bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of Calcium Transport Pathways in Kidney and Intestine During Lactation
Authors: Dimke Henrik , Beggs Megan R , Alexander R. Todd 
Published: November 2023

Significant alterations in maternal calcium balance occur postpartum during lactation. Demineralization of the skeleton mobilizes calcium, in order to supply this mineral to the neonate via breast milk. Changes in intestinal and renal calcium transport processes also promote this. However, the specific molecular alterations in transcellular and paracellular calcium transport pathways across renal and intestinal epithelia during lactation have not been fully delineated. To ascertain which changes occur during lactation, female mice were divided into 3 groups, non‐pregnant controls, lactating mice with litters maintained for 12 days after birth, or mice undergoing involution with litters removed after birth and maintained for 12 days. Induction of lactogenesis was confirmed histologically in mammary fat pads. Bodyweight increased in lactating mice as did urinary volume, measured during 9‐hr collections performed on day 11. Both total urinary excretion and fractional excretion of calcium and magnesium were elevated during lactation. Enlargement of the intestinal tract was confirmed visually in lactating animals. Renal 1‐alpha hydroxylase and 24‐OHase mRNA expression increased dramatically. This was accompanied by significant increases in the gene expression of Trpv6 and Calbindin‐D9K in duodenum and proximal large bowel of lactating mice. No significant alterations in the calcium permeable claudins (−2, −12 or −15) were observed in any intestinal segments. A similar gene expression profile was observed in kidney, which showed no change in claudins (−2, −12, −14, −15, −16 or −19), while marked increases in Trpv5 and Calbindin‐D28K were observed. Increased Calbindin‐D28K protein expression was found by immunohistochemical analysis, while no changes in claudin‐2 were noted, in line with observations from mRNA expression analysis. In conclusion, proteins driving transcellular calcium transport pathways increase during lactation, likely due to increased Vitamin D dependent‐stimulation, while proteins mediating paracellular transport appear unchanged. Increased urinary mineral excretion can be explained on the basis of intestinal hyperabsorption and bone demineralization, although selective calcium uptake is maintained in kidney.Support or Funding InformationThis work was funded by grants from the Novo Nordisk Foundation (HD), the Carlsberg Foundation (HD), the A.P. Møller Foundation (HD), the Lundbeck foundation (HD), the Kidney Foundation of Canada (RTA), and the Canadian Institute of Health Research (CIHR) (RTA). A Clinician Scientist Award from CIHR and an Alberta Innovates Health Solutions Clinical Investigator Award supports Dr. R.T. Alexander.
Effects of PYY on IL‐6 Signaling in Primary Human Myotubes
Authors: Blum Jamie Elaine , Gheller Brandon JF , Roman Heather , Thalacker‐Mercer Anna 
Published: November 2023

RationaleLife expectancy is increasing and as a result, the older adult population in the United States is growing. Diminishing skeletal muscle function can impact the lives of older adults by limiting mobility and independence. Inflammation is thought to contribute to the development skeletal muscle metabolic dysfunction. Thus, identifying and understanding biological molecules that affect inflammatory signaling will inform interventions designed to support preservation of healthy skeletal muscle metabolism. Peptide Tyrosine Tyrosine (PYY), a novel skeletal muscle factor identified by our laboratory, is a potential mediator of inflammation in skeletal muscle. The effects of PYY in skeletal muscle need to be elucidated. Skeletal muscle gene expression of PYY is down‐regulated with aging and in individuals whose skeletal muscles do not hypertrophy in response to resistance exercise training. Exogenous administration of PYY decreases inflammatory signaling in various cell‐lines.HypothesisBased on observations from microarray analysis conducted on RNA from skeletal muscle biopsies as well as preliminary data from our laboratory, we hypothesized that PYY down‐regulates inflammatory signaling in human primary skeletal muscle cells.MethodsBiopsies were taken from the vastus lateralis muscle of two healthy, young females. Primary human skeletal muscle progenitor cells were isolated. Cells were pooled and cultured in Ham's F12 supplemented with 20% HI FBS and bFGF (GM). Once cells reached 75% confluence (t=0 h) all cultures were switched to differentiation medium [(DM), Ham's F12 + 2% HI Equine Serum] without (CONT) or with 500 pM PYY3‐36 (pPYY). At t=72 h, the medium on CONT and pPYY plates was replaced with DM + 0.5% Fatty Acid Free BSA (FAF‐BSA) without or with 100 ng/ml IL‐6 (CONTinflame and pPYY inflame) for 24 h. A subset of the cells from all treatment groups were exposed to a 20 min 200nM insulin challenge (CONT+I, pPYY+I, CONTinflame+I, and pPYY inflame+I) prior to harvesting the cells for RNA. Gene expression was measured using quantitative PCR.ResultsDifferentiating primary human myoblasts that were pretreated with PYY3‐36 then incubated in IL‐6 and insulin challenged had a −1.87 and −1.79 fold change in AMPK and STAT3 mRNA expression, respectively (pPYYinflame+I vs. CONTinflame+I, p<0.05). This difference however, was not observed in cells pretreated with PYY3‐36 and incubated in IL‐6 without an insulin challenge (CONTinflame vs. pPYYinflame, p>0.05) or in cells incubated without IL‐6 (CONT vs. pPYY, p>0.05 and CONT+I vs. pPYY+I, p>0.05).ConclusionsPYY pretreatment appears to have a protective effect against IL‐6 inflammatory signaling when an anabolic stimulus is given; PYY down‐regulates IL‐6 signaling through both AMPK and STAT3 pathways in the presence of insulin. Ongoing research in our laboratory is clarifying the role of the insulin stimulus in facilitating this effect of PYY.Support or Funding InformationCornell Start‐up
High Glucose Promotes the Pro‐Migratory Phenotype of Retinal Pigment Epithelial Cells through Increased Oxidative Stress
Authors: Farnoodian Mitra , Sorenson Christine M , Sheibani Nader 
Published: November 2023

Defects in the outer blood‐retinal barrier have significant impact on pathogenesis of diabetic retinopathy and macular edema. However, the detailed mechanisms involved remain largely unknown. This is, in part, attributed to the lack of suitable animal and cell culture models, including those of mouse origin for such studies. The mouse models provide the ability to test the impact of various genes with important functions in ocular homeostasis in diabetes and its complications. We recently reported a method for culture of mouse retinal pigment epithelial (RPE) cells from wild type and transgenic mice. The RPE cells are responsible for maintaining the integrity of outer blood‐retinal barrier whose dysfunction during diabetes has significant impact on vision. Here we determined the impact of high glucose conditions on the function of RPE cells. We showed that high glucose conditions minimally impacted the proliferation and apoptosis of both human and mouse RPE cells, but these cells were more migratory and exhibited increased levels of oxidative stress. High glucose minimally affected the cell‐matrix and cell‐cell interactions of RPE cells. However, the expression of integrins and extracellular matrix proteins including tenascin C, thrombospondin‐1 and pigmented epithelium derived factor were increased under high glucose conditions. These cells also exhibited increased nuclear localization of anti‐oxidant transcription factor Nrf2 and reduced phagocytic activity under high glucose conditions. Furthermore, high glucose minimally affected the level vascular endothelial growth factor and activity of AKT, MAPK, and Src kinase signaling pathways. However, incubation of RPE cells under high glucose conditions with anti‐oxidant N‐acetylcysteine restored their normal migratory activity. Thus, high glucose promotes a pro‐inflammatory and oxidative state of RPE cells without a significant effect on their proliferation and apoptosis. The increased migration of RPE cells under high glucose condition is consistent with increased susceptibility to choroidal neovascularization with diabetes.Support or Funding InformationNIH R24 EY022883; NIH P30 EY016665; NIH R21 EY023024
Nuclear Lemur Tyrosine Kinase‐2 Regulates RNA Polymerase II Dependent Transcription in Prostate Cancer
Authors: Shah Kalpit , Bradbury Neil 
Published: November 2023

The Androgen Receptor (AR), a DNA‐binding transcription factor plays a key role in the development and maintenance of prostate epithelia by modulating expression of growth‐promoting genes. AR has been proposed to regulate gene expression by enhancing the efficiency of RNA polymerase II (RNAPII) dependent transcription elongation. Dysregulation of this AR‐dependent transcriptional activity has been implicated, not only in prostate cancer but also androgen‐independent prostate cancer. Recently, we showed that protein expression of Lemur Tyrosine Kianse‐2 (LMTK2), a serine‐threonine kinase is downregulated in prostate cancer. Importantly, our study ascribed a novel role for LMTK2, as a negative regulator of AR‐dependent transcriptional activity in prostate epithelial cells. However, the mechanism through which LMTK2 is able to regulate AR remains to be determined. Here, we show that LMTK2 is not only present in cytoplasmic fraction but also, within the nucleus of mammalian cells where it co‐localizes with AR, newly transcribed mRNA and with RNA polymerase II (RNAPII). Interestingly, our data reveals that LMTK2 colocalizes with elongating RNAPII, phosphorylated on serine 2 of the carboxyl‐terminal domain. This presents an interesting possibility that LMTK2 might be able to regulate transcription of AR‐dependent genes by interacting with the elongating RNAPII. As such, our study identifies LMTK2 as not only a novel regulator of AR but also, possibly an important player regulating the transcription machinery in mammalian cells.Support or Funding InformationNational Institute of Health (1R01HL102208‐01A1) and Cystic Fibrosis Foundation Grants (BRADBU12XXO)
TRPC3 serves as a link between osmosensitivity and water transport in the mouse collecting duct
Authors: Tomilin Viktor , Mamenko Mykola , Zaika Oleg , Pochynyuk Oleh M 
Published: November 2023

Collecting duct serves as the primary site of controlled water reabsorption driven by osmotic gradient in the kidney. Cells of the collecting duct sense the changes of the extracellular osmolarity and react by elevating intracellular calcium via a yet unidentified mechanism. Moreover, the relation between osmosensitivity and water transport is poorly understood. Ca2+ permeable TRPC3 channel has been proposed to participate in mechanosensitive responses in different cell types. Previous evidence demonstrates prominent expression of this channel in the collecting duct cells. Using ratiometric Fura 2‐based [Ca2+]i imaging in freshly isolated split‐opened mouse collecting ducts, we found that pharmacological inhibition of TRPC3 with Pyr3 significantly reduces cellular responses to hypotonicity. Consistently, genetic TRPC3 deletion markedly impairs sensitivity of the collecting duct cells to decreases in extracellular osmolarity. Furthermore, collecting duct cells of TRPC3 −/− mice fail to properly regulate their volume in response to acute hypotonic challenge. Stimulation of water reabsorption with water deprivation exacerbates differences in collecting duct osmosensitivity between wild type and TRPC3 −/− mice and also transiently reduces urinary concentration ability in the mutant animals. We conclude that TRPC3‐mediated Ca2+ influx during hypotonicity is important for proper stimulation of water reabsorption in the collecting duct.Support or Funding InformationSupported by AHA and NIH grants
Exploring an autoinhibitory domain in the electrogenic Na/HCO3 transporter NBCe1‐B
Authors: Lee Seong‐Ki , Boron Walter F. 
Published: November 2023

The SoLute Carrier (SLC) superfamily of membrane transport proteins comprises over 300 genes organized into 52 families. One of the SLC groups is the SLC4 family, the members of which mainly transport HCO3−, or a species related to HCO3− (e.g., CO3=), together with another “coupled” ion across the plasma membrane. Here we study a member of the SLC4 family, the electrogenic Na/HCO3 cotransporter NBCe1 (SLC4A4). This transporter is widely expressed in cells throughout the body. NBCe1 has 5 known splice variants (A—E). A—C variants have been extensively studied. Whereas NBCe1‐A (predominantly expressed in the kidney) is very fast, NBCe1‐B (expressed throughout the body) and NBCe1‐C (mainly expressed in the brain) are very slow. The critical determinant of speed is the extreme NH2‐terminus (eNt), which in NBCe1‐A comprises the first 41 amino acids (aa). In NBCe1‐B and NBCe1‐C, this cassette is replaced by a cassette of 85 aa. Truncation of the first 85 aa of NBCe1‐B/C enhances activity. Thus, it has been suggested that this 85aa cassette in NBCe1‐B/C includes an autoinhibitory domain (AID).Recently, Shcheynikov et al. (PNAS, 2015) showed that removing 23 aa between residues 40 and 62 stimulates NBCe1‐B. The deleted region contains a unique cluster (residues 40–48) of 9 consecutive positively charged residues. We hypothesize that this positively charged cluster normally inhibits NBCe1‐B. We assess NBCe1 activity by two‐electrode voltage‐clamping of Xenopus oocytes expressing NBCe1‐B constructs in which we systematically mutated or deleted elements of the cluster. We also deleted 3 to 9 aa just before the cluster. All constructs traffic well to the plasma membrane. We show that [1] removing or mutating the cluster of positive charges robustly stimulates NBCe1‐B activity (i.e., relieve autoinhibition), whereas [2] deleting aa before the cluster does not.Our findings suggest that positively charged residues between residues 40 and 48 of NBCe1‐B are a necessary component of the AID, whereas the preceding 9 residues are not a necessary part of the AID.
The Role of a Common HFE Gene Variant in Brain Iron Accumulation
Authors: Duck Kari Ann , Neely Elizabeth B , Simpson Ian A , Connor James R 
Published: November 2023

HFE is an essential protein for regulating iron transport into cells. Two common mutations of HFE, C282Y and H63D, result in loss of this regulation and cause accumulation of iron within the cell. The cellular iron accumulation can reach toxic levels. While much has been studied regarding the role of HFE in iron uptake, it has remained unclear what role the protein plays in the transport of iron into the brain. In fact, it has long been believed that the brain is protected against iron overload by the blood‐brain barrier (BBB). Recently, however, our laboratory and others have correlated HFE gene variants with neurodegenerative diseases, specifically amyotrophic lateral sclerosis (ALS) and Alzheimer's Disease (AD). A critical review of historic literature, recent MRI studies and a novel more representative mouse model generated in our laboratory all suggest there is elevated iron in the brain associated with HFE gene variants. We generated a mouse model in which the wild‐type (WT) HFE gene is replaced by the H67D gene variant (mouse homologue of the human H63D gene variant). There are elevated levels of brain iron in 3 month old mice with the H67D mutation when compared to their controls, but this difference in levels is decreased at 6 months and no longer present by 12 months of age. Here, we performed an in vivo study to identify brain iron uptake and distribution patterns in 3 month old WT and H67D mice. After injection with 59Fe‐transferrin, mice were sacrificed at 24 hours and 5 days post‐injection. 59Fe levels measured in brain homogenate and isolated microvessels showed no significant differences between genotypes. There was, however, increased 59Fe accumulation in the livers of H67D mice. This study makes two novel discoveries. First, there is iron accumulation in the microvessels in the brain supporting the concept put forth recently by our group that the BBB is not a conduit but a regulatory site for brain iron uptake. Secondly, the similar rate of uptake for iron in the HFE mutant mice, coupled with the increased amount of iron suggests that the activity of the HFE protein is limited at the BBB. The increased iron in the brain in the H67D mice may be the result of altered handling of the iron including decreased export. We cannot rule out the lack of difference in uptake is a compensatory process already in place because of the higher iron levels and this notion is under investigation. The apparent ability of the BBB, unlike the liver for example, to adapt to the HFE gene variant reinforces the importance of understanding the mechanisms and regulation of brain iron uptake and how the pathway is modulated in the diseased brain.Support or Funding InformationThis work was supported by NIH P01 AG021190 and P01 HD39386.
Functional analysis of N‐glycosylation in the intestinal peptide transporter PEPT1
Authors: Daniel Hannelore , Stelzl Tamara 
Published: November 2023

The intestinal peptide transporter PEPT1 (SLC15A1) mediates electrogenic proton‐coupled influx of dietary di‐ and tripeptides as well as of peptidomimetics into epithelial cells. Western blot analyses performed with protein isolates of mouse intestine revealed the existence of diverse PEPT1 glycoforms (Wuensch, T., et al. Am J Physiol Gastrointest Liver Physiol 305: G66‐73, 2013). In small intestine PEPT1 exhibits a mass of ~95 kDa, while in colon the transporter appears with a mass ~105 kDa. Enzymatic deglycosylation with PNGaseF uniformly reduced the PEPT1 mass in both gut segments to ~65 kDa. These mass differences addressed the question of whether glycosylation has an effect on function. We thus assessed whether different glycosylation states alter PEPT1 transport activity. By targeted mutagenesis, putative N‐glycosylation sites in murine PEPT1 were disrupted and mutant transporters heterologously expressed in Xenopus laevis oocytes. Replacement of six asparagine residues (N) at positions N50, N406, N439, N510, N515 and N532 by glutamine (Q) resulted in a decrease of the PEPT1 mass by around 35 kDa. Electrophysiology revealed all glycosylation‐deficient transporters to be functional with comparable expression levels in oocyte membranes. Remarkably, in the mutant transporter N50Q affinity for glycyl‐sarcosine (Gly‐Sar) decreased from 0.66 ±0.12 mM to 1.33 ± 0.23 mM while maximal inward currents increased 2.5‐fold from 0.85 ± 0.21 nA to 2.11 ± 0.21 nA. Tracer flux studies with [14C]‐Gly‐Sar confirmed the reduction in substrate affinity of the N50Q mutant while maximal influx rate increased from 2.32 ± 0.30 to 4.47 ± 0.32 pmol/min/oocyte. In the human protein (hPEPT1) the N50Q mutant as well presented a lower Gly‐Sar affinity (0.8 ± 0.1 mM in WT versus 1.2 ± 0.2 mM in mutant) and a maximal inward current increased from 1.6 ± 0.2 to 4.1 ± 1.4 nA. Since neither the surface density of the mutant transporter N50Q was changed, nor its conductance, we assume that N‐glcyosylation at N50 constraints the movement of the N‐terminal domain during the transport cycle. Attachment of N‐linked oligosaccharides at sequon N50, with an identified overall mass of 3–5 kDa, seems to attenuate PEPT1 mobility while the absence of the glycans could accelerate theSupport or Funding InformationThis work was funded by GRK 1482 by Deutsche Forschungsgemeinschaft
Feeding status and the rate of crop emptying in the hissing cockroach, Gromphadorhina portentosa.
Authors: Medina Rubie , Contreras Heidy 
Published: November 2023

Crop emptying has been studied in only a few species of insects. Generally, it is known that there are a variety of factors that can affect the retention time of a meal in the crop: 1) the availability of food; 2) the nutritional status of the organism, and 3) regulatory mechanisms aimed at meeting the varying nutrient demands of the animal. In this experiment we wanted to investigate whether or not feeding status of fed vs. unfed cockroaches affected the rate at which food moved through the gut. Roaches in the unfed group were starved for 7 days. They were then fed a control diet which had been dyed blue. All roaches were allowed consume 2% of their initial body weight. After roaches had consumed this volume of food, they were placed in a chamber without food for different time periods (0, 30, 60, 120, and 240 minutes). A total of 8 roaches were placed under each time treatment. Cockroaches were then sacrificed using liquid nitrogen. Their digestive system was dissected and measurements were taken to determine how far food had moved through the gut. A similar protocol was used to determine the rate of food movement through the gut of fed roaches. Previous studies suggest that unfed insects retain food in the crop for longer periods of time compared to fed animals. We therefore hypothesize that G. portentosa will show a decreased rate of food movement out of the crop and into the mid gut compared to fed roaches.Support or Funding InformationULV Faculty Research Grant
ER Stress‐BiP Axis Regulates Endothelial Barrier Dysfunction and Inflammation in Acute Lung Injury
Authors: Leonard Antony , Rahman Arshad , Grose Valerie , Slavin Spencer , Fazal Fabeha 
Published: November 2023

Recent Studies have provided evidence of associations between endoplasmic reticulum (ER) stress and human diseases. Aberrant regulation of ER stress is associated with many chronic inflammatory diseases including type 2 diabetes, inflammatory bowel disease (IBD), cancer, rheumatoid arthritis, atherosclerosis, and chronic obstructive pulmonary disease (COPD). However, the role of ER stress and ER chaperone BiP/GRP78 in acute inflammatory injury, particularly in the context of lung endothelium, is poorly defined. We provide novel evidence that implicates ER stress‐BiP axis as a critical determinant of EC barrier disruption and inflammation associated with acute lung injury (ALI). Our data show that stimulation of endothelial cells (EC) with thrombin caused an increase in BiP levels and pretreatment of cells with ER stress inhibitor 4‐phenylbutyric acid (4‐PBA) prevented this response. Inhibition of ER stress by this approach also decreased thrombin‐induced levels of proinflammtory mediators ICAM‐1, IL‐6, and IL‐8. Consistent with this, pretreatment of EC with AB5 cytotoxin family member subtilase (SubAB), which selectively cleaves and inactivates BiP inhibited thrombin‐induced elevation of intracellular Ca2+, a critical requirement in the mechanism of EC permeability and inflammation. Importantly, BiP inactivation also protected against thrombin‐induced VE‐cadherin disassembly and EC permeability. We determined the in vivo relevance of BiP in ALI by monitoring the effect of SubAB on lung inflammation and injury in an aerosolized bacterial lipopolysaccharide (LPS) inhalation mouse model of ALI. We found a marked decrease in LPS‐induced lung PMN sequestration and microvascular leakage in mice treated with SubAB. Together these data identify ER‐BiP axis as a novel regulator of EC dysfunction in ALI.Support or Funding Information(Supported by: NIH (HL096907 and HL116632)