Ser2]- and [SerP2] incretin analogs: comparison of dipeptidyl peptidase IV resistance and biological activities in vitro and in vivo

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide) are incretin hormones that reduce postprandial glycemic excursions via enhancing insulin release but are rapidly inactivated by enzymatic N-terminal truncation. As such, efforts have been made to improve their plasma stability by synthetic modification or by inhibition of the responsible protease, dipeptidyl peptidase (DP) IV. Here we report a parallel comparison of synthetic GIP and GLP-1 with their Ser2- and Ser(P)2-substituted analogs, examining receptor binding and activation, metabolic stability, and biological effects in vivo. Both incretins and their Ser2-substituted analogs showed similar EC50s (0.16-0.52 nm) and IC50s (4.3-8.1 nm) at their respective cloned receptors. Although both phosphoserine 2-modified (Ser(PO3H2); Ser(P)) peptides were able to stimulate maximal cAMP production and fully displace receptor-bound tracer, they showed significantly right-shifted concentration-response curves and binding affinities. Ser2-substituted analogs were moderately resistant to DP IV cleavage, whereas [Ser(P)2]GIP and [Ser(P)2] GLP-1 showed complete resistance to purified DP IV. It was shown that the Ser(P) forms were dephosphorylated in serum and thus in vivo act as precursor forms of Ser2-substituted analogs. When injected subcutaneously into conscious Wistar rats, all peptides reduced glycemic excursions (rank potency: [Ser(P)2]incretins > or = [Ser2] incretins > native hormones). Insulin determinations indicated that the reductions in postprandial glycemia were at least in part insulin-mediated. Thus it has been shown that despite having low in vitro bioactivity using receptor-transfected cells, in vivo potency of [Ser(P)2] incretins was comparable with or greater than that of native or [Ser2]peptides. Hence, Ser(P)2-modified incretins present as novel glucose-lowering agents.     

A standardized kinesin nomenclature

In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered.     

Microtubule depolymerization can drive poleward chromosome motion in fission yeast

Prometaphase kinetochores interact with spindle microtubules (MTs) to establish chromosome bi-orientation. Before becoming bi-oriented, chromosomes frequently exhibit poleward movements (P-movements), which are commonly attributed to minus end-directed, MT-dependent motors. In fission yeast there are three such motors: dynein and two kinesin-14s, Pkl1p and Klp2p. None of these enzymes is essential for viability, and even the triple deletion grows well. This might be due to the fact that yeasts kinetochores are normally juxtapolar at mitosis onset, removing the need for poleward chromosome movement during prometaphase. Anaphase P-movement might also be dispensable in a spindle that elongates significantly. To test this supposition, we have analyzed kinetochore dynamics in cells whose kinetochore-pole connections have been dispersed. In cells recovering from this condition, the maximum rate of poleward kinetochore movement was unaffected by the deletion of any or all of these motors, strongly suggesting that other factors, like MT depolymerization, can cause such movements in vivo. However, Klp2p, which localizes to kinetochores, contributed to the effectiveness of P-movement by promoting the shortening of kinetochore fibers.     

Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.     

GIP-overexpressing mice demonstrate reduced diet-induced obesity and steatosis, and improved glucose homeostasis

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone that potentiates glucose-stimulated insulin secretion during a meal. Since GIP has also been shown to exert β-cell prosurvival and adipocyte lipogenic effects in rodents, both GIP receptor agonists and antagonists have been considered as potential therapeutics in type 2 diabetes (T2DM). In the present study, we tested the hypothesis that chronically elevating GIP levels in a transgenic (Tg) mouse model would increase adipose tissue expansion and exert beneficial effects on glucose homeostasis. In contrast, although GIP Tg mice demonstrated enhanced β-cell function, resulting in improved glucose tolerance and insulin sensitivity, they exhibited reduced diet-induced obesity. Adipose tissue macrophage infiltration and hepatic steatosis were both greatly reduced, and a number of genes involved in lipid metabolism/inflammatory signaling pathways were found to be down-regulated. Reduced adiposity in GIP Tg mice was associated with decreased energy intake, involving overexpression of hypothalamic GIP. Together, these studies suggest that, in the context of over-nutrition, transgenic GIP overexpression has the potential to improve hepatic and adipocyte function as well as glucose homeostasis.     

The neuronal nicotinic acetylcholine receptors alpha 4* and alpha 6* differentially modulate dopamine release in mouse striatal slices

Striatal dopamine (DA) plays a major role in the regulation of motor coordination and in the processing of salient information. We used voltammetry to monitor DA-release evoked by electrical stimulation in striatal slices, where interneurons continuously release acetylcholine. Use of the α6-selective antagonist α-conotoxin MII[E11A] and α4 knockout mice enabled identification of two populations of DA-ergic fibers. The first population had a low action potential threshold, and action potential-evoked DA-release from these fibers was modulated by α6. The second population had a higher action potential threshold, and only α4(non-α6) modulated action potential-evoked DA-release. Striatal DA-ergic neurons fire in both tonic and phasic patterns. When stimuli were applied in a train to mimic phasic firing, more DA-release was observed in α4 knockout versus wild-type mice. Furthermore, block of α4(non-α6), but not of α6, increased DA release evoked by a train. These results indicate that there are different classes of striatal DA-ergic fibers that express different subtypes of nicotinic receptors.