (p<0.001), severe CAV (p<0.003), graft failure (p<0.009) and failure due to CAV (p<0.001). Interestingly, although 3‐month scores added to the predictive value, a majority of patients were already classified correctly by the end of the first month post‐transplant (56–76% correctly predicted), and even information available as early as 10 days possessed good predictive value (56–71% correctly predicted). The TA score is a valid predictor of atherothrombosis, and TA detected 10‐days post‐transplant is already predictive of long‐term outcome.
Optical characterization of vascular tissue constructs made with soluble vs. homogenized collagen
Authors: Levitz David , Hinds Monica T. , Hanson Stephen R. , Jacques Steven L.
Published: June 2021

The ability of optical imaging techniques like optical coherence tomography (OCT) to non‐destructively characterize engineered tissues has generated much interest recently. We are testing the hypothesis that OCT data can characterize cellular remodeling of vascular constructs made from 2 types of scaffold matrix: soluble collagen and homogenized collagen. Smooth muscle cells were seeded into the 2 types of collagen matrices at a density of 106 cells/ml. The constructs were allowed to remodel for 5 days, with OCT imaging of triplicate samples occurring every 24 hours. From the OCT data, the attenuation and reflectivity were evaluated by fitting the data to a theoretical model that relates the construct optical properties to the OCT signal. The fitted optical properties were compared to the construct volume. Construct cellularity was assessed by histology. Our preliminary results showed the soluble collagen constructs compacted over time, while the homogenized constructs did not. In agreement with the compaction data and histology, the measured optical properties from the OCT images showed an increase in both attenuation and reflectivity of the soluble constructs, but not in the homogenized constructs. These results highlight the potential of OCT to non‐destructively characterize engineered tissues and may have important implications in regenerative medicine. This study was supported by NIH RO1 HL084013.
Differences in cellular characteristics of locus coeruleus neurons from WKY rats may be indices for stress hyperresponsivity
Authors: Piel David , Valentino Rita J. , Beck Sheryl G.
Published: June 2021

The Wistar‐Kyoto (WKY) rat exhibits increased neuroendocrine, behavioral and autonomic responses to stressors and therefore provides a well characterized model of stress hyperreactivity. The locus coeruleus (LC) norepinephrine system is implicated in the cognitive, attention and behavioral aspects of the stress response. Whole cell electrophysiology in horizontal slices containing the LC was conducted to assess differences in cellular characteristics, firing rate, response to corticotrophin releasing factor (CRF) receptor activation and glutamate activity in WKY and control SD rats. Cells were filled with biocytin and the morphology of the LC neurons assessed using the Neurolucida program. No differences were found in the cell characteristics. In neurons recorded from SD rats, 93% of the neurons were spontaneously active, whereas only 60% of the WKY neurons were active. The response to CRF (10 nM) was an increase in firing rate in the WKY rats, but in SD rats the increase was non‐significant due to large variability. The frequency of glutamate miniature excitatory postsynaptic potentials was greater in SD than WKY rats. Morphological assessment revealed that the WKY neurons had a greater soma size and dendrite length. These results highlight differences in LC neuron physiology and morphology that may underlie differences in stress reactivity between the WKY and SD rats. Supported by N0014‐03‐0311.
Effects of green tea on lipids, blood pressure and vasorelaxation in rats with hypercholesterolaemia‐induced hypertension
Authors: Song Liyang , Koo Marcel WL , Lau Chu‐Pak , Cheung Bernard MY
Published: June 2021

Objective: In this study, the effects of green tea on hypercholesterolemia‐induced hypertension, vascular responses in isolated aortic rings, and serum lipid profile in rats were examined.

Methods: Male Sprague‐Dawley rats were divided into 4 groups. The normal control group was fed with a normal diet while the remaining 3 groups were given a diet high in cholesterol. Two of the hypercholesterol diet treated groups were administered with green tea or tea powder solution. The treatments were given for 2, 4 or 8 weeks.

Results: High cholesterol diet treatment for 2 and 4 weeks induced significantly higher total serum cholesterol level (p<0.01) and blood pressure (p<0.05). There was no increase in blood pressure in tea and tea powder treated groups when compared to the normal control group. The blood pressure of the tea leaf group, and tea powder group was significantly lowered than the disease control group at 4 weeks and 4 to 8 weeks, respectively. Tea and tea powder solutions at physiologically attainable concentrations enhanced vasorelaxation induced by physiological concentrations of acetylcholine (10−8 M ‐ 10−7 M) in rat aortic rings.

Conclusions: Green tea lowered blood pressure that was increased by hypercholesterolemic diet. It decreased serum total cholesterol and prevented reduction of HDL‐cholesterol level, thus tea consumption may exert beneficial effects on the cardiovascular system.
Opposing effects of the kinin B1 and B2 receptors in LPS‐induced hypotension.
Authors: Duchene Johan , Francis Stephanie , Lecomte Florence , Stidwill Raymond , Singer Mervyn , Cayla Cecile , Bader Michael , Perretti Mauro , Ahluwalia. Amrita
Published: June 2021

Lipopolysaccharide (LPS)‐induced hypotension is absent in kinin B1/B2 receptor double knockout mice (B1B2dKO; Cayla et al., FASEB J. 2007) however, the mechanisms of this effect are uncertain. We now show that whilst LPS (12.5 mg/kg) induced hypotension is absent at 4 h it is present at 12 h in B1B2dKO mice (n = 6, p<0.05). LPS has no effect in B1KO mice (n = 6). These differences were not due to altered [NO] since LPS‐induced increases in plasma [NO] and tissue iNOS were similar in all genotypes (n = 6). However prostaglandins (PG) PGE2 and PGF1α.were elevated at 4 h (3 and 7‐fold respectively returning to basal by 12 h) in WT mice; an effect absent in B1KO mice, and delayed (evident at 12 h but not 4 h) in B1B2dKO mice (6 and 8‐fold). This effect was due to changes in COX activity since LPSinduced changes in COX‐1/2 expression were similar in all genotypes. Absence of raised [PG] was associated with reduced vascular dysfunction in isolated resistance arteries.

Our data show that absence of B1 receptor abolishes LPS‐induced hypotension; however inactivation of both B1 and B2 receptors reverse this protective effect. We propose that during sepsis, B1 receptor activation causes early and substantial pathogenic PG production; whilst B2 receptor activation inhibits this response highlighting the potential of B1 receptor blockade combined with selective B2 receptor agonism in treatment of sepsis.

Supported by The Wellcome Trust
Inhibition of matrix metalloproteinase (MMP)‐dependent epidermal growth factor receptor (EGFR) transactivation prevents the development of hypertension in high fructose diet fed rats
Authors: Nagareddy Prabhakara Reddy , Vasudevan Harish , McClure Brian , McNeill John H.
Published: June 2021

The metabolic syndrome is a cluster of cardiovascular risk factors among which are insulin resistance (IR) and hypertension. Despite major research efforts, it remains uncertain what causes the hypertension. Recent studies have suggested a link between disease progression and activation of growth factor receptor signaling pathways. A key event in this pathway is transactivation of growth factor receptors, such as the EGFR by MMPs. This transactivation can be triggered by agonists such as catecholamines, endothelin‐I and angiotensin II, all of which are elevated in models of hypertension including high fructose diet fed rats. We hypothesize that the increased activity of MMPs (MMP‐7) and subsequent transactivation of the EGFR contributes to the development of hypertension in high fructose diet fed rats. Male Wistar rats were fed with either normal rat chow or a 60% fructose diet for 10 weeks and treated daily with doxycycline (MMP inhibitor, 20 mg/kg) and AG 1478 (EGFR inhibitor, 5 mg/kg) orally for 4 weeks. Fructose fed rats developed IR, endothelial dysfunction and hypertension. Western and substrate zymographic analysis indicated increased expression and activity of MMP‐7 in cardiovascular tissues of untreated fructose rats. Inhibition of either MMP or the EGFR activity prevented endothelial dysfunction and development of hypertension without affecting IR. Our results suggest that the MMP‐EGFR pathway may be a potential target for the treatment of hypertension in metabolic syndrome. Funded by HSFC, CIHR & MSFHR.
C6‐ceramide‐coated catheters promote re‐endothelialization of stretch‐injured arteries
Authors: O’Neill Sean Michael , Olympia Dina K , Fox Todd , Brown J. Tony , Stover Thomas C. , Houck Kristy , Wilson Ronald , Waybill Peter , Kozak Mark , Kester Mark
Published: June 2021

The objective of the present study is to elucidate the biological mechanism(s) by which direct, acute application of growth arresting ceramide analogues leads to a reduction in arterial injury after angioplasty. Immunohistochemical studies in rabbit and porcine coronary, carotid, iliac, and renal models suggest that C6‐ceramide limits arterial stenosis without inhibiting endothelial wound healing responses. C6‐ceramide‐coated balloon catheters reduce internal elastica injury, with a corresponding reduction in medial fracture length in a 28‐day porcine coronary artery stretch model. We investigated if differential metabolism of exogenous ceramide by coronary endothelial and smooth muscle cells could explain the apparent discrepancy between the anti‐proliferative actions of ceramide and the pro‐wound healing responses to ceramide. Human coronary artery endothelial cells, in contrast to human coronary artery smooth muscle cells, preferentially express ceramide kinase and form ceramide‐1‐phoshphate which promoted endothelial cell survival. The combinatorial actions of C6‐ceramide‐coated catheters to limit vascular smooth muscle proliferation and promote re‐endothelialization argue for therapeutic efficacy as adjuncts to stent‐based modalities or as a stand‐alone treatment. These studies were funded by NIH RO1 HL076789 to MK as well as by Reva Medical, San Diego California.
Differential effects of metformin on smooth muscle contractile properties of duodenum, uterus and bladder
Authors: Peuler Jacob D , Nass Samuel M , Phelps Laura E , Petrinec Nicholas J
Published: June 2021

The widely‐used antidiabetic agent metformin frequently causes gastrointestinal (GI) side effects. When taken orally, metformin reaches millimolar levels in GI tissues. Previously, we found that such levels exert contrasting effects on contractile properties of intact duodenal tissues isolated from male rats, i.e. inhibition of spontaneously‐occurring contractions and those induced by 5‐hydroxytryptamine (5HT) and high potassium (K) but enhancement of those induced by acetylcholine (ACh). In the present study, we found that the same levels of metformin exerted similar effects in duodenal tissues isolated from female rats but different effects in uterine and urinary bladder tissues from the same rats. In uterus, metformin inhibited K‐induced contractions but failed to meaningfully alter spontaneous contractions and those induced by 5HT and ACh. In bladder, metformin enhanced ACh‐induced contractions but failed to meaningfully alter spontaneous contractions and those induced by 5HT and K. Such a unique set of tissue specificities should greatly facilitate future efforts to unravel mechanisms responsible for metformin's various effects. Supported by Midwestern University.
Neurokinin‐1 Receptor antagonist treatment alleviates CLP‐induced polymicrobial sepsis in mice, acting via NFkappaB
Authors: HEGDE AKHIL Kumar , Moochhala Shabbir M , Bhatia Madhav
Published: June 2021

We have earlier shown a beneficial effect of substance P (SP) receptor blocking on lung inflammation in sepsis by using SP receptor antagonist SR140333. To further understand the mechanism, we evaluated the effect of SR140333 on plasma SP levels and lung nuclear transcription factor NFκB in sepsis. Sepsis was induced in male Swiss mice by cecal ligation and puncture (CLP). Sham operated animals received the same surgical procedure except cecal ligation and puncture. Vehicle or SR140333 (1 mg/kg; s.c.) was administered to CLP mice either 30 min before or 1 h after the CLP. 8 h after surgery, plasma SP levels were estimated and lung tissue was analyzed for binding activity of NFκB. CLP induced sepsis resulted in a significant increase in plasma SP levels in vehicle treated mice compared to the corresponding levels in sham mice. Prophylactic and therapeutic administration of SR140333 showed a significant reduction in plasma SP levels. NFκB is known to be responsible for the gene regulation of various mediators in lung inflammation. We observed a significant increase in the binding activity of NFκB in lung in CLP operated mice. SR140333 treatment lowered the binding activity of NFκB indicating a role for this transcription factor downstream of SP in inducing lung inflammation. These results show that SR140333 therapy may be beneficial in the treatment of lung inflammation in sepsis by acting via NFκB. Hegde A et al J Leukoc Biol 2007, 82:678–85.
Effect of chronic nadolol treatment on isoproterenol induced mouse tracheal relaxation
Authors: Lin Rui , Knoll Brian J. , Bond Richard A.
Published: June 2021

Upregulation of Gi protein has been observed in animal models of asthma, and is associated with the impairment of isoproterenol (ISO) induced tracheal relaxation. Using an antigen driven mouse model of asthma, we investigated the effect of chronic nadolol treatment on ISO induced tracheal relaxation. The relaxant response of tracheal rings was tested in organ chamber. PKCα translocation and PLCβ1 expression in lung or bronchi homogenates were also evaluated by western blotting. ISO induced relaxations were impaired in contracted trachea from mice sensitized and challenged with ovalbumin (s/c mice) compared to trachea from control mice. Twelve hours incubation with 0.3 μg/ml pertussis toxin (PTX) resulted in a 20% increase of ISO induced relaxation in s/c mice, which was not observed in control mice. However, chronic nadolol treatment diminished this effect of PTX on s/c mice. Chronic nadolol treatment also decreased PLCβ1 in trachea and major bronchi homogenates though no effect was observed in the elevated translocation of PKCα in s/c mice. Our results suggested that chronic nadolol treatment may decrease β2‐AR‐Gi coupling and Gq signaling in s/c mice.
Engineering of Human Cytochrome P450 2B6 for Enhanced Expression, Stability, and Functional Studies of the Wild‐Type and Genetic Variants
Authors: Kumar Santosh , Talakad Jyothi C. , Sun Ling , Halpert James R.
Published: June 2021

Despite the emerging importance of human P450 2B6 in xenobiotic metabolism and increasing number of genetic variants, thorough biochemical and biophysical characterization has been impeded due to low expression and stability. Recently, an N‐terminal modified 2B6 (2B6dH) mutant was engineered termed L264F, which showed enhanced expression and stability and retained functional integrity compared with 2B6dH. To further enhance the expression and stability of 2B6dH construction is underway of V234I, E254A, Y325Q, P334S, I427M, and Q473K, which involves changing the residues from the one found in the less stable 2B6dH to the more stable 2B1dH. 2B6dH mutants that show increased stability will be combined with L264F, and the resulting mutant with the highest expression and stability will be used as a template for directed evolution to model a thermostable 2B6dH. To investigate the functional role of the 2B6 variant, K262R, which shows altered activity with several drugs, the mutant was constructed in 2B6dH and L264F backgrounds. K262R and K262R/L264F were then characterized for stability and for functional properties using model and drug substrates/inhibitors. K262R showed decreased protein stability and altered substrate metabolism and inhibitor binding compared with 2B6dH. Structural analysis to understand how K262R alters enzyme functions is underway. [NIH grant ES03619 and Center grant ES06676]
Extracellular protons activate human ENaC by relieving sodium self‐inhibition
Authors: Collier Daniel M , Snyder Peter M
Published: June 2021

The Epithelial Sodium Ion Channel (ENaC) is exposed to a wide range of proton concentrations in the kidney and lung. We tested whether protons alter ENaC activity. In Xenopus oocytes expressing human αβγENaC, amiloride sensitive current was altered by pH in the physiologically relevant range (pH 8.5‐6.0) with a pH50 of 7.2. Compared to pH 7.4, pH 8.5 decreased current by 18.4%, whereas changing to pH 6.5 increased current by 29.7%. We hypothesized that pH might regulate ENaC by altering sodium self‐inhibition. Consistent with this hypothesis, we found that external sodium was required for pH to alter ENaC outward current. Moreover, we found that the response to pH was altered by mutations in the extracellular domain that disrupt sodium self‐inhibition. αH255R increased sodium self‐inhibition and caused an increase in the size of the pH activated currents. Conversely, γH233R, which decreased sodium self‐inhibition, was insensitive to external pH. Our results suggest that protons activate human ENaC in part by relieving sodium self‐inhibition.
Activation of PPARγ by rosiglitazone (Ro) inhibits intestinal Cl− secretion
Authors: Bajwa Poonam J. , Lee Jimmy W. , Straus Daniel S. , Lytle Christian
Published: June 2021

Thiazolidinedione drugs Ro and pioglitazone (Po) are PPARγ agonists in widespread clinical use as insulin‐sensitizing agents in type 2 diabetes. The effects of Ro and Po on intestinal Cl− secretion was evaluated in mice and in cultured human intestinal cells (HT29‐Cl.19A). Oral administration of Ro (20 mg/kg/day) to mice for 8 days reduced cAMP‐evoked electrogenic Cl− secretion by the terminal ileum and distal colon by 80±14%. In the proximal colon both Ca2+ and cAMP‐evoked Cl− secretion were inhibited by 96±28% and 91±19%, respectively. In a mouse model of secretory diarrhea, Ro reduced cholera toxin‐induced fluid secretion by 52±15%. Treatment of HT29 cells with 10 μM Ro for 3 days reduced the secretory Isc evoked by either Ca2+ (64±22%) and cAMP (52±13%). The inhibitory effect of Ro became apparent only after 3 days. Similar effects were observed with Po. Experiments with 8cpt‐cAMP indicated that inhibition occurs distal to cAMP generation. Membrane permeabilization experiments revealed that Ro inhibits both cAMP‐evoked basolateral K+ conductance and apical CFTR Cl− conductance. Western blot analysis of HT29 cells indicated that chronic Ro treatment reduced CFTR channel expression by 60±20%. We conclude that Ro suppresses intestinal Cl secretion through a PPARγ‐dependent genomic mechanism that downregulates expression of apical CFTR Cl− channels and reduces expression or activity of basolateral K+ channels.
Lipopolysaccharides modify amiloride‐sensitive Na+ transport processes across H441 lung epithelial cells
Authors: Baines Deborah L , Albert Anthony P , Woollhead Alison M
Published: June 2021

Bacterial infections modify the ion transport processes that control lung fluid homeostasis. We investigated the effects of bacterial lipopolysaccharides (LPS) on amiloride‐sensitive Na+ transport across H441 cells. Monolayers were cultured at air interface on permeable supports for 7 days, mounted in Ussing chambers and bathed in physiological saline. Mean resistance (Rt) was 296 ± 69 Ωcm2, n = 5. Incubation with 15μgml−1 LPS for 8 hours caused a reduction in amiloride‐sensitive Isc (10 μM amiloride) from 29 ± 4 to 20 ± 3 μ Acm−2, p<0.05 n = 5. There was no change in amiloride‐insensitive Isc or resistance. Pre‐treatment of cells with 20μM PD98059 (MAPK inhibitor) prevented the effect of LPS (29 ± 3.0 μAcm−2, p<0.05 n = 6). LPS also shifted the amiloride concentration effect curve from an EC50 for amiloride of 6.8 × 10−7 M in control cells to 6.4 × 10−6 M in LPS treated cells p<0.05, n = 4. Using cell‐attached patch recordings we identified two constitutively active amiloride‐sensitive cation channels in the apical membrane of H441 cells. A 5pS highly Na+ selective ENaC–like channel and an 18pS non‐selective cation channel (NSC). Treatment with LPS increased the frequency of patches containing the less amiloride‐sensitive NSC by over 50%. We suggest that LPS alters the expression of ENaC/NSC channels in H441 cells resulting in a reduced amiloride‐sensitivity and Na+ conductance. Supported by The Wellcome Trust & BBSRC.
Different effects of Cyclosporine‐A and FK506 on aquaporin‐2 expression
Authors: Edemir Bayram , Rinschen Markus Matthias , Klokkers Jens , Schlatter Eberhard
Published: June 2021

Immunosuppressive drugs like Cyclosporine‐A (CyA) and FK506, both calcineurin inhibitors, are used to silence the immune system after transplantation. Both drugs have often complex side effects. For example long term treatment with CyA is followed by down‐regulation of aquaporin‐2 (AQP2) at least in the rat kidney. To investigate if CyA directly influences the expression of AQP2 we used primary cultured rat inner medullary collecting duct cells (IMCD), which endogenously express AQP2 and AVP‐mediated regulation of AQP2. IMCD‐cells were treated for different time‐periods with different concentrations of CyA or FK506. We used real‐time PCR to analyze mRNA expression levels of AQP2. Treatment with 0.5 μM, 1 μM or 5 μM CyA for 30 min and for 48 h was followed by concentration and time dependent down‐regulation of AQP2 expression. In contrast treatment with FK506 (1 μM, 30 min and 48 h) showed no effect on AQP2 expression. CyA had no effect on expression and activity of the tonicity element binding protein (TonEBP), a factor involved in AQP2 mRNA expression. But CyA suppressed also the cAMP induced expression of AQP2. In conclusion our results showed that CyA directly down‐regulates the expression of AQP2 in IMCD cells. Our data indicate that the underlying mechanism is calcineurin and TonEBP independent and that the down‐regulation of AQP2 is due to a disturbance of cAMP signaling induced by CyA.
Enhanced endothelial cell BKCa channel activity following chronic hypoxia in the rat
Authors: Riddle Melissa Anne , Paffett Michael , Walker Benjimen
Published: June 2021

Chronic hypoxia (CH) results in endothelium‐dependent blunting of myogenic reactivity and hyperpolarized endothelial cell (EC) resting membrane potential (Em) in rat resistance arteries. We previously demonstrated that myogenic reactivity is restored in CH arterioles by luminal administration of the large conductance Ca2+ activated K+ (BKCa) channel inhibitor iberiotoxin (IBTX), suggesting a role of EC BKCa channels in blunted vasoconstriction. To test the hypothesis that EC BKCa channel activity is enhanced following CH, we performed whole cell patch‐clamp experiments on freshly dispersed aortic ECs from control and CH (48 hr; 0.5 atm) rats. Whole K+ currents were significantly greater in ECs from CH compared to control rats. In addition, although IBTX was without effect in control cells, it normalized K+ current in EC from CH rats to levels of the control group. Furthermore, the BKCa activator NS1619 increased whole cell outward K+ currents only in the CH group In contrast to the electrophysiological data, BKCa α and β subunits were detected similarly by immunofluorescence in ECs from both groups. These results suggest that CH exposure results in activation of existing EC BKCa channels resulting in altered vasoreactivity.
Responses of cerebellar fastigial nucleus neurons to whole‐body rotations in vertical planes.
Authors: Miller Derek M , Cotter Lucy A , Gandhi Neeraj J , Schor Robert H , Huff Nicholas O , Shulman Joshua A , Raj Shiv G , Yates Bill J
Published: June 2021

The rostral portion of the fastigial nucleus (rFN) of the cerebellum has been demonstrated to participate in cardiovascular and respiratory regulation. This deep cerebellar nucleus is also known to receive and process vestibular inputs. Although a number of studies conducted in nonhuman primates have reported the responses of rFN units to body movements, little is known about the vestibular inputs to rFN neurons in conscious cats, a common animal model used in studies of vestibulo‐autonomic regulation. In the present study, activity was recorded from rFN neurons in conscious felines during rotations in vertical planes at frequencies of 0.02–2 Hz and amplitudes up to 15°. None of the units had firing related to eye movements. The response vector orientations for most neurons were closer to the pitch (head‐up or head‐down) plane than the roll (ear‐down) plane. The responses of over 60% of the neurons had gains that were relatively constant (increasing <3‐fold per decade) and phases that were near stimulus position across rotation frequencies. Since vestibulo‐autonomic reflexes are mainly elicited by graviceptive inputs produced by pitch rotations, the response properties of rFN neurons suggest that they could mediate these responses. Supported by NIH grants R01‐DC00693 and R01‐DC03732.
Chronic MC3/4R activation does not mimic the actions of leptin on baroreceptor sensitivity and heart rate regulation in diabetic rats
Authors: da Silva Alexandre A. , do Carmo Jussara M. , Freeman J. Nathan , Hall John E.
Published: June 2021

We recently showed that leptin restores baroreflex sensitivity (BRS), cardiac sympathetic tone (CST) and intrinsic heart rate (IHR) in streptozotocin (STZ) diabetic rats. Since most actions of leptin require functional melanocortin 3 and 4 receptor (MC3/4R) signaling, we tested whether chronic MC3/4R activation with Melanotan II (MTII) mimics the cardiovascular actions of leptin in diabetic rats. After a 5‐day control period, STZ was injected to create diabetes. Seven days after STZ injection, an ICV infusion of MTII (10 ng/hr) was started and maintained for 10 days. MAP and HR (measured by telemetry), cardiac sympathetic‐vagal balance, IHR (measured after propranolol and atropine injections) and BRS (phenylephrine and sodium nitroprusside injections) were determined during control, 7 days after STZ injection and on the 7th day of MTII infusion. Animals were pair‐fed the same amount consumed by leptin treated rats from our previous studies. Chronic MTII infusion plus pair‐feeding failed to normalize blood glucose and the bradycardia caused by diabetes (∼400 mg/dl and −90 bpm) and did not restore IHR (337±6, 276±7 and 272±8 bpm, for control, diabetes, and MTII respectively), CST (−75±8, −47±10, −49±12 bpm) or BRS (−2.6±0.3, −1.7±0.3 and −1.9±0.3 bpm/mmHg) to normal values. These results suggest that chronic MC3/4‐R activation can be offset by compensatory mechanisms normally suppressed by leptin. (NHLBI, AHA
Opposite regulation of ANP and renin in hyperleptinemic rats
Authors: Yuan Kuichang , Yu Jiahua , Kim Sun Young , Han Jeong Hee , Park Byung‐Hyun , Kim Sung Zoo , Kim Suhn Hee
Published: June 2021

The kidney expresses the leptin receptor, and various renal effects of leptin have been reported. This study is aimed to investigate the effects of leptin on regulation of body fluid. Sprague‐Dawley rats were received adenovirus containing the rat leptin cDNA (AdCMV‐leptin) or b‐galactosidase gene (AdCMV‐bGal) and their metabolism was observed for 2–4weeks. Food intake and body weight were markedly decreased in hyperleptinemic rats. However, no significant differences in water balance and urinary excretions of electrolytes were observed between hyperleptinemic rats and pair‐feeding rats. Plasma renin activity was increased whereas plasma ANP level was decreased. No significant change in ANP binding density in kidney was observed. The ratio between plasma cAMP and cGMP levels was decreased. In isolated perfused beating atria from hyperleptinemic rats, no differences in basal ANP release, contractility and ECF translocation were observed. The response of isoproterenol‐induced decrease of ANP release was similar. These results suggest that leptin may stimulate renin secretion rate from kidney, which was followed by water retention and suppression of ANP release. Supported by KRF‐2005‐204‐E0007 and the KOSEF (R01‐2006‐000‐10554‐0).
Momordica charantia (bitter melon) improves hepatic insulin signaling
Authors: Nerurkar Pratibha Vivek , Lee Yun Kyung , Adeli Khosrow , Nerurkar Vivek R
Published: June 2021

Momordica charantia (bitter melon, BM) has traditionally been used in the treatment of diabetes and its complications. Insulin resistance is characterized by resistance to insulin‐stimulated glucose uptake, leading to enhanced very low density lipoprotein (VLDL) secretion, and significant down‐regulation of hepatic insulin signaling as documented by attenuated phosphorylation of i54 nsulin receptor (IR), IR substrates (IRS‐1/IRS‐2) and PI3K, PKB/Akt. Our recent studies demonstrated that BM juice (BMJ) can significantly reduce apoB secretion, in human hepatoma cells, HepG2. The aim of our study was to evaluate the effects of BMJ on plasma apoB levels and hepatic insulin signaling cascade in mice fed high fat diet (HFD). Four to six weeks old female C57BL/6 mice were randomized into three groups of control, receiving regular rodent chow with 11 kcal% fat; high fat diet (HFD), receiving chow containing 58 kcal% fat; and HDF + 1.5% lyophilized BMJ. Our data indicates that BMJ lowers plasma apoB‐100 and apoB‐48 in HFD‐fed mice by improving the phosphorylation status of insulin receptor and its downstream signaling molecules. Investigating the mechanisms mediated by BMJ to ameliorate diabetic dyslipidemia may lead to identification of new molecular targets for dietary therapies. [Grant support by RCMI, NIH/NCRR, G12RR00361; Hawaii Community Foundation, 20041652 and NIH/NCCAM 1R21AT003719‐01A1].
Plasma cholesterol reduction in hamsters fed all barley, oat or wheat breads with and without soluble cellulose
Authors: Hong Yun‐Jeong , Kim Yookyung , Turowski Maciej , Anderson Kerr , Yokoyama Wallace
Published: June 2021

All oat and barley breads, commercially unavailable, were developed and evaluated for their cholesterol lowering properties. The two cereal grains, oat and barley, contain significant amounts of beta‐glucan soluble fiber. FDA has allowed heart health claims if serving size meets the prescribed minimal amounts of whole grain oat or barley. Oat and barley breads were formulated by the addition of 5% hydroxypropylmethylcellulose (HPMC) as a gluten replacer and as an additional source of soluble fiber. The breads were freeze dried and ground into a powder that was incorporated into diets. Hamsters fed a high fat diet including 0.1% cholesterol were fed diets containing wheat, oat and barley breads with and without HPMC. Compared to the animals fed the control diet containing 5% microcrystalline cellulose wheat, oat and barley fed animals had about 30% lower LDL cholesterol. The addition of 5% additional HPMC further reduced LDL cholesterol 50% in diets containing oat and barley but not wheat compared to control. The plasma cholesterol profile of oat bread containing soluble cellulose resembled plasma from animals fed low fat (chow) diets. Liver weight and lipids, fecal sterols and bile acids from cereal grain only and HPMC supplemented hamsters were also correlated with diet. Whole grain and flours containing bran and germ, including wheat, were effective in lowering plasma cholesterol.
Expression profiling of hepatic genes associated with lipid metabolism in nephrotic rats
Authors: Guan Youfei , Zhou Yunfeng , Zhang Xiaoyan , Chen Lihong , Wei Mingfen , Zhang Yahua
Published: June 2021

Hyperlipidemia is one of the major features of nephrotic syndrome (NS) and the underlying mechanisms remain largely uncharacterized. The present study was designed to examine the gene profile associated with lipid metabolism in the livers of nephrotic rats. NS was made in male rats (n=6) receiving sequential i.p injections of puromycin aminonucleoside. An Affymetrix assay, the quantitative PCR, and immunoblot identified 12 genes associated with cholesterol metabolism and 9 genes associated with fatty acid metabolism. Eight genes involved in cholesterol metabolism including Apo A‐I, Acly, Acat, Mpd, Fdps, Ss, Lss, and Nsdhl were significantly upregulated. Four genes involved in fatty acid biosynthesis including Acc, FAS, ELOVL 2 and 6 and three genes critical for triglyceride biosynthesis including Gpam, Agpat 3 and Dgat 1 were significantly induced, while two genes involved in fatty acid oxidation including Dci and MCAD were downregulated. Expression of several genes leading to SREBP‐1 activation was also altered. Collectively, induction of genes involved in cholesterol biosynthesis may be responsible for hypercholesterolemia, while upregulation of genes participated in fatty acid and triglyceride biosynthesis, and reduction of genes involved in fatty acid oxidation may contribute to hypertriglyceridemia in nephrotic rats. Overactivation of SREBP‐1 pathway may represent an underlying mechanism.
Neural circuits involved in glucoprivation‐evoked inhibition of metabolism in brown adipose tissue (BAT)
Authors: Madden Christopher J. , Morrison Shaun F.
Published: June 2021

Glucoprivation suppresses sympathetic activation of metabolism in BAT, however the neural circuits responsible for this effect are not known. In urethane/chloralose anesthetized rats (n=10), cooling (core temperature: 35.8 ± 0.2°C) resulted in an increase in BAT sympathetic nerve activity (SNA: +569 ± 92% control). The glucoprivic agent, 2‐deoxy‐D‐glucose (2‐DG; 200mg/ml/kg, iv) reversed the cooling evoked activation of BAT SNA (nadir within 5 minutes of 2‐DG: 113 ± 11% of control, n=4). Microinjection of 5‐thio‐D‐glucose (5‐TG) into the rostral ventrolateral medulla (RVLM) ipsilateral to the recorded BAT nerve also reversed the cooling evoked activation of BAT SNA (nadir: 100 ± 2% of control at 4.6 ± 1.8 minutes after the microinjection of 5‐TG, n=6). In three rats transection of the neuraxis at the pontomedullary junction increased BAT SNA (+983 ± 277% of control). Administration of 2‐DG (200mg/ml/kg, iv) did not attenuate this activation. These results suggest that neurons within the RVLM may play a role in the glucoprivic inhibition of BAT SNA and metabolism via neural pathways which include neurons located rostral to the medulla. Supported by: DK065401 (CJM) and DK57838 (SFM).