Bi-analyte SERS with isotopically edited dyes.

Isotopically substituted rhodamine dyes provide ideal probes for the study of single-molecule surface enhanced Raman scattering (SM-SERS) events through multiple-analyte techniques. Isotopic editing should, in principle, provide probes that have identical chemical properties (and surface chemistries); while exhibiting at the same time distinct Raman features which enable us to identify single-molecule SERS events. We present here a specific example of two-analyte SM-SERS based on the isotopic substitution of a methyl ester rhodamine dye. The dyes are carefully characterized (in both standard and SERS conditions) to confirm experimentally their similar chemical properties. We then demonstrate their utility for bi-analyte SERS (BiASERS) experiments and, as an example, highlight the transition from a single, to a few, to many molecules in the statistics of SM-SERS signals.

Spatial and temporal variability of cytotoxic metabolites in populations of the New Zealand sponge Mycale hentscheli.

Intraspecific variation in the composition of three cytotoxic secondary metabolites from the New Zealand marine sponge Mycale hentscheli collected at two sites in central New Zealand was quantified by 1H NMR techniques. A total of 275 sponges were analyzed bimonthly over 15 mo to compare intersite (approximately 100 km) and intrasite (approximately 100 m) spatial and temporal variations in the metabolites. Biological and physical characteristics of sponge size, morphology, depth, and temperature were recorded at each site. Metabolite concentrations were found to vary in space and time. Metabolite composition was site-specific; mycalamide A, pateamine, and peloruside A were present at Pelorus Sound, whereas pateamine was absent from sponges at Kapiti Island. Pateamine and peloruside A concentrations in sponges at Pelorus Sound varied seasonally; no such patterns were observed at Kapiti Island. Relationships of compound concentration with volume and depth were complex. High levels of peloruside A in Pelorus Sound sponges from between 8 and 10 m depth coincided with a density boundary layer and chlorophyll a maximum.

Peloruside A enhances apoptosis in H-ras-transformed cells and is cytotoxic to proliferating T cells.

Peloruside A (peloruside), a compound isolated from the marine sponge Mycale hentscheli , inhibits growth of human (HL-60) and mouse (32D-ras) myeloid leukemic cells, as well as non-transformed 32D cells. Using the MTT cell proliferation assay and trypan blue dye exclusion tests, little difference was seen in growth inhibition between 32D and 32D- ras cells; however, peloruside was more cytotoxic to the oncogene-transformed cells. Peloruside also blocked 32D- ras cells more readily in G2/M of the cell cycle, leading to apoptosis. Annexin-V/propidium iodide staining of 32D and 32D- ras cells showed that 1.6 microM peloruside induced significant cell death by 36 hours in 32D cells (16% survival), but to comparable levels as early as 14 hours in 32D- ras cells (11% survival). There was no evidence for activation of either of the initiator caspases-8 or -9 by 0.1 microM peloruside following 12 hours of exposure. Peloruside inhibited T cell proliferation and IL-2 and IFN gamma production in both the mixed lymphocyte reaction and following CD3 cross-linking, and this effect was shown to be a non-specific cytotoxic effect. It is concluded that peloruside preferentially targets oncogene-transformed cells over non-transformed cells by inducing transformed cells to undergo apoptosis.

Induction of apoptosis by the marine sponge (Mycale) metabolites, mycalamide A and pateamine.

The marine sponge metabolites mycalamide A (mycalamide) and pateamine are extremely cytotoxic. While mycalamide has been shown to inhibit protein synthesis, the mechanism by which these compounds induce cell death is unknown. Using DNA laddering, Annexin-V staining, and morphological analysis, we demonstrate that both metabolites induce apoptosis in several different cell lines. Furthermore, both mycalamide and pateamine were more potent inducers of apoptosis in the 32D myeloid cell line after transformation with either the ras or bcr-abl oncogenes. This increased sensitivity was also observed in response to the protein synthesis inhibitors cycloheximide and puromycin, and cytosine-beta-D-arabinofuranoside (Ara-C), an inducer of DNA damage. We propose, therefore, that in 32D cells where Ras signalling has been altered either by constitutive expression of oncogenic ras or by Bcr/abl-mediated perturbation of upstream signalling events, increased susceptibility to apoptosis by a range of stimuli is conferred.

The novel cytotoxic sponge metabolite peloruside A, structurally similar to bryostatin-1, has unique bioactivity independent of protein kinase C.

A novel secondary sponge metabolite, peloruside A (peloruside), isolated from the marine sponge Mycale sp. (New Zealand), was tested for its cytotoxic effects on mammalian cells in culture. The macrolide structure of peloruside is similar to that of the protein kinase C (PKC) activator, bryostatin-1 (bryostatin), both containing a pyranose ring adjacent to a gemdimethyl moiety. Peloruside is a potent inhibitor of cell proliferation. Treatment of different mammalian cell lines with peloruside for 48-96 h gave IC50 values ranging from 4 to 15 nM, using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium (MTT) cell proliferation assay. Peloruside was shown to be both cytostatic and cytotoxic by trypan blue dye exclusion tests. Peloruside induced apoptosis in a dose-dependent manner in murine (32D) and human (HL-60) myeloid cell lines, revealed by DNA laddering in agarose gels and flow cytometric analysis of annexin-V- and propidium iodide-stained cells. Treatment of HL-60 cells caused vacuolisation, partial substrate adherence, and the appearance of multi-lobed nuclei, suggesting the induction of a differentiation pathway. Vacuolisation was also observed in a human lung cancer cell line (H441). Opening of the pyranose ring of peloruside by sodium borohydride reduction increased the 48 h IC50 value by 26-fold in 32D cells, suggesting a similar active site to that proposed for bryostatin. However, unlike bryostatin, peloruside failed to bind to PKC in HL-60 cells and was unable to synergize with the calcium ionophore, ionomycin, or with interleukin-2, to activate T-lymphocytes in culture. In summary, although structurally similar to bryostatin, peloruside is a potent inhibitor of cell proliferation, has apoptosis-inducing properties and has a unique mode of action independent of PKC.

Mycalamide D, a new cytotoxic amide from the New Zealand marine sponge Mycale species.

A new mycalamide, mycalamide D (3), has been isolated from the New Zealand marine sponge Mycale sp. This new metabolite, in which the C13-O-methyl group of mycalamide A (1) is replaced by a hydrogen atom, was found to be cytotoxic to a range of mammalian cell lines, with a potency approximately 20-fold less than that of 1.

Peloruside A: a potent cytotoxic macrolide isolated from the new zealand marine sponge Mycale sp.

A novel, polyoxygenated, pyranose ring containing 16-membered macrolide peloruside A (1) exhibiting cytotoxic activity in the nanomolar range was isolated from the New Zealand marine sponge Mycale sp. The structure of 1 and relative stereochemistry of the 10 stereogenic centers were determined on a 3 mg sample using a variety of spectroscopic methods. Compound 1 was isolated along with the previously reported cytotoxins mycalamide A (2) and pateamine (3) from a single specimen of this sponge.

Pyrroindomycins, novel antibiotics produced by Streptomyces rugosporus sp. LL-42D005. I. Isolation and structure determination.

Pyrroindomycins A and B were isolated from fermentations of culture LL-42D005, a strain of Streptomyces rugosporus. Pyrroindomycins possess potent antimicrobial activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci. Their structures have been determined by using 1- and 2-D NMR, mass spectroscopy and chemical degradations. Pyrroindomycins are the first natural products that contain the highly unsaturated pyrroloindole moiety.

Glycothiohexide alpha, a novel antibiotic produced by "Sebekia" sp., LL-14E605. II. Isolation and physical-chemical characterization.

Glycothiohexide alpha was recovered from the fermentation broth of a "Sebekia" sp. by mixed solvent extraction, selective precipitation and adsorption chromatography on Diaion HP-20. The amount of glycothiohexide alpha present in the crude preparation was enriched by photolysis. Purification of glycothiohexide alpha was accomplished by repetitive countercurrent chromatography.

Glycothiohexide alpha, a novel antibiotic produced by "Sebekia" sp., LL-14E605. III. Structural elucidation.

The chemical structure of a novel thiopeptide antibiotic, glycothiohexide alpha (1), isolated from the fermentation broth of a "Sebekia" species was determined based on extensive 2D NMR studies, as well as, IR, UV, and mass spectral data. The chemical structure of glycothiohexide alpha is closely related to nosiheptide (3) and antibiotic S-54832A.