Detection of mRNA degradation intermediates in tissues using the 3'-end poly(A)-tailing polymerase chain reaction method

It has become increasingly clear that mRNA stability is an important determinant of mRNA abundance in virtually all organisms. Although our understanding of prokaryotic lower eukaryotic mRNA stability mechanisms has progressed considerably, little is known about mammalian mRNA stability mechanisms, particularly at the tissue and animal levels. This is due largely to the lack of suitable methods to approach the problem. In this study, we have developed and refined the 3'-end poly(A)-tailing polymerase chain reaction (PCR) method to detect degradation intermediates in vivo. Using an in vitro transcribed RNA as a template, we found that the method could be used to detect a homogeneous pool of RNA down to 0.1 ng. The addition of 10 microg of total RNA from tissues decreased the sensitivity limit to 4 ng. Detection limits of the technique were determined precisely by varying the concentrations of in vitro transcribed RNA in a constant amount of total RNA and varying the concentration of total RNA while maintaining a constant amount of in vitro transcribed RNA. Our overall results showed that the poly(A)-tailing PCR method could be used to detect specific RNA species of approximately 1000 nt in a pool of heterogeneous RNA in the range of 1 in 2500 to 1 in 10,000. To our knowledge, this is the most sensitive method to date for identifying mRNA degradation intermediates. Employing sense strand gene-specific primers in this method, we have discovered the class II and class III P-glycoprotein (Pgp) mRNA degradation intermediates in normal rat tissues. This method should serve as an additional tool to help us understand mRNA decay mechanisms in tissues and at animal levels.     

Structural understanding of stabilization patterns in engineered bispecific Ig‐like antibody molecules

Bispecific immunoglobulin‐like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin‐beta receptor (LTβR) binding Fv domain of an anti‐LTβR/anti‐TNF‐related apoptosis inducing ligand receptor‐2 (TRAIL‐R2) bispecific immunoglobulin‐like antibody. We further describe a new hierarchical structure‐guided approach toward engineering of antibody‐like molecules to enhance their thermal and chemical stability. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles

Alteration of the mouse genome through homologous recombination in embryonic stem (ES) cells is the most accurate and versatile way to dissect gene function in a vertebrate model. Most often, a selectable marker is used to create a knockout allele by replacing an essential part of the gene. However, knockout strategies are limited because the mutation is present constitutively. Conditional approaches based on the Cre-loxP site-specific recombination (SSR) system address this limitation; however, it requires that all parts of the targeted gene remain in ES cells. Here we report success with a "knockout-first" strategy that ablates gene function by insertion of RNA processing signals without deletion of any of the target gene. Incorporation of site-specific recombination target sites creates a multipurpose allele for both knockout and conditional applications.     

Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities

Influenza A virus specificity for the host is mediated by the viral surface glycoprotein hemagglutinin (HA), which binds to receptors containing glycans with terminal sialic acids. Avian viruses preferentially bind to alpha2-3-linked sialic acids on receptors of intestinal epithelial cells, whereas human viruses are specific for the alpha2-6 linkage on epithelial cells of the lungs and upper respiratory tract. To define the receptor preferences of a number of human and avian H1 and H3 viruses, including the 1918 H1N1 pandemic strains, their hemagglutinins were analyzed using a recently described glycan array. The array, which contains 200 carbohydrates and glycoproteins, not only revealed clear differentiation of receptor preferences for alpha2-3 and/or alpha2-6 sialic acid linkage, but could also detect fine differences in HA specificity, such as preferences for fucosylation, sulfation and sialylation at positions 2 (Gal) and 3 (GlcNAc, GalNAc) of the terminal trisaccharide. For the two 1918 HA variants, the South Carolina (SC) HA (with Asp190, Asp225) bound exclusively alpha2-6 receptors, while the New York (NY) variant, which differed only by one residue (Gly225), had mixed alpha2-6/alpha2-3 specificity, especially for sulfated oligosaccharides. Only one mutation of the NY variant (Asp190Glu) was sufficient to revert the HA receptor preference to that of classical avian strains. Thus, the species barrier, as defined by the receptor specificity preferences of 1918 human viruses compared to likely avian virus progenitors, can be circumvented by changes at only two positions in the HA receptor binding site. The glycan array thus provides highly detailed profiles of influenza receptor specificity that can be used to map the evolution of new human pathogenic strains, such as the H5N1 avian influenza.     

Heterocyclic ketones as inhibitors of histone deacetylase

Several heterocyclic ketones were investigated as potential inhibitors of histone deacetylase. Nanomolar inhibitors such as 22 and 25 were obtained, the anti-proliferative activity of which were shown to be mediated by HDAC inhibition.     

Epidemiologic and environmental characterization of the Re-emergence of St. Louis Encephalitis Virus in California, 2015–2020

St. Louis encephalitis virus (SLEV) is an endemic flavivirus in the western and southeastern United States, including California. From 1938 to 2003, the virus was detected annually in California, but after West Nile virus (WNV) arrived in 2003, SLEV was not detected again until it re-emerged in Riverside County in 2015. The re-emerging virus in California and other areas of the western US is SLEV genotype III, which previously had been detected only in Argentina, suggesting a South American origin. This study describes SLEV activity in California since its re-emergence in 2015 and compares it to WNV activity during the same period. From 2015 to 2020, SLEV was detected in 1,650 mosquito pools and 26 sentinel chickens, whereas WNV was detected concurrently in 18,108 mosquito pools and 1,542 sentinel chickens from the same samples. There were 24 reported human infections of SLEV in 10 California counties, including two fatalities (case fatality rate: 8%), compared to 2,469 reported human infections of WNV from 43 California counties, with 143 fatalities (case fatality rate: 6%). From 2015 through 2020, SLEV was detected in 17 (29%) of California’s 58 counties, while WNV was detected in 54 (93%). Although mosquitoes and sentinel chickens have been tested routinely for arboviruses in California for over fifty years, surveillance has not been uniform throughout the state. Of note, since 2005 there has been a steady decline in the use of sentinel chickens among vector control agencies, potentially contributing to gaps in SLEV surveillance. The incidence of SLEV disease in California may have been underestimated because human surveillance for SLEV relied on an environmental detection to trigger SLEV patient screening and mosquito surveillance effort is spatially variable. In addition, human diagnostic testing usually relies on changes in host antibodies and SLEV infection can be indistinguishable from infection with other flaviviruses such as WNV, which is more prevalent.     

11-(Ethylthio)undecanoic acid. A myristic acid analogue of altered hydrophobicity which is functional for peptide N-myristoylation with wheat germ and yeast acyltransferase

The covalent attachment of myristic acid to the NH2-terminal glycine residue of proteins is catalyzed by the enzyme myristoyl CoA:protein N-myristoyltransferase (NMT). Using synthetic octapeptide substrates we have identified and characterized an NMT activity in wheat germ lysates used for cell-free translation of exogenous mRNAs. C-12 and C-14 fatty acids are efficiently transferred to the peptides by this plant NMT, but C-10 and C-16 fatty acids are not. Glycine is required as the NH2-terminal residue: peptides with an NH2-terminal alanine were not substrates. Peptides with proline, aspartic acid, or tyrosine residues adjacent to the NH2-terminal glycine were also not myristoylated. Serine in the fifth position reduced the peptide's Km up to 4000-fold. We have chemically synthesized a sulfur analogue of myristate, 11-(ethylthio)undecanoic acid. Its CoA ester is as good a substrate as myristoyl-CoA for both wheat germ and yeast NMT. Peptides linked to 11-(ethylthio)undecanoic acid are less hydrophobic than the corresponding myristoylpeptides. 11-(Ethylthio)-undecanoic acid may, therefore, help define the role of myristic acid in targeting of acyl proteins within cells.     

Ca2+-dependent cross-linking processes in human platelets

When platelet cytoplasmic Ca²⁺ is increased by the ionophore A23187 in the presence of the protease inhibitor leupeptin, there is the coincident appearance of a cross-linked polymer and the partial disappearance of monomeric protein and glycoprotein units. In the absence of leupeptin only 30% of the polymer was formed. The disappearance of monomeric protein bands, as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is prevented by histamine, which as a pseudodonor amine is a known inhibitor of transglutaminase-catalyzed cross-linking. [¹⁴C]Histamine, at a tracer concentration, is incorporated into the polymer as well as into myosin, glycoproteins IIB and III, actin and tropomyosin. The lose of monomeric protein bands is mostly due to their conversion into polymers. Control measurements show that leupeptin effectively inhibited platelet Ca²⁺-dependent proteases. The cross-linking processes bringing about the observed increase in polymer formation are thus the result of a Ca²⁺-dependent platelet transglutaminase activity. The latter is located in the platelet cytosol and has been identified as platelet factor XIII on the basis of its specific cross-linking of fibrin. Platelet factor XIII, upon activation, may function physiologically to couple membrane proteins to cytoplasmic structural proteins. Thus, a new concept is proposed for the stabilization of platelet membranes and platelets as they form the hemostatic plug.