Wide distribution of the cysteine string proteins in Drosophila tissues revealed by targeted mutagenesis

The “cysteine string protein” (CSP) genes of higher eukaryotes code for a novel family of proteins characterized by a “J” domain and an unusual cysteine-rich region. Previous studies had localized the proteins in neuropil and synaptic terminals of larval and adult Drosophila and linked the temperature-sensitive paralysis of the mutants described here to conditional failure of synaptic transmission. We now use the null mutants as negative controls in order to reliably detect even low concentrations of CSPs by immunohistochemistry, employing three monoclonal antibodies. In wild-type flies high levels of cysteine string proteins are found not only in apparently all synaptic terminals of the embryonic, larval, and adult nervous systems, but also in the “tall cells” of the cardia, in the follicle cells of the ovary, in specific structures of the female spermatheca, and in the male testis and ejaculatory bulb. In addition, low levels of CSPs appear to be present in all tissues examined, including neuronal perikarya, axons, muscles, Malpighian tubules, and salivary glands. Western blots of isolated tissues demonstrate that of the four isoforms expressed in heads only the largest is found in non-neural organs. The wide expression of CSPs suggests that at least some of the various phenotypes of the null mutants observed at permissive temperatures, such as delayed development, short adult lifespan, modified electroretinogram, and optomotor behavior, may be caused by the lack of CSPs outside synaptic terminals.     

Osteoprotegerin production by breast cancer cells is suppressed by dexamethasone and confers resistance against TRAIL‐induced apoptosis

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF‐κB ligand (RANKL) and TNF‐related apoptosis‐inducing ligand (TRAIL). While RANKL is essential for osteoclastogenesis and facilitates breast cancer migration into bone, TRAIL promotes breast cancer apoptosis. We analyzed the expression of OPG and TRAIL and its modulation in estrogen receptor‐positive MCF‐7 cells and receptor‐negative MDA‐MB‐231 cells. In both cells, OPG mRNA levels and protein secretion were dose‐ and time‐dependently enhanced by interleukin (IL)‐1β and suppressed by dexamethasone. In contrast to MCF‐7 cells, MDA‐MB‐231 abundantly expressed TRAIL mRNA, which was enhanced by IL‐1β and inhibited by dexamethasone. TRAIL activated pro‐apoptotic caspase‐3, ‐7, and poly‐ADP‐ribose polymerase and decreased cell numbers of MDA‐MB‐231, but had no effect on MCF‐7 cells. Gene silencing siRNA directed against OPG resulted in a 31% higher apoptotic rate compared to non‐target siRNA‐treated MDA‐MB‐231 cells. Furthermore, TRAIL induced significantly less apoptosis in cells cultured in conditioned media (containing OPG) compared to cells exposed to TRAIL in fresh medium lacking OPG (P < 0.01) and these protective effects were reversed by blocking OPG with its specific ligand RANKL (P < 0.05). The association between cancer cell survival and OPG production by MDA‐MB‐231 cells was further supported by the finding, that modulation of OPG secretion using IL‐1β or dexamethasone prior to TRAIL exposure resulted in decreased and increased rate of apoptosis, respectively (P < 0.05). Thus, OPG secretion by breast cancer cells is modulated by cytokines and dexamethasone, and may represent a critical resistance mechanism that protects against TRAIL‐induced apoptosis. J. Cell. Biochem. 108: 106–116, 2009. © 2009 Wiley‐Liss, Inc.     

Lipid droplet autophagy in the yeast Saccharomyces cerevisiae

Cytosolic lipid droplets (LDs) are ubiquitous organelles in prokaryotes and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAGs) and steryl esters, which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy is identified, which includes the core autophagic machinery but not Atg11 or Atg20. Thus LD autophagy is distinct from endoplasmic reticulum–autophagy, pexophagy, or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, is required for LD formation in yeast.     

Gibberellin-like substances and indole type auxins in developing grains of normal- and high-lysine genotypes of barley

Changes in gibberellin-like activity and content of indole type auxins were investigated during grain development of the two high-lysine barley (Hordeum vulgare L.) genotypes Sv 73608 and Risø 1508, and their corresponding normal cultivars Mona and Bomi. A peak in gibberellin-like activity was found in developing grains of the normal cultivars about 18 days after anthesis, whereas the grains of the high-lysine genotypes showed a two to five times higher maximum about 3–4 days later. The auxin content of the cultivar Bomi showed a maximum between the 22nd and the 29th day after anthesis, whereas, throughout their development the grains of the mutant Risø 1508 exhibited only about ¹/10 of the maximum level of auxin found in the grains of Bomi. The normal cultivar Mona also displayed higher contents of auxin than the high-lysine genotypes Sv 73608, particularly at the later stages of grain growth, but the differences in concentration were considerable smaller than for the pair ‘Bomi’—‘Risø 1508’. It is suggested that auxins play an important role in the development of barley grains.     

Evolutionary dynamics for bimatrix games: A Hamiltonian system?

We review some properties of the evolutionary dynamics for asymmetric conflicts, give a simplified approach to them, and present some new results on the stability and bifurcations occurring in these conservative systems. In particular, we compare their dynamics to those of Hamiltonian systems     

Evolutionary stability in strategic models of single-locus frequency-dependent viability selection

The dynamic stability of an evolutionarily stable strategy (ESS) is analyzed for a diploid species under individual viability selection. An individual's viability depends on the genotypic frequencies at a single autosomal locus through a payoff matrix determined by phenotypic behaviours (i.e. strategies). It is shown that an ESS of this payoff matrix is dynamically stable if there are at most three alleles — an intuitive result that strengthens the importance of static game-theoretic methods in genetic models.Author for correspondence     

A technique for the electron microscopic autoradiography of Al adenosine receptors in brain tissue

Summary A procedure for the electron microscopic autoradiography of Al adenosine receptors is described. Fresh tissue slices from rat hippocampus were incubated with the radioactive adenosine analogs: Cyclohexyl[³H]adenosine, 5-N-ethylcarboxamido[³H]adenosine or [¹²⁵I]-iodohydroxyphenylisopropyladenosine. Various fixation agents were tested with respect to the retention of these ligands by the tissue. While most of the ligands were lost in aldehyde fixation they were retained by osmium tetroxide probably via a crosslinking reaction. The final method of choice was an aldehyde prefixation (in the case of [¹²⁵I]-iodohydroxyphenylisopropyladenosine with 4% buffered paraformaldehyde) during which more than 90% of the nonspecifically bound ligands were washed out while 40% of the specifically bound ligands remained. Subsequent fixation with osmium tetroxide (1%) allowed a standard protocoll for dehydration and embedding to be used with only minimal (less than 5%) further loss of the ligands. Electron microscopic autoradiography provided evidence for a specific distribution of the binding sites for [¹²⁵I]-iodohydroxyphenylisopropyladenosine.Abbreviations GA Glutardialdehyde - PFA Paraformaldehyde - OsO₄ Osmiumtetroxide - CHA Cyclohexyladenosine - NECA N-ethyl-carboxamidoadenosine - PIA Phenylisopropyladenosine - I-HPIA Iodohydroxyphenylisopropyladenosine - HPIA Hydroxyphenylisopropyladenosine     

Proliferation pattern and early differentiation of the optic lobes in Drosophila melanogaster

Summary The larval and early pupal development of the optic lobes in Drosophila is described qualitatively and quantitatively using [³H]thymidine autoradiography on 2-m plastic sections. The optic lobes develop from 30–40 precursor cells present in each hemisphere of the freshly hatched larva. During the first and second larval instars, these cells develop to neuroblasts arranged in two epithelial optic anlagen. In the third larval instar and in the early pupa these neuroblasts generate the cells of the imaginal optic lobes at discrete proliferation zones, which can be correlated with individual visual neuropils.The different neuropils as well as the repetitive elements of each neuropil are generated in a defined temporal sequence. Cells of the medulla are the first to become postmitotic with the onset of the third larval instar, followed by cells of the lobula complex and finally of the lamina at about the middle of the third instar. The elements of each neuropil connected to the most posterior part of the retina are generated first, elements corresponding to the most anterior retina are generated last.The proliferation pattern of neuroblasts into ganglion mother cells and ganglion cells is likely to include equal as well as unequal divisions of neuroblasts, followed by one or two generations of ganglion mother cells. For the lamina the proliferation pattern and its temporal coordination with the differentiation of the retina are shown.     

Metagenomic analysis of a stable trichloroethene-degrading microbial community

Dehalococcoides bacteria are the only organisms known to completely reduce chlorinated ethenes to the harmless product ethene. However, Dehalococcoides dechlorinate these chemicals more effectively and grow more robustly in mixed microbial communities than in isolation. In this study, the phylogenetic composition and gene content of a functionally stable trichloroethene-degrading microbial community was examined using metagenomic sequencing and analysis. For phylogenetic classification, contiguous sequences (contigs) longer than 2500 bp were grouped into classes according to tetranucleotide frequencies and assigned to taxa based on rRNA genes and other phylogenetic marker genes. Classes were identified for Clostridiaceae, Dehalococcoides, Desulfovibrio, Methanobacterium, Methanospirillum, as well as a Spirochete, a Synergistete, and an unknown Deltaproteobacterium. Dehalococcoides contigs were also identified based on sequence similarity to previously sequenced genomes, allowing the identification of 170 kb on contigs shorter than 2500 bp. Examination of metagenome sequences affiliated with Dehalococcoides revealed 406 genes not found in previously sequenced Dehalococcoides genomes, including 9 cobalamin biosynthesis genes related to corrin ring synthesis. This is the first time that a Dehalococcoides strain has been found to possess genes for synthesizing this cofactor critical to reductive dechlorination. Besides Dehalococcoides, several other members of this community appear to have genes for complete or near-complete cobalamin biosynthesis pathways. In all, 17 genes for putative reductive dehalogenases were identified, including 11 novel ones, all associated with Dehalococcoides. Genes for hydrogenase components (271 in total) were widespread, highlighting the importance of hydrogen metabolism in this community. PhyloChip analysis confirmed the stability of this microbial community.