bluuebunny/crossref_metadata_2025_split huggingface dataset:
Influence of CPZ and DMSO on the mechanism regulation of the plasma membrane Ca2+ ATPase of human erythrocyte
Authors: Domínguez‐Solís Carlos Augusto , Plenge‐Tellechea Fernando 
Published: June 2021

The calcium pumping ATPase of the erythrocyte plasma membrane (hPMCA4b) is the principal active system that expulse intracellular calcium into extracellular space in eukaryotic cells. PMCA is regulated by calmodulin (CaM), incrementing its ATP affinity. The use of molecules like chlorpromazine (CPZ) and dimethylsulfoxide (DMSO), inhibitors to hPMCA4b activity (non specific), were used in this study as a molecular tools that could help to understand the regulating mechanism of biochemical activation by CaM activation and their relationship with them. These molecules inhibit the PMCA activity, affecting its calcium homeostasis. CPZ is a phenotiazine that interfere with CaM functionality and it is commonly used in the treatment of mental disease. Erythrocytes membranes were used as the model biological system in order to assess the PMCA due to the absence of other calcium ATPases.PMCA activities in absence and presence of CaM and activated by trypsin exhibited inhibition by DMSO and CPZ. The ATPase activated with CaM in presence of CPZ does not interfere with the mechanism of regulation because there is no protection against the effect inhibitory of CPZ compared with the native PMCA. DMSO interacts with the regulation mechanism in the PMCA activated with CaM since there is the same profile of protection against the inhibition in comparison with the native enzyme. The recovery values of the activity in presence of CPZ and DMSO indicated the possible dilution of CPZ by the solvent.Supported by the grants of Fondos Mixtos CONACYT‐Edo. De Chihuahua and PROMEP‐CAEC.
Haematocrit and urine volume response to acute intravenous saline and oral water loading in normal volunteers
Authors: Chuen M. Jennyfer Ng Kam , Lip Gregory Y. H. , MacFadyen Robert J. 
Published: June 2021

Introduction: Haematocrit (Hct) is widely recognized as a marker of haemodilution. Acute oral water (OW) and intravenous saline (IVS) have variable effects on blood volume. We aimed to test the utility of repeated measures of Hct in mapping this response.Methods: 30 normal volunteers (16 male; age 48.7 ± 8.6, range 48–63; body mass index 24.7 ± 2.9 Kg/m2, range 19.1–31.9) were given in random order, no intervention (C), OW (1.5 L over 20 minutes) or IVS (1.5 L over 20 minutes) on 3 separate days. Hct was measured at 0, 0.5, 1, 2, 4, 6 and 8 h, and urine volume was measured at 4 and 8 h post intervention.Results: Baseline Hct was similar before all treatments [C 40.8 (3.5); OW 41.5 (3.4); IVS 41.9 (4.3); p=0.083] and at 8h [C 39.4 (3.0); OW 39.5 (2.9); IVS 38.7 (3.3); p= 0.118]. On C, there was a significant decrease in Hct between 0 and 0.5 h [41.0 (3.4) versus 38.9 (2.8); p<0.0001]. The fall in Hct was significant and greater for IVS compared to OW, but not between C and either IVS or OW (Figure 1). Urine output rose significantly following both OW and IVS compared to C, with larger increase following OW (Figure 2).Conclusion: The small relative rise in Hct following OW compared to C suggesting a transient reduction in blood volume is in keeping with previous studies. Urine volume response following OW is in contrast to that seen with Hct changes. The mediators of this differential response are unclear.Funding: Haemostasis Thrombosis and Vascular Biology Unit charitable funds.
CVL‐projecting NTS neurons receive input from a distinct subset of vagal afferents
Authors: Aicher Sue A , Hermes Sam M 
Published: June 2021

The vagus nerve contains primary visceral afferents that convey information from cardiovascular, pulmonary, and gastrointestinal tissues to the nucleus tractus solitarii (NTS). We previously showed that two tract tracers, cholera toxin B subunit (CTb) and isolectin B4 (IB4), label distinct populations of vagal afferents. In this study, we tested whether these fibers contact different populations of NTS neurons, including NTS neurons that project to the caudal ventrolateral medulla (CVL). To label CVL‐projecting NTS neurons, the retrograde tracer Fluorogold (2% in 0.9% saline; 50 nl, Fluorochrome Inc.) was injected into CVL; vagal afferents were labeled with injections of CTb and IB4 (2% solution, 1–4 ul) into the vagus nerve. Male Sprague‐Dawley rats (350–425 g) were used and surgeries were performed under isoflurane anesthesia. Seven days later, rats were perfused and vibratome sections through NTS were examined using dual immunocytochemistry for Fluorogold (rabbit anti‐FG, Chemicon) and either CTb (goat anti‐CTb, List Biological Laboratories) or IB4 (goat anti‐IB4, Vector Laboratories). Primary antibodies were visualized using fluorescent secondary antibodies. By confocal microscopy, we found that CVL‐projecting NTS neurons were more than twice as likely to receive somatic input from CTb‐labeled afferents (79%) than IB4‐labeled afferents (34%). In somatic nerves, CTb and IB4 label myelinated and unmyelinated afferents, respectively. Therefore, our findings suggest that CVL‐projecting NTS neurons receive input mainly from myelinated vagal afferents that contain a distinct population of transporters, neurotransmitters, and receptors.Support: NIH HL56301
Determination of MLSS: two tests are better than one
Authors: Yaeger Daniel B. , Stavrianeas Stasinos 
Published: June 2021

The Maximum Lactate Steady State (MLSS) is often estimated using a hybrid VO2max/Lactate Threshold (LT) test even though the literature suggests that it yields a poor estimate of MLSS. We hypothesized that a second incremental test (SIT) improves estimation of MLSS. Trained cyclists (n=5) completed 2 incremental tests on a calibrated computer‐controlled cycle ergometer. After performing a VO2peak test the LTinitial was identified as visual deflection from baseline. In the SIT workload increased in 10 W increments from 30 W below to 30 W above the LTinitial. The LTSIT was then identified using the visual deflection method. To verify the LTSIT as the MLSS, participants performed three 30‐minute bouts at 95%, 100%, and 105% of LTSIT intensity. Although the three intensities differed in workload by only 10 ± 1 W, Heart Rate, VO2, and blood lactate levels were different between the three workloads at all times (p < 0.01). The LTSIT was 99 ± 4% of MLSS, whereas the LTinitial was 93 ± 10% of MLSS intensity. Furthermore, the SIT corresponded to 100% of MLSS for 2 of the cyclists and was within ± 5% of MLSS for all 5 cyclists. We conclude that the SIT greatly improves estimation of MLSS as compared to a single‐test protocol.Supported by NSF‐DUE‐CCLI #0511219, the Mary Stuart Rogers Foundation, and an APS Undergraduate Summer Research Fellowship (DBY).
Superficial and deep aponeuroses movements in the human medial gastrocnemius during isometric plantarflexion contraction
Authors: Kinugasa Ryuta , Shin Dongsuk , Hodgson John A , Edgerton Reggie V , Sinha Shantanu 
Published: June 2021

In pinnate muscle such as medial gastrocnemius (MG), the aponeuroses are located on both sides of the muscle. It has been reported that superficial (SA) and deep aponeuroses (DA) of the MG are stretched homogenously along its length by isometric contraction. However, it is impossible to elongate the entire aponeuroses, because the proximal edge of each aponeurosis attaches to the femur. We hypothesized that at certain locations each aponeurosis is shortened by isometric contraction. Velocity encoded‐phase contrast MRI was used to measure velocity and displacement of each aponeurosis in 4 subjects during repeated plantarflexion (20 and 40% loads). The velocities of each aponeurosis increased and was positive during the contraction phase, declined to zero at peak torque, and then became negative during the relaxation phase, suggesting both moved proximally during contraction and moved distally during relaxation. The displacement moved the least in middle region of the SA and proximal region of the DA along its length. These results indicate that the SA lengthened in the proximal region and shortened in the distal region, and the DA shortened in the proximal region. However, the displacement of the distal region may have been overestimated since the length of this region was of the same order as the fascicle length.This work was supported by National Institute on Arthritis and Musculoskeletal and Skin Diseases Grant.
Nitric oxide synthase inibition and thermoregulatory control of local sweat rate in exercising humans
Authors: Mack Gary W. , Welch Garrett , Foote Kristopher M. 
Published: June 2021

The role of nitric oxide (NO) in modulating human thermoregulatory sweating was examined in 10 subjects (5 male/5 female). Three intradermal microdialysis probes were placed in the dorsal forearm. The control site was perfused with 0.9% saline while the two remaining sites were perfused with a nitric oxide inhibitor: 10 mM NG‐nitro‐L‐arginine (L‐NAME) or 10 mM NG‐monomethyl‐L‐arginine (L‐NMMA). Local sweat rate (SR) and skin blood flow (SkBF) were monitored directly over the path of the intradermal microdialysis probe while arterial blood pressure was measured in the opposite arm non‐invasively. Thermoregulatory sweating was induced by cycle ergometer exercise (60% VO2max) in a warm environment (30°C). Thermoregulatory control of SR was described by the following four parameters: resting sweat rate (SRREST), the increase in esophageal temperature required to initiate a significant rise in SR (ΔTesTHRESHOLD), the slope of the initial linear rate of rise in SR per unit rise in Tes (SR‐ΔTesSLOPE), and peak SR (SRPEAK). In general, NOS inhibition attenuated thermoregulatory sweating during dynamic exercise in a warm environment as shown in the Table below.L‐NMMA appeared to be more effective, than L‐NAME, in reducing the SR response. We conclude that NO has a subtle but physiologically active role in thermoregulatory control of sweating. (supported by NIH‐RO1‐HL039818)
Energy efficiency and substrate partitioning in individuals with atherogenic lipoprotein profile
Authors: Faghihnia Nastaran , Siri Patty W. , Krauss Ronald M. , Brooks George A. 
Published: June 2021

Individuals with an atherogenic lipoprotein profile (ALP), characterized by increased triglyceride, low HDL, and small dense LDL particles, were found to require greater caloric restriction to achieve the same amount of weight loss compared to unaffected subjects (Krauss, R, unpublished data, 2006).PURPOSE:We sought to determine the physiological basis for the greater caloric restriction required for weight loss in subjects with ALP.METHOD:We studied seven men with ALP (ALP+) and six unaffected controls (ALP−) during rest (30 min), exercise (20 min) and postexercise recovery (30 min) by indirect calorimetry to determine energy efficiency and substrate partitioning after an overnight fast. Delta efficiency (DE) was calculated as Δ work accomplished/Δ energy expended between workloads of 10, 50, 100, and 150 watts.RESULTS:ALP+ had higher DE than ALP− at 50–10 watts (P < 0.05). Respiratory exchange ratios (RER) were significantly (P < 0.05) elevated in ALP+ vs ALP– during rest (0.86 +/− 0.01 vs 0.81 +/− 0.02), exercise (0.99 +/− 0.02 vs 0.92 +/− 0.02) and postexercise recovery (1.1 +/− 0.05 vs 0.95 +/− 0.05, respectively).CONCLUSION:Increased efficiency at low exercise intensity and reduced lipid oxidation during rest, exercise and postexercise recovery in ALP+ may explain their increased resistance to weight loss. Supported by NIH grant AR 42906, National Dairy Council, and Atherosclerosis Research at CHORI.
Podoplanin is required for human lymphatic endothelial cell migration in response to VEGF stimulation
Authors: Navarro Angels , Perez Ricardo E , Rezaiekhaligh MO , Mabry Sherry M , Ekekezie Ikechukwu I 
Published: June 2021

Podoplanin, a transmembrane protein with expression in endothelial cells restricted to those of lymphatics, has been associated with increased tumor migratory and invasive behavior. However, its role, if any, in endothelial cell migration is unknown.METHODS: Confluent monolayers of siRNA‐podoplanin silenced versus control human lung lymphatic microvascular endothelial cells (HMVEC‐LLy) were scratch‐wounded and cell migration analyzed in basal media (0.5% FBS), VEGF‐containing media (10 ng/ml in basal media) or complete media (5% FBS and growth factors). Pictures were acquired at regular intervals over 2–3 days. Cell polarization was assessed 2–3 hours after wounding by immunocytochemistry for α‐tubulin and pericentrin.RESULTS: By 48 hours, wounds in control HMVEC‐LLy exposed to either 10 ng/ml VEGF or complete media are 100% healed versus only 40% at 72hrs for siRNA‐podoplanin HMVEC‐LLy. Moreover, reorientation of microtubule‐organizing center (MTOC) towards the front of the cell in response to VEGF is observed to be impeded in these cells.CONCLUSION: Podoplanin is required for a full migratory response to VEGF in HMVEC‐LLy. Complete reorientation of the MTOC in response to VEGF, necessary for directional migration, is achieved only if the cells express podoplanin.Funded by: Children's Mercy Hospital
Thrombin activates calcium independent phospholipase A2 (iPLA2) in lung microvascular endothelial cells
Authors: Rastogi Prerna , McHowat Jane 
Published: June 2021

The alveolar‐capillary barrier comprises of type 1 pneumocytes of the alveolar wall, endothelial cells of the capillaries and the basement membrane. Its function is to prevent blood entering the alveoli and to facilitate efficient gaseous exchange. Any disruption in the lining of the lung microveasculature is potentially deleterious. This study examines thrombin stimulation of human lung microvascular endothelial cells (HMVEC‐L) and the resultant increase in iPLA2 activity. Incubation of HMVEC‐L with thrombin resulted in a fourfold increase in iPLA2 activity that remained elevated for up to 60 minutes. Pretreatment of HMVEC‐L with bromoenol lactone (BEL, 2μM, 10 min, a selective iPLA2 inhibitor) but not with PX‐18 (2μM, 10 min, an sPLA2 inhibitor), or AACOCF3 (2μM, 10 min, a cPLA2 inhibitor) attenuated thrombin‐activated iPLA2. Activation of iPLA2 resulted in a rapid increase in arachidonic acid and PGI2 release that was also inhibited by BEL pretreatment. Thrombin stimulation also resulted in a four‐fold increase in neutrophil adherence to the apical surface of HMVEC‐L that was inhibited by BEL pretreatment of HMVEC‐L and pretreatment of the neutrophils with a PAF receptor antagonist. Taken together, these results demonstrate that iPLA2 inhibition has the potential to block the several inflammatory responses produced by thrombin in the lung microvasculature.Supported by AHA, Pre‐Doctoral Fellowship (PR)
The tight junction proteins ZO‐1 and occludin undergo novel MLCK‐independent redistribution during TNF‐induced cell shedding
Authors: Marchiando Amanda M , Guan Yanfang , Hecht Aaron L. , Angus Elizabeth M. , Montrose Marshall H. , Turner Jerrold R. , Watson Alastair JM 
Published: June 2021

Small intestinal epithelial cells are continuously shed from the villus at steady state. Small discontinuities, or epithelial gaps, occur during in vivo cell shedding. Our aims were to determine if tumor necrosis factor (TNF) increased rates of shedding and gap formation, the role of myosin light chain kinase (MLCK) in shedding, and whether the tight junction contributes to barrier maintenance at these sites.Fluorescent‐tagged ZO‐1 or occludin expression in intestinal epithelia was driven by the villin promoter. Transgenic mice were injected with TNF (7.5μg, i.p.). Shedding was evaluated by video confocal microscopy. TNF increased shedding by 27‐fold (p<0.007), beginning 60 min after TNF, and caused reorganization of both ZO‐1 and occludin. Over the 10 min during which shedding occurred, both ZO‐1 and occludin migrated downward along lateral membranes and condensed beneath shedding nuclei to form a funnel that moved apically with the extruding cell. Extracellular probes (e.g. lucifer yellow) approached but did not cross the line of ZO‐1 and occludin, suggesting maintenance of the mucosal barrier. Tight junction remodelling was not altered via enzymatic MLCK inhibition or in MLCK knockout mice, suggesting that this process may not require MLCK.We conclude that ZO‐1 and occludin undergo novel MLCK‐independent redistribution at sites of barrier maintenance during TNF‐induced cell shedding.Supported by NIH.
Induced JunD in intestinal epithelial cells represses CDK4 transcription through dimerization with ATF‐2 following polyamine depletion
Authors: Xiao Lan , Rao Jaladanki N. , Zou Tongtong , Liu Lan , Chen Jie , Yu Tingxi , Turner Douglas J. , Wang Jian‐Ying 
Published: June 2021

Intestinal epithelium is a rapidly self‐renewing tissue and its homeostasis is regulated by polyamines. Decreasing polyamines increases JunD inhibiting intestinal epithelial cell (IEC) renewal through CDK4 repression, but the mechanism by which JunD represses CDK4 remains elusive. ATF‐2 is an ATF/CREB transcription factor and its expression is regulated by polyamines. Here, we determined if JunD forms dimers with ATF‐2 repressing CDK4 transcription following polyamine depletion in IECs.Methods: In vivo JunD binding was detected by immunoprecipitation assay (IP), while in vitro dimerization was examined by GST pull‐down assays. CDK4 transcription was measured by CDK4‐promoter luciferase reporter gene assays. ATF‐2 function was investigated by RNA interference.Results: Induced JunD formed complexes with ATF‐2 as measured by IP, which was confirmed by GST pull‐down assays. Studies using deletion‐ and point‐mutations of ATF‐2 revealed that JunD/ATF‐2 dimer formation depended on the C‐terminal leucine zipper motif located between 380th to 413th amino acids in ATF‐2 sequence. JunD/ATF‐2 dimers were increased following polyamine depletion, which were associated with CDK4‐promoter inhibition. ATF‐2 silencing prevented CDK4 repression induced by JunD and enhanced IEC renewal.Conclusions: Induced JunD represses CDK4 transcription through dimerization with ATF‐2 following polyamine depletion.
Susceptibility of the colon to psychological stress‐induced changes is increased by exposure to previous inflammation
Authors: Santiago Olga I , Cruz Myrella L , Cuevas Marielly , Appleyard Caroline B 
Published: June 2021

Stress has been associated with exacerbation of symptoms in disorders such as Irritable Bowel Syndrome.AIM:To determine the effects of prior inflammation on subsequent stress‐induced changes in colonic cytokine and bacterial levels.METHODS:Four groups of female rats were used: normal (untreated); control‐WAS (water avoidance stress: for 3 hrs, during 3 consecutive days); control‐colitis, animals received trinitrobenzene sulfonic acid (TNBS 30mg in 50% ETOH ic) to induce an acute colitis which was allowed to heal for 2 weeks; and colitis‐WAS, the animals received TNBS as above, then WAS 2 weeks later. All animals were evaluated for weight change, macroscopic damage and histology. Colonic tissues were inoculated on blood, MacConkey or lactobacillus agar. Colonic tissue levels of IL‐10, IL‐4 and TGF were measured by ELISA.RESULTS:Stress increased weight loss in animals that previously had colitis compared to controls (p<0.05). These animals also showed increased macroscopic and microscopic damage, and mast cell numbers. Colitis‐WAS had the lowest levels of lactobacillus, but little change in total bacterial load. Colitis‐WAS had decreased levels of IL‐4 and IL‐10, and increased levels of TGF compared with control‐WAS.CONCLUSION:Previous inflammation increases the susceptibility of the colon to subsequent stress, with alterations in the microflora and immune response. F31 DK074378 & 2G12RR03050
Epidermal growth factor preserves intestinal integrity and decreases mortality in a murine model of Pseudomonas aeruginosa pneumonia
Authors: Clark Jessica Ann , Lawrance Christopher P. , Dunne William M. , Coopersmith Craig M. 
Published: June 2021

Exogenous epidermal growth factor (EGF) attenuates intestinal injury and decreases mortality in a peritonitis model. However, the role of EGF in sepsis caused by extraabdominal infection is unknown.We hypothesized that EGF preserves intestinal integrity and decreases mortality in P. aeruginosa pneumonia‐induced sepsis.FVB/N mice were intratracheally injected with 40 μl of 0.6 McF P. aeruginosa and treated with or without EGF (150 ug/kg/d i.p.; n=9/group). Controls were injected with saline (n=5). All mice received antibiotics. At 24 hr, intestines were evaluated for apoptosis by caspase‐3 staining, proliferation by BrdU staining, and villus length. Survival studies were done in a separate cohort of mice (n=5–10/group).Compared to shams, septic mice had increased intestinal apoptosis (36 ± 3 vs. 17 ± 3 cells/100 crypts; p<0.001) and decreased proliferation (502 ± 51 vs. 959 ± 43 cells/100 crypts; p<0.001), while EGF normalized both to sham levels (21 ± 1 vs. 17 ± 3 and 886 ± 64 vs. 959 ± 43 cells/100 crypts, respectively; p=ns). Septic mice had shorter villi compared to shams, and EGF treatment resulted in normalization to sham villi lengths (333 ± 15 vs. 415 ± 30 vs. 432 ± 19 μ m; p<0.01 and p=ns). At 7 days, septic EGF treated mice had a survival advantage compared to septic mice (92% vs. 50%; p=0.05).EGF preserves intestinal integrity and improves survival in pneumonia‐induced sepsis; thus, the protective effects of EGF are not specific to the anatomical site or type of infection. EGF may be a novel therapeutic agent for the treatment of sepsis.
Ferroportin residue C326 is critical for its interaction with hepcidin
Authors: Nemeth Elizabeta , Fernandes Augustine , Ganz Tomas 
Published: June 2021

The hormone hepcidin binds to the Fe exporter Fpn and induces its internalization and degradation, decreasing Fe efflux from cells into plasma. Fpn mutations that confer resistance to hepcidin cause classical hemochromatosis with parenchymal Fe loading. We analyzed mutations C326S, C326T, N144D and N144T to address the mechanism of Fpn interaction with hepcidin. Human Fpn‐EGFP construct was subjected to site‐directed mutagenesis and analyzed in HEK293T cells. The Fpn mutants localized properly to the cell surface and exported Fe normally. However, they were either completely (C326S/T) or partially (N144D/T) resistant to hepcidin‐mediated internalization, and exported Fe even in the presence of hepcidin. 125I‐hepcidin binding was completely (C236S/T) or partially (N144D/T) abrogated.C326 and C205 were reported to be cell surface thiols. A non‐permeable thiol‐dependent crosslinker linked hepcidin to Fpn but not in C326S/T mutants, and only partly in N144D/T mutants. Alkylation of Fpn‐expressing cells ablated 125I‐hepcidin binding. We next examined whether hepcidin binding depended on a transient SS‐bond formation between C205 and C326. C205S/T substitutions, however, only partially inhibited hepcidin uptake and crosslinking. Bacitracin had no effect on hepcidin uptake or Fpn internalization indicating that isomerisation of a C205‐C326 disulfide bond was not necessary. Thus the C326 thiol is located in or near the binding site of hepcidin and is essential for hepcidin binding and subsequent internalization of ferroportin. Our observations help explain the severity of hemochromatosis in patients with C326X mutations.