Murine Hepatitis Virus (mhv) Replication: A Molecular Study

L-2 cells infected with murine coronavirus (MHV) strains MHV-3 and JHM shut off host cell protein synthesis and synthesized polypeptides with apparent molecular weights 180,000 (p180), 56,000 (p56), 24,000, (p24) and 22,000 (p22) after a 15 minute pulse with {lcub}(\u2735)S{rcub}-methionine. During a 2 hour chase period performed late in infection (5.5 h PI) a fifth polypeptide of molecular weight 50,000 (p50) was detected. The corresponding p56 and p50 polypeptides of the two viruses were enriched in the basic amino acid arginine. Pulse-chase and peptide mapping experiments confirmed a precursor-product relationship between the p56 and p50 proteins. MHV-3 virions, however, showed no trace of the p50 protein. Moreover, of the total five detected polypeptides only three, p22, p56 and p180 were labeled in MHV-3-infected cells during a short two minute pulse with {lcub}(\u2735)S{rcub}-methionine and, are therefore, presumed to be immediate translation products. Pulse-chase and peptide mapping experiments confirmed that p24 is derived from p22 by post-translational modification. Further analyses with the glycosylation inhibitor tunicamycin provided strong evidence that conversion of p22 to p24 apparently does not involve N-linked glycosylation.;Analysis of {lcub}(\u2735)S{rcub}-methionine-labeled viral proteins by SDS polyacrylamide gel electrophoresis on five different murine hepatitis strains: A59, JHM, MHV-1, MHV-3, and MHV-S revealed that the proteins of each strain of MHV have very similar molecular weights. The strong degree of interstrain homology was confirmed by comparative HPLC peptide mapping of the viral nucleocapsid proteins.;Concurrent with the study of MHV polypeptides, intracellular viral-specific RNAs were also examined by using {lcub}(\u2732)P{rcub}-complementary DNA prepared against the A59 nucleocapsid-encoding mRNA in a Northern Transfer procedure as well as by in vitro labeling of MHV-infected cultures in the presence of actinomycin D and subsequent analyses in DMSO-urea agarose gels. The results demonstrate that MHV-infected cells generate six overlapping, polyadenylated, subgenomic RNAs all of which share common sequences and thus provide evidence for a nested set arrangement of coronavirus mRNAs. Hence coronavirus replication differs from that of other well characterized single-stranded, plus-sense RNA animal viruses such as that of the picornaviruses and alphaviruses

Stochastic Sensing of Nanomolar Inositol 1,4,5-Trisphosphate with an Engineered Pore

AbstractThe introduction of a ring of arginine residues near the constriction in the transmembrane β barrel of the staphylococcal α-hemolysin heptamer yielded a pore that could be almost completely blocked by phosphate anions at pH 7.5. Block did not occur with other oxyanions, including nitrate, sulfate, perchlorate, and citrate. Based on this finding, additional pores were engineered with high affinities for important cell signaling molecules, such as the Ca2+-mobilizing second messenger inositol 1,4,5-trisphosphate (IP3), that contain phosphate groups. One of these engineered pores, PRR-2, provides a ring of fourteen arginines that project into the lumen of the transmembrane barrel. Remarkably, PRR-2 bound IP3 with low nanomolar affinity while failing to bind another second messenger, adenosine 3′, 5′-cyclic monophosphate (cAMP). The engineered α-hemolysin pores may be useful as components of stochastic sensors for cell signaling molecules

High temperature ion channels and pores

The present invention includes an apparatus, system and method for stochastic sensing of an analyte to a protein pore. The protein pore may be an engineer protein pore, such as an ion channel at temperatures above 55.degree. C. and even as high as near 100.degree. C. The analyte may be any reactive analyte, including chemical weapons, environmental toxins and pharmaceuticals. The analyte covalently bonds to the sensor element to produce a detectable electrical current signal. Possible signals include change in electrical current. Detection of the signal allows identification of the analyte and determination of its concentration in a sample solution. Multiple analytes present in the same solution may also be detected

Carga laboral y notificación de eventos adversos en un centro hospitalario, Chimbote 2022

La investigación tenía como objetivo determinar la relación entre la carga laboral y la notificación de eventos adversos por el personal de salud de un Centro Hospitalario, Chimbote – 2022, para lo cual fue necesario el uso de la investigación aplicada y el diseño no experimental, siendo la población censal de 40 trabajadores, la técnica y el instrumento usado para el análisis de ambas variables fue la encuesta y el cuestionario, se halló que la dimensión de carga laboral y notificación de eventos adversos es percibida como regular (50%), finalmente se halló que el índice de relación fue de 0.722, por lo mencionado se determina que las variables se encuentran relacionadas

Semisynthetic Nanoreactor for Reversible Single-Molecule Covalent Chemistry

Protein engineering has been used to remodel pores for applications in biotechnology. For example, the heptameric alpha-hemolysin pore (alpha HL) has been engineered to form a nanoreactor to study covalent chemistry at the single -molecule level. Previous work has been confined largely to the chemistry of cysteine side chains or, in one instance, to an irreversible reaction of an unnatural amino acid side chain bearing a terminal alkyne. Here, we present four different alpha HL pores obtained by coupling either two or three fragments by native chemical ligation (NCL). The synthetic alpha HL monomers were folded and incorporated into heptameric pores. The functionality of the pores was validated by hemolysis assays and by single-channel current recording. By using NCL to introduce a ketone amino acid, the nanoreactor approach was extended to an investigation of reversible covalent chemistry on an unnatural side chain at the single -molecule level

Prolonged Residence Time of a Noncovalent Molecular Adapter, β-Cyclodextrin, within the Lumen of Mutant α-Hemolysin Pores

Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the α-hemolysin (αHL) pore, thereby offering a basis for the detection of a variety of organic molecules with αHL as a sensor element. β-Cyclodextrin (βCD) resides in the wild-type αHL pore for several hundred microseconds. The residence time can be extended to several milliseconds by the manipulation of pH and transmembrane potential. Here, we describe mutant homoheptameric αHL pores that are capable of accommodating βCD for tens of seconds. The mutants were obtained by site-directed mutagenesis at position 113, which is a residue that lies near a constriction in the lumen of the transmembrane β barrel, and fall into two classes. Members of the tight-binding class, M113D, M113N, M113V, M113H, M113F and M113Y, bind βCD ∼10(4)-fold more avidly than the remaining αHL pores, including WT-αHL. The lower K (d) values of these mutants are dominated by reduced values of k(off). The major effect of the mutations is most likely a remodeling of the binding site for βCD in the vicinity of position 113. In addition, there is a smaller voltage-sensitive component of the binding, which is also affected by the residue at 113 and may result from transport of the neutral βCD molecule by electroosmotic flow. The mutant pores for which the dwell time of βCD is prolonged can serve as improved components for stochastic sensors

Stochastic Study of the Effect of Ionic Strength on Noncovalent Interactions in Protein Pores

Salt plays a critical role in the physiological activities of cells. We show that ionic strength significantly affects the kinetics of noncovalent interactions in protein channels, as observed in stochastic studies of the transfer of various analytes through pores of wild-type and mutant α-hemolysin proteins. As the ionic strength increased, the association rate constant of electrostatic interactions was accelerated, whereas those of both hydrophobic and aromatic interactions were retarded. Dramatic decreases in the dissociation rate constants, and thus increases in the overall reaction formation constants, were observed for all noncovalent interactions studied. The results suggest that with the increase of salt concentration, the streaming potentials for all the protein pores decrease, whereas the preferential selectivities of the pores for either cations or anions drop. Furthermore, results also show that the salt effect on the rate of association of analytes to a pore differs significantly depending on the nature of the noncovalent interactions occurring in the protein channel. In addition to providing new insights into the nature of analyte-protein pore interactions, the salt-dependence of noncovalent interactions in protein pores observed provides a useful means to greatly enhance the sensitivity of the nanopore, which may find useful application in stochastic sensing

Unzipping Kinetics of Double-Stranded DNA in a Nanopore

We studied the unzipping kinetics of single molecules of double-stranded DNA by pulling one of their two strands through a narrow protein pore. PCR analysis yielded the first direct proof of DNA unzipping in such a system. The time to unzip each molecule was inferred from the ionic current signature of DNA traversal. The distribution of times to unzip under various experimental conditions fit a simple kinetic model. Using this model, we estimated the enthalpy barriers to unzipping and the effective charge of a nucleotide in the pore, which was considerably smaller than previously assumed.Comment: 10 pages, 5 figures, Accepted: Physics Review Letter

Prolonged Residence Time of a Noncovalent Molecular Adapter, β-Cyclodextrin, within the Lumen of Mutant α-Hemolysin Pores

Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the α-hemolysin (αHL) pore, thereby offering a basis for the detection of a variety of organic molecules with αHL as a sensor element. β-Cyclodextrin (βCD) resides in the wild-type αHL pore for several hundred microseconds. The residence time can be extended to several milliseconds by the manipulation of pH and transmembrane potential. Here, we describe mutant homoheptameric αHL pores that are capable of accommodating βCD for tens of seconds. The mutants were obtained by site-directed mutagenesis at position 113, which is a residue that lies near a constriction in the lumen of the transmembrane β barrel, and fall into two classes. Members of the tight-binding class, M113D, M113N, M113V, M113H, M113F and M113Y, bind βCD ∼10(4)-fold more avidly than the remaining αHL pores, including WT-αHL. The lower K (d) values of these mutants are dominated by reduced values of k(off). The major effect of the mutations is most likely a remodeling of the binding site for βCD in the vicinity of position 113. In addition, there is a smaller voltage-sensitive component of the binding, which is also affected by the residue at 113 and may result from transport of the neutral βCD molecule by electroosmotic flow. The mutant pores for which the dwell time of βCD is prolonged can serve as improved components for stochastic sensors