Studies on Mitomycins,Carcinostatic Antibiotics

Mitomycin A (C₁₆H₁₉O₆N₃) and mitomycin C (C₁₅H₁₈O₅N₄) are pigments which have the quinoid structure. When treated with aqueous ammonia, mitomycin A is converted to mitomycin C. Acid hydrolysis of mitomycin C gave three degradation products, namely, C₁₄H₁₅O₅N₄, C₁₄H₁₅O₆N₃ and C₁₃H₁₄O₅N₂. Acetylation with acetic anhydride and pyridine and methylation with methyl iodide gave monoacetyl and monomethyl derivatives of mitomycin C respectively, though diacetate of demethyl derivatives were obtained when boiled with acetic anhydride.     

Synthetic circular RNA switches and circuits that control protein expression in mammalian cells

Synthetic messenger RNA (mRNA) has been focused on as an emerging application for mRNA-based therapies and vaccinations. Recently, synthetic circular RNAs (circRNAs) have shown promise as a new class of synthetic mRNA that enables superior stability and persistent gene expression in cells. However, translational control of circRNA remained challenging. Here, we develop ‘circRNA switches’ capable of controlling protein expression from circRNA by sensing intracellular RNA or proteins. We designed microRNA (miRNA) and protein-responsive circRNA switches by inserting miRNA-binding or protein-binding sequences into untranslated regions (UTRs), or Coxsackievirus B3 Internal Ribosome Entry Site (CVB3 IRES), respectively. Engineered circRNAs efficiently expressed reporter proteins without inducing severe cell cytotoxicity and immunogenicity, and responded to target miRNAs or proteins, controlling translation levels from circRNA in a cell type-specific manner. Moreover, we constructed circRNA-based gene circuits that selectively activated translation by detecting endogenous miRNA, by connecting miRNA and protein-responsive circRNAs. The designed circRNA circuits performed better than the linear mRNA-based circuits in terms of persistent expression levels. Synthetic circRNA devices provide new insights into RNA engineering and have a potential for RNA synthetic biology and therapies.     

Involvement of protein phsophatase-1 in cytoskeletal organization of cultured endothelial cells

The phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effect of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vien endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non-selective kinase inhibitor, K-252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP-1. The catalytic subunit of PP-1 was also identified in HUVECs by Western blotting with its monoclonal antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton. © 1995 Wiley-Liss, Inc.     

Novel anticancer agents, kayeassamins A and B from the flower of Kayea assamica of Myanmar

The CHCl(3)-soluble fraction of 70% EtOH extract of the flower of Kayea assamica completely killed human pancreatic PANC-1 cancer cells preferentially under nutrient-deprived conditions at 1 microg/mL. Bioassay-guided fractionation and isolation afforded two novel compounds, kayeassamins A (1) and B (2). Their structures were elucidated using extensive spectroscopic methods and the modified Mosher method. Each compound showed 100% preferential cytotoxicity (PC(100)) against PANC-1 cells under nutrient-deprived conditions at 1 microM. Furthermore, both compounds inhibited the migration of PANC-1 cells in the wound closure assay.     

Relationship between the distribution of euphausiids and baleen whales in the Antarctic (35°E – 145°W)

Simultaneous whale sighting and hydroacoustic surveys were conducted from a research vessel in the Antarctic to examine the relationship between the distribution of euphausiids and baleen whales. High densities of minke whales and large aggregations of euphausiids were observed along the ice edge over the continental slope in the southeast region of area IV and in the southwest region of area V. The results suggest that the continental slope zone that coincides with the ice edge would be an important minke whale feeding area. Minke whales were rarely sighted in the offshore region even if euphausiids were abundant. Distributions of humpback whales were correlated with high euphausiid density zones, regardless of the bottom topographic features. Several groups of blue whales were sighted in the small area along the ice edge where euphausiids were abundant, but sightings were too few to draw any general conclusion about the relationship between blue whales and euphausiids. Both baleen whales and euphausiids were scarce in the area east of 170°W where sea ice covered the continental shelf and slope zone.     

Random Amplified Polymorphic DNA (RAPD) Variation among Populations of the Insular Endemic Plant Campanula microdonta(Campanulaceae)

RAPD variation was examined in nine populations of Campanulamicrodonta Koidz., endemic to the Izu Islands, Japan. Ninety-eightbands were obtained for all populations, 94% of which were polymorphicat least within a population. Shannon's H values were calculated;these have frequently been used in RAPD studies to estimategenetic diversity. The values within populations did not correlatewith the allozyme gene diversity estimated by a previous studyor with distance from the Japanese mainland. The possible reasonsfor this discrepancy are different selection regimes betweenthe two markers, higher RAPD mutation rates, and each marker'sdifferent coverage of genomes. Cluster analysis of genetic similaritiessuggested that colonization of each island probably occurredonce, except for Miyake Island, where immigration has occurredat least twice. Copyright 2001 Annals of Botany Company AMOVA, Campanula microdonta Koidz., insular endemic plant, genetic diversity, population genetic structure, RAPD     

Oscillations of cytoplasmic concentrations of Ca2+ and K+ in fused L cells

Summary Using Ca²⁺- and K⁺-selective microelectrodes, the cytosolic free Ca²⁺ and K⁺ concentrations were measured in mouse fibroblastic L cells. When the extracellular Ca²⁺ concentration exceeded several micromoles, spontaneous oscillations of the intracellular free Ca²⁺ concentration were observed in the submicromolar ranges. During the Ca²⁺ oscillations, the membrane potential was found to oscillate concomitantly. The peak of cyclic increases in the free Ca²⁺ level coincided in time with the peak of periodic hyperpolarizations. Both oscillations were abolished by reducing the extracellular Ca²⁺ concentration down to 10^–7 m or by applying a Ca²⁺ channel blocker, nifedipine (50 m). In the presence of 0.5mm quinine, an inhibitor of Ca²⁺-activated K⁺ channel, sizable Ca²⁺ oscillations still persisted, while the potential oscillations were markedly suppressed. Oscillations of the intracellular K⁺ concentration between about 145 and 140mm were often associated with the potential oscillations. The minimum phase of the K⁺ concentration was always 5 to 6 sec behind the peak hyperpolarization. Thus, it is concluded that the oscillation of membrane potential results from oscillatory increases in the intracellular Ca²⁺ level, which, in turn, periodically stimulate Ca²⁺-activated K⁺ channels.     

The synergic modeling for the binding of fluoroquinolone antibiotics to the hERG potassium channel

The fluoroquinolone antibiotic binding site in the hERG potassium channel was examined for the residues involved and their position in the tetrameric channel. The blocking effect of the two fluoroquinolones levofloxacin and sparfloxacin to tandem dimers of the hERG mutants were evaluated electrophysiologically. The results indicated that two Tyr652s in the neighboring subunits and one or two Phe656s in the diagonal subunits contributed to the blockade in the case of both compounds, and Ser624 was also involved. The docking studies suggested that the protonated carboxyl group in the compounds strongly interacts with Phe656 as a π acceptor.     

Structural dynamics of proximal heme pocket in HemAT-Bs associated with oxygen dissociation

HemAT from Bacillus subtilis (HemAT-Bs) is a heme-containing O(2) sensor protein that acts as a chemotactic signal transducer. Binding of O(2) to the heme in the sensor domain of HemAT-Bs induces a conformational change in the protein matrix, and this is transmitted to a signaling domain. To characterize the specific mechanism of O(2)-dependent conformational changes in HemAT-Bs, we investigated time-resolved resonance Raman spectra of the truncated sensor domain and the full-length HemAT-Bs upon O(2) and CO dissociation. A comparison between the O(2) and CO complexes provides insights on O(2)/CO discrimination in HemAT-Bs. While no spectral changes upon CO dissociation were observed in our experimental time window between 10ns and 100μs, the band position of the stretching mode between the heme iron and the proximal histidine, ν(Fe-His), for the O(2)-dissociated HemAT-Bs was lower than that for the deoxy form on time-resolved resonance Raman spectra. This spectral change specific to O(2) dissociation would be associated with the O(2)/CO discrimination in HemAT-Bs. We also compared the results obtained for the truncated sensor domain and the full-length HemAT-Bs, which showed that the structural dynamics related to O(2) dissociation for the full-length HemAT-Bs are faster than those for the sensor domain HemAT-Bs. This indicates that the heme proximal structural dynamics upon O(2) dissociation are coupled with signal transduction in HemAT-Bs.