We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca 2؉ -ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca 2؉ -ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca 2؉ /H ؉ exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 m from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.

Azonazine, a unique hexacyclic dipeptide was isolated from a Hawaiian marine sediment-derived fungus eventually identified as, Aspergillus insulicola. Its absolute configurations, 2R, 10R, 11S, 19R were established using NMR, HRESIMS, and CD data plus insights derived from molecular models. A possible route for its biogenesis is proposed and biological properties were explored against cancer cell lines and in an NFκB inhibition assay.We believe that scientific study of fungi (Ascomycota) within the genus Aspergillus (Class: Eurotiomycetes, Family: Trichocomaceae) is important. Though more than 200 Aspergillus strains have been isolated from a host of terrestrial ecological niches, they provide a steady stream of diverse small molecules.1 Recent understanding the genomics of chemically Correspondence to: Phillip Crews, phil@chemistry.ucsc.edu. Supporting Information Available: Extraction scheme and bioactivity data, NMR data, possible configurations and biosynthetic pathway of azonazine. This material is available free of charge via the Internet at http://pubs.acs.org. We began a campaign to emphasize chemical study on the >20 marine-derived Aspergillus strains (from sponges and sediments) housed in the UCSC repository. The prospect of encountering orphan 13 biosynthetic pathways was part of the rationale for this investigation. The results reported below represent a first step and involve the characterization and biological evaluation of a novel hexacyclic dipeptide, azonazine from A. insulicola related to ochraceopetaliformis (syn: A. flocculosisi). 14 This project was initiated by scanning our collection of Aspergillus cultures to identfy those possessing strain uniqueness accompanied by activity in a cytotoxicity soft agar-based disk diffusion assay. 15 One top candidate was A. insulicola (strain no. 088708a, identifed by molecular taxonomy evaluations shown in SI Table S5) obtained from a Hawiaan shallow water sediment. The EtOAc crude extract of the initial small-scale culture (125 mL, in Czapek-Dox liquid medium, pH adusted to 7.0, prepared with artificial seawater) exhibited potent activity (See Supporting Information (SI) Table S2) against murine Colon 38 cell lines and selectivity against human prostate adenocarcinoma (LNCaP) vs human leukemia cell lines (CEM). A scale up culture (10 L, same media) was harvested at 21 days (shaking at 150 rpm) and worked up by passing it through HP-20 resin. The resin was subsequently washed using water (fraction coded: Hp1, 362 mg), 50% methanol/water (Hp2, 1327 mg), methanol (Hp3, 443 mg) and isopropanol (Hp4, 12.8 mg). Each fraction was subjected to LCMS analysis and the Hp3 was selected for further purification via RP-HPLC (30-50% acetonitrile / 0.1% formic acid-water, 50 mins), and 42 fractions (F1-F42) were collected. Further purification of fraction F11 (3.5mg) via RP-HPLC yielded azonazine (1.1mg) and, insulicolide A (2.2 mg). 16 NIH Public AccessEarly on we recognized that azonazine, 17 obtained as a colorless powder and possessing molecular formula C 2...