Phosphorylation of the large RNA Polymerase II subunit C-terminal domain (CTD) is believed to be important in promoter clearance and for recruiting protein factors that function in messenger RNA synthesis and processing. P-TEFb is a protein kinase that targets the (CTD). The goal of this study was to identify chromatin modifications and associations that require P-TEFb activity in vivo. We knocked down the catalytic subunit of P-TEFb, Cdk9, in Drosophila melanogaster using RNA interference. Cdk9 knockdown flies die during metamorphosis. Phosphorylation at serine 2 and serine 5 of the CTD heptad repeat were both dramatically reduced in knockdown larvae. Hsp 70 mRNA induction by heat shock was attenuated in Cdk9 knockdown larvae. Both mono- and trimethylation of histone H3 at lysine 4 were dramatically reduced, suggesting a link between CTD phosphorylation and histone methylation in transcribed chromatin in vivo. Levels of the chromo helicase protein CHD1 were reduced in Cdk9 knockdown chromosomes, suggesting that CHD1 is targeted to chromosomes through P-TEFb-dependent histone methylation. Dimethylation of histone H3 at lysine 36 was significantly reduced in knockdown larvae, implicating CTD phosphorylation in the regulation of this chromatin modification. Binding of the RNA Polymerase II elongation factor ELL was reduced in knockdown chromosomes, suggesting that ELL is recruited to active polymerase via CTD phosphorylation.
Tight junctions (TJs) are essential for normal function of epithelia, restricting paracellular diffusion and contributing to the maintainance of cell surface polarity. Superficial cells of the urothelium develop TJs, the basis for the paracellular permeability barrier of the bladder against diffusion of urinary solutes. Focusing on the superficial cell layer of stratified cell cultures of an immortalized human ureteral cell line, TEU-2 cells, we have examined the presence of TJ and TJ-associated proteins. TEU-2 cells were treated with calcium chloride and fetal bovine serum culture conditions used to induce stratification that resembles the normal transitional epithelial phenotype. Cultures were examined for TJ and TJ-associated proteins by confocal immuno-fluorescence microscopy and evaluated for TJ mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). TEU-2 cultures exhibited immunoreactivity at intercellular margins for claudins 1, 4, 5, 7, 14 and 16 whereas claudins 2, 8 and 12 were intracellular. RT-PCR corroborated the presence of these claudins at the mRNA level. The TJ-associated proteins occludin, JAM-1, and zonula occludens (ZO-1, ZO-2 and ZO-3) were localized at cell margins. We have found that numerous TJs and TJ-associated proteins are expressed in stratified TEU-2 cultures. Further, we propose TEU-2s provide a useful ureteral model for future studies on the involvement of TJs proteins in the normal and pathological physiology of the human urinary system.
Interstitial cystitis (IC) is associated with increased activated mast cell numbers in the bladder and impairment of the barrier function of the urothelium. We stimulated immortalized urothelial cells derived from the inflamed region of IC bladders (SR22A or SM28 abn) or from healthy bladders (PD07i or PD08i) with tryptase and measured phospholipase A(2) (PLA(2)) activity and the resultant release of arachidonic acid and prostaglandin E(2) (PGE(2)). Tryptase stimulation of either PD07i or SR22A resulted in similar increases in PLA(2) activity and arachidonic acid release. However, tryptase stimulation of SR22A and SM28 abn did not result in a significant increase in PGE(2) release compared with the increase in PGE(2) release from tryptase-stimulated PD07i and PD08i cells. Expression of mRNA for cyclooxygenase-2 and PGE synthase was lower and mRNA for 15-hydroxyprostaglandin dehydrogenase was higher in SR22A compared with PD07i, suggesting that both decreased synthesis and increased metabolism are responsible for the lack of a PGE(2) response in tryptase-stimulated SR22A cells. Since PGE(2) is a cytoprotective eicosanoid, the failure to produce this metabolite in cells isolated from the IC bladder may represent an increased susceptibility to damage by proinfammatory stimuli.
SR22 cells are from the inflamed area of the bladder of a patient with interstitial cystitis (IC), a disease characterized by increased activated mast cells in the bladder wall and compromised urothelial function. Since the urinary content of tryptase is increased in IC, this study determined whether there was impairment of prostaglandin E2 (PGE2) release from SR22 cells in response to tryptase. Responses in SR22 cells were compared to responses observed in PD07i cells derived from a healthy bladder. Interestingly, although PAR‐2 density was significantly lower in SR22 cells, tryptase stimulation of either SR22 or PD07i resulted in a similar increase in phospholipase A2 activity and arachidonic acid release. However, SR22 cells did not show any increase in PGE2 release in response to tryptase, whereas there was a significant increase in PGE2 release from tryptase‐stimulated PD07i. The levels of COX‐1 and COX‐2 mRNA expression were equivalent, but mRNA expression of PGE synthase was considerably lower and of hydroxyprostaglandin dehydrogenase was considerably higher in SR22 compared to PD07i, suggesting that both decreased production and increased metabolism could responsible for the lack of PGE2 response. Since PGE2 is cytoprotective, these data may explain why IC is associated with impaired ability to resist cell damage and warrants further investigation.Supported by DK66119 (JM)
The article presents data from a study of 161 child with pneumonia. Two groups of patients with uncomplicated pneumonia and pneumonia with pleural pulmonary complication were formed. The study of coagulation hemostasis was realized in the studied groups. It was found that in children with uncomplicated pneumonia levels of SFMC and D-dimer were significantly higher than in normal conditions that indicates activation of coagulation hemostasis. When complicated by pneumonia hemostasis matches the current intravascular coagulation: thrombocytosis, increased levels of fibrinogen, hypoor hypercoagulation on aPTT, changing TV thrombinemia (SFMC is 5 times higher than normal), and increased level of D-dimer, inhibition of fibrinolysis, which indicates more pronounced activation of coagulation in children with pneumonia.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.