bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of PKCα Alters the Renal Immune Response in Angiotensin II‐dependent Hypertension
Authors: Borchart Matthew R. , Pulous Fadi E. , Wang Yirong , Sun Michael A. , Himmel Nathaniel J. , Vanderford Thomas H. , Blount Mitsi A.
Published: November 2023

Angiotensin II (Ang II) stimulation of T‐cell proliferation and accumulation plays an important role in the development of hypertension and end‐stage kidney damage. Unfortunately, the signaling mechanisms behind the immune response within the kidney remains underexplored. One pathway that is important to both Ang II and T‐cell response is mediated by PKCα. To determine the role of PKCα in facilitating T‐cell infiltration in the kidney during Ang II‐dependent hypertension, PKCα null (KO) mice were treated with either saline or Ang II and compared to PKCα control (WT) mice treated similarly. After 14 days, systolic blood pressure, water intake and urine output were increased in all Ang II‐treated mice. Kidney damage and collagen deposition is elevated with ANG II treatment and appears more extensive in KO mice. Ang II‐treatment significantly increased spleen weight in WT mice only; suggesting that the absence of PKCα attenuates increased immune cell production. Histological examination of immune cell infiltration of the renal cortex revealed that Ang II‐treatment increased CD4+ and CD8+ T‐cells but not macrophage invasion in both WT and KO mice and suggests that CD4+ T‐cell recruitment is higher in Ang II‐treated WT compared to treated KO mice. Immune cell infiltration and cytokine production was also measured via flow cytometry and results suggest that PKCα is important for Ang II‐meditated cytokine release in renal tissues. Collectively, these data suggest that the absence of PKCα decreases inflammatory response to Ang II‐induced hypertension in the kidney.

Support or Funding Information

NIDDK R25‐DK101390
Screening Bioactivity of Virus Surface Glycoprotein Peptides using C. elegans Electropharyngiogram
Authors: Hincks Joshua B , Monrean Billyann B , Lu Brianna , Taylor Barbara E , Hueffer Karsten , Harris Michael B
Published: November 2023

The rabies virus surface glycoprotein contains an amino acid sequence similar to neurotoxin peptides known to bind to and inhibit the function of nicotinic acetylcholine receptors (nAChR). In preliminary experiments, we have shown that a peptide fraction of the neurotoxin‐like‐domain of the rabies virus inhibits signaling of central nervous system nAChRs. It has been shown, in the nematode Caenorhabditis elegans, that defined motor neuron MC acts as a neurogenic pacemaker for pharyngeal pumping and that nAChR are critical to excitation of the pharynx. We propose to use pharyngeal pumping in C. elegans as a screening assay for bioactivity of variants of virus glycoprotein peptides. In the present study we either administered a 29 amino acid peptide fraction (or a scrambled peptide) by microinjection through the cuticle proximal to the pharynx, or incubated animals for 90 minutes in concentrated peptide (5 or 2.5 mM) or M9 buffer solution. Following treatments, we assessed pharyngeal pumping frequency during 2‐minute observation periods using a non‐invasive electropharyngeogram (NemaMetrix Katalyst 100). Pharyngeal pumping was absent in 5 of 6 animals receiving active peptide injection, while pharyngeal pumping in those receiving scrambled peptide was identical to untreated animals. Pharyngeal pumping was similarly absent or greatly attenuated in animals incubated in active peptide compared to controls. Results indicate that internal and external exposure to a peptide proposed to inhibit nAChR attenuate pharyngeal pumping in C. elegans in a manner expected by nAChR antagonism. Results support the bioactivity of this virus peptide fraction and the utility of C. elegans electropharyngeogram as a screen for bioactivity of variants of this peptide.

Support or Funding Information

Work reported in this publication was supported by the National Heart Lung and Blood Institute and National Institute of General Medical Sciences of the National Institutes of Health under awards 1R15HL126105, 1SC2GM112570 and three linked awards number RL5GM118990, TL4GM118992 and 1UL1GM118991. The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health or any other funding body.
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 Information

This 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 Information

Research 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.

Objective

To describe the diffusion pattern from blood to cerebrospinal fluid (CSF) and to explore the possibility to be synthesized in CNS.

Methods

We 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.

Results

CSF 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.

Conclusions

The 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 Information

This 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 Information

This 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 Information

Supported 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 Information

The 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 Information

NSF CAREER award IBN IOS‐0845741 to Dane A Crossley II
Novel Function of Insulin‐Like Peptides: Signaling in Sickness‐Induced Anorexia (SIA)
Authors: Cambron Lizzette D , Greenlee Kendra J
Published: November 2023

Often, when an organism becomes ill, it stops feeding. Yet, somehow most animals are able to regain their health and overcome the illness. This phenomenon is called sickness‐induced anorexia (SIA). SIA is hypothesized to be an adaptive function that reallocates energy toward immune function. Most animals exhibit SIA, yet what triggers it and the mechanisms tying it to immunity remain unknown. One potential mediator of SIA is insulin signaling. Both vertebrates and invertebrates share a conserved insulin‐signaling pathway, but how this pathway regulates feeding behavior and dietary intake is not yet well understood. In insects, the storage tissue known as the fat body plays a key role in assessing nutritional status, such as amino acid content, to secrete insulin‐like peptides (ILP). Although various ILP have been found in insects, their physiological functions are largely unknown. Much like insulin in vertebrates, ILP have been shown to block synthesis of blood sugar (trehalose) from dietary sugars and amino acids and promote the consumption of glycogen reserves. When carbohydrate levels are high, fat body cells signal to ILP‐producing cells via the target of rapamycin (TOR) pathway to secrete ILP. This study aims to elucidate a mechanism between ILP secretion and SIA and to discover novel functions of ILP using the tobacco hornworm caterpillar, Manduca sexta, an insect that has been shown to exhibit SIA. To determine if ILP secretion is required for SIA, ILP will be manipulated in both the presence and absence of an immune challenge. ILP levels will be manipulated by either blocking with wortmannin, a downstream inhibitor of insulin‐signaling, or increasing with bovine insulin, which has been shown to have similar effects as ILP in insects. During SIA from a bacterial infection, blocking or increasing ILP are expected to result respectively in no glycogen consumption and glycogen exhaustion. With the lack of nutrient availability from either feeding or glycogen storage, we expect survival of a bacterial infection to decrease. However, if ILP secretion is blocked then recovered by bovine insulin injection, we would expect survival to increase. As a result of these changes, fat body content and trehalose concentrations are expected to change. Clarifying what mediates SIA would have a broad impact on nutritional, immunological, and developmental studies in all types of organisms.
Tissue‐Specific Seasonal Changes in Mitochondrial Membrane Composition and Respiratory Function in the Golden‐Mantled Ground Squirrel
Authors: Heim Ashley B. , Rosales Susana A. , Chicco Adam J. , Florant Gregory L.
Published: November 2023

Mammals that hibernate, such as the golden‐mantled ground squirrel (Callospermophilus lateralis; GMGS), cease to feed, reduce metabolic rate, and lower body temperature during the winter months, surviving almost exclusively on the oxidation of lipids from endogenous fat stores. Whether mitochondria, the cellular sites of oxidative metabolism, undergo adaptive changes in response to hypometabolism and decreasing ambient temperature to facilitate this remarkable phenotype is unclear. It has been postulated that changes in mitochondrial membrane composition reported in response to cold exposure in some species may facilitate maintenance of respiratory enzyme function with decreasing temperature. However, no studies to date have investigated the function and membrane fatty acid composition of mitochondria from different tissues from the warm summer months, to fall and winter in a hibernator. We hypothesize that tissue‐specific differences in mitochondrial respiration occur across seasons in the GMGS that may parallel distinct changes in mitochondrial membrane composition. We compared the respiratory capacity, oxidative enzyme activity, and membrane composition of GMGS mitochondria isolated from liver, heart, skeletal muscle, and brown adipose tissue (BAT) from summer, fall, and winter (torpid) seasons. Maximal mitochondrial oxidative phosphorylation (OXPHOS)‐supported respiration was determined by high‐resolution respirometry at 37°C, 25°C, and 15°C in the presence of saturating concentrations of ADP and respiratory substrates. Mitochondrial membrane fatty acid composition was determined by gas chromatography on phospholipid fractions obtained from isolated mitochondria. Maximal OXPHOS assayed at 37°C tended to increase from summer to winter in liver, heart and BAT, and decrease in skeletal muscle, with tissue‐ and season‐specific changes in pyruvate versus fatty acid (palmitoylcarnitine) oxidation capacity. OXPHOS capacity was uniformly suppressed by decreasing assay temperature in all tissue mitochondria from torpid GMGS in the winter, despite widely variable changes in mitochondrial membrane composition across tissues and seasons. Interestingly, a consistent relative decrease in mitochondrial membrane docosahexaenoic acid (DHA; 22:6n3) was seen from summer to winter in all tissues, the biological relevance of which merits further investigation. Taken together, these findings argue against a consistent relationship between changes in mitochondrial membrane composition and respiratory function across seasons in GMGS, but highlight distinct tissue‐ and season‐specific differences that may have important biological effects that remain to be elucidated.
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 Information

WJI acknowledges support from the Sara and Frank McKnight Fund for Biochemical Research and an NIH Early Independence Award (DP5OD021365).
Initiation And Elongation Of RNA Polymerase II Are Regulated By Multiple OGlcNAcylation Events
Authors: Lewis Brian A
Published: November 2023

RNA pol II is thought to be regulated at two positions on a gene and this regulation determines whether a gene is transcribed. The first is at the promoter, where pol II and the general transcriptional machinery are recruited to form a pre‐initiation complex (PIC). Upon receiving the appropriate signal, pol II initiates transcription and elongates into the gene body. The current dogma states that a hypophosphorylated RNA pol II (pol IIA) is the initiation‐competent species. However, this pol IIA was shown to also contain glcnacylated pol II. Thus, although the O‐GlcNAc modification of the RNA polymerase II CTD was described twenty years ago it has never been clear what the initiation‐specific species of RNA pol II is, or whether O‐GlcNAcylation regulates PIC formation.

The second position is located immediately downstream of the transcriptional start site (TSS). In the late 1980s, a transcriptionally engaged, paused pol II was found on several genes, roughly at +50 relative to the TSS. Recent genome‐wide approaches have shown that paused pol II exists on approximately 40% of promoters in the genome and thus understanding the regulation of pausing is a major issue in eukaryotic transcription. It is not known nor has it been suspected that O‐GlcNAcylation plays a role in regulating these events.

We have made significant advances in showing functional requirements for O‐GlcNAcylation during both transcription initiation and elongation. Firstly, we have collected several lines of evidence that O‐GlcNAcylation events are required for transcription initiation and the recruitment of RNA pol II to promoters. Notably, OGT and OGA chemical inhibitors block transcription in vitro and inhibition of O‐GlcNAcylation prevents pol II entry into the promoter in vitro and in vivo. Secondly, we have found that chemical inhibition of the O‐GlcNAc aminidase (OGA) has pausing and elongation defects in vivo. In vitro elongation assays also showed a functional requirement for OGA activity in both crude nuclear extract and partially purified systems. Lastly, we identified a 600 kDa complex containing OGA and the pausing/elongation factor DSIF.

These data indicate that O‐GlcNAcylation is essential for RNA pol II promoter recruitment and that RNA pol II goes through a cycling of O‐GlcNAcylation at the promoter, where before initiation pol II is in a glcnacylated state, and upon initiation, the O‐GlcNAc is removed. Our data also suggest a novel role of dynamic cycling of O‐GlcNAcylation in RNA pol II elongation control by showing a functional and physical coupling of OGA with the pol II elongation and the pausing machinery. In conclusion, our work has brought to light an entirely new level of regulation of both transcription initiation and elongation, mediated by O‐GlcNAcylation enzymes. These results are possibly manifestations of a direct link between the regulation of RNA pol II activity genome‐wide and the nutrient state of the cell.

Support or Funding Information

CCR/NCI/NIH Intramural Research Program
Role of CD36 in ROS‐induced Calcium Influx in Human Lung Microvascular Endothelial Cells
Authors: Reyes Jose , Suresh Karthik , Servinsky Laura , Pearse David Bennett , Shimoda Larissa
Published: November 2023

Rationale

Acute respiratory distress syndrome (ARDS) is characterized by disruption of the endothelial barrier in lung microvascular endothelial cells (LMVEC). Reactive oxygen species (ROS) and increased intracellular calcium concentration ([Ca2+]i) have been implicated in the endothelial barrier dysfunction in ARDS, but the mechanisms linking these processes are not fully understood. We previously showed that exogenous ROS (H2O2) increases intracellular calcium in human LMVEC (HLMVEC) and that the calcium channel TRPV4 contributes to H2O2‐induced Ca2+ influx in HLMVEC. Additionally, our prior data suggest that phosphorylation of TRPV4 by the Src kinase Fyn is necessary for ROS‐induced Ca2+ influx. In endothelial cells, Fyn is known to be tethered to the cell membrane by CD36, a fatty acid scavenger receptor. In mouse LMVEC, we have shown that loss of CD36 significantly attenuates ROS‐induced Ca2+ influx, but the role of CD36 in ROS‐induced Ca2+ influx in HLMVEC is not known. We hypothesized that CD36 is present in HLMVEC, and that it acts as a scaffold for Fyn; thus, we hypothesized that direct inhibition of CD36 would not alter ROS‐induced Ca2+ influx in HLMVEC.

Methods

We measured [Ca2+]i by loading HLMVEC with the ratiometric calcium sensor Fura‐2AM and measuring the change in [Ca2+]i following exposure to H‐2O2 (250 μM) while in a temperature‐controlled flow chamber. Following excitation at 340 and 380 nM, emissions at 510 nM were recorded. Immunoflourescence was performed using fluorescently labeled AcLDL and DAPI nuclear stain in HLMVEC.

Results

CD36 was present in HLMVEC by both immunoblot and immunofluorescence analysis. To determine the effects of direct inhibition of CD36, we treated cells with the CD36 inhibitor succinimidyl oleate (SSO; 100 μM), which has previously been shown, in macrophages, to inhibit fatty acid uptake by CD36 but not the interaction between CD36 and Fyn. To ensure that adequate inhibition had occurred, we assessed uptake of Acetylated‐LDL in HLMVEC with and without SSO treatment. SSO significantly inhibited AcLDL uptake by HLMVEC. Untreated HLMVEC exhibit a large increase in [Ca2+]i following exposure to H2O‐2 that was not attenuated in HLMVEC treated with SSO. These data suggest that while CD36 is necessary for ROS‐induced Ca2+ influx, this mechanism, in HLMVEC, does not occur via direct binding and activation of CD36 by lipid moieties generated by increased intracellular ROS.
Hemoglobin Induced Lipid Peroxidation as an Initiating Factor of Pulmonary Hypertension
Authors: Irwin David , Loomis Zoe L , Redinius Katherine , Eigenberger Paul , Lisk Christina , Buehler Paul
Published: November 2023

Pulmonary hypertension (PH) is a common etiology of both inherited and acquired forms of hemolytic diseases. It is suspected that cell free hemoglobin (CFH) may initiate vascular remodeling and increased pulmonary pressures, however, the mechanisms behind this pathology remain unclear. It is understood that CFH increases reactive oxygen species and lipid peroxidation through reactions which are exacerbated in pro‐oxidative microenvironments. Recently, oxidative stress has been implicated in NFkB activation through a Toll like receptor 9 (TLR9) mediated pathway; it has been proposed that TLR9 is activated by either nuclear or mitochondrial DNA that has been liberated by stress induced cellular trauma. In this regard, the purpose of this investigation was to test whether hemoglobin induced oxidative stress triggers TLR9 mediated inflammation. We hypothesized that Hb induced lipid peroxidation is linked to TLR9 activation, resulting in IL6 induction and a corresponding activation of the smooth muscle cell proliferation indicative of pulmonary hypertension.

Methods

Rat pulmonary artery endothelial cells and smooth muscle cells (RPAEC and RPASMC) or HEK TLR9 reporter cells were incubated for 6–24h with HbFe2+ and HbFe3+ in the presence or absence of glucose oxidase (GOX). Lipid peroxidation, TLR9 activity, IL6 expression, and SMC activation were determined using CLICK chemistry, NFkB inducible enzymatic assay, qRTPCR and MTT assay, respectively.

Results

HbFe2+ and HbFe3+ (with or without GOX) induced lipid peroxidation in RPAEC and reporter cell TLR9 activation; IL6 expression in RPAEC was increased when incubated with HbFe2+ and HbFe3+; RPASMC demonstrated a more proliferative state when co‐cultured with RPAEC treated with HbFe2+ and HbFe3+. Glucose oxidase augmented the Hb induced phenotypes in all experiments.

Conclusions

Hb‐induced lipid peroxidation is associated with TLR9 activation, IL6 induction, and SMC activation. As such, CFH may be an initiating factor and mediator of pulmonary hypertension associated with hemolytic diseases, thus providing clinicians with a potential therapeutic target.

Support or Funding Information

NIH NLBI 1RO1HL125642‐01A1
The Role of PP5 in the FKBP51 Inhibition of Isoc Current
Authors: Hamilton Caleb L , Kadeba Pierre I , Abney Kevin A , Honkanen Richard , Scammell Jonathan G , Cioffi Donna L
Published: November 2023

Background

In acute respiratory distress syndrome, inflammatory signals increase calcium influx into endothelial cells through store‐operated calcium entry (SOCE). SOCE plays a major role in the promotion of inter‐endothelial cell gap formation. SOCE is comprised of two major currents: the calcium release activated current (Icrac) and the store‐operated calcium current (Isoc). Our laboratory has shown that FK506 binding protein 51 (FKBP51) inhibits Isoc current in pulmonary artery endothelial cells (PAECs). However, the mechanism(s) by which FKBP51 inhibits Isoc is unknown. FKBP51 has been shown to mediate its effects in neuronal cells through dephosphorylation events of the microtubule associated protein tau. We propose that a similar cellular event may be responsible for the action of FKBP51 on Isoc. FKBP51 has no endogenous phosphatase activity and is likely dependent on other cellular phosphatases for downstream dephosphorylation of tau. In particular, the role of the protein phosphatase five (PP5) might be implicated because, like FKBP51, it is a co‐chaperone of heat shock protein 90 (HSP90) that has been shown to decreases tau phosphorylation in vivo. We have shown that HSP90 and FKBP51 both co‐precipitate with the Isoc heterocomplex in PAECs. We hypothesize that the FKBP51‐dependent inhibition in Isoc requires PP5.

Methods

We utilize immunocytochemistry and a membrane‐cytoskeletal preparation to determine the localization of PP5 in PAECs. Whole‐cell patch clamp electrophysiology was used to measure Isoc in HEK293 cells and PAECs. Dexamethasone treatment was used to increase FKBP51 expression. CRISPR/Cas9 was used to generate a PP5 knockout HEK293 cell line.

Results

We show that a subset of PP5 is located at the cell‐cell borders of PAECs and in both the cell membrane and spectrin‐actin cytoskeleton fractions associated with the Isoc heterocomplex. Additionally, we show that dexamethasone treatment leads to decreased Isoc in HEK293 cells. However, in the CRISPR/Cas9 knockout PP5 HEK293 cells, the dexamethasone‐mediated decrease in Isoc was prevented.

Conclusion

Together, these findings suggest a role for PP5 in the FKBP51‐mediated inhibition of Isoc.

Support or Funding Information

Supported by NIH 5R00HL089361.
Hypocapnia aggravates hypoxia‐induced inhibition of alveolar Na‐transport
Authors: Mairbaurl Heimo , Sahinoz Melis , Baloglu Emel
Published: November 2023

Hypercapnia that is often associated with ARDS inhibits alveolar fluid reabsorption by PKC‐dependent mechanisms. Also Hypocapnia seems to reduce lung fluid clearance. In both cases inhibition was associated with internalization of Na/K‐ATPase. Hypocapnia is a consequence of hyperventilation in hypoxia. Hypoxia also inhibits alveolar fluid clearance by decreasing Na/K‐ATPase‐ and Na‐channel dependent transepithelial Na transport. It is not known, however, how altered CO2 affects hypoxia‐induced alveolar reabsorption. To answer this question we exposed primary rat alveolar epithelial cells to acute and 24h of hypoxia with 5% and 3% CO2, and measured trans‐monolayer ion transport as short circuit currents (ISC) in Ussing chambers. We show here that inhibition of alveolar reabsorption by hypoxia and hypocapnia was additive. This effect was small after 30 min but well pronounced after 24h of treatment. Ion transport at both CO2‐levels was not affected by acetazolamide, an inhibitor of carbonic anhydrase. In contrast to earlier reports we found no hypocapnia‐induced inhibition of Na/K‐ATPase, i.e. ISC after apical permeabilization with amphotericin A, although there was a decrease in whole‐cell α1‐Na/K‐ATPase protein measured by Western blot. However, we found a decrease in amiloride‐sensitive ISC indicating inhibition of epithelial Na‐channels (ENaC), which was associated with a decrease in apical αENaC protein in hypoxia, which was enhanced by hypocapnia. Our results indicate that hypocapnia aggravates hypoxia‐induced inhibition of alveolar reabsorption which may enhance the risk of formation alveolar edema in hypoxia.

Support or Funding Information

TLRC‐H, DZL
Alterations in the Expression of Profilin1/pVASP‐S157 and Cofilin1/pVASP‐S239 in Perinatal Inflammation/Neonatal Hyperoxia‐Induced Lung Injury
Authors: Ali Mehboob , Heyob Kathryn M , Rogers Lynette K
Published: November 2023

Cell proliferation and migration have been shown to be regulated by actin dynamics/cytoskeletal remodeling. Profilin1, cofilin1 and VASP (phosphorylated at serine‐157 (pVASP‐S157) or serine‐239 (pVASP‐S239)) are the major regulators of actin remodeling within the cell. Infants that develop Bronchopulmonary Dysplasia (BPD) exhibit decreased alveolarization resulting from altered cellular proliferation and migration during septal formation. Using our mouse model of BPD, we tested the hypothesis that changes in expression and cellular localization of profilin1, cofilin1 and phosphorylated VASP play a key role in decreases in secondary septal formation and thus alveolarization.

Human lung autopsy tissues from infants that died with BPD or controls were assessed for total F‐actin content and expression of profilin1, colfilin1, and phosphorylated VASP by immunofluorescence. Lung tissues were obtained from our murine model of maternal LPS exposure followed by 7 days of 85% hyperoxia. Murine tissues were harvested at 7 days of life and lung homogenates were assessed for profilin1, colfilin1, and VASP phosphorylation by western blot.

A significant decrease in F‐actin content, pVASP‐157, pVASP‐S239, and profilin1 and an increase in cofilin1 were observed in BPD tissues compared to controls. Western blots from lung tissue homogenates indicated increases in cofilin1, profilin1, and pVASP‐S239 but a decrease pVASP‐S157 at 7 days.

In conclusion, we propose that these alterations in expression and phosphorylation of ABP may be contributing to the decrease in cell migration and septal formation in immature lungs injured by hyperoxia exposure. The differences between the findings in humans and mice are likely to be due to the early age of the mice, prior to onset of severe symptoms. Further investigations are needed to define the specific cell types affected and the role of actin binding proteins in lung development.

Support or Funding Information

This work was supported by the National Institutes of Health, NCCIH/ODS (LKR R01AT006880).