Arabidopsis Aux/IAA genes are involved in brassinosteroid-mediated growth responses in a manner dependent on organ type

We examined whether auxin/indole-3-acetic acid (Aux/IAA) proteins, which are key players in auxin-signal transduction, are involved in brassinosteroid (BR) responses. iaa7/axr2-1 and iaa17/axr3-3 mutants showed aberrant BR sensitivity and aberrant BR-induced gene expression in an organ-dependent manner. Two auxin inhibitors were tested in terms of BR responses. Yokonolide B inhibited BR responses, whereas p-chlorophenoxyisobutyric acid did not inhibit BR responses. DNA microarray analysis revealed that 108 genes were up-regulated, while only eight genes were down-regulated in iaa7. Among the genes that were up- or down-regulated in axr2, 22% were brassinolide-inducible genes, 20% were auxin-inducible genes, and the majority were sensitive neither to BR nor to auxin. An inhibitor of BR biosynthesis, brassinazole, inhibited auxin induction of the DR5-GUS gene, which consists of a synthetic auxin-response element, a minimum promoter, and a beta-glucuronidase. These results suggest that Aux/IAA proteins function in auxin- and BR-signaling pathways, and that IAA proteins function as the signaling components modulating BR sensitivity in a manner dependent on organ type.     

Roles of NO Signaling in Long-Term Memory Formation in Visual Learning in an Insect

Many insects exhibit excellent capability of visual learning, but the molecular and neural mechanisms are poorly understood. This is in contrast to accumulation of information on molecular and neural mechanisms of olfactory learning in insects. In olfactory learning in insects, it has been shown that cyclic AMP (cAMP) signaling critically participates in the formation of protein synthesis-dependent long-term memory (LTM) and, in some insects, nitric oxide (NO)-cyclic GMP (cGMP) signaling also plays roles in LTM formation. In this study, we examined the possible contribution of NO-cGMP signaling and cAMP signaling to LTM formation in visual pattern learning in crickets. Crickets that had been subjected to 8-trial conditioning to associate a visual pattern with water reward exhibited memory retention 1 day after conditioning, whereas those subjected to 4-trial conditioning exhibited 30-min memory retention but not 1-day retention. Injection of cycloheximide, a protein synthesis inhibitor, into the hemolymph prior to 8-trial conditioning blocked formation of 1-day memory, whereas it had no effect on 30-min memory formation, indicating that 1-day memory can be characterized as protein synthesis-dependent long-term memory (LTM). Injection of an inhibitor of the enzyme producing an NO or cAMP prior to 8-trial visual conditioning blocked LTM formation, whereas it had no effect on 30-min memory formation. Moreover, injection of an NO donor, cGMP analogue or cAMP analogue prior to 4-trial conditioning induced LTM. Induction of LTM by an NO donor was blocked by DDA, an inhibitor of adenylyl cyclase, an enzyme producing cAMP, but LTM induction by a cAMP analogue was not impaired by L-NAME, an inhibitor of NO synthase. The results indicate that cAMP signaling is downstream of NO signaling for visual LTM formation. We conclude that visual learning and olfactory learning share common biochemical cascades for LTM formation.     

Pifithrin-μ, an Inhibitor of Heat-Shock Protein 70, Can Increase the Antitumor Effects of Hyperthermia Against Human Prostate Cancer Cells

Hyperthermia (HT) improves the efficacy of anti-cancer radiotherapy and chemotherapy. However, HT also inevitably evokes stress responses and increases the expression of heat-shock proteins (HSPs) in cancer cells. Among the HSPs, HSP70 is known as a pro-survival protein. In this study, we investigated the sensitizing effect of pifithrin (PFT)-μ, a small molecule inhibitor of HSP70, when three human prostate cancer cell lines (LNCaP, PC-3, and DU-145) were treated with HT (43°C for 2 h). All cell lines constitutively expressed HSP70, and HT further increased its expression in LNCaP and DU-145. Knockdown of HSP70 with RNA interference decreased the viability and colony-forming ability of cancer cells. PFT-μ decreased the viabilities of all cell lines at one-tenth the dose of Quercetin, a well-known HSP inhibitor. The combination therapy with suboptimal doses of PFT-μ and HT decreased the viability of cancer cells most effectively when PFT-μ was added immediately before HT, and this combination effect was abolished by pre-knockdown of HSP70, suggesting that the effect was mediated via HSP70 inhibition. The combination therapy induced cell death, partially caspase-dependent, and decreased proliferating cancer cells, with decreased expression of c-Myc and cyclin D1 and increased expression of p21^WAF1/Cip, indicating arrest of cell growth. Additionally, the combination therapy significantly decreased the colony-forming ability of cancer cells compared to therapy with either alone. Furthermore, in a xenograft mouse model, the combination therapy significantly inhibited PC-3 tumor growth. These findings suggest that PFT-μ can effectively enhance HT-induced antitumor effects via HSP70 inhibition by inducing cell death and arrest of cell growth, and that PFT-μ is a promising agent for use in combination with HT to treat prostate cancer.     

Inhibition of chromosome separation in fertilized starfish eggs by kalihinol F, a topoisomerase I inhibitor obtained from a marine sponge

Kalihinol F, a naturally occurring diterpene from a marine sponge, Acanthella sp., inhibited chromosome separation in fertilized starfish (Asterina pectinifera) eggs but allows the first cleavage to occur, thereby forming unseparated metaphase chromosomes which were elongated between the two daughter cells. The chromosomes were eventually torn off in the embryonic cells. Most of the cells gradually lost the chromosomes during the cell cycle progression. The embryonic development halted at the morula stage just before the onset of blastulation. The mitotic failure occurred when kalihinol F was applied to a fertilized egg during the second meiotic process, but not after the completion of the second meiotic division. Kalihinol F inhibited topoisomerase I activity in vitro, but had no effects on activities of DNA polymerases alpha, beta, and gamma, and of topoisomerase II. These results suggest that the topoisomerase I plays an essential role in meiosis II in this species.     

A Protein Encoded by din1, a Dark-Inducible and Senescence-Associated Gene of Radish, Can Be Imported by Isolated Chloroplasts and Has Sequence Similarity to Sulfide Dehydrogenase and Other Small Stress Proteins

In an attempt to isolate cDNA clones for dark-inducible chloroplastproteins, we screened a cDNA library which was prepared fromradish cotyledons by a two-step method. The source plants weregrown under continuous light for 14 d and kept in darkness for24 h. One of the selected clones, S2D12, corresponded to thedin1 gene which we previously reported as a dark-inducible,senescence-associated gene [Azumi and Watanabe (1991) PlantPhysiol. 95: 577]. A 22 kDa polypeptide was produced from thecDNA in an in vitro expression system in the presence of [³⁵S]methionine.This polypeptide was capable of being imported by isolated chloroplasts,processed to a smaller mature form and localized in the stromalfraction. As the amino acid sequence of the putative matureprotein has no homology to any known chloroplast protein, din1was suggested to be the first gene for a chloroplast proteinwhich is negatively controlled by light. The putative matureprotein has similarity to sulfide dehydrogenase from Wolinellasuccinogenes and other small stress proteins; glpE and pspEfrom Escherichia coli and hsp67B2 from Drosophila melanogaster. ¹ The nucleotide sequence data in this paper has been submittedto EMBL, GenBank and DDBJ Data Libraries under the acces sionnumber AB004242 ² Present address: The Institute of Physical and Chemical Research(RIKEN), 2-1 Hirosawa, Wako-shi, Saitama, 351-01 Japan