Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.

Leinamycin (1) was isolated from the culture broth of a Streptomyces sp. in 1989, 1-3 and its structure was elucidated by spectroscopic analysis, 1 X-ray crystallography, 4 and chemical synthesis. 5 This antibiotic contains an unusual 1,3-dioxo-1,2dithiolane moiety, which is connected to the 18-membered lactam through a spiro linkage, and appeared to be a new class of natural product. 1 exhibited significant antitumor activity in some murine tumor models. 2 We previously reported that 1 induces single-strand scission of DNA in the presence of thiol cofactors in vitro. We report here the detailed chemistry of thiol-activation and DNA-cleavage induced by 1.Addition of 1.5 equiv of 2-mercaptoethanol (2-ME) to a solution of 1 in MeOH/10 mM phosphate buffer (pH 7) (1/9, v/v) resulted in a rapid conversion to a major product with several minor products. Other thiols including ethanethiol, dithiothreitol, cysteine, and glutathione afforded approximately equal amounts of 2. We isolated 2 from the reaction mixture with reverse-phase HPLC (70%, yield), but the full characterization of 2 failed due to its instability in DMSO. Treatment of 2 with K 2 CO 3 and iodomethane afforded a stable methyl ester 2a (68%, yield). Characterization of 2a by 1D and 2D-NMR experiments established an unexpected structure, in which the 1,3-dioxo-1,2-dithiolane moiety and the 6,7-olefin were missing and a new 3,7-sulfide linkage and 6-hydroxyl group were observed. Treatment of 1 with 2-ME in a MeOH-rich solvent, MeOH/0.5 M phosphate buffer (pH 7) (99/1, v/v), afforded the methanol adduct 2b 6 as the main product.After reaction of 1 with calf thymus DNA in the presence of 2-ME (drug/DNAbp/2ME ) 1/20/1.5), the DNA was purified by ethanol precipitation. The DNA showed a UV spectrum characteristic of the complex with a chromophore. Although it was stable at 4°C, gradual release of the chromophore from the DNA was observed at 37°C. The rate and efficiency of the release increased with further an increase in the temperature. On a preparative scale, we isolated the released chromophore 3 from the leinamycin-treated calf thymus DNA (75% yield from 1). In the 13 C NMR spectrum of 3 all of the resonances were comparable to those of 2, and five additional resonances were found, suggesting the addition of a purine residue to 2. In the 1 H-NMR spectrum, all of the resonances were also comparable to those of 2. One additional nonexchangeable resonance at 7.77 ppm (1H) was found. The only nucleobase that gives one nonexchangeable resonance is guanine. Observation of the NOE between the guanine H-8 and the 6-CH 3 is consistent with alkylation of the C-6 carbon by the N-7 of guanine. These spectroscopic data revealed that 3 is a leinamycin-N7 guanine adduct. This was supported by the molecular formula that was established as the sodium salt C 27 H 31 O 7 N 7 S 2 Na: HRFABMS (M + Na) + m/z 652.1600, calcd 652.1624. 1 did not react with guanosine nucleotide monomer or single stranded DNA, suggesting that the alkylation of DNA could be attributable to the unique...

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Leinamycin is a recently discovered antitumor antibiotic with an unusual 1,3-dioxo-1,2-dithiolane structure. It preferentially inhibits the incorporation of [3H]thymidine into the acid-insoluble fraction of Bacillus subtilis. In vitro, leinamycin causes single-strand cleavage of supercoiled double-helical pBR322 DNA in the presence of thiol cofactors. Scavengers of oxygen radical did not supress the DNA-cleaving activity. Thiol-activated leinamycin binds calf thymus DNA at 4 degrees C and thermal treatment of the leinamycin-DNA adduct released a chemically modified leinamycin from the complex. The lack of cytotoxicity and DNA-cleaving activity for S-deoxyleinamycin indicates that the 1,3-dioxo-1,2-dithiolane moiety is essential for the activity of leinamycin. Thus, the primary cellular target of leinamycin appears to be DNA. It binds DNA and causes single-strand break at low concentrations, which may account for the potent antitumor activity.

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A new inhibitor of protein kinase C (PKC), UCN-01, was isolated from the culture broth of Streptomyces sp. N-126. Wehave found that this strain also produces UCN-02 which is a stereoisomer of UCN-01.The inhibitors have the molecular formula C28H26N4O4 and have an indolo[2,3-#]carbazole chromophore. Their structures have been elucidated by mass and NMR spectra. UCN-01has been shown to inhibit PKCand protein kinase A (PKA) with IC5o values of 0.0041 and 0.042^m, respectively, and UCN-02has been shown to inhibit PKCand PKAwith IC50 values of 0.062 and 0.25^m, respectively. UCN-01and UCN-02also showedthe cytotoxic effect on the growth of HeLaS3 cells.In the course of our screening program for new selective inhibitors of protein kinase C (PKC)1~3), a Streptomyces strain N-1264) was found to produce new selective inhibitors of PKC, UCN-01and UCN-02. UCN-01is a novel compound that has a hydroxyl group at C-7 of staurosporine5>6), and UCN-02is a stereoisomer at C-7 of UCN-01. They have been shown to inhibit PKCat extremely low concentration and showed the cytotoxic effect on the growth of HeLa S3 cells. In this paper, their purification, physico-chemical properties, structures and biological activities are described.Isolation and Purification TLC and HPLCanalysis were used to monitor UCN-01 and UCN-02 during isolation from the culture broth. The whole broth (30 liters) was filtered and the solid cake was extracted with acetone. Acetone extract was diluted with equivalent volume of water and then the extract was mixed with the filtrate of the broth. The mixed solution was applied on a column (2 liters) of non-ionic porous resin, Diaion HP-20 (Mitsubishi Chemical Industries Limited). After washing with water and then with aqueous methanol (2 : 1) to remove impurities, the column was eluted with acetone. The acetone eluate was evaporated to remove acetone. The aqueous solution was adjusted to pH10 and extracted with ethyl acetate. The resulting organic phase was extracted with 0.1 n hydrochloric acid and after the separation the aqueous phase was immediately adjusted to pH 10 by addition of NaOHand extracted with ethyl acetate. The ethyl acetate extract was applied to silica gel (Wakogel C-200) column chromatography with chloroform -methanol as a elution solvent. The 97 %-chloroform eluate was concentrated to give a pale yellow syrup, which was recrystallized from methanol to obtain 200 mg of

RP-1776, a novel cyclic peptide, was isolated from the culture broth of Streptomyces sp. KYI 1784. RP-1776 selectively inhibited the binding of PDGFBB to the extracellular domain of the PDGF /^receptor with an IC50 value of 11±6/im. Detailed binding experiments suggested that RP-1776 directly interacts with PDGF BB. RP-1776 inhibited the phosphorylation of the PDGFj3-receptor induced by PDGFBB. These results suggested that RP-1776 antagonizes the signaling of PDGFBB probably through the inhibition of PDGFBB binding to the PDGF^-receptor. Platelet-derived growth factor (PDGF) is a potent mitogen and chemotactic molecule for various cells, released from activated platelets. h2) PDGFis also expressed in embryonal tissues, and has neurotrophic activity for neuronal cells in rat brain, suggesting involvement of