Inhibiting ERK Activation with CI-1040 Leads to Compensatory Upregulation of Alternate MAPKs and Plasminogen Activator Inhibitor-1 following Subtotal Nephrectomy with No Impact on Kidney Fibrosis

Extracellular-signal regulated kinase (ERK) activation by MEK plays a key role in many of the cellular processes that underlie progressive kidney fibrosis including cell proliferation, apoptosis and transforming growth factor β1-mediated epithelial to mesenchymal transition. We therefore assessed the therapeutic impact of ERK1/2 inhibition using a MEK inhibitor in the rat 5/6 subtotal nephrectomy (SNx) model of kidney fibrosis. There was a twentyfold upregulation in phospho-ERK1/2 expression in the kidney after SNx in Male Wistar rats. Rats undergoing SNx became hypertensive, proteinuric and developed progressive kidney failure with reduced creatinine clearance. Treatment with the MEK inhibitor, CI-1040 abolished phospho- ERK1/2 expression in kidney tissue and prevented phospho-ERK1/2 expression in peripheral lymphocytes during the entire course of therapy. CI-1040 had no impact on creatinine clearance, proteinuria, glomerular and tubular fibrosis, and α-smooth muscle actin expression. However, inhibition of ERK1/2 activation led to significant compensatory upregulation of the MAP kinases, p38 and JNK in kidney tissue. CI-1040 also increased the expression of plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasmin-dependent matrix metalloproteinases. Thus inhibition of ERK1/2 activation has no therapeutic effect on kidney fibrosis in SNx possibly due to increased compensatory activation of the p38 and JNK signalling pathways with subsequent upregulation of PAI-1.     

Discovery of dihydrothieno- and dihydrofuropyrimidines as potent pan Akt inhibitors

Herein we report the discovery and synthesis of a novel series of dihydrothieno- and dihydrofuropyrimidines (2 and 3) as potent pan Akt inhibitors. Utilizing previous SAR and analysis of the amino acid sequences in the binding site we have designed inhibitors displaying increased PKA and general kinase selectivity with improved tolerability compared to the progenitor pyrrolopyrimidine (1). A representative dihydrothieno compound (34) was advanced into a PC3-NCI prostate mouse tumor model in which it demonstrated a dose-dependent reduction in tumor growth and stasis when dosed orally daily at 200 mg/kg.     

Threatened populations of the Australian squirrel glider (Petaurus norfolcensis) show evidence of evolutionary distinctiveness on a Late Pleistocene timescale

The squirrel glider Petaurus norfolcensis occurs across a broad Australian latitudinal range that includes gaps in distribution and potential biogeographic barriers, creating the potential for evolution of distinct entities within this species. Because of the species’ threatened status in the southern part of its range, we tested for the presence of geographically based independent evolutionary units among gliders sampled from southern, and northern coastal populations, using sequences of mitochondrial cytochrome b DNA (mtDNA) and a set of five nuclear microsatellites in 258 individuals. Our analyses suggest that an initial northward colonisation in the early- to mid-Pleistocene was followed by isolation by distance and, eventually, divergence between the sampled coastal and southern populations in the mid- to late-Pleistocene. We propose that the previously large and diverse southern populations have declined coincidentally with the replacement of wet forests by open sclerophyll woodlands during the preceding few million years. By contrast coastal populations further north appear to have been expanding and at present have an effective population size several times greater than southern populations. These results suggest that the two forms are on different evolutionary trajectories and should be treated separately for conservation purposes. It is highly desirable that loss of southern populations be prevented to maintain the unique genetic diversity accumulated over a considerable evolutionary timescale.     

The aryl hydrocarbon receptor is required for optimal resistance to Listeria monocytogenes infection in mice

The aryl hydrocarbon receptor (AhR) is part of a powerful signaling system that is triggered by xenobiotic agents such as polychlorinated hydrocarbons and polycyclic aromatic hydrocarbons. Although activation of the AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin or certain polycyclic aromatic hydrocarbons can lead to immunosuppression, there is also increasing evidence that the AhR regulates certain normal developmental processes. In this study, we asked whether the AhR plays a role in host resistance using murine listeriosis as an experimental system. Our data clearly demonstrate that AhR null C57BL/6J mice (AhR(-/-)) are more susceptible to listeriosis than AhR heterozygous (AhR(+/-)) littermates when inoculated i.v. with log-phase Listeria monocytogenes. AhR(-/-) mice exhibited greater numbers of CFU of L. monocytogenes in the spleen and liver, and greater histopathological changes in the liver than AhR(+/-) mice. Serum levels of IL-6, MCP-1, IFN-gamma, and TNF-alpha were comparable between L. monocytogenes-infected AhR(-/-) and AhR(+/-) mice. Increased levels of IL-12 and IL-10 were observed in L. monocytogenes-infected AhR(-/-) mice. No significant difference was found between AhR(+/-) and AhR(-/-) macrophages ex vivo with regard to their ability to ingest and inhibit intracellular growth of L. monocytogenes. Intracellular cytokine staining of CD4(+) and CD8(+) splenocytes for IFN-gamma and TNF-alpha revealed comparable T cell-mediated responses in AhR(-/-) and AhR(+/-) mice. Previously infected AhR(-/-) and AhR(+/-) mice both exhibited enhanced resistance to reinfection with L. monocytogenes. These data provide the first evidence that AhR is required for optimal resistance but is not essential for adaptive immune response to L. monocytogenes infection.     

Discovery and characterization of a novel inhibitor of matrix metalloprotease-13 that reduces cartilage damage in vivo without joint fibroplasia side effects

Matrix metalloproteinase-13 (MMP13) is a Zn(2+)-dependent protease that catalyzes the cleavage of type II collagen, the main structural protein in articular cartilage. Excess MMP13 activity causes cartilage degradation in osteoarthritis, making this protease an attractive therapeutic target. However, clinically tested MMP inhibitors have been associated with a painful, joint-stiffening musculoskeletal side effect that may be due to their lack of selectivity. In our efforts to develop a disease-modifying osteoarthritis drug, we have discovered MMP13 inhibitors that differ greatly from previous MMP inhibitors; they do not bind to the catalytic zinc ion, they are noncompetitive with respect to substrate binding, and they show extreme selectivity for inhibiting MMP13. By structure-based drug design, we generated an orally active MMP13 inhibitor that effectively reduces cartilage damage in vivo and does not induce joint fibroplasias in a rat model of musculoskeletal syndrome side effects. Thus, highly selective inhibition of MMP13 in patients may overcome the major safety and efficacy challenges that have limited previously tested non-selective MMP inhibitors. MMP13 inhibitors such as the ones described here will help further define the role of this protease in arthritis and other diseases and may soon lead to drugs that safely halt cartilage damage in patients.     

Proteolytic adaptor for transfer-messenger RNA-tagged proteins from alpha-proteobacteria

We have identified an analog of SspB, the proteolytic adaptor for transfer-messenger RNA (tmRNA)-tagged proteins, in Caulobacter crescentus. C. crescentus SspB shares limited sequence similarity with Escherichia coli SspB but binds the tmRNA tag in vitro and is required for optimal proteolysis of tagged proteins in vivo.     

Phylogenetic topology mapped onto dietary ecospace reveals multiple pathways in the evolution of the herbivorous niche in African Bovidae

Understanding the evolutionary history of the herbivore niche within African bovids has traditionally relied on examining anatomical adaptations to diet, particularly those related to digestive strategy. More recently, mesowear and stable isotope analyses have been used to great effect to reconstruct dietary preferences. We use these dietary proxies to construct a morphology‐free dietary ecospace and examine the topology of the phylogenetic relationships of African bovids mapped onto this ecospace. The reconstructed dietary ecospace provides evidence for four distinct dietary classes: species with C3‐ or C4‐dominated diets that produce low or high occlusal relief, likely related to diets high or low in abrasives, respectively. We detected no evidence for a discrete mixed feeder category; the species often categorized as such represent the end members of groups of species with either C3‐ or C4‐ dominated diets. Our analysis reveals high variability within the C4 grazing ecospace, and phylogenetic evidence indicates at least two pathways to grazing, likely related to the abrasive qualities of ingested food, which may be determined by the moisture content or the height of consumed grasses. These different pathways probably contribute to the high diversity of African grazers, both today and in the fossil record. C3 browsers (non‐frugivores) also display a high degree of variation, but there are no species associated with highly abrasive diets and there is evidence for only a single evolutionary pathway. We find evidence for only one evolutionary route towards frugivory, which includes species with diets that produce both high and low occlusal reliefs. The cause of abrasive wear in frugivores may be related to grit and/or the hard parts of fruits, but this requires further examination.     

Why and how might genetic and phylogenetic diversity be reflected in the identification of key biodiversity areas?

‘Key biodiversity areas'' are defined as sites contributing significantly to the global persistence of biodiversity. The identification of these sites builds from existing approaches based on measures of species and ecosystem diversity and process. Here, we therefore build from the work of Sgró et al. (2011 Evol. Appl. 4, 326–337. (doi:10.1111/j.1752-4571.2010.00157.x)) to extend a framework for how components of genetic diversity might be considered in the identification of key biodiversity areas. We make three recommendations to inform the ongoing process of consolidating a key biodiversity areas standard: (i) thresholds for the threatened species criterion currently consider a site''s share of a threatened species'' population; expand these to include the proportion of the species'' genetic diversity unique to a site; (ii) expand criterion for ‘threatened species'' to consider ‘threatened taxa’ and (iii) expand the centre of endemism criterion to identify as key biodiversity areas those sites holding a threshold proportion of the compositional or phylogenetic diversity of species (within a taxonomic group) whose restricted ranges collectively define a centre of endemism. We also recommend consideration of occurrence of EDGE species (i.e. threatened phylogenetic diversity) in key biodiversity areas to prioritize species-specific conservation actions among sites.     

ADP regulates SNF1, the Saccharomyces cerevisiae homolog of AMP-activated protein kinase

The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.