bluuebunny/crossref_metadata_2025 huggingface dataset:
Critical role of increased angiotensin II in the maternal placenta in a human transgenic rat model of preeclampsia
Authors: Brosnihan K. Bridget , Dechend Ralf , Hering Lydia
Published: June 2021

The pregnant female human angiotensinogen (hAGN) transgenic rat (TGR) mated with the male hrenin (hREN) TGR has been shown to be a model of preeclampsia with increased blood pressure, proteinuria, and placenta alterations of edema and necrosis. The reverse mating of female hREN x male hAGN does not show preeclamptic features. Because the maternal influence is critical in determining whether preeclampsia occurs, we characterized the Ang peptide content of the mesometrial triangle (MT) of the maternal placenta and the fetal labyrinth placenta (Pla). Three groups of pregnant rats from the following matings [SD x SD, hRENxhAGN, and hAGNxhREN] were studied on day 21 of gestation. Animals were sedated with ether and the placentas were removed, dissected into MT and Pla, and snapfrozen until analysis. The Figure shows that Ang‐(1‐7) is the predominate peptide in the MT and Pla of both TGR pregnant groups, is signficantly increased in both TGR pregnant rats, and does not differ between maternal or fetal placenta or TGR pregnant groups. In the hAGNxhREN TGR Ang II is significantly increased in the Pla and MT, but in the hRenxhAGN Ang II is not changed in the MT. The marked contrast in the expression of Ang II in the MT of the preeclamptic model vs the non‐preeclamptic groups suggests that the maternal placenta could be critical in contributing to the preeclamptic condition. This research was supported by NHLBI P01‐HL51952.
Effect of prenatal Betamethasone (B) exposure on sodium excretion in response to intrarenal infusions of Angiotensin II (Ang II) and its antagonist in male sheep.
Authors: Bi Jianli , Contag Stephen , Figueroa Jorge , Chappell Mark , Rose James C
Published: June 2021

Prenatal exposure to clinically relevant doses of Betamethasone (B) reduces nephron number in adult sheep, but its effects on renal function are unknown. Therefore, we evaluated if prenatal B exposure alters sodium excretion (UNa) in response to intrarenal infusions (IRI) of angiotensin II (AII) with or without its antagonists (Candesartan (C) [type 1] and PD123319 (PD) [type 2] receptor blockers) in adult male sheep.

1.5 year old male sheep which were exposed to B (n=6) or vehicle (V, n=6) at 80‐81 days gestation and born at term received renal arterial infusions of AII (1ng/kg/min) with or without antagonists for 24 hours. Na excretion (UNa) was measured during the infusions. Analysis of variance (ANOVA) and T‐test were used for data analysis.

AII infusion decreased UNa from 0.39±0.07 to 0.14±0.04mM/hr/Kg (p<0.01) in B sheep, and from 0.37±0.06 to 0.18±0.05 in V sheep. AII plus PD decreased UNa in V sheep but not in B sheep (decreases were ‐0.27±0.09 in V sheep and ‐0.09 ±0.04mM/hr/Kg in B sheep, p=0.059). In presence of C, UNa initially decreased in both groups (from 0.39±0.09 to 0.13±0.03 in V and from 0.26±0.07 to 0.12±0.03mM/hr/Kg in B sheep), and then increased. The increase was 0.39± 0.09 in vehicle sheep and 0.25±0.05 in B (p=0.05).

The data suggest that the natriuretic response to stimulation of the AII type 2 receptor is converted to an antinatriuresis by prenatal B exposure. Supported by NIH grants HD 047584 and HD17644
The increase in renal sodium excretion in response to Angiotensin II infusion in exercised female rats is dependent on a rise in renal perfusion pressure.
Authors: Janssen Karmon M , Knoblich Penny R
Published: June 2021

Prior studies in this lab have shown that chronically exercised female spontaneously hypertensive rats (SHR) and Wistar‐Kyoto (WKY) rats excrete a greater amount of sodium in response to angiotensin II infusion than do sedentary rats. The current study determined if the difference in renal sodium excretion persisted when the renal perfusion pressure (RPP) was held constant. Female SHR and female WKY rats were separated into sedentary and exercise groups at 4 weeks of age. The exercise group voluntarily exercised for at least eight weeks using an exercise wheel and time/distance monitor. At 13 weeks of age or older, rats were anesthetized and catheters were placed into the jugular vein, and carotid and femoral arteries. A noose, placed around the abdominal aorta, was continually adjusted to maintain a constant RPP during Ang II infusion. Ang II was infused at 0, 0.125, 0.5, and 2µg/ml saline, each for 15 minutes. MAP and RPP were measured continually. Urine was collected during each 15 minute period and analyzed for sodium excretion. Sodium excretion did not significantly increase with the Ang II infusions when RPP was held constant, and no difference was found between the exercised and sedentary rats. The increase in renal sodium excretion observed in exercised female rats compared to sedentary appears to be due to a difference in the pressure natriuresis response. Funding: Department of Biological Sciences, MSU, M.
The effects of guanylyl cyclase‐G on ischemia‐reperfusion induced intestinal damage
Authors: Lo Hui‐Chen , Yang Ruey‐Bing
Published: June 2021

Recently, we demonstrated that guanylyl cyclase (GC)‐G, a novel membrane bound receptor GC, is expressed in the kidneys and plays critical apoptotic and inflammatory roles during renal ischemia and reperfusion (I/R). Using GC‐G gene knockout (KO) mice, we further investigated the role of mGC‐G gene on modulating the intestinal I/R‐induced intestinal injury and systemic and local inflammatory responses. Sham operation and 45 min intestinal ischemia with 3h, 6h, 12h, or 24h reperfusion were performed in both male C57BL/6 wild‐type (WT) and KO mice, respectively. Our results showed that KO mice had significantly decreased villus density and height, crypt depth, jejunal DNA content, and plasma nitric oxide (NO) compared to WT mice. The villus height was significantly decreased at 3h reperfusion and was reversed at 6h and 12h reperfusion in WT and KO mice, respectively. Plasma NO was significantly decreased in WT mice and increased in KO mice with intestinal I/R. Intestinal I/R induced elevation in plasma and jejunal IL‐6 were approximately 2‐ to 3‐fold greater in WT mice than in KO mice. In contrast, jejunal IL‐6 mRNA was significantly greater in KO mice than in WT mice and was 2‐ to 12‐fold greater in KO mice than in WT mice during the intestinal I/R period. Moreover, intestinal I/R significantly elevated caspase‐3 activity, an indicator of apoptosis, in the jejunum in WT mice, not in KO mice. In summary, our results suggest that mGC‐G plays specific roles in maintaining jejunal morphology and inducing systemic and local inflammatory responses in mice with intestinal I/R.
Curing multiple organ dysfunction syndrome (MODS) by adjusting intestinal tract dysbacteriosis using Radix codonopsis Polysaccharides
Authors: Yang Jingyun , Wang Molin , Ma Shuxia , Yang Jixin , Li Liqiu , Kang Yu‐Ming
Published: June 2021

Radix Codonopsis Polysaccharide (RCP) is a plant extraction, which got beneficial effects on multiple organ dysfunction syndrome (MODS). We chose to hypothesize that RCP would treat intestinal infection and endotoxemia of MODS by adjusting intestinal tract dysbacteriosis, repairing intestinal barrier function, and controlling translocation of bacteria and toxins. MODS was induced in 60 Sprage‐Dawley rats by intestinal ischemia‐reperfusion. These rats were treated with RCP and Bacillus bifidus praeparatum (LZCL) respectively. Bacteria quantity and location, plasma endotoxin, intestinal contents level of SIgA and number of CD 4+, CD 8+ cells were measured. RCP group and LZCL group fostered the adoption of helpful bacteria, while suppressed the growth of harmful bacteria. RCP reduced bacterial translocation, controlled endotoxemia, and improved other related pathological and immunological targets. Compared with the control group, bacterial translocation of treated group was alleviated, and serum endotoxin was reduced. The level of SIgA in treated group was higher than that in control group. The model group exhibited serious dysbacteriosis. Bacteria and toxins translocation are the key factors in the development of MODS. These results suggest that RCP could prevent MODS though regulating intestinal dysbacteriosis.
Probiotics prevent death caused by Citrobacter rodentium infection in neonatal mice via T cells
Authors: Gareau Mélanie G , Wine Eytan , Reardon Colin , Perdue Mary H , Sherman Philip M
Published: June 2021

Intestinal infections cause morbidity and mortality, particularly in young children. C. rodentium challenge of mouse pups is used as a model to study neonatal enteric infections.

AIMS

Determine if neonatal mice become sick after infection with C. rodentium and if probiotics can ameliorate disease.

METHODS

Mouse pups were infected with C. rodentium by orogastric gavage at 14d of age and some pups pre‐treated with probiotics daily 1 wk prior to infection and onward. For others, the dam was given probiotics in drinking water to determine if probiotics act indirectly. Immune‐deficient (Rag1−/−; JH) pups were used to study the role of adaptive immunity in mediating the effects of probiotics on infection.

RESULTS

At 10d post‐infection (PI), infected pups lost weight and displayed colonic epithelial cell hyperplasia, compared to sham‐infected animals. By 12 days PI, death occurred in infected pups (C. rodentium; 75% vs sham; 0%). Pre‐treatment with probiotics prevented death (probiotics; 17%), reduced epithelial hyperplasia and weight loss. Probiotics did not prevent death when administered either to the mother or Rag1−/− pups, but JH, B cell deficient pups survived.

CONCLUSION

C. rodentium infection causes death in mouse pups. Probiotics prevented these effects, but only in the presence of T cells. Probiotics may serve as an option for therapy in newborns at high risk of developing infections.
Polyamines regulate integrin mediated signaling during IEC‐6 cell attachment and spreading
Authors: Elias Bertha C. , Ray Ramesh M. , Johnson Leonard R.
Published: June 2021

Polyamines have been shown to play a pivotal role in normal cellular processes including cell division, migration and apoptosis. Migration is a two‐step process that consists of cell attachment followed by spreading. Cells depleted of polyamines are slow to attach and spread during integrin β3 activation, however the signaling mechanism involved is still unclear. In this study, we have examined the role of polyamines in the signaling process. Rat intestinal epithelial cells (IEC‐6) were plated on fibronectin‐coated plates in the presence or absence of α‐difluoromethylornithine (DFMO) to inhibit polyamine synthesis. Their attachment over time was observed microscopically, and cell lysates were analyzed by Western blot analysis. We observed that (a) polyamine depletion quantitatively decreased the attachment of cells (b) control cells began to spread within 2 h, while spreading was delayed in polyamine‐depleted cells (c) focal adhesion kinase (FAK) as well as cSrc autophosphorylation was greatly reduced in polyamine‐depleted cells leading to a decrease in Rac‐1 activity during attachment and spreading (d) the Tiam1 inhibitor, NSC‐23766 inhibited Rac‐1 activity. We conclude that polyamines regulate the integrin/Src/FAK complex formation leading to activation of Rac‐1 GEFs. (Supported by NIDDK grant DK‐052784)
BNP Alters Myosin Type II Distribution in the Mouse Intestinal Villi
Authors: Addisu Anteneh , Gower William R. , Dietz John R.
Published: June 2021

B‐type natriuretic (BNP) decreases absorption from the GI tract. Since non‐muscle myosin type II is known to regulate epithelial tight junctions and paracellular absorption, we determined if BNP effects distribution of non‐muscle myosin in intestinal villi. We treated mice with a 10ng/g intravenous bolus of BNP followed by a 1ng/g/minute infusion for 30 minutes or vehicle. The tissue was immunostained for non‐muscle myosin type II and examined using fluorescence and electron microscopy (EM). Fluorescence microscopy revealed markedly enhanced distribution of non‐muscle myosin at the crypts and villus core of the jejunum and ileum of BNP treated mice as compared to vehicle. EM revealed that the microvilli of BNP treated mice lost the distinctive appearance of contracting microvilli as compared to vehicle. Such an appearance was previously shown to lead to decreased paracellular permeability of epithelia. The result supports the hypothesis that elevated plasma BNP to pathophysiological levels makes the tight junctions of intestinal microvilli less permeable to water and small solutes. We postulate that decreased paracellular water and salt movement while allowing continued trans‐cellular transport at the apical membrane of intestinal microvilli may be how the failing heart attempts to modulate preload by regulating water and salt absorption (Sup: VA Merit Award to W. Gower & NSF/IGERT, Grant to A. Addisu).
Regulation of NADPH oxidase 5 activity by PKC and MAPK pathways
Authors: Pandey Deepesh , Fulton David
Published: June 2021

NADPH oxidase 5 (Nox5) is a unique Nox isoform in that its activity is independent of the protein:protein interactions that coordinate the activation of other Nox isoforms and is instead regulated by cytosolic Ca2+ levels. Previously, we showed that PMA induces the sustained activation of Nox5 that was dependent on the phosphorylation of specific serine and threonine residues which control the calcium‐sensitivity of the enzyme. However, the specific kinase(s) mediating the phosphorylation of Nox5 are not known. We hypothesize that Nox5 is activated by the coordinated phosphorylation of MAPK and PKC pathways and that different isoforms of PKC are required for site‐specific phosphorylation. Inhibition of Mek1 using PD98059 and U0126 significantly reduced the phosphorylation and activity of Nox5 in response to PMA. Dominant negative MEK1 and knockdown of endogenous MEK1 using shRNA also inhibited Nox5 activity in response to PMA. However, a constitutively active form of MEK1 failed to increase Nox5 activity in the absence of PMA stimulation. These results suggest that the MEK‐Erk1/2 pathway is necessary but not sufficient to regulate Nox5 activity. Future studies will identify whether Erk or other kinases can directly phosphorylate and activate Nox5.
Impact of Hydroxyl Radicals on Calcium Handling and Myofilament sensitivity in Isolated Myocardium
Authors: Hiranandani Nitisha , Janssen Paul ML
Published: June 2021

Objective

Hydroxyl radicals (OH*) are involved in the pathogenesis of reperfusion injury, which is observed in many clinical situations including acute heart failure, stroke and myocardial infarction. Two different sub‐cellular defects are involved in the phenotypical OH*‐injury, deranged calcium handling and alterations of myofilament responsiveness.

Method

We temporarily delineated the impact of these two mechanisms at different time points on OH* injury. In order to elucidate the injury response, we measured the amplitude and kinetics of calibrated intracellular Ca2+ transients and twitch contractions simultaneously using ionophoretically loaded bis fura‐2 and potassium contracture.

Results

After the exposure of OH* in rabbit trabeculae injury initially there is a marked increase in resting tension (6.27± 0.71 to 46.4 ± 3.49 mN/mm2) in parallel with an increase in diastolic calcium (136.6 ± 7.65 to 779.4 ± 64.63 nM) (n=8). We observed these parameters until a new steady state level was reached. At this new baseline, diastolic calcium returned to near‐normal levels to pre‐OH* levels whereas diastolic force declines, but not all the way to pre‐OH* levels, indicating a sustained injury. We also observed myofilament sensitivity before and after OH* injury through potassium contracture. Myofilament response was increased at the new baseline after OH* injury as compared to the pre‐OH* state.

Conclusion

This indicates the acute injury that occurs after OH* exposure is mainly if not entirely is due to calcium overload in acute phase while sustained myocardial dysfunction is mainly due to the altered/increased myofilament responsiveness.
Kudzu Polyphenols Improve Glucose Regulation in OB/OB Mice
Authors: Peng Ning , Prasain Jeevan K. , Carlson Scott H. , Dai Yanying , Wyss J. Micheal
Published: June 2021

Our previous studies demonstrated that Radix puerariae, the primary polyphenol in kudzu root, can decrease arterial pressure and improve glucose regulation in spontaneously hypertensive rats and CD‐1 mice. This study tested the hypothesis that dietary kudzu root extract supplementation could also improve blood pressure, glycemic control and insulin sensitivity in ob/ob mice, an animal model that mimics some components of type 2 diabetes. Two‐month‐old male ob/ob and lean control mice were placed on a polyphenol‐free diet with or without 0.2% kudzu root extract added (23% puerarin). After 3 months on the diet, oral glucose tolerance was tested. In the ob/ob mice, kudzu root extract supplementation significantly improved glucose tolerance. Total and peak responses were significantly lower in ob/ob (but not lean control) mice receiving the supplement (peak response in ob/ob 281 mg/dl [kuzu], 401 mg/dl [control]). Following 4 additional months on the diets glucose and insulin tolerance were tested. In ob/ob (but not lean control mice), dietary kudzu root extract supplementation improved glucose tolerance and insulin sensitivity. The supplementation consistently decreased arterial pressure by 6‐10 mm Hg in both ob/ob and lean control mice. These results suggest that kudzu polyphenols improve glycemic control and insulin sensitivity in ob/ob mice and thus may provide similar protection in type 2 diabetics.
Traditional ‘rate‐limiting’ molecular indices of gluconeogenesis do not correlate with flux through the pathway in vivo
Authors: Ramnanan Christopher Jerome , Rivera Noelia , Edgerton Dale , Neal Doss , Farmer Ben , Lautz Margaret , Cherrington Alan
Published: June 2021

Hyperglycemia in type 2 diabetes is due, in part, to elevated gluconeogenesis (GNG). This has been attributed, based largely on in vitro models, to increased mRNA transcription of supposed rate‐limiting enzymes PEPCK and G6Pase. However, in vivo metabolic substrate analysis in man and dog indicate that GNG flux to G6P varies minimally within the physiological range of insulin. We propose that GNG mRNA and protein expression would be decreased during hyperinsulinemic (2.0 mU/kg/min), euglycemic conditions in 24 h fasted conscious dogs subjected to a pancreatic clamp, but that the time course of expression would not correlate with functional GNG flux to G6P. Arterial and hepatic sinusoidal insulin levels increased 8‐fold and glucagon was clamped at basal levels. At 0.5 h, GNG flux to G6P was decreased 50% due to the suppression of lipolysis and a switch from net hepatic lactate uptake to output. Liver FOXO1 phosphorylation was increased 1.9‐fold and GNG enzyme mRNA expression was decreased ~40‐50% at 0.5 h but PEPCK protein levels did not change. By 4 h, GNG mRNA expression was decreased ~80%, and PEPCK protein was reduced by 45%, but GNG flux to G6P had stabilized at near‐basal levels. Thus, while insulin can inhibit GNG gene transcription and protein expression in vivo, these changes do not result in functional changes in substrate flux in the GNG pathway, indicating PEPCK was not a rate‐limiting marker for GNG in vivo.
Endotoxemia induces a differential metabolic response in the heart and liver as determined by metabolomic analysis
Authors: Bateman Ryon M , Obata Reiichiro , Nagahata Yoshiko , Ohmura Mitsuyo , Hishiki Takako , Suematsu Makoto
Published: June 2021

The systemic inflammatory response to bacterial infection (sepsis) alters microvascular and mitochondrial function; yet, little is known about effects on metabolism. The objective was to determine how intermediary carbohydrate metabolism changes during the onset of sepsis. C57bl/6 mice (30‐35g) were injected intraperitoneally with lipopolysaccharide (LPS, 40mg/kg). Six hours post LPS, C13‐pyruvate (a key intermediate metabolite) was administered subcutaneously. At 20,40 and 60 minutes, heart and liver tissues were harvested and stored at ‐80C. Capillary electrophoresis ‐ mass spectrometry (CE‐MS) quantified metabolic intermediates. Labeled metabolites were normalized to non‐labeled metabolite levels. Pyruvate was preferentially metabolized by the heart, but uptake was delayed in both liver and heart relative to control. Lactate, TCA intermediates (citrate, a‐ketoglutarate, malate), G1P and UDP‐glucose increased in the endotoxemic heart. By contrast, glutamate and B‐hydroxybutyrate increased in endotoxemic liver. Fluxome analysis of pyruvate metabolism suggests the endotoxemic heart stores glucose, while the liver generates alternative energy sources. Funding source, Mext Japan.
Key role of CFTR for all modes of intestinal HCO3− secretion
Authors: Singh Anurag Kumar , Zheng Wen , Sjöblom Markus , Seidler Ursula
Published: June 2021

Background

CF patients suffer from a variety of gastrointestinal problems, which may all be directly or indirectly linked to the inability of the intestinal mucosa to secrete HCO3− and to inhibit Na+/H+ mediated fluid absorption and proton secretion.

Aim

To delineate the dependency of different modes of intestinal HCO3− secretion on CFTR expression.

Methods and Results

We studied acid‐, agonist‐, and HCO3− stimulated, as well as Cl−‐dependent HCO3− secretion in the CFTRtm1cam and WT murine duodenum in vivo. NHE3 and Slc26a6‐deficient mice were used for selected questions. In WT mice, luminal acid, forskolin, heat‐stable E. coli enterotoxin (STa), PG E2, carbachol, and an increase of blood HCO3− all stimulated duodenal HCO3− secretion in anesthetized WT but not in CFTRtm1cam mice. Pharmacological inhibition or genetic ablation of NHE3 resulted in a significantly higher basal HCO3− secretory rate, which was electroneutral and therefore due to an unmasking of apical Cl−/HCO3− exchange activity. Accordingly, Slc26a6 as well as Slc26a3 ablation attenuated S1611‐induced JHCO3‐. In the absence of CFTR, electroneutral NaCl absorptive rates were similar to wt rates, but S1611 induced virtually no increase in HCO3− secretion.

Conclusion

This indicates that the apical anion exchangers Slc26a6 and Slc26a3 need proton recycling via NHE3 to operate in the Cl− absorptive mode, and Cl− exit via CFTR to operate in the HCO3− secretory mode.
β‐adrenergic activation of Cl secretion and K secretion in guinea pig distal colon acts through distinct signaling pathways
Authors: Halm Susan , Zhang Jin , Halm Dan
Published: June 2021

Cl and K secretion measured as short‐circuit current (Isc) in isolated mucosa from guinea pig distal colon were stimulated by epinephrine (epi). Cl secretory Isc was transient lasting ~5min, while the negative Isc consistent with K secretion persisted indefinitely. 2'5′‐dideoxyadenosine (ddAdo; inhibits adenylyl cyclase, AC) reduced Cl sec whereas rolipram (inhibits phosphodiesterase) caused an increase; neither altered K sec. Peptide‐YY (PYY; acts via receptor inhibiting AC) reduced Cl sec without altering K sec. Pertussis toxin (inhibits G‐protein‐αi) eliminated PYY inhibition. cAMP levels in colonic mucosa increased at 30s after epi followed by a decrease at 10min, but not to control levels. Forskolin [0.1μM] (stimulates AC and K sec) increased cAMP comparable to epi 10min. PYY inhibited the rise in cAMP at 30s epi; ddAdo blunted the rise in cAMP. 8Br‐cAMP [100μM] stimulated K sec, and at 1mM increased Cl sec. Signaling could act via the cAMP‐dependent effectors protein kinase A (PKA) or Epac. H‐89 (inhibits PKA) reduced Cl sec, but not K sec. 8CPT‐2Me‐cAMP (Epac selective analog) partially stimulated K sec at 100μM. Epi did not activate Rap‐1 (Epac sensitive G‐protein). Thus, β‐adrenergic stimulation activated Cl secretion via a cAMP dependent pathway involving PKA, and activated K secretion via a pathway independent of PKA and Rap1 but possibly involving cAMP‐dependent exchange factor Epac. [NIH DK65845]
Stretch‐Induced Transcriptional Regulation of the Large Conductance Calcium‐Activated Potassium Channel (BK)‐β4 Subunit in Human Mesangial Cells (MC)
Authors: Irsik Debra L , Liu Liping , Grimm P Richard , Holtzclaw J David , O'Neil Roger G , Sansom Steven C
Published: June 2021

Renal nephropathy is a complication of Type 2 Diabetes. In hyperinsulinemic mice, hyperfiltration transmits stretch to MC. A stretch‐induced increase in [Ca2+]i can activate the calcineurin/ Nuclear Factor of activated T cells (NFAT), a transcription factor that associates with GATA in smooth muscle. In physiological conditions, the pore‐forming BK‐α interacts with the β1 to regulate mesangial contractile tone. However, the mouse glomerulus also contains the β4 and only the glomerular β4 is up‐regulated (immunohistochemical staining) in hyperinsulinemic mice. Our underlying hypothesis is that stretch due to hyperfiltration increases the expression of β4 and that BK‐α /β4 causes a sustained elevation in [Ca2+], causing MC expansion. Using nested RT PCR, following 10% cyclical stretching (24 h; Flexercell), β4 expression was 6‐fold > (p< 0.05) and β1 was 50% < control. β4 protein level doubled after 2 hours of stretch. Using the Transcription Element Search System of the University of Pennsylvania, (TESS) we identified 6 GATA and 12 NFAT sites on the β4 promoter. Through ChIP, we confirmed that NFATc3 and GATA4 bind to the β4 promoter 1760 bases upstream of the transcription start site, yielding a 175 bp fragment identified by sequencing as β4.

Conclusion

(1) Stretching MC results in transcriptional up‐regulation of β4 and down‐regulation of β1 and (2) NFATc3 and GATA4 bind β4 at a consensus promoter site.
Expression, function and regulation of NH2‐terminal Sgk1 variants
Authors: Raikwar Nandita S , Kelley Elizabeth A , Snyder Peter M , Thomas Christie P
Published: June 2021

Sgk1 is an aldosterone induced kinase that regulates epithelial sodium channel (ENaC)‐mediated Na+ transport. We identified N‐terminal variant Sgk1 isoforms: Sgk1_i2 and Sgk1_i3 that arise by alternate transcription initiation from human Sgk1 transcripts, Sgk1_v2 and Sgk1_v3 respectively. These isoforms are more resistant to proteosomal degradation as compared to Sgk1. Furthermore, aldosterone stimulates Sgk1_v3 mRNA expression in a collecting duct cell line, mpkCCDc14, indicating that this 5' variant may have a role in mineralocorticoid‐regulated Na+ reabsorption. When expressed together with ENaC subunits in FRT epithelia, Sgk1_i2 or Sgk1_i3 stimulate amiloride‐sensitive Na+ transport an effect that appears to be mediated via the kinase domain of Sgk1. Collectively, the data demonstrate that N‐terminal variants of Sgk1 are more stable, functionally more active and are expressed and regulated in collecting duct epithelia raising the possibility that these variants may have a physiological role.

NHLBI and VA Merit
Inhibition of ENaC activity by the Influenza Virus M2 proton channel
Authors: Lazrak Ahmed , Iles Karen , Noah Diane L , Noah James , Matalon Sadis
Published: June 2021

The mechanisms by which replicating influenza viruses decrease the activity of ENaC have not been elucidated. Herein we show that the influenza transmembrane protein (M2), a hydrogen channel, decreases levels and activity of ENaC in Xenopus oocytes, H441, and A549 cells transfected with a M2‐GFP cDNA. The regions of M2 protein responsible for this effect were localized to two separate portions of C‐terminus, between amino acids 52‐62 and 78‐82. This effect did not require M2 activation by low pH, and was prevented by MG‐132, a proteasome inhibitor. Furthermore, M2 has no effect when co‐expressed with ENaC bearing the Liddle mutations. Surface biotinylation of oocytes indicates that ENaC levels in cell membranes are reduced in the presence of M2. Pre‐treatment of both Xenopus oocytes and cells with GSH‐ester prevented the deleterious effects of M2 on ENaC. Together, these data suggest that M2 protein increases free radical production which results in ENaC endocytosis and proteasomal degradation by promoting increased ENaC ubiquitination. These results are the first indication that M2 regulates host ion channel function and suggest a mechanism for the influenza‐induced rhinorrhea and life‐threatening alveolar edema in humans.

This Research was supported by NIH HL311979 grant to Sadis Matalon and NIH AI071393 grant to James Noah.
PKC isozymes differentially regulate BKCa channels in pulmonary arterial smooth muscle
Authors: Barman Scott A , White Richard E , Meadows Mary L , Zhu Shu
Published: June 2021

Large conductance, voltage‐ and calcium‐activated potassium (BKCa) channel modulation is important in the regulation of pulmonary arterial pressure. Although protein kinases influence BKCa channel activity in vascular smooth muscle, little is known about the effect of protein kinase C(PKC) on BKCa channel activity in pulmonary arterial smooth muscle. A novel finding from our laboratory showed that a purified PKC (2nM) catalytic isozyme mixture activated BKCa channels in pulmonary arterial smooth muscle cells (PASMC) from the Sprague‐Dawley rat (SDR). Based on the presence of western blot expression in SDR PASMC, we assessed the direct effect of specific catalytic PKC isozymes on BKCa channels in SDR PASMCs using the inside‐out patch configuration. PKC[epsilon](3nM) activated BKCa channels significantly, but neither PKC[alpha](5nM) nor PKC[delta](8nM) showed any stimulatory effect on BKCa channels when 100nM Ca2+ was present. The effect of PKC[alpha] and PKC[delta] on BKCa channels was also examined when high (100[mu]M) Ca2+ was present in the inside‐out patches. PKC[alpha] caused short‐term inhibition of channel activity, while PKC[delta] had no inhibitory effect on Ca2+ ‐mediated BKCa channel activation. These results suggest that different signaling pathways involving specific PKC isozymes exist in pulmonary arterial smooth muscle to regulate BKCa channel function. Supported by NIH and AHA.
Effects of endurance running training and diet control on bone development and metabolism in young male rat
Authors: Chang Ming‐Yao , Huang Tsang‐Hai , Yang Ai‐Lun , Lin Che‐Fu , Lin Hsin‐Shin
Published: June 2021

Objective

The aim of this study was to investigate how endurance running and diet control affect growing bone.

Methods

Forty‐eight male Wistar rats (7 weeks old) were randomly assigned into four groups, which were control group (CON, n=12), high intensity running group (HIGH, n=12), low intensity running group (LOW, n=12), and diet control group (DCON, n=12). Animals in the High and Low groups underwent treadmill running 5 days/week for 8weeks. The DCON rats received mean quantity of chow consumed by exercise groups. Blood and bone samples were collected and stored for further analysis of serum marker, dynamic histology and geometry.

Results

After an eight‐week exercise training, body weight gain of the CON group was significantly higher than other three groups. Regarding dynamic histology assay, MS/BS (mineral surface/bone surface) did not show difference among groups. The CON group was numeric but not significantly higher in long bone cross‐sectional parameters. Tibiae length were higher in the CON group than other groups (p<.05). Serum calcium, phosphorus and insulin‐like growth factor‐I (IGF‐I) was significant lower in the DCON group (p<.05).

Conclusions

1) Similar to human studies, endurance running rats showed equal or somewhat lower values in bone tissue measurements, but no negative results revealed on bone turnover. 2) Diet control might cause an un‐equivalent physiology status in non‐exercise rats, which may not be appropriate to be set as a normal control group.

Grant supported by National Science Council (NSC‐97‐2410‐H‐006‐083‐MY2, Taiwan).
Oxidative stress upregulates renal Angiotensin II type I receptors via activation of transcription factor nuclear factor‐kappaB
Authors: Javkhedkar Apurva Arvind , Banday Anees Ahmad , Lokhandwala Mustafa F
Published: June 2021

Regulation of redox homeostasis is associated with normal functioning of cells, whereas oxidative stress is seen with various cardiovascular disorders like hypertension. Angiotensin II (Ang II), acting via Ang II type I (AT1) receptors, has known effects on the renal proximal tubular sodium transport, thus plays an important role in regulating blood pressure. Oxidative stress is also known to activate redox sensitive transcription factor NF‐kappaB and evidences indicate that AT1 receptor gene posses binding site for this transcription factor. Herein, we aim to investigate if oxidative stress upregulates renal AT1 receptors through NF‐kappaB activation. Human proximal tubular (HK‐2) cells were incubated with a pro oxidant L‐buthionine sulfoximine (BSO), an inhibitor of glutathione, for 48 hrs.

Results

Compared to control, there was an increase in levels of protein nitrotyrosination indicating oxidative stress in BSO treated HK‐2 cells. There was also activation of NF‐kappaB, upregulation of AT1 receptor protein, and increased Ang II‐induced Ca2+ signaling. Furthermore, treatment with pyrrolidine dithiocarbamate, a NF‐kappaB inihibitor, blocked NF‐kappaB activation, abolished AT1 receptor upregulation and exaggerated Ang II induced Ca2+ signaling.

Conclusion

These results suggest that oxidative stress upregulates renal AT1 receptors via activation of transcription factor NF‐kappaB.

Grant: AHA‐SDG‐0835428N
VACM‐1, a cul 5 Gene, Regulates Aquaporin‐1 Levels
Authors: Stodola Joseph L , Burnatowska‐Hledin Maria
Published: June 2021

VACM‐1/cul 5 cDNA expression in Cos‐1 cells inhibits cell growth. The mechanism of this regulation involves MAPK phosphorylation, but the direct target of VACM‐1 activity is not known. After identifying VACM‐1 as a regulator of AQP1 protein and mRNA levels in vitro, we sought to elucidate effects of VACM‐1 expression on AQP1 in vivo in response to osmotic stress. In rats deprived of water for twenty‐four hours, VACM‐1 mRNA and protein levels were significantly higher in the heart ventricle but not the heart atrium when compared to the control group. Twenty‐four hour water deprivation also increased modification of VACM‐1 by the ubiquitin‐like protein Nedd8 in the heart ventricle and mesenteric arteries. Although water deprivation did not change mesenteric AQP1 levels significantly, AQP1 protein levels were inversely correlated with the ratio of VACM‐1 to the Nedd8 modified VACM‐1 protein. In summary, these results suggest that VACM‐1/cul 5 protein may regulate endothelial AQP1 concentration both in vivo and in vitro. This work was funded by the Arnold and Mabel Beckman Foundation.
Subunit composition of the Sin3 histone deacetylase complex is altered by histone deacetylase inhibitors
Authors: Smith Karen T , Martin‐Brown Skylar , Florens Laurence , Washburn Michael P , Workman Jerry L
Published: June 2021

Histone deacetylase inhibitors are currently in clinical development for several types of cancer. However, the mechanisms through which HDAC inhibitors alter gene expression and inhibit cancer progression are poorly understood. HDAC inhibitors catalytically inhibit the activity of HDACs by chelating a zinc molecule in the active site of the HDAC. However, it is not known what the fate of the multisubunit HDAC containing chromatin complexes are after HDAC inhibitor treatment. To address this, we purified the Sin3/HDAC1/2 complex from 293T cells treated with the HDAC inhibitor SAHA. We discovered that SAHA causes the loss of the PHD‐finger containing ING2 subunit from the Sin3/HDAC1/2 chromatin modifying complex. However, total nuclear levels of ING2 did not change after HDAC inhibitor treatment. Furthermore, this dissociation also occurred in vitro with specific HDAC inhibitors. The occupancies of some Sin3/HDAC subunits at target promoters were reduced after SAHA treatment however ING2 occupancy was not changed. Currently we are determining if the dissociation of ING2 from the complex leads to the loss of binding of the Sin3/HDAC complex at target promoters and assessing the downstream consequences on gene expression. This research is supported by the NIH and the American Cancer Society.
All‐trans retinoic acid prevents KLF4 deacetylation through inducing dissociation of KLF4 with HDAC2 in vascular smooth muscle cells
Authors: Meng Fang , Han Mei , Zheng Bin , Wen Jin‐kun
Published: June 2021

We previously showed that KLF4 is required for the expression of VSMC differentiation marker genes induced by ATRA. Histone deacetylase 2 (HDAC2) interacts with KLF4 and regulates its acetylation and transcriptional activity. In this report, we show that KLF4 acetylation is regulated by its interaction with HDAC2 in ATRA‐induced VSMCs. To determine whether ATRA affects KLF4 modifications, the phosphorylation and acetylation of KLF4, which regulate KLF4 functions, were analyzed. Immunoprecipitation assay showed that level of phosphorylated KLF4 markedly increased in VSMCs treated with 10 µmol/L of ATRA . Similarly, KLF4 acetylation level was also significantly enhanced after VSMCs were treated with ATRA. To investigate the mechanism by which ATRA stimulates KLF4 acetylation, the interaction of KLF4 in vitro and in vivo with HDAC2, which is highly expression in ATRA‐treated VSMCs, was detected by cross‐CoIP and GST pull‐down. Western blot analysis showed that association of HDAC2 with KLF4 decreased in a time‐dependent manner after ATRA stimulation. Meanwhile, the increase in HDAC2 phosphorylation was observed in VSMCs treated with ATRA . These results suggest that ATRA increases KLF4 acetylation via inhibiting its interaction with HDAC2 and suppressing KLF4 deacetylation by HDAC2 in VSMCs. Furthermore, we showed for the first time that JNK mediates ATRA‐induced HDAC2 phosphorylation.
Transcriptional Regulation of Ceruloplasmin by FOXO1A and IL‐6
Authors: Hollenbach Andrew Durrell , Miller Patrick J. , Sidhu Alpa
Published: June 2021

Ceruloplasmin (Cp), a six‐copper containing protein synthesized and secreted mainly by the liver, is an important extracellular anti‐oxidant and is part of the acute phase reaction during inflammation. The increase in hepatic expression and serum concentration of Cp during inflammation, infection, and trauma is primarily mediated by the inflammatory cytokine IL‐6. IL‐6 is known to positively regulate the transcription of Cp through its proximal promoter region. In addition, IL‐6 treatment localizes FOXO1A, a winged‐helix transcription factor, to the nucleus leading to its participation in IL‐6 induced gene expression. Microarray analysis showed that the expression of Cp increased 16 fold in the presence of a constitutively active FOXO1A. Consistent with this data we demonstrate for the first time that Cp is a direct transcriptional target of FOXO1A in an IL‐6 dependent manner. Through in vitro and in vivo mutational analysis, we identify the exact DNA recognition site for FOXO1A in the Cp proximal promoter region. We also demonstrate for the first time that both IL‐6 and FOXO1A are required for the transcriptional activity of Cp in HepG2 cells, identifying an additional role of FOXO1A in the inflammatory process.

Funding: National Institutes of Health Centers of Biomedical Research Excellence (1 P20 RR020152) and the Louisiana Cancer Research Consortium Competitive Advantage Fund
Joint associations of polymorphism, methylation and expression of serotonin receptor 2A (HTR2A) with Chronic Fatigue Syndrome
Authors: Falkenberg Virginia Rebecca , Unger Elizabeth R , Rajeevan Mangalathu S
Published: June 2021

Understanding the impact of genetic and epigenetic variations on gene expression is essential for understanding the genetic basis of complex diseases. The rs6311 (‐1438G/A) promoter polymorphism in the serotonin receptor 2A (HTR2A) gene is associated with Chronic Fatigue Syndrome (CFS). This polymorphism creates a binding site for the transcription factor E47 and disrupts a CpG methylation site resulting in a heritable difference in methylation. We analyzed methylation levels in HTR2A with DNA from peripheral blood mononuclear cells (PBMC) using pyrosequencing. A total of 17 CpG sites were examined in 88 subjects derived from a population‐based study of CFS. Methylation of three CpG sites at ‐1439, ‐1420, and ‐1224 were correlated with genotype at ‐1438G/A and/or CFS. Position ‐1420 is in a glucocorticoid response element, providing a molecular link for the interaction of glucocorticoid and serotonergic signaling systems. Position ‐1224 harbors an Sp1 binding site. Increased HTR2A expression in CFS appeared to originate from the preferential expression of the A allele compared to non‐fatigued control subjects. We constructed a model of HTR2A expression in CFS based on genetic and epigenetic variation at critical transcription factor binding sites. This integrated functional genomics approach may be highly relevant to defining the molecular determinants of human phenotypes. This study was funded by the CDC.
B. subtilis Elongation Factor Tu binds Calcium ions
Authors: Dominguez Delfina C , Lopes Rosana , Palomino Javier
Published: June 2021

ABSTRACT

An essential step toward the understanding of the role of calcium in prokaryotes is the identification of intracellular calcium‐binding proteins. In this report we present evidence that Bacillus subtilis elongation factor EF‐Tu is a calcium‐binding protein.

A genomic analysis of EF‐hand related sequences in the genus Arabidopsis showed that elongation factors have EF‐hand domains. Considering the high degree of conservation across species and based on previous findings in our laboratory suggesting EF‐Tu may be a CaBP (unpublished results) we investigated further the B. subtilis EF‐Tu. We performed multiple sequence alignment and motif discovery of bacterial EF‐Tu and D. discoideum calmodulin sequences and the results suggested the presence of a possible calcium binding site in B. subtilis EF‐Tu. Secondary structure prediction indicated that the conserved acidic amino acids were found within a loop flanked by two helices. A recombinant B. subtilis EF‐Tu was partially purified. The protein was identified by Western blot assay using anti‐EF‐Tu antibodies and two‐dimensional electrophoresis followed by liquid chromatography and mass spectrometry. Calcium binding was examined, in vivo and in vitro, by 45Ca‐autoradiography and immunodetection using monoclonal anti‐calmodulin and polyclonal anti‐EF‐hand antibodies the combined results indicated the B. subtilis EF‐Tu bindis calcium ions in vivo and in vitro. Addition of calcium chelators EGTA/BAPTA or CaCl2 to cell cultures affected expression of B. subtilis EF‐Tu.
Positional Effects of Monofluorinated Phenylalanines on Histone Acetyltransferase on Stability, Activity and Specificity
Authors: Voloshchuk Natalya , Zhu Anita Y , Snydacker David , Montclare Jin Kim
Published: June 2021

To explore the impact of globally incorporated fluorinated aromatic analogs on functional proteins, we investigate the effects of the monofluorinated phenylalanine analogs para‐fluorophenylalanine (pFF), meta‐fluorophenylalanine (mFF), and ortho‐fluorophenylalanine (oFF) on the stability, activity and specificity of the histone acetyltransferase (HAT) protein, tGCN5. We selected this set of fluorinated amino acids because each analog bears the same overall polarity and size while altering the direction of the dipole and side chain shape. Our experiments demonstrate a positional effect of single fluorine substitution on tGCN5 in which pFF provides minimal perturbance to structure and activity followed by mFF and oFF. Surprisingly, all monofluorophenylalanines lead to an enhanced selectivity for the target histone H3 relative to the non‐histone p53 substrate. Based on these results, tGCN5 labeled with pFF appears to maintain stability and function, while improving its specificity for its target histone H3 substrate.
Influence of selenoprotein methionine‐R‐sulfoxide reductase B1 on the lifespan of fruit flies
Authors: Shchedrina Valentina A , Vorbruggen Gerd , Lee Byung Cheon , Gladyshev Vadim N
Published: June 2021

Selenoproteins are a group of proteins (25 in mammals) that have a rare amino acid, selenocysteine. Selenoproteins serve critical functions in redox regulation. The Drosophila melanogaster has only 3 selenoproteins. D. melanogaster in which selenoprotein synthesis is disrupted has normal lifespan and is resistant to oxidative stress. Here, the GAL4‐UAS system was used to drive the expression of a recombinant selenoprotein, mouse methionine sulfoxide reductase 1 (mMsrB1), in the whole body and the nervous system of flies. mMsrB1 catalyzes the reduction of oxidized methionine residues in proteins and its function is implicated in the regulation of aging. The expression of mMsrB1 was confirmed by 75Se metabolic labeling and enzyme activity assays. mMsrB1‐expressing flies had a significant decrease in lifespan (up to 50%), but showed an increased resistance to oxidative stress. These data suggest that expression of a recombinant selenoprotein is toxic to fruit flies. This finding is consistent with the fact that the use of selenoproteins was reduced in insects via selenoprotein gene loss or their conversion to Cys‐containing homologs. This work was supported by the NIH grant AG021518 (to VNG).
Vanadate inhibition of fungal and bacterial histidine acid phosphatases
Authors: Ullah Abul H.J. , Sethumadhavan Kandan , Mullaney Edward J
Published: June 2021

The fungal phyA protein, which was first identified as an acid optimum phosphomonoesterase (EC 3.1.3.8), could double duty as a vanadate haloperoxidase (EC 1.11.1.10) provided the acid phosphatase activity is shutdown by vanadate. To understand how vanadate inhibits both phytate and pNPP degrading activities of fungal phyA phytase and bacterial APPA2 phytase, we performed kinetic experiments in presence and absence of orthovanadate and metavanadate under various acidic pH. Orthovanadate was found to be a potent inhibitor at pH 2.5 to 3.0. A 50% activity of fungal phytase was inhibited at 0.56 μM by orthovanadate. However, metavanadate preferentially inhibited the bacterial APPA2 phytase (50% inhibition at 8 μM) over the fungal phytase (50% inhibition at 40 μM). While in bacterial phytase the Km was not affected by ortho‐ or metavanadate, the Vmax was reduced. In fungal phytase, both the Km and Vmax was lowered. The vanadate exists as anion at pH 3.0 and below and binds to the active center of phytases that has cluster of positively charged Arg, Lys, and His residues below the enzymes' isoelectric point (pI). The active site fold of haloperoxidase was shown to be very similar to fungal phytase. The vanadate anions binding to cationic residues in the active site at acidic pH thus serves as a molecular switch to turn off phytase activity and turn on haloperoxidase activity.