Melanogenesis Inhibitors from the Endophytic Fungus Aspergillus amstelodami

Two new compounds, named 3,4‐dimethoxyphenyl α‐d ‐ribofuranoside ( 1 ) and 3β‐(β‐d ‐glucopyranosyloxy)olean‐12‐ene‐23,28,30‐trioic acid ( 2 ), together with thirteen known compounds, were isolated from the white beans culture of the marine derived endophytic fungus Aspergillus amstelodami. Structure elucidation of the new compounds was carried out by one‐, two‐dimensional spectroscopy, and high resolution electrospray ionization mass. The antimelanogenic and anti‐allergic activity of the isolated compounds were investigated. Compounds 4 , 7 , 1 , 3 , 11 , 6 and 9 selectively suppressed melanin production in B16 melanoma cells, using arbutin as a positive control. Their IC₅₀ values were 30.8±5.57, 38.5±6.08, 52.6±6.64, 98.0±1.16, 100.4±3.05, 112.0±0.22 and 144.7±2.35 μm , respectively, while that of arbutin was 151.7±1.27 μm . The tested compounds did not show any significant anti‐allergic activity in RBL‐2H3 cells, as compared to quercetin.     

Effect of salt on the activity of Streptomyces prolyl aminopeptidase

A salt-tolerant prolyl aminopeptidase from Streptomyces aureofaciens TH-3 (TH-3PAP) was purified from a culture supernatant. The gene encoding TH-3PAP was cloned and sequenced. The primary structure of TH-3PAP showed 65% identity with that of PAP from Streptomyces lividans (SLPAP) and possessed a conserved catalytic motif, GxSxGG, which is conserved in the alpha/beta hydrolase fold family. The characterization of the recombinants TH-3PAP and SLPAP indicated a difference: in 4.0 M NaCl, TH-3PAP showed enzyme activity, whereas SLPAP was inactive. Next, we constructed chimeras between TH-3PAP and SLPAP using an in vivo DNA shuffling system and a sandwich chimera (sc-PAP), whose region from 63 to 78 amino acids of TH-3PAP was substituted with that of SLPAP. Comparison of the biochemical properties between TH-3PAP and the salt-sensitive sc-PAP suggested that the fine tuning of the N-terminal conformation of TH-3PAP by hydrophobic interaction is important for the salt tolerance mechanism of the enzyme.     

Arabidopsis Bax inhibitor-1 promotes sphingolipid synthesis during cold stress by interacting with ceramide-modifying enzymes

Bax inhibitor-1 (BI-1) is a widely conserved cell death suppressor localized in the endoplasmic reticulum membrane. Our previous results revealed that Arabidopsis BI-1 (AtBI-1) interacts with not only Arabidopsis cytochrome b ₅ (Cb5), an electron transfer protein, but also a Cb5-like domain (Cb5LD)-containing protein, Saccharomyces cerevisiae fatty acid 2-hydroxylase 1, which 2-hydroxylates sphingolipid fatty acids. We have now found that AtBI-1 binds Arabidopsis sphingolipid Δ8 long-chain base (LCB) desaturases AtSLD1 and AtSLD2, which are Cb5LD-containing proteins. The expression of both AtBI-1 and AtSLD1 was increased by cold exposure. However, different phenotypes were observed in response to cold treatment between an atbi-1 mutant and a sld1sld2 double mutant. To elucidate the reasons behind the difference, we analyzed sphingolipids and found that unsaturated LCBs in atbi-1 were not altered compared to wild type, whereas almost all LCBs in sld1sld2 were saturated, suggesting that AtBI-1 may not be necessary for the desaturation of LCBs. On the other hand, the sphingolipid content in wild type increased in response to low temperature, whereas total sphingolipid levels in atbi-1 were unaltered. In addition, the ceramide-modifying enzymes AtFAH1, sphingolipid base hydroxylase 2 (AtSBH2), acyl lipid desaturase 2 (AtADS2) and AtSLD1 were highly expressed under cold stress, and all are likely to be related to AtBI-1 function. These findings suggest that AtBI-1 contributes to synthesis of sphingolipids during cold stress by interacting with AtSLD1, AtFAH1, AtSBH2 and AtADS2.     

Structure-activity relationship for inhibition of 5alpha-reductase by triterpenoids isolated from Ganoderma lucidum

In humans, 5alpha-reductase is involved in the development of benign prostatic hyperplasia. Triterpenoids isolated from ethanol extracts of Ganoderma lucidum (Fr.) Krast (Ganodermataceae) inhibited 5alpha-reductase activity. The presence of the C-3 carbonyl group and of the C-26-alpha,beta-unsaturated carbonyl group was characteristic of almost all inhibitors isolated from G. lucidum.     

A new cell line from the wax moth Galleria mellonella Linne (lepidoptera: Pyralididae)

Summary A cell line derived from the larval-fat body tissues of the wax moth, Galleria mellonella Linne, was established in MGM-450 medium. The cells grew in suspension and were mainly spherical in shape. Population doubling time was between 1.4 and 1.7 d over a range of 15 to 35°C, and the maximum growth rate was at 25°C. The chromosome number ranged from 70–239, with a mode of 170. The cells were sensitive to 20-hydroxyecdysone, which stimulated their growth and induced morphological changes. The cell line was designated GaMe-LF1.     

Electrical stimulation of cultured lepidopteran dorsal vessel tissue: an experiment for development of bioactuators

An insect dorsal vessel (DV) is well suited for a bioactuator since it is capable of contracting autonomously, and its tissue and cells are more environmentally robust under culturing conditions compared with mammalian tissue. In this study, electrical pulse stimulation was examined so as to regulate a bioactuator using the DV tissue. The DV tissue of a larva of Ctenoplusia agnate was assembled on a micropillar array, which was stimulated after culturing for about 3 wk. The contraction of the DV tissue was evaluated by image analysis to measure lateral displacements at the micropillar top. As a result, suitable stimulation conditions in a 35-mm petri dish were determined as: applied voltage of 10 V with 20-ms duration. Next, the time lag between the onset of electrical stimulus and the onset of mechanical contraction (electromechanical delay (EMD)) was estimated. A light-emitting diode (LED) was connected serially with the petri dish, and the LED flashed when electrical pulses were given. Movie images were analyzed in which electrical pulses made the DV tissue contract and the LED flashed virtually simultaneously; from these, the EMD was estimated as approximately 50 ms. These results suggest that the electrical pulse stimulation is capable of regulating the DV tissue, and the micropillar array is a useful biological tool to investigate physiological properties of muscle tissue.     

RAD18 promotes DNA double-strand break repair during G1 phase through chromatin retention of 53BP1

Recruitment of RAD18 to stalled replication forks facilitates monoubiquitination of PCNA during S-phase, promoting translesion synthesis at sites of UV irradiation-induced DNA damage. In this study, we show that RAD18 is also recruited to ionizing radiation (IR)-induced sites of DNA double-strand breaks (DSBs) forming foci which are co-localized with 53BP1, NBS1, phosphorylated ATM, BRCA1 and γ-H2AX. RAD18 associates with 53BP1 and is recruited to DSB sites in a 53BP1-dependent manner specifically during G1-phase, RAD18 monoubiquitinates KBD domain of 53BP1 at lysine 1268 in vitro. A monoubiquitination-resistant 53BP1 mutant harboring a substitution at lysine 1268 is not retained efficiently at the chromatin in the vicinity of DSBs. In Rad18-null cells, retention of 53BP1 foci, efficiency of DSB repair and post-irradiation viability are impaired compared with wild-type cells. Taken together, these results suggest that RAD18 promotes 53BP1-directed DSB repair by enhancing retention of 53BP1, possibly through an interaction between RAD18 and 53BP1 and the modification of 53BP1.