Protein kinase Cdelta participates in insulin-induced activation of PKB via PDK1

PKCdelta has been shown to be activated by insulin and to interact with insulin receptor and IRS. PKB(Akt) plays an important role in glucose transport and glycogen synthesis. In this study, we investigated the possibility that PKCdelta may be involved in insulin-induced activation of PKB. Studies were conducted on primary cultures of rat skeletal muscle. PKB was activated by insulin stimulation within 5min and reached a peak by 15-30min. Insulin also increased the physical association between PKCdelta with PKB and with PDK1. The insulin-induced PKCdelta-PKB association was PI3K dependent. PKB-PKCdelta association was accounted for by the involvement of PDK1. Overexpression of dominant negative PKCdelta abrogated insulin-induced association of PKCdelta with both PKB and PDK1. Blockade of PKCdelta also decreased insulin-induced Thr308 PKB phosphorylation and PKB translocation. Moreover, PKCdelta inhibition reduced insulin-induced GSK3 phosphorylation. The results indicate that insulin-activated PKCdelta interacts with PDK1 to regulate PKB.     

Interaction between AT1 and AT2 receptors during postinfarction left ventricular remodeling

The relative contribution of the angiotensin II type 1 and 2 receptors (AT1-R and AT2-R) in postmyocardial infarction (MI) remodeling remains incompletely understood. We studied five groups of C57Bl/6 mice after 1 h of left anterior descending artery occlusion-reperfusion: 1) wild type, untreated (n = 12); 2) wild type, treated with the AT1-R blocker losartan (10-20 mg.kg(-1).day(-1) in drinking water) from day 1 to day 28 post-MI (n = 10); 3) cardiac overexpression of the AT2-R [AT2-transgenic (TG); n = 14]; 4) AT2-TG treated with losartan (n = 13); and 5) AT2-TG and null for the AT1a-R [AT2-TG/AT1 knockout (KO); n = 10]. Cardiac magnetic resonance imaging (CMR) measured ejection fraction and left ventricular end-diastolic and end-systolic volume (EDVI and ESVI) and mass indexed to weight on days 0, 1, 7, and 28 post-MI. Infarct size was measured on day 1 by late gadolinium-enhanced CMR. Regional myocyte hypertrophy and collagen content were measured on day 28 post-MI. Infarct size was similar among groups. Systolic blood pressure was lowest in AT2-TG/AT1KO. By day 28 post-MI, when corrected for baseline differences, EDVI and ESVI were higher and ejection fraction was lower in wild type than other groups. Ejection fraction was highest and EDVI and mass index were lowest in AT2-TG/AT1KO at day 28. The AT2-TG/AT1KO demonstrated less fibrosis in adjacent regions. Regional myocyte hypertrophy was similar in all groups. The AT1-R and AT2-R are intricately intertwined in post-MI remodeling. Pharmacological blockade of AT1-R is equivalent to AT2-R overexpression in attenuating post-MI remodeling. Genetic knockout of the AT1a-R is additive to AT2-R overexpression, due, at least in part, to blood pressure lowering.     

Potent 4-amino-5-azaindole factor VIIa inhibitors

The 4-amino-5-azaindole as an amidino-benzimidazole replacement is described. A series of potent and selective analogs were discovered and showed desirable ex vivo efficacy as measured by PT.     

Efforts toward oral bioavailability in factor VIIa inhibitors

Efforts toward developing orally bioavailable factor VIIa inhibitors starting from parenteral lead compound 1 are described. SAR resulted in improved physicochemical properties, leading to enhanced oral absorption in rat.     

Factor VIIa inhibitors: gaining selectivity within the trypsin family

Within the trypsin family of coagulation proteases, obtaining highly selective inhibitors of factor VIIa has been challenging. We report a series of factor VIIa (fVIIa) inhibitors based on the 5-amidino-2-(2-hydroxy-biphenyl-3-yl)-benzimidazole (1) scaffold with potency for fVIIa and high selectivity against factors IIa, Xa, and trypsin. With this scaffold class, we propose that a unique hydrogen bond interaction between a hydroxyl on the distal ring of the biaryl system and the backbone carbonyl of fVIIa lysine-192 provides a basis for enhanced selectivity and potency for fVIIa.     

Expression of a Mutant p53 Results in an Age-Related Demographic Shift in Spontaneous Lung Tumor Formation in Transgenic Mice

Background

Mutations in the P53 gene are among the most common genetic abnormalities in human lung cancer. Codon 273 in the sequence-specific DNA binding domain is one of the most frequently mutated sites.

Methodology

To investigate the role of mutant p53 in lung tumorigenesis, a lung specific p53(273H) transgenic mouse model was developed. Rates of lung cancer formation in the transgenic animals and their littermates were evaluated by necropsy studies performed in progressive age cohorts ranging from 4 to 24 months. In order to establish the influence of other common genetic abnormalities in lung tumor formation in the animals, K-Ras gene mutation and p16INK4a (p16) promoter methylation were evaluated in a total of 281 transgenic mice and 189 non-transgenic littermates.

Principal Findings

At the age extremes of 4–12 and 22–24 months no differences were observed, with very low prevalence of tumors in animals younger than 12 months, and a relatively high prevalence at age 22 months or older. However, the transgenic mice had a significant higher lung tumor rate than their non-transgenic counterparts during the age of 13–21 months, suggesting an age-related shift in lung tumor formation induced by the lung-specific expression of the human mutant p53. Histopathology suggested a more aggressive nature for the transgenic tumors. Older mice (>13 months) had a significantly higher rate of p16 promoter methylation (17% v 82%). In addition, an age related effect was observed for K-Ras codons 12 or 13 mutations, but not for codon 61 mutations.

Conclusions/Significance

These results would suggest that the mutant p53(273H) contributes to an acceleration in the development of spontaneous lung tumors in these mice. Combination with other genetic and epigenetic alterations occurring after the age of 13 months is intimately linked to its oncogenic potential.     

Local and landscape effects on butterfly density in northern Idaho grasslands and forests

Understanding butterfly response to landscape context can inform conservation management and planning. We tested whether local-scale resources (host and nectar plants, canopy cover) or landscape context, measured at two scales, better explained the densities of four butterfly species. The density of Coenonympha tullia, which has host plants strongly associated with grassland habitats, was positively correlated with the amount of grassland in 0.5- and 1-km radius landscapes and only occurred in forests when they bordered grasslands. For the other species, Celastrina ladon, Cupido amyntula, and Vanessa cardui, local-scale resources better explained butterfly densities, emphasizing the importance of local habitat quality for butterflies. These three species also used host plants that were distributed more heterogeneously within and among habitat types. Our findings demonstrate the importance of host plant spatial distributions when determining the scale at which butterfly density relates to resources, and we recommend that both these distributions and landscape context be evaluated when developing butterfly monitoring programs, managing for species of concern, or modeling potential habitat.     

Pseudacteon decapitating flies (Diptera: Phoridae): Are they potential vectors of the fire ant pathogens Kneallhazia (=Thelohania) solenopsae (Microsporidia: Thelohaniidae) and Vairimorpha invictae (Microsporidia: Burenellidae)?

Fire ant decapitating flies in the genus Pseudacteon were tested for their potential as hosts or vectors of two microsporidian pathogens of the red imported fire ant, Solenopsis invicta. Decapitating flies that attacked or were reared from S. invicta workers infected by Kneallhazia (=Thelohania) solenopsae or Vairimorpha invictae were tested for either pathogen by polymerase chain reaction (PCR) tests or visual examination for spores using phase microscopy. Three species of fire ant decapitating flies acquired the pathogen, Kneallhazia solenopsae. K. solenopsae was detected in 58% of pooled samples of Pseudacteon obtusus flies and 44% of pooled samples of Pseudacteon cultellatus that developed in K. solenopsae-infected fire ant workers. K. solenopsae was also found in 17% of pooled samples of field-collected Pseudacteon curvatus. In contrast, the microsporidium V. invictae was not detected in P. obtusus reared from V. invictae-infected S. invicta workers. Neither K. solenopsae nor V. invictae were detected in any of the hovering or ovipositing flies in the laboratory exposures, indicating no mechanical acquisition of the microsporidia occurred during oviposition activity. Greater than 92% of the P. obtusus that developed in K. solenopsae-infected ants survived and emerged as adults, thus indicating no detrimental effects of the microsporidium on pupal development and emergence. These results indicate that Pseudacteon decapitating flies may be able to vector K. solenopsae but not V. invictae among fire ants. Further tests are planned to determine if flies containing K. solenopsae are capable of transferring this pathogen either during oviposition or by being consumed by fire ant larvae.     

Dose-rate effects of protons on in vivo activation of nuclear factor-kappa B and cytokines in mouse bone marrow cells

The objective of this study was to determine the kinetics of nuclear factor-kappa B (NF-κB) activation and cytokine expression in bone marrow (BM) cells of exposed mice as a function of the dose rate of protons. The cytokines included in this study are pro-inflammatory [i.e., tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and IL-6] and anti-inflammatory cytokines (i.e., IL-4 and IL-10). We gave male BALB/cJ mice a whole-body exposure to 0 (sham-controls) or 1.0 Gy of 100 MeV protons, delivered at 5 or 10 mGy min⁻¹, the dose and dose rates found during solar particle events in space. As a reference radiation, groups of mice were exposed to 0 (sham-controls) or 1 Gy of ¹³⁷Cs γ rays (10 mGy min⁻¹). After irradiation, BM cells were collected at 1.5, 3, 24 h, and 1 month for analyses (five mice per treatment group per harvest time). The results indicated that the in vivo time course of effects induced by a single dose of 1 Gy of 100 MeV protons or ¹³⁷Cs γ rays, delivered at 10 mGy min⁻¹, was similar. Although statistically significant levels of NF-κB activation and pro-inflammatory cytokines in BM cells of exposed mice when compared to those in the corresponding sham controls (Student’s t-test, p < 0.05 or <0.01) were induced by either dose rate, these levels varied over time for each protein. Further, only a dose rate of 5 mGy min⁻¹ induced significant levels of anti-inflammatory cytokines. The results indicate dose-rate effects of protons.     

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes ^3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl⁺ that has been successfully used in other fluorescence quenching assays ^5,13. Tl⁺ permeates the lipid bilayer slowly ⁸ but passes readily through conducting gramicidin channels ^1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results ¹².Download video file.^{(69M, mov)}