bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcriptional Regulation of LMTK2, a Serine/Threonine kinase implicated in Cystic Fibrosis
Authors: Dey Isha , Bradbury Neil 
Published: November 2023

Lemur Tyrosine Kinase 2 (LMTK2) is a transmembrane Serine/Threonine kinase primarily found in the membranes of the endocytic compartments, and participates in intracellular trafficking by directly binding to myosin VI. The Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride selective anion channel, has been shown to be a substrate of LMTK2 kinase activity. We have previously shown that LMTK2 gene knockdown reduces CFTR recycling and channel activity at the plasma membrane of polarized epithelial cells. In contrast, overexpressing LMTK2 increases CFTR activity. Thus, increasing LMTK2 expression or activity might have a therapeutic potential in patients with cystic fibrosis; a disease associated with defective overall ion transport due to loss of CFTR at the plasma membrane. However, the precise regulation of LMTK2 activity and protein levels is poorly understood. Examination of the promoter region for the LMTK2 gene predicts a consensus PKC response element (5′‐TGAGTCA‐3′). This sequence serves as the binding site for AP‐1 transcription factor complex, which is transactivated by PKC. In this study, we investigated the mechanism of regulation of LMTK2 expression. Our data indicate that the PKC activator Phorbol 12‐myristate 13‐acetate (PMA) regulates LMTK2 expression in a dose‐dependent manner. This effect is seen at both the protein and the mRNA levels, as quantified by Western Blot and Real‐time PCR respectively. In contrast, treating bronchial epithelial cells with PMA in presence of PKC inhibitors like Staurosporine and Calphostin C eliminates the PMA induced increase in LMTK2 protein and RNA. So, we hypothesise that PMA response on LMTK2 expression involves PKC‐mediated transactivation of the AP‐1 complex. We checked for the expression of nine different isoforms of PKC in these cells, and showed that the expression of atypical isoforms (PKC zeta and PKC iota) is the highest. Our current studies are aimed at determining which isoform(s) of PKC are responsible for the PMA response on LMTK2 expression; and also at quantifying the difference in the nuclear localization of the proteins in the AP‐1 complex, upon treating our experimental cells with PMA, with or without PKC inhibitors. Elucidating the factors involved in the PMA response would give an indepth understanding of the signaling pathway of transcriptional regulation of LMTK2. Since there are no available enhancers of LMTK2 activity, this novel finding would allow us to explore the possibility of using LMTK2 as a potential therapeutic target in cystic fibrosis, by designing activators that would increase the transcription and hence protein expression of LMTK2.Support or Funding InformationThis work is funded by the National Institutes of Health (1R01HL102208‐01A1) and Cystic Fibrosis Foundation grants (BRADBU12XX0) to Neil A. Bradbury.
The SH2 Domain of the Src Family Kinase FYN Binds to Partitioning Defective Protein 3 (PARD3)
Authors: Hinkle Karen , Haswell Abigail , Ballif Bryan 
Published: November 2023

The Src Family of tyrosine Kinases (SFKs) are activated downstream of stimulated growth factor receptors to regulate a host of cellular processes including proliferation, migration, and survival. Aberrantly regulated SFKs are implicated in a variety of cancers, making the study of their regulation important. Using a quantitative mass spectrometry (SILAC) approach in HEK293 cells, we identified a novel SFK binding partner, partition defective 3 (PARD3), a protein known to regulate epithelial cell polarity. PARD3 is known to be phosphorylated at pY 1127 by the SFKs SRC and YES downstream of EGF receptor signaling; however, binding of SFKs to PARD3 has not previously been reported. The goal of this study was to validate and further characterize PARD3‐SFK binding. Pulldown experiments using GST‐SH2 fusion constructs for the SFK FYN with HEK293 cell extracts followed by immunoblotting for PARD3 confirmed that PARD3 binds to FYN's SH2 domain. ScanSite and PhosphoSite analysis were used to predict specific phosphotyrosine residues in PARD3 that may be responsible for FYN SH2‐PARD3 binding. This study suggests that SFK signaling regulates PARD3 function.Support or Funding InformationResearch reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103449. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.
MAp44: diffusion from blood to cerebrospinal fluid and intrathecal synthesis
Authors: Dorta‐Contreras Alberto J , Padilla‐Docal Bárbara , González Ivonne M Iglesias , Martínez‐Larrarte Jose P , Castillo‐González William , González‐Losada Cristóbal , González‐Argote Javier , Jensenius Jens Christian 
Published: November 2023

MAp44 is a MBL associated protein also related with pattern recognition molecules of the complement system and regulating the lectin pathway of complement activation.ObjectiveTo describe the diffusion pattern from blood to cerebrospinal fluid (CSF) and to explore the possibility to be synthesized in CNS.MethodsWe measured MAp44 in 60 serum and cerebrospinal fluid (CSF) samples from control patients without neuroinflammation by a sandwich ELISA. Albumin was measured in both biological fluids by immunodiffusion technique.ResultsCSF and serum values follow a normal distribution by Kolmogorov‐Smirnov test. CSF MAp44 is possible to be 13 times more concentrated than the expected value according to their molecular weight.ConclusionsThe dynamic of the Map44 diffusion in comparison with albumin according to their Q values indicate that this protein can diffuse from blood to CSF but also it is possible to be synthesized in CNS.
Stimulation of the electrogenic Na/HCO3 transporter NBCe1‐B by phosphomimetic mutants of IRBIT
Authors: Lee Seong‐Ki , Boron Walter F. 
Published: November 2023

In oocyte expression studies, Shirakabe et al. (PNAS, 2006) showed that the co‐expression of the soluble protein IRBIT with the electrogenic Na/HCO3 cotransporter NBCe1‐B markedly stimulates the activity of NBCe1‐B. They also showed that the first 62 amino acids of the −NH2 terminus of NBCe1‐B are required for interaction of NBCe1‐B with IRBIT. Other studies suggested that serine phosphorylation of IRBIT (pS68, pS71, and pS74 or pS68, pS71, pS74, and pS77 depending on the study) within a PEST domain of IRBIT is essential for IRBIT function. One of the studies found that the upstream region of the PEST domain has protein phosphatase‐1 (PP1) binding site and IRBIT becomes weaker (sub‐IRBIT) or stronger (super‐IRBIT), depending on whether the PP1 binding site is optimized or deleted. We also previously showed that, whereas sub‐IRBIT does not stimulate NBCe1‐B activity at all, super‐IRBIT enhances NBCe1‐B activity ~30% better than does wild‐type IRBIT.We hypothesize that phosphomimetic mutants of the sub‐IRBIT can rescue the activity of the impotent sub‐IRBIT. We assess NBCe1 activity by two‐electrode voltage‐clamping of Xenopus oocytes expressing NBCe1‐B alone, or NBCe1‐B with a phosphomimetic mutant of IRBIT. We show that [1] phosphomimetic mutations in the PEST domain render IRBIT constitutively active, and [2] on the background of the most active phosphomimetic IRBIT mutant, the triple dephosphomimetic mutant S68A/S71A/S74A still robustly stimulates NBCe1‐B.Our findings suggest that [1] negative charges (not necessarily phosphorylation) within the PEST domain are required for IRBIT potency, and [2] the previously proposed model that IRBIT activity requires phosphorylation at IRBIT residues S68/S71/S74 may not be correct.
Alcohol Consumption Increases Claudin‐18 Turnover in Alveolar Epithelial Cells
Authors: Dorsainvil White StevenClaude H. , Molina Samuel A. , Capaldo Christopher T. , Schlingmann Barbara L. , Koval Michael H. 
Published: November 2023

Alveolar epithelial cell (AEC) tight junctions are crucial for maintaining barrier function between fluid filled tissues and the airspace in the lung. Failure of alveolar tight junctions leads to acute respiratory distress syndrome (ARDS), a frequently lethal condition characterized by airspace flooding in the lung. Chronic alcohol abuse is a great risk factor for ARDS. Alcohol consumption impairs alveolar barrier function by modulation of tight junction proteins, called claudins. Claudins are a family of tetraspan transmembrane proteins that confer specific barrier properties to epithelial cell monolayers by regulating tight junction permeability. We used a rodent model of chronic alcohol ingestion to investigate the effects of alcohol on claudins and barrier function. Primary AECs from alcohol and control fed rats were cultured on Transwell permeable supports and allowed to differentiate into model type I cells. Transepithelial resistance measurements (TER) and dye flux assays were used to determine barrier function in cultured AECs. Tight junction morphology was assessed by immunofluorescence. In response to alcohol, alveolar tight junctions showed altered claudin composition leading to an increase in paracellular leak and formation of projections perpendicular to tight junctions (“spikes”) when immunostained for claudin‐18. Quantification of cells containing tight junction spikes revealed that alcohol increases the portion of cells containing tight junction spikes, which correlated with decreased barrier function. Live cell imaging of AECs expressing YFP‐tagged claudin‐18 confirmed that vesicles containing tagged claudin‐18 fused with and pinched off of tight junction spikes. Therefore, we hypothesized that these tight junction spikes are sites of enhanced protein turnover which might then result in decreased barrier function. As endocytosis is an important regulator of claudin turnover, we examined the effect of treating cells with the inhibitor Dynasore that targets dynamin, a protein which has a role in the scission of endocytic vesicles. Treatment of AECs with Dynasore resulted in a significant decrease in the number of cells containing claudin‐18 spikes and also caused a concomitant decrease in paracellular flux of a 10 kDa marker, Texas Red dextran. Our data support a role for tight junction spikes in regulating paracellular permeability and suggest that dynamin or dynamin‐related proteins may be useful therapeutic targets to stabilize alveolar barrier function and impair the development of ARDS.Support or Funding InformationThis work was supported by Emory Alcohol and Lung Biology Center/National Institutes of Health (NIH) grant P50‐AA013757 (MK), R01‐HL116958 (MK), T32‐AA‐013528 (SAM), the German Academic Exchange Service (DAAD) (BS), R25‐GM099644 (SDW), and Emory+Children's Center of Excellence for Cystic Fibrosis Research (MK).
The Flavonol Quercetin Improves Endometrial Barrier Function through the Expression of Tight Junction Proteins
Authors: Deachapunya Chatsri , Poonyachoti Sutthasinee 
Published: November 2023

Quercetin, the most abundant dietary flavonoid, shows the promotive and protective effects on intestinal tight junction (TJ) barrier function; however, little has been known about its effects in endometrium. The present study aims to investigate the potential effect of quercetin on endometrial TJ barrier function using the immortalized porcine glandular endometrial epithelial cell culture. The TJ barrier function was determined by transepithelial electrical resistance (TER) and fluorescein isothiocyanate‐dextran flux (FD‐4 and FD‐40) across the monolayer. The mRNA expression of TJ proteins, claudin‐2, ‐4, ‐5 and cytoskeletal zona occludens‐1 (ZO‐1) was evaluated by real‐time reverse transcription‐polymerase chain reaction. Quercetin (1, 10 and 100 μM) showed a concentration‐dependent increase in TER with markedly observed in the first 1–2 h. However, treatment with quercetin for 48 h had no significant change in the TER but a markedly decrease in FD4 and FD40 fluxes. Treatment with quercetin (10 μM) for 48 h also increased claudin‐2, ‐4 ‐5 and ZO‐1 mRNA whereas higher concentration only increased the ZO‐1 expression. Thus the increased claudin‐4, ‐5 and ZO‐1 appeared to promote the TJ assembly leading to decreased paracellular permeability. The present results indicate the promotive effects of quercetin on endometrial barrier function and possibly innate immune defense protecting against invading pathogens.Support or Funding InformationThis work was supported by Srinakharinwirot University Research Grant.
Effect of Chronic Alcohol Exposure on Physiological and Molecular Aspects of Pancreatic Acinar Cells Ascorbic Acid Transport
Authors: Subramanian Veedamali S , Srinivasan Padmanabhan , Said Hamid M 
Published: November 2023

Vitamin C (ascorbic acid, AA) is required for normal cellular metabolism including that of pancreatic acinar cells (PAC). The vitamin is a potent antioxidant and acts as a cofactor for several important enzymes, while low intracellular level of AA leads to oxidative stress. PAC obtain the vitamin from their surroundings via transport across their cell membrane. A negative effect of chronic alcohol exposure on uptake of AA that is important for reducing cellular oxidative stress may contribute to the known deleterious effects of alcohol on the pancreas. Recent studies have shown that such alcohol exposure affects PAC uptake of other micronutrients, however its effect on PAC AA uptake is not clear. We address this issue in the present study using mouse‐derived pancreatic acinar 266‐6 cells (in vitro exposure; 50 mM ethanol for 96hrs) and mice fed alcohol chronically and controls were pair‐fed the same diet without alcohol (in vivo exposure; 25%, v/v for 4 weeks). We first determined the relative expression of the two known AA transporters in mouse and human PAC, the sodium‐dependent vitamin C transporter‐1 and ‐2 (SVCT‐1 and SVCT‐2; products of the SLC23A1 and SLC23A2 genes, respectively) and found SVCT‐2 to be the predominant transporter in PAC. Chronic exposure of 266‐6 cells to alcohol significantly inhibited AA uptake, and this inhibition was associated with a marked reduction in SVCT‐2 expression at the protein, mRNA, and heterogenous nuclear RNA (hnRNA) levels. Also, chronic alcohol feeding of mice led to a significant reduction in the level of expression of SVCT‐2 protein, mRNA and hnRNA in PAC. These findings suggest the possible involvement of transcriptional mechanism(s) in mediating the chronic alcohol effect and this observation was supported by the significant epigenetic changes (histone modifications) affecting the Slc23a2 promoter (a decrease in euchromatin H3K4me3 and an increase in heterochromatin H3K27me3) that we found in the alcohol exposed 266‐6 cells compared to controls. No significant changes in the methylation status of the predicted CpG island within the Slc23a2 promoter was found. Collectively, these findings show that chronic alcohol exposure inhibits AA uptake by PAC and that the effect is in part mediated at the level of transcription of the Slc23a2 gene and involves epigenetic mechanism(s).Support or Funding InformationSupported by grants from the DVA and the NIH (AA18071, DK56061, and DK58057).
The choroid plexus epithelium displays conventional cell polarization
Authors: Praetorius Jeppe , Damkier Helle Hasager , Christensen Inga Baasch 
Published: November 2023

The choroid plexus epithelium (CPE) has been studied extensively as a model cell in e.g. high rate fluid secretion because of the direct access to the luminal surface. The high‐rate secretion is orchestrated by an unusual plasma membrane distribution of key transport proteins in the CPE cells. Par example, the ubiquitously expressed Na, K‐ATPase accumulates in the luminal membrane domain of CPE cells. The same has been observed for other normally basolateral proteins such as NKCC1 and NHE1, but not for all basolateral proteins (e.g. AE2). The observation led to the hypotheses that the CPE cells exhibit an inverse cell polarity, despite a conventional localization of structural polarity elements in epithelial cells: basal membrane facing the interstitium, tight junctions close to the luminal surface and luminal specializations such as microvilli and cilia. In this study, we investigated the expression and subcellular distribution of basic cell polarity markers in the CPE cells. Immunohistochemical analysis revealed a basolateral domain accumulation of the membrane lipid PtdIns(3,4,5)P3, Scrib, E‐cadherin, and partitioning‐defective protein (Par) 1. Luminal membrane domain accumulation was observed for the atypical protein kinase aPKC, the plasma membrane target snare syntaxin‐3, whereas Par3 was localized at the tight junction area. These markers represent the molecular complexes known to determine luminal and basolateral identity and were, thus, all located as expected for mature and normally polarized epithelial cells. We conclude that these central polarity proteins are distributed normally in the choroid plexus cells, and that cell polarity as such does not seem to be involved in the unusual localizations of key transport proteins in these cells.Support or Funding InformationThe work was supported by the Danish Medical Research Council and Aarhus University Ideas programme.
Function of the Ang II system during the ontogeny of Archosaurs
Authors: Crossley Dane Alan , Eme John , Tate Kevin , Mueller Casey 
Published: November 2023

Development of cardiovascular regulation in embryonic birds and reptiles has become an area of growing interest over the past 20 yrs. Embryonic cardiovascular control is predominantly dependent on humoral mechanisms across these vertebrate groups. This is evident in the absence of autonomic tone in many species of birds and reptiles, as well as elevated levels of circulating catecholamines. In addition to circulating catecholamines, other humoral regulators have been identified to contribute to baseline regulation of the embryonic cardiovascular system, including angiotensin II (Ang II). Initial work has illustrated that in two representative, extant archosaurs, the domestic chicken (Gallus domesticus) and the American alligator, Ang II produces a dose dependent increase in arterial pressure that is mediated, in part by alpha‐adrenergic receptor stimulation. While the agonist produced a clear cardiovascular response in these species, the response is unaltered by known Ang II receptor antagonists. Direct inhibition was unsuccessful, but indirect manipulation of the Ang II system by decreasing angiotensin converting enzyme function produced noted reductions in arterial pressure, altered organ system size, and altered baroreflex function. This suggests Ang II is an important tonic regulator of cardiovascular function in Archosaurs during development.Support or Funding InformationNSF CAREER award IBN IOS‐0845741 to Dane A Crossley II
The Meadow Jumping Mouse as a Laboratory Model of Hibernation
Authors: Israelsen William J. , Cong Qian , Roorbach Madeline D. 
Published: November 2023

Hibernating mammals exhibit great metabolic flexibility. They experience seasonal obesity and employ heterothermy as a survival strategy. During hibernation in cold climates, small mammalian hibernators enter a state of metabolic torpor and profound hypothermia. The significant (>95%) reduction of metabolic rate during torpor allows the hibernator to subsist on body fat for up to six months. The physiological flexibility of hibernators and their ability to suppress metabolic rate and survive hypothermia are of interest to human medicine; however, the molecular mechanisms underlying these phenotypes are not well understood and most current hibernating model organisms are not conducive to genetic approaches due to long generation times. The goal of this research has been to establish the meadow jumping mouse (Zapus hudsonius) as a convenient laboratory model of hibernation. Meadow jumping mice are small North American rodents with a ‘true’ hibernation phenotype: they fatten prior to hibernation and undergo multi‐day bouts of torpor that are interrupted by short interbout arousals. Crucially, meadow jumping mice breed multiple times per summer in the wild, and they cue their transition from summer reproduction to fall fattening and hibernation largely on day length. We established a breeding colony using wild‐caught animals that were trapped in Massachusetts in the summers of 2014 and 2015. The animals remain in a lean breeding condition year‐round when housed at long photoperiod (16 hours light, 8 hours dark). Obesity and torpor can be induced by exposure to short photoperiod (8 hours light, 16 hours dark) and reduced temperature. We have generated the first meadow jumping mouse transcriptome data and expect that completion of ongoing genome sequencing efforts will increase the utility of this model. The meadow jumping mouse appears to be suitable for investigation of hibernation metabolism in the laboratory, and its short generation time may open the door to future genetic studies of hibernation phenotypes.Support or Funding InformationWJI acknowledges support from the Sara and Frank McKnight Fund for Biochemical Research and an NIH Early Independence Award (DP5OD021365).