The effect of pramlintide on hormonal, metabolic or symptomatic responses to insulin-induced hypoglycaemia in patients with type 1 diabetes.

BACKGROUND: Pramlintide, a human amylin analogue, is a potential new adjunctive therapy to insulin for patients with type 1 diabetes and insulin-using patients with type 2 diabetes. Early clinical trials have shown a transient increased risk of hypoglycaemia in some patients at the time of initiating pramlintide therapy. This may be the result of combining the postprandial glucose, lowering effect of pramlintide with the existing hypoglycaemic potential of insulin without appropriate adjustment of insulin doses. However, the possibility that pramlintide may exert an independent detrimental effect on the physiological responses to insulin-induced hypoglycaemia needs to be excluded. METHODS: We conducted three separate randomized, placebo-controlled studies in patients with type 1 diabetes treated with adjunctive pramlintide. These studies utilized pramlintide at high doses (either 0.1-1 mg pramlintide daily or 0.1-0.8 mg pramlintide four times a day for 5 or 6 days) as well as doses closer to those anticipated for therapeutic usage (30, 100 or 300 microg three times daily for 14 days), and examined the hormonal, metabolic and symptomatic responses to an insulin-infusion hypoglycaemic challenge conducted at baseline and after days of therapy. RESULTS AND CONCLUSION: Pramlintide had no effect on the counter-regulatory hormonal, metabolic and symptomatic responses to hypoglycaemia. These findings demonstrated that pramlintide, when used as adjunctive therapy to insulin in patients with type 1 diabetes, has no independent effect on the response to hypoglycaemia.

Crystal structure of the N-terminal domain of Oxytricha nova telomere end-binding protein alpha subunit both uncomplexed and complexed with telomeric ssDNA.

Oxytricha nova telomere end-binding protein specifically recognizes and caps single strand (T(4)G(4))(n) telomeric DNA at the very 3'-ends of O. nova macronuclear chromosomes. Proteins homologous to the N-terminal domain of OnTEBP alpha subunit have now been identified in Oxytricha trifallax, Stylonychia mytilis, Euplotes crassus, Schizosaccharomyces pombe, and Homo sapiens, suggesting that this protein is widely distributed in eukaryotes. We describe here the crystal structures of the N-terminal single-stranded DNA (ssDNA)-binding domain of O. nova telomere end-binding protein alpha subunit both uncomplexed and complexed with single strand telomeric DNA. These structures show how the N-terminal domain of alpha alone, in the absence of the beta subunit and without alpha dimerization, can bind single-stranded telomeric DNA in a sequence-specific and 3'-end-specific manner. Furthermore, comparison of the uncomplexed and complexed forms of this protein shows that the ssDNA-binding site is largely pre-organized in the absence of ssDNA with modest, but interesting, rearrangements of amino acid side-chains that compose the ssDNA-binding site. The structures described here extend our understanding of structures of O. nova telomeric complexes by adding uncomplexed and complexed forms of monomeric alpha to previously described structures for (alpha 56/ssDNA)(2) dimer and alpha 56/beta 28/ssDNA ternary complexes. We believe that each of these four structures represent intermediates in an ordered assembly/disassembly pathway for O. nova telomeric complexes.

Crystal structure of the Oxytricha nova telomere end binding protein complexed with single strand DNA.

Telomeres are specialized protein-DNA complexes that compose the ends of eukaryotic chromosomes. Telomeres protect chromosome termini from degradation and recombination and act together with telomerase to ensure complete genome replication. We have determined the crystal structure of the two-subunit Oxytricha nova telomere end binding protein (OnTEBP) complexed with single strand telomeric DNA at 2.8 A resolution. The structure reveals four oligonucleotide/oligosaccharide-binding folds, three of which form a deep cleft that binds the ssDNA, and a fourth that forms an unusual protein-protein interaction between the alpha and beta subunits. This structure provides a molecular description of how the two subunits of OnTEBP recognize and bind ssDNA to form a sequence-specific, telomeric nucleoprotein complex that caps the very 3' ends of chromosomes.

Kinetics of appearance and disappearance of classes of bacterial ice nuclei support an aggregation model for ice nucleus assembly.

The kinetics of appearance and disappearance of three classes of ice nuclei in Pseudomonas syringae was investigated under conditions where high-level expression of the ice nucleation phenotype was obtained. The appearance of types 1, 2, and 3 ice nuclei, catalyzing nucleation at -2 to -5, -5 to -7, and -7 to -10 degrees C, respectively, was investigated during low-temperature induction in wild-type strains and in a unique, detergent-sensitive mutant that contained no type 3 ice nuclei when grown at 32 degrees C. Nuclei appeared in the following order: type 3, then type 2 and type 1. The disappearance of classes of ice nuclei was monitored during high-temperature treatment of fully induced cells; nuclei disappeared in the order type 1, type 2, and type 3. Although analysis of nucleation events is complicated by masking and unmasking of ice sites in the same cells, these temporal sequences of ice nucleus appearance or disappearance are consistent with an aggregation model for ice nucleus assembly (A. G. Govindarajan and S. E. Lindow, Proc. Natl. Acad. Sci. USA 85:1334-1338, 1988; G. Warren and P. Wolber, Mol. Microbiol. 5:239-243, 1991).