Dexamethasone modulates ErbB tyrosine kinase expression and signaling through multiple and redundant mechanisms in cultured rat hepatocytes

Glucocorticoids paradoxically exert both stimulatory and inhibitory effects on the proliferation of cultured rat hepatocytes. We studied the effects of dexamethasone, a synthetic glucocorticoid, on the proliferation of cultured rat hepatocytes. The timing of growth factor addition modified the action of high-dose dexamethasone (10(-6) M) on DNA synthesis. When we added transforming growth factor-alpha at the time of plating, 10(-6) M dexamethasone weakly stimulated DNA synthesis by 26% relative to cells cultured in dexamethasone-free media. When we delayed growth factor addition until 24-48 h after plating, 10(-6) M dexamethasone inhibited DNA synthesis by 50%. Using immunological methods, we analyzed the expression and signaling patterns of the ErbB kinases in dexamethasone-treated cells. High-dose dexamethasone stabilized the expression of epidermal growth factor receptor (EGFr) and ErbB3, and it suppressed the de novo expression of ErbB2 that occurs during the third and fourth day of culture in 10(-8) M dexamethasone. High-dose dexamethasone by 72 h suppressed basal and EGF-associated phosphorylation of ERK and Akt. The reduction in ERK1/2 phosphorylation correlated with suppression of a culture-dependent increase in Son-of sevenless 1 (Sos1) and ERK1/2 expression. High-dose dexamethasone in hepatocytes stabilized or upregulated several inhibitory effectors of EGFr/ErbB2 and ERK, including receptor-associated late transducer (RALT) and MKP-1, respectively. Thus 10(-6) M dexamethasone exerts a time-dependent and redundant inhibitory effect on EGFr-mediated proliferative signaling in hepatocytes, targeting not only the ErbB proteins but also their various positive and negative effectors.     

Localization and behaviors in null mice suggest that ASIC1 and ASIC2 modulate responses to aversive stimuli

Acid‐sensing ion channels (ASICs) generate H⁺‐gated Na⁺ currents that contribute to neuronal function and animal behavior. Like ASIC1, ASIC2 subunits are expressed in the brain and multimerize with ASIC1 to influence acid‐evoked currents and facilitate ASIC1 localization to dendritic spines. To better understand how ASIC2 contributes to brain function, we localized the protein and tested the behavioral consequences of ASIC2 gene disruption. For comparison, we also localized ASIC1 and studied ASIC1^?/? mice. ASIC2 was prominently expressed in areas of high synaptic density, and with a few exceptions, ASIC1 and ASIC2 localization exhibited substantial overlap. Loss of ASIC1 or ASIC2 decreased freezing behavior in contextual and auditory cue fear conditioning assays, in response to predator odor and in response to CO₂ inhalation. In addition, loss of ASIC1 or ASIC2 increased activity in a forced swim assay. These data suggest that ASIC2, like ASIC1, plays a key role in determining the defensive response to aversive stimuli. They also raise the question of whether gene variations in both ASIC1 and ASIC2 might affect fear and panic in humans .     

Interlayer micromechanics of the aortic heart valve leaflet

While the mechanical behaviors of the fibrosa and ventricularis layers of the aortic valve (AV) leaflet are understood, little information exists on their mechanical interactions mediated by the GAG-rich central spongiosa layer. Parametric simulations of the interlayer interactions of the AV leaflets in flexure utilized a tri-layered finite element (FE) model of circumferentially oriented tissue sections to investigate inter-layer sliding hypothesized to occur. Simulation results indicated that the leaflet tissue functions as a tightly bonded structure when the spongiosa effective modulus was at least 25 % that of the fibrosa and ventricularis layers. Novel studies that directly measured transmural strain in flexure of AV leaflet tissue specimens validated these findings. Interestingly, a smooth transmural strain distribution indicated that the layers of the leaflet indeed act as a bonded unit, consistent with our previous observations (Stella and Sacks in J Biomech Eng 129:757–766, 2007) of a large number of transverse collagen fibers interconnecting the fibrosa and ventricularis layers. Additionally, when the tri-layered FE model was refined to match the transmural deformations, a layer-specific bimodular material model (resulting in four total moduli) accurately matched the transmural strain and moment-curvature relations simultaneously. Collectively, these results provide evidence, contrary to previous assumptions, that the valve layers function as a bonded structure in the low-strain flexure deformation mode. Most likely, this results directly from the transverse collagen fibers that bind the layers together to disable physical sliding and maintain layer residual stresses. Further, the spongiosa may function as a general dampening layer while the AV leaflets deforms as a homogenous structure despite its heterogeneous architecture.     

Possible Race and Gender Divergence in Association of Genetic Variations with Plasma von Willebrand Factor: A Study of ARIC and 1000 Genome Cohorts

The synthesis, secretion and clearance of von Willebrand factor (VWF) are regulated by genetic variations in coding and promoter regions of the VWF gene. We have previously identified 19 single nucleotide polymorphisms (SNPs), primarily in introns that are associated with VWF antigen levels in subjects of European descent. In this study, we conducted race by gender analyses to compare the association of VWF SNPs with VWF antigen among 10,434 healthy Americans of European (EA) or African (AA) descent from the Atherosclerosis Risk in Communities (ARIC) study. Among 75 SNPs analyzed, 13 and 10 SNPs were associated with VWF antigen levels in EA male and EA female subjects, respectively. However, only one SNP (RS1063857) was significantly associated with VWF antigen in AA females and none was in AA males. Haplotype analysis of the ARIC samples and studying racial diversities in the VWF gene from the 1000 genomes database suggest a greater degree of variations in the VWF gene in AA subjects as compared to EA subjects. Together, these data suggest potential race and gender divergence in regulating VWF expression by genetic variations.     

STUDIES ON TOXICITY

With yeast subjected to the action of phenol or of phenol and alcohol the curves of survivors as measured by resistance to staining with methylene blue when plotted against time do not in general prove to be logarithmic. It is believed that such resemblances as have been found between such curves and monomolecular reaction or logarithmic curves are superficial and fortuitous. Any method therefore of evaluating disinfecting power based upon such a concept must prove misleading. Variations in resistance of individual cells and the distribution of such variations must be regarded as of fundamental importance in accounting for rates of death of microorganisms.     

Localization and interaction of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors of lamprey spinal neurons.

Small volumes of N-Methyl-D-Aspartate (NMDA) and non-NMDA excitatory amino acid receptor agonists were applied to localized regions of the dendritic trees of lamprey spinal neurons along their medial-lateral axis to obtain a spatial map of glutamate receptor distribution. Voltage clamp and frequency domain methods were used to obtain quantitative kinetic data of the voltage dependent ionic channels located both on the soma and on highly branched dendritic membranes. Pressure pulses of NMDA applied to the most peripheral regions of the dendritic tree elicited large somatic impedance increases, indicating that the most peripheral dendrites are well supplied with NMDA receptors. Experiments done with kainate did not elicit somatic responses to agonist applications on peripheral dendrites. The data obtained are consistent with the hypothesis that the activation of NMDA receptors by exogenous glutamate is significantly modified by the simultaneous activation of non-NMDA receptors, which shunts the NMDA response. The non-NMDA shunting hypothesis was tested by a combined application of kainate and NMDA to mimic the action of glutamate showing that the shunting effect of non-NMDA receptor activation virtually abolished the marked voltage dependency typical of NMDA receptor activation. These data were interpreted with a compartmental neuronal model having both NMDA and non-NMDA receptors.     

Predictive neural coding of reward preference involves dissociable responses in human ventral midbrain and ventral striatum

Food preferences are acquired through experience and can exert strong influence on choice behavior. In order to choose which food to consume, it is necessary to maintain a predictive representation of the subjective value of the associated food stimulus. Here, we explore the neural mechanisms by which such predictive representations are learned through classical conditioning. Human subjects were scanned using fMRI while learning associations between arbitrary visual stimuli and subsequent delivery of one of five different food flavors. Using a temporal difference algorithm to model learning, we found predictive responses in the ventral midbrain and a part of ventral striatum (ventral putamen) that were related directly to subjects' actual behavioral preferences. These brain structures demonstrated divergent response profiles, with the ventral midbrain showing a linear response profile with preference, and the ventral striatum a bivalent response. These results provide insight into the neural mechanisms underlying human preference behavior.     

Unexpected diversity of ferredoxin-dependent thioredoxin reductases in cyanobacteria

Thioredoxin reductases control the redox state of thioredoxins (Trxs)—ubiquitous proteins that regulate a spectrum of enzymes by dithiol–disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. We have recently found that some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack NTR and FTR but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeply-rooted thioredoxin reductase or DTR), whose electron donor remained undefined. Here, we demonstrate that Fdx functions in this capacity and report the crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. Thereby, our data demonstrate that this cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. Our experiments further show that the redox-sensitive peptide CP12 is modulated in vitro by the FFTR/Trx system, demonstrating that FFTR functionally substitutes for FTR in light-linked enzyme regulation in Gloeobacter. Altogether, we demonstrate the FFTR is spread within the cyanobacteria phylum and propose that, by substituting for FTR, it connects the reduction of target proteins to photosynthesis. Besides, the results indicate that FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments.