Glucose promotes pancreatic islet beta-cell survival through a PI 3-kinase/Akt-signaling pathway

The concentration of glucose in plasma is an important determinant of pancreatic beta-cell mass, whereas the relative contributions of hypertrophy, proliferation, and cell survival to this process are unclear. Glucose results in depolarization and subsequent calcium influx into islet beta-cells. Because depolarization and calcium (Ca(2+)) influx promote survival of neuronal cells, we hypothesized that glucose might alter survival of islet beta-cells through a similar mechanism. In the present studies, cultured mouse islet beta-cells showed a threefold decrease in apoptosis under conditions of 15 mM glucose compared with 2 mM glucose (P < 0.05). MIN6 insulinoma cells incubated in 25 mM glucose for 24 h showed a threefold decrease in apoptosis compared with cells in 5 mM glucose (1.7 +/- 0.2 vs. 6.3 +/- 1%, respectively, P < 0.001). High glucose (25 mM) enhanced survival-required depolarization and Ca(2+) influx and was blocked by phosphatidylinositol (PI) 3-kinase inhibitors. Glucose activation of the protein kinase Akt was demonstrated in both insulinoma cells and cultured mouse islets by means of an antibody specific for Ser(473) phospho-Akt and by an in vitro Akt kinase assay. Akt phosphorylation was dependent on PI 3-kinase but not on MAPK. Transfection of insulinoma cells with an Akt kinase-dead plasmid (Akt-K179M) resulted in loss of glucose-mediated protection, whereas transfection with a constitutively active Akt enhanced survival in glucose-deprived insulinoma cells. The results of these studies defined a novel pathway for glucose-mediated activation of a PI 3-kinase/Akt survival-signaling pathway in islet beta-cells. This pathway may provide important targets for therapeutic intervention.     

Design,Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors

Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K_i and IC₅₀ values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC₅₀ values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a).     

Juvenile hormone catabolism and oviposition in the codling moth, Cydia pomonella, as functions of age, mating status, and hormone treatment.

In vitro catabolism of juvenile hormone (JH) in haemolymph of adult female Cydia pomonella was ascribed mainly to juvenile hormone esterase (JHE) activity. No significant differences were noted between virgin and mated females 0-96 h post-emergence. Changes in JHE activity did not appear dependent upon fluctuations in JH titre; conversely, changes in JHE activity could not explain the changes in JH titres. Maximal JHE activity was recorded at 24 h (331.47 +/- 47.25 pmol/h/microl; 355.93 +/- 36.68 pmol/h/microl, virgin; mated insects, respectively) and preceded the peak in JH titres at 48 h. Topical application of JH II (10 ng-10 microg) or fenoxycarb (50 ng) enhanced JHE activity up to 640 and 56%, respectively. Treatment upon emergence with 10 microg JH II induced enzymic activity for less than 24 h, and when 10 microg JH II or 50 ng fenoxycarb were applied, circulating JH titres returned to control levels within 24 h. Oviposition was highly sensitive to exogenous JH and declined significantly with dosages >100 pg. To allow a degree of oocyte maturation before JH treatment, the hormone was administered at 6, 12, 24, or 48 h post-emergence and/or females were mated. Neither measure "protected" the system; oviposition declined immediately after JH application.     

Cardiac mitochondrial preconditioning by Big Ca2+-sensitive K+ channel opening requires superoxide radical generation

ATP-sensitive K+ channel opening in inner mitochondrial membranes protects hearts from ischemia-reperfusion (I/R) injury. Opening of the Big conductance Ca2+-sensitive K+ channel (BK(Ca)) is now also known to elicit cardiac preconditioning. We investigated the role of the pharmacological opening of the BK(Ca) channel on inducing mitochondrial preconditioning during I/R and the role of O2-derived free radicals in modulating protection by putative mitochondrial (m)BK(Ca) channel opening. Left ventricular (LV) pressure (LVP) was measured with a balloon and transducer in guinea pig hearts isolated and perfused at constant pressure. NADH, reactive oxygen species (ROS), principally superoxide (O2(-*)), and m[Ca2+] were measured spectrophotofluorometrically at the LV free wall using autofluorescence and fluorescent dyes dihydroethidium and indo 1, respectively. BK(Ca) channel opener 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimid-axolone (NS; NS-1619) was given for 15 min, ending 25 min before 30 min of global I/R. Either Mn(III)tetrakis(4-benzoic acid)porphyrin (TB; MnTBAP), a synthetic dismutator of O2(-*), or an antagonist of the BK(Ca) channel paxilline (PX) was given alone or for 5 min before, during, and 5 min after NS. NS pretreatment resulted in a 2.5-fold increase in developed LVP and a 2.5-fold decrease in infarct size. This was accompanied by less O2(-*) generation, decreased m[Ca2+], and more normalized NADH during early ischemia and throughout reperfusion. Both TB and PX antagonized each preconditioning effect. This indicates that 1) NS induces a mitochondrial-preconditioned state, evident during early ischemia, presumably on mBK(Ca) channels; 2) NS effects are blocked by BK(Ca) antagonist PX; and 3) NS-induced preconditioning is dependent on the production of ROS. Thus NS may induce mitochondrial ROS release to initiate preconditioning.     

Comparative analyses of quaternary arrangements in homo‐oligomeric proteins in superfamilies: Functional implications

A comprehensive analysis of the quaternary features of distantly related homo‐oligomeric proteins is the focus of the current study. This study has been performed at the levels of quaternary state, symmetry, and quaternary structure. Quaternary state and quaternary structure refers to the number of subunits and spatial arrangements of subunits, respectively. Using a large dataset of available 3D structures of biologically relevant assemblies, we show that only 53% of the distantly related homo‐oligomeric proteins have the same quaternary state. Considering these homologous homo‐oligomers with the same quaternary state, conservation of quaternary structures is observed only in 38% of the pairs. In 36% of the pairs of distantly related homo‐oligomers with different quaternary states the larger assembly in a pair shows high structural similarity with the entire quaternary structure of the related protein with lower quaternary state and it is referred as “Russian doll effect.” The differences in quaternary state and structure have been suggested to contribute to the functional diversity. Detailed investigations show that even though the gross functions of many distantly related homo‐oligomers are the same, finer level differences in molecular functions are manifested by differences in quaternary states and structures. Comparison of structures of biological assemblies in distantly and closely related homo‐oligomeric proteins throughout the study differentiates the effects of sequence divergence on the quaternary structures and function. Knowledge inferred from this study can provide insights for improved protein structure classification and function prediction of homo‐oligomers. Proteins 2016; 84:1190–1202. © 2016 Wiley Periodicals, Inc.     

Rest-inserted loading rapidly amplifies the response of bone to small increases in strain and load cycles.

We hypothesized that a 10-s rest interval (at zero load) inserted between each load cycle would increase the osteogenic effects of mechanical loading near previously identified thresholds for strain magnitude and cycle numbers. We tested our hypothesis by subjecting the right tibiae of female C57BL/6J mice (16 wk, n = 70) to exogenous mechanical loading within a peri-threshold physiological range of strain magnitudes and load cycle numbers using a noninvasive murine tibia loading device. Bone responses to mechanical loading were determined via dynamic histomorphometry. More specifically, we contrasted bone formation induced by cyclic vs. rest-inserted loading (10-s rest at zero load inserted between each load cycle) by first varying peak strains (1,000, 1,250, or 1,600 micro epsilon) at fixed cycle numbers (50 cycles/day, 3 days/wk for 3 wk) and then varying cycle numbers (10, 50, or 250 cycles/day) at a fixed strain magnitude (1,250 micro epsilon). Within the range of strain magnitudes tested, the slope of periosteal bone formation rate (p.BFR/BS) with increasing strain magnitudes was significantly increased by rest-inserted compared with cyclical loading. Within the range of load cycles tested, the slope of p.BFR/BS with increasing load cycles of rest-inserted loading was also significantly increased by rest-inserted compared with cyclical loading. In sum, the data of this study indicate that inserting a 10-s rest interval between each load cycle amplifies bone's response to mechanical loading, even within a peri-threshold range of strain magnitudes and cycle numbers.     

Design and activity of AP endonuclease-1 inhibitors

Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE-1) is a critical component of base excision repair that excises abasic lesions created enzymatically by the action of DNA glycosylases on modified bases and non-enzymatically by hydrolytic depurination/depyrimidination of nucleobases. Many anticancer drugs generate DNA adducts that are processed by base excision repair, and tumor resistance is frequently associated with enhanced APE-1 expression. Accordingly, APE-1 is a potential therapeutic target to treat cancer. Using computational approaches and the high resolution structure of APE-1, we developed a 5-point pharmacophore model for APE-1 small molecule inhibitors. One of the nM APE-1 inhibitors (AJAY-4) that was identified based on this model exhibited an overall median growth inhibition (GI₅₀) of 4.19 μM in the NCI-60 cell line panel. The mechanism of action is shown to be related to the buildup of abasic sites that cause PARP activation and PARP cleavage, and the activation of caspase-3 and caspase-7, which is consistent with cell death by apoptosis. In a drug combination growth inhibition screen conducted in 10 randomly selected NCI-60 cell lines and with 20 clinically used non-genotoxic anticancer drugs, a synergy was flagged in the SK-MEL-5 melanoma cell line exposed to combinations of vemurafenib, which targets melanoma cells with V600E mutated BRAF, and AJAY-4, our most potent APE-1 inhibitor. The synergy between AJAY-4 and vemurafenib was not observed in cell lines expressing wild-type B-Raf protein. This synergistic combination may provide a solution to the resistance that develops in tumors treated with B-Raf-targeting drugs.

Electronic supplementary material

The online version of this article (doi:10.1007/s12154-015-0131-7) contains supplementary material, which is available to authorized users.     

CD8+ T Cell Fate and Function Influenced by Antigen-Specific Virus-Like Nanoparticles Co-Expressing Membrane Tethered IL-2

A variety of adjuvants fostering humoral immunity are known as of today. However, there is a lack of adjuvants or adjuvant strategies, which directly target T cellular effector functions and memory. We here determined whether systemically toxic cytokines such as IL-2 can be restricted to the site of antigen presentation and used as ‘natural adjuvants’. Therefore, we devised antigen-presenting virus-like nanoparticles (VNP) co-expressing IL-2 attached to different membrane-anchors and assessed their potency to modulate CD8⁺ T cell responses in vitro and in vivo. Efficient targeting of IL-2 to lipid rafts and ultimately VNP was achieved by fusing IL-2 at its C-terminus to a minimal glycosylphosphatidylinositol (GPI)-anchor acceptor sequence. To identify optimal membrane-anchor dimensions we inserted one (1Ig), two (2Ig) or four (4Ig) immunoglobulin(Ig)-like domains of CD16b between IL-2 and the minimal GPI-anchor acceptor sequence of CD16b (GPI). We found that the 2IgGPI version was superior to all other evaluated IL-2 variants (IL-2v) in terms of its i) degree of targeting to lipid rafts and to the VNP surface, ii) biological activity, iii) co-stimulation of cognate T cells in the absence of bystander activation and iv) potency to induce differentiation and acquisition of CD8⁺ T cell effector functions in vitro and in vivo. In contrast, the GPI version rather favored memory precursor cell formation. These results exemplify novel beneficial features of membrane-bound IL-2, which in addition to its mere T cell stimulatory capacity include the induction of differential effector and memory functions in CD8⁺ T lymphocytes.     

Phylogeographical Structure in Mitochondrial DNA of Legume Pod Borer (Maruca vitrata) Population in Tropical Asia and Sub-Saharan Africa

This study was undertaken to assess the genetic diversity and host plant races of M. vitrata population in South and Southeast Asia and sub-Saharan Africa. The cytochrome c oxidase subunit 1 (cox1) gene was used to understand the phylogenetic relationship of geographically different M. vitrata population, but previous studies did not include population from Southeast Asia, the probable center of origin for Maruca, and from east Africa. Extensive sampling was done from different host plant species in target countries. Reference populations from Oceania and Latin America were used. An amplicon of 658 bp was produced by polymerase chain reaction, and 64 haplotypes were identified in 686 M. vitrata individuals. Phylogenetic analysis showed no difference among the M. vitrata population from different host plants. However, the results suggested that M. vitrata has formed two putative subspecies (which cannot be differentiated based on morphological characters) in Asia and sub-Saharan Africa, as indicated by the high pairwise F_ST values (0.44–0.85). The extremely high F_ST values (≥0.93) of Maruca population in Latin America and Oceania compared to Asian and African population seem to indicate a different species. On the continental or larger geographical region basis, the genetic differentiation is significantly correlated with the geographical distance. In addition, two putative species of Maruca, including M. vitrata occur in Australia, Indonesia and Papua New Guinea. The negative Tajima’s D and Fu’s F_S values showed the recent demographic expansion of Maruca population. The haplotype network and Automatic Barcode Gap Discovery analyses confirmed the results of phylogenetic analysis. Thus, this study confirmed the presence of three putative Maruca species, including one in Latin America, one in Oceania (including Indonesia) and M. vitrata in Asia, Africa and Oceania. Hence, the genetic differences in Maruca population should be carefully considered while designing the pest management strategies in different regions.