Iriomoteolide-3a, a cytotoxic 15-membered macrolide from a marine dinoflagellate amphidinium species.

A 15-membered macrolide, iriomoteolide-3a (1), with an allyl epoxide has been isolated from a marine benthic dinoflagellate Amphidinium sp. (strain HYA024), and the structure was assigned by detailed analyses of 2D NMR data. Relative and absolute configurations were elucidated on the basis of conformational studies of 1 and its acetonide (2) and modified Mosher's method of 1, respectively. Iriomoteolide-3a (1) and the acetonide (2) exhibited potently cytotoxic activity against antitumor cells.

Amphidinolides B6 and B7, cytotoxic macrolides from a symbiotic dinoflagellate Amphidinium species.

Two 26-membered macrolides, amphidinolides B6 ( 2) and B7 ( 1), have been isolated from a marine symbiotic dinoflagellate Amphidinium sp., and the structures were elucidated on the basis of detailed analyses of 2D NMR data. The relative and absolute configurations for 1 and 2 were assigned by comparison of NMR data and CD data with those of known amphidinolides.

Iriomoteolides-1b and -1c, 20-membered macrolides from a marine dinoflagellate Amphidinium species.

Two 20-membered macrolides, iriomoteolides-1b ( 1) and -1c ( 2), have been isolated from a marine dinoflagellate Amphidinium sp. (strain HYA024), and the structures were elucidated on the basis of detailed analyses of 2D NMR data and chemical correlation.

Iriomoteolide-1a, a potent cytotoxic 20-membered macrolide from a benthic dinoflagellate amphidinium species.

A potent cytotoxic 20-membered macrolide, iriomoteolide-1a (1), has been isolated from a benthic dinoflagellate Amphidinium sp. (strain HYA024), and the structure was elucidated on the basis of detailed analyses of 2-D NMR data. The relative and absolute stereochemistries were assigned by the combination of conformational analyses using NMR data and modified Mosher's method of 1.

Characterization of Oryza sativa telomerase reverse transcriptase and possible role of its phosphorylation in the control of telomerase activity.

Telomerase reverse transcriptase (TERT) has been characterized in the dicotyledon Arabidopsis thaliana. A TERT homolog has now been identified in the monocotyledon rice (Oryza sativa L.) on the basis of its predicted homology to the A. thaliana enzyme (AtTERT). At least five alternatively spliced transcripts of the rice TERT (OsTERT) gene were detected. The full-length OsTERT protein shares structural features with TERTs of other species, including a calculated molecular size of 144 kDa, an isoelectric point of 9.6, and conserved sequence motifs. Phylogenetic analysis showed that OsTERT clusters with AtTERT and is more related to the human and mouse enzymes than to those of yeast and ciliated protozoa, consistent with the evolutionary relations among these eukaryotes. Telomerase activity was abundant in shoot apices and cultured cells but was low or absent in leaves or roots of rice plants, whereas similarly spliced OsTERT transcripts were detected in all tissues examined and cultured cells. Similar to mouse and human TERT proteins, OsTERT contains two putative phosphorylation sites for Akt kinase. Incubation of a rice cell extract with Akt or with protein phosphatase 2A potentiated or inhibited telomerase activity, respectively, whereas Akt did not affect the activity in Arabidopsis cell extract. In addition, the kinase activated the telomerase in a leaf extract. The mechanism of telomerase regulation in rice thus appears to differ from that in Arabidopsis (which is mediated predominantly at the level of AtTERT transcription), possibly reflecting the taxonomic distance between monocotyledons and dicotyledons.

Characterization of NADPH-dependent oxidoreductase induced by auxin in rice.

An NADPH-dependent oxidoreductase (NADPH-DO) with an isoelectric point of 5.2 and a molecular mass of 35 kDa was isolated from suspension-cultured rice cells. NADPH-DO was inducible by auxin and gibberellin. NADPH-DO mRNA was expressed in roots and leaf sheaths of rice seedlings, but not in leaf blades. The levels of NADPH-DO mRNA and protein in suspension-cultured cells were increased by auxin; they were further raised by the application of zinc. In contrast, NADPH-DO mRNA and protein accumulation in intact roots was stimulated by auxin, but in this case the stimulation was inhibited by zinc. Auxin-induced callus growth and root formation in intact rice plants were further enhanced by zinc. These results indicate that high levels of NADPH-DO are necessary for auxin- and zinc-induced callus formation, and imply that zinc plays a role in the regulation of NADPH-DO induction by auxin.

Methylmalonate-semialdehyde dehydrogenase is induced in auxin-stimulated and zinc-stimulated root formation in rice.

Proteins induced in rice by auxin and zinc were determined by proteome analysis. Cultured suspension cells of rice were treated with 2,4-dichlorophenoxyacetic acid and ZnSO4 and then proteins were separated by two-dimensional polyacrylamide gel electrophoresis; seven proteins were found to be induced by auxin and zinc. Of these seven, methylmalonate-semialdehyde dehydrogenase (MMSDH) was elevated by treatment with auxin alone. MMSDH was detected in cultured suspension cells, root and leaf sheath, but not in leaf blades. MMSDH responded to auxin and gibberellin, but did not respond to brassinolide and cytokinin. Furthermore, the amount of MMSDH in slr1, a constitutive gibberellin response mutant, was 2-fold that of wild type. MMSDH mRNA and protein were stimulated in root formation induced by auxin and/or zinc over a 4-week period. These results suggest that MMSDH may be necessary for root formation in rice induced by auxin and/or zinc.

Identification of Ku70 and Ku80 homologues in Arabidopsis thaliana: evidence for a role in the repair of DNA double-strand breaks.

In higher organisms such as mammals and plants, DNA double-strand breaks (DSBs) are repaired preferentially by non-homologous end joining (NHEJ) rather than by homologous recombination. The NHEJ pathway is mediated by Ku, a heterodimer of approximately 70 and 80 kDa subunits, which contributes to various aspects of the metabolism of DNA ends in eukaryotic cells. On the basis of their predicted sequence similarity to human Ku70 and Ku80, cDNAs encoding the first plant homologues of these proteins (AtKu70 and AtKu80, respectively) have now been isolated from Arabidopsis thaliana. AtKu70 and AtKu80 share 28.6 and 22.5% amino acid sequence identity with human Ku70 and Ku80, respectively. Yeast two-hybrid analysis demonstrated that AtKu70 and AtKu80 form a heterodimer, and electrophoretic mobility-shift assays revealed that this heterodimer binds to double-stranded telomeric and non-telomeric DNA sequences, but not to single-stranded DNA. The AtKu heterodimer also possesses single-stranded DNA-dependent ATPase and ATP-dependent DNA helicase activities. Reverse transcription and the polymerase chain reaction revealed that AtKu70 and AtKu80 genes are expressed widely but at low levels in plant tissues. The expression of these two genes in cultured cells was markedly increased in response to the generation of DSBs by bleomycin or methylmethane sulfonate. These results suggest that the evolutionarily conserved Ku70-Ku80 heterodimer functions in DSB repair by the NHEJ pathway in A. thaliana.

MAP2 is required for dendrite elongation, PKA anchoring in dendrites, and proper PKA signal transduction.

Microtubule-associated protein 2 (MAP2) is a major component of cross-bridges between microtubules in dendrites, and is known to stabilize microtubules. MAP2 also has a binding domain for the regulatory subunit II of cAMP-dependent protein kinase (PKA). We found that there is reduction in microtubule density in dendrites and a reduction of dendritic length in MAP2-deficient mice. Moreover, there is a significant reduction of various subunits of PKA in dendrites and total amounts of various PKA subunits in hippocampal tissue and cultured neurons. In MAP2-deficient cultured neurons, the induction rate of phosphorylated CREB after forskolin stimulation was much lower than in wild-type neurons. Therefore, MAP2 is an anchoring protein of PKA in dendrites, whose loss leads to reduced amount of dendritic and total PKA and reduced activation of CREB.

Novel mutations in C-terminal channel region of the ryanodine receptor in malignant hyperthermia patients.

Malignant hyperthermia (MH) is a pharmacogenetical complication of general anesthesia resulting from abnormal Ca2+-induced Ca2+ release (CICR) via the type 1 ryanodine receptor (RyR1) in skeletal muscles. In this study, we analyzed the genomic DNAs prepared for determination of all the 106 exons of the RyR1 gene from blood samples donated by two MH patients with extremely high CICR rates in their biopsied skeletal muscles and a clear history of MH incidence. Two novel point mutations were found in the exons 96 and 101 with alterations in the coded amino acids within the C-terminal channel region, i.e., Pro4668 to Ser and Leu4838 to Val. The latter mutation was found in both MH patients. Rabbit RyR1 channels carrying corresponding mutations were expressed in CHO cells for functional assay. It was found that the L to V but not the P to S mutation of the RyR1 resulted in enhanced Ca2+ release activity. These results indicate that the L4838V mutation is responsible for the MH incidence. The L4838V mutation is unique because it is the mutation first found within a hydrophobic transmembrane segment of the channel region and should provide further information on the function of the RyR1 as well as for genetic diagnosis of MH.