Absolute Configuration of 4-Chloroisoleucine in Cyclolithistide A: An Antifungal Peptide Lactone Discovered in Marine Lithistid Sponges.

Cyclolithistide A is a peptide lactone isolated from marine lithistid sponges. Its entire structure, including absolute configurations, has been reported except the relative and absolute configurations of its characteristic residue, 4-chloroisoleucine (4-CIle). We synthesized four isomers of 4-CIle from furfural-derived N-Boc imine and propionaldehyde. Analysis of the acid hydrolysate of cyclolithistide A and the synthetic samples of 4-CIle after derivatization with l- and d-FDAA permitted us to propose the absolute configuration of the 4-chloroisoleucine residue in cyclolithistide A as 2S,3R,4R.

Mice with gene alterations in the GH and IGF family.

Much of our understanding of GH's action stems from animal models and the generation and characterization of genetically altered or modified mice. Manipulation of genes in the GH/IGF1 family in animals started in 1982 when the first GH transgenic mice were produced. Since then, multiple laboratories have altered mouse DNA to globally disrupt Gh, Ghr, and other genes upstream or downstream of GH or its receptor. The ability to stay current with the various genetically manipulated mouse lines within the realm of GH/IGF1 research has been daunting. As such, this review attempts to consolidate and summarize the literature related to the initial characterization of many of the known gene-manipulated mice relating to the actions of GH, PRL and IGF1. We have organized the mouse lines by modifications made to constituents of the GH/IGF1 family either upstream or downstream of GHR or to the GHR itself. Available data on the effect of altered gene expression on growth, GH/IGF1 levels, body composition, reproduction, diabetes, metabolism, cancer, and aging are summarized. For the ease of finding this information, key words are highlighted in bold throughout the main text for each mouse line and this information is summarized in Tables 1, 2, 3 and 4. Most importantly, the collective data derived from and reported for these mice have enhanced our understanding of GH action.

Oshimalides A and B, Sesterterpenes of the Manoalide Class from a Luffariella sp. Deep-Sea Marine Sponge: Application of Asymmetric Dihydroxylation in Structure Elucidation.

Oshimalides A (1) and B (2) were isolated from a Luffariella sp. marine sponge. The absolute configurations of the stereogenic centers in the cyclohexenone ring were determined by the modified Mosher's analysis of the reduction product. The absolute configuration of the stereogenic center in the dihydropyran ring was assigned by analysis of the 1H NMR data of the vicinal diols which were prepared by AD-mix reagents stereoselectively.

Homophymamide A, Heterodetic Cyclic Tetrapeptide from a Homophymia sp. Marine Sponge: A Cautionary Note on Configurational Assignment of Peptides That Contain a Ureido Linkage.

A previously unreported heterodetic cyclic peptide, homophymamide A (1), was isolated from a Homophymia sp. marine sponge. The structure of homophymamide A was determined to be a lower homologue of anabaenopeptins by spectroscopic analysis, chemical degradation, and chemical synthesis. Analysis of the acidic hydrolysate showed that the racemization of Lys took place, leading us to pose a cautionary note on the configurational assignment of peptides that contain a ureido bond.

Structure Elucidation of Calyxoside B, a Bipolar Sphingolipid from a Marine Sponge Cladocroce sp. through the Use of Beckmann Rearrangement.

Marine sponges are an excellent source of biologically active secondary metabolites. We focus on deep-sea sponges for our discovery study. A marine sponge Cladocroce sp. exhibited cytotoxic activity in the bioactivity screening. From this sponge a previously unreported cytotoxic glycosphingolipid, calyxoside B, was isolated and the structure of this compound was elucidated by analyses of MS and NMR spectra and chemical derivatization. We converted the ketone in the middle of a long aliphatic chain into an oxime to which was applied Beckmann rearrangement to afford two positional isomers of amides. The products were subjected to acidic hydrolysis followed by LC-MS analysis, permitting us to assign unequivocally the position of the ketone. Calyxoside B shows cytotoxicity against HeLa cells with an IC50 value of 31 µM and also weakly stimulated the production of cytokines in mice.

Metachromins X and Y from a marine sponge Spongia sp. and their effects on cell cycle progression.

New sesquiterpene quinones, metachromins X (1) and Y (2), together with the known metachromins C (3), J (4), and T (5), were isolated as inhibitors of cell cycle progression in the HeLa/Fucci2 cells. The structure of 1 was assigned by spectroscopic data and confirmed by a total synthesis. The planar structure of 2 was determined by interpretation of spectroscopic data, whereas its absolute configuration was analyzed by a combination of chiral HPLC and CD spectroscopy. Metachromins X (1) and C (3) arrested the cell cycle progression of HeLa/Fucci2 cells at S/G2/M phase.

Isolation and identification of N6-isopentenyladenosine as the cytotoxic constituent of a marine sponge Oceanapia sp.

N6-Isopentenyladenosine (i6A) was isolated from a marine sponge Oceanapia sp. as the major cytotoxic constituent along with N6-isopentenyladenosine 5'-monophosphate (i6AP) which was inactive. The structures of i6A and i6AP were assigned by a combination of the analysis of NMR spectroscopy and mass spectrometry. This is the first isolation of i6A and i6AP from a marine sponge.

An exception among diatoms: unique organization of genes involved in isoprenoid biosynthesis in Rhizosolenia setigera CCMP 1694.

The marine diatom Rhizosolenia setigera is unique among this group of microalgae given that it is only one of a handful of diatom species that can produce highly branched isoprenoid (HBI) hydrocarbons. In our efforts to determine distinguishing molecular characteristics in R. setigera CCMP 1694 that could help elucidate the underlying mechanisms for its ability to biosynthesize HBIs, we discovered the occurrence of independent genes encoding for two isopentenyl diphosphate isomerases (RsIDI1 and RsIDI2) and one squalene synthase (RsSQS), enzymes that catalyze non-consecutive steps in isoprenoid biosynthesis. These genes are peculiarly fused in all other genome-sequenced diatoms to date, making their organization in R. setigera CCMP 1694 a clear distinguishing molecular feature. Phylogenetic and sequence analysis of RsIDI1, RsIDI2, and RsSQS revealed that such an arrangement of individually transcribed genes involved in isoprenoid biosynthesis could have arisen through a secondary gene fission event. We further demonstrate that inhibition of squalene synthase (SQS) shifts the flux of exogenous isoprenoid precursors towards HBI biosynthesis suggesting the competition for isoprenoid substrates in the form of farnesyl diphosphate between the sterol and HBI biosynthetic pathways in this diatom.

3D reconstruction of endoplasmic reticulum in a hydrocarbon-secreting green alga, Botryococcus braunii (Race B).

MAIN CONCLUSION: Based on 3D sections through cells of Botryococcus braunii, the structure of three domains of endoplasmic reticulum, and their spatial and functional relationships to other organelles are clarified. Oil production by photosynthetic microalgae has attracted attention since these oils can be converted into renewable, carbon-neutral fuels. The green alga B. braunii accumulates large amounts of hydrocarbons, 30-50% of cell dry weight, in extracellular spaces rather than its cytoplasm. To advance the knowledge of hydrocarbon biosynthesis and transport pathways in this alga, we utilized transmission EM combined with rapid freezing and image reconstruction. We constructed detailed 3D maps distinguishing three ER domains: rdER with ribosomes on both sides, rsER with ribosomes on one side, and sER without ribosomes. The rsER and sER domains were especially prominent during the oil body formation and oil secretion stages. The ER contacted the chloroplasts, oil bodies, or plasma membrane via the rsER domains, oriented with the ribosome-free surface facing the organelles. We discuss the following transport pathway for hydrocarbons and their precursors in the cytoplasm: chloroplast â†’ endoplasmic reticulum (ER) â†’ oil bodies â†’ ER â†’ plasma membrane â†’ secretion. This study represents the first 3D study of the three-domain classification (rdER, rsER and sER) of the ER network among eukaryotic cells. Finally, we propose the novel features of the ERs in plant cells that are distinct from the latest proposed model for the ERs in mammalian cells.

Colony-wise Analysis of a Theonella swinhoei Marine Sponge with a Yellow Interior Permitted the Isolation of Theonellamide I.

There are several examples of marine organisms whose metabolic profiles differ among conspecifics inhabiting the same region. We have analyzed the metabolic profile of each colony of a Theonella swinhoei marine sponge with a yellow interior and noticed the patchy distribution of one metabolite. This compound was isolated and its structure was studied by a combination of spectrometric analyses and chemical degradation, showing it to be a congener in the theonellamide class of bicyclic peptides. Theonellamides had previously been isolated by us only from T. swinhoei with a white interior and not from those with a yellow interior.

Miuramides A and B, Trisoxazole Macrolides from a Mycale sp. Marine Sponge That Induce a Protrusion Phenotype in Cultured Mammalian Cells.

Morphology-guided cell-based screening of the extract of a Mycale sp. marine sponge led to the isolation of two trisoxazole macrolides, miuramides A (1) and B (2), which induced characteristic morphological changes in 3Y1 cells. The structure of 1 including absolute configuration was elucidated by a combination of the analysis of spectroscopic data, derivatization, and degradation. Both compounds exhibit potent cytotoxicity against 3Y1 cells.

Poecillastrin H, a Chondropsin-Type Macrolide with a Conjugated Pentaene Moiety, from a Characella sp. Marine Sponge.

Poecillastrin H (1), a chondropsin-type macrolide with a conjugated pentaene moiety, was isolated from the Characella sp. marine sponge. The planar structure of 1 was elucidated by analysis of spectroscopic data. The absolute configuration of the ß-hydroxyaspartic acid residue (ß-OHAsp) was determined to be d- threo by Marfey's analysis, and the mode of lactone ring formation through the OHAsp residue was determined by chemical degradation. Poecillastrin H was extremely sensitive toward light and showed potent cytotoxic activity against 3Y1 cells with an IC50 value of 4.1 nM.

Isolation and characterization of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene from Botryococcus braunii, race B.

The B race of a green microalga Botryococcus braunii Kützing produces triterpene hydrocarbons that is a promising source for biofuel. In this algal race, precursors of triterpene hydrocarbons are provided from the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The terminal enzyme of this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is regarded as one of the key enzymes that affect yields of products in terpene biosynthesis. In order to better understand the MEP pathway of the alga, cDNA and genomic clones of HDR were obtained from B. braunii Showa strain. B. braunii HDR (BbHDR) is encoded on a single copy gene including a 1509-bp open reading frame that was intervened by 6 introns. The exon-intron structure of BbHDR genes did not show clear relation to phylogeny, while its amino acid sequence reflected phyla and classes well. BbHDR sequence was distinctive from that of the HDR protein from Escherichia coli in the residues involved in hydrogen-bond network that surrounds substrate. Introduction of BbHDR cDNA into an E. coli HDR deficient mutant resulted in recovery of its auxotrophy. BbHDR expression level was upregulated from the onset of liquid culture to the 24th day after inoculation with a 2.5-fold increase and retained its level in the subsequent period.

Lactomycins A-C, Dephosphorylated Phoslactomycin Derivatives that Inhibit Cathepsin B, from the Marine-derived Streptomyces sp. ACT232.

Three new polyketides, lactomycins A (1)-C (3), were isolated from the culture broth of a marine-derived Streptomyces sp. ACT232 as cathepsin B inhibitors. Their structures were determined by a combination of NMR and MS data analyses to be the dephosphorylated derivatives of a phoslactomycin class of metabolites. Lactomycins exhibited cathepsin B inhibitory activity (IC50 0.8 to 4.5 µg/mL). Even though the biosynthetic gene clusters found in the genome of the current strain have high similarity to those of phoslactomycin, neither phoslactomycins nor leustroducsins were detected by LC-MS analyses of the crude extract.

Isolation and Characterization of Cyclic C33 Botryococcenes and a Trimethylsqualene Isomer from Botryococcus braunii Race B.

Three cyclic C33 botryococcenes and one new trimethylsqualene isomer were isolated from the B race, Showa (Berkeley) strain of Botryococcus braunii, which is known to produce large amounts of isoprenoid hydrocarbons ranging in carbon number from 30 to 34. Their purity was determined by GC-MS, and structures were characterized by 1D and 2D NMR. One of these molecules, cyclic C33-1 botryococcene (5), has an unusual connection of a methylenecyclohexane ring to the molecule backbone not seen before in botryococcenes. This report further adds to our knowledge of the wide range of isoprenoid hydrocarbon structures produced by B. braunii.

The Incidence of Oral and Oropharyngeal Cancers in Betel Quid-Chewing Populations in South Myanmar Rural Areas.

Oral cancer is a very common disease in South and Southeast Asia. Betel quid (BQ)- chewing and tobaccosmoking habits are etiological factors for oral cancer patients in these regions. We conducted an oral cancer screening in BQ-chewing endemic rural areas in South Myanmar for the early detection of oral cancer in BQ-chewing and smoking individuals. We examined 105 subjects who were at high risk of oral cancer due to their oral habits (BQ users and/or smokers). Three carcinoma cases were detected, and there were 8 dysplasia cases. The carcinoma detection rate was 2.9%, and the carcinoma and precancerous lesion detection rate was 10.5%. In Myanmar, oral cancer screening has been conducted sporadically on a voluntary basis, and nationwide surveys have never been performed. There are also few reports of oral cancer screening for high-risk groups among the general population in Myanmar. Our present findings highlight the need for further screening and surveys. Education on betel quid chewing- and tobacco- related oral diseases and screening for the early detection of oral cancer are of the utmost importance in the control and prevention of oral cancer.

Attenuation of Bleomycin-Induced Pulmonary Fibrosis in Rats with S-Allyl Cysteine.

Pulmonary fibrosis is a complex disease with high mortality and morbidity. As there are currently no effective treatments, development of new strategies is essential for improving therapeutic outcomes. S-allyl cysteine (SAC) is a constituent of aged garlic extract that has demonstrated efficacy as an antioxidant and anti-inflammatory agent. The current study examines the effects of SAC on pulmonary fibrosis induced by a single intratracheal instillation of bleomycin (2.5 mg/kg). SAC was administered to rats as 0.15% SAC-containing diet from seven days prior to instillation up until the conclusion of the experiment (14 days post-instillation). SAC significantly reduced collagen mRNA expression and protein deposition (33.3 ± 2.7 µg/mg and 28.2 ± 2.1 µg/mg tissue in vehicle- and SAC-treated rats, respectively), and decreased fibrotic area, as assessed histologically. In the rats' lungs, SAC also attenuated the increased expression of transforming growth factor-ß1 (TGF-ß1), a central regulator of myofibroblast recruitment, activation, and differentiation. While bleomycin instillation increased the number of myofibroblasts within the lung mesenchymal area, this change was significantly reduced by SAC treatment. SAC may exert efficacy as an anti-fibrotic by attenuating myofibroblast differentiation through TGF-ß1-mediated fibroproliferative processes. Thus, our results indicate SAC may be useful for the prevention or treatment of pulmonary fibrosis.

Astaxanthin ameliorates ferric nitrilotriacetate-induced renal oxidative injury in rats.

Daily intake of vegetables can reduce the risk of cancer and lifestyle-related diseases. However, supplementary intake of ß-carotene alone has been reported to increase the risk of lung cancer in male cigarette smokers and people who were exposed to asbestos. The mechanism of the antioxidative properties of carotenoids in vivo, especially under oxidative stress conditions, still remains unclear. To investigate the antioxidant properties of dietary compounds, we examined the effects of chemically modified astaxanthin (Ax-C-8) using a rat model of ferric nitrilotriacetate (Fe-NTA)-induced renal oxidative injury. Ax-C-8 demonstrated lethally toxic effects on the rats in a dose-dependent manner. Following supplementation with Ax-C-8 (0.02%, w/w) for 30 days, the rats were euthanized 1, 4 and 24 h after injection of Fe-NTA. After 4 h, Ax-C-8 pretreatment suppressed the elevation of creatinine and blood urea nitrogen and protected the rats from renal tubular necrosis and the formation of 4-hydroxy-2-nonenal-modified proteins. After 24 h, pretreatment with Ax-C-8 maintained the renal antioxidant enzyme levels and renal tubules. Here, we demonstrate the antioxidant effects of Ax-C-8 against Fe-NTA-induced oxidative injury in rats receiving a regular diet. These data suggest that dietary intake of astaxanthin may be useful for the prevention of renal tubular oxidative damage.

Biosynthetic Gene Cluster for Surugamide†A Encompasses an Unrelated Decapeptide, Surugamide†F.

Genome mining is a powerful method for finding novel secondary metabolites. In our study on the biosynthetic gene cluster for the cyclic octapeptides surugamides A-E (inhibitors of cathepsin B), we found a putative gene cluster consisting of four successive non-ribosomal peptide synthetase (NRPS) genes, surA, surB, surC, and surD. Prediction of amino acid sequence based on the NRPSs and gene inactivation revealed that surugamides A-E are produced by two NRPS genes, surA and surD, which were separated by two NRPS genes, surB and surC. The latter genes are responsible for the biosynthesis of an unrelated peptide, surugamide F. The pattern of intercalation observed in the sur genes is unprecedented. The structure of surugamide F, a linear decapeptide containing one 3-amino-2-methylpropionic acid (AMPA) residue, was determined by spectroscopic methods and was confirmed by solid-phase peptide synthesis.

Nazumazoles D-F, Cyclic Pentapeptides That Inhibit Chymotrypsin, from the Marine Sponge Theonella swinhoei.

Nazumazoles D-F (1-3) were isolated from the marine sponge Theonella swinhoei. The compounds gave extremely broad peaks by reversed-phase HPLC using an ODS column. HPLC using a gel permeation column was instrumental for the separation of the three compounds. Their planar structures were determined by interpretation of NMR data to be cyclic pentapeptides. Nazumazoles D-F contained one residue each of α-keto-l-norvaline (l-Knv) {or α-keto-d-leucine (l-Kle)}, l-alanyloxazole (l-Aox), d-Abu (or d-Ser), N-α-CHO-ß-l-Dpr, and cis-4-methyl-l-proline. The absolute configuration of each amino acid residue was determined by Marfey's method in combination with conversion of the α-keto-ß-amino acid to the α-amino acid by oxidation. Nazumazoles D-F are not cytotoxic against P388 cells at 50 µM, but inhibit chymotrypsin.