C/EBP-β Regulates Endoplasmic Reticulum Stress–Triggered Cell Death in Mouse and Human Models

Endoplasmic reticulum (ER) stress elicits the unfolded protein response (UPR), initially aimed at coping with the stress, but triggering cell death upon further stress. ER stress induces the C/EBP-® variant Liver-enriched Activating Protein (LAP), followed by the dominant-negative variant, Liver Inhibitory Protein (LIP). However, the distinct role of LAP and LIP in ER stress is unknown. We found that the kinetics of the ER stress-induced expression of LIP overlapped with that of the cell death in mouse B16 melanoma cells. Furthermore, inducible over-expression of LIP augmented ER stress-triggered cell death whereas over-expression of LAP attenuated cell death. Similar results were obtained in human 293T cells. Limited vasculature in tumors triggers hypoxia, nutrient shortage and accumulation of toxic metabolites, all of which eliciting continuous ER stress. We found that LAP promoted and LIP inhibited B16 melanoma tumor progression without affecting angiogenesis or accelerating the cell cycle. Rather, LAP attenuated, whereas LIP augmented tumor ER stress. We therefore suggest that C/EBP-® regulates the transition from the protective to the death–promoting phase of the UPR. We further suggest that the over-expression of LAP observed in many solid tumors promotes tumor progression by attenuating ER stress–triggered tumor cell death.     

The Genetic,Morphological, and Physiological Characterization of a Dark Larval Cuticle Mutation in the Butterfly,Bicyclus anynana

Studies on insect melanism have greatly contributed to our understanding of natural selection and the ultimate factors influencing the evolution of darkly pigmented phenotypes. Research on several species of melanic lepidopteran larvae have found that low levels of circulating juvenile hormone (JH) titers are associated with a melanic phenotype, suggesting that genetic changes in the JH biosynthetic pathway give rise to increased deposition of melanin granules in the cuticle in this group. But does melanism arise through different molecular mechanisms in different species? The present study reports on a Bicyclus anynana (Lepidoptera: Nymphalidae) dark larvae single locus mutation, in which larvae exhibit a darker cuticle relative to wild type. Unlike other lepidopteran melanic larvae mutations, this one is autosomal recessive and does not appear to involve a deficiency in JH titers. Unlike JH deficiency mutants, dark larvae mutants display similar growth rates and sexual behaviors as wild type, and topical application of a JH analogue failed to rescue the wild type cuticular coloration. Finally, transmission electron microscopy showed that sclerotization or deposition of diffuse melanin, rather than deposition of melanin granules, produces the dark coloration found in the cuticle of this species. We conclude that different molecular mechanisms underlie larval melanism in different species of Lepidoptera.     

Experimental and theoretical analysis of neuron-transistor hybrid electrical coupling: the relationships between the electro-anatomy of cultured Aplysia neurons and the recorded field potentials

Understanding the mechanisms that generate field potentials (FPs) by neurons grown on semiconductor chips is essential for implementing neuro-electronic devices. Earlier studies emphasized that FPs are generated by current flow between differentially expressed ion channels on the membranes facing the chip surface, and those facing the culture medium in electrically compact cells. Less is known, however, about the mechanisms that generate FPs by action potentials (APs) that propagate along typical non-isopotential neurons. Using Aplysia neurons cultured on floating gate-transistors, we found that the FPs generated by APs in cultured neurons are produced by current flow along neuronal compartments comprising the axon, cell body, and neurites, rather than by flow between the membrane facing the chip substrate and that facing the culture medium. We demonstrate that the FPs waveform generated by non-isopotential neurons largely depends on the morphology of the neuron.     

Synthesis and SAR studies of diarylpyrrole anticoccidial agents

2-(4-Fluorophenyl)-3-(4-pyridinyl)-5-substituted pyrroles were prepared and evaluated as anticoccidial agents in both in vitro and in vivo assays. Among the compounds evaluated, the dimethylamine-substituted pyrrole 19a is the most potent inhibitor of Eimeria tenella PKG (cGMP-dependent protein kinase). Further SAR studies on the side chain of the 2-pyrrolidine nitrogen did not enhance in vivo anticoccidial activity.     

CD48 is an allergen and IL-3-induced activation molecule on eosinophils

Eosinophils are involved in a variety of allergic, parasitic, malignant, and idiopathic disorders by releasing a variety of factors including specific granule proteins, lipid mediators, and proinflammatory and immunoregulatory cytokines and chemokines. In addition, they interact with various cell types in the inflamed tissue. Yet, the mechanism of eosinophil activation is still poorly understood. Recently, we described the expression and function of the CD2-subfamily of receptors and especially 2B4 on human eosinophils. In this study we focus on CD48, the high-affinity ligand of 2B4. CD48 is a GPI-anchored protein involved in cellular activation, costimulation, and adhesion, but has not been studied on eosinophils. We demonstrate that human eosinophils from atopic asthmatics display enhanced levels of CD48 expression and that IL-3 up-regulates CD48 expression. Furthermore, cross-linking CD48 on human eosinophils triggers release of eosinophil granule proteins. Assessment of CD48 expression in a murine model of experimental asthma revealed that CD48 is induced by allergen challenge and partially regulated by IL-3. Additionally, anti-IL-3 reduces CD48 expression and the degree of airway inflammation. Thus, CD48 is an IL-3-induced activating receptor on eosinophils, likely involved in promoting allergic inflammation.     

Molecular basis of intramolecular electron transfer in proteins during radical-mediated oxidations: computer simulation studies in model tyrosine-cysteine peptides in solution

Experimental studies in hemeproteins and model Tyr/Cys-containing peptides exposed to oxidizing and nitrating species suggest that intramolecular electron transfer (IET) between tyrosyl radicals (Tyr-O(·)) and Cys residues controls oxidative modification yields. The molecular basis of this IET process is not sufficiently understood with structural atomic detail. Herein, we analyzed using molecular dynamics and quantum mechanics-based computational calculations, mechanistic possibilities for the radical transfer reaction in Tyr/Cys-containing peptides in solution and correlated them with existing experimental data. Our results support that Tyr-O(·) to Cys radical transfer is mediated by an acid/base equilibrium that involves deprotonation of Cys to form the thiolate, followed by a likely rate-limiting transfer process to yield cysteinyl radical and a Tyr phenolate; proton uptake by Tyr completes the reaction. Both, the pKa values of the Tyr phenol and Cys thiol groups and the energetic and kinetics of the reversible IET are revealed as key physico-chemical factors. The proposed mechanism constitutes a case of sequential, acid/base equilibrium-dependent and solvent-mediated, proton-coupled electron transfer and explains the dependency of oxidative yields in Tyr/Cys peptides as a function of the number of alanine spacers. These findings contribute to explain oxidative modifications in proteins that contain sequence and/or spatially close Tyr-Cys residues.     

The Fe(III) and Ga(III) coordination chemistry of 3-(1-hydroxymethylidene) and 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione: novel tetramic acid degradation products of homoserine lactone bacterial quorum sensing molecules

Bacteria use small diffusible molecules to exchange information in a process called quorum sensing (QS). An important class of quorum sensing molecules used by Gram-negative bacteria is the family of N-acylhomoserine lactones (HSL). It was recently discovered that a degradation product of the QS molecule 3-oxo-C₁₂-homoserine lactone, the tetramic acid 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, is a potent antibacterial agent, thus implying roles for QS outside of simply communication. Because these tetramic acids also appear to bind iron with appreciable affinity it was suggested that metal binding might contribute to their biological activity. Here, using a variety of spectroscopic tools, we describe the coordination chemistry of both the methylidene and decylidene tetramic acid derivatives with Fe(III) and Ga(III) and discuss the potential biological significance of such metal binding.     

Evaluation of Three Approaches for Real-Time Monitoring of Roller Compaction with Near-Infrared Spectroscopy

Three different approaches have been evaluated for monitoring ribbon density through real-time near-infrared spectroscopy measurements. The roll compactor was operated to produce microcrystalline cellulose (MCC) ribbons of varying densities. The first approach used the slope of the spectra which showed a variation through the ribbon that could be attributed to density. A second qualitative approach was also developed with a principal component analysis (PCA) model with spectra taken in-line during the production of ribbons in an ideal roll pressure range. The PCA (i.e., real-time) density scans show that the model was able to qualitatively capture the density responses resulting from variation in process parameters. The third approach involved multivariate partial least squares (PLS) calibration models developed at wavelength regions of 1,120–1,310 and 1,305–2,205 nm. Also, various PLS models were developed using three reference methods: caliper, pycnometer, and in-line laser. The third approach shows a quantitative difference between the model-predicted and the measured densities. Models developed at high-wavelength region showed highest accuracy compared with models at low-wavelength region. All the PLS models showed a high accuracy along the spectra collected throughout the production of the ribbons. The three methods showed applicability to process control monitoring by describing the changes in density during in-line sampling.