Characterization of phytochelatin synthase produced by the primitive red alga Cyanidioschyzon merolae

Phytochelatins (PCs), non-protein peptides with the general structure [(γ-Glu-Cys)n-Gly (n≥ 2)], are involved in the detoxification of toxic heavy metals mainly in higher plants. The synthesis of the peptides is mediated by phytochelatin synthase (PCS), which is activated by a range of heavy metals. CmPCS, a PCS-like gene found in the genomic DNA of the primitive red alga Cyanidioschyzon merolae, was isolated and a recombinant protein (rCmPCS) fused with a hexahistidine tag at the N-terminus of CmPCS was produced. The finding that this protein mediated PC synthesis from glutathione in a metal-dependent way clearly establishes that rCmPCS is functional. The maximum activity was attained at a reaction temperature of 50 °C, considerably higher than the temperature required for the maximal activity of PCS isolated from the higher plant Silene cucubalus, probably due to the alga being a thermophile. CmPCS showed optimal pH in a slightly higher region than higher plant PCSs, probably due to the less effective charge relay network in the catalytic triad. In addition, the pattern of enzyme activation by metal ions was specific to rCmPCS, with Ag+, Cu2+, and Hg2+ showing only limited activation. In contrast to other eukaryotic PCSs, CmPCS has an extra domain in the N-terminal region from residues 1 to 109, and contains fewer cysteine residues in the C-terminal domain. These differences may be responsible for the metal specificity of the activation of CmPCS. Although the enzyme preparation lost PCS activity progressively when stored at 4 °C, the inclusion of Cd2+ in the preparation effectively prevented the reduction of activity. Furthermore, Cd2+ effectively restored the activity of the inactivated enzyme. These results indicate that Cd2+ ions bind the enzyme to maintain the structural integrity of the peptides.     

Quantitative evaluation of each catalytic subsite of cathepsin B for inhibitory activity based on inhibitory activity-binding mode relationship of epoxysuccinyl inhibitors by X-ray crystal structure analyses of complexes

To quantitatively estimate the inhibitory effect of each substrate-binding subsite of cathepsin B (CB), a series of epoxysuccinyl derivatives with different functional groups bound to both carbon atoms of the epoxy ring were synthesized, and the relationship between their inhibitory activities and binding modes at CB subsites was evaluated by the X-ray crystal structure analyses of eight complexes. With the common reaction in which the epoxy ring of inhibitor was opened to form a covalent bond with the SgammaH group of the active center Cys29, the observed binding modes of the substituents of inhibitors at the binding subsites of CB enabled the quantitative assessment of the inhibitory effect of each subsite. Although the single blockage of S1' or S2' subsite exerts only the inhibitory effect of IC50 = approximately 24 microM (k2 = approximately 1250 M(-1) s(-1)) or approximately 15 microM (k2 = approximately 1800 M(-1) s(-1)), respectively, the synchronous block of both subsites leads to IC50 = approximately 23 nM (k2 = 153,000 - 185,000 M(-1) s(-1)), under the condition that (i) the inhibitor possesses a P1' hydrophobic residue such as Ile and a P2' hydrophobic residue such as Ala, Ile or Pro, and (ii) the C-terminal carboxyl group of a P2' residue is able to form paired hydrogen bonds with the imidazole NH of His110 and the imidazole N of His111 of CB. The inhibitor of a Pn' > or = 3' substituent was not potentiated by collision with the occluding loop. On the other hand, it was suggested that the inhibitory effects of Sn subsites are independent of those of Sn' subsites, and the simultaneous blockage of the funnel-like arrangement of S2 and S3 subsites leads to the inhibition of IC50 = approximately 40 nM (k2 = approximately 66,600 M(-1) s(-1)) regardless of the lack of Pn' substituents. Here we present a systematic X-ray structure-based evaluation of structure-inhibitory activity relationship of each binding subsite of CB, and the results provide the structural basis for designing a more potent CB-specific inhibitor.     

E3 ubiquitin ligase synoviolin is involved in liver fibrogenesis

Background and Aim

Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis.

Methods

The expression and localization of synoviolin in the liver were analyzed in CCl₄-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno^+/− mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno^+/− mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno^−/− mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno^−/− MEF cells.

Results

In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno^+/− mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno^+/− mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno^−/− MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum.

Conclusion

Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.     

A comparative study on testicular microstructure and relative sperm production in gorillas, chimpanzees, and orangutans

We performed histological analyses for comparing testicular microstructure between the gorilla, chimpanzee, and orangutan. Testicular samples were obtained by autopsy or biopsy from 10 gorillas, 11 chimpanzees, and 7 orangutans from several zoos and institutes. The seminiferous epithelia were thick in the chimpanzee and orangutan but thin in the gorilla. Leydig cells in the interstitial tissue were abundant in the gorilla. The acrosomic system was extremely well developed in the orangutans. Our study reveals that the cycle of seminiferous epithelium in orangutan testis can be divided into ten stages, whereas that in human, chimpanzee, and gorilla testes can be divided into only six stages. Phylogenetic analyses of the number of divisions may indicate that the seminiferous epithelium of our common ancestor has changed since the orangutan diverged from it. Furthermore, we performed comparative analyses of testicular microstructure to estimate relative sperm production among these three animals, and proposed a new indicator (namely the spermatogenic index, SI) closely related to sperm production. The SI indicated that a chimpanzee usually produces about 223 times more sperm than a gorilla and about 14 times more than an orangutan. Our data demonstrate the significance of the SI for estimating sperm production, thus aiding our understanding of the reproductive strategy as well as testis weight and relative testis size in investigated primates.     

Occurrence and diversity of mesophilic Shewanella strains isolated from the North-West Pacific Ocean

Although bacteria of the genus Shewanella belong to one of the readily cultivable groups of "Gammaproteobacteria", little is known about the occurrence and abundance of these microorganisms in the marine ecosystem. Studies revealed that of 654 isolates obtained from marine invertebrates (ophiuroid Amphiopholis kochii, sipuncula Phascolosoma japonicum, and holothurian Apostichopus japonicus, Cucumaria japonica), seawater and sediments of the North-West Pacific Ocean (i.e. the Sea of Japan and Iturup Is, Kurile Islands), 10.7% belonged to the genus Shewanella. The proportion of viable Shewanella species varied from 4% to 20% depending on the source of isolation. From the isolation study, representative strains of different phenotypes (from seventy presumptive Shewanella strains) were selected for detailed characterization using phenotypic, chemotaxonomic, and phylogenetic testing. 16S rDNA sequence-based phylogenetic analysis confirmed the results of tentative identification and placed the majority of these strains within only a few species of the genus Shewanella with 98-99% of 16S rDNA sequences identity mainly with S. japonica and S. colwelliana, suggesting that the strains studied might belong to these species. Numerically dominant strains of S. japonica were metabolically active and produced proteinases (gelatinases, caseinases), lipases, amylases, agarases, and alginases. Shewanella strains studied demonstrated weak antimicrobial and antifungal activities that might be an indication of their passive role in the colonization on living and non-living surfaces.     

GFS9/TT9 contributes to intracellular membrane trafficking and flavonoid accumulation in Arabidopsis thaliana

Flavonoids are the most important pigments for the coloration of flowers and seeds. In plant cells, flavonoids are synthesized by a multi‐enzyme complex located on the cytosolic surface of the endoplasmic reticulum, and they accumulate in vacuoles. Two non‐exclusive pathways have been proposed to mediate flavonoid transport to vacuoles: the membrane transporter‐mediated pathway and the vesicle trafficking‐mediated pathway. No molecules involved in the vesicle trafficking‐mediated pathway have been identified, however. Here, we show that a membrane trafficking factor, GFS9, has a role in flavonoid accumulation in the vacuole. We screened a library of Arabidopsis thaliana mutants with defects in vesicle trafficking, and isolated the gfs9 mutant with abnormal pale tan‐colored seeds caused by low flavonoid accumulation levels. gfs9 is allelic to the unidentified transparent testa mutant tt9. The responsible gene for these phenotypes encodes a previously uncharacterized protein containing a region that is conserved among eukaryotes. GFS9 is a peripheral membrane protein localized at the Golgi apparatus. GFS9 deficiency causes several membrane trafficking defects, including the mis‐sorting of vacuolar proteins, vacuole fragmentation, the aggregation of enlarged vesicles, and the proliferation of autophagosome‐like structures. These results suggest that GFS9 is required for vacuolar development through membrane fusion at vacuoles. Our findings introduce a concept that plants use GFS9‐mediated membrane trafficking machinery for delivery of not only proteins but also phytochemicals, such as flavonoids, to vacuoles.     

Physiologic and metagenomic attributes of the rhodoliths forming the largest CaCO3 bed in the South Atlantic Ocean

Rhodoliths are free-living coralline algae (Rhodophyta, Corallinales) that are ecologically important for the functioning of marine environments. They form extensive beds distributed worldwide, providing a habitat and nursery for benthic organisms and space for fisheries, and are an important source of calcium carbonate. The Abrolhos Bank, off eastern Brazil, harbors the world''s largest continuous rhodolith bed (of ∼21 000 km²) and has one of the largest marine CaCO₃ deposits (producing 25 megatons of CaCO₃ per year). Nevertheless, there is a lack of information about the microbial diversity, photosynthetic potential and ecological interactions within the rhodolith holobiont. Herein, we performed an ecophysiologic and metagenomic analysis of the Abrolhos rhodoliths to understand their microbial composition and functional components. Rhodoliths contained a specific microbiome that displayed a significant enrichment in aerobic ammonia-oxidizing betaproteobacteria and dissimilative sulfate-reducing deltaproteobacteria. We also observed a significant contribution of bacterial guilds (that is, photolithoautotrophs, anaerobic heterotrophs, sulfide oxidizers, anoxygenic phototrophs and methanogens) in the rhodolith metagenome, suggested to have important roles in biomineralization. The increased hits in aromatic compounds, fatty acid and secondary metabolism subsystems hint at an important chemically mediated interaction in which a functional job partition among eukaryal, archaeal and bacterial groups allows the rhodolith holobiont to thrive in the global ocean. High rates of photosynthesis were measured for Abrolhos rhodoliths (52.16 μmol carbon m⁻²s⁻¹), allowing the entire Abrolhos rhodolith bed to produce 5.65 × 10⁵ tons C per day. This estimate illustrates the great importance of the Abrolhos rhodolith beds for dissolved carbon production in the South Atlantic Ocean.     

Decomposition of Organic Mercurial Compounds by Mercury-resistant Bacteria

Isopentyl isovalerate and anisic acid were first isolated and identified from the Japanese peppermint oil. The former compound possesses characteristic apple-like oder. α-Bourbonene, menthofurolactone, and β-caryophyllene epoxide were also isolated and identified from the oil of Shubi, a newly registered Japanese peppermint.     

Microbial Degradation of Natural Rubber Vulcanizates

An actinomycete, Nocardia sp. strain 835A, grows well on unvulcanized natural rubber and synthetic isoprene rubber, but not on other types of synthetic rubber. Not only unvulcanized but also various kinds of vulcanized natural rubber products were more or less utilized by the organism as the sole source of carbon and energy. The thin film from a latex glove was rapidly degraded, and the weight loss reached 75% after a 2-week cultivation period. Oligomers with molecular weights from 10⁴ to 10³ were accumulated during microbial growth on the latex glove. The partially purified oligomers were examined by infrared and ¹H nuclear magnetic resonance and ¹³C nuclear magnetic resonance spectroscopy, and the spectra were those expected of cis-1, 4-polyisoprene with the structure, OHC—CH₂—[—CH₂—C(—CH₃)=CH —CH₂—]_n—CH₂—C(=O)— CH₃, with average values of n of about 114 and 19 for the two oligomers.     

Structure-based drug design of 1,3,6-trisubstituted 1,4-diazepan-7-ones as selective human kallikrein 7 inhibitors

A novel series of 1,3,6-trisubstituted 1,4-diazepan-7-ones were investigated as human kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Based on the X-ray co-crystal structure of compound 1 bound to human KLK7, the derivatives of this scaffold were designed, synthesized, and evaluated. Through structure-activity relationship studies focused on the side chain located in the prime site region of the enzyme, representative compounds 15, 33a, and 35a were identified as highly potent and selective inhibitors of human KLK7.