TMKS8A, an antibacterial and cytotoxic chlorinated α-lapachone, from a sea slug-derived actinomycete of the genus Streptomyces.

TMKS8A (1), a new chlorinated α-lapachone derivative, along with five known related metabolites, A80915 C (2), SF2415B1 (3), chlorinated dihydroquinone 3 (4), SF2415B3 (5), and A80915 C (6), were identified from the culture extract of Streptomyces sp. TMKS8, which was isolated from a sea slug, Paromoionchis tumidus. The structure of 1 was determined by the analysis of NMR and MS spectral data, assisted by NMR chemical shift prediction using DFT-based calculation. The absolute configuration was determined to be R by comparison of experimental and calculated ECD spectra. Compound 1 displayed antimicrobial activity against Gram-positive bacteria with MIC values ranging from 6.25 to 12.5 µg ml-1 and cytotoxicity against murine leukemia P388 cells with IC50 9.8 µM.

New polyketides and diterpenoid derivatives from the fungus Penicillium sclerotiorum GZU-XW03-2 and their anti-inflammatory activity.

Two new compounds, named penisclerotiorin A (1) and diaporthein C (8), and a new natural product, penidepsidone A (4), together with five known compounds (2, 3, 5-7) were isolated from the fungus Penicillium sclerotiorum GZU-XW03-2. Their structures were assigned using spectroscopic methods, quantum chemical calculations, and single-crystal X-ray diffraction analysis. Penisclerotiorin A (1) that belongs to the highly oxidized diphenyl ether is rare found in natural sources, and it was the sixth example of highly oxidized diphenyl ether analogues in natural sources. Penidepsidone A (4) is a new natural product and no any NMR spectral data were reported to date, in this paper, we firstly used the NMR calculations to confirm the intact structure by comparison of the experimental NMR data. Diaporthein C (8) represents the third example of pimarane diterpenes bearing a double bond at C-8 and C-9. In the bioassays, all of the isolates (1-8) were tested for their anti-inflammatory effects on the production of nitric oxide in lipopolysaccharide-induced microglial cells (RAW 264.7 cells). Compounds 2, 3 and 6 showed potent anti-inflammatory effects than the positive control (indomethacin, IC50, 24.0 µM) with IC50 values of 11.52, 8.13 and 21.27 µM, respectively.

Ferritin from the Pacific abalone Haliotis discus hannai: Analysis of cDNA sequence, expression, and activity.

Ferritin plays an important role in iron homeostasis due to its ability to bind and sequester large amounts of iron. In this study, the gene encoding a ferritin (HdhFer2) was cloned from Pacific abalone (Haliotis discus hannai). The full-length cDNA of HdhFer2 contains a 5'-UTR of 121 bp, an ORF of 516 bp, and a 3'-UTR of 252 bp with a polyadenylation signal sequence of AATAAA and a poly(A) tail. It also contains a 31 bp iron-responsive element (IRE) in the 5'-UTR position, which is conserved in many ferritins. HdhFer2 consists of 171 amino acid residues with a predicted molecular weight (MW) ∼19.8 kDa and a theoretical isoelectric point (PI) of 4.84. The deduced amino acid sequence of HdhFer2 contains two ferritin iron-binding region signatures (IBRSs). HdhFer2 mRNA was detected in a wide range of tissues and was dominantly expressed in the gill. Infection with the bacterial pathogen Vibrio anguillarum significantly upregulated HdhFer2 expression in a time-dependent manner. Recombinant HdhFer2 (rHdhFer2) purified from Escherichia coli was able to bind ferrous iron in a concentration-dependent manner. In summary, these results suggest that HdhFer2 is a crucial protein in the iron-withholding defense system, and plays an important role in the innate immune response of abalone.

Characterization and expression analysis of EF hand domain-containing calcium-regulatory gene from disk abalone: calcium homeostasis and its role in immunity.

The complete amino acid sequence of a calcium-regulatory gene (denoted as Ab-CaReg I) was identified from the disk abalone Haliotis discus discus cDNA library. The Ab-CaReg I is composed of 176 amino acids and the calculated molecular mass and isoelectric point were 20 and 4.2, respectively. The sequence homology of Ab-CaReg I was 28-30 and 18-27% of known calmodulin and troponin C, respectively. Four characteristic calcium-binding EF hand motifs with some modifications at conserved positions of known homologous calmodulin genes were observed in the sequence. The tissue-specific transcription analysis and variation of mRNA transcription level of Ab-CaReg I in gills and mantle after animals were immersed in seawater containing 2000 ppm CaCl(2) was quantified by SYBR Green real-time PCR analysis. Transcription variation of Ab-CaReg I in hemocytes and gills followed by bacteria challenge (Vibrio alginolyticus, Vibrio parahaemolyticus and Listeria monocytogenes) was used to investigate Ab-CaReg I in immune responses. Transcripts of Ab-CaReg I mRNA were mainly detected in hemocytes, mantle, muscle, gills, digestive tract and hepatopancreas with highest expression in hemocytes. The CaCl(2) immersion significantly altered the Ab-CaReg I mRNA transcription level by 3 h, compared to animals in normal seawater (control). The mRNA expression of Ab-CaReg I in gills and hemocytes was upregulated significantly to 11-fold and 4-fold in 3 h compared to control (uninfected), respectively, in bacteria-challenged abalones. The results suggest that Ab-CaReg I could be effectively induced to maintain internal Ca(2+) homeostasis of the animal due to influx of Ca(2+) in the cells by external stimuli such as a high dose of Ca(2+) and pathogens like bacteria.

Transcriptional up-regulation of disk abalone selenium dependent glutathione peroxidase by H(2)O(2) oxidative stress and Vibrio alginolyticus bacterial infection.

Selenium dependent glutathione peroxidase (Se-GPx) belongs to the family of selenoprotein, which acts mainly as an antioxidant in the cellular defence system. We have identified Se-GPx full length cDNA from disk abalone (Haliotis discus discus) designated as AbSe-GPx. It has a characteristic codon at (223)TGA(225) that corresponds to selenocysteine (Sec) amino acid as U(75). The full length cDNA consists of 675 bp, an open reading frame encoding 225 amino acids. Sequence characterization revealed that AbSe-GPx contains a characteristic GPx signature motif 2 ((97)LGFPCNQF(104)), an active site motif ((183)WNFEKF(188)) and essential residues for the enzymatic function. Additionally, the eukaryotic selenocysteine insertion sequence (SECIS) is conserved in the 3' UTR. The AbSe-GPx amino acid sequence exhibited the highest level of identity (46%) with insect (Ixodes scapularis) GPx, and shares 41% with bivalve (Unio tumidus) Se-GPx. The RT-PCR analysis revealed that AbSe-GPx mRNA was expressed constitutively in gill, mantle, gonad, abductor muscle, digestive tract, and hemocytes in a tissue specific manner. AbSe-GPx mRNA expression was significantly up-regulated in gill and digestive tract tissues after H(2)O(2) injection and Vibrio alginolyticus infection. However, AbSe-GPx expression was not up-regulated after Aroclor 1,254 injection. These results indicate that AbSe-GPx mRNA is expressed at a basal level in abalone tissues, which can be up-regulated transcriptionally by H(2)O(2) oxidative stress and Vibrio alginolyticus infection. Therefore, AbSe-GPx may be involved in a protective role against H(2)O(2) oxidative stress and immune defence against bacterial infection.