bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of a prior exhausting exercise on peak O2 uptake, lactate and ventilatory thresholds during ramp exercise in men
Authors: Esposito Fabio , Vanni Rosalba , Alfieri Paola , Cè Emiliano , Veicsteinas Arsenio
Published: June 2021

Aim of the study was to evaluate the effects of a prior exhausting exercise on maximum performance, lactate threshold (LaT) and first (V̇et1) and the second (V̇et2) ventilatory thresholds. Eight young males performed a maximum ramp (25 W·min−1) cycloergometric test before and 5 hours after an exhausting exercise. During tests, breath‐by‐breath measurements of gas exchange, power output and blood lactate concentration ([La−]) were obtained. LaT together with V̇et1, V̇et2 and the amplitude of the isocapnic buffering range of exercise (V̇et2 − V̇et1) were then calculated. After exhaustion it resulted that: i) the peak power significantly decreased from 247±11 W to 234±12 W (P<0.05), and a lower V̇o2 peak (2.77±0.15 L·min−1 vs. 2.65±0.14 L·min−1, P<0.05) was observed; ii) LaT occurred at higher V̇o2 (1.96±0.10 L·min−1 and 2.15±0.15 L·min−1 before and after exhaustion, respectively; P<0.05), whereas V̇et1 was similar in both tests; iii) V̇et2 appeared at lower V̇o2 (2.63±0.16 L·min−1 and 2.47±0.17 L·min−1 before and after exhaustion, respectively; P<0.05), and iv) V̇et2 − V̇ET1 decreased significantly by 21±4% (P<0.05). These results show that prior exhausting exercise affected the metabolic and respiratory response of a following exercise and are compatible with the hypothesis that prior exhausting exercise alters acid‐base status and the activity of the enzymes involved in CO2 transport and buffering systems. The study was supported by a grant of Fondazione CARIPLO.
The effects of patent foramen ovale (PFO) on pulmonary gas exchange during incremental exercise
Authors: Lovering Andrew Thomas , Stickland Michael K. , Amann Markus , Murphy Joan C. , Hokanson John S. , Eldridge Marlowe W.
Published: June 2021

The prevalence of a probe‐patent foramen ovale (PFO) reported in autopsy studies vary between 20 and 35%. We hypothesized that individuals with a PFO (PFO+) would have greater pulmonary gas exchange inefficiency during exercise than subjects without a PFO (PFO−) because a PFO could act as a right‐to‐left shunt. PFO+ (n=8) and PFO− (n=8) performed two incremental cycle ergometer exercise tests to max, separated by one hour, while breathing either room air or hypoxic gas (FIO2=0.12). Using saline contrast echocardiography, both PFO− and PFO+ demonstrated significant intrapulmonary shunt (bubbles appearing in the left atrium (LA) after 5 heart beats) during both exercise conditions. However, at rest and during both exercise conditions in PFO+, contrast bubbles were also observed traveling through the foramen ovale as small, intermittent boluses entering directly into the LA within 3 heart beats. These findings suggest, qualitatively, a very small intracardiac shunt at rest and during exercise in PFO+. Consequently, the alveolar‐to‐arterial oxygen difference was not significantly different between PFO+ and PFO− at either peak normoxic (24.2 ± 7.8 vs. 17.5 ± 10.8 mm Hg, p= 0.18) or hypoxic (22.7 ± 2.9 vs. 20.2 ± 4.9 mm Hg, p= 0.25) exercise. We conclude that a PFO does not further exacerbate pulmonary gas exchange inefficiency occurring during exercise in healthy humans. Support: HL‐15469 & T32 HL07654; AHA 0550176Z.
The menstrual cycle and exercise‐induced cutaneous blood flow
Authors: Ware Stuart K. , Ware Jason L.
Published: June 2021

The study purpose was to determine if menstrual cycle (mc) phase impacts the cutaneous vascular response to exercise in young women. Thirteen healthy women ages 20‐28 yrs were studied (mean 23.3 years). None of the women were taking oral contraceptives and all reported having had regular menstrual cycles for the past year. The exercise consisted of 20 min of brisk treadmill walking at a room temp of 32 C and 45% relative humidity. Skin blood flow (SBF) at the forearm and back was recorded before and throughout the exercise. A dual‐channel laser Doppler flowmeter was used to measure SBFs, and the values were stored in computer and later analyzed by commercial software. Using ANOVA mean SBFs for both skin locations were compared before and at 2 min intervals during exercise. One set of SBFs was taken at the mid‐follicular phase (F) of the mc. Another set was recorded at the mid‐luteal phase (L). The order in which the mc phases were studied was randomized. The vascular responses at the two phases were then compared to determine if mc phase altered the thermoregulatory response. Results showed vasodilation at each skin location during both mc phases. When the two phases were compared, it was found that the response was most pronounced during the L phase (12% greater SBF at forearm, P = 0.08; 23% greater SBF at back, P = 0.03). The different vascular responses to exercise are likely accounted for by the different hormonal conditions present.
Maximal cutaneous blood flow is not affected by endurance training: implications for physiological interpretation of cutaneous blood flow
Authors: Ray Chester A , Kuipers Nathan T , Kearney Matthew L
Published: June 2021

Cutaneous blood flow can be altered by non‐sympathetic influences such as hydration level, acclimatization, and training level. It is currently unknown what effect these factors have on maximal cutaneous blood flow. The purpose of this study was to investigate the effect of endurance training on maximal cutaneous blood flow. Fifteen healthy sedentary subjects (age, 25±1 years) had maximal cutaneous blood flow measured before and after eight weeks of endurance training or eight weeks of sedentary control. Arterial blood pressure (Finometer), brachial blood flow (Doppler ultrasound), and forearm cutaneous vascular conductance (laser Doppler flowmetry; flux/mean arterial pressure) were measured for 5 min at room temperature (22±1ºC). The arm was then heated for 30 min followed by an additional 30 min of measurements. A water perfused tube‐lined sleeve was used to heat the forearm skin to 41±1ºC. Exercise training increased VO2peak by 19.3%. Brachial blood flow and cutaneous vascular conductance increased with heating. Training did not alter brachial blood flow at rest (47±11 ml/min before training and 41±6 ml/min after training) or during maximal heating (217±28 ml/min before training and 217±28 ml/min after training). Training did not alter cutaneous vascular conductance at rest (0.010±0.003 ml/min/mmHg before training and 0.010±0.002 ml/min/mmHg after training) or during maximal heating (0.047±0.007 ml/min/mmHg before training and 0.051±0.009 ml/min/mmHg after training). These results suggest that endurance training does not alter maximal forearm cutaneous blood flow in humans.

Research supported by NIH DC006459, HL077670, & M01RR10732
In‐magnet bicycling exercise: a novel 31P MRS window on the energetics of human locomotion
Authors: Jeneson Jeroen , Schmitz Joep , Nicolay Klaas
Published: June 2021

The clinical standard test of patient fitness is the upright bicycle exercise test. For a number of reasons, no proper equivalent human MR exercise test has been available. Past 31P MR studies employing single limb exercise regimens generally failed to put any significant demands on the cardiovascular system (1). As such, a comprehensive understanding of skeletal muscle performance during whole body activity has been lacking. Here, we report on 31P MRS studies employing a novel ergometer that for the first time offers true in‐magnet human bicycling exercise testing. Heart rates directly following exercise were of 150 + 15 bpm. In addition to 31P MRS study of ATP metabolism over a 100‐fold dynamic range of ATP turnover at near‐constant pH, it allows for non‐invasive 31P MRS study of glycogenolysis through the dynamics of hexose monophosphate (HMP) resonances. Here (but not previously (2)) we routinely observed HMP accumulations of up to 10 mM within 2 minutes after termination of exercise at high workloads indicating massive activation of glycogenolysis during the preceding exercise. Yet intramuscular pH typically did not fall below 6.8 during exercise confirming our previous observation of unique homeostatic robustness of quadriceps muscle involved in two‐legged exercise (2).
Proteomics of Pathological Protein in Neurodegenerative Diseases
Authors: Montine Thomas
Published: June 2021

Several neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and enigmatic disease on Guam that shares features of both called Parkinson‐Dementia Complex (PDC) are proposed to derive from pathological protein that share a number of potentially inter‐related biochemical features including misfolding, decreased surfactant solubility, aggregation into amyloid or inclusions, and extensive post‐translational modifications. We have employed stable isotope dilution techniques coupled with liquid chromatography followed by tandem mass spectrometry as a means of discovering those proteins that adopt a pathological phenotype in these diseases. Our first set of experiments used laser capture microdissected (LCM) to obtain neurofibrillary tangles (NFTs) from brain sections of patients with AD, or neocortical Lewy Bodies from individuals Dementia with Lewy bodies. Our work has confirmed the presence of many proteins previously associated with these structures, and identified new protein constituents subsequently confirmed by immunohistochemistry. Our second set of experiments used reduced surfactant solubility to enrich for those proteins that may adopt pathological configurations in diseased tissue. We discovered approximately 100 proteins with reduced surfactant solubility that were not identified previously in any of these diseases, and discovered some surfactant insoluble proteins already known in one disease in another. For example, we discovered surfactant‐insoluble alpha‐synuclein in PDC frontal cortex despite the lack of Lewy bodies. Finally, we have used these datasets to screen for post‐translational modifications on these pathologic proteins. These results demonstrate the power of proteomic‐based discovery tools in the comprehensive assessment of pathological protein in neurodegenerative diseases.
POSH decreases ROMK1 channel activity through stimulating clatharin‐independent and dynamin‐dependent endocytosis.
Authors: Lin Daohong , Yue Peng , Sun Peng , Jin Yan , Roos Marcel , Caplan Michael , Wang Wenhui
Published: June 2021

Through a yeast‐two‐hybrid‐screen we detected an interaction between POSH and the C‐terminus of ROMK1. Immunostaining reveals that POSH is highly expressed in the renal cortical collecting duct (CCD). Moreover, immunoprecipitation of renal tissue lysate with ROMK antibody can pull‐down POSH. To study the effect of POSH expression on ROMK1 channels, we used two‐electrode voltage clamp to measure the K current in oocytes injected with ROMK1 or ROMK1+POSH. Co‐injection of 1, 2.5, 5 and 10 ng POSH RNA decreased K current in oocytes injected with 5 ng ROMK1 RNA by 12%, 25%, 45% and 50%, respectively. The effect of POSH on ROMK1 channels was specific because POSH did not inhibit Na current in oocytes injected with ENaC‐alpha, beta and gamma subunits. The inhibitory effect of POSH on ROMK1 channels requires the RING domain of POSH since deleting the RING domain abolished the inhibitory effect of POSH on ROMK1 channels. Also, the inhibitory effect of POSH on ROMK channels was absent in oocytes injected with dominant negative dynamin. However, POSH still decreased the K current in oocytes injected with a ROMK1 mutant in which amino acid residues 373–378 that contain the clathrin‐dependent tyrosine‐based internalization signal were deleted. We conclude that POSH‐induced inhibition of ROMK channels is due to stimulation of the dynamin‐dependent and clathring‐independent endocytosis.
Translocation of caveolin into the endoplasmic reticulum and transport of cholesterol to caveolae requires a functional “flip domain”
Authors: Guerin Theresa , Smart Eric J
Published: June 2021

We previously demonstrated that a caveolin‐chaperone complex transports newly synthesized cholesterol from the endoplasmic reticulum through the cytoplasm to caveolae. We also showed that the depletion of caveolae with cholesterol oxidase induces caveolin to translocate to the lumen of the ER. We hypothesized that the amino acid sequence, VxKxWF, functions as a “flip domain” to facilitate translocation of caveolin into the ER. To test this hypothesis, tryptophan‐98 was changed to phenylalanine. A myc‐tag was added to the C‐terminal ends of wild type caveolin (wt‐myc) and the mutant caveolin (flip‐myc) and both constructs were stably expressed in Swiss 3T3 cells. Both wt‐myc and flip‐myc caveolin localized to caveolae as determined by subcellular fractionation and indirect immunofluorescence. Treatment of cells with cholesterol oxidase caused wt‐myc caveolin to translocate from caveolae to the ER and flip‐myc caveolin to translocate from the caveolae to the cytosol. When wt‐myc and flip‐myc were expressed in L1210 cells, both proteins formed a complete caveolin‐chaperone but only wt‐myc rapidly transported newly synthesized cholesterol from the ER to caveolae. We conclude that tryptophan‐98 in the putative caveolin “flip domain” is necessary for the translocation of caveolin into the ER and for the rapid transport of cholesterol to caveolae.
Role of Chylomicron Formation and Secretion in the Release of Diabetic Hormones GLP‐1 and GIP
Authors: Lu Wendell J , Yang Qing , D'Alessio David , Tso Patrick
Published: June 2021

Glucagon‐like peptide‐1 (GLP‐1) and glucose‐dependent insulinotropic polypeptide (GIP) are incretins produced in the intestine that play a central role in glucose metabolism and insulin secretion. Plasma GLP‐1 and GIP concentrations are low and difficult to assay in rodents. We have established a novel method of assaying GLP‐1 and GIP in vivo using the intestinal lymph fistula model. These studies further utilize this model to investigate the mechanism of lipid‐stimulated secretion of these incretins.

Using this model, we have determined that peak GLP‐1 and GIP levels are 5–6 times higher in lymph than portal vein plasma. We further explored the role of chylomicron formation and secretion during lipid‐stimulated secretion of these incretins. Pluronic L‐81 (L‐81), a surfactant inhibiting chylomicrons, was given concurrently with Intralipid. The presence of L‐81 caused a significant decrease in lymph triglyceride level (P < 0.001). L‐81 plus Intralipid was shown to reduce lymph GLP‐1 secretion by 30 min (P < 0.001) and 60 min (P < 0.05), respectively, and the total GLP‐1 output over 5 hours was also significantly blunted (P < 0.046) compared to Intralipid stimulation alone. The effect of L‐81 was even greater with lipid‐stimulated GIP secretion which was completely abolished, and the decrease in total GIP output was more apparent (P < 0.008). We also demonstrated there was no direct correlation between free fatty acids levels and incretins secretion. We conclude chylomicron formation and secretion may be critical in the release of incretins in vivo during lipid ingestion, and GLP‐1 and GIP exhibit independent mechanisms of lipid‐stimulated secretion.
Ectopic expression of an RNA‐binding protein delays the maturation of female and male secondary sex organs
Authors: Schneider Andrew , Clark Brad , Tessier Charles , Ross Jeff
Published: June 2021

We have discovered an RNA‐binding protein (CRD‐BP) that is expressed in multiple tissues throughout embryonic development, but cannot be detected in most adult tissues, including normal human breast tissue. Conversely, CRD‐BP is detectable in 40–60% of human breast cancers. To determine whether or not ectopic CRD‐BP expression is capable of inducing breast tumors, we generated Tet‐On transgenic mice in which CRD‐BP expression was doxycycline‐inducible in mammary epithelial cells. Early characterization of the mammary glands of the Tet‐On transgenic mice has revealed that when CRD‐BP is induced from pre‐adolescence (3 weeks old) to early adulthood (8 weeks old), the mammary ductal tree fails to elongate properly. Moreover, growths or buds along the length of the ducts give them a rough‐looking appearance, when compared to wild‐type mammary glands not expressing CRD‐BP. We have also identified a developmental phenotype in male Tet‐On transgenic mice. The seminal vesicles of CRD‐BP expressing males produce minimal seminal vesicle secretions, resulting in infertility. We conclude that this developmentally regulated RNA‐binding protein can compromise post‐natal sexual development. This research is funded by an NIH RO1 grant.
Rescue of Growth Retardation and Perinatal Lethality of Nedd4‐1 Null Mice by Loss of Maternally Inherited Grb10
Authors: Yang Baoli , Cao Xiao Renee , Lill Nancy L , Shi Peijun Peter , Sweezer Eileen M , Place Trenton , Kirby Pat A , Shao Dingwu , Kinney Thomas S , Stokes John B
Published: June 2021

Nedd4‐1, a HECT type ubiquitin ligase, has been proposed to be involved in the regulation of type 1 insulin‐like growth factor receptor (IFG‐1R) from in vitro data. Mice with Nedd4‐1 disrupted were generated in this study. At birth, the body weight of −/− mice was about 1/3 of that of +/+ littermates and they died right after birth. Immature or delayed development was seen throughout, consistent with the notion that the IGF‐1R pathway is inhibited. Mouse embryonic fibroblasts (MEFs) isolated from Nedd4‐1 −/− embryos showed reduced mitogenic activity. IGF‐1R, presented on the plasma membrane of +/+ MEF, was not detected on Nedd4‐1 −/− MEF. Knock‐down of Grb10 using siRNA in the −/− MEF restored IGF‐1R expression. In addition, Nedd4‐1 −/− lethality was rescued by the maternal inheritance of Grb10 disruption (Grb10 −m/+p). Thus, the growth inhibitory effect on IGF‐1R in the absence of Nedd4‐1 is mediated through Grb10.
Increased Hsp32 Expression and Endothelial Dysfunction in Human
Authors: Rafiee Parvaneh , Nelson Victoria , Otterson Mary , Binion David
Published: June 2021

INTRODUCTION

Endothelial dysfunction in IBD is associated with loss of NO generation and sustained levels of reactive oxygen species (ROS), which is linked to a loss of iNOS gene transcription. Hsp32 is associated with loss of NO generation via inhibition of iNOS gene expression in various disease, but little is known about its expression in IBD. We characterize Hsp32 expression in control and IBD endothelial cells, microvessels and submucosal gut tissues, to define the molecular mechanisms underlying the loss of NO generation,

METHODS: Hsp32 and iNOS expression were assessed in freshly resected human gut tissues (normal and IBD) and human intestinal microvascular endothelial cells (HIMEC) by immunohistochemistry, RT‐PCR and Western blotting.

RESULTS: Hsp32 expression was significantly increased in Ulcerative colitis (UC) and Crohn's disease (CD) compared to control tissue (n=9 for each group). Whereas the level of iNOS was decreased in the same samples. Strong staining for Hsp32 and a very weak iNOS staining in UC and CD microvessels compared to control tissue was observed. TNF/LPS resulted in increased iNOS expression in HIMEC.

CONCLUSION: Increased Hsp32 expression in IBD microvessels may limit NO production leading to endothelial dysfunction. Hsp32 induction during chronic stress is an epigenetic mechanism for altered endothelial gene transcription and homeostasis in human IBD.
Modulation of the human paraoxonase gene family members by various effectors in Caco‐2 cells
Authors: Précourt Louis‐Philippe , Delvin Edgard , Amre Devendra , Sinnett Daniel , Seidman Ernest , Levy Emile
Published: June 2021

The human paraoxonase (PON) gene family is composed of three members (PON1, PON2, PON3) aligned next to each other on chromosome 7. They are highly conserved in mammals, display similarities in their structural properties (~65% identity atthe amino acid level) and seem to play an important part in oxidative stress. Recently, PONs have been detected in the gastrointestinal tract, but their physiological roles and regulation remain obscure. Our objective was to examine the modulation of PON1, PON2 and PON3 in the Caco‐2 cell line. Fe2+‐ascorbate‐mediated lipid peroxidation promoted MDA formation without affecting sucrase activity, transepithelial resistance, DNA and protein content, as well as cell viability. The addition of the oxygen radical‐generating system reduced the gene expression of PON1, PON2 and PON3. Pre‐incubation with the antioxidant BHT or Trolox restrained the production of MDA and the inhibitory effect on PON mRNA levels. Similarly, the administration of CAPE, an inhibitor of NF‐kB activity, reversed the negative influence of inflammatory agents, including LPS, TNF‐α and INF‐γ. Rosiglitazone and DHA enhanced the gene expression of PON1 and PON3 without any influence on PON2 mRNA levels. Our data indicate that various effectors may impact on the gene expression of PON1, PON2 and PON3, sometimes with a distinct modulation.

Supported by Crohn's and Colitis Foundation of Canada.
IFN‐γ‐producing γδT Cells Promote Liver Injury in Virus Infected Liver
Authors: AJUEBOR MAUREEN , Jin Y. , Gremillion G. , Chen Q. , Adegboyega P.
Published: June 2021

γδT cells are a small population of CD3(+) innate immune T cells that are few in the liver (vs. the mucosal & brain), hence their role in viral liver infections is largely unknown. Adenovirus (AdV) was used to induce viral liver infection in male C57BL6 mice. Liver injury in response to AdV infection was paralleled by significant increases in the influx of γδT cells into the liver (by FACS) at days 1 and 6 (vs. control). Moreover, γδT cells were specifically localized in the hepatocytes. By FACS, we observed that AdV directly infect hepatic γδT cells to mediate increased IFN‐γ production by γδT cells. In addition, AdV infection mediated increased hepatic levels of IFN‐γ‐inducible/hepatocyte‐derived CXCR3 ligands. Both IFN‐γ KO and γδT cell KO mice were associated with significant reductions in (i) liver damage and (ii) hepatic levels of CXCR3 ligands (vs. WT mice) post‐AdV. Furthermore, CXCR3 Ab treatment significantly reduced liver‐infiltrating γδ T cells and hepatic injury post‐AdV. These results support our hypothesis that AdV directly infects/activates hepatic γ[delta]T cells to induce IFN‐γ production. IFN‐γ‐producing γ[delta]T cells subsequently act on hepatocytes in an autocrine manner to induce the production of hepatic CXCR3 ligands, which inturn exert liver damage by promoting/sustaining the influx of IFN‐γ‐producing γ[delta]T cells.
Gated pore mutation affects ferritin iron in vivo
Authors: Hasan Mohammad Rubayet , Theil Elizabeth C.
Published: June 2021

Ferritins, spherical protein cages, concentrate iron and oxidants as ferric oxy biominerals. Eight pores, formed by junctions of three subunits, have crucial roles in iron removal from ferritin minerals based on the coincidence of pore unfolding (X‐ray crystallography, CD spectroscopy in mutant and wild type proteins) and changes in iron removal rates in frog model ferritins. Sequence similarity between human and frog ferritins is 65%, but quaternary structure and pore gates are the same. To examine the effects of pore mutations in human cell lines we made the analogous mutation in human H ferritin: L138P (L134P in horse/frog numbering). We observed, as in the frog mutant, there was no effect on cage assembly (gel filtration), and the initial rate of Fe removal from the mineral was faster (4.9‐fold, P<0.01), with 50% Fe removed from L138P in 2.2 min vs. 46.6 min from the wild type (WT). To study the pores in vivo, HeLa cells were transiently transfected with plasmids encoding H‐WT or H‐L138P ferritin, with comparable increases in ferritin protein (8 fold). When the retention of Fe in ferritin (cpm/ng ferritin protein) was compared, using cells incubated two hours with 59Fe‐transferrin, H‐L138P had only 25% (P<0.01) as much 59Fe as WT; the background used was 59Fe in endogenous ferritin (mock transfection). The data on ferritin pores in cultured cells, together with a study identifying a ferritin binding peptide with effects similar to L134 mutation in vitro, (Liu et al, JBC, 282:31821), suggest that cytoplasmic factors regulate recovery of iron from ferritin in vivo by pore folding/unfolding. Part support (NIH Grant DK20251).
Hepcidin‐regulated and hepcidin–independent iron release pathways in hepatocytes and macrophages
Authors: Valore Erika V , Ganz Tomas
Published: June 2021

Ferroportin (Fpn), a multi‐transmembrane protein, is the only known cellular exporter of Fe. In Fpn‐transfected cells or in macrophages, the iron‐regulatory hormone hepcidin (Hepc) binds to Fpn, induces its internalization and degradation, and thereby inhibits the release of Fe from cells. It is not clear whether the same mechanisms also regulate Fe efflux from hepatocytes. We examined Fe release from primary hepatocytes, hepatocyte cell lines (HepG2 and Hep3B), and macrophage cell lines (J774 and RAW264) with or without 7.5 μg/ml Hepc. After loading with 55Fe‐nitriloacetic acid, radioactive Fe release was monitored over 72h. In primary hepatocytes, Hep3B cells and macrophage cell lines, Hepc inhibited the total Fe release by up to 50% compared to control. The media were analyzed by gel‐filtration. For all cell types, 55Fe eluted in two fractions. The major fraction, ≈70 kD, eluted with transferrin, and its 55Fe content was decreased by Hepc‐treating the cells. A second fraction was > 220 kD and its 55Fe content was unaffected by Hepc‐treating the cells. On SDS‐PAGE, the 55Fe‐containing >220 kD fraction was unaffected by reducing conditions but was degraded by boiling. In summary, hepatocytes and hepatocyte‐derived cell lines exhibit Hepc‐regulated and presumably Fpn‐mediated Fe efflux. One or more alternative pathways for Fe export, not regulated by Hepc, also exist in hepatocytes and macrophage cell lines.

Support provided by the Will Rogers Fund and NIH RO1 DK 065029
Ferropotin Inhibits HIV‐1 Transcription
Authors: Nekhai Sergei , Foster Altreisha , Xu Min , Niu Xiaomei , Knutson Mitchel D , Gordeuk Victor R
Published: June 2021

HIV‐1 transcription is induced by viral transcriptional activator, Tat protein that recruits transcriptional co‐activators, including CDK9/cyclin T1 to the HIV‐1 promoter. We recently showed that Tat is a substrate for CDK2 and that mutations in the Ser16 and Ser 46 residues of Tat that were phosphorylated by CDK2, prevented HIV‐1 transcription and viral replication. We also recently showed that iron chelators ICL670 and 311 inhibited on Tat‐induced HIV‐1 transcription by inhibiting cellular activities of CDK2 and CDK9. Thus our previous studies suggest that a decrease in cellular iron might have a protective effect against HIV‐1. Here, we analyzed the effect of iron exporter, ferroportin on HIV‐1 transcription and viral replication. Expression of ferroportin in 293T cells dramatically inhibited HIV‐1 transcription. Induction of ferroportin expression in THP‐1 cells inhibited one round of replication of pseudotyped HIV‐1. Previously, ferroportin Q248H mutation was shown to have a potential protective effect against iron deficiency in children exposed to repeated inflammatory conditions. We analyzed the effect of hepcidin on the expression of ferroportin and found that the Q248H mutant was less sensitive to lower concentrations of hepcidin. This finding suggested that Q248H mutation might be protective against HIV‐1 infection under conditions of mild inflammation. We are currently investigating the direct effect of hepcidin and ferroportin Q248H mutation on HIV‐1 transcription.
Survey of the GLUT2 translocation pore for substrate selectivity determinants
Authors: Witkowska Kate , Cheeseman Chris I.
Published: June 2021

GLUT2 plays a major role in the absorption of nutrient hexoses, being expressed constitutively in the BLM and transiently in the apical membrane. This study's objective was to characterize motifs within TM 7 of GLUT2 which determine substrate specificity. When expressed in Xenopus oocytes, hGLUT1 transports fructose (FRU) at a rate of only 0.1% relative to glucose (GLU), while in GLUT2 they are equivalent. Mutating the NAV motif to NAI in GLUT1 increased FRU transport to approximately 0.9% (2.5 pmoles/oocyte/30 min with 100 μM substrate) relative to that of GLU. Mutating the molecular filter QLS in hGLUT1 to HVA also increased the relative rate of FRU transport (up to 0.5%) but to a lesser extent than the NAI mutation. Further, the combined hGLUT1 HVA‐NAI mutant had a relative FRU transport rate of 1.1 % (6 pmoles/oocyte/30 min at 100 μM substrate). Kinetic analysis for the hGLUT1 HVA‐NAI revealed that the Km for GLU was comparable to that of hGLUT1wt (1.5mM), while the Km for FRU was comparable to that for hGLUT2 (~70 mM).

Thus, we have successfully introduced hGLUT2‐like FRU kinetics into hGLUT1, without affecting the GLU transport by this isoform. These results point to the presence of, at least two, selectivity filters within class I hGLUTs. NAV/NAI is the exofacial access filter, while QLS/HVA seems to allow FRU translocation beyond the substrate binding site without influencing GLU binding and/or translocation.
Cerebral Hypoperfusion Increases Neuronal Nitric Oxide Synthase Abundance in the Ovine Fetal Brainstem
Authors: Shah Maitreyee , Wood Charles E
Published: June 2021

Nitric oxide (NO) is potent vasorelaxant in vascular smooth muscle and serves as a neurotransmitter in the brainstem, influencing the neural pathways that control cardiovascular function. Hypotension, and the resultant cerebral hypoperfusion, stimulates a series of cellular responses that modify the reflex responses to subsequent periods of hypotension. This study was performed to test the influence of brachiocephalic occlusion (BCO) on the abundance of nNOS in the fetal brainstem. Fifteen chronically catheterized, late gestation fetal sheep were subjected to a 10 minute period of cerebral hypoperfusion followed by reperfusion. Fetuses were euthanized 0, 0.5, 3, 8, and 18 hours after the onset of BCO (n=3 at each timepoint). Brain regions were rapidly dissected, snap‐frozen, and later processed for isolation of protein for Western Blot analysis. We tested for an upregulation in fetal brainstem nNOS abundance in response to brachiocephalic occlusion using anti‐nNOS antiserum (Transduction Labs) and anti‐β‐actin (Sigma) for normalization of protein loading. After normalization with β‐actin, nNOS protein abundance appeared to increase 3 hours after the onset of a 10‐min period of BCO.

When analyzed by one‐way ANOVA, the apparent increase was not statistically significant (p=0.079). Analysis of a smaller data set, in which the 18 hour values were excluded yielded a statistically significant change (p=0.022). Analysis of the individual means by Bonferroni test revealed that the nNOS abundance was greater at 3 hours than at earlier or later times. We conclude that nNOS abundance is increased in fetal brainstem after a period of cerebral hypoperfusion, and we speculate that increased nNOS activity increases brainstem NO responses to subsequent periods of hypotension.
Differentiation of Umbilical Cord Matrix Cells into Hepatocytes
Authors: Lemming Matthew T. , Fry Madeline , Hoynowski Steven M. , Rastovic Milica , Mitchell Kathy E.
Published: June 2021

Stem cells from the human umbilical cord matrix (HUCM) cells express many proteins important for regulation of embryonic and adult stem cells and can be differentiated into cells from mesodermal and ectodermal lineages. Here we describe differentiation of HUCM stem cells into hepatocytes, an endodermal cell‐type. HUCM‐derived hepatocytes stored glycogen, indocyanine green and took up low density lipoprotein while the undifferentiated HUCM cells did not. HUCM‐derived hepatocytes expressed hepatocyte markers: albumin, HNF3‐beta, HNF4‐alpha, and glutamine synthetase, which are not expressed by undifferentiated HUCM cells. Smooth muscle actin and nanog, proteins normally expressed in undifferentiated HUCM cells, were no longer expressed. Transcription factors and P450's that are associated with liver metabolism were expressed in the HUCM‐derived hepatocytes and further induced by treatment with drugs. Changes in gene expression determined by real‐time PCR showed that stem cell gene expression is decreased in HUMC‐derived hepatocytes while .expression of genes associated with metabolic pathways in the liver were increased. These findings suggest that this abundant source of stem cells could be used for drug development in addition to cell‐based therapy for liver disease. Funded by NIH P20 RR016475 and the State of Kansas.
Analysis of Gene Expression in Human Umbilical Cord Matrix Cells Before and After Differentiation into Ectodermal, Mesodermal and Endodermal Lineages
Authors: Lemming Matthew T. , Fry Madeline , Rastovic Milica , Mitchell Kathy E.
Published: June 2021

ES‐like stem cells from the human umbilical cord matrix (HUCM) express the ES transcription factors Oct‐4 and nanog. HUCM cells also express adult stem cell markers so appear to be intermediate between ES and tissue‐specific adult stem cells. HUCM cells can be differentiated into endodermal, mesodermal and ectodermal lineages making them candidates for cell‐based therapies. We explored changes in gene expression after differentiation of HUCM cells into different cell lineages. In particular, we determined changes in expression of genes associated with undifferentiated embryonic stems (Oct4 and nanog); adult stem cells (CD146, CD90 and smooth muscle actin) and immune complexes (HLA‐ABC and MHC II). We found that Oct4 and nanog expression was decreased to non‐detectable levels after differentiation of HUCM cells and that smooth muscle actin was initially greatly diminished, becoming disorganized and eventually below the level of detection. HLA‐ABC and MHC‐II are not detectable in undifferentiated HUCM cells or in some cases HLA‐ABC expression was very weak. Expression of MHC‐II remained below the level of detection and HLA‐ABC remained very low after in vitro differentiation. This work will establish whether HUCM cells are potential candidates for allogenic cell‐based therapies. Funded by NIH Grant Number P20 RR016475, NIH (R01NS036124) and the State of Kansas.
Coordinated changes of mitochondrial biogenesis and antioxidant enzymes during osteogenic differentiation of human mesenchymal stem cells
Authors: Wei Yau‐Huei , Chen Chien‐Tsun , Shih Yu‐Ru V. , Kuo Tom K , Lee Oscar K
Published: June 2021

We investigated the changes of mitochondrial biogenesis and bioenergetic function of human mesenchymal stem cells (hMSCs) during differentiation. Upon osteogenic induction, the copy number of mitochondrial DNA, protein subunits of the respiratory enzymes, oxygen consumption rate, and intracellular ATP content were increased, indicating the upregulation of aerobic metabolism. On the other hand, undifferentiated hMSCs showed higher levels of glycolytic enzymes and lactate production rate, suggesting that hMSCs rely more on glycolysis for energy supply compared with hMSC‐differentiated osteoblasts. In addition, we observed a dramatic decrease of intracellular reactive oxygen species (ROS) as a consequence of upregulation of two antioxidant enzymes, Mn‐SOD and catalase. Finally, we found that H2O2 and mitochondrial inhibitors retarded the osteogenic differentiation. These findings suggest an energy production transition from glycolysis to oxidative phosphorylation in hMSCs upon osteogenic induction. Meanwhile, antioxidant enzymes were concurrently upregulated to prevent the accumulation of intracellular ROS. Together, our findings suggest that coordinated regulation of mitochondrial biogenesis and antioxidant enzymes occurs synergistically during osteogenic differentiation of hMSCs.
Changes in claudin isoform expression in the gill during salinity shifts and smoltification of Atlantic salmon
Authors: Madsen Steffen Sondergaard , Tipsmark Christian Kolbaek
Published: June 2021

Permeability changes of the gill epithelium of euryhaline teleosts are imperative during salinity acclimation. This study examined the expression patterns of 5 claudin isoforms in the salmon gill. These are membrane proteins being major determinants of selective and gross tight junction permeability. Nucleotide sequences representing proteins of this family were identified in expressed sequence tag libraries from Atlantic salmon gill and classified according to the established teleosts nomenclature. Five isoforms: 10e, 27a, 28a, 28b and 30a, were identified in the gill, and tissue specific expression was confirmed by RT‐QPCR. We examined the expression profile during the parr‐smolt transformation (PST) in freshwater and during acclimation to sea water (SW). During PST, claudin 10e expression peaked in May, coinciding with optimal SW tolerance. The other claudin isoforms were not influenced during PST. SW‐transfer induced a 5‐fold increase in expression of claudin 10e, reduced the expression of 27a and 30a and had no overall effect on 28a and 28b isoforms. The study demonstrates for the first time that SW acclimation involves differential regulation of claudin gene expression in the salmon gill, some of which are induced in preparation to SW migration during smolting. We suggest discrete functional roles of the identified claudins in the gill. Supported by The National Danish Research Agency.
Metalloproteinase Action on the Tight Junction in Epithelial Cells.
Authors: Ebers Andrew M. , Jenkins James T. , Koehler Christopher L. , King Jonathan M.
Published: June 2021

This study focuses on epithelial barrier function after exposure to recombinant MMP‐9 or proinflammatory cytokines TNFα and IFNγ in MDCK cells. The aim is to elucidate the mechanisms by which select metalloproteinases (those that are elevated after cytokine treatment) lead to modification of the tight junction. Sequences from tight junction proteins that contained putative MMP‐9 substrates were produced by solid‐phase peptide synthesis, treated with MMP‐9, and analyzed for fragmentation by HPLC. Limited proteolysis experiments were conducted for tight junction proteins to determine if MMP‐9 cleaved these substrates. These studies indicated increased fragmentation of occludin. Physiological measures including TER and paracellular flux confirm that MMP‐9 directly alters TJ function. Alteration of barrier function was not due to apoptosis; only modest caspase‐3 activation following cutokine exposure was observed. In addition to MMP‐9, MT1‐MMP may be involved in barrier dysfunction. Real‐time RT‐PCR and Western blotting demonstrated a dose dependent increase in MT1‐MMP expression following cytokine exposure. Based on these results, both MMP‐9 and MT1‐MMP expression are upregulated in response to inflammatory stressors. MT1‐MMP and MMP‐9 action likely modify epithelial barrier function by direct TJ cleavage. This work was supported by NIH Grant DK065652, HHMI Foundation and the TU‐SURF program.
CFTR is involved in airway epithelial cell migration.
Authors: O’Grady Scott M , Maniak Peter J.
Published: June 2021

Migration of Calu‐3 cells was investigated by measuring changes in impedance as cells moved onto an electrode imbedded within a tissue culture chamber slide. Experiments were performed in serum‐free, HCO3 buffered MEM maintained in a humidified 5% CO2 incubator at 37 °C. Once cells reached confluence, a circular lesion (250 μm diameter) was produced by electroporation of cells in contact with the electrode. Alternating current (∼1 μA) at high frequency was then continuously applied to measure impedance and monitor migration of cells from the margin into the lesioned area. Under serum free conditions, maximum impedance (∼10 kΩ) was restored after 10 hours. When cells were treated with EGF (5 μg/ml), maximum impedance was restored within 2.5 hours. Inhibition of actin polymerization with cytochalasin D completely inhibited migration. Moreover, pretreatment with EGF (5 μg/ml) and 50 μM CFTRinh–172, delayed restitution by 22 hours compared to EGF treated controls. Additionally, constitutive knockdown (>95%) of CFTR expression using shRNA also delayed recovery by >30 hours compared to cells expressing an altered shCFTR construct containing 4 bp substitutions that prevent targeting of CFTR. The results of these experiments indicate that functional activity of CFTR is necessary for Calu‐3 cell migration and suggests a role for CFTR in epithelial cell restitution.
Differential Modulation by Src Tyrosine Kinase of HCN Channels
Authors: Yu Han‐Gang , Li Chenhong , Lin Yen‐Chang , Zhang Qi
Published: June 2021

Distinct biophysical properties of HCN pacemaker channel isoforms and the pacemaker current, I(f), in various heart regions are attributed to the non‐uniform mRNA expression and different post‐translational modulation of HCN channel isoforms. Using whole‐cell patch clamp recordings of currents from HCN channels expressed in HEK293 cells, we report here that 15–30min incubation of 10μM PP2 (a selective inhibitor for Src tyrosine kinases) inhibited HCN2 due to a hyperpolarizing shift of the voltage dependent activation. The mid‐points of activation (V1/2) were −95.3±1.2 mV in the absence of PP2 and −104.1±1.2 mV in the presence of PP2 (n=7, p<0.05) with no changes in slope factor. The activation kinetics of HCN2 were slowed. At −125mV, time constants were 526±16 ms in the absence and 765±18 ms in the presence of PP2 (n=7, p<0.05). On the other hand, the gating properties of HCN1 were not significantly affected by PP2 (with PP2, V1/2= 72±3 mV, τ(−125mV) = 45.8±3.7 ms; without PP2, V1/2= 73±5 mV, τ(−125mV) = 44.8±2.1 ms, n=8, p>0.05). Co‐immunoprecipitation experiments revealed that both HCN1 and HCN2 were associated with the constitutively active Src tyrosine kinase (Src529) and the channel proteins were phosphorylated on tyrosine residues using a phosphotyrosine specific antibody, . Our data demonstrated a differential response of HCN channel isoforms, HCN1 and HCN2, to PP2.
Daily genistein injections stimulate intestinal chloride secretion in male and female mice via differing mechanisms.
Authors: Al‐Nakkash Layla , Jensen Jesse , Hale Nathan , Batia Lyn
Published: June 2021

Intestinal chloride (Cl) secretion in male and female mice (C57BL/6J) was determined following injection with either 600 mg/kg genistein/day (600G) or genistein‐free (0G, DMSO vehicle control) for either 1 or 2 weeks. After injections, jejuna were isolated and intestinal Cl secretion measured with Ussing chamber short circuit current (Isc, μA/cm2). After 1 week basal Isc was significantly increased in females injected with 600G (198.8±18.9, n=6, P<0.05) compared to 0G (128.3±12.8, n=4). Basal Isc was significantly increased in males injected with 600G (183.6±7.6, n=7) compared to 0G (100.8±18.2, n=7) only after 2 weeks. Cl‐free ringer reduced the basal Isc by 55%, suggesting a major Cl component. Forskolin‐stimulated Isc (10μM bilateral) was significantly increased (P<0.05) in both female and male mice injected with 600G compared to those injected with 0G (n=4–9). Similar data were obtained after 2 weeks of injections. The estrogen‐receptor antagonist ICI‐182780 (25 mg/kg body weight) concomitantly injected with 600G for 2 weeks, significantly decreased basal Isc in males (by 52%, n=3, P<0.01) but not females (by 5%, n=4). Villi length and crypt depth were similar in males and females injected with 600G or 0G. These data suggest that male and female mice respond similarly to 600G (longer duration for males), but the effects are mediated via different mechanisms. Support: CFF and NIH Area grants to L. A.
P2Y Purinoceptor Mediates Ca2+ Signaling in Duodenocytes and Involves Duodenal Mucosal Bicarbonate Secretion
Authors: Dong Hui , Ko Kwang Hyun , Lee Jonathan , Dong Xiao , Chow Jimmy
Published: June 2021

Purinergic receptors have been identified in most epithelial tissues, including gastrointestinal tract. Although the role of P2Y purinoceptors and their signaling in the intestinal epithelial Cl− secretion have been studied extensively, little is known about the expression and function of P2Y purinoceptors in the duodenum, especially their role in duodenal mucosal bicarbonate secretion (DMBS). Duodenal mucosae from C57BL mice were stripped of seromuscular layers and mounted in Ussing chambers. Both short‐circuit current (Isc) and HCO3 − secretion were measured in these tissues. Acid‐stimulated DMBS was also measured in vivo. Cytoplasmic free Ca2+ ([Ca2+]cyt) was determined by digital imaging in SCBN cells, a duodenal epithelial cell line. UTP (10 μM) significantly induced duodenal Isc and HCO3− secretion, which were abolished by the pretreatment of the tissues with either suramin (300 μM), a P2 receptor antagonist or 2‐APB (100 μM), a store‐operated channel inhibitor. Acidstimulated DMBS in vivo was significantly blocked by suramin (1 mM). Both ATP and UTP at 10 μM raised comparable [Ca2+]cyt in SCBN cell in the presence or the absence of extracellular Ca2+, which were prevented by the pretreatment with either suramin or 2‐APB. However, ADP at 10 μM raised smaller [Ca2+]cyt in these cells. These results suggest that activation of P2Y purinoceptors in the duodenal epithelium may increase [Ca2+]cyt by inducing intracellular Ca2+ release and extracellular Ca2+ entry via SOC, and thereby stimulate duodenal Cl− and HCO3− secretion (Supported by Cystic Fibrosis Foundation).
Basolateral but not apical localization of sodium‐dependent vitamin C transporter 2 (SVCT2) in mouse small intestine epithelial cells
Authors: Kuo Shiu‐Ming , Sigurdson Wade J
Published: June 2021

EGFP‐tagged human SVCT2 was previously shown to be basolaterally localized in cultured intestinal epithelial cells, Caco‐2. To confirm the localization of endogenous SVCT2, we performed immunofluorescence staining of cryosections from mouse small intestine (duodenum and ileum) using polyclonal anti‐SVCT2 antibodies and Alexa Fluor® 488‐conjugated goat anti‐rabbit IgG as the secondary antibodies. To ensure the presence of proper reference points to the tissue/cell structure, carbocyanine dye, DiI, was used to concurrently label the whole section. Through two‐color confocal microscopic fluorescence imaging, we found that similar to Na,K‐ATPase (visualized by using rabbit anti‐Na+/K+ ATPase α IgG), SVCT2 was basolaterally localized and no apical localization was detected in the mouse intestinal epithelial cells. Consistent with the known wide‐spread SVCT2 expression in the body, SVCT2 was also found in other cell types in small intestine sections. SVCT2+/− mice showed greatly diminished SVCT2 expression in intestinal sections although the Na,K‐ATPase and DiI stainings of sections were comparable to that of the wildtype mice.
Characterization of stratification and tight junction formation in cultured human urothelial cells
Authors: Rickard Alice , Marentette John , McHowat Jane
Published: June 2021

The urothelium is composed of stratified transitional epithelium of 3 cell types: basal, intermediate, and superficial. It has been proposed that urothelial cells acquire their mature characteristics through differentiation. The most differentiated, superficial cells develop tight junctions (TJs) that restrict urine movement and abnormalities in barrier function may be involved in syndromes such as interstitial cystitis. To further our understanding of urothelial differentiation, we used cells from normal human ureter and examined the zonula occludens (ZOs), “undercoat” scaffolding proteins in the cytosol that interact with claudins and actin filaments. Cell monolayers were stratified and differentiated using Ca and fetal bovine serum (FBS) and the process was studied using impedance, videomicroscopy, and confocal immunohistochemistry. With addition of Ca and FBS, impedance quickly increased by 2.5 fold. Withdrawal of the supplements returned impedance to baseline, suggesting the formation of tight junctions, rather than increased cell proliferation and immunohistochemistry demonstrated the appearance of ZO‐1 at cell junctions minutes after supplement addition. Observation by videomicroscopy suggested that cell division continued to occur after the cells had differentiated. Taken together, these studies increase our understanding of urothelial development.

Supported by DK66119 (JM)