Studies on the secondary structure of single-stranded RNA from the bacteriophage MS2. II Analysis of the RNase IV cleavage products

Single-stranded RNA from the bacteriophage MS2 was cleaved into two unequal fragments using the Escherichia coli endonuclease RNase IV. The fragments were purified by sucrose gradient centrifugation and secondary structure maps of the purified fragments were prepared after spreading the RNAs in 0·5 mmMgCl₂. Comparison of these maps with those of native RNA permitted the identification of the 5′ and 3′ ends of the maps of native single-stranded RNA. In addition, the location of the cleavage site with respect to the secondary and tertiary structure of the RNA suggests that the conformation of the RNA around this site may be important in determining the specificity of cleavage by the enzyme.The approximate location of individual viral genes within the secondary structure map has been obtained by comparing the map of native RNA with known sequence data. A new model is proposed to explain the role of secondary structure, as seen in the electron microscope, in the regulation of the synthesis of coat protein and the viral subunit of the MS2 replicase.     

A new substrate for formylkynurenine formamidase: N',N alpha-diformylkynurenine.

Commercially available N′-formyl-l-kynurenine has been incorrectly identified. Evidence is presented to demonstrate that the identity of this substance is N′,N^α? diformyl-l-kynurenine. The diformylkynurenine reacts readily with formylkynurenine formamidase, but the product does not serve as substrate for kynureninase. The assay of the formamidase, therefore, can be done in the presence of, and without interference by, kynureninase with diformylkynurenine as substrate.     

Inhibition of Pancreatic β-Cell Ca2+/Calmodulin-dependent Protein Kinase II Reduces Glucose-stimulated Calcium Influx and Insulin Secretion,Impairing Glucose Tolerance

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is caused by Ca²⁺ entry via voltage-dependent Ca²⁺ channels. CaMKII is a key mediator and feedback regulator of Ca²⁺ signaling in many tissues, but its role in β-cells is poorly understood, especially in vivo. Here, we report that mice with conditional inhibition of CaMKII in β-cells show significantly impaired glucose tolerance due to decreased GSIS. Moreover, β-cell CaMKII inhibition dramatically exacerbates glucose intolerance following exposure to a high fat diet. The impairment of islet GSIS by β-cell CaMKII inhibition is not accompanied by changes in either glucose metabolism or the activities of K_ATP and voltage-gated potassium channels. However, glucose-stimulated Ca²⁺ entry via voltage-dependent Ca²⁺ channels is reduced in islet β-cells with CaMKII inhibition, as well as in primary wild-type β-cells treated with a peptide inhibitor of CaMKII. The levels of basal β-cell cytoplasmic Ca²⁺ and of endoplasmic reticulum Ca²⁺ stores are also decreased by CaMKII inhibition. In addition, CaMKII inhibition suppresses glucose-stimulated action potential firing frequency. These results reveal that CaMKII is a Ca²⁺ sensor with a key role as a feed-forward stimulator of β-cell Ca²⁺ signals that enhance GSIS under physiological and pathological conditions.     

The Crystal Structure of Shiga Toxin Type 2 with Bound Disaccharide Guides the Design of a Heterobifunctional Toxin Inhibitor

Shiga toxin type 2 (Stx2a) is clinically most closely associated with enterohemorrhagic E. coli O157:H7-mediated hemorrhagic colitis that sometimes progresses to hemolytic-uremic syndrome. The ability to express the toxin has been acquired by other Escherichia coli strains, and outbreaks of food poisoning have caused significant mortality rates as, for example, in the 2011 outbreak in northern Germany. Stx2a, an AB₅ toxin, gains entry into human cells via the glycosphingolipid receptor Gb₃. We have determined the first crystal structure of a disaccharide analog of Gb₃ bound to the B₅ pentamer of Stx2a holotoxin. In this Gb₃ analog, α-GalNAc replaces the terminal α-Gal residue. This co-crystal structure confirms previous inferences that two of the primary binding sites identified in the B₅ pentamer of Stx1 are also functional in Stx2a. This knowledge provides a rationale for the synthesis and evaluation of heterobifunctional antagonists for E. coli toxins that target Stx2a. Incorporation of GalNAc Gb₃ trisaccharide in a heterobifunctional ligand with an attached pyruvate acetal, a ligand for human amyloid P component, and conjugation to poly[acrylamide-co-(3-azidopropylmethacrylamide)] produced a polymer that neutralized Stx2a in a mouse model of Shigatoxemia.