bluuebunny/crossref_metadata_2025_split huggingface dataset:
In vitro acute effects of free fatty acids on protein degradation in young rat skeletal muscle
Authors: Góis Lidiany , Zanon Neusa Maria , Migliorini Renato Helios , Kettelhut Isis do Carmo 
Published: June 2021

Previous reports from literature have suggested that plasma free fatty acids (FFA) may reduce the rates of proteolysis, sparing muscle proteins in situations like fasting and diabetes. Here we investigated the acute effects of palmitic acid on total protein degradation and the activity of proteolytic pathways in rat skeletal muscle incubated in vitro. Soleus muscles were removed from Wistar male rats (70–80g) and incubated in Krebs‐Ringer buffer in the presence of palmitic acid (0,3–2,0mM) bound to albumin 1–2%. The rates of total proteolysis and proteolytic pathways were determined by release of tyrosine in the medium. The presence of palmitic acid (1,0mM) induced a clear reduction of total proteolysis (nmol Tyr/mg.2h) (0,340±0,014; 0,242±0,007; n=8), which was accompanied by a 50% reduction in the activity of calcium‐dependent proteolytic process. No changes in lysosomal and ATP‐Ub‐proteasome processes were observed. The inhibition of palmitic acid oxidation (50%) by etomoxir (75μM) and dichloroacetate (7mM) or the presence of 2‐bromopalmitate, a non‐metabolizable analog of fatty acids, did not alter the antiproteolytic effect observed. The present results show that palmitic acid exerts an acute inhibitory effect on total proteolysis by reducing the activity of calcium‐dependent pathway in rat skeletal muscle and this effect seems not depend on fatty acid oxidation.Financial Support: FAPESP and CNPq
The effects of nesfatin‐1, an anorexigenic peptide, on blood pressure and lights out locomotor activity
Authors: Yosten Gina Louise Cecilia , White Meghan M. , Samson Willis K. 
Published: June 2021

Here we describe the effects of Nesfatin‐1 (NES) on mean arterial blood pressure and locomotor activity in normotensive rats. Cannulas were inserted into the right lateral ventricle of male rats. After 5 days, the left ascending carotid artery was cannulated with PE‐50 tubing. The next day, mean arterial pressure (MAP) was recorded in conscious, unrestrained animals for 30 minutes following intracerebroventricular (i.c.v.) injection of 20, 60, or 180 pmol NES or saline. For behavior, naïve rats with i.c.v. cannulas were habituated in a behavior box (Opto‐Max, Columbus Instruments) for 1 hour (16:00). At 17:00, 20, 60, or 180 pmol NES or saline was administered. The lights were turned off at 18:00 and activity was recorded until 19:00.There was an initial significant increase in MAP after i.c.v. injection of all three doses of NES which lasted approximately 15–20 min. Behavioral readings indicated that the rats were very active in the initial 15 minutes after i.c.v. injection of NES, but then entered a phase of inactivity, which lasted until Lights Out, when they became active again. The highest dose of NES induced a state of almost complete inactivity, which lasted beyond Lights Out. These results indicate that the rise in MAP could reflect the initial behavioral activity. Furthermore, the anorexigenic effect of NES may also be secondary to its action to reduce spontaneous locomotor activity.
Receptor for activated C kinase1 (RACK1) binds with G beta1 and restricts focal adhesion kinase activation and restoration of endothelial barrier function
Authors: Knezevic Nebojsa , Kini Vidisha , Malik Asrar B , Mehta Dolly 
Published: June 2021

We showed that sustained activation of focal adhesion kinase (FAK) is required for reversing the increase in endothelial permeability induced by inflammatory mediator thrombin. However, mechanism that sustain FAK activation is not known and therefore will help in identifying signaling cues responsible for restoring endothelial barrier function. Gβ1 and receptor for activated C kinase (RACK1) belong to large family of WD40 repeat proteins and act as scaffold proteins to orchestrate downstream signaling events. We therefore investigated the contribution of RACK1 and Gβ1 in regulating FAK activation and recovery of endothelial barrier function. We show that RACK1 is associated with Gβ1 in untreated cells. Thrombin induced a time‐dependent reversible dissociation of RACK1 from Gβ1, enabling it to interact with upstream FAK regulator Fyn. Inhibiting endogenous expression of Gβ1 had no effect on thrombin‐induced increase in endothelial permeability but markedly impaired endothelial barrier repair. Knockdown of Fyn in endothelial cells also prevented barrier recovery post‐thrombin challenge, linking Gβ1 to the regulation of Fyn function. In contrast, knock down of RACK1 increased basal phosphorylation of FAK and potentiated endothelial barrier recovery. Thus, these data demonstrate that RACK1 prevents the access of Gβ1 from forming a complex with Fyn, which is required for FAK activation and restoration of endothelial barrier function.Supported by NIH grants: HL71794 and HL84153
Ca2+ pulsars: spatially restricted, IP3R‐mediated Ca2+ release important for endothelial function
Authors: Ledoux Jonathan , Taylor Mark S , Bonev Adrian D , Hannah Rachael M , Tallini Yvonne N , Kotlikoff Michael I , Nelson Mark T 
Published: June 2021

Intracellular Ca2+ is essential for endothelial functions such as the control of vascular tone. Using confocal microscopy, endothelial Ca2+ signaling was investigated in cut‐open mesenteric arteries from mice expressing the Ca2+ indicator GCaMP2 exclusively in the endothelium. Spontaneous and local Ca2+ release (“Ca2+ pulsars”) from endothelial IP3 receptors (IP3R) have been identified and are also present in pressurized arteries. The Ca2+ pulsars show distinct kinetics from ryanodine receptor–dependent Ca2+ sparks and from IP3R‐mediated Ca2+ puffs. The endothelium‐dependent vasodilator acetylcholine increased the frequency of Ca2+ pulsars. The Ca2+ pulsars are localized in the endothelial projections through the internal elastic lamina to the vascular smooth muscle. Immunocytochemistry also identified IP3R and KCa3.1 channels in the myoendothelial projections. While block of KCa3.1 channels with ChTX depolarized the endothelial membrane by ≈10 mV, inhibition of Ca2+ pulsars with CPA (10 μM) prevented ChTX‐induced depolarization. Moreover, exposure to CPA induced a depolarization of the endothelium similar to ChTX (≈11 mV). Our results suggest a novel IP3R‐dependent mechanism regulating endothelium‐smooth muscle communication.Supported by NIH (HL44455 & HL63722) and CIHR.
MUC1 interacts with CAML: A protein involved in Ca2+ signaling
Authors: Lillehoj Erik P. , Guang Wei 
Published: June 2021

Objective: MUC1 is a transmembrane mucin glycoprotein expressed on the surface of lymphocytes and epithelial cells. Its cytoplasmic COOH‐terminus (CT) is evolutionarily conserved and mediates intracellular signaling. The objective of this study was to identify proteins involved in MUC1 CT signaling.Methods: We searched for proteins binding to the MUC1 CT using a yeast two‐hybrid system and confirmed the results by coimmunoprecipitation (coIP) analysis.Results: Of the positive clones encoding proteins that interacted with the MUC1 CT, the majority coded for CAML (calcium modulating cyclophilin ligand), a protein involved in Ca2+ signaling. The MUC1 CT interacted with CAML as revealed by growth on selective media and in situ X‐alpha‐galactosidase activity of yeast double transformants or progeny from matings of single transformants. Binding of the MUC1 CT to CAML was confirmed by reciprocal coIPs (MUC1 CT IP + CAML IB and CAML IP + MUC1 CT IB). By deletion mutagenesis, only the MUC1 CT and not its extracellular tandem repeat region or SEA domain interacted with CAML. Likewise, the region of CAML binding to MUC1 was localized to its NH2‐terminus.Conclusion: Our results have identified CAML as a novel binding partner of the MUC1 CT and suggest that MUC1 plays a role in Ca2+ signaling.
Soybean product inhibits K88 enterotoxigenic Escherichia coli binding to enterocytes
Authors: Lin Jun , He Dong , Van Hout Amber , Kaushik Radhey , Francis David , Erickson Alan 
Published: June 2021

Strains of enterotoxigenic Escherichia coli expressing the K88 fimbrial adhesin (K88 ETEC) induce colibacillosis by binding to enterocytes in the small intestine of newborn and weaned piglets and causing secretory diarrhea. Current vaccination protocols are ineffective at preventing K88 ETEC infections during the postweaning period. Consequently, new procedures for preventing and treating postweaning colibacillosis caused by K88 ETEC are needed. Previous studies have shown that extracts of fermented soy products have the potential to prevent ETEC disease by blocking the binding of ETEC to enterocytes. In this study, we evaluated extracts of the fermented soy product, Dochi, for its ability to block K88 ETEC binding to enterocytes. We found that Dochi extracts were able significantly inhibit binding of K88 ETEC to brush borders isolated from porcine enterocytes (50% inhibition, P < 0.05) and cultured porcine intestinal epithelial (IPEC‐J2) cells (60% inhibition, P < 0.05). The inhibitory compound in the Dochi extract was found to elute during fractionation on a Superose 12 FPLC size exclusion column at an approximate molecular weight of 1300 Da. Future studies will be focused on determining the chemical nature of the inhibitory compound and on determining if the inhibitory compound can be used to prevent or treat K88 ETEC‐induced colibacillosis in weaned piglets.Supported by South Dakota State University Research/Scholarship Support Fund, the South Dakota Agricultural Experiment Station, and the South Dakota Center for Infectious Disease Research and Vaccinology.
Continuous testosterone administration for 5 months reduces markers of bone turnover in older men
Authors: Dillon Edgar Lichar , Urban Randall J. , Angel James A. , Casperson Shanon L. , Paddon‐Jones Douglas , Sheffield‐Moore Melinda 
Published: June 2021

Men and women undergo a progressive reduction in bone mineral density (BMD) with advancing age. Maintenance of circulating testosterone concentrations is beneficial to BMD in older men. We investigated whether continuous and monthly cycled administration of testosterone improves markers of bone metabolism in older men with low normal endogenous testosterone.Older men (68.92 ± 1.59 yrs, serum total testosterone 362 ± 23 ng/dL, n=13) completed a 5‐month randomized double blinded placebo controlled protocol. Subjects received either continuous testosterone (weekly injections, 100 mg testosterone‐enanthate) for 5 months (TE, n=4), monthly cycled testosterone/placebo treatment (MO, n=4), or weekly placebo (PL, n=5). Fasting blood and 24‐h urine was collected each month and DEXA scans were obtained at 0 and 5 months to assess changes in markers of bone turnover and BMD.Serum n‐telopeptide decreased in TE and showed a trend for decrease in MO when compared to PL. Serum osteocalcin decreased significantly in TE but not in MO when compared to PL. There were no changes in urinary deoxypyrrodinoline, bone‐specific alkaline phosphatase, intact‐parathyroid hormone, or calcitonin in any of the groups. Pelvic BMD decreased in PL and MO but not in TE.These data show beneficial effects of continuous testosterone therapy on markers of bone turnover in older men with low‐normal endogenous testosterone concentrations.
Inhibition of Adenosine Monophosphate‐activated Protein Kinase (AMPK) ameliorates the effects of Interferon gamma (IFN gamma) on epithelial barrier function in T84 cells
Authors: Scharl Michael , Barrett Kim E , McCole Declan F 
Published: June 2021

IFNγ plays a major role in the pathogenesis of inflammatory bowel disease (IBD). Treatment of intestinal epithelial cells with IFNγ leads to decreased transepithelial resistance (TER), increased epithelial permeability and a downregulation of tight junction proteins. AMPK has been shown to reduce energy consuming processes during inflammation. Here, we investigated if AMPK is involved in IFNγ‐induced effects on epithelial barrier function.Protein analysis was performed by Western blot. TER was assessed by voltohmeter and confirmed with Ussing chambers.IFNγ increased AMPK phosphorylation in T84 cells (1000 U/ml, 6h). The AMPK inhibitor, Compound C (50 μM), reversed the IFNγ‐induced decrease in TER (p<0.001, n=5) across T84 monolayers at 72h treatment. In contrast, activation of AMPK with AICA‐Riboside‐5′‐Phosphate (1 mM, 24–72h) had no effect on TER. AMPK inhibition also prevented the IFNγ induced downregulation of the tight junction protein, occludin (p<0.05, n=3, 72h).In summary, these data show involvement of AMPK in IFNγ‐induced alterations in epithelial integrity. However, AMPK activity alone is not sufficient to generate such effects. These findings indicate a novel role for AMPK in the regulation of epithelial barrier function, and may have implications for inflammatory conditions associated with metabolic stress, such as IBD.Supported by the Crohn's and Colitis Foundation of America and NIH.
A new population of bone marrow cells restores lung growth in a model of Bronchopulmonary Dysplasia (BPD)
Authors: Balasubramaniam Vivek , Ryan Sharon L , Seedorf Gregory J , Markham Neil E , Yoder Mervin C , Abman Steven H 
Published: June 2021

BPD is characterized by impaired lung growth that persists in adulthood. We have shown neonatal hyperoxia reduces bone marrow progenitor cells & causes abnormal lung structure in mice. The role of progenitor cells in lung growth is controversial. We isolated a new population of bone marrow derived angiogenic cells (BMDAC). We hypothesize that BMDACs can home & engraft in the lung, & promote vascular & alveolar growth after neonatal hyperoxic lung injury.Methods: BMDACs from transgenic Tie2‐GFP mice, were cultured on a feeder cell layer, & characterized by FACS. Neonatal mice were exposed to hyperoxia (FiO2=0.8) X 10 days. BMDAC (10^5 cells) were injected into the right ventricle prior to room air recovery X 10 days. Lung morphometry & immunofluorescence was done.Results: BMDAC displayed a phenotype of a myeloid progenitor: CD45+/CD34‐/CD38+/CD11b‐/CXCR4+/Ly6c‐/F4/80+. Hyperoxia impaired lung structure, which persisted in room air recovery in controls. BMDAC treatment improved septation by 78% & vessel density by 134%(p<0.01), equal to room air raised mice. BMDAC engraftment was 12%, & localized to vessel wall & interstitium, in association with Type II cells. BMADCs did not co‐express smooth muscle, endothelial, epithelial or pericyte markers. We conclude BMDACs are a novel cell population that home to sites of injury in the infant lung & stimulate alveolar & vascular growth after neonatal hyperoxia.
Effect of dietary flavonoids on hormone‐stimulated ion transport in renal principal cells
Authors: Creason Lacey Beth , Muchatuta Monalisa Ndanatsei , Platt Laura Keese , Blazer‐Yost Bonnie L 
Published: June 2021

Flavonoids are a class of compounds found in plants such as parsley, cocoa, soybeans, tea, and grapes. Flavonoids have anti‐oxidant properties, can act as retroviral inhibitors, and have cardiovascular benefits. We hypothesize that the cardiovascular benefits are, in part, due to an effect on salt and water homeostasis via the renal epithelial Na+ channel (ENaC). Quercetin, apigenin, and genistein are the dietary flavonoids of interest. Insulin and aldosterone regulate ENaC through a PI3‐kinase dependant pathway. A PI3‐kinase inhibitor, LY294002, was designed from the quercetin structure. Therefore, we hypothesize that flavonoids may inhibit ENaC activity. We used electrophysiological techniques to study the effects of flavonoids on ion transport in the mouse principle cells of the kidney cortical collecting duct (mpkCCD) cell line. All three flavonoids lower basal ion transport in mpkCDD cells. Neither genistein nor apigenin inhibit insulin stimulated ion transport. Following ADH stimulation, both flavonoids inhibit anion secretion via CFTR, but have no statistical effect on ADH‐stimulated Na+ reabsorption. In summary, in mpkCCD cells, the flavonoids lower basal, amiloride sensitive Na+ transport, but do not alter peptide‐hormone stimulated Na+ reabsorption. The flavonoids do, however, inhibit ADH‐stimulated anion secretion.Funding: IUPUI Undergraduate Research Opportunities Program.
TGF‐β1 Effect on Cortical Collecting Duct Cells
Authors: Feng Zhuang , Somanna Naveen K , Hamm Lee L , Hering‐Smith Kathleen S 
Published: June 2021

ENaC in collecting duct (CD) cells is involved in the regulation of Na balance and blood pressure. Prostasin, an endogenous protease, has been shown to activate ENaC, which can be mimicked by trypsin. Basolateral (BL) TGF‐β1 has been shown to inhibit Na current (Ieq), mRNA expression of prostasin and ENaC subunits. The purpose of this study was to characterize the CD response to TGF‐β1, in particular the involvement of proteolytic processes and the paracellular pathway. M‐1 cells were studied on permeable supports. The activity of secreted serine proteases was measured by an amidolytic assay. BL TGF‐β1 inhibited Ieq in the range of 2–40 ng/ml. The most marked effect was observed at concentrations ≥20 ng/ml, with >60% inhibition at 24 hours. However, transepithelial resistance Rte also decreased significantly after 2 hours, and to > 50% inhibition at 24 hours, an effect not expected with inhibition of ENaC only. The effects of TGF‐β1 on Ieq and Rte were not reversed by trypsin. Proteolytic activity in the apical media was not altered by TGF‐β1. In conclusion, BL TGF‐β1 significantly decreases Na transport in CD cells but also increases paracellular permeability. The lack of response to trypsin in TGF‐β1 treated cells implies that the down‐regulation of prostasin is not the major factor in the inhibition of Na current by TGF‐β1.Supported by research grants from VA and NIH.
Tx2‐6 toxin from Phoneutria nigriventer spider improves relaxation induced by electrical stimulation of rat cavernosum strips
Authors: Nunes Kenia Pedrosa , Cordeiro Marta Nascimento , Richardson Michael , Tostes Rita , De Lima Maria Elena , Webb R. Clinton , Leite Romulo 
Published: June 2021

Background: Accidents involving the spider P. nigriventer are characterized by different symptoms including priapism. Some toxins from poisonous animals exert their toxic effects by modifying the properties of ion channels. We have previously demonstrated that Tx2‐6, a peptide extracted from the venom of P. nigriventer, improves erectile function in anesthetized rats. Here we investigated the mechanism involved in the Tx2‐6 potentiation of penile erection.Methods: Rat cavernosum strips were incubated with bretylium (3 x 10–5M) and contracted with phenylephrine (10–5 M). Relaxation responses were evoked by electrical field stimulation (EFS) or sodium nitroprusside (SNP) before and after 5 min incubation with Tx2–6 (10–8 M). The effect of Tx2‐6 on relaxation induced by EFS was also tested in the presence of atropine (10–6 M), conotoxin GVIA (10–6 M) or nifedipine (10–7 M).Results: Relaxation induced by SNP was not affected by Tx2‐6. EFS‐induced relaxation was significantly potentiated by Tx2‐6 and this effect was not affected by atropine, whereas blockade of N‐ or L‐Type Ca+2 channels abolished the potentiation of the relaxation induced by Tx2‐6.Conclusion: Tx2‐6 potentiates the relaxation of cavernosum tissue via Na+ and Ca+2 channels, facilitating the nitric oxide/cGMP pathway. Tx2‐6 has great potential for development of drugs to treat erectile dysfunction.SUPPORT: CNPq, FAPEMIG, NIH
Pulmonary venous hypertension causes differential expression of developmental genes in the lung
Authors: Hoffmann Julia , Kuebler Wolfgang M 
Published: June 2021

Pulmonary venous hypertension (PVH) is a frequent consequence of left sided heart disease and causes extensive lung vascular remodeling which leads to right ventricular failure. Functional genomics underlying this structural remodeling in PVH are unknown but present potential targets for therapeutic strategies. Developmental genes have been implicated in the pathophysiology of lung cancer and asthma but their role in vascular remodeling and pulmonary hypertension is unclear.PVH was induced in rats by supracoronary aortic banding 8 weeks prior to investigations. For genomic analyses, RNA was extracted from isolated lungs of control and PVH rats and whole rat genome microarrays were performed.Out of 31100 arrayed genes, 231 were significantly up‐ or downregulated more than 3‐fold in PHV. These comprised genes involved in vascular regulation and inflammation such as ETB receptor, heme oxygenase 1 and interleukin‐1 alpha but also developmental genes like frizzled related protein, Jun D and semaphorin 6A. Differential expression of selected genes was verified by RT‐PCR and confirmed by Western Blot and 2D‐PAGE.PVH causes differential expression of genes involved in vascular regulation and development. Functional effects of this genomic regulation may contribute to the pathology of PVH and suggest a potential role for developmental genes in vascular remodeling.Sponsored by EU‐IP “Pulmotension”
Endothelin receptor (ETR) blockade combined with phosphodiesterase‐5 (PDE5) inhibition prevents right ventricular hypoxia in Pulmonary Hypertension
Authors: Mouchaers Koen Thijs Bert , Wong Yeun‐Ying , Schalij Ingrid , Jaspers Richard T , van Nieuw Amerongen Geerten P , Vonk‐Noordegraaf Anton , van der Laarse Willem J 
Published: June 2021

BackgroundPulmonary hypertension (PH) may cause right ventricular (RV) hypoxia, which may contribute to RV failure.GoalTo study the effects of current PH therapies on the presence of hypoxia in the RV.MethodsPH was induced in Wistar rats by monocrotaline (MCT) (40 mg/kg). From day 14 to 28, rats were treated with Bosentan (ETR antagonist, 300 mg/kg/day), Sildenafil (PDE5 inhibitor, 10 mg/kg/day) or both (all n=3). Healthy rats served as controls (n=3). 1.5 hour prior to sacrifice hypoxyprobe (pimonidazole, 60 mg/kg) was administered i.v. Cryosections (5 μm) of the hearts were incubated with hypoxyprobe antibody and for succinate dehydrogenase (SDH) activity. RV capillary density and cardiomyocyte cross‐sectional area (CSA) were determined.ResultsPH significantly increased RV pimonidazole binding, and cardiomyocyte CSA, and decreased capillary density, and SDH activity (all p<0.05 vs control). Single treatment with Bosentan or Sildenafil had no effect, whereas combination treatment normalized all parameters (p<0.05 vs MCT rats). Pimonidazole binding related linearly to CSA (r=0.6), SDH activity (r=−0.7), and capillary density (r=−0.8) (all p<0.05).ConclusionHypoxia in the RV of PH rats is caused by cardiomyocyte hypertrophy, a lowering in mitochondrial activity and a diminished capillary density, and can be prevented by combining ETR blockade and PDE5 inhibition.