scholarly journals Stimulation of ribosomal frameshifting by antisense LNA

2010 ◽  
Vol 38 (22) ◽  
pp. 8277-8283 ◽  
Author(s):  
Chien-Hung Yu ◽  
Mathieu H. M. Noteborn ◽  
René C. L. Olsthoorn
Keyword(s):  
Rna Stability ◽  
Three Dimensional ◽  
Fixed Ratio ◽  
Essential Element ◽  
Dimensional Structure ◽  
Ribosomal Frameshifting ◽  
Dna Oligonucleotides ◽  
Ribosomal Tunnel ◽  
Slippery Sequence

Abstract Programmed ribosomal frameshifting is a translational recoding mechanism commonly used by RNA viruses to express two or more proteins from a single mRNA at a fixed ratio. An essential element in this process is the presence of an RNA secondary structure, such as a pseudoknot or a hairpin, located downstream of the slippery sequence. Here, we have tested the efficiency of RNA oligonucleotides annealing downstream of the slippery sequence to induce frameshifting in vitro. Maximal frameshifting was observed with oligonucleotides of 12–18 nt. Antisense oligonucleotides bearing locked nucleid acid (LNA) modifications also proved to be efficient frameshift-stimulators in contrast to DNA oligonucleotides. The number, sequence and location of LNA bases in an otherwise DNA oligonucleotide have to be carefully manipulated to obtain optimal levels of frameshifting. Our data favor a model in which RNA stability at the entrance of the ribosomal tunnel is the major determinant of stimulating slippage rather than a specific three-dimensional structure of the stimulating RNA element.

scholarly journals Nanofiber-Mâché Hollow Ball Mimicking the Three-Dimensional Structure of a Cyst

Polymers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 2273
Author(s):  
Wan-Ying Huang ◽  
Norichika Hashimoto ◽  
Ryuhei Kitai ◽  
Shin-ichiro Suye ◽  
Satoshi Fujita
Keyword(s):  
Three Dimensional ◽  
Hollow Structure ◽  
Dimensional Structure ◽  
The Novel ◽  
Fibrous Scaffold ◽  
Hydrogel Beads ◽  
Inner Surface ◽  
Intracranial Epidermoid ◽  
Hollow Ball

The occasional malignant transformation of intracranial epidermoid cysts into squamous cell carcinomas remains poorly understood; the development of an in vitro cyst model is urgently needed. For this purpose, we designed a hollow nanofiber sphere, the “nanofiber-mâché ball.” This hollow structure was fabricated by electrospinning nanofiber onto alginate hydrogel beads followed by dissolving the beads. A ball with approximately 230 mm3 inner volume provided a fibrous geometry mimicking the topography of the extracellular matrix. Two ducts located on opposite sides provided a route to exchange nutrients and waste. This resulted in a concentration gradient that induced oriented migration, in which seeded cells adhered randomly to the inner surface, formed a highly oriented structure, and then secreted a dense web of collagen fibrils. Circumferentially aligned fibers on the internal interface between the duct and hollow ball inhibited cells from migrating out of the interior, similar to a fish bottle trap. This structure helped to form an adepithelial layer on the inner surface. The novel nanofiber-mâché technique, using a millimeter-sized hollow fibrous scaffold, is excellently suited to investigating cyst physiology.

scholarly journals Protein Folding: Search for Basic Physical Models

2003 ◽  
Vol 3 ◽  
pp. 623-635 ◽  
Author(s):  
Ivan Y. Torshin ◽  
Robert W. Harrison
Keyword(s):  
Protein Folding ◽  
Tertiary Structure ◽  
Three Dimensional ◽  
Physical Models ◽  
Dimensional Structure ◽  
Computational Techniques ◽  
Linear Sequence ◽  
Physical Forces

How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context ofin vivoprotein folding (which has been studied only for a few proteins), the roles of the fundamental physical forces in thein vitrofolding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces). Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

scholarly journals Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

2008 ◽  
Vol 190 (6) ◽  
pp. 2056-2064 ◽  
Author(s):  
Jonathan E. Ulmer ◽  
Yap Boum ◽  
Christopher D. Thouvenel ◽  
Hannu Myllykallio ◽  
Carol Hopkins Sibley
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Thymidylate Synthase ◽  
Three Dimensional ◽  
Pyrimidine Ring ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Catalytic Residue ◽  
Amino Acid Residues ◽  
Insight Into

ABSTRACT A novel FAD-dependent thymidylate synthase, ThyX, is present in a variety of eubacteria and archaea, including the mycobacteria. A short motif found in all thyX genes, RHRX7-8S, has been identified. The three-dimensional structure of the Mycobacterium tuberculosis ThyX enzyme has been solved. Building upon this information, we used directed mutagenesis to produce 67 mutants of the M. tuberculosis thyX gene. Each enzyme was assayed to determine its ability to complement the defect in thymidine biosynthesis in a ΔthyA strain of Escherichia coli. Enzymes from selected strains were then tested in vitro for their ability to catalyze the oxidation of NADPH and the release of a proton from position 5 of the pyrimidine ring of dUMP. The results defined an extended motif of amino acids essential to enzyme activity in M. tuberculosis (Y44X24 H69X25R95HRX7 S105XRYX90R199 [with the underlined histidine acting as the catalytic residue and the underlined serine as the nucleophile]) and provided insight into the ThyX reaction mechanism. ThyX is found in a variety of bacterial pathogens but is absent in humans, which depend upon an unrelated thymidylate synthase, ThyA. Therefore, ThyX is a potential target for development of antibacterial drugs.

scholarly journals In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

2003 ◽  
Vol 77 (6) ◽  
pp. 3669-3679 ◽  
Author(s):  
Caterina Trozzi ◽  
Linda Bartholomew ◽  
Alessandra Ceccacci ◽  
Gabriella Biasiol ◽  
Laura Pacini ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
In Vitro Selection ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

Rational humanization of the powerful antithrombotic anti-GPIbα antibody: 6B4

2006 ◽  
Vol 96 (11) ◽  
pp. 671-684 ◽  
Author(s):  
Alexandre Fontayne ◽  
Karen Vanhoorelbeke ◽  
Inge Pareyn ◽  
Isabel Van Rompaey ◽  
Muriel Meiring ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Three Dimensional ◽  
Vector System ◽  
Dimensional Structure ◽  
Fab Fragment ◽  
Variable Regions ◽  
Significant Prolongation ◽  
Von Willebrand ◽  
First Time

SummaryFab-fragments of the monoclonal antibody 6B4, raised against human glycoprotein Ibα (GPIbα), have a powerful antithrombotic effect in baboons by blocking the GPIbα binding site for von Willebrand factor (VWF), without significant prolongation of the skin bleeding time. In order to bring this antibody to the clinic,we here humanized for the first time an anti-human GPIbα by variable-domain resurfacing guided by computer modeling. First, the genes coding for the variable regions of the heavy and light chains of 6B4 were cloned and sequenced. Based on this,a three-dimensional structure of the Fv-fragment was constructed by using homology-based modeling, and with this and comparison with antibodies with known structure,”murine” putative immunogenic residues which are exposed, were changed for “human-like” residues. The humanized Fab-fragment, h6B4-Fab, was constructed in the pKaneo vector system, expressed and purified and showed in vitro an unaltered, even slightly higher binding affinity for its antigen than the murine form as determined by different ELISA set-ups and surface plasmon resonance. Finally, injection of doses of 0.1 to 1.5 mg/kg of h6B4-Fab in baboons showed that both pharmacokinetics and ex-vivo bio-activity of the molecule were to a large extent preserved.In conclusion, the method used here to humanize 6B4 by resurfacing resulted in a fully active derivative, which is now ready for further development.

scholarly journals Role of YidC in folding of polytopic membrane proteins

2004 ◽  
Vol 165 (1) ◽  
pp. 53-62 ◽  
Author(s):  
Shushi Nagamori ◽  
Irina N. Smirnova ◽  
H. Ronald Kaback
Keyword(s):  
Membrane Proteins ◽  
Three Dimensional ◽  
In Vitro Transcription ◽  
Dimensional Structure ◽  
Lipid Phase ◽  
Lactose Permease ◽  
Primary Role ◽  
Protein Insertion

YidC of Echerichia coli, a member of the conserved Alb3/Oxa1/YidC family, is postulated to be important for biogenesis of membrane proteins. Here, we use as a model the lactose permease (LacY), a membrane transport protein with a known three-dimensional structure, to determine whether YidC plays a role in polytopic membrane protein insertion and/or folding. Experiments in vivo and with an in vitro transcription/translation/insertion system demonstrate that YidC is not necessary for insertion per se, but plays an important role in folding of LacY. By using the in vitro system and two monoclonal antibodies directed against conformational epitopes, LacY is shown to bind the antibodies poorly in YidC-depleted membranes. Moreover, LacY also folds improperly in proteoliposomes prepared without YidC. However, when the proteoliposomes are supplemented with purified YidC, LacY folds correctly. The results indicate that YidC plays a primary role in folding of LacY into its final tertiary conformation via an interaction that likely occurs transiently during insertion into the lipid phase of the membrane.

scholarly journals Complete In Vitro Assembly of the Reovirus Outer Capsid Produces Highly Infectious Particles Suitable for Genetic Studies of the Receptor-Binding Protein

2001 ◽  
Vol 75 (11) ◽  
pp. 5335-5342 ◽  
Author(s):  
Kartik Chandran ◽  
Xing Zhang ◽  
Norman H. Olson ◽  
Stephen B. Walker ◽  
James D. Chappell ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Cell Attachment ◽  
Molecular Genetic ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
Dependent Manner ◽  
Viral Attachment ◽  
Structure Assembly

ABSTRACT Mammalian reoviruses, prototype members of theReoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins—ς1, μ1, and ς3—to enter host cells. ς1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of ς1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by “recoating” genome-containing core particles that lacked ς1, μ1, and ς3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to ς1. The recoated particles bound to and infected cultured cells in a ς1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant ς1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the ς1 protein. Additional experiments showed that recoated particles containing ς1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound ς1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of ς1 with respect to its structure, assembly into particles, and roles in entry.

scholarly journals Characterisation of three novel CYP11B1 mutations in classic and non-classic 11β-hydroxylase deficiency

2014 ◽  
Vol 170 (5) ◽  
pp. 697-706 ◽  
Author(s):  
Seher Polat ◽  
Alexandra Kulle ◽  
Züleyha Karaca ◽  
Ilker Akkurt ◽  
Selim Kurtoglu ◽  
...  
Keyword(s):  
Expression System ◽  
Three Dimensional ◽  
Gene Mutations ◽  
Dimensional Structure ◽  
Hydroxylase Deficiency ◽  
Mutant Proteins ◽  
Maximum Reaction ◽  
Functional Consequences ◽  
21 Hydroxylase Deficiency

BackgroundCongenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine diseases. Steroid 11β-hydroxylase (P450c11) deficiency (11OHD) is the second most common form of CAH.AimThe aim of the study was to study the functional consequences of three novelCYP11B1gene mutations (p.His125Thrfs*8, p.Leu463_Leu464dup and p.Ser150Leu) detected in patients suffering from 11OHD and to correlate this data with the clinical phenotype.MethodsFunctional analyses were done by using a HEK293 cellin vitroexpression system comparing WT with mutant P450c11 activity. Mutant proteins were examinedin silicoto study their effect on the three-dimensional structure of the protein.ResultsTwo mutations (p.His125Thrfs*8 and p.Leu463_Leu464dup) detected in patients with classic 11OHD showed a complete loss of P450c11 activity. The mutation (p.Ser150Leu) detected in a patient with non-classic 11OHD showed partial functional impairment with 19% of WT activity.ConclusionFunctional mutation analysis enables the correlation of novelCYP11B1mutations to the classic and non-classic 11OHD phenotype respectively. Mutations causing a non-classic phenotype show typically partial impairment due to reduced maximum reaction velocity comparable with non-classic mutations in 21-hydroxylase deficiency. The increasing number of mutations associated with non-classic 11OHD illustrate that this disease should be considered as diagnosis in patients with otherwise unexplained hyperandrogenism.

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