scholarly journals In Vitro and In Vivo Studies of the RNA Conformational Switch in Alfalfa Mosaic Virus

2009 ◽  
Vol 84 (3) ◽  
pp. 1423-1429 ◽  
Author(s):  
Shih-Cheng Chen ◽  
René C. L. Olsthoorn
Keyword(s):  
Mosaic Virus ◽  
Binding Affinity ◽  
Linear Array ◽  
Alfalfa Mosaic Virus ◽  
In Vivo Studies ◽  
Conformational Switch ◽  
Stem Loop ◽  
Viral Replicase

ABSTRACT The 3′ termini of Alfalfa mosaic virus (AMV) RNAs adopt two mutually exclusive conformations, a coat protein binding (CPB) and a tRNA-like (TL) conformer, which consist of a linear array of stem-loop structures and a pseudoknot structure, respectively. Previously, switching between CPB and TL conformers has been proposed as a mechanism to regulate the competing processes of translation and replication of the viral RNA (R. C. L. Olsthoorn et al., EMBO J. 18:4856-4864, 1999). In the present study, the switch between CPB and TL conformers was further investigated. First, we showed that recognition of the AMV 3′ untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding enzyme) in vitro is more efficient when the distribution is shifted toward the TL conformation. Second, the recognition of the 3′ UTR by the viral replicase was similarly dependent on the ratio of CBP and TL conformers. Furthermore, the addition of CP, which is expected to shift the distribution toward the CPB conformer, inhibited recognition by the CCA-adding enzyme and the replicase. Finally, we monitored how the binding affinity to CP is affected by this conformational switch in the yeast three-hybrid system. Here, disruption of the pseudoknot enhanced the binding affinity to CP by shifting the balance in favor of the CPB conformer, whereas stabilizing the pseudoknot did the reverse. Together, the in vitro and in vivo data clearly demonstrate the existence of the conformational switch in the 3′ UTR of AMV RNAs.

In Vivo and in Vitro Translation of the RNAS of Alfalfa Mosaic Virus

1977 ◽  
pp. 387-411 ◽  
Author(s):  
L. van Vloten-Doting ◽  
J. Bol ◽  
L. Neeleman ◽  
T. Rutgers ◽  
D. van Dalen ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Alfalfa Mosaic Virus ◽  
In Vitro Translation

scholarly journals A Bifunctional Adsorber Particle for the Removal of Hydrophobic Uremic Toxins from Whole Blood of Renal Failure Patients

Toxins ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 389 ◽  
Author(s):  
Marieke Sternkopf ◽  
Sven Thoröe-Boveleth ◽  
Tobias Beck ◽  
Kirsten Oleschko ◽  
Ansgar Erlenkötter ◽  
...  
Keyword(s):  
Adsorption Capacity ◽  
Binding Affinity ◽  
Whole Blood ◽  
Uremic Toxins ◽  
In Vivo Studies ◽  
Uremic Toxin ◽  
High Binding ◽  
High Binding Affinity

Hydrophobic uremic toxins accumulate in patients with chronic kidney disease, contributing to a highly increased cardiovascular risk. The clearance of these uremic toxins using current hemodialysis techniques is limited due to their hydrophobicity and their high binding affinity to plasma proteins. Adsorber techniques may be an appropriate alternative to increase hydrophobic uremic toxin removal. We developed an extracorporeal, whole-blood bifunctional adsorber particle consisting of a porous, activated charcoal core with a hydrophilic polyvinylpyrrolidone surface coating. The adsorption capacity was quantified using analytical chromatography after perfusion of the particles with an albumin solution or blood, each containing mixtures of hydrophobic uremic toxins. A time-dependent increase in hydrophobic uremic toxin adsorption was depicted and all toxins showed a high binding affinity to the adsorber particles. Further, the particle showed a sufficient hemocompatibility without significant effects on complement component 5a, thrombin-antithrombin III complex, or thrombocyte concentration in blood in vitro, although leukocyte counts were slightly reduced. In conclusion, the bifunctional adsorber particle with cross-linked polyvinylpyrrolidone coating showed a high adsorption capacity without adverse effects on hemocompatibility in vitro. Thus, it may be an interesting candidate for further in vivo studies with the aim to increase the efficiency of conventional dialysis techniques.

Coumarins and Quinolones as Effective Multiple Targeted Agents Versus Covid-19: An in Silico Study

2021 ◽  
Vol 17 ◽  
Author(s):  
Mojgan Nejabat ◽  
Razieh Ghodsi ◽  
Farzin Hadizadeh
Keyword(s):  
Binding Affinity ◽  
In Silico ◽  
Oral Absorption ◽  
Antiviral Agent ◽  
In Vivo Studies ◽  
Reference Drug ◽  
Viral Proteases ◽  
In Silico Study

Background: The Covid-19 virus emerged a few months ago in China and infections rapidly escalated into a pandemic. Objective: To date, there is no selective antiviral agent for the management of pathologies associated with covid-19 and the need for an effective agent against it is essential. Method: In this work two home-made databases from synthetic quinolines and coumarins were virtually docked against viral proteases (3CL and PL), human cell surface proteases (TMPRSS2 and furin) and spike proteins (S1 and S2). Chloroquine, a reference drug without a clear mechanism against coronavirus was also docked on mentioned targets and the binding affinities compared with title compounds. Result: The best compounds of synthetic coumarins and quinolines for each target were determined. All compounds against all targets showed binding affinity between -5.80 to -8.99 kcal/mol in comparison with the FDA-approved drug, Chloroquine, with binding affinity of -5.7 to -7.98 kcal/mol. Two compounds, quinoline-1 and coumarin-24, were found to be effective on three targets – S2, TMPRSS2 and furin – simultaneously, with good predicted affinity between -7.54 to -8.85 kcal/mol. In silico ADME studies also confirmed good oral absorption for them. Furthermore, PASS prediction was calculated and coumarin-24 had higher probable activity (Pa) than probable inactivity (Pi) with acceptable protease inhibitory as well as good antiviral activity against Hepatitis C virus (HCV), Human immunodeficiency virus (HIV) and influenza. Conclusion: Quinoline-1 and Coumarin-24 have the potential to be used against Covid-19. Hence these agents could be useful in combating covid-19 infection after further in vitro and in vivo studies.

scholarly journals Structural and Functional Analysis of the cis-Acting Elements Required for Plus-Strand RNA Synthesis of Bamboo Mosaic Virus

2005 ◽  
Vol 79 (14) ◽  
pp. 9046-9053 ◽  
Author(s):  
Jen-Wen Lin ◽  
Hsiao-Ning Chiu ◽  
I-Hsuan Chen ◽  
Tzu-Chi Chen ◽  
Yau-Heiu Hsu ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Minus Strand ◽  
Internal Deletion ◽  
Stem Loop ◽  
Cis Acting ◽  
Rna Accumulation

ABSTRACT Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome. The secondary structure of the 3′-terminal sequence of the minus-strand RNA has been predicted by MFOLD and confirmed by enzymatic structural probing to consist of a large, stable stem-loop and a small, unstable stem-loop. To identify the promoter for plus-strand RNA synthesis in this region, transcripts of 39, 77, and 173 nucleotides (Ba-39, Ba-77, and Ba-173, respectively) derived from the 3′ terminus of the minus-strand RNA were examined by an in vitro RNA-dependent RNA polymerase assay for the ability to direct RNA synthesis. Ba-77 and Ba-39 appeared to direct the RNA synthesis efficiently, while Ba-173 failed. Ba-77/Δ5, with a deletion of the 3′-terminal UUUUC sequence in Ba-77, directed the RNA synthesis only to 7% that of Ba-77. However, Ba-77/Δ16 and Ba-77/Δ31, with longer deletions but preserving the terminal UUUUC sequence of Ba-77, restored the template activity to about 60% that of the wild type. Moreover, mutations that changed the sequence in the stem of the large stem-loop interfered with the efficiency of RNA synthesis and RNA accumulation in vivo. The mutant with an internal deletion in the region between the terminal UUUUC sequence and the large stem-loop reduced the viral RNA accumulation in protoplasts, but mutants with insertions did not. Taken together, these results suggest that three cis-acting elements in the 3′ end of the minus-strand RNA, namely, the terminal UUUUC sequence, the sequence in the large stem-loop, and the distance between these two regions, are involved in modulating the efficiency of BaMV plus-strand viral RNA synthesis.

Expression of Alfalfa Mosaic virus RNA 4 cDNA transcripts in Vitro and in Vivo

Virology ◽  
1985 ◽  
Vol 146 (2) ◽  
pp. 177-187 ◽  
Author(s):  
L. Sue Loesch-Fries ◽  
Nancy P. Jarvis ◽  
Karen J. Krahn ◽  
Steven E. Nelson ◽  
Timothy C. Hall
Keyword(s):  
Mosaic Virus ◽  
Alfalfa Mosaic Virus ◽  
Virus Rna

scholarly journals IN VITRO AND IN VIVO INACTIVATION OF TOP AND BOTTOM FRACTION OF ALFALFA MOSAIC VIRUS (AMV)

Uirusu ◽  
1969 ◽  
Vol 19 (4) ◽  
pp. 164-165
Author(s):  
Tsutomu MATSUMOTO ◽  
Daiki MURAYAMA
Keyword(s):  
Mosaic Virus ◽  
Alfalfa Mosaic Virus ◽  
Bottom Fraction

scholarly journals Brome Mosaic Virus RNA Syntheses In Vitro and in Barley Protoplasts

2003 ◽  
Vol 77 (10) ◽  
pp. 5703-5711 ◽  
Author(s):  
K. Sivakumaran ◽  
M. Hema ◽  
C. Cheng Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Previous Analysis ◽  
Brome Mosaic Virus ◽  
Minus Strand ◽  
Stem Loop ◽  
Virus Rna ◽  
Different Levels

ABSTRACT The RNA replicase extracted from Brome mosaic virus (BMV)-infected plants has been used to characterize the cis-acting elements for RNA synthesis and the mechanism of RNA synthesis. Minus-strand RNA synthesis in vitro requires a structure named stem-loop C (SLC) that contains a clamped adenine motif. In vitro, there are several specific requirements for SLC recognition. We examined whether these requirements also apply to BMV replication in barley protoplasts. BMV RNA3s with mutations in SLC were transfected into barley protoplasts, and the requirements for minus- and plus-strand replication were found to correlate well with the requirements in vitro. Furthermore, previous analysis of replicase recognition of the Cucumber mosaic virus (CMV) and BMV SLCs indicates that the requirements in the BMV SLC are highly specific. In protoplasts, we found that BMV RNA3s with their SLCs replaced with two different CMV SLCs were defective for replication. In vitro results generated with the BMV replicase and minimal-length RNAs generally agreed with those of in vivo BMV RNA replication. To extend this conclusion, we determined that, corresponding with the process of infection, the BMV replicases extracted from plants at different times after infection have different levels of recognition of the minimal promoters for plus- and minus-strand RNA syntheses.

scholarly journals P1101A BIFUNCTIONAL ADSORBER PARTICLE FOR THE REMOVAL OF HYDROPHOBIC UREMIC TOXINS FROM WHOLE BLOOD OF RENAL FAILURE PATIENTS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Marieke Sternkopf ◽  
Thimoteus Speer ◽  
Claudia Göttsch ◽  
Vera Jankowski ◽  
Joachim Jankowski ◽  
...  
Keyword(s):  
Adsorption Capacity ◽  
Binding Affinity ◽  
Whole Blood ◽  
Uremic Toxins ◽  
In Vivo Studies ◽  
Uremic Toxin ◽  
High Binding ◽  
High Binding Affinity

Abstract Background and Aims Hydrophobic uremic toxins accumulate in patients with chronic kidney disease, contributing to a highly increased cardiovascular risk. The clearance of these uremic toxins using current hemodialysis techniques is limited due to their hydrophobicity and their high binding affinity to plasma proteins. Adsorber techniques may be an appropriate alternative to increase hydrophobic uremic toxin removal. Method We developed an extracorporeal, whole-blood bifunctional adsorber particle consisting of a porous, activated charcoal core with a hydrophilic polyvinylpyrrolidone surface coating. The adsorption capacity was quantified using analytical chromatography after perfusion of the particles with an albumin solution or blood, each containing mixtures of hydrophobic uremic toxins. Results A time-dependent increase in hydrophobic uremic toxin adsorption was depicted and all toxins showed a high binding affinity to the adsorber particles. Further, the particle showed a sufficient hemocompatibility without significant effects on complement component 5a, thrombin-antithrombin III complex, or thrombocyte concentration in blood in vitro, although leukocyte counts were slightly reduced. Conclusion In conclusion, the bifunctional adsorber particle with cross-linked polyvinylpyrrolidone coating showed a high adsorption capacity without adverse effects on hemocompatibility in vitro. Thus, it may be an interesting candidate for further in vivo studies with the aim to increase the efficiency of conventional dialysis techniques.

scholarly journals Targeting dysregulation signatures of p53-MDM2 interactions to identify newer small molecule inhibitors for cancer therapy: Insight from computational direction

2021 ◽  
Author(s):  
Prosper Obed Chukwuemeka ◽  
Haruna Isiyaku Umar ◽  
Opeyemi Iwaloye ◽  
Oluwaseyi Matthew Oretade ◽  
Christopher Busayo Olowosoke ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Therapeutic Option ◽  
Pharmacophore Model ◽  
In Vivo Studies ◽  
Hydrogen Bond Acceptor ◽  
Potential Inhibitors

Abstract Dysregulation of the p53-MDM2 interactions has been implicated in majority of human tumors presenting a target for finding small molecule inhibitors. In this study, a training set of 17 experimentally tested inhibitors of MDM2 was used to develop series of pharmacophore models among which a four-featured (AHRR_1) model with one hydrogen bond acceptor, one hydrophobic group and two aromatic ring features and characterized by a survival score of 4.176 was considered significant among the top ranked generated hypothesis. Further, the model was validated by an external set of actives and decoy molecules and was found to exhibit encouraging statistical attributes (such as AUC > 0.7, BEDROC > 0.5 and EF > 1.0 etc). The model was used to screen the ZINC compound database, from the database, the top best 1375 hits satisfying the pharmacophore model was were docked to MDM2 protein to identify the likely interactions of the compounds as well as their binding affinity with MDM2. Further, druglikeness and pharmacokinetic properties screening on top-ranked compounds with higher binding affinity than reference inhibitors revealed four compounds (ZINC02639178, ZINC38933175, ZINC77969611, and ZINC06752762) with suitable pharmacological properties including low ligand toxicity. Investigation of the dynamic behaviour of each candidate inhibitors in complex with MDM2 via molecular dynamic simulation suggested ZINC02639178 and ZINC06752762 as the most potential inhibitors. Thus, these compounds may emerged as therapeutic option for cancer treatment after extensive in vitro and in vivo studies.

scholarly journals Disruption of Microtubule Organization and Centrosome Function by Expression of Tobacco Mosaic Virus Movement Protein

2006 ◽  
Vol 80 (12) ◽  
pp. 5807-5821 ◽  
Author(s):  
Jacqueline Ferralli ◽  
Jamie Ashby ◽  
Monika Fasler ◽  
Vitaly Boyko ◽  
Manfred Heinlein
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Movement Protein ◽  
Infected Plant ◽  
In Vivo Studies ◽  
Microtubule Organization

ABSTRACT The movement protein (MP) of Tobacco mosaic virus mediates the cell-to-cell transport of viral RNA through plasmodesmata, cytoplasmic cell wall channels for direct cell-to-cell communication between adjacent cells. Previous in vivo studies demonstrated that the RNA transport function of the protein correlates with its association with microtubules, although the exact role of microtubules in the movement process remains unknown. Since the binding of MP to microtubules is conserved in transfected mammalian cells, we took advantage of available mammalian cell biology reagents and tools to further address the interaction in flat-growing and transparent COS-7 cells. We demonstrate that neither actin, nor endoplasmic reticulum (ER), nor dynein motor complexes are involved in the apparent alignment of MP with microtubules. Together with results of in vitro coprecipitation experiments, these findings indicate that MP binds microtubules directly. Unlike microtubules associated with neuronal MAP2c, MP-associated microtubules are resistant to disruption by microtubule-disrupting agents or cold, suggesting that MP is a specialized microtubule binding protein that forms unusually stable complexes with microtubules. MP-associated microtubules accumulate ER membranes, which is consistent with a proposed role for MP in the recruitment of membranes in infected plant cells and may suggest that microtubules are involved in this process. The ability of MP to interfere with centrosomal γ-tubulin is independent of microtubule association with MP, does not involve the removal of other tested centrosomal markers, and correlates with inhibition of centrosomal microtubule nucleation activity. These observations suggest that the function of MP in viral movement may involve interaction with the microtubule-nucleating machinery.

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