bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sleep loss changes neuropeptidase expression and activty in rat brain (876.3)
Authors: Visniauskas Bruna , Dalio Fernanda , Simões Priscila , Mazzacoratti Maria da Graça , Oliveira Vitor , Tufik Sergio , Chagas Jair 
Published: June 2021

The effect of sleep deprivation on proteolytic activity in the central nervous system has not been frequently addressed in the literature. We have started to study the changes on peptidase activities in animal models of paradoxical sleep deprivation (PSD), mainly focusing on metallopeptidases like thimet oligopeptidase (EP24.15) known to process neuropeptides. In the present study, we demonstrated that PSD in rats induces changes both in the expression and activity of EP24.15 in the male rat hypothalamus and striatum tissue extracts. In the hypothalamus, the significant decrease in activity and mRNA levels, after PSD, was only totally reversed after 96h of sleep recovery. In the striatum, albeit significative, changes in mRNA and activity do not parallel changes on protein content. Neurotensin, GnRH, dynorphins, enkephalins, orphanin/nociceptin are among the many neuropeptides whose balance can be modified by the modifications on EP24.15 activity and can be, at least partially, related with many of the physiological changes observed in PSD and during the sleep recovery periods.Grant Funding Source: Supported by FAPESP, CNPq and AFIP
Kidney oxygenation in the subacute phase of ischemia‐reperfusion injury (890.1)
Authors: Evans Roger , Ow Connie , Abdelkader Amany , Hilliard Lucinda , Malpas Simon , Koeners Maarten 
Published: June 2021

We determined the status of kidney tissue oxygenation during the subacute phase of renal ischemia reperfusion injury in 10‐12 week old male Sprague‐Dawley rats. Inner medullary oxygen tension (PO2) was determined using a carbon paste electrode attached to a telemetry transmitter, for 24 hours before and for 5 days after 60 min of bilateral renal ischemia (n=4) or sham (n=4) surgery. In a second protocol, renal tissue PO2 was measured by Clark electrode under thiobutabarbital anesthesia, either 1 or 5 days after recovery from renal ischemia (n = 6 and 3 respectively) or sham surgery (n = 5 and 3 respectively). Inner medullary tissue PO2 measured by telemetry was increased by 45 ± 15% 24 h after reperfusion, but then progressively fell over the next 5 days. Tissue PO2 measured by Clark electrode was well maintained 24 h after ischemia. However, outer medullary PO2 was 69% less 5 days after ischemia than after sham surgery. Our observations suggest that widespread renal hypoxia may not be obligatory in the subacute phase of renal ischemia‐reperfusion injury. However, medullary hypoxia may evolve over a period of days following reperfusion.Grant Funding Source: Supported by the National Health and Medical Research Council of Australia
Rosiglitazone increases aquaporin 2 transcription and apical expression in renal cells: possible implication for treating nephrogenic diabetes insipidus? (892.10)
Authors: Procino Giuseppe , Milano Serena , Nicoletti Maria , Gerbino Andrea , Mastrofrancesco Lisa , Carmosino Monica , Svelto Maria 
Published: June 2021

Aquaporin 2 (AQP2) is the vasopressin‐regulated water channel palying a crucial role in urine concentration in the kidney. Rosiglitazone (RGZ), an agonist of the peroxisome proliferator‐activated receptor gamma (PPARγ), increases AQP2 expression in normal rats. Here we tested whether RGZ could also modulate AQP2 intracellular trafficking in renal cells.Wild type C57BL‐6 mice were treated for 3 days with 20mg/Kg RGZ to confirm the transcriptional effect on AQP2 in mice. In parallel, we used mouse MCD4 renal collecting duct cells to study the effect of RGZ on AQP2 trafficking to the apical membrane. Apical surface biotinylation and confocal analysis were used to semiquantify the effect. Single cell live microfluorimetry and FRET were used to measure changes in Ca2+ and cAMP intracellular concentrations, respectively.In mice, RGZ induced a twofold increase of AQP2 compared to control mice.In MCD4 cells, stimulation with 50µM RGZ for 30 min robustly increased AQP2 trafficking to the apical plasma membrane to an extent comparable to that elicited by forskolin stimulation. Interestingly, RGZ was able to increasely AQP2 phosphorylation at Ser256 concomitant with intracellular Ca++ increase and without apparent intracellular cAMP elevation.Taken together these results suggest that RGZ might be useful to increase AQP2 transcription and apical trafficking in renal cells bypassing the stimulation of vasopressin receptor which is defective in pathological conditions as in Nephrogenic Diabetes Insipidus.Grant Funding Source: Telethon GGP12040/ AIFA MRAR08P011
MATE1 sequesters organic cations within an intracellular, bafilomycin‐sensitive compartment (892.39)
Authors: Martinez Lucy , Evans Kristen , Dantzler William , Wright Stephen 
Published: June 2021

OC secretion across renal proximal tubules (RPTs) involves basolateral OCT2‐mediated uptake from the blood, followed by apical MATE1/2‐mediated efflux into the tubule filtrate. Whereas OCT2 is an electrogenic OC uniporter, MATE is an OC/H exchanger. Epifluorescence microscopy of hMATE1‐expressing CHO cells revealed accumulation of the fluorescent OC, N,N,N‐trimethyl‐2‐[methyl(7‐nitrobenzo[c][l,2,5]oxadiazol‐4‐yl)amino]ethanaminium (NBD‐MTMA) in the cytoplasm and in a smaller, punctate compartment; accumulation in OCT2 expressing cells was restricted to the cytoplasm. A second intracellular compartment was also evident in the multicompartmental kinetics of NBD‐MTMA efflux from MATE1‐CHO cells. Punctate accumulation (20 min) was markedly reduced by coexposure of MATE1‐CHO cells with 1 μM bafilomycin (BAF) and eliminated with 10 μM BAF. BAF had no effect on the initial rate of MATE1‐mediated uptake of NBD‐MTMA (10‐120 sec) suggesting that its effect involved collapse of endosomal pH gradients via inhibition of the V‐type H‐ATPase. The 15 min accumulation of the prototypic OC, [³H]TEA by isolated single non‐perfused rabbit RPTs was also reduced 40% by coexposure to 1 μM BAF. Thus, the native expression in RPTs of MATE protein within endosomes can increase steady‐state OC accumulation. Normal rates of apical endosome turnover may provide a significant parallel pathway for apical OC effluxGrant Funding Source: NIH grant 5R01DK080801
Deletion of caveolin‐1 decreased the basolateral K channel activity in the early distal convoluted tubule (DCT1) and increased urinary Mg2+ excretion (893.18)
Authors: Zhang Chengbiao , Wang Lijun , Su Xiaotong , Wang Wen‐Hui 
Published: June 2021

We used the patch clamp technique to examine the basolateral K channel in the isolated DCT1 of wt and cav‐1 knockout (KO) mice. We identified a 40 pS K channel in the DCT1 of wt (18 patches with K channels from total 35 patches) and of KO mice (25 patches with K channels in total 66 patches). Although K channel Po in DCT1 was similar in wt and KO mice, K channel number per patch was significantly lower in KO than those in wt mice. The whole‐cell K currents in the DCT1 of KO mice were also significantly lower than those of wt and the K reversed potential in DCT1 was less negative in KO mice. This suggests that deletion of cav‐1 decreased the basolateral K channel activity. The expression of c‐Src stimulates KCNJ10 channel, a major basolateral K channel in DCT1, only in cav‐1 cotransfected HEK‐cells but not in un transfected cells. Since the basolateral K channels provide the driving force for apical Mg2+ absorption, we used metabolic cage to examine renal Mg2+ transport in wt and KO mice. Although dietary Mg2+ intake was similar in wt and KO mice, the urinal Mg2+ excretion was significantly higher in KO mice than those of wt mice. Also, plasma Mg2+ concentration was lower (1.2 mg/dl) in KO than that of wt mice (2.2 mg/Dl ). We conclude that deletion of cav‐1 diminished c‐Src‐induced stimulation of KCNJ10 channels and depolarized the cell membrane potential in the DCT1 thereby impairing Mg2+ reabsorption in the DCT and causing hypomagnesaemia . Grant Funding Source: NIH
The role of a UT‐A urea transporter in human small intestine (897.2)
Authors: McGrane Alison , Courtney Danielle , Winter Desmond , Baird Alan , Stewart Gavin 
Published: June 2021

Urea transporters (UTs) encoded by the Slc14a1 (UT‐B) and Slc14a2 (UT‐A) genes facilitate the movement of urea across plasma membranes. Urea flux into the gastrointestinal tract enhances the growth of bacterial populations by providing a source of nitrogen, thus maintaining the symbiotic relationship between humans and their intestinal bacteria (Stewart 2011). A previous study identified the UT‐B1 urea transporter within the human colon, localized to the basolateral membrane of colonocytes (Collins et al. 2010). The aim of this present study was to investigate for the expression of UT‐A urea transporters in the human gastrointestinal tract. Using primers for UT‐A1, RT‐PCR analysis detected the expected RNA‐derived 682bp signal primarily in the small intestine, compared to stomach and colon. Using different samples of small intestine cDNA (AMS Biotechnology), UT‐A1 expression was shown to be higher in the jejunum and ileum than the duodenum. Western blotting experiments were then performed using human ileum tissue samples. Using a characterised rat UT‐A1 antibody (L194) (Nielsen et al. 1996), a 120kDa glycosylated UT‐A1 protein could be detected in protein samples from ileostomy patients. In contrast, ileum samples from surgical resection tissue showed UT‐A1 signals of around 170kDa. This study shows that UT‐A1 urea transporters are expressed in the human ileum. Since high levels of bacteria exist in this region, this suggests a physiological function for UT‐A1 in the urea nitrogen salvaging mechanism that is crucial for human gastrointestinal health.Grant Funding Source: Supported by Science Foundation Ireland (SFI) and University College Dublin (UCD)
Regulation of PSCA expression by m‐Numb in the stomach (904.2)
Authors: Takahashi Tetsufumi , Suzuki Hidekazu , Matsunaga Kayoko , Imai Takao , Shibata Shinsuke , Tabuchi Yoshiaki , Okano Hideyuki , Nakamura Masahiko , Tsuchimoto Kanji 
Published: June 2021

Prostate stem cell antigen (PSCA) is a GPI‐anchored cell surface protein expressed in the normal epithelium of several tissues including the isthmus region of the gastric mucosa. Recently, a genome‐wide association study revealed several SNPs in the PSCA‐gene to be associated with gastric cancer and duodenal ulcer risk. However, the underlying regulatory mechanisms of PSCA are still unclear.Methods: Msi1‐KO mice with lowered expression of m‐Numb in the stomach and wild‐type ICR mice were used for in vivo studies. In vitro studies were carried out with the mouse gastric epithelial cell line MGE507. Overexpression and suppression of m‐Numb in MGE507 cells was achieved by transfection with lentiviral particles carrying an m‐Numb expression vector and shRNA expression vector, respectively. Gene expression was analysed with qRT‐PCR, and western blotting was used to measure protein levels.Results: In Msi1‐KO mice, a significantly reduced expression‐ and protein level of PSCA was found when comparing the gastric mucosa to wild‐type ICR mice. Expectedly, the protein level of m‐Numb was also verified as lower in the KO‐line. Silencing m‐Numb expression in MGE507 cells resulted in a reduction in protein level of PSCA, and overexpression of m‐Numb led to an increased PSCA‐level.Conclusion: PSCA gene expression and protein level was regulated by m‐Numb in the mouse stomach and in mouse gastric epithelial cells.
Mechanism and regulation of biotin uptake by mouse and human pancreatic beta cells/islets: physiological and molecular aspects (909.2)
Authors: Ghosal Abhisek , Sekar Thillai , Said Hamid 
Published: June 2021

Biotin is essential for normal cellular function of all pancreatic cells. Little, however, is known about how pancreatic beta cells obtain biotin and the molecular identity of the system involved and its regulation. We addressed these issues using mouse‐derived beta‐TC‐6 cells as well as freshly isolated mouse and human primary pancreatic islets. Our results showed biotin uptake by beta‐TC‐6 cells is carrier‐mediated, Na‐dependent, cis‐inhibited by structural analogs and by pantothenic acid, and is saturable (apparent Km of 17 µM). Similarly, freshly isolated mouse and human primary pancreatic islets took in biotin via a carrier‐mediated Na‐dependent mechanism. These studies suggest the involvement of the Na‐dependent multivitamin transporter, SMVT; this was confirmed in studies using gene specific knockdown (shRNA) approach. Uptake of biotin was also adaptively‐regulated by extracellular vitamin levels through a transcriptional mechanism (s). Maintaining (96 hr) pancreatic beta cells in hyper‐glycemic condition (26 mM) did not affect biotin uptake. Exposer to specific cytokines (IL‐1β+TNF‐+IFN‐), whose level increases in diabetes, led to a significant inhibition in biotin uptake. The results show for the first time that biotin uptake by pancreatic beta cells is via a cytokine sensitive carrier‐mediated process that involves the SMVT system.Grant Funding Source: Supported by DVA and NIH (DK58057, AA‐18071 and DK56061
Tear analysis of the anterior cruciate ligament using a novel staining method (914.8)
Authors: Gillespie Michael , Jorgensen Adam , Wilkes Daniel , Wisco Jonathan 
Published: June 2021

The purpose of this project is to create data driven, three‐dimensional fiber maps of various grade‐III sprained human anterior cruciate ligaments. In order to increase visibility of these fibers, we have created a stain made from powdered sugar and blue dye that reveals their fine architecture. Application of the stain to human cadaveric ACL and calcaneal tendon has revealed otherwise undistinguishable fiber boundaries. We performed the following six steps on each of five un‐embalmed human cadaveric knees. 1) MRI imaging, 2) Induced valgus force to simulate common ACL tear, 3) MRI re‐imaging, 4) Dissection to reveal ACL, 5) Stain ACL, 6) MicroScribe both torn halves. With our newly developed staining technique, the intricate surface architecture of torn human cadaveric ACL was exposed to a degree never before possible. This allowed for more complete and accurate MicroScribe 3D digitizing and the creation of maps that exhibit the intricate details of how and where ACLs typically tear. A more thorough understanding of ACL tear microstructure will offer insight into why current repair techniques are ineffective and how they can be improved. The overarching goal is the development of an effective repair method to replace the current method of reconstruction. This would eliminate the many post‐operative complications that accompany ACL reconstruction.Grant Funding Source: Supported by Mentoring Research Grant, College of Life Sciences, Brigham Young University
Lymphoid formation of mucous membrane of a human rectum (916.5)
Authors: Maul Ya , Aubakirov A. , Suleimenova F. 
Published: June 2021

Mucous membrane of a rectum constantly is subject to a significant antigene influence taking into account the reservoir function of the organ. However, problems of interrelation of lymphoid tissue associated with mucous membrane of the organ with the epithelium integument and with other tissue structures were not studied enough.Aim: to study histomorphological interrelation of lymphoid tissue localised in the mucous membrane of a rectum with epithelium cover of the organ and adjusting tissue structures.Material and methods of investigation: Human rectum from the period of birth till the eighty seven years. Morphometric, histomorphological, statistic methods have been used.Results: In the process of research we revealed some interesting facts: A great number of postcapillary venules with the migrating lymphocytes through their walls.In the anorectal zone of the anal section a lymphoid tissue is represented as a continuous “ring” consisting of the lymphoid nodes and diffuse lymphoid tissue.In the mucous membrane of the rectum we revealed periarterial macrophagal‐lymphocyte clutches.The clutches had the form of ovoid or ellipse.Stratified flat non‐cornified epithelium similar to the esophageal epithelium is revealed in the area of anal columns and sinuses. In this section of the rectum there is not a lymphoid tissue or it is shown as single nodes.Conclusions: In the mucous membrane of the rectum the presens of well‐ developed lymphoid apparatus was proved morphologically that allows to consider the rectum as the peripheric organ of immunogenesis Besides, we suggest a continuous “ring” of the lymphoid tissue localized in the anorectal zone of the anal section to classify as the analogue of “Fabritsious bad” of a man.
Characterization of calcium phosphate composite two solution bone cements (922.20)
Authors: Chari Jonathan , Rodriguez Lucas , Aghyarian Shant , Rodrigues Danieli 
Published: June 2021

Introduction: Acrylic bone cements are used in a variety of orthopedic applications including vertebral decompression and implant augmentation. However, they are bioinert with no ability to integrate with bone. We hypothesize that correction of this drawback can be achieved through the addition of the calcium phosphate (CaP), Brushite, providing a porous scaffold for bone interdigitation while retaining desirable mechanical, rheological, and handling properties.Methods: Composite two solution cement samples were prepared by Brushite addition yielding a pre‐mixed cement with up to 75% of the polymer replaced. Viscosity was assessed using frequency sweeps on non‐setting cements with a DHR‐3 Rheometer. Compression tests followed the ASTM F451 standard using a Bionix MTS 370. To study injectability, a pneumatic gun was used to extrude 1mL of cement through the system and the time measured.Results: CaP addition was shown to be feasible with no deleterious effect on cement mechanical properties. Rheology showed that the variation in viscosities between samples is not significant (p<0.05) and pseudoplasticity was sustained. Injectability of the cement formulations varied considerably, while some showed “plugging” after the cartridge was emptied.Significance: The versatility of the pre‐mixed two solution bone cement allows for the incorporation of CaP fillers thereby enhancing interdigitation with bone.Grant Funding Source: Supported by The University of Texas System, UT Transform Award (Dr. Danieli Rodrigues)
Structural and chemical perspectives of RNA polymerase II transcriptional fidelity (939.5)
Authors: Wang Dong 
Published: June 2021

Maintaining a high transcriptional fidelity is essential to life. A long‐standing key question has been how RNA polymerase II (Pol II) recognizes DNA template and substrate and achieves such a high transcriptional fidelity. Here we use a combined structural, computational, and chemical biology approaches to gain novel insights of the molecular basis and chemical interactions in controlling Pol II transcriptional fidelity. We focused on the three key checkpoint steps in controlling transcriptional fidelity: specific nucleotide selection and incorporation, differentiation of RNA transcript extension of a matched over mismatched 3’‐RNA terminus, and preferential removal of misincorporated nucleotides from the 3’‐RNA end (proofreading). Our recent work on the systematic evaluations of individual chemical interactions and the roles of sugar integrity in controlling Pol II transcriptional fidelity will be discussed.Grant Funding Source: NIH GM085136, NIH GM102362
Regulation of acetyl‐CoA homeostasis and global histone acetylation (942.1)
Authors: Galdieri Luciano , Zhang Man , Vancura Ales 
Published: June 2021

Acetyl‐CoA is a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Concentration of acetyl‐CoA affects histone acetylation and links intermediary metabolism and transcriptional regulation. We have identified acetyl‐CoA carboxylase, encoded in yeast by the ACC1 gene, as the key regulator of acetyl‐CoA homeostasis. Acc1p catalyzes the carboxylation of acetyl‐CoA to malonyl‐CoA, the first and rate‐limiting reaction in the de novo synthesis of fatty acids. Attenuated expression of ACC1 results in increased cellular level of acetyl‐CoA, increased histone acetylation and altered transcriptional regulation. The glucose sensor Snf1p, the yeast ortholog of the mammalian AMP‐activated protein kinase (AMPK), phosphorylates and inactivates Acc1p. Inactivation of Snf1p results in an increased conversion of acetyl‐CoA to malonyl‐CoA, reduced pool of cellular acetyl‐CoA, and globally decreased histone acetylation. Collectively, our results demonstrate that signaling pathways that affect acetyl‐CoA homeostasis regulate global histone acetylation and transcriptional regulation. Our current experiments test the hypothesis that global histone acetylation functions as a signal reflecting acetyl‐CoA homeostasis and metabolic state of the cell.Grant Funding Source: Supported by NIH
Tryptophan fluorescence quenching kinetic studies of steps of open complex formation by E. coli RNA polymerase (945.1)
Authors: Xia Xue , Murray Jan , Record Thomas , Yang Zao 
Published: June 2021

To initiate transcription, E. coli RNA polymerase (RNAP) binds promoter DNA, bends the start site region into the active site cleft, opens it using binding free energy, and places the start site base of the template strand in the active site. Structural studies of the open complex show that bending is accompanied by insertion of the ‐11 base into a pocket on the specificity (σ70) subunit. We are using stopped flow fluorescence measurements of tryptophan (W) quenching to investigate when in the mechanism of open complex formation this base‐flipping step occurs, and to characterize the kinetics of this step for different promoters for comparison with other kinetic assays for formation of closed and open complexes. For the λPR promoter at 42°C, tryptophan quenching kinetics fit two exponentials, as expected for the rapid‐equilibrium formation of an ensemble of closed complexes, including the base flipped species, followed by the slower irreversible opening step; rates of these steps are comparable to those determined by other assays. The kinetics are complicated by slower decays which appear to be photobleaching.Grant Funding Source: NIH GM103061