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1.
Dong-Mei Wang Dheeraj Soni Sushil C. Regmi Stephen M. Vogel Chinnaswamy Tiruppathi 《Molecular biology of the cell》2022,33(7)
TGF–β-activated kinase 1 (TAK1) plays crucial roles in innate and adaptive immune responses and is required for embryonic vascular development. However, TAK1’s role in regulating vascular barrier integrity is not well defined. Here we show that endothelial TAK1 kinase function is required to maintain and repair the injured lung endothelial barrier. We observed that inhibition of TAK1 with 5Z-7-oxozeaenol markedly reduced expression of β-catenin (β-cat) and VE-cadherin at endothelial adherens junctions and augmented protease-activated receptor-1 (PAR-1)- or toll-like receptor-4 (TLR-4)-induced increases in lung vascular permeability. In inducible endothelial cell (EC)-restricted TAK1 knockout (TAK1i∆EC) mice, we observed that the lung endothelial barrier was compromised and in addition, TAK1i∆EC mice exhibited heightened sensitivity to septic shock. Consistent with these findings, we observed dramatically reduced β-cat expression in lung ECs of TAK1i∆EC mice. Further, either inhibition or knockdown of TAK1 blocked PAR-1- or TLR-4-induced inactivation of glycogen synthase kinase 3β (GSK3β), which in turn increased phosphorylation, ubiquitylation, and degradation of β-cat in ECs to destabilize the endothelial barrier. Importantly, we showed that TAK1 inactivates GSK3β through AKT activation in ECs. Thus our findings in this study point to the potential of targeting the TAK1-AKT-GSK3β axis as a therapeutic approach to treat uncontrolled lung vascular leak during sepsis. 相似文献
2.
Nikolai Otmakhov Elena V. Gorbacheva Shaurav Regmi Ryohei Yasuda Andy Hudmon John Lisman 《PloS one》2015,10(3)
Over-activation of excitatory NMDA receptors and the resulting Ca2+ overload is the main cause of neuronal toxicity during stroke. CaMKII becomes misregulated during such events. Biochemical studies show either a dramatic loss of CaMKII activity or its persistent autonomous activation after stroke, with both of these processes being implicated in cell toxicity. To complement the biochemical data, we monitored CaMKII activation in living hippocampal neurons in slice cultures using high spatial/temporal resolution two-photon imaging of the CaMKIIα FRET sensor, Camui. CaMKII activation state was estimated by measuring Camui fluorescence lifetime. Short NMDA insult resulted in Camui activation followed by a redistribution of its protein localization: an increase in spines, a decrease in dendritic shafts, and concentration into numerous clusters in the cell soma. Camui activation was either persistent (> 1–3 hours) or transient (~20 min) and, in general, correlated with its protein redistribution. After longer NMDA insult, however, Camui redistribution persisted longer than its activation, suggesting distinct regulation/phases of these processes. Mutational and pharmacological analysis suggested that persistent Camui activation was due to prolonged Ca2+ elevation, with little impact of autonomous states produced by T286 autophosphorylation and/or by C280/M281 oxidation. Cell injury was monitored using expressible mitochondrial marker mito-dsRed. Shortly after Camui activation and clustering, NMDA treatment resulted in mitochondrial swelling, with persistence of the swelling temporarily linked to the persistence of Camui activation. The results suggest that in living neurons excitotoxic insult produces long-lasting Ca2+-dependent active state of CaMKII temporarily linked to cell injury. CaMKII function, however, is to be restricted due to strong clustering. The study provides the first characterization of CaMKII activation dynamics in living neurons during excitotoxic insults. 相似文献
3.
Regmi Sudip Choi Yoon Seok Kim Young Kyun Khan Md Maruf Lee Sang Hun Choi Yun Hee Cho Seung Sik Jin Ying-Yu Yoo Jin Cheol Suh Joo-Won 《Bioprocess and biosystems engineering》2020,43(2):249-259
Bioprocess and Biosystems Engineering - The β-glucanase produced from Bacillus sp. CSB55 not only depicts the potent industrial characteristics but also relates as bio-industrial catalyst... 相似文献
4.
Suhrid Banskota Jaya Gautam Sushil C. Regmi Pallavi Gurung Myo-Hyeon Park Seung Joo Kim Tae-gyu Nam Byeong-Seon Jeong Jung-Ae Kim 《PloS one》2016,11(1)
5-Hydroxytryptamine (5-HT) induces proliferation of cancer cells and vascular cells. In addition to 5-HT production by several cancer cells including gastrointestinal and breast cancer, a significant level of 5-HT is released from activated platelets in the thrombotic environment of tumors, suggesting that inhibition of 5-HT signaling may constitute a new target for antiangiogenic anticancer drug discovery. In the current study we clearly demonstrate that 5-HT-induced angiogenesis was mediated through the 5-HT1 receptor-linked Gβγ/Src/PI3K pathway, but not through the MAPK/ERK/p38 pathway. In addition, 5-HT induced production of NADPH oxidase (NOX)-derived reactive oxygen species (ROS). In an effort to develop new molecularly targeted anticancer agents against 5-HT action in tumor growth, we demonstrate that BJ-1108, a derivative of 6-amino-2,4,5-trimethylpyridin-3-ol, significantly inhibited 5-HT-induced angiogenesis. In addition, BJ-1108 induced a significant reduction in the size and weight of excised tumors in breast cancer cell-inoculated CAM assay, showing proportionate suppression of tumor growth along with inhibition of angiogenesis. In human umbilical vein endothelial cells (HUVECs), BJ-1108 significantly suppressed 5-HT-induced ROS generation and phosphorylation of PI3K/Akt but not of Src. Unlike NOX inhibitors, BJ-1108, which showed better antioxidant activity than vitamin C, barely suppressed superoxide anion induced by mevalonate or geranylgeranyl pyrophosphate which directly activates NOX without help from other signaling molecules in HUVECs, implying that the anti-angiogenic action of BJ-1108 was not mediated through direct action on NOX activation, or free radical scavenging activity. In conclusion, BJ-1108 inhibited 5-HT-induced angiogenesis through PI3K/NOX signaling but not through Src, ERK, or p38. 相似文献
5.
OBJECTIVE: To study the role of fine needle aspiration cytology (FNAC) and ancillary studies in the diagnosis of tubercular epididymitis or epididymo-orchitis. STUDY DESIGN: Forty patients with tubercular epididymitis or epididymoorchitis diagnosed on FNAC underwent a detailed clinical workup, imaging and microbiologic studies before being started on antitubercular treatment (ATT). One patient underwent orchiectomy. RESULTS: Clinically, the disease presented in patients of all ages usually as a scrotal swelling or rarely as a scrotal sinus (3) or abscess (3) or as part of disseminated tuberculosis (2). Three patients gave a history of previous tuberculosis. Scrotal sonography confirmed the involvement of the epididymis, testis or spermatic cord in each case. FNAC was diagnostic in 27 aspirates (epithelioid cell granulomas with caseation) but nondiagnostic in the rest. Tubercular etiology was confirmed directly by detection of acid-fast bacilli (AFB) on FNA smears in 24 (60%) patients and urine samples in 11 and indirectly in 9 patients with negative AFB by using a combination of a positive Mantoux test (5 of 9), presence of caseating granulomas on FNA smears (7 of 9) and therapeutic response to ATT (9 of 9). CONCLUSION: FNA as a minimally invasive technique plays a prime role in the diagnosis of tubercular epididymitis and epididymoorchitis. It provides adequate material for cytologic and microbiologic examination and helps to avoid unnecesary orchiectomy. 相似文献
6.
RACK1 is a WD-repeat protein that forms signal complexes at appropriate locations in the cell. RACK1 homologues are core components of ribosomes from yeast, plants and mammals. In contrast, a cryo-EM analysis of trypanosome ribosomes failed to detect RACK1, thus eliminating an important translational regulatory mechanism. Here we report that TbRACK1 from Trypanosoma brucei associates with eukaryotic translation elongation factor-1a (eEF1A) as determined by tandem MS of TAP-TbRACK1 affinity eluates, co-sedimentation in a sucrose gradient, and co-precipitation assays. Consistent with these observations, sucrose gradient purified 80S monosomes and translating polysomes each contained TbRACK1. When RNAi was used to deplete cells of TbRACK1, a shift in the polysome profile was observed, while the phosphorylation of a ribosomal protein increased. Under these conditions, cell growth became hypersensitive to the translational inhibitor anisomycin. The kinetoplasts and nuclei were misaligned in the postmitotic cells, resulting in partial cleavage furrow ingression during cytokinesis. Overall, these findings identify eEF1A as a novel TbRACK1 binding partner and establish TbRACK1 as a component of the trypanosome translational apparatus. The synergy between anisomycin and TbRACK1 RNAi suggests that continued translation is required for complete ingression of the cleavage furrow. 相似文献
7.
Because CaMKII is the critical Ca2+ sensor that triggers long-term potentiation (LTP), understanding its activation and deactivation is important. A major advance has been the development of a FRET indicator of the conformational state of CaMKII called Camui. Experiments using Camui have demonstrated that the open (active) conformation increases during LTP induction and then decays in tens of seconds, with the major fast component decaying with a time-constant of ~ 6 sec (tau1). Because this decay is faster if autophosphorylation of T286 is prevented (the autophosphorylation prolongs activity by making the enzyme active even after Ca2+ falls), it seemed likely that the fast decay is due to the T286 dephosphorylation. To test this interpretation, we studied the effect of phosphatase inhibitors on the single-spine Camui signal evoked by two-photon glutamate uncaging. We applied inhibitors of PP1 and PP2A, two phosphatases that are present at synapses and that have been shown to dephosphorylate CaMKII in vitro. The inhibitors increased the basal Camui activation state, indicating their effectiveness in cells. However, in no case did we find that tau1 was prolonged, contrary to what would be expected if the decay was phosphatase-dependent. This could either mean that decay was due to some unknown phosphatase or that the decay was not due to dephosphorylation. To distinguish between these possibilities, we expressed pseudo-phosphorylated Camui (T286D) (plus additional mutations [T/A] that prevented inhibitory 305/306 phosphorylation). This form had an elevated basal activation state, but was further activated during glutamate uncaging; importantly the activation state decayed with tau1 nearly the same as that of WT Camui. Therefore, the data strongly indicate that tau1 is not due to T286 dephosphorylation. We conclude that, although Camui is an excellent tool for observing CaMKII signaling, further experimentation is needed to determine how CaMKII is turned off by its dephosphorylation. 相似文献
8.
Informed Consent in Health Research: Challenges and Barriers in Low‐and Middle‐Income Countries with Specific Reference to Nepal
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Pramod R. Regmi Nirmal Aryal Om Kurmi Puspa Raj Pant Edwin van Teijlingen Sharada P. Wasti 《Developing world bioethics》2017,17(2):84-89
Obtaining ‘informed consent’ from every individual participant involved in health research is a mandatory ethical practice. Informed consent is a process whereby potential participants are genuinely informed about their role, risk and rights before they are enrolled in the study. Thus, ethics committees in most countries require ‘informed consent form’ as part of an ethics application which is reviewed before granting research ethics approval. Despite a significant increase in health research activity in low‐and middle‐income countries (LMICs) in recent years, only limited work has been done to address ethical concerns. Most ethics committees in LMICs lack the authority and/or the capacity to monitor research in the field. This is important since not all research, particularly in LMICs region, complies with ethical principles, sometimes this is inadvertently or due to a lack of awareness of their importance in assuring proper research governance. With several examples from Nepal, this paper reflects on the steps required to obtain informed consents and highlights some of the major challenges and barriers to seeking informed consent from research participants. At the end of this paper, we also offer some recommendations around how can we can promote and implement optimal informed consent taking process. We believe that paper is useful for researchers and members of ethical review boards in highlighting key issues around informed consent. 相似文献
9.
Savitha Kalidas Igor Cestari Severine Monnerat Qiong Li Sandesh Regmi Nicholas Hasle Mehdi Labaied Marilyn Parsons Kenneth Stuart Margaret A. Phillips 《Eukaryotic cell》2014,13(4):504-516
Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and β) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts. 相似文献