Evaluating Nitrogen-Containing Biosynthetic Products Produced by Saltwater Culturing of Several California Littoral Zone Gram-Negative Bacteria.

The biosynthetic potential of marine-sediment-derived Gram-negative bacteria is poorly understood. Sampling of California near-shore marine environments afforded isolation of numerous Gram-negative bacteria in the Proteobacteria and Bacteriodetes phyla, which were grown in the laboratory to provide extracts whose metabolites were identified by comparative analyses of LC-mass spectrometry and MSn data. Overall, we developed an assemblage of seven bacterial strains grown in five different media types designed to coax out unique secondary metabolite production as a function of varying culture conditions. The changes in metabolite production patterns were tracked using the GNPS MS2 fragmentation pattern analysis tool. A variety of nitrogen-rich metabolites were visualized from the different strains grown in different media, and strikingly, all of the strains examined produced the same new, proton-atom-deficient compound, 1-methyl-4-methylthio-ß-carboline (1), C13H12N2S. Scale-up liquid culture of Achromobacter spanius (order: Burkholderiales; class: Betaproteobacteria) provided material for the final structure elucidation. The methods successfully combined in this work should stimulate future studies of molecules from marine-derived Gram-negative bacteria.

Assessing the trypanocidal potential of natural and semi-synthetic diketopiperazines from two deep water marine-derived fungi.

Human African trypanosomiasis (HAT, commonly known as African sleeping sickness) is categorized as a neglected disease, as it afflicts >50,000 people annually in sub-saharan Africa, and there are few formal programs in the world focused on drug discovery approaches for this disease. In this study, we examined the crude extracts of two fungal strains (Aspergillus fumigatus and Nectria inventa) isolated from deep water sediment which provided >99% growth inhibition at 1microg/mL of Trypanosoma brucei, the causative parasite of HAT. A collection of fifteen natural products was supplemented with six semi-synthetic derivatives and one commercially available compound. Twelve of the compounds, each containing a diketopiperazine core, showed excellent activity against T. brucei (IC(50)=0.002-40microM), with selectivity over mammalian cells as great as 20-fold. The trypanocidal diketopiperazines were also tested against two cysteine protease targets Rhodesain and TbCatB, where five compounds showed inhibition activity at concentrations less than 20microM. A preliminary activity pattern is described and analyzed.

Four Classes of Structurally Unusual Peptides from Two Marine-Derived Fungi: Structures and Bioactivities.

The structures and biological properties of peptides produced by two genera of marine-derived fungi, an atypical Acremonium sp., and a Metarrhizium sp. were explored. The Acremonium strain was isolated from a marine sponge and has previously been shown by our group to produce peptides from the efrapeptin and RHM families. The isolation and structure elucidation of the new linear pentadecapeptides efrapeptins Eα (1), H (2) and N-methylated octapeptides RHM3 (3) and RHM4 (4) were carried out through a combination of 1D and 2D NMR techniques and tandem MS. Additional known efrapeptins E, F, G and the known syctalidamides A and B were also isolated. The absolute configurations of 1 - 4 are proposed to be the same as the original compound families. The marine-sponge derived Metarrhizium sp. was shown to produce destruxin cyclic depsipeptides including A, B, B2, desmethyl B, E chlorohydrin and E2 chlorohydrin. Efrapeptins Eα (1), F and G each displayed IC(50)s of 1.3 nM against H125 cells, and destruxin E2 chlorohydrin displayed an IC(50) of 160 nM against HCT-116 cells. An initial therapeutic assessment suggested a continuous (168 h) exposure of at least 2 ng/ml, or a daily (24 h) exposure of at least 300 ng/ml for H125 cells treated with efrapeptin G, and a continuous (168 h) exposure of at least 190 ng/ml for HCT-116 cells treated with destruxin E2 chlorohydrin, will cause 90% tumor cell death in vitro.

Estrogen-triggered delays in mammary gland gene expression during the estrous cycle: evidence for a novel timing system.

During the estrous cycle and beginning in estrus, the mammary gland undergoes pregnancy-like development that depends on transcriptional regulation by the estrogen and progesterone receptors (ER, PR) and Pax-2 as well as the action of the growth factors Wnt-4 and RANKL. In this report, we first describe the decay and delayed expression of ERalpha, PR, and Pax-2 proteins as well as depression of Wnt-4 and RANKL mRNA coincident with the strong estrogen surge in proestrus. In time-course studies using ovari-ectomized mice, a single estrogen injection replicated these delays and caused an 18 h delay in Wnt-4 expression. Molecular time-delay systems are at the core of cellular cycles, most notably the circadian clock, and depend on proteasome degradation of transcriptional regulators that exhibit dedicated timing functions. The cytoplasmic dynamics of these regulators govern delay duration through negative transcription/translation feedback loops. A proteasome inhibitor, PS-341, blocked estrogen-stimulated ERalpha, PR, and Pax-2 decay and proteasome chymotryptic activity, assayed using a fluorogenic substrate, was elevated in proestrus correlating with the depletion of the transcription factors. The 18-h delay in Wnt-4 induction corresponded to the turnover time of Pax-2 protein in the cytoplasm and was eliminated in Pax-2 knockout mammary tissue, demonstrating that Pax-2 has a unique timing function. The patterns of estrogen-triggered ERalpha, PR, and Pax-2 turnover were consistent with a negative transcriptional feedback. Retarding the expression of ERalpha, PR, and Pax-2 may optimize preparations for pregnancy by coordinating expression of critical receptors and transcription factors with rising estrogen and progesterone levels in estrus. The estrogen surge in proestrus has no defined mammotropic function. This study provides the first evidence that it is a synchronizing signal triggering proteasome-dependent turnover of mammary gland ERalpha, PR, and Pax-2. We hypothesize that the delays reflect a previously unrecognized timing system, which is present in all ovarian target tissues.

SLIT/ROBO2 signaling promotes mammary stem cell senescence by inhibiting Wnt signaling.

WNT signaling stimulates the self-renewal of many types of adult stem cells, including mammary stem cells (MaSCs), but mechanisms that limit this activity are poorly understood. Here, we demonstrate that SLIT2 restricts stem cell renewal by signaling through ROBO2 in a subset of basal cells to negatively regulate WNT signaling. The absence of SLIT/ROBO2 signaling leads to increased levels of nuclear ß-catenin. Robo2 loss does not increase the number of stem cells; instead, stem cell renewal is enhanced in the absence of SLIT/ROBO2 signaling. This is due to repressed expression of p16(INK4a), which, in turn, delays MaSC senescence. Together, our studies support a model in which SLITs restrict the expansion of MaSCs by countering the activity of WNTs and limiting self-renewal.

SLIT/ROBO1 signaling suppresses mammary branching morphogenesis by limiting basal cell number.

In the field of breast biology, there is a growing appreciation for the "gatekeeping function" of basal cells during development and disease processes yet mechanisms regulating the generation of these cells are poorly understood. Here, we report that the proliferation of basal cells is controlled by SLIT/ROBO1 signaling and that production of these cells regulates outgrowth of mammary branches. We identify the negative regulator TGF-ß1 upstream of Robo1 and show that it induces Robo1 expression specifically in the basal layer, functioning together with SLIT2 to restrict branch formation. Loss of SLIT/ROBO1 signaling in this layer alone results in precocious branching due to a surplus of basal cells. SLIT2 limits basal cell proliferation by inhibiting canonical WNT signaling, increasing the cytoplasmic and membrane pools of ß-catenin at the expense of its nuclear pool. Together, our studies provide mechanistic insight into how specification of basal cell number influences branching morphogenesis.

Pinpointing pseurotins from a marine-derived Aspergillus as tools for chemical genetics using a synthetic lethality yeast screen.

A new compound of mixed polyketide synthase-nonribosomal peptide synthetase (PKS/NRPS) origin, 11- O-methylpseurotin A ( 1), was identified from a marine-derived Aspergillus fumigatus. Bioassay-guided fractionation using a yeast halo assay with wild-type and cell cycle-related mutant strains of Saccharomyces cerevisiae resulted in the isolation of 1, which selectively inhibited a Hof1 deletion strain. Techniques including 1D and 2D NMR, HRESIMS, optical rotation, J-based analysis, and biosynthetic parallels were used in the elucidation of the planar structure and absolute configuration of 1. A related known compound, pseurotin A ( 2), was also isolated and found to be inactive in the yeast screen.

Using jasplakinolide to turn on pathways that enable the isolation of new chaetoglobosins from Phomospis asparagi.

The isolation and structure elucidation of three new secondary metabolites, chaetoglobosin-510 (1), -540 (2), and -542 (3), are described. These compounds were produced by cultures of the marine-derived fungus Phomopsis asparagi, challenged with the known F-actin inhibitor jasplakinolide. Chaetoglobosin-542 (3) displayed antimicrofilament activity and was cytotoxic toward murine colon and leukemia cancer cell lines.