Doxorubicin induces EGF receptor-dependent downregulation of gap junctional intercellular communication in rat liver epithelial cells

Exposure of rat liver epithelial cells to doxorubicin, an anthraquinone derivative widely employed in cancer chemotherapy, led to a dose-dependent decrease in gap junctional intercellular communication (GJC). Gap junctions are clusters of inter-cellular channels consisting of connexins, the major connexin in the cells used being connexin-43 (Cx43). Doxorubicin-induced loss of GJC was mediated by activation of extracellular signal-regulated kinase (ERK)-1 and ERK-2, as demonstrated using inhibitors of ERK activation. Furthermore, activation of the epidermal growth factor (EGF) receptor by doxorubicin was responsible for ERK activation and the subsequent attenuation of GJC. Inhibition of GJC, however, was not by direct phosphorylation of Cx43 by ERK-1/2, whereas menadione, a 1,4-naphthoquinone derivative that was previously demonstrated to activate the same EGF receptor-dependent pathway as doxorubicin, resulting in downregulation of GJC, caused strong phos-phorylation of Cx43 at serines 279 and 282. Thus, ERK-dependent downregulation of GJC upon exposure to quinones may occur both by direct phosphorylation of Cx43 and in a phosphorylation-independent manner.     

TMEM126A is a mitochondrial located mRNA (MLR) protein of the mitochondrial inner membrane

Background

Hereditary optic neuropathies (HONs) are a heterogeneous group of disorders that affect retinal ganglion cells (RGCs) and axons that form the optic nerve. Leber's Hereditary Optic Neuropathy and the autosomal dominant optic atrophy related to OPA1 mutations are the most common forms. Nonsyndromic autosomal recessive optic neuropathies are rare and their existence has been long debated. We recently identified the first gene responsible for these conditions, TMEM126A. This gene is highly expressed in retinal cellular compartments enriched in mitochondria and supposed to encode a mitochondrial transmembrane protein of unknown function.

Methods

A specific polyclonal antibody targeting the TMEM126A protein has been generated. Quantitative fluorescent in situ hybridization, cellular fractionation, mitochondrial membrane association study, mitochondrial sub compartmentalization analysis by both proteolysis assays and transmission electron microscopy, and expression analysis of truncated TMEM126A constructs by immunofluorescence confocal microscopy were carried out.

Results

TMEM126A mRNAs are strongly enriched in the vicinity of mitochondria and encode an inner mitochondrial membrane associated cristae protein. Moreover, the second transmembrane domain of TMEM126A is required for its mitochondrial localization.

Conclusions

TMEM126A is a mitochondrial located mRNA (MLR) that may be translated in the mitochondrial surface and the protein is subsequently imported to the inner membrane. These data constitute the first step toward a better understanding of the mechanism of action of TMEM126A in RGCs and support the importance of mitochondrial dysfunction in the pathogenesis of HON.

General significance

Local translation of nuclearly encoded mitochondrial mRNAs might be a mechanism for rapid onsite supply of mitochondrial membrane proteins.     

Behavioral analyses of sugar processing in choice, feeding, and learning in larval Drosophila

Gustatory stimuli have at least 2 kinds of function: They can support immediate, reflexive responses (such as substrate choice and feeding) and they can drive internal reinforcement. We provide behavioral analyses of these functions with respect to sweet taste in larval Drosophila. The idea is to use the dose-effect characteristics as behavioral "fingerprints" to dissociate reflexive and reinforcing functions. For glucose and trehalose, we uncover relatively weak preference. In contrast, for fructose and sucrose, preference responses are strong and the effects on feeding pronounced. Specifically, larvae are attracted to, and feeding is stimulated most strongly for, intermediate concentrations of either sugar: Using very high concentrations (4 M) results in weakened preference and suppression of feeding. In contrast to such an optimum function regarding choice and feeding, an asymptotic dose-effect function is found for reinforcement learning: Learning scores reach asymptote at 2 M and remain stable for a 4-M concentration. A similar parametric discrepancy between the reflexive (choice and feeding) and reinforcing function is also seen for sodium chloride (Niewalda T, Singhal S, Fiala A, Saumweber T, Wegener S, Gerber B, in preparation). We discuss whether these discrepancies are based either on inhibition from high-osmolarity sensors upon specifically the reflexive pathways or whether different sensory pathways, with different effective dose-response characteristics, may have preferential access to drive either reflex responses or modulatory neurons mediating internal reinforcement, respectively.     

Activation of a novel long-chain free fatty acid generation and export system in mitochondria of diabetic rat hearts

A number of reports indicate that a long-chain free fatty acid export system may be operating in mitochondria. In this study, we sought evidence of its existence in rat heart mitochondria. To determine its potential role, we also sought evidence of its activation or inhibition in streptozotocin (STZ)-induced diabetic rat heart mitochondria. If confirmed, it could be a novel mechanism for regulation of long-chain fatty acid oxidation (FAO) in mitochondria. To obtain evidence of its existence, we tested whether heart mitochondria presented with palmitoyl-carnitine can generate and export palmitate. We found that intact mitochondria indeed generate and export palmitate. We have also found that the rates of these processes are markedly higher in STZ-diabetic rat heart mitochondria, in which palmitoyl-carnitine oxidation is also increased. Since mitochondrial thioesterase-1 (MTE-1) hydrolyzes acyl-CoA to CoA-SH + free fatty acid, and uncoupling protein-3 (UCP-3), reconstituted in liposomes, transports free fatty acids, we examined whether these proteins are also increased in STZ-diabetic rat heart mitochondria. We found that both of these proteins are indeed increased. Gene expression profile analysis revealed striking expression of mitochondrial long-chain fatty acid transport and oxidation genes, accompanying overexpression of MTE-1 and UCP-3 in STZ-diabetic rat hearts. Our findings provide the first direct evidence for the existence of a long-chain free fatty acid generation and export system in mitochondria and its activation in STZ-diabetic rat hearts in which FAO is enhanced. We suggest that its activation may facilitate, and inhibition may limit, enhancement of FAO. fatty acid oxidation; diabetes; lipotoxic cardiomyopathy; gene array     

Expression and functional properties of α7 acetylcholine nicotinic receptors are modified in the presence of other receptor subunits

Although α7 nicotinic receptors are predominantly homopentamers, previous reports have indicated that α7 and β2 subunits are able to form heteromers. We have studied whether other nicotinic receptor subunits can also assemble with α7 subunits and the effect of this potential association. Coexpression of α7 with α2, α3, or β4 subunits reduced to about half, surface α‐bungarotoxin binding sites and acetylcholine‐gated currents. This is probably because of inhibition of membrane trafficking, as the total amount of α7 subunits was similar in all cases and a significant proportion of mature α7 receptors was present inside the cell. Only β4 subunits appeared to directly associate with α7 receptors at the membrane and these heteromeric receptors showed some kinetic and pharmacological differences when compared with homomeric α7 receptors. Finally, we emulated the situation of bovine chromaffin cells in Xenopus laevis oocytes by using the same proportion of α3, β4, α5, and α7 mRNAs, finding that α‐bungarotoxin binding was similarly reduced in spite of increased currents, apparently mediated by α3β4(α5) receptors.     

Multisite Phosphorylation of the Guanine Nucleotide Exchange Factor Cdc24 during Yeast Cell Polarization

Background

Cell polarization is essential for processes such as cell migration and asymmetric cell division. A common regulator of cell polarization in most eukaryotic cells is the conserved Rho GTPase, Cdc42. In budding yeast, Cdc42 is activated by a single guanine nucleotide exchange factor, Cdc24. The mechanistic details of Cdc24 activation at the onset of yeast cell polarization are unclear. Previous studies have suggested an important role for phosphorylation of Cdc24, which may regulate activity or function of the protein, representing a key step in the symmetry breaking process.

Methodology/Principal Findings

Here, we directly ask whether multisite phosphorylation of Cdc24 plays a role in its regulation. We identify through mass spectrometry analysis over thirty putative in vivo phosphorylation sites. We first focus on sites matching consensus sequences for cyclin-dependent and p21-activated kinases, two kinase families that have been previously shown to phosphorylate Cdc24. Through site-directed mutagenesis, yeast genetics, and light and fluorescence microscopy, we show that nonphosphorylatable mutations of these consensus sites do not lead to any detectable consequences on growth rate, morphology, kinetics of polarization, or localization of the mutant protein. We do, however, observe a change in the mobility shift of mutant Cdc24 proteins on SDS-PAGE, suggesting that we have indeed perturbed its phosphorylation. Finally, we show that mutation of all identified phosphorylation sites does not cause observable defects in growth rate or morphology.

Conclusions/Significance

We conclude that lack of phosphorylation on Cdc24 has no overt functional consequences in budding yeast. Yeast cell polarization may be more tightly regulated by inactivation of Cdc42 by GTPase activating proteins or by alternative methods of Cdc24 regulation, such as conformational changes or oligomerization.     

Population sizes and within-deme movement of Trimerotropis saxatilis (Acrididae), a grasshopper with a fragmented distribution

Capture-mark-recapture studies were initiated in 1990 on four Missouri populations of the lichen grasshopper, Trimerotropis saxatilis. This grasshopper lives only on glade habitat, predominantly in the Ozark Mountains. Genetic data suggest that no gene flow occurs among T. saxatilis populations. Lichen grasshopper population size (both present and historical), and the likelihood of movement within and between glades, are the subjects of this study. Population sizes on all glades were found to be small (<280 individuals) and to vary from year to year. Inbreeding effective sizes were found to be much larger than census sizes. On one of the sites, Graham Cave Glade, population size was calculated for 5 years; in 3 of those years (1991, 1993 and 1994) our studies of this population also tested for movement of T. saxatilis individuals among different regions of the moderately subdivided glade. Maintenance of Graham Cave Glade (burning and clearing) was initiated after the 1991 capture-mark-recapture season. Comparisons of before-and after-burning intraglade movement probabilities did not show a significant difference. Grasshoppers more frequently remained in the part of the glade where they were previously captured, but were able to move about the moderately subdivided glade. The presence of a closed-canopy forest, rather than distance, appears to be an effective dispersal barrier.