Search

Abstract Background Sodium absorption by semicircular canal duct (SCCD) epithelial cells is thought to contribute to the homeostasis of the volume of vestibular endolymph. It was previously shown that the epithelial cells could absorb Na+ under control of a glucocorticoid hormone (dexamethasone) and the absorptive transepithelial current was blocked by amiloride. The most commonly-observed target of amiloride is the epithelial sodium channel (ENaC), comprised of the three subunits α-, β- and γ-ENaC. However, other cation channels have also been observed to be sensitive in a similar concentration range. The aim of this study was to determine whether SCCD epithelial cells absorb only Na+ or also K+ through an amiloride-sensitive pathway. Parasensory K+ absorption could contribute to regulation of the transduction current through hair cells, as found to occur via vestibular transitional cells [S. H. Kim and D. C. Marcus. Regulation of sodium transport in the inner ear. Hear.Res. doi:10.1016/j.heares.2011.05.003, 2011]. Results We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6197), whole-cell patch clamp and transepithelial recordings in primary cultures of rat SCCD. α-, β- and γ-ENaC were all previously reported as present. The selectivity of the amiloride-sensitive transepithelial and cell membrane currents was observed in Ussing chamber and whole-cell patch clamp recordings. The cell membrane currents were carried by Na+ but not K+, but the Na+ selectivity disappeared when the cells were cultured on impermeable supports. Transepithelial currents across SCCD were also carried exclusively by Na+. Conclusions These results are consistent with the amiloride-sensitive absorptive flux of SCCD mediated by a highly Na+-selective channel, likely αβγ-ENaC. These epithelial cells therefore absorb only Na+ via the amiloride-sensitive pathway and do not provide a parasensory K+ efflux from the canals via this pathway. The results further provide caution to the culture of epithelial cells on impermeable surfaces.

Abstract Background The ionic composition of the luminal fluid in the vestibular labyrinth is maintained within tight limits by the many types of epithelial cells bounding the lumen. Regulatory mechanisms include systemic, paracrine and autocrine hormones along with their associated intracellular signal pathways. The epithelium lining the semicircular canal duct (SCCD) is a tissue that is known to absorb sodium and calcium and to secrete chloride. Findings Transport function was assessed by measurements of short circuit current (Isc) and gene transcript expression was evaluated by microarray. Neither ATP nor UTP (100 microM) on the apical side of the epithelium had any effect on Isc. By contrast, basolateral ATP transiently increased Isc and transepithelial resistance dropped significantly after basolateral ATP and UTP. P2Y2 was the sole UTP-sensitive purinergic receptor expressed. Isc was reduced by 42%, 50% and 63% after knockdown of α-ENaC, stimulation of PKC and inhibition of PI3-K, while the latter two increased the transepithelial resistance. PKCdelta, PKCgamma and PI3-K were found to be expressed. Conclusions These observations demonstrate that ion transport by the SCCD is regulated by P2Y2 purinergic receptors on the basolateral membrane that may respond to systemic or local agonists, such as ATP and/or UTP. The sodium absorption from endolymph mediated by ENaC in SCCD is regulated by signal pathways that include the kinases PKC and PI3-K. These three newly-identified regulatory components may prove to be valuable drug targets in the control of pathologic vestibular conditions involving dysfunction of transport homeostasis in the ear, such as Meniere's disease.

Aims: This study established the in vitro anti-lymphoma pharmacodynamic actions of the endocannabinoids (anandamide-AEA and 2-arachidonoylglycerol-2AG) on canine non-Hodgkin lymphoma (NHL) and human NHL cells., Main Methods: The expression of cannabinoid (CB 1 and CB 2 ) receptors in various canine NHL cells {1771, CLBL-1, CLL-1, peripheral blood mononuclear cells (PBMCs)} was studied using Quantitative real-time PCR (RT-qPCR). Anti-lymphoma cell viability assay was performed to assess the effect of endocannabinoids on various canine and human NHL cells (1771, CLBL-1, CLL-1, Ramos cells). The spectrophotometric and fluorometric procedures evaluated oxidative stress, inflammation, apoptosis, and mitochondrial function markers. SAS® and Prism-V La Jolla, CA, USA, were used for statistical analysis., Key Findings: The current study validated the presence of CB 1 and CB 2 receptors in the canine NHL cells. There was a significantly higher expression of CB 1 and CB 2 receptors in B-cell lymphoma (BCL) cells (1771, CLBL-1, Ramos) compared to canine T-cell lymphoma (TCL) cells (CL-1). AEA and 2AG dose and time-dependently exhibited significant but differential anti-lymphoma effects on canine and human NHL cells. Anti-lymphoma pharmacodynamic actions of the endocannabinoids in the canine 1771 NHL cells revealed a significant alteration in the markers of oxidative stress, inflammation, and a decrease in mitochondrial function without altering the apoptotic markers., Significance: Establishing the anti-lymphoma pharmacodynamic actions of endocannabinoids may provide new therapeutic interventions and expedite cannabinoid research., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations., Experimental Design: Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression., Results: We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra-S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism., Conclusions: Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress. See related commentary by Viny, p. 573., (©2021 The Authors; Published by the American Association for Cancer Research.)

Environmental chemicals can interfere with the endocrine axis hence they are classified as endocrine disrupting chemicals (EDCs). Bisphenol S (BPS) is used in the manufacture of consumer products because of its superior thermal stability and is thought to be a safe replacement chemical for its analog bisphenol A (BPA). However, the safety profile of these compounds alone or in the presence of other EDCs is yet to be fully investigated. Also, the estrogenic chemical 17α-ethinyl estradiol (EE2) and a constituent of female oral contraceptives for women, is present in water supplies. To simulate concurrent exposure of the population to chemical mixtures, we investigated the effects of BPA, BPS, EE2, and their combinations on sex steroid secretion in the growing male rat gonad. Prepubertal and pubertal male rats at 21 and 35 days of age were provided test chemicals in drinking water (parts per billion) for 14 days. At termination of exposure, some individual chemical effects were modified by exposure to chemical combinations. Single chemical exposures markedly decreased androgen secretion but their combination (e.g., BPA + BPS + EE2) caused the opposite effect, i.e., increased Leydig cell T secretion. Also, the test chemicals acting alone or in combination increased testicular and Leydig cell 17β-estradiol (E2) secretion. Chemical-induced changes in T and E2 secretion were associated with altered testicular expression of the cholesterol side-chain cleavage (Cyp11a1) and 17β-hydroxysteroid dehydrogenase (Hsd17β) enzyme protein. Additional studies are warranted to understand the mechanisms by which single and chemical combinations impact function of testicular cells and disrupt their paracrine regulation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

COVID-19 has severely impacted socioeconomically disadvantaged populations. To support pandemic control strategies, geographically weighted negative binomial regression (GWNBR) mapped COVID-19 risk related to epidemiological and socioeconomic risk factors using South Korean incidence data (January 20, 2020 to July 1, 2020). We constructed COVID-19-specific socioeconomic and epidemiological themes using established social theoretical frameworks and created composite indexes through principal component analysis. The risk of COVID-19 increased with higher area morbidity, risky health behaviours, crowding, and population mobility, and with lower social distancing, healthcare access, and education. Falling COVID-19 risks and spatial shifts over three consecutive time periods reflected effective public health interventions. This study provides a globally replicable methodological framework and precision mapping for COVID-19 and future pandemics., Competing Interests: The authors declare no conflict of interest. The views expressed here are those of the authors and do not necessarily reflect that of any government agency or institution.

Aims: Inflammation is considered to be one of the crucial pathological factors associated with the development of Alzheimer's disease, although supportive experimental evidence remains undiscovered. Therefore, the current study was carried out to better understand and establish the pathophysiological involvement of chronic inflammation in a double transgenic mouse model of Alzheimer's disease., Main Methods: We analyzed amyloid-beta deposition, oxidative stress, biochemical, neurochemical and immunological markers in a 10month old (APΔE9) mouse model. Memory functions were assessed by behavioral testing followed by measurement of synaptic plasticity via extracellular field recordings., Key Findings: Substantial increases in amyloid-beta levels, beta-secretase activity, and oxidative stress, along with significant neurochemical alterations in glutamate and GABA levels were detected in the brain of APΔE9 mice. Interestingly, marked elevations of pro-inflammatory cytokines in whole brain lysate of APΔE9 mice were observed. Flow cytometric analysis revealed a higher frequency of CD4+ IL-17a and IFN-γ secreting T-cells in APΔE9 brain, indicating a robust T-cell infiltration and activation. Behavioral deficits in learning and memory tasks, along with impairment in long-term potentiation and associated biochemical changes in the expression of glutamatergic receptor subunits were evident., Significance: Thus, this study establishes the role by which oxidative stress, alterations in glutamate and GABA levels and inflammation increases hippocampal and cortical neurotoxicity resulting in the cognitive deficits associated with Alzheimer's disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Although epigenetic abnormalities have been described in Huntington's disease (HD), the causal epigenetic mechanisms driving neurodegeneration in HD cortex and striatum remain undefined. Using an epigenetic pathway-targeted drug screen, we report that inhibitors of DNA methyltransferases (DNMTs), decitabine and FdCyd, block mutant huntingtin (Htt)-induced toxicity in primary cortical and striatal neurons. In addition, knockdown of DNMT3A or DNMT1 protected neurons against mutant Htt-induced toxicity, together demonstrating a requirement for DNMTs in mutant Htt-triggered neuronal death and suggesting a neurodegenerative mechanism based on DNA methylation-mediated transcriptional repression. Inhibition of DNMTs in HD model primary cortical or striatal neurons restored the expression of several key genes, including Bdnf, an important neurotrophic factor implicated in HD. Accordingly, the Bdnf promoter exhibited aberrant cytosine methylation in mutant Htt-expressing cortical neurons. In vivo, pharmacological inhibition of DNMTs in HD mouse brains restored the mRNA levels of key striatal genes known to be downregulated in HD. Thus, disturbances in DNA methylation play a critical role in mutant Htt-induced neuronal dysfunction and death, raising the possibility that epigenetic strategies targeting abnormal DNA methylation may have therapeutic utility in HD.

Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for T(m)CG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Bisulfite genomic sequencing provides a single-molecule view of cytosine methylation states. After deamination, each cloned molecule contains a record of methylation within its sequence. The full power of this technique is harnessed by treating nuclei with an exogenous DNMT prior to DNA extraction. This exogenous methylation marks regions of accessibility and footprints nucleosomes, as well as other DNA-binding proteins. Thus, each cloned molecule records not only the endogenous methylation present (at CG sites, in mammals), but also the exogenous (GC, when using the Chlorella virus protein M.CviPI). We term this technique MAPit, methylation accessibility protocol for individual templates.

The ATP-dependent chromatin remodeling complex SWI/SNF regulates transcription and has been implicated in promoter nucleosome eviction. Efficient nucleosome disassembly by SWI/SNF alone in biochemical assays, however, has not been directly observed. Employing a model system of dinucleosomes rather than mononucleosomes, we demonstrate that remodeling leads to ordered and efficient disassembly of one of the two nucleosomes. An H2A/H2B dimer is first rapidly displaced, and then, in a slower reaction, an entire histone octamer is lost. Nucleosome disassembly by SWI/SNF did not require additional factors such as chaperones or acceptors of histones. Observations in single molecules as well as bulk measurement suggest that a key intermediate in this process is one in which a nucleosome is moved toward the adjacent nucleosome. SWI/SNF recruited by the transcriptional activator Gal4-VP16 preferentially mobilizes the proximal nucleosome and destabilizes the adjacent nucleosome., (Copyright 2010 Elsevier Inc. All rights reserved.)

Cells devote considerable resources to nutrient homeostasis, involving nutrient surveillance, acquisition, and storage at physiologically relevant concentrations. Many Saccharomyces cerevisiae transcripts coding for proteins with nutrient uptake functions exhibit peak periodic accumulation during M phase, indicating that an important aspect of nutrient homeostasis involves transcriptional regulation. Inorganic phosphate is a central macronutrient that we have previously shown oscillates inversely with mitotic activation of PHO5. The mechanism of this periodic cell cycle expression remains unknown. To date, only two sequence-specific activators, Pho4 and Pho2, were known to induce PHO5 transcription. We provide here evidence that Mcm1, a MADS-box protein, is essential for PHO5 mitotic activation. In addition, we found that cells simultaneously lacking the forkhead proteins, Fkh1 and Fkh2, exhibited a 2.5-fold decrease in PHO5 expression. The Mcm1-Fkh2 complex, first shown to transactivate genes within the CLB2 cluster that drive G(2)/M progression, also associated directly at the PHO5 promoter in a cell cycle-dependent manner in chromatin immunoprecipitation assays. Sds3, a component specific to the Rpd3L histone deacetylase complex, was also recruited to PHO5 in G(1). These findings provide (i) further mechanistic insight into PHO5 mitotic activation, (ii) demonstrate that Mcm1-Fkh2 can function combinatorially with other activators to yield late M/G(1) induction, and (iii) couple the mitotic cell cycle progression machinery to cellular phosphate homeostasis.

Non-invasive methods for mapping chromatin structure are necessary for creating an accurate view of genome function and dynamics in vivo. Ectopic induction of cytosine-5 DNA methyltransferases (C5 MTases) in Saccharomyces cerevisiae is a powerful technique for probing chromatin structure with minimal disruption to yeast physiology. Accessibility of MTases to their cognate sites is impaired based on the strength and span of the protein-DNA interaction to be probed. Methylated cytosines that resist chemical deamination are detected positively by the PCR-based technique of bisulfite genomic sequencing. PCR amplicons can be sequenced directly yielding an average m(5)C frequency or accessibility of each target site within the population, a technique termed methyltransferase accessibility protocol (MAP). More recently, the sequencing of cloned molecules in MAP for individual templates (MAPit) enables assignment of the methylation status of each target site along a continuous DNA strand from a single cell. The unique capability to score methylation at multiple sites in single molecules permits detection of inherent structural variability in chromatin. Here, MAPit analysis of the repressed and induced PHO5 promoter of budding yeast, using a C5 MTase with dinucleotide recognition specificity, reveals considerable cell-to-cell heterogeneity in chromatin structure. Substantial variation is observed in the extent to which the MTase gains entry to each of the nucleosomes positioned at PHO5, suggesting differences in their intrinsic thermodynamic stability in vivo. MAPit should be readily adaptable to the analysis of chromatin structure and non-histone protein-DNA interactions in a variety of model systems.

Wrapping DNA into chromatin provides a wealth of regulatory mechanisms that ensure normal growth and development in eukaryotes. Our understanding of chromatin structure, including nucleosomes and non-histone protein-DNA interactions, has benefited immensely from nuclease and chemical digestion techniques. DNA-bound proteins, such as histones or site-specific factors, protect DNA against nuclease cleavage and generate large nucleosomal or small regulatory factor footprints. Chromatin subject to distinct modes of regulation often coincides with sites of nuclease hypersensitivity or nucleosome positioning. An inherent limitation of cleavage-based analyses has been the inability to reliably analyze regions of interest when levels of digestion depart from single-hit kinetics. Moreover, cleavage-based techniques provide views that are averaged over all the molecules in a sample population. Therefore, in cases of occupancy of multiple regulatory elements by factors, one cannot define whether the factors are bound to the same or different molecules in the population. The recent development of DNA methyltransferase-based, single-molecule MAP-IT technology overcomes limitations of ensemble approaches and has opened numerous new avenues in chromatin research. Here, we review the strengths, limitations, applications and future prospects of MAP-IT ranging from structural issues to mechanistic questions in eukaryotic chromatin regulation., ((c) 2008 Wiley-Liss, Inc.)