Academic literature on the topic 'Kinetics of mRNAs expression'
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Journal articles on the topic "Kinetics of mRNAs expression"
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Hao, Shengli, and David Baltimore. "RNA splicing regulates the temporal order of TNF-induced gene expression (167.5)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 167.5. http://dx.doi.org/10.4049/jimmunol.188.supp.167.5.
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Abstract When cells are induced to express inflammatory genes by treatment with TNF the mRNAs for the induced genes appear in three distinct waves, defining gene Groups I, II and III or early, intermediate and late genes. To examine the basis for these different kinetic classes, we developed a PCR-based procedure to distinguish pre-mRNA synthesis from mRNA synthesis. It showed that the three Groups initiate transcription virtually simultaneously but that delays in splicing characterize Groups II and III. We examined the elongation times and polyadenylation timing, concluding that pre-mRNA synthesis is coordinate but splicing differences directly regulate the timing of mRNA production. Coupled to previous measurements of the half-life of mRNAs, the kinetic parameters can be combined in a general mathematical model of pre-mRNA conversion to mRNA that reproduces the experimental kinetics without recourse to differences in transcription.This model could be a generally relevant description of sequential transcriptional events in response to inducers.
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Bette, M., M. K. Schäfer, N. van Rooijen, E. Weihe, and B. Fleischer. "Distribution and kinetics of superantigen-induced cytokine gene expression in mouse spleen." Journal of Experimental Medicine 178, no. 5 (November 1, 1993): 1531–39. http://dx.doi.org/10.1084/jem.178.5.1531.
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The polyclonal stimulation of T cells by bacterial superantigens is involved in the pathogenesis of the toxic shock syndrome in certain staphylococcal and streptococcal infections. Here we describe the onset and kinetics of superantigen-induced cytokine production in situ in spleens of normal BALB/c mice monitored at the level of cytokine mRNA expression by in situ hybridization. Messenger RNAs for interleukin 2 (IL-2), interferon gamma, and tumor necrosis factors (TNF) alpha and beta were not expressed at detectable levels in spleens of unstimulated animals but became visible already 30 min after intraperitoneal application of 50 micrograms staphylococcal enterotoxin B. All mRNA levels showed peak expression approximately 3 h after injection and a slow decrease up to 24 h after injection. Expression of the mRNAs was restricted to the T cell-dependent area of the periarteriolar lymphatic sheets of the spleen. Interestingly, TNF-alpha mRNA showed a biphasic response, the early appearing mRNA had the same localization as the other mRNAs, whereas after 3 h TNF-alpha mRNA showed a broader distribution indicating a second cell population producing TNF-alpha. The expression of IL-2 and TNF proteins in the serum increased in parallel to the observed mRNA changes with a slight delay. The presence of macrophages was not required for the expression of the cytokine mRNAs in the spleen as the expression was unchanged in macrophage-depleted mice. Only the second phase of TNF-alpha mRNA expression was abrogated in such animals. The expression of all mRNAs was completely suppressed by prior administration of cyclosporin A. These data show that nonphagocytic cells are the essential superantigen-presenting cells in vivo and indicate that at least part of the pathogenetic TNF-alpha is T cell derived.
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Granelli-Piperno, A., L. Andrus, and R. M. Steinman. "Lymphokine and nonlymphokine mRNA levels in stimulated human T cells. Kinetics, mitogen requirements, and effects of cyclosporin A." Journal of Experimental Medicine 163, no. 4 (April 1, 1986): 922–37. http://dx.doi.org/10.1084/jem.163.4.922.
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Northern and dot blotting with a panel of DNA probes were used to monitor the levels of specific mRNAs in mitogen-stimulated human T cells. The induction of IL-2 and IFN mRNAs required the synergistic action of PMA and either PHA or OKT3 mAb. In contrast, several nonlymphokine genes, the protooncogenes c-fos and c-myc, and the IL-2-R gene, were induced by either PHA or PMA alone. PHA increased the background levels of a 70 kD heat shock protein mRNA, but did not affect the observed background of c-myb mRNA. For all mRNAs that were induced, isolated CD4 and CD8 T cell subsets behaved similarly. Exogenous IL-2 had little (IFN) or no (IL-2) effect on lymphokine mRNAs, but significantly increased c-myc, IL-2-R and heat shock protein mRNAs. Therefore, the stimuli for lymphokine mRNAs differed from those required for several inducible nonlymphokine genes. IL-2 and IFN mRNAs exhibited some important similarities with c-myc, however. The levels of IL-2, IFN, and c-myc mRNA followed similar kinetics, peaking at 3 h in restimulated blasts and at 12 h in unstimulated T cells. The subsequent downregulation of lymphokine and c-myc mRNAs was retarded by cycloheximide. The induction of IL-2, IFN, and c-myc mRNAs was blocked by the immunosuppressive drug CsA, but not by the inactive analog CsH, and this block occurred at the level of nuclear transcription. Since the exogenous stimuli for lymphokine and c-myc gene expression differ, we suggest that intracellular controls must be shared to account for the similarities in their kinetics of expression and CsA sensitivity.
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Liu, C. C., S. Rafii, A. Granelli-Piperno, J. A. Trapani, and J. D. Young. "Perforin and serine esterase gene expression in stimulated human T cells. Kinetics, mitogen requirements, and effects of cyclosporin A." Journal of Experimental Medicine 170, no. 6 (December 1, 1989): 2105–18. http://dx.doi.org/10.1084/jem.170.6.2105.
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A pore-forming protein (PFP; perforin) and various serine esterases (SE) have been identified in the cytoplasmic granules of CTL and NK cells. Perforin and several SE have recently been cloned. Northern blotting analysis was performed here using cDNA probes specific for human perforin and two SE (SE 1/HS and SE 2/GB) to monitor the levels of specific mRNAs in mitogen-stimulated primary human T cells. These mRNAs were rapidly induced by IL-2 with optimal responses at 300 U/ml. After IL-2 treatment, mRNAs for perforin, SE 1, and SE 2 peaked at 12-24 h and decreased after 48 h. The three mRNAs were also induced in T cells treated with a combination of PMA plus lectin, OKT3 mAb, or plastic-adherent accessory cells. However, the induction induced by PMA/mitogen followed a slower kinetics, peaking at 48 h. In general, we found that SE 1 mRNA was more readily induced by IL-2, while SE 2 responded better to PMA/mitogen. Similar patterns of mRNA expression were observed for both unprimed T cells and PHA-primed T blasts. After stimulation with IL-2 and PMA/mitogen, the T8+ subset was shown to be the main producer of perforin, SE 1, and SE 2. Low levels of all three mRNAs, however, were also detected in the T4+ subset. The induction of all three mRNAs by either IL-2 or PMA/mitogen was partially blocked by the immunosuppressive drug cyclosporin A (CsA), but not by the biologically inactive analogue cyclosporin H. Together, these results point to some similarities and differences with upregulation of granule mediator mRNAs relative to lymphokine mRNAs. Both sets of genes require two signals for their induction by mitogens. In contrast to lymphokines, there is a strong response of granule mRNAs to IL-2, and the induction of these transcripts is only partially blocked by CsA.
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Barton, P. J., A. J. Harris, and M. E. Buckingham. "Myosin light chain gene expression in developing and denervated fetal muscle in the mouse." Development 107, no. 4 (December 1, 1989): 819–24. http://dx.doi.org/10.1242/dev.107.4.819.
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We have investigated the accumulation of mRNA transcripts of the atrial (or embryonic) myosin light chain MLC1A (MLC1emb), and the two adult fast muscle myosin light chains (MLC1F and MLC3F) during fetal skeletal muscle development in the mouse. In 15-day fetal muscle, MLC1A is the predominant mRNA detectable, by 18 days MLC1F has become the major transcript and MLC3F mRNA is detectable for the first time. By 12 days after birth, MLC1A transcripts are undetectable and MLC1F and MLC3F are similar in abundance. In fetuses treated with beta-bungarotoxin and which therefore develop in the absence of functional nerve, MLC1A and MLC1F undergo normal transitions but MLC3F mRNA accumulation is significantly retarded. This demonstrates that these myosin light chain mRNAs accumulate with differing kinetics, and that MLC3F mRNA accumulation is nerve-dependent during fetal development. The results are discussed in terms of secondary muscle fibre formation, and in relation to the independent regulation of MLC1F and MLC3F mRNAs which are transcribed from the same gene.
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Rende, Francesca, Ilaria Cavallari, Alberto Corradin, Micol Silic-Benussi, Frederic Toulza, Gianna M. Toffolo, Yuetsu Tanaka, et al. "Kinetics and intracellular compartmentalization of HTLV-1 gene expression: nuclear retention of HBZ mRNAs." Blood 117, no. 18 (May 5, 2011): 4855–59. http://dx.doi.org/10.1182/blood-2010-11-316463.
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AbstractHuman T-cell leukemia virus type 1 (HTLV-1) codes for 9 alternatively spliced transcripts and 2 major regulatory proteins named Tax and Rex that function at the transcriptional and posttranscriptional levels, respectively. We investigated the temporal sequence of HTLV-1 gene expression in primary cells from infected patients using splice site-specific quantitative RT-PCR. The results indicated a two-phase kinetics with the tax/rex mRNA preceding expression of other viral transcripts. Analysis of mRNA compartmentalization in cells transfected with HTLV-1 molecular clones demonstrated the strict Rex-dependency of the two-phase kinetics and revealed strong nuclear retention of HBZ mRNAs, supporting their function as noncoding transcripts. Mathematical modeling underscored the importance of a delay between the functions of Tax and Rex, which was supported by experimental evidence of the longer half-life of Rex. These data provide evidence for a temporal pattern of HTLV-1 expression and reveal major differences in the intracellular compartmentalization of HTLV-1 transcripts.
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Snaar, Sabine P., Pauline Verdijk, Hans J. Tanke, and Roeland W. Dirks. "Kinetics of HCMV immediate early mRNA expression in stably transfected fibroblasts." Journal of Cell Science 115, no. 2 (January 15, 2002): 321–28. http://dx.doi.org/10.1242/jcs.115.2.321.
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Compelling evidence supports an intimate link in time and space between eukaryotic pre-mRNA synthesis and processing and nucleocytoplasmic transport of mature mRNA. In this study, we analyzed the kinetic behavior of these processes in a quantitative manner. We used FISH and confocal scanning laser microscopy to detect transcripts produced by an inducible human cytomegalovirus immediate early (HCMV-IE) expression system. Upon induction, a large amount of pre-mRNA accumulated in nuclear foci at or near their transcription sites and, at later time, throughout the nucleoplasm. Inhibition of RNA polymerase II activity resulted in a rapid decrease in the number of transcripts in the nuclear RNA foci (half time ∼two minutes), indicating that accumulated transcripts were rapidly spliced and then released. The dispersed nucleoplasmic transcripts exited the nucleus with a half time of ∼10 minutes. Both processes were temperature dependent, suggesting that mRNA export is an active process. RNA polymerase II activation revealed that production of mature HCMV IE mRNAs required less than five minutes. Transcripts radiated from the gene at an average speed of ∼0.13 μm2/sec from this time on. Thus, it appears that these processes are tightly linked in time and space, with the splicing reaction as a rate-limiting factor.
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Feng, Pinghui, David N. Everly, and G. Sullivan Read. "mRNA Decay during Herpesvirus Infections: Interaction between a Putative Viral Nuclease and a Cellular Translation Factor." Journal of Virology 75, no. 21 (November 1, 2001): 10272–80. http://dx.doi.org/10.1128/jvi.75.21.10272-10280.2001.
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ABSTRACT During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating mRNA decay, it helps determine the levels and kinetics of viral and cellular gene expression. In vivo, Vhs shows a strong preference for mRNAs, as opposed to non-mRNAs, and degrades the 5′ end of mRNAs prior to the 3′ end. In contrast, partially purified Vhs is not restricted to mRNAs and causes cleavage of target RNAs at various sites throughout the molecule. To explain this discrepancy, we searched for cellular proteins that interact with Vhs using theSaccharomyces cerevisiae two-hybrid system. Vhs was found to interact with the human translation initiation factor, eIF4H. This interaction was verified by glutathioneS-transferase pull-down experiments and by coimmunoprecipitation of Vhs and epitope-tagged eIF4H from extracts of mammalian cells. The interaction was abolished by several point mutations in Vhs that abrogate its ability to degrade mRNAs in vivo. The results suggest that Vhs is a viral mRNA degradation factor that is targeted to mRNAs, and to regions of translation initiation, through an interaction with eIF4H.
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Zhao, Renbin, Kurt Gish, Maureen Murphy, Yuxin Yin, Daniel Notterman, William H. Hoffman, Edward Tom, David H. Mack, and Arnold J. Levine. "Analysis of p53-regulated gene expression patterns using oligonucleotide arrays." Genes & Development 14, no. 8 (April 15, 2000): 981–93. http://dx.doi.org/10.1101/gad.14.8.981.
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Oligonucleotide microarrays were employed to quantitate mRNA levels from a large number of genes regulated by the p53 transcription factor. Responses to DNA damage and to zinc-inducible p53 were compared for their transcription patterns in cell culture. A cluster analysis of these data demonstrates that genes induced by γ radiation, UV radiation, and the zinc-induced p53 form distinct sets and subsets with a few genes in common to all these treatments. Cell type- or cell line-specific p53 responses were detected. When p53 proteins were induced with zinc, the kinetics of induction or repression of mRNAs from p53-responsive genes fell into eight distinct classes, five different kinetics of induction, and three different kinetics of repression. In addition, low levels of p53 in a cell induced or repressed only a subset of genes observed at higher p53 levels. The results of this study demonstrate that the nature of the p53 response in diverse mRNA species depends on the levels of p53 protein in a cell, the type of inducing agent or event, and the cell type employed. Of 6000 genes examined for p53 regulatory responses, 107 induced and 54 repressed genes fell into categories of apoptosis and growth arrest, cytoskeletal functions, growth factors and their inhibitors, extracellular matrix, and adhesion genes.
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Wu, X., G. J. Dolecki, and J. B. Lefkowith. "GRO chemokines: a transduction, integration, and amplification mechanism in acute renal inflammation." American Journal of Physiology-Renal Physiology 269, no. 2 (August 1, 1995): F248—F256. http://dx.doi.org/10.1152/ajprenal.1995.269.2.f248.
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We recently observed that cytokine-induced neutrophil chemoattractant (CINC), a GRO chemokine, contributes to neutrophil migration into the inflamed glomerulus in rat. Therefore, we sought to clarify how expression of the GRO chemokines, CINC and macrophage inflammatory protein-2 (MIP-2), is regulated in mesangial cells in vitro and the kidney in vivo. Mesangial cells expressed both GRO chemokine mRNAs in response to mediators of acute renal inflammation [interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and lipopolysaccharides (LPS)], but not chronic renal inflammation (transforming growth factor-beta 1), with CINC mRNA expression predominating over MIP-2. The kinetics of GRO chemokine mRNA expression in response to both IL-1 beta and TNF-alpha (but not LPS) paralleled those defined for polymorphonuclear leukocyte (PMN) migration during nephritis in vivo. IL-1 beta and TNF-alpha displayed nonparallel concentration-response relationships for GRO chemokine mRNA expression, and together were synergistic together rather than additive. Expression of GRO chemokine mRNAs in response to both cytokine agonists, however, was inhibited by genistein, a tyrosine kinase inhibitor. GRO chemokine mRNAs were rapidly expressed in inflamed glomeruli during immune complex glomerulonephritis with MIP-2 predominating over CINC. Expression of both chemokines was substantially inhibited by complement, leukocyte, and PMN depletion. In sum, GRO chemokines are expressed coordinately by mesangial cells and inflamed glomeruli and appear both to transduce the response to mediators of acute inflammation into a chemotactic signal and to amplify this response both temporally and quantitatively.(ABSTRACT TRUNCATED AT 250 WORDS)
Dissertations / Theses on the topic "Kinetics of mRNAs expression"
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BENDER, Cecilia. "Study of Human T-Lymphotropic Virus type 2 mRNA kinetics of expression and identification of a novel splicing site." Doctoral thesis, Università degli Studi di Verona, 2010. http://hdl.handle.net/11562/343864.
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L’analisi di espressione dei retrovirus Human T-cell lymphotropic viruses (HTLV-1 e 2) durante il processo di infezione è essenziale per capire l’influenza che i prodotti dei geni virali hanno sui processi di proliferazione, ciclo e signalling cellulari.
Studi recenti condotti su cellule infettate con HTLV-1 hanno ermesso di delineare la cinetica di espressione dei diversi trascritti virali. Per quanto concerne HTLV-2, il profilo di espressione degli mRNA virali in cellule infettate risulta ancora poco studiato e le relative cinetiche non sono state ancora delineate.
Scopo di questo studio è stato quindi quello di analizzare l’espressione dei diversi trascritti di HTLV-2 mediante la messa a punto di analisi real time RT-PCR per la loro determinazione quantitativa. In particolare sono state studiate e quantificate le
cinetiche di espressione dei trascritti di HTLV-2 in linee cellulari infettate e in linfociti da sangue periferico (PBMC) provenienti da pazienti infetti.
In modo analogo ad altri retrovirus, HTLV-2 esprime diversi prodotti genici dalla stessa regione codificante, attraverso diverse strategie che sottointendono un complesso pattern di splicing alternativo. L’espressione di HTLV-2 è basata su tre principali classi di mRNA: a) “unspliced” mRNA, che codifica per le proteine Gag, Pol e Proteasi; b) “singly” spliced mRNA che codifica per Env e per le proteine accessorie p28 e p22/p20; c) “doubly spliced” mRNA che codifica per le proteine Tax, Rex e le
proteine accessorie p10, p11 ed 1-2-B. Recentemente inoltre è stato descritto un nuovo prodotto proteico, APH-2, che viene codificato dallo strand negativo di HTLV-2 e sembra essere coinvolto nei processi di silenziamento trascrizionale del virus in cellule infettate. Il profilo di espressione e le cinetiche dei diversi trascritti virali sono stati quantificati mediante la metodica di Real Time RT-PCR, utilizzando sonde e primers specifici per le giunzioni di splicing. Il profilo temporale di ogni trascritto di HTLV-2 è stato studiato utilizzando tre diversi sistemi cellulari: linee stabilmente infettate, cellule trasfettate in modo transiente, e infine PBMC provenienti da soggetti infettati da HTLV-
2B messi in colturea e stimolati con IL-2.
I risultati ottenuti in questo studio hanno portato alla quantificazione di tutti i trascritti virali di HTLV-2. Sono stati inizialmente riscontrati diversi livelli di espressione di env nei due sottotipi HTLV-2 A e B. La diversa espressione del trascritto
env in linee cellulari stabilente infettate, ha indotto ad approfondire il complesso meccanismo di splicing tra gli esoni 1 e 2, portando all’identificazione di un nuovo sito accettore di splicing (3’SS). Si è potuto così determinare che questo nuovo sito
accettore viene utilizzato nell’espressione del trascritto env “singly splicing” e dei trascritti bicistronici tax/rex, p10/p11 e 1-2-B “doubly splicing” derivati da splicing alternativo all’interno della regione genica pX di HTLV-2. I risultati sperimentali hanno
quindi permesso di evidenziare che il nuovo sito 3’SS è presente in entrambi i sottotipi A e B di HTLV-2 e che la nuova isoforma di env, chiamata env 1-2b, è preferenzialmente espressa nel sottotipo 2B mentre nel sottotipo 2A l’espressione di env
utilizza più efficientemente il sito canonico di splicing.
L’analisi della cinetica di espressione del mRNA totale, ha mostrato un andamento caratterizzato da una bassa trascrizione iniziale, seguita da un aumento con un progressivo picco di espressione dopo 21-24 ore dall’inizio della coltura.
Il trascritto “full lenght” gag/pol risulta essere il più espresso durante il periodo analizzato, comportandosi come un gene tardivo e raggiungendo quindi un picco di espressione dopo 21-48 ore. L’env canonico è espresso a livelli molto bassi nelle linee
stabilmente infettate e non e’ rilevabile in PBMC di pazienti infettati da HTLV-2B. Al contrario, l’isoforma env 1-2b viene efficientemente espressa, sia in linee cellulari che in PBMC ex-vivo e presenta un profilo di espressione tardivo seguito da un graduale e costante aumento, raggiungendo il picco massimo di espressione tra 21 e 48 ore. Infine il trascritto di regolazione tax/rex viene espresso precocemente da HTLV-2B sia in linee
stabilmente infettate che in ex-vivo PBMC.
Le cinetiche del trascritto 1-2-B indicano che questo gene viene espresso ad alti livelli negli intervalli di tempo precoci mentre il mRNA per le proteine accessorie p10 e p11 risulta essere poco espresso o inferiore alla soglia di detezione. Tra gli mRNA che
subiscono “singly splicing” e che codificano per le proteine accessorie, l’isoforma p28,p22/p20-II è maggiormente espressa nei due sottotipi HTLV-2A e B rispetto all’isoforma p28, p22/p20-I.
Il trascritto APH-2 viene espresso ad alti livelli sia in cellule stabilmente infettate che in cellule trasfettate e presenta un andamento tardivo in questi sistemi cellulari. Tuttavia nei PBMC ottenuti da pazienti infetti non e' stato possibile determinarne un chiaro pattern di espressione in quanto questo trascritto risulta essere molto variabile.
Questo studio ha messo in evidenza che i trascritti di HTLV-2 presentano cinetiche di espressione specifiche e diversificate. I risultati ottenuti hanno permesso di descrivere un preciso pattern di produzione temporale degli mRNA, con geni precoci quali tax/rex e 1-2-B e geni tardivi quali gag/pol, env e 28,p22/p20-II. L’espressione precoce di tax/rex indica che questa proteina è necessaria nelle fasi iniziali del ciclo virale al fine di transattivare e regolare i geni virali e cellulari. I geni strutturali gag/pol
e env sono invece espressi tardivamente quando i livelli d’espressione dei geni precoci sono in fase di dimunuizione, il che indica quindi un chiaro “switch” temporale tra geni precoci e tardivi. Questo studio ha inoltre evidenziato un nuovo sito accettore di splicing alternativo nella regione pX di HTLV-2.
In conclusione, questo studio ha permesso di stabilire che i livelli dei geni di HTLV-2 e le loro cinetiche di espressione sono strettamente regolati. Inoltre, queste ricerche hanno messo in evidenza che l’utilizzo di nuovi siti accettori gioca un ruolo
fondamentale nell’ espressione preferenziale di proteine virali specifiche alle diverse fasi del ciclo virale di HTLV-2.The analysis of HTLV expression during the infection process is essential to understand the influence of viral gene products on proliferation, cell cycle and signalling. Recent studies carried out on HTLV-1-infected cells have provided information on the relative abundance and timing of expression of different viral transcripts. In the case of HTLV-2, very little information has been so far obtained on the profile of viral gene expression in infected cells, and the kinetics of transcript expression have not yet been investigated. The aim of the study was to further investigate HTLV-2 transcripts expression, by developing new quantitative analyses to measure the levels of expression of different HTLV-2 mRNAs. In particular the kinetics of HTLV-2 transcripts at different stages of virus gene expression and their quantitation in infected cell lines and in PBMCs from infected subjects, have been evaluated. Similarly to other retroviruses, HTLV-2 expresses multiple gene products from the same coding region by choosing different strategies, including a complex pattern of alternative splicing. HTLV-2 expression produces three major classes of mRNAs: unspliced mRNA for Gag, Protease and Pol proteins; singly spliced mRNAs for Env and accessory proteins p28 and p22/p20; and doubly spliced mRNAs for the regulatory Tax, Rex and the accessory p10, p11 and 1-2-B proteins. More recently, the new APH-2 protein coded by the negative strand of HTLV-2, and possibly involved in the transcriptional silencing of the virus in infected cells was described. The expression profile and kinetics of the different transcripts were analysed by RT-PCR assays using a series of splice-junction-specific primers and probes. The time courses of each HTLV-2 transcript in three different cellular systems, namely stably infected cells lines, transiently transfected cells and ex-vivo IL-2 stimulated PBMCs from infected subjects were investigated. The results obtained led to the quantitation of all known HTLV-2 mRNAs. Preliminary data showed different levels of env transcript in HTLV-2 subtypes A and B. Evidence for a differential env expression in HTLV-2B stably infected cells, prompted further investigation on the complex pattern of splicing between exons 1 and 2 and allowed the identification of a novel 3’ acceptor site (SS) of splicing. This novel 3’SS is used to express alternative spliced isoforms within the pX terminal region of HTLV-2, including the singly spliced env and the three doubly spliced bicistronic tax/rex, p10/p11 and 1-2-B transcripts. Results demonstrated that the novel 3’SS was utilised in both HTLV-2 subtypes, and that the novel env isoform, named env 1-2b, was preferentially expressed in 2B subtypes, while 2A subtypes used more efficiently the canonical splice site. The kinetics of total mRNA HTLV-2 expression in these three cellular systems presented a general pattern characterised by an initial low transcript level and a subsequent increase to reach a peak of expression after 21-24 hours in culture. The full length gag/pol mRNA was the most abundant one during the time course, and behaved as a late gene that peaked after 21 to 48 hours. The canonical env transcript was expressed at very low rates in stably infected cells, and it was undetectable in PBMCs from infected subjects and in cells transfected with the HTLV-2 proviral clone. By contrast, env 1-2b isoform was efficiently expressed in stably infected cells as well as in ex-vivo infected PBMCs, behaving as a late gene showing a gradual and steady increase in copy number and reaching maximum expression at 21 to 48 hours. The regulatory tax/rex gene was expressed early and with a high/intermediate transcription rate in HTLV-2B subtypes in both stably infected cells and ex-vivo PBMCs. In this study the kinetics of expression of the yet unkwnon 1-2-B gene was investigated and found to be expressed at high levels at early time points, whereas the doubly spliced mRNA of accessory p10/p11 genes were poorly expressed or under detection limit. Among the singly spliced mRNAs coding for other accessory proteins, the p28, p22/p20-II isoform was found to be highly expressed in both HTLV-2 A and B subtypes as compared to its alternative p28, p22/p20-I form. The APH-2 negative transcript was efficiently expressed at high levels in both stably infected and transfected cells and behaved as a late gene. However, in ex-vivo PBMCs its expression level and kinetics pattern appeared to be variable and a clear pattern of expression was not assessed. In conclusion, this study demonstrated that HTLV-2 transcripts of both A and B subtypes are differentially expressed. A temporal pattern of mRNA production, with early, tax/rex and 1-2-B and late, gag/pol, env, p28,p22/p20-II genes was established. The tax/rex early expression indicates that this protein is necessary at the beginning of the viral cycle to transactivate and regulate viral and cellular genes. Most structural genes were expressed late when the transcription of early genes was already decreasing, thus showing that a temporal switch was occurring between early to late genes production. This study also provided new clues on the selective use of alternative 3’SS within the pX region of HTLV-2 for both subtypes A and B. Overall these findings indicate that the control of HTLV-2 viral gene expression is highly regulated both in its kinetics and expression. Moreover, it suggests that the use of multiple acceptor sites might play an important role on the preferential expression of specific proteins in the different phases of the viral cycle.
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Taylor, David C. "SELEX targeting mRNAs : the hunt for novel riboregulators /." free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3013032.
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Macdonald, Murdo. "Expression of mRNAs encoding FMRFamide-related peptides in adult and embryo Helix aspersa." Thesis, University of St Andrews, 1993. http://hdl.handle.net/10023/14454.
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The gastropod mollusc Helix aspersa is known to contain at least seven FMRFamide-related peptides (FaRPs), neuropeptides which fall into two broad classes, distinguished by their primary structure and their physiological actions. We have sought to use the techniques available to us through molecular biology to study the structure and expression of the nucleic acids (RNA and DNA) which encode these peptides in this organism. The two classes of peptide, tetra- and heptapeptides, were found by us to be apparently separated by the stage of mRNA generation: the precursor polypeptides encoded by these mRNAs were also found to have differing structures. Expression of mRNAs specific for the FaRPs were studied during embryogenesis, where there appears to be regulated expression of these mRNAs. In situ hybridization analyses of the central nervous system of adult Helix revealed expression of FaRP-specific mRNAs to be limited to a small number of discrete neurons: it was again observed that there was an apparent distinction between the tetra and extended FaRPs, no ceils being identified in our studies to express both types of mRNA. Confocal scanning microscopy indicated that the distribution of the mRNA, which appeared to be limited to the cytoplasm of cell bodies expressing the peptides, was non-uniform, probably reflecting a functional characteristic of the cells concerned.
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Drummond, D. R. "The stability, movement and expression of natural and synthetic mRNAs injected into Xenopus oocytes." Thesis, University of Warwick, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.373064.
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Chang, Chih Chien Anne. "Developmental expression of GABAA[subscript]/BZ receptor subunit mRNAs in olivocerebellar circuitry of the mouse /." The Ohio State University, 1994. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487848891512785.
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Rende, Francesca. "Kinetics and regulation of HTLV-1 gene expression." Doctoral thesis, Università degli studi di Padova, 2011. http://hdl.handle.net/11577/3421976.
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ABSTRACT
Human T-Lymphotropic virus type 1 (HTLV-1) is the causative agent of two distinct pathologies, adult T-cell leukemia/lymphoma (ATLL), an aggressive malignancy of mature CD4+ T-cells, and tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM), a demyelinating neurodegenerative disease.
The HTLV-1 expression strategy is characterized by the production of plus- and minus-strand transcripts, alternative splicing and polycistronic translation. This strategy greatly increases the coding potential of the virus, resulting in expression of several regulatory and accessory genes (Tax, Rex, p12, p13, p21rex, p30tof and HBZ) in addition to the structural proteins and virion-associated enzymes common to all retroviruses (Gag, Pro, Pol and Env).
In spite of over 30 years of studies, several key features of the HTLV-1 life cycle and pathogenicity remain obscure. In particular, it is still unclear whether HTLV-1 gene expression is characterized by latency patterns, whether the different viral genes follow distinct kinetics of expression and, if this is the case, which molecular mechanisms control these phenomena.
The work described in the present thesis was aimed at understanding these aspects of HTLV-1 regulation. To this end we optimized a Real Time RT-PCR method using splice-site-specific primers to quantitate the different HTLV-1 transcripts and their kinetics of expression in peripheral blood mononuclear cells (PBMCs) isolated from HTLV-1-infected individuals and in cells transfected with HTLV-1 molecular clones. Results indicated that expression of HTLV-1 mRNAs follows a distinct timing upon reactivation of viral expression, with the mRNA coding for the Tax and Rex regulatory proteins acting as an early "master" transcript preceding expression of the other viral transcripts.
Although it is commonly accepted that Rex acts at a post-transcriptional level controlling the nuclear export and stability of viral mRNAs coding for the virion-associated proteins, the Rex-dependency of tax/rex, p12, p13, p21rex, p30tof and hbz transcripts has not been investigated so far. To test if the kinetics of HTLV-1 gene expression might be dependent on Rex function and to determine the Rex-dependence of individual HTLV-1 mRNAs, we generated a Rex knock-out HTLV-1 molecular clone and analyzed the nucleo-cytoplasmic compartmentalization of the viral mRNAs. Results demonstrated the strict Rex-dependency of the “two-phase” kinetics and revealed strong nuclear retention of hbz mRNAs, supporting their function as non-coding transcripts. Furthermore our results revealed that the Rex-responsiveness of the different HTLV-1 mRNAs is determined by a novel 72-nucleotides cis-acting regulatory sequence located upstream of exon 3.
Mathematical modelling underscored the importance of a temporal delay between the Tax and Rex functions, which was supported by experimental evidence of a delayed accumulation and longer half-life of Rex compared to Tax.
These data provide evidence for a temporal pattern of HTLV-1 gene expression, reveal major differences in the intracellular compartmentalization of HTLV-1 transcripts and, importantly, provide clues to a long-standing paradox of HTLV-1 regulation, i.e. the different Rex-dependence of viral transcripts in spite of the presence of the Rex-responsive element (RxRE) in the 3' untranslated region of all viral mRNAs.RIASSUNTO Il virus T-linfotropico umano di tipo 1 (HTLV-1) è l’agente eziologico di due distinte patologie, la leucemia/linfoma a cellule T dell’adulto (ATLL, adult T-cell leukemia/lymphoma), un'aggressiva neoplasia a carico dei linfociti T CD4+ maturi, e della paraparesi spastica tropicale/mielopatia associata ad HTLV-1 (TSP/HAM, tropical spastic paraparesis/HTLV-1-associated myelopathy), una patologia degenerativa del sistema nervoso centrale. La strategia di espressione genica di HTLV-1, caratterizzata dalla produzione di trascritti a partire da promotori localizzati sia nel filamento positivo che in quello negativo del genoma virale, da splicing alternativo e da traduzione bicistronica, incrementa notevolmente la capacità codificante di HTLV-1, con la conseguente espressione di numerosi geni regolatori ed accessori (Tax, Rex, p12, p13, p21rex, p30tof e HBZ) in aggiunta alle proteine strutturali e agli enzimi associati al virione, comuni a tutti i retrovirus (Gag, Pro, Pol ed Env). Nonostante oltre 30 anni di studi, diversi aspetti chiave del ciclo vitale di HTLV-1 e della sua patogenicità rimangono tutt'oggi non noti. In particolare, non è ancora chiaro se l'espressione genica di HTLV-1 sia caratterizzata da stadi di latenza, se i diversi geni virali presentino cinetiche di espressione distinte e quali meccanismi molecolari possano controllare questi fenomeni. Gli studi descritti nella presente tesi sono stati mirati a comprendere questi aspetti della regolazione genica di HTLV-1. A questo scopo abbiamo sviluppato un protocollo di Real Time RT-PCR associato all'impiego di primer specifici per le diverse giunzioni di splicing al fine di quantificare i diversi trascritti codificati da HTLV-1 e di analizzarne le cinetiche di espressione sia in cellule mononucleate di sangue periferico isolate da individui infettati con HTLV-1, che in cellule trasfettate con cloni molecolari di HTLV-1. I risultati ottenuti indicano che l'espressione degli mRNA codificati da HTLV-1 segue una precisa cinetica dopo riattivazione dell'espressione virale: l'mRNA codificante le proteine regolatrici Tax e Rex agisce come trascritto precoce che precede l'espressione degli altri geni virali. Sebbene sia comunemente accettato che Rex eserciti la sua funzione a livello post-trascrizionale controllando l'esporto nucleare e la stabilità degli mRNA che codificano le proteine associate al virione, fino ad oggi non è mai stata investigata la Rex-dipendenza dei trascritti p12, p13, p21rex, p30tof e hbz. Al fine di testare se le cinetiche di espressione genica di HTLV-1 osservate potessero dipendere dalla funzione di Rex e al fine di determinare la Rex-dipendenza dei singoli mRNA virali, abbiamo generato un clone molecolare di HTLV-1 knock-out per Rex e analizzato la compartimentalizzazione nucleo-citoplasmatica dei trascritti virali. I risultati ottenuti hanno dimostrato la stretta Rex-dipendenza delle cinetiche di espressione a "due fasi" ed hanno rivelato una forte ritenzione nucleare degli mRNA codificanti HBZ, supportando la loro funzione come trascritti non codificanti. Inoltre, i risultati ottenuti hanno dimostrato che la responsività a Rex dei differenti mRNA virali potrebbe essere determinata dalla presenza di una sequenza regolatoria di 72 nucleotidi che agisce in cis, localizzata a monte dell'esone 3. Infine, analisi matematiche hanno sottolineato l'importanza di un ritardo temporale tra le funzioni di Tax e di Rex, supportata dall'evidenza sperimentale di un ritardo nell'accumulo e di un'emivita più prolungata di Rex rispetto a Tax. I dati ottenuti in questo studio forniscono l'evidenza di una regolazione temporale dell'espressione genica di HTLV-1, rivelano una differente compartimentalizzazione degli mRNA virali e offrono una possibile spiegazione di un paradosso ancora irrisolto della regolazione di HTLV-1, ovvero la differente Rex-dipendenza dei trascritti virali, nonostante la presenza della sequenza responsiva a Rex (RxRE, Rex-responsive element) nella regione 3' non tradotta di tutti i trascritti virali.
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Crutchfield, Gerald L. "Kruppel-Like Transcription Factor 6 & 7 mRNAs (KLF6 & KLF7) Expression in the Developing Zebrafish." University of Akron / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=akron1572200378181869.
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Guo, Fang. "Regulation of expression of SSTR1 and SSTR2 somatostatin receptor mRNAs in the arcuate nucleus by growth hormone." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1995. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/MQ37318.pdf.
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Ohmori, Sachiko, Kazumi Kanda, Hirohito Mitsuyama, Takashi Miyazaki, Xia Cao, Fukushi Kambe, and Hisao Seo. "Tail-suspension Induces Altered Expression of GAPDH and ZAKI-4β mRNAs in Rat Hindlimbs : Implication for Disuse Muscle Atrophy." Research Institute of Environmental Medicine, Nagoya University, 2003. http://hdl.handle.net/2237/7561.
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Parkin, Neil T. "Regulation of gene expression by the 5' untranslated region of eukaryotic mRNAS : c-myc and HIV-1 as examples." Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74327.
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The 5$ sp prime$ untranslated region (UTR) of c-myc and human immunodeficiency virus type 1 (HIV-1) mRNAs were used as models in a variety of in vitro and in vivo systems in order to study the role of this region in the control of eukaryotic gene expression. Using an ultraviolet light-induced crosslinking assay, a 55 kilodalton protein was identified in extracts of HeLa, mouse erythroleukemia, and other cell lines, which interacts specifically with a purine-rich RNA sequence in the 5$ sp prime$ UTR of c-myc. The function of this protein in control of c-myc expression is not known, but may be implicated in the process of transcriptional elongation. The 5$ sp prime$ UTR of HIV-1 mRNAs was shown to inhibit strongly the translation of a heterologous mRNA; this inhibition was dependent on the secondary structure predicted to form in this region, and on the accessibility of the cap structure to initiation factors. The structural requirements in the HIV-1 5$ sp prime$ UTR for trans-activation by the viral tat gene product were examined by mutagenesis studies; the base-pairing in the stem-loop structure, the sequence of the loop, and the presence of a three nucleotide bulge were found to be critical features necessary for complete trans-activation. These findings indicate that the 5$ sp prime$ UTR can have important effects on the expression of eukaryotic genes.
Books on the topic "Kinetics of mRNAs expression"
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Taubhorn, Katja geb Gebhardt. Differentielle Expression Nebenhoden-spezifischer mRNAs beim Hund. Hannover: [s.n.], 1999.
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Drummond, Douglas R. The stability, movement and expression of natural and synthetic mRNAs injected into 'Xenopus' oocytes. [s.l.]: typescript, 1985.
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Bioprocess engineering: Kinetics, mass transport, reactors, and gene expression. New York: Wiley, 1994.
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Garetz, Susan Lynn. Variation in expression of Na+K+ATPase ł and ø subunit mRNAs in rat tissues and nervous system cell lines. [New Haven: s.n.], 1989.
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Kumar, Ashesh. In vivo kinetics of early cytokine expression in the lactobacillus cell wall extract-based mouse model of Kawasaki disease. Ottawa: National Library of Canada, 2002.
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Johnston, Ian G. Expression cloning of two novel mRNAs using an antibody directed against synaptic glycoproteins. 1992.
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Foster, Jane Allyson. Differential expression of heat shock mRNAs in neural cell types in the unstressed and hyperthermic rabbit brain. 1996.
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Clarke, Andrew. Temperature and reaction rate. Oxford University Press, 2017. http://dx.doi.org/10.1093/oso/9780199551668.003.0007.
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All other things being equal, physiological reaction rate increases roughly exponentially with temperature. Organisms that have adapted over evolutionary time to live at different temperatures can have enzyme variants that exhibit similar kinetics at the temperatures to which they have adapted to operate. Within species whose distribution covers a range of temperatures, there may be differential expression of enzyme variants with different kinetics across the distribution. Enzymes adapted to different optimum temperatures differ in their amino acid sequence and thermal stability. The Gibbs energy of activation tends to be slightly lower in enzyme variants adapted to lower temperatures, but the big change is a decrease in the enthalpy of activation, with a corresponding change in the entropy of activation, both associated with a more open, flexible structure. Despite evolutionary adjustments to individual enzymes involved in intermediary metabolism (ATP regeneration), many whole-organism processes operate faster in tropical ectotherms compared with temperate or polar ectotherms. Examples include locomotion (muscle power output), ATP regeneration (mitochondrial function), nervous conduction and growth.
Book chapters on the topic "Kinetics of mRNAs expression"
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Shim, Jae Youn, Byung Hun Lee, and Hye Yoon Park. "Visualization of Single mRNAs in Live Neurons." In Imaging Gene Expression, 47–61. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9674-2_4.
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Celis, Julio E. "Expression of mRNAs Microinjected Into Somatic Cells." In Ciba Foundation Symposium 103 - Cell Fusion, 220–38. Chichester, UK: John Wiley & Sons, Ltd., 2008. http://dx.doi.org/10.1002/9780470720844.ch14.
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Mali, Pekka, Antti Kaipia, Marko Kangasniemi, Jorma Toppari, Minna Sandberg, Eero Vuorio, Pamela C. Yelick, Norman B. Hecht, and Martti Parvinen. "Expression of Nucleoprotein mRNAs During Rat Spermiogenesis." In Nuclear Structure and Function, 89–93. Boston, MA: Springer US, 1990. http://dx.doi.org/10.1007/978-1-4613-0667-2_18.
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Gallie, Daniel R. "Translational control of cellular and viral mRNAs." In Post-Transcriptional Control of Gene Expression in Plants, 145–58. Dordrecht: Springer Netherlands, 1996. http://dx.doi.org/10.1007/978-94-009-0353-1_7.
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Roy, Bijoyita. "Effects of mRNA Modifications on Translation: An Overview." In Methods in Molecular Biology, 327–56. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1374-0_20.
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AbstractThe mRNA epitranscriptome imparts diversity to gene expression by installing chemical modifications. Advances in detection methods have identified chemical modifications in eukaryotic, bacterial, and viral messenger RNAs (mRNAs). The biological functions of modifications in mRNAs still remain to be understood. Chemical modifications are introduced in synthetic mRNAs meant for therapeutic applications to maximize expression from the synthetic mRNAs and to evade the host immune response. This overview provides a background of chemical modifications found in mRNAs, with an emphasis on pseudouridine and its known effects on the mRNA life cycle, its potential applications in synthetic mRNA, and the methods used to assess its effects on mRNA translation.
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Merrick, William C., and Donald D. Anthony. "Initiation Mechanisms Used in the Translation of Bicistronic mRNAs." In Translational Regulation of Gene Expression 2, 391–403. Boston, MA: Springer US, 1993. http://dx.doi.org/10.1007/978-1-4615-2894-4_19.
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Ben-Yishay, Rakefet, and Yaron Shav-Tal. "Detection of mRNAs Anchored to the Nuclear Envelope During Export Inhibition in Living Cells." In Imaging Gene Expression, 151–63. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9674-2_10.
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Jacques, N., M. Chevrier-Miller, J. Guillerez, and M. Dreyfus. "Culture Conditions Affect Differently the Translation of Individual Escherichia Coli mRNAs." In Post-Transcriptional Control of Gene Expression, 145–55. Berlin, Heidelberg: Springer Berlin Heidelberg, 1990. http://dx.doi.org/10.1007/978-3-642-75139-4_15.
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Rodgers, R. J., and H. F. Rodgers. "Localization of mRNAs That Encode Steroidogenic Enzymes in Bovine Ovaries." In Signaling Mechanisms and Gene Expression in the Ovary, 213–17. New York, NY: Springer New York, 1991. http://dx.doi.org/10.1007/978-1-4612-3200-1_19.
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Lee, Kai-Fai, and Shuk-Mei Ho. "Hammerhead ribozymes mediated down-regulation of rat metallothionein mrnas expression." In Metallothionein IV, 273–80. Basel: Birkhäuser Basel, 1999. http://dx.doi.org/10.1007/978-3-0348-8847-9_38.
Full textConference papers on the topic "Kinetics of mRNAs expression"
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Cai, Xiaoyu, Marcio de Queiroz, Glen Meades, and Grover Waldrop. "Modeling the Negative Feedback Mechanism in the Enzyme Carboxyltransferase." In ASME 2011 Dynamic Systems and Control Conference and Bath/ASME Symposium on Fluid Power and Motion Control. ASMEDC, 2011. http://dx.doi.org/10.1115/dscc2011-6171.
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The enzyme acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis in all organisms. The E. coli form of the carboxyltransferase subunit was recently found to regulate its own activity and expression by binding its own mRNA. By binding acetyl-CoA or the mRNA encoding its own subunits, Carboxyltransferase is able to sense the metabolic state of the cell and attenuate its own translation and enzymatic activity using a negative feedback mechanism. In this paper, this network of interactions is modeled mathematically using mass action kinetics. Numerical simulations of the model show agreement with experimental results.
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Wang, Sihong, Kenneth R. Diller, and Shanti J. Aggarwal. "Heat Shock Protein 70 Expression Kinetics." In ASME 2002 International Mechanical Engineering Congress and Exposition. ASMEDC, 2002. http://dx.doi.org/10.1115/imece2002-33678.
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HSP70 is well known for its major role in cardiac ischemia protection. The purpose of this study was to determine the HSP70 expression kinetics for new protocol design in cardiac surgery, based on HSP70 protection function in clinical applications. Bovine aortic endothelial cells (BAEC) were used in experiments. Cells were heated at 42°C at different time intervals up to 5 hours and subsequently incubated at 37°C for up to 48 hours. Western blot and quantitative protein analysis were performed to measure HSP70 expression. The expression kinetics is a function of thermal stress time as well as poststress time. At least three stages were identified for the kinetics curve: increasing, maximum plateau and decreasing regions. The peak HSP70 concentration is 10 times the basal level for western blot analysis in BAECs. Two hours incubator heating followed by twelve hours post-heating falls in the plateau region. This research result provides information applicable to evaluation of energy sources and heating methods to induce optimal HSP70 expression in a target tissue.
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Coutinho-Camillo, Cláudia M., Silvia V. Lourenço, Ines N. Nishimoto, Luiz P. Kowalski, and Fernando A. Soares. "Abstract 3160: Expression of apoptosis-regulating miRNAs and target mRNAs in oral squamous cell carcinoma." In Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL. American Association for Cancer Research, 2012. http://dx.doi.org/10.1158/1538-7445.am2012-3160.
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Meyer, M., C. Arolt, D. Beutner, M. Odenthal, JP Klußmann, and A. Quaas. "Expression analysis of mRNAs of extracellular matrix components in aggressive entities of salivary gland carcinomas." In 100 JAHRE DGHNO-KHC: WO KOMMEN WIR HER? WO STEHEN WIR? WO GEHEN WIR HIN? Georg Thieme Verlag KG, 2021. http://dx.doi.org/10.1055/s-0041-1728927.
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Holliday, Casey J., Randall F. Ankeny, Hanjoong Jo, and Robert M. Nerem. "Discovery of Side- and Shear-Dependent miRNAs and mRNAs in Human Aortic Valvular Endothelial Cells." In ASME 2011 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2011. http://dx.doi.org/10.1115/sbc2011-53315.
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Aortic valve (AV) disease is diagnosed by severe symptoms, such as calcification, and typically treated by AV replacement and repair surgeries. The mechanism by which AV disease occurs, specifically the role of the endothelium remains relatively unknown. It is known that disease preferentially occurs on the fibrosa, or aortic side, where it is exposed to disturbed, oscillatory flow, whereas the ventricularis, or side facing the left ventricle, experiences pulsatile, laminar shear and remains non-calcified [1, 2]. Research shows that regulation of miRNAs, short nucleotide segments targeting mRNAs, coincides with cardiovascular pathologies [3] though expression profiles of miRNAs and the mRNAs they modulate in human AV endothelial cells (HAVECs) have not been reported. We hypothesize that disturbed flow conditions present on the fibrosa stimulate ECs to modify expression of genes and miRNAs to induce a pro-inflammatory phenotype.
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Levene, Richard B., Francis M. Booyse, Juan Chediak, Therodore S. Zimmerman, David M. Livingston, and Dennis C. Lynch. "ABNORMAL EXPRESSION OF VON WILLEBRAND FACTOR BY ENDOTHELIAL CELLS FROM A PATIENT WITH TYPE IIA VON WILLEBRAND’ S DISEASE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642915.
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Studies were conducted to characterize the biosynthesis of von Willebrand factor (vWf) by cultured endothelial cells (EC) derived from the umbilical vein of a patient with type HA von Willebrand’ s disease. The patient’ s EC, compared with those from normal individuals, produced vWf which had decreased amounts of large multimers and an increase in rapidly migrating satellite species, features which are characteristic of plasma vWf from patients with type IIA von Willebrand’ s disease. The typQ IIA EC produced a full spectrum of vWf multimers in both cell lysates and post-culture medium, although the relative amounts of the larger species were decreased. The large multimers were degraded in conjunction with the appearance of rapidly migrating satellites which contained =170 kDa proteolytic fragments. Kinetic studies demonstrated that the =170 kDa species is not a primary translation product Normal metabolically labeled vWf, incubated with either the patient’ s EC or medium conditioned by these cells, was not similarly degraded. These results demonstrated that this patient’ s clinical phenotype is due to abnormal proteolysis and not to a primary failure of subunit oligomerization. Moreover, the increased degradation is attributable to increased proteolytic sensitivity of an abnormal vWf molecule rather than to pathologically elevated levels of endogenous proteases. Experiments using monoclonal antibodies which recognize either N- or C-associated epitopes have localized the defect to the N-terminal portion of the vWf molecule, which is believed to be involved in the inter-dimer polymerization reaction. The type DA EC also contained a single vWf mRNA species which comigrated with that from normal EC. However, the type HA EC contained 8-10 fold more vWf mRNA than their normal counterparts. These results suggest that the functional defect in this patient is caused by a subtle mutation in the vWf coding sequence leading to increased proteolytic sensitivity of its protein product
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DeGorordo, A., DC Files, P. Kesari, L. Johnston, N. Aggarwal, VK Sidhaye, F. D'Alessio, LS King, and MT Crow. "Acute Lung Injury Causes Increased Expression of mRNAs Encoding Atrophy and Autophagy Genes in Skeletal Muscle and the Diaphragm." In American Thoracic Society 2009 International Conference, May 15-20, 2009 • San Diego, California. American Thoracic Society, 2009. http://dx.doi.org/10.1164/ajrccm-conference.2009.179.1_meetingabstracts.a4186.
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Tran, Huy, and Andre Ribeiro. "Effects of inducer intake kinetics on the dynamics of gene expression." In European Conference on Artificial Life 2013. MIT Press, 2013. http://dx.doi.org/10.7551/978-0-262-31709-2-ch181.
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Murphy, R., S. A. Gharib, R. Sehmi, G. M. Gauvreau, and T. S. Hallstrand. "Kinetics of Airway Gene Expression in Atopic Asthmatics Following Inhaled Allergen Challenge." In American Thoracic Society 2022 International Conference, May 13-18, 2022 - San Francisco, CA. American Thoracic Society, 2022. http://dx.doi.org/10.1164/ajrccm-conference.2022.205.1_meetingabstracts.a3477.
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Grabarek, Beniamin, Magdalena Mistarz, Mateusz Maziarz, Michał Szurgot, and Weronika Wieczorek. "Cyclosporine A and adalimumab change the expression profile of mRNAs amd miRNAs related with the histaminergic system in keratinocytes exposed to LPS." In 6th International Electronic Conference on Medicinal Chemistry. Basel, Switzerland: MDPI, 2020. http://dx.doi.org/10.3390/ecmc2020-07411.
Full textReports on the topic "Kinetics of mRNAs expression"
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Stern, David, and Gadi Schuster. Manipulation of Gene Expression in the Chloroplast. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575289.bard.
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The steady-state level of a given mRNA is determined by its rates of transcription and degradation. The stabilities of chloroplast mRNAs vary during plant development, in part regulating gene expression. Furthermore, the fitness of the organelle depends on its ability to destroy non-functional transcripts. In addition, there is a resurgent interest by the biotechnology community in chloroplast transformation due to the public concerns over pollen transmission of introduced traits or foreign proteins. Therefore, studies into basic gene expression mechanisms in the chloroplast will open the door to take advantage of these opportunities. This project was aimed at gaining mechanistic insights into mRNA processing and degradation in the chloroplast and to engineer transcripts of varying stability in Chlamydomonas reinhardtii cells. This research uncovered new and important information on chloroplast mRNA stability, processing, degradation and translation. In particular, the processing of the 3' untranslated regions of chloroplast mRNAs was shown to be important determinants in translation. The endonucleolytic site in the 3' untranslated region was characterized by site directed mutagensis. RNA polyadenylation has been characterized in the chloroplast of Chlamydomonas reinhardtii and chloroplast transformants carrying polyadenylated sequences were constructed and analyzed. Data obtained to date suggest that chloroplasts have gene regulatory mechanisms which are uniquely adapted to their post-endosymbiotic environment, including those that regulate RNA stability. An exciting point has been reached, because molecular genetic studies have defined critical RNA-protein interactions that participate in these processes. However, much remains to be learned about these multiple pathways, how they interact with each other, and how many nuclear genes are consecrated to overseeing them. Chlamydomonas is an ideal model system to extend our understanding of these areas, given its ease of manipulation and the existing knowledge base, some of which we have generated.
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Sessa, Guido, and Gregory Martin. A functional genomics approach to dissect resistance of tomato to bacterial spot disease. United States Department of Agriculture, January 2004. http://dx.doi.org/10.32747/2004.7695876.bard.
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The research problem. Bacterial spot disease in tomato is of great economic importance worldwide and it is particularly severe in warm and moist areas affecting yield and quality of tomato fruits. Causal agent of spot disease is the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv), which can be a contaminant on tomato seeds, or survive in plant debris and in association with certain weeds. Despite the economic significance of spot disease, plant protection against Xcvby cultural practices and chemical control have so far proven unsuccessful. In addition, breeding for resistance to bacterial spot in tomato has been undermined by the genetic complexity of the available sources of resistance and by the multiple races of the pathogen. Genetic resistance to specific Xcvraces have been identified in tomato lines that develop a hypersensitive response and additional defense responses upon bacterial challenge. Central goals of this research were: 1. To identify plant genes involved in signaling and defense responses that result in the onset of resistance. 2. To characterize molecular properties and mode of action of bacterial proteins, which function as avirulence or virulence factors during the interaction between Xcvand resistant or susceptible tomato plants, respectively. Our main achievements during this research program are in three major areas: 1. Identification of differentially expressed genes during the resistance response of tomato to Xcvrace T3. A combination of suppression subtractive hybridization and microarray analysis identified a large set of tomato genes that are induced or repressed during the response of resistant plants to avirulent XcvT3 bacteria. These genes were grouped in clusters based on coordinate expression kinetics, and classified into over 20 functional classes. Among them we identified genes that are directly modulated by expression of the type III effector protein AvrXv3 and genes that are induced also during the tomato resistance response to Pseudomonas syringae pv. tomato. 2. Characterization of molecular and biochemical properties of the tomato LeMPK3MAP kinase. A detailed molecular and biochemical analysis was performed for LeMPK3 MAP kinase, which was among the genes induced by XcvT3 in resistant tomato plants. LeMPK3 was induced at the mRNA level by different pathogens, elicitors, and wounding, but not by defense-related plant hormones. Moreover, an induction of LeMPK3 kinase activity was observed in resistant tomato plants upon Xcvinfection. LeMPK3 was biochemically defined as a dual-specificity MAP kinase, and extensively characterized in vitro in terms of kinase activity, sites and mechanism of autophosphorylation, divalent cation preference, Kₘand Vₘₐₓ values for ATP. 3. Characteriztion of molecular properties of the Xcveffector protein AvrRxv. The avirulence gene avrRxvis involved in the genetic interaction that determines tomato resistance to Xcvrace T1. We found that AvrRxv functions inside the plant cell, localizes to the cytoplasm, and is sufficient to confer avirulence to virulent Xcvstrains. In addition, we showed that the AvrRxv cysteine protease catalytic core is essential for host recognition. Finally, insights into cellular processes activated by AvrRxv expression in resistant plants were obtained by microarray analysis of 8,600 tomato genes. Scientific and agricultural significance: The findings of these activities depict a comprehensive and detailed picture of cellular processes taking place during the onset of tomato resistance to Xcv. In this research, a large pool of genes, which may be involved in the control and execution of plant defense responses, was identified and the stage is set for the dissection of signaling pathways specifically triggered by Xcv.
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Stern, David B., and Gadi Schuster. Manipulation of Gene Expression in the Chloroplast: Control of mRNA Stability and Transcription Termination. United States Department of Agriculture, December 1993. http://dx.doi.org/10.32747/1993.7568750.bard.
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Chloroplasts are the site of photosynthesis and of other essential biosynthetic activities in plant cells. Chloroplasts are semi-autonomous organelles, since they contain their own genomes and protein biosynthetic machinery, but depend on the coordinate expression of nuclear genes to assemble macromolecular complexes. The bioeingineering of plants requires manipulation of chloroplast gene expression, and thus a knowledge of the molecular mechanisms that modulate mRNA and protein production. In this proposal the heterotrophic green alga Chlamydomonas reinhardtii has been used as a model system to understand the control and interrelationships between transcription termination, mRNA 3' end processing and mRNA stability in chloroplasts. Chlamydomonas is a unique and ideal system in which to address these issues, because the chloroplast can be easily manipulated by genetic transformation techniques. This research uncovered new and important information on chloroplast mRNA 3' end formation and mRNA stability. In particular, the 3' untranslated regions of chloroplast mRNAs were shown not to be efficient transcription terminators. The endonucleolytic site in the 3' untranslated region was characterized by site directed mutagensis and the role of several 3' untranslated regions in modulating RNA stability and translation has been studied. This information will allow us to experimentally manipulate the expression of chloroplast genes in vivo by post-transcriptional mechanisms, and should be widely applicable to other higher plant systems.
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Sun, Lina, Yanan Han, Hua Wang, Huanyu Liu, Shan Liu, Hongbin Yang, Xiaoxia Ren, and Ying Fang. MicroRNAs as Potential Biomarkers for the Diagnosis of Inflammatory Bowel Disease: A Systematic Review and Meta-analysis. INPLASY - International Platform of Registered Systematic Review and Meta-analysis Protocols, February 2022. http://dx.doi.org/10.37766/inplasy2022.2.0027.
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Review question / Objective: The purpose of this systematic review was to systematically review the clinical studies regarding miRNAs as diagnostic biomarkers for inflammatory bowel disease and assess the overall diagnostic accuracy of miRNAs. Condition being studied: The symptoms of inflammatory bowel disease (IBD) are highly variable. The diagnosis of IBD must be made through medical history, physical, laboratory, radiologic, endoscopic, and histological examinations. However, these diagnostic techniques are not specific and sometimes even equivocal. Therefore, reliable biomarkers are urgently needed in the diagnosis of IBD. Several clinical and preclinical researches have shown that dysregulated microRNAs (miRNAs) play a crucial role in IBD development. miRNAs, as single-stranded noncoding RNAs that contain 22-24 nucleotides, can post-transcriptionally regulate gene expression by blocking mRNA translation or degrading target mRNAs. miRNAs are widely involved in physiological and pathological cellular processes, such as differentiation, proliferation and apoptosis. Besides, they are stable, noninvasive, and resistant to degradation by ribonucleases, making them valuable targets in the diagnosis, monitoring, prognosis, and treatment of diseases. To date, inconsistent results have been found about miRNA expression profiling in the patients with IBD. Moreover, the diagnostic accuracy of miRNAs for IBD has not been reported in any meta-analysis.
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Woodson, William, Shimon Mayak, and Haim Rabinowitch. Physiological and Molecular Characterization of the Response to Ethylene during Senescence of Carnation Genotypic Variants. United States Department of Agriculture, July 1995. http://dx.doi.org/10.32747/1995.7613011.bard.
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The senescence of carnation (Dianthus caryophyllus L.) flowers is associated with increased production of the phytohormone ethylene, which in turn serves to initiate and regulate the processes involved in programmed petal death. We investigated the regulation of ethylene production and petal senescence in carnation. Several carnation genotypes were identified that exhibited extended vase-life in comparison to flowers from typical commercial cultivars. The capacity of these genotypes to produce ethylene during postharvest vase-life and to respond to exogenous ethylene was investigated. Several genotypes, represented by 'Sandrosa' and 87-37G produced little ethylene durig their postharvest vase-life and as a result failed to exhibit the symptoms (in-rolling and wilting) typical of flowers producing elevated levels of ethylene. These genotypes were further separated by their capacity to respond to exogenous ethylene by both increased ethylene synthesis and premature petal senescence. In one case a genotype (799) was identified that was not capable of responding to exogenous ethylene by either increased ethylene production or premature petal senescence. The regulation of ethylene production during petal senescence was investigated both at the enzyme and gene levels. A full length cDNA was identified for the petal senescence-related ACC synthase gene. Utilizing this, and other ethylene biosynthetic pathway cDNA probes, an increase in both ACC synthase and ACC oxidase mRNAs were detected following ethylene treatment. An increase in ACC oxidase mRNA and enzyme activity was detected within 2-3 h following ethylene treatment, indicating the expression of this gene is an early response to ethylene. An investigation into the expression of novel proteins during petal senescence revealed a number of polypeptides increased in abundance and possibly play a role in the regulation or biochemical processes of senescence. One polypeptide of 70 kDa was identified as being encoded by the previously characterized gene SR12 and possibly represents a b-galactosidase involved in the remobilization of carbohydrates during senescence.
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Funkenstein, Bruria, and Cunming Duan. GH-IGF Axis in Sparus aurata: Possible Applications to Genetic Selection. United States Department of Agriculture, November 2000. http://dx.doi.org/10.32747/2000.7580665.bard.
Full textAbstract:
Many factors affect growth rate in fish: environmental, nutritional, genetics and endogenous (physiological) factors. Endogenous control of growth is very complex and many hormone systems are involved. Nevertheless, it is well accepted that growth hormone (GH) plays a major role in stimulating somatic growth. Although it is now clear that most, if not all, components of the GH-IGF axis exist in fish, we are still far from understanding how fish grow. In our project we used as the experimental system a marine fish, the gilthead sea bream (Sparus aurata), which inhabits lagoons along the Mediterranean and Atlantic coasts of Europe, and represents one of the most important fish species used in the mariculture industry in the Mediterranean region, including Israel. Production of Sparus is rapidly growing, however, in order for this production to stay competitive, the farming of this fish species has to intensify and become more efficient. One drawback, still, in Sparus extensive culture is that it grows relatively slow. In addition, it is now clear that growth and reproduction are physiological interrelated processes that affect each other. In particular sexual maturation (puberty) is known to be closely related to growth rate in fish as it is in mammals, indicating interactions between the somatotropic and gonadotropic axes. The goal of our project was to try to identify the rate-limiting components(s) in Sparus aurata GH-IGF system which might explain its slow growth by studying the ontogeny of growth-related genes: GH, GH receptor, IGF-I, IGF-II, IGF receptor, IGF-binding proteins (IGFBPs) and Pit-1 during early stages of development of Sparus aurata larvae from slow and fast growing lines. Our project was a continuation of a previous BARD project and could be divided into five major parts: i) obtaining additional tools to those obtained in the previous project that are necessary to carry out the developmental study; ii) the developmental expression of growth-related genes and their cellular localization; iii) tissue-specific expression and effect of GH on expression of growth-related genes; iv) possible relationship between GH gene structure, growth rate and genetic selection; v) the possible role of the IGF system in gonadal development. The major findings of our research can be summarized as follows: 1) The cDNAs (complete or partial) coding for Sparus IGFBP-2, GH receptor and Pit-1 were cloned. Sequence comparison reveals that the primary structure of IGFBP-2 protein is 43-49% identical to that of zebrafish and other vertebrates. Intensive efforts resulted in cloning a fragment of 138 nucleotides, coding for 46 amino acids in the proximal end of the intracellular domain of GH receptor. This is the first fish GH receptor cDNA that had been cloned to date. The cloned fragment will enable us to complete the GH - receptor cloning. 2) IGF-I, IGF-II, IGFBP-2, and IGF receptor transcripts were detected by RT-PCR method throughout development in unfertilized eggs, embryos, and larvae suggesting that these mRNAs are products of both the maternal and the embryonic genomes. Preliminary RT-PCR analysis suggest that GH receptor transcript is present in post-hatching larvae already on day 1. 3) IGF-1R transcripts were detected in all tissues tested by RT-PCR with highest levels in gill cartilage, skin, kidney, heart, pyloric caeca, and brain. Northern blot analysis detected IGF receptor only in gonads, brain and gill cartilage but not in muscle; GH increased slightly brain and gill cartilage IGF-1R mRNA levels. 4) IGFBP-2 transcript were detected only in liver and gonads, when analyzed by Northern blots; RT-PCR analysis revealed expression in all tissues studied, with the highest levels found in liver, skin, gonad and pyloric caeca. 5) Expression of IGF-I, IGF-II, IGF-1R and IGFBP-2 was analyzed during gonadal development. High levels of IGF-I and IGFBP-2 expression were found in bisexual young gonads, which decreased during gonadal development. Regardless of maturational stage, IGF-II levels were higher than those of IGF-L 6) The GH gene was cloned and its structure was characterized. It contains minisatellites of tandem repeats in the first and third introns that result in high level of genetic polymorphism. 7) Analysis of the presence of IGF-I and two types of IGF receptor by immunohistochemistry revealed tissue- and stage-specific expression during larval development. Immunohistochemistry also showed that IGF-I and its receptors are present in both testicular and ovarian cells. Although at this stage we are not able to pinpoint which is the rate-limiting step causing the slow growth of Sparus aurata, our project (together with the previous BARD) yielded a great number of experimental tools both DNA probes and antibodies that will enable further studies on the factors regulating growth in Sparus aurata. Our expression studies and cellular localization shed new light on the tissue and developmental expression of growth-related genes in fish.
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Yaron, Zvi, Martin P. Schreibman, Abigail Elizur, and Yonathan Zohar. Advancing Puberty in the Black Carp (Mylopharyngodon Piceus) and the Striped Bass (Morone Saxatilis). United States Department of Agriculture, August 1993. http://dx.doi.org/10.32747/1993.7568102.bard.
Full textAbstract:
The black carp (bc)GtH IIb cDNA was amplified and isolated, cloned and sequenced. Comparison of the bcGtH IIb deduced a.a. sequence with that of GtH IIb from other teleosts revealed high homology to cyprinid species and a lower homology to salmonid or perciform fish. The gene coding for the GtH IIb was isolated and sequenced. Three bc recombinant phages which hybridized to the goldfish GtH Ib cDNA probe were isolated and are currently being characterized. The region coding for the mature GtH IIb was expressed in a bacterial expression vector resulting in the production of a recombinant protein. In vitro folding resulted in a protein only 1.3% of which displaced the native common carp GtH II in a RIA. Therefore, the common carp GtH RIA was utilized for the physiological studies at the current phase of the project. Two non-functional sites were identified along the brain-pituitary gonadal axis in the immature black carp. The pituitary is refractory to GnRH stimulation due to a block proximal to the activation of PKA and PKC probably at the level of GnRH receptors. The gonads, although capable of producing steroids, are refractory to gonadotropic stimulation but do respond to cAMP antagonists, indicating a block at the GtH receptor level. Attempts to advance puberty in 2 and 3 y old black carp showed that testosterone (T) stimulates GtH synthesis in the pituitary and increases its sensitivity to GnRh. A 2 month treatment combining T+GnRH increased the circulating GFtH level in 3 y old fish. Addition of domperidone to such a treatment facilitated both the accumulation of GtH in the pituitary and its response to GnRH. The cDNA of striped bass GtH a, Ib and IIb subunits were amplified, isolated, cloned and sequenced, and their deduced a.a. sequences were compared with those of other teleosts. A ribonuclease protection assay was developed for a sensitive and simultaneous determination of all GtH subunits, and of b-actin mRNAs of the striped bass. GnRH stimulated dramatically the expression of the a and GtH IIb subunits but the level of GtH Ib mRNA increased only moderately. These findings suggest that GtH-II, considered in salmonids to be involved only in final stages of gametogenesis, can be induced by GnRH to a higher extent than GtH-I in juvenile striped bass. The native GtH II of the striped bass was isolated and purified, and an ELISA for its determination was developed. The production of all recombinant striped bass GtH subunits is in progress using the insect cell (Sf9) culture and the BAC-TO-BAC baculovirus expression system. A recombinant GtH IIb subunit has been produced already, and its similarity to the native subunit was confirmed. The yield of the recombinant glycoprotein can reach 3.5 mg/ml after 3 days culture. All male striped bass reach puberty after 3 y. However, precocious puberty was discovered in 1 and 2 y old males. Females become vitellogenic during their 4th year. In immature 2 y old females, T treatment elevates the pituitary GtH II content while GnRH only potentiates the effect. However, in males GnRH and not T affects GtH accumulation in the pituitary. Neither GnRH, nor T treatment resulted in gonadal growth in 2 y old striped bass, indicating that either the accumulated GtH II was not released, or if released, the gonads were refractory to GtH stimulation, similar to the situation in the immature black carp. In 3 y old female striped bass, 150 day GnRHa treatment resulted in an increase in GSI, while T treatment, with or without GnRHa, resulted in a decrease in oocyte diameter, similar to the effect seen in the black carp. Further attempts to advance puberty in both fish species should take into account the positive effect of T on pituitary GtH and its negative effect of ovarian growth.
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Meidan, Rina, and Robert Milvae. Regulation of Bovine Corpus Luteum Function. United States Department of Agriculture, March 1995. http://dx.doi.org/10.32747/1995.7604935.bard.
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The main goal of this research plan was to elucidate regulatory mechanisms controlling the development, function of the bovine corpus luteum (CL). The CL contains two different sterodigenic cell types and therefore it was necessary to obtain pure cell population. A system was developed in which granulosa and theca interna cells, isolated from a preovulatory follicle, acquired characteristics typical of large (LL) and small (SL) luteal cells, respectively, as judged by several biochemical and morphological criteria. Experiments were conducted to determine the effects of granulosa cells removal on subsequent CL function, the results obtained support the concept that granulosa cells make a substaintial contribution to the output of progesterone by the cyclic CL but may have a limited role in determining the functional lifespan of the CL. This experimental model was also used to better understand the contribution of follicular granulosa cells to subsequent luteal SCC mRNA expression. The mitochondrial cytochrome side-chain cleavage enzyme (SCC), which converts cholesterol to pregnenolone, is the first and rate-limiting enzyme of the steroidogenic pathway. Experiments were conducted to characterize the gene expression of P450scc in bovine CL. Levels of P450scc mRNA were higher during mid-luteal phase than in either the early or late luteal phases. PGF 2a injection decreased luteal P450scc mRNA in a time-dependent manner; levels were significantly reduced by 2h after treatment. CLs obtained from heifers on day 8 of the estrous cycle which had granulosa cells removed had a 45% reduction in the levels of mRNA for SCC enzymes as well as a 78% reduction in the numbers of LL cells. To characterize SCC expression in each steroidogenic cell type we utilized pure cell populations. Upon luteinization, LL expressed 2-3 fold higher amounts of both SCC enzymes mRNAs than SL. Moreover, eight days after stimulant removal, LL retained their P4 production capacity, expressed P450scc mRNA and contained this protein. In our attempts to establish the in vitro luteinization model, we had to select the prevulatory and pre-gonadotropin surge follicles. The ratio of estradiol:P4 which is often used was unreliable since P4 levels are high in atretic follicles and also in preovulatory post-gonadotropin follicles. We have therefore examined whether oxytocin (OT) levels in follicular fluids could enhance our ability to correctly and easily define follicular status. Based on E2 and OT concentrations in follicular fluids we could more accurately identify follicles that are preovulatory and post gonadotropin surge. Next we studied OT biosynthesis in granulosa cells, cells which were incubated with forskolin contained stores of the precursor indicating that forskolin (which mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release. While studying in vitro luteinization, we noticed that IGF-I induced effects were not identical to those induced by insulin despite the fact that megadoses of insulin were used. This was the first indication that the cells may secrete IGF binding protein(s) which regonize IGFs and not insulin. In a detailed study involving several techniques, we characterized the species of IGF binding proteins secreted by luteal cells. The effects of exogenous polyunsaturated fatty acids and arachidonic acid on the production of P4 and prostanoids by dispersed bovine luteal cells was examined. The addition of eicosapentaenoic acid and arachidonic acid resulted in a dose-dependent reduction in basal and LH-stimulated biosynthesis of P4 and PGI2 and an increase in production of PGF 2a and 5-HETE production. Indomethacin, an inhibitor of arachidonic acid metabolism via the production of 5-HETE was unaffected. Results of these experiments suggest that the inhibitory effect of arachidonic acid on the biosynthesis of luteal P4 is due to either a direct action of arachidonic acid, or its conversion to 5-HETE via the lipoxgenase pathway of metabolism. The detailed and important information gained by the two labs elucidated the mode of action of factors crucially important to the function of the bovine CL. The data indicate that follicular granulosa cells make a major contribution to numbers of large luteal cells, OT and basal P4 production, as well as the content of cytochrome P450 scc. Granulosa-derived large luteal cells have distinct features: when luteinized, the cell no longer possesses LH receptors, its cAMP response is diminished yet P4 synthesis is sustained. This may imply that maintenance of P4 (even in the absence of a Luteotropic signal) during critical periods such as pregnancy recognition, is dependent on the proper luteinization and function of the large luteal cell.
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Bar-Joseph, Moshe, William O. Dawson, and Munir Mawassi. Role of Defective RNAs in Citrus Tristeza Virus Diseases. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575279.bard.
Full textAbstract:
This program focused on citrus tristeza virus (CTV), the largest and one of the most complex RNA-plant-viruses. The economic importance of this virus to the US and Israeli citrus industries, its uniqueness among RNA viruses and the possibility to tame the virus and eventually turn it into a useful tool for the protection and genetic improvement of citrus trees justify these continued efforts. Although the overall goal of this project was to study the role(s) of CTV associated defective (d)-RNAs in CTV-induced diseases, considerable research efforts had to be devoted to the engineering of the helper virus which provides the machinery to allow dRNA replication. Considerable progress was made through three main lines of complementary studies. For the first time, the generation of an engineered CTV genetic system that is capable of infecting citrus plants with in vitro modified virus was achieved. Considering that this RNA virus consists of a 20 kb genome, much larger than any other previously developed similar genetic system, completing this goal was an extremely difficult task that was accomplished by the effective collaboration and complementarity of both partners. Other full-length genomic CTV isolates were sequenced and populations examined, resulting in a new level of understanding of population complexities and dynamics in the US and Israel. In addition, this project has now considerably advanced our understanding and ability to manipulate dRNAs, a new class of genetic elements of closteroviruses, which were first found in the Israeli VT isolate and later shown to be omnipresent in CTV populations. We have characterized additional natural dRNAs and have shown that production of subgenomic mRNAs can be involved in the generation of dRNAs. We have molecularly cloned natural dRNAs and directly inoculated citrus plants with 35S-cDNA constructs and have shown that specific dRNAs are correlated with specific disease symptoms. Systems to examine dRNA replication in protoplasts were developed and the requirements for dRNA replication were defined. Several artificial dRNAs that replicate efficiently with a helper virus were created from infectious full-genomic cDNAs. Elements that allow the specific replication of dRNAs by heterologous helper viruses also were defined. The T36-derived dRNAs were replicated efficiently by a range of different wild CTV isolates and hybrid dRNAs with heterologous termini are efficiently replicated with T36 as helper. In addition we found: 1) All CTV genes except of the p6 gene product from the conserved signature block of the Closteroviridae are obligate for assembly, infectivity, and serial protoplast passage; 2) The p20 protein is a major component of the amorphous inclusion bodies of infected cells; and 3) Novel 5'-Co-terminal RNAs in CTV infected cells were characterized. These results have considerably advanced our basic understanding of the molecular biology of CTV and CTV-dRNAs and form the platform for the future manipulation of this complicated virus. As a result of these developments, the way is now open to turn constructs of this viral plant pathogen into new tools for protecting citrus against severe CTV terms and development of virus-based expression vectors for other citrus improvement needs. In conclusion, this research program has accomplished two main interconnected missions, the collection of basic information on the molecular and biological characteristics of the virus and its associated dRNAs toward development of management strategies against severe diseases caused by the virus and building of novel research tools to improve citrus varieties. Reaching these goals will allow us to advance this project to a new phase of turning the virus from a pathogen to an ally.
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Bercovier, Herve, and Ronald P. Hedrick. Diagnostic, eco-epidemiology and control of KHV, a new viral pathogen of koi and common carp. United States Department of Agriculture, December 2007. http://dx.doi.org/10.32747/2007.7695593.bard.
Full textAbstract:
Original objectives and revisions-The proposed research included these original objectives: field validation of diagnostic tests (PCR), the development and evaluation of new sensitive tools (LC-PCR/TaqManPCR, antibody detection by ELISA) including their use to study the ecology and the epidemiology of KHV (virus distribution in the environment and native cyprinids) and the carrier status of fish exposed experimentally or naturally to KHV (sites of virus replication and potential persistence or latency). In the course of the study we completed the genome sequence of KHV and developed a DNA array to study the expression of KHV genes in different conditions. Background to the topics-Mass mortality of koi or common carp has been observed in Israel, USA, Europe and Asia. These outbreaks have reduced exports of koi from Israel and have created fear about production, import, and movements of koi and have raised concerns about potential impacts on native cyprinid populations in the U.S.A. Major conclusions-A suite of new diagnostic tools was developed that included 3 PCR assays for detection of KHV DNA in cell culture and fish tissues and an ELISA assay capable of detecting anti-KHV antibodies in the serum of koi and common carp. The TKPCR assay developed during the grant has become an internationally accepted gold standard for detection of viral DNA. Additionally, the ELISA developed for detecting serum anti-KHV antibodies is now in wide use as a major nonlethal screening tool for evaluating virus status of koi and common carp populations. Real time PCR assays have been able to detect viral DNA in the internal organs of survivors of natural and wild type vaccine exposures at 1 and 10³ genome equivalents at 7 months after exposure. In addition, vaccinated fish were able to transmit the virus to naive fish. Potential control utilizing hybrids of goldfish and common carp for production demonstrated they were considerably more resistant than pure common carp or koi to both KHV (CyHV-3). There was no evidence that goldfish or other tested endemic cyprinids species were susceptible to KHV. The complete genomic sequencing of 3 strains from Japan, the USA, and Israel revealed a 295 kbp genome containing a 22 kbp terminal direct repeat encoding clear gene homologs to other fish herpesviruses in the family Herpesviridae. The genome encodes156 unique protein-coding genes, eight of which are duplicated in the terminal repeat. Four to seven genes are fragmented and the loss of these genes may be associated with the high virulence of the virus. Viral gene expression was studies by a newly developed chip which has allowed verification of transcription of most all hypothetical genes (ORFs) as well as their kinetics. Implications, both scientific and agricultural- The results from this study have immediate application for the control and management of KHV. The proposal provides elements key to disease management with improved diagnostic tools. Studies on the ecology of the virus also provide insights into management of the virus at the farms that farmers will be able to apply immediately to reduce risks of infections. Lastly, critical issues that surround present procedures used to create “resistant fish” must be be resolved (e.g. carriers, risks, etc.). Currently stamping out may be effective in eradicating the disease. The emerging disease caused by KHV continues to spread. With the economic importance of koi and carp and the vast international movements of koi for the hobby, this disease has the potential for even further spread. The results from our studies form a critical component of a comprehensive program to curtail this emerging pathogen at the local, regional and international levels.