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71.
Megha Ghildiyal Jia Xu Hervé Seitz Zhiping Weng Phillip D. Zamore 《RNA (New York, N.Y.)》2010,16(1):43-56
In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character—as is found in small interfering RNAs (siRNAs)—directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide. 相似文献
72.
Meenakshi Gaur Carissa Ritner Rich Sievers Anissa Pedersen Megha Prasad Harold S. Bernstein Yerem Yeghiazarians 《Cytotherapy》2010,12(6):807-817
Background aimsHeart failure therapy with human embryonic stem cell (hESC)-derived cardiomyocytes (hCM) has been limited by the low rate of spontaneous hCM differentiation. As others have shown that p38 mitogen-activated protein kinase (p38MAPK) directs neurogenesis from mouse embryonic stem cells, we investigated whether the p38MAPK inhibitor, SB203580, might influence hCM differentiation.MethodsWe treated differentiating hESC with SB203580 at specific time-points, and used flow cytometry, immunocytochemistry, quantitative real-time (RT)–polymerase chain reaction (PCR), teratoma formation and transmission electron microscopy to evaluate cardiomyocyte formation.ResultsWe observed that the addition of inhibitor resulted in 2.1-fold enrichment of spontaneously beating human embryoid bodies (hEB) at 21 days of differentiation, and that 25% of treated cells expressed cardiac-specific α-myosin heavy chain. This effect was dependent on the stage of differentiation at which the inhibitor was introduced. Immunostaining and teratoma formation assays demonstrated that the inhibitor did not affect hESC pluripotency; however, treated hESC gave rise to hCM exhibiting increased expression of sarcomeric proteins, including cardiac troponin T, myosin light chain and α-myosin heavy chain. This was consistent with significantly increased numbers of myofibrillar bundles and the appearance of nascent Z-bodies at earlier time-points in treated hCM. Treated hEB also demonstrated a normal karyotype by array comparative genomic hybridization and viability in vivo following injection into mouse myocardium.ConclusionsThese studies demonstrate that p38MAPK inhibition accelerates directed hCM differentiation from hESC, and that this effect is developmental stage-specific. The use of this inhibitor should improve our ability to generate hESC-derived hCM for cell-based therapy. 相似文献
73.
Laser backscattering from biological tissues depends on their composition and blood flow. The onset of the tissue abnormalities is associated with the change in composition at a specific location which may affect laser backscattering. The objective of the present work is to detect the compositional changes in tissue-equivalent phantom of fat, prepared by mixing paraffin wax with wax colors, and to characterize these in terms of their optical parameters. For this purpose these phantoms are scanned by a multi-probe non-contact automatic laser scanning system and images of the normalized backscattered intensity (NBI) are constructed. By scanning the background subtracted image of the phantom the location of the abnormality and its size from the full width at half maximum (FWHM) are determined. The data obtained by ultrasonic technique for localization of the abnormalities are in agreement with that as obtained by the present method. The optical parameters of the abnormality are obtained by matching the measured surface profiles of the abnormality with that of the profile obtained by Monte Carlo simulation. This analysis shows the possibility of detection of changes at the onset stage in tissues as required for planning of the photodynamic therapy. 相似文献
74.
Parameet Kumar Manas K. Sen Devendra S. Chauhan Vishwa M. Katoch Sarman Singh Hanumanthappa K. Prasad 《PloS one》2010,5(4)
Background
The nonspecific clinical presentation and paucibacillary nature of tuberculous pleuritis remains a challenge for diagnosis. Diagnosis of tuberculous pleural effusion depends on the demonstration of the presence of tubercle bacilli in the sputum, pleural fluid, or pleural biopsy specimen, or demonstration of granuloma in pleura by histological examination. We examined the clinical utility of the diagnosis of pleural tuberculosis using the in house N-PCR assay, AFB smear microscopy and culture. Besides pleural fluid the inclusion of sputum in the efficacy of diagnosis of pleural tuberculosis was scrutinized.Methodology/Principal Findings
Pleural fluid and sputum samples of 58 tuberculous and 42 non-tuberculous pleural effusion patients were processed for AFB smear microscopy, culture and the N-PCR assay. Mycobacteria were detected exclusively in tuberculous pleural effusion samples. None of the non-tuberculous pleural effusion samples were positive for mycobacteria. Comparative analysis showed that the N-PCR assay had the highest sensitivity. Inclusion of sputum along with pleural fluid increased N-PCR sensitivity from 51.7 to 70.6% (p<0.0001).This improved sensitivity was reflected in AFB smear microscopy and isolation by culture. The sensitivity enhanced on inclusion of sputum from 3.4 (p = 0.50) to 10.3% (p = 0.038) for AFB smear microscopy and for isolation of mycobacteria from 10.3(p = 0.03) to 22.4% (p = 0.0005). Thirteen isolates were obtained from 58 pleural tuberculosis patients. Eleven mycobacterial isolates were identified as M.tuberculosis and two as M.fortuitum and M.chelonae. Complete concordance was seen between the biochemical identification of isolates and the N-PCR identification of mycobacterial species prior to isolation.Conclusions/Significance
To the best of our knowledge this is the first PCR based report on utility of sputum for diagnosis of pleural tuberculosis. The present study demonstrates that a combination of pleural fluid with sputum sample and N-PCR improved the diagnosis of pleural tuberculosis. 相似文献75.
Protein kinase C and mitogen-activated protein (MAP) kinase are expressed in all smooth muscle cells and believed to be important in several physiologically relevant properties of this muscle. Our goal was to determine if protein kinase C and MAP kinase are activated by a simple increase in cellular Ca(2+) and to determine if protein kinase C is an upstream activator of MAP kinase. These studies were performed in the Triton X-100 detergent-skinned preparation of the swine carotid artery, which allows control of the intracellular environment without influence from membrane or receptor-mediated modulation. The p42 and p44 isoforms of MAP kinase were activated in a concentration-dependent fashion by an increase in Ca2+. This was shown by in-the-gel kinase assay and direct measurement of MAP kinase phosphotransferase activity. Protein kinase C was also activated by an increase in Ca2+, as shown by a novel assay that measures total active protein kinase C in the tissue. Inhibition of protein kinase C activity completely abolished MAP kinase activity. Additionally, inhibition of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) also abolished MAP kinase activity. Using intact swine carotid arteries, we showed p42 and p44 MAP kinase to be activated by both histamine and phorbol dibutyrate, but only the p42 isoform was calcium-sensitive. Our results suggest that a Ca(2+)-dependent isoform of protein kinase C and CaM kinase II are upstream activators of MAP kinase in the swine carotid artery. 相似文献
76.
Three skeletal muscles viz., gastrocnemius, pectoralis and diaphragm from rats acclimated to a low temperature (4 +/- 1 degrees C; 16 hr daily; maximum for 8 weeks) exhibit an increased myosin ATPase activity. An analysis of native myosin from these muscles under non-dissociating conditions reveals two myosin isozymes instead of a single isozyme expressed in control muscles. Isoelectric focusing (IEF) coupled with two dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) confirms an increased phosphorylation of myosin light chain 2 (MLC2) in muscles from cold acclimated rats. 相似文献
77.
Leoncini L Del Vecchio MT Spina D Megha T Lazzi S Bellan C Tosi P 《Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology》1999,21(1):70-74
OBJECTIVE: To review the value of biopathologic factors in single lymphomatous patients across the boundaries of histologic classification. STUDY DESIGN: In a series of previous studies, based on a large collection of biopsy samples, the value of the above biopathologic characteristics in individual lymphomatous patients was quantitatively evaluated. RESULTS: The relationships between apoptotic index and growth fraction, in light of the expression of oncogenes, which regulate cell birth and death, were of particular value in determining the growth pattern of different lymphoma cases across the boundaries of histologic classification. CONCLUSION: The study of mechanisms that regulate cell proliferation and death might have therapeutic implications as the proper therapeutic approach should be based on detailed knowledge of the kinetic and molecular characteristics of each tumor. 相似文献
78.
Aerobic organisms experience oxidative stress due to generation of reactive oxygen species during normal aerobic metabolism.
In addition, several chemicals also generate reactive oxygen species which induce oxidative stress. Thus oxidative stress
constitutes a major threat to organisms living in aerobic environments. Programmed cell death or apoptosis is a physiological
mechanism of cell death, that probably evolved with multicellularity, and is indispensable for normal growth and development.Dictyostelium discoideum, an eukaryotic developmental model, shows both unicellular and multicellular forms in its life cycle and exhibits apparent
caspase-independent programmed cell death, and also shows high resistance to oxidative stress. An attempt has been made to
investigate the biochemical basis for high resistance ofD. discoideum cell death induced by different oxidants. Dose-dependent induction of cell death by exogenous addition of hydrogen peroxide
(H2O2),in situ generation of H2O2 by hydroxylamine, and nitric oxide (NO) generation by sodium nitroprusside treatment inD. discoideum were studied. The AD50 doses (concentration of the oxidants cusing 50% of the cells to die) after 24 h of treatment were found to be 0.45 mM, 4
mM and 1 mM, respectively. Studies on enzymatic antioxidant status ofD. discoideum when subjected to oxidative stress, NO and nutrient stress reveal that superoxide dismutase and catalase were unchanged;
a significant induction of glutathione peroxidase was observed. Interestingly, oxidative stress-induced lipid membrane peroxidative
damage could not be detected. The results shed light on the biochemical basis for the observed high resistance to oxidative
stress inD. discoideum. 相似文献
79.
Parashar D Chauhan DS Sharma VD Chauhan A Chauhan SV Katoch VM 《Applied and environmental microbiology》2004,70(6):3751-3753
For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria. 相似文献
80.
Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB 总被引:2,自引:0,他引:2
Chopra P Singh B Singh R Vohra R Koul A Meena LS Koduri H Ghildiyal M Deol P Das TK Tyagi AK Singh Y 《Biochemical and biophysical research communications》2003,311(1):112-120
The regulation of cellular processes by the modulation of protein phosphorylation/dephosphorylation is fundamental to a large number of processes in living organisms. These processes are carried out by specific protein kinases and phosphatases. In this study, a previously uncharacterized gene (Rv0018c) of Mycobacterium tuberculosis, designated as mycobacterial Ser/Thr phosphatase (mstp), was cloned, expressed in Escherichia coli, and purified as a histidine-tagged protein. Purified protein (Mstp) dephosphorylated the phosphorylated Ser/Thr residues of myelin basic protein (MBP), histone, and casein but failed to dephosphorylate phospho-tyrosine residue of these substrates, suggesting that this phosphatase is specific for Ser/Thr residues. It has been suggested that mstp is a part of a gene cluster that also includes two Ser/Thr kinases pknA and pknB. We show that Mstp is a trans-membrane protein that dephosphorylates phosphorylated PknA and PknB. Southern blot analysis revealed that mstp is absent in the fast growing saprophytes Mycobacterium smegmatis and Mycobacterium fortuitum. PknA has been shown, whereas PknB has been proposed to play a role in cell division. The presence of mstp in slow growing mycobacterial species, its trans-membrane localization, and ability to dephosphorylate phosphorylated PknA and PknB implicates that Mstp may play a role in regulating cell division in M. tuberculosis. 相似文献