High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A.

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2-5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site. LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actin-dependent and the other actin-independent, underlie polarity development. The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function. Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways. Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site. Thus, actin filaments are also required for maintenance of an axis of cell polarity.

Jasplakinolide's inhibition of the growth of prostate carcinoma cells in vitro with disruption of the actin cytoskeleton.

BACKGROUND: Jasplakinolide, a cyclodepsipeptide produced by an Indo-Pacific sponge, Jaspis johnstoni, has been reported to inhibit the growth of breast cancer cells. PURPOSE: The effects of jasplakinolide on the proliferation of three human immortalized prostate carcinoma cell lines (PC-3, LNCaP, and TSU-Pr1) were studied. The growth-inhibitory effect of jasplakinolide on the PC-3 cell line was studied in detail to elucidate its mechanism of action. METHODS: Cell counts were used to study growth inhibition. A protein-based microplate assay was used to assess the time of exposure needed to cause persistent growth inhibition and to study the effects of jasplakinolide analogues. Metabolic changes were assessed by following the incorporation of radiolabeled precursors. The effects of jasplakinolide on the cytoskeleton were studied by fluorescent microscopy, using rhodamine phalloidin (RP) and antibodies to cytoskeletal components. Changes in RP binding were quantified by extracting bound fluorescent material from fixed cells and measuring the amount of fluorescence in a spectrofluorometer. RESULTS: The growth of PC-3, LNCaP, and TSU-Pr1 cells was potently inhibited by exposure to jasplakinolide for 48 hours; doses of jasplakinolide that led to 50% growth inhibition were 65 nM for PC-3 cells, 41 nM for LNCaP cells, and 170 nM for TSU-Pr1 cells. In PC-3 cells, exposure to 160 nM for 48 hours led to total growth inhibition, which persisted for several days even after drug removal. Several jasplakinolide analogues also inhibited the growth of PC-3 cells, although analogues in which the rigidity of the macrolide ring was altered were ineffective. No early changes in the synthesis of DNA, RNA, or protein or in intracellular adenosine triphosphate levels were seen in the PC-3 cells after exposure to jasplakinolide. Growth inhibition by jasplakinolide was accompanied by striking morphologic changes. Exposure for several doublings led to multinucleated cells. Further investigation of these changes in the PC-3 cells revealed a dramatic and early disruption of the actin cytoskeleton and a statistically significant decrease in RP binding. The doses of jasplakinolide, the time of exposure, and the pattern of growth inhibition by structural analogues corresponded with the changes seen in actin distribution. CONCLUSIONS: Jasplakinolide represents a novel marine natural product with potent in vitro antiproliferative activity against human prostate carcinoma cell lines, and it appears to target the actin cytoskeleton. IMPLICATIONS: Jasplakinolide is a potential candidate for further preclinical development and a lead structure for a novel class of therapeutic agents that can disrupt the actin cytoskeleton in mammalian cells.

Synthesis and antitumor activity of ester-modified analogues of bengamide B.

Bengamide B, a novel sponge-derived marine natural product with broad spectrum antitumor activity, was not suitable for further preclinical development because of its difficult synthesis and very poor water solubility. Bengamide B produced a 31% T/C at its solubility-limited maximum intravenous dose of 33 micromol/kg in MDA-MB-435 breast carcinoma implanted subcutaneously as a xenograft in nude mice. Compound 8a, a bengamide B analogue with three structural changes (t-Bu alkene substituent, unsubstituted lactam nitrogen, and inverted lactam 5'-myristoyloxy group), was as potent as bengamide B in vitro and more efficacious than bengamide B in vivo. A series of ester-modified analogues based on 8a were synthesized and tested in vitro and in vivo (MDA-MB-435). The cyclohexyl- and phenethyl-substituted esters, 8c and 8g, respectively, had in vitro and in vivo activities similar to that of 8a and enhanced water solubility (ca. 1 mg/mL). Consequently, 8c and 8g were tested in the MDA-MB-435 xenograft model at 100 micromol/kg and produced 29% and 57% tumor regression, respectively.

Comparison of the intrasubject repeatability of auditory brain stem and middle latency responses elicited in young children.

The auditory brain stem response (ABR) and middle latency response (MLR) were studied in 48 young children (96 ears). The responses were elicited using low intensity stimuli (30-dB nHL clicks) and simultaneously were recorded on a dual time base. Both the ABR and MLR were elicited in 70 ears. In 12 ears, just one response was recorded (ABR in eight ears and the MLR in four ears). In 14 ears, neither response was recorded. Test-retest analysis on the same subject demonstrated that the ABR was more repeatable and easier to identify than the MLR. The test-retest difference was determined for the amplitude and latency of the ABR and MLR waveforms. The test-retest latency difference for wave Pa was found to be 3.6 times larger than for wave V. The normalized test-retest amplitude difference for P phi-Na, Na-Pa, and Pa-Nb was found to be two to three times larger than for wave V. These data support the conclusion that the ABR, rather than the MLR, should be used to measure hearing in young children. The authors also advocate using minimal high pass (HP) filtering when recording the ABR in a sedated or sleeping child. Muscle artifact was not found to be a problem. The authors suggest the use of minimal HP filtering so that phase-shift distortion is minimized and a larger response amplitude can be recorded.

Buzonamine, a new alkaloid from the defensive secretion of the millipede, Buzonium crassipes.

GC-MS analysis of the defensive secretion from the millipede Buzonium crassipes showed three volatile components, beta-pinene (35%), limonene (6%) and a new alkaloid, buzonamine (59%). Buzonamine had an HRMS molecular ion at m/z=221.1785 (calculated for C(14)H(23)NO, 221.1781), 14 carbons in the 13C-NMR and 23 hydrogens by SFORD, DEPT and APT experiments. All distinct 1H nuclei were assigned to 13C resonances with 2D 1H-13C COSY data, and the final structure was determined by 1H-1H COSY, 1H-13C HMBC and nOe experiments. Buzonamine contains four rings including an epoxy group and a tertiary nitrogen. beta-Pinene, limonene or buzonamine, kept the mound nesting ant, Formica obscuripes, from eating mealworms (Tenebrio molitor) treated with 1mg of the chemical. A 150mg millipede expels 4mg of secretion.

Unraveling the Numerous Biosynthetic Products of the Marine Sediment-Derived Fungus, Aspergillus insulicola.

A new tripeptide, pre-sclerotiotide F (3), was isolated from a marine sediment-derived fungus, Aspergillus insulicola, along with five known compounds, one of which was new at the time of isolation, scerotiotide F (4). The absolute configuration elucidation of the new compound was determined using a combination of NMR, HR-ESI-MS, and optical rotation analyses. Cytotoxicities were measured in vitro against selected cancer cells. The effects of pre-sclerotiotide F (3) and sclerotiotide F (4) on LPS-induced NF-κB and iNOS expression were also measured.

Biosynthetically diverse compounds from a saltwater culture of sponge-derived Aspergillus niger.

The new compound, asperic acid (1), and the known compounds hexylitaconic acid (2), malformin C (3), pyrophen (4), and asperazine (5) were isolated from the saltwater culture of Aspergillus niger derived from a Caribbean sponge, Hyrtios proteus. The structure elucidation of asperic acid is presented.

13C-NMR assignments and cytotoxicity assessment of zoanthoxanthin alkaloids from zoanthid corals.

Zoanthoxanthin alkaloids can vary among skeletal types A, B, and C. Three type C zoanthoxanthins were examined, including a new compound 1, previously reported paragracine [2], and zoanthoxanthin 3. Their nmr and cytotoxic properties are reported. We have used 2D nmr data to complete the assignments for 1 and suggest that these benchmark nmr assignments will allow future investigators to establish new metabolites of this class as a member of families A, B, or C with only a 13C APT and a 1H-1H nmr spectra.

Novel marine sponge amino acids, 10. Xestoaminols from Xestospongia sp.

The study of the anthelminthic components from a Fiji sponge Xestospongia sp. has yielded new amino alcohols, xestoaminols A-C. Xestoaminol B, (2S*)-aminotetradeca-11, 13-dien-(3R*)-ol [2], is isomeric to known Xestospongia products, (2S)-aminotetradeca-5,7-dien-(3R)-ol [6] and (2S)-aminotetradeca-5,7-dien-(3S)-ol [7], recently reported by Gulavita and Scheuer. Xestoaminols A [1] and C [3] are, respectively, the dihydro and tetrahydro derivatives of xestoaminol B. A combined nmr and molecular mechanics study on the oxazolidinone of xestoaminol A provided the basis for the relative stereochemistry assigned at C-2 and C-3 in xestoaminol A. This compound was extremely active in assays testing for action against parasites, microbes, and reverse transcriptase.

Haliclostanone sulfate and halistanol sulfate from an Indo-Pacific Haliclona sponge.

Two steroids, haliclostanone sulfate (1) and halistanol sulfate (3), were isolated from a Haliclona sp. marine sponge. The structure of the new compound 1 was established based on its spectroscopic data and the properties of a pentaacetate derivative 2. Compound 1 is unique in that it possesses the rare cis C/D ring junction precedented only in marine sterols, contignasterol (4) and xestobergsterols A (5), B (6), and C (7).

Dihydrotubastrines: phenethylguanidine analogues from the Indo-Pacific marine sponge Petrosia cf. contignata.

Two phenethylguanidine derivatives, 7,8-dihydrotubastrine (1) and 4-deoxy-7,8-dihydrotubastrine (2), along with the sterol xestobergsterol A (3), were isolated from the marine sponge Petrosia cf. contignata. The structures of the new natural products 1 and 2 were based on spectroscopic data and comparison to the literature properties for semisynthetic 1. This is the first example from this compound class with a saturated acyclic C2 unit.

Sesterterpenes from a common marine sponge, Hyrtios erecta.

Eleven sesterterpenes have been characterized from the biotoxic extracts of Hyrtios erecta. Their structures and stereochemistry were established by spectral properties or results from chemical interconversions. Especially valuable for establishing stereochemical features were 13C-nmr methyl shift correlations and analysis of 1H-nmr couplings and chemical shifts. The new scalarane sesterterpenes included 2, 3, 7, 9, and 10a (but 9 and 10a might be artifacts), and they were accompanied by five previously described scalaranes, 1, 4, 5a, 6, and 8, along with hyrtial (11), a novel norsesterterpene. Many of these sesterterpenes exhibited interesting biological activity.

Cyclic polyketide peroxides and acyclic diol analogues from the sponge Plakortis lita.

The sponge Plakortis lita from Papua New Guinea is a source of three cyclic peroxides-ethyl plakortide Z (3), ethyl didehydroplakortide Z (4), and methyl didehydroplakortide Z (5)-and three acyclic diol analogues-ethyl seco-plakortide Z (6), epi-ethyl seco-plakortide Z (7), and ethyl didehydro-seco-plakortide Z (8). The absolute stereochemistry at the three chiral sites of 3 was assigned by preparing 6, which was investigated using the refined Mosher's method. Compounds 4, 5, and 6 were also concluded to have the same absolute stereochemistry as 3. The cyclic peroxides were generally cytotoxic, while the acyclic analogues were devoid of activity. Compound 3 was equally active in vitro against solid tumor and L-1210 leukemia cell lines. Alternatively, 4 was observed in vitro to be moderately solid-tumor selective but did not exhibit in vivo activity against solid tumors in mice.

Homoscalarane sesterterpenes from Lendenfeldia frondosa.

The marine sponge Lendenfeldia frondosa, collected from the Solomon Islands, has yielded homoscalarane sesterterpenes. Two new metabolites, epihomoscalaralactone IIA [5] and homoscalarate II [10], were accompanied by two known metabolites, homoscalaralactone IIA [1] and homoscalaralactone IIB [7]. These structures were established by analysis of 2D nmr data, trends in 13C-nmr shifts, and comparison of experimental with molecular-mechanics-calculated nmr J's. Each of the alcohols 1, 2, and 3 was converted to its corresponding acetate, 2, 6, and 8, respectively. Compound 6 exhibited moderate anti-inflammatory activity.

The arenarans, sesquiterpene ethers from the marine sponge Dysidea arenaria.

Two new cyclic sesquiterpenes, arenarans A [1] and B [2], were isolated from a Thai collection of Dysidea arenaria. Their chemical and cytotoxic properties are described.

Anthelmintic polyfunctional nitrogen-containing terpenoids from marine sponges.

The study of the anthelmintic terpenoid components from the Fiji sponge Axinyssa fenestratus (senior synonym of Leucophloeus fenestratus) and two Thailand sponges, Acanthella cavernosa and Topsentia sp., has yielded several new nitrogen-containing sesqui- and diterpenes of known carbon skeletons. Amorphane sesquiterpenes from A. fenestratus included (1R, 6S, 7S, 10S)-10-isothiocyanato-4-amorphene [(+)-1] and new metabolites (1R*, 4S*, 6R*, 7S*)-4-isothiocyanato-9-amorphene [2], 10-isothiocyanato-4,6-amorphadiene [3], and (4S*, 10S*)-10-isothiocyanato-5-amorphen-4-ol [4]. The amorphene (+)-1 of this study may be antipodal to (-)-1 previously isolated from a Hawaiian sponge. A similar relationship may exist at C-1, C-6, C-7 between (+)-2 of this study and (-)-11 isolated from a Palauian sponge. Another known sesquiterpene, axisonitrile 3 [5] was obtained from Topsentia sp. The diterpenes obtained from A. cavernosa included known kalihinols X [6] and Y [8] and new kalihinols J [7] and I [9]. Those terpenoids with potent antiparasite activity include 1, 2, 3-5, and 7-9.

The structures and cytotoxic properties of polyketide peroxides from a Plakortis sponge.

A morphologically distinct Fijian sponge, Plakortis sp., has yielded two new peroxides, plakortolide E [5] and plakoric acid [12]. After standing for approximately one year, plakortolide E rearranged to plakortolide ether [10]. The structures of plakortolide E [5] and plakortolide ether [10] were established from 2D nmr data and by analogy to a known compound, plakortolide [3]. The stereochemistry of the bicyclic ring substituents of 5 was established using nOe and NOESY nmr data along with comparisons to 3. The absolute stereochemistry at the three chiral sites of 5 was assigned by preparing acyclic compounds 6-9, and both 8 and 9 were investigated using the modified Mosher's method. This represents the first absolute stereochemistry determination for a sponge-derived polyketide peroxide. The characterization of plakoric acid [12] was based on spectral analogies to known polyketides such as plakortin. Plakortolide E [5] exhibited selective potency against the melanoma and breast tumor cell lines in the in vitro 60-cell line panel of the National Cancer Institute.

New and known diketopiperazines from the Caribbean sponge, Calyx cf. podatypa.

The cyanobacteria-containing Caribbean sponge, Calyx cf. podatypa, was collected from three sites in the Bahamas. In each of the three collections, a polar solvent partition fraction contained six known compounds including five diketopiperazines [1-4,6] and phenylacetic acid, along with a new diketopiperazine, cyclo-(4-methyl-R-proline-S-norvaline) [5]. Interestingly, all six diketopiperazines are proline-derived cyclic dipeptides. This is the first example for this class of peptide derivative to be isolated from a Calyx sponge. Parallel studies of one of the sponge collections in which the ectosome (cyanobacteria-rich) was separated from the endosome (no cyanobacteria) revealed no significant differences in their content of diketopiperazines.

Jasplakinolide induces apoptosis in various transformed cell lines by a caspase-3-like protease-dependent pathway.

To clarify the mechanisms underlying the antiproliferative effects of jasplakinolide, a cyclic depsipeptide from marine sponges, we examined whether jasplakinolide induces apoptosis in a variety of transformed and nontransformed cells. Jasplakinolide inhibited proliferation of human Jurkat T cells, resulting in cell death. This was accompanied by chromatin condensation and DNA cleavage at the linker regions between the nucleosomes. When caspase-3-like activity in the cytosolic extracts of Jurkat T cells was examined with a fluorescent substrate, DEVD-MAC (N-acetyl-Asp-Glu-Val-Asp-4-methyl-coumaryl-7-amide), the activity in the cells treated with jasplakinolide was remarkably increased in a time-dependent manner. Pretreatment of Jurkat T cells with the caspase inhibitor zVAD [benzyloxycarbonyl(Cbz)-Val-Ala-beta-Asp(OMe)-fluoromethylketone] or DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-1-aldehyde) prevented the induction of apoptosis by jasplakinolide. Moreover, exposure of various murine transformed cell lines to jasplakinolide resulted in cell death, which was inhibited by zVAD. Although it has been well established that murine immature thymocytes are sensitive to apoptosis when exposed to various apoptotic stimuli, these cells as well as mature T lymphocytes were resistant to jasplakinolide-induced apoptosis. The results suggest that jasplakinolide induces apoptotic cell death through a caspase-3-like protease-dependent pathway. Another important outcome is that transformed cell lines were more susceptible to jasplakinolide-induced apoptosis than normal nontransformed cells.

New structures and bioactivity patterns of bengazole alkaloids from a choristid marine sponge.

New bengazoles 3-9 as inseparable mixtures are reported from the sponge Jaspis cf. coricea collected in Papua New Guinea. These compounds contain a new variation of the unusual bisoxazole core present in bengazole A [1]. The B-ring oxazole hexatetraol side chain is varyingly substituted with either myristic acid or 13-methylmyristic acid. Hydrolysis of three different bengazole mixtures each yielded an identical tetraol, bengazole Z [11].