New polyether triterpenoids from Laurencia viridis and their biological evaluation

The red seaweed Laurencia viridis is a rich source of secondary metabolites derived from squalene. New polyethers, such as iubol (2), 22-hydroxy-15(28)- dehydrovenustatriol (3), 1,2-dehydropseudodehydrothyrsiferol (4), and secodehydrothyrsiferol (5) have been isolated and characterized from this alga. The structures were determined through the interpretation of NMR spectroscopic data and the relative configuration was proposed on the basis of NOESY spectrum and biogenetic considerations. All new compounds exhibited significant cytotoxic activity against a panel of cancer cell lines.     

Extracts from Cladiella australis, Clavularia viridis and Klyxum simplex (Soft Corals) are Capable of Inhibiting the Growth of Human Oral Squamous Cell Carcinoma Cells

Many biomedical products have already been obtained from marine organisms. In order to search more therapeutic drugs against cancer, this study demonstrates the cytotoxicity effects of Cladiella australis, Clavularia viridis and Klyxum simplex extracts on human oral squamous cell carcinoma (SCC4, SCC9 and SCC25) cells using cell adhesion and cell viability assay. The morphological alterations in SCCs cells after treatment with three extracts, such as typical nuclear condensation, nuclear fragmentation and apoptotic bodies of cells were demonstrated by Hoechst stain. Flow cytometry indicated that three extracts sensitized SCC25 cells in the G₀/G₁ and S-G₂/M phases with a concomitant significantly increased sub-G₁ fraction, indicating cell death by apoptosis. This apoptosis process was accompanied by activation of caspase-3 expression after SCC25 cells were treated with three extracts. Thereby, it is possible that extracts of C. australis, C. viridis and K. simplex cause apoptosis of SCCs and warrant further research investigating the possible anti-oral cancer compounds in these soft corals.     

Influence of Environmental Factors on the Paralytic Shellfish Toxin Content and Profile of Alexandrium catenella (Dinophyceae) Isolated from the Mediterranean Sea

Laboratory experiments were designed to study the toxin content and profile of the Alexandrium catenella strain ACT03 (isolated from Thau Lagoon, French Mediterranean) in response to abiotic environmental factors under nutrient-replete conditions. This dinoflagellate can produce various paralytic shellfish toxins with concentrations ranging from 2.9 to 50.3 fmol/cell. The toxin profile was characterized by carbamate toxins (GTX3, GTX4 and GTX5) and N-sulfocarbamoyl toxins (C1, C2, C3 and C4). C2 dominated at 12–18 °C, but only for salinities ranging from 10 to 25 psu, whereas GTX5 became dominant at temperatures ranging from 21 to 30 °C at almost all salinities. There was no significant variation in the cellular toxin amount from 18 °C to 27 °C for salinities ranging between 30 and 40 psu. At salinities of 10 to 25 psu, the toxin concentrations always remained below 20 fmol/cell. Toxin content was stable for irradiance ranging from 10 to 70 μmol photons/m²/s then slightly increased. Overall, the toxin profile was more stable than the toxin content (fmol/cell), except for temperature and/or salinity values different from those recorded during Alexandrium blooms in Thau Lagoon.     

Isolation,Structure Elucidation and Total Synthesis of Lajollamide A from the Marine Fungus Asteromyces cruciatus

The marine-derived filamentous fungus Asteromyces cruciatus 763, obtained off the coast of La Jolla, San Diego, USA, yielded the new pentapeptide lajollamide A (1), along with the known compounds regiolone (2), hyalodendrin (3), gliovictin (4), ¹N-norgliovicitin (5), and bis-N-norgliovictin (6). The planar structure of lajollamide A (1) was determined by Nuclear Magnetic Resonance (NMR) spectroscopy in combination with mass spectrometry. The absolute configuration of lajollamide A (1) was unambiguously solved by total synthesis which provided three additional diastereomers of 1 and also revealed that an unexpected acid-mediated partial racemization (2:1) of the L-leucine and L-N-Me-leucine residues occurred during the chemical degradation process. The biological activities of the isolated metabolites, in particular their antimicrobial properties, were investigated in a series of assay systems.     

Isolation and Purification of a Peptide from Bullacta exarata and Its Impaction of Apoptosis on Prostate Cancer Cell

Bullacta exarata was hydrolyzed with trypsin to prepare peptides; Hydrolysates were isolated by ultrafiltration and purified using G-25 gel filtration. The purity of the Bullacta exarata was demonstrated by HPLC and its peptide sequence analysis was detected. The effects of BEPT II and BEPT II-1 on the proliferation of PC-3 cells were examined using a MTT assay. BEPT II and BEPT II-1 significantly inhibited the proliferation of PC-3 cells in a time- and dose-dependent manner. Annexin V/PI double staining studies showed exposing PC-3 cells to 5, or 15 mg/mL BEPT II-1 for 24 h increased the percentage of the early stage of apoptotic cells from 11.22% to 22.09%. In addition, typical morphologic changes were observed in the cells with acridine orange/ethidium bromide staining. These data support that BEPT II-1 has anticancer properties and merits further investigation to understand the mechanisms of BEPT II-1-induced apoptosis in PC-3 cells.     

Nitric Oxide Synthase in the Central Nervous System and Peripheral Organs of Stramonita haemastoma: Protein Distribution and Gene Expression in Response to Thermal Stress

Nitric oxide (NO) is generated via the oxidation of l-arginine by the enzyme NO synthase (NOS) both in vertebrates and invertebrates. Three NOS isoforms, nNOS, iNOS and eNOS, are known in vertebrates, whereas a single NOS isoform is usually expressed in invertebrates, sharing structural and functional characteristics with nNOS or iNOS depending on the species. The present paper is focused on the constitutive Ca²⁺/calmodulin-dependent nNOS recently sequenced by our group in the neogastropod Stramonita haemastoma (ShNOS). In this paper we provide new data on cellular distribution of ShNOS in the CNS (pedal ganglion) and peripheral organs (osphradium, tentacle, eye and foot) obtained by WB, IF, CM and NADPHd. Results demonstrated that NOS-like proteins are widely expressed in sensory receptor elements, neurons and epithelial cells. The detailed study of NOS distribution in peripheral and central neurons suggested that NOS is both intracellular and presynaptically located. Present findings confirm that NO may have a key role in the central neuronal circuits of gastropods and in sensory perception. The physiological relevance of NOS enzymes in the same organs was suggested by thermal stress experiments demonstrating that the constitutive expression of ShNOS is modulated in a time- and organ-dependent manner in response to environmental stressors.     

Identification and Expression of Two Novel Cytochrome P450 Genes,CYP6CV1 and CYP9A38, in Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Cnaphalocrocis medinalis Güenée can cause severe losses in rice. Cytochrome P450s play crucial roles in the metabolism of allelochemicals in herbivorous insects. Two novel P450 cDNAs, CYP6CV1 and CYP9A38, were cloned from the midgut of C. medinalis. CYP6CV1 encodes a protein of 500 amino acid residues, while CYP9A38-predicted protein has 531 amino acid residues. Both cDNA-predicted proteins contain the conserved functional domains for all P450s. Phylogenetic analyses showed that CYP6CV1 is grouped in the cluster containing CYP6B members, while CYP9A38 is in the cluster including CYP9 members. However, both clusters are contained in the same higher lineage. Homologous analysis revealed that CYP6CV1 is most similar to CYP6B8, CYP6B7, CYP6B6, CYP6B2, and CYP6B4 with the highest amino acid identity of 41%. CYP9A38 is closest to CYP9A17, CYP9A21, CYP9A20, and CYP9A19 with the highest amino acid identity of 66%. Studies of temporal expression profiles revealed that CYP9A38 showed a steady increase in mRNA level during the five instar stages, but a low-expression level in pupae, and then presented at a high-expression level again in adults. Similar expression patterns were obtained with CYP6CV1. In the fifth instar larvae, CYP6CV1 was mainly expressed in midgut and fat bodies, whereas CYP9A38 was mainly expressed in midgut. Expression studies also revealed a 3.20-fold over-expression of CYP6CV1 and 3.54-fold over-expression of CYP9A38 after larval exposure to host rice resistance. Our results suggest that both CYP6CV1 and CYP9A38 may be involved in detoxification of rice phytochemicals.     

Isolation and Expression Analysis of CYP9A11 and Cytochrome P450 Reductase Gene in the Beet Armyworm (Lepidoptera: Noctuidae)

Cytochrome P450 monooxygenases (CYPs), as an enzyme superfamily, is widely distributed in organisms and plays a vital function in the metabolism of exogenous and endogenous compounds by interacting with its obligatory redox partner, CYP reductase (CPR). A novel CYP gene (CYP9A11) and CPR gene from the agricultural pest insect Spodoptera exigua were cloned and characterized. The complete cDNA sequences of SeCYP9A11 and SeCPR are 1,931 and 3,919 bp in length, respectively, and contain open reading frames of 1,593 and 2,070 nucleotides, respectively. Analysis of the putative protein sequences indicated that SeCYP9A11 contains a heme-binding domain and the unique characteristic sequence (SRFALCE) of the CYP9 family, in addition to a signal peptide and transmembrane segment at the N-terminal. Alignment analysis revealed that SeCYP9A11 shares the highest sequence similarity with CYP9A13 from Mamestra brassicae, which is 66.54%. The putative protein sequence of SeCPR has all of the classical CPR features, such as an N-terminal membrane anchor; three conserved domain flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) domain; and characteristic binding motifs. Phylogenetic analysis revealed that SeCPR shares the highest identity with HaCPR, which is 95.21%. The SeCYP9A11 and SeCPR genes were detected in the midgut, fat body, and cuticle tissues, and throughout all of the developmental stages of S. exigua. The mRNA levels of SeCYP9A11 and SeCPR decreased remarkably after exposure to plant secondary metabolites quercetin and tannin. The results regarding SeCYP9A11 and SeCPR genes in the current study provide foundation for the further study of S. exigua P450 system.     

Lipopolysaccharides from Commensal and Opportunistic Bacteria: Characterization and Response of the Immune System of the Host Sponge Suberites domuncula

Marine sponges harbor a rich bacterioflora with which they maintain close relationships. However, the way these animals make the distinction between bacteria which are consumed to meet their metabolic needs and opportunistic and commensal bacteria which are hosted is not elucidated. Among the elements participating in this discrimination, bacterial cell wall components such as lipopolysaccharides (LPS) could play a role. In the present study, we investigated the LPS chemical structure of two bacteria associated with the sponge Suberites domuncula: a commensal Endozoicomonas sp. and an opportunistic Pseudoalteromonas sp. Electrophoretic patterns indicated different LPS structures for these bacteria. The immunomodulatory lipid A was isolated after mild acetic acid hydrolysis. The electrospray ionization ion-trap mass spectra revealed monophosphorylated molecules corresponding to tetra- and pentaacylated structures with common structural features between the two strains. Despite peculiar structural characteristics, none of these two LPS influenced the expression of the macrophage-expressed gene S. domuncula unlike the Escherichia coli ones. Further research will have to include a larger number of genes to understand how this animal can distinguish between LPS with resembling structures and discriminate between bacteria associated with it.     

Gene Cloning,Expression and Characterization of a Novel Xylanase from the Marine Bacterium,Glaciecola mesophila KMM241

Marine xylanases are rather less studied compared to terrestrial xylanases. In this study, a new xylanase gene, xynB, was cloned from the marine bacterium, Glaciecola mesophila KMM241, and expressed in Escherichia coli. xynB encodes a multi-domain xylanase XynB of glycoside hydrolase (GH) family 8. The recombinant XynB comprises an N-terminal domain (NTD) with unknown function and a catalytic domain, which is structurally novel among the characterized xylanases of GH family 8. XynB has the highest identity (38%) to rXyn8 among the characterized xylanases. The recombinant XynB showed maximal activity at pH 6–7 and 35 °C. It is thermolabile and salt-tolerant. XynB is an endo-xylanase that demands at least five sugar moieties for effective cleavage and to hydrolyze xylohexaose and xylopentaose into xylotetraose, xylotriose and xylobiose. NTD was expressed in Escherichia coli to analyze its function. The recombinant NTD exhibited a high binding ability to insoluble xylan and avicel and little binding ability to chitosan and chitin. Since the NTD shows no obvious homology to any known carbohydrate-binding module (CBM) sequence in public databases, XynB may contain a new type of CBM.     

Characterization of ACE Inhibitory Peptides from Mactra veneriformis Hydrolysate by Nano-Liquid Chromatography Electrospray Ionization Mass Spectrometry (Nano-LC-ESI-MS) and Molecular Docking

Food-derived bioactive compounds are gaining increasing significance in life sciences. In the present study, we identified angiotensin I-converting enzyme (ACE)-inhibitory peptides from Mactra veneriformis hydrolysate using a nano-LC-MS/MS method. Mactra veneriformis hydrolysate was first separated into four fractions (F1–F4) based on molecular weight by ultrafiltration. The fraction with molecular weight lower than 1 kDa (F1) showed the highest ACE inhibitory activity. F1 was then analyzed by a high throughput nano-LC-MS/MS method and sequences of peptides in F1 were calculated accordingly. The 27 peptides identified as above were chemically synthesized and tested for ACE-inhibitory activity. The hexapeptide VVCVPW showed the highest potency with an IC₅₀ value of 4.07 μM. We then investigated the interaction mechanism between the six most potent peptides and ACE by molecular docking. Our docking results suggested that the ACE inhibitory peptides bind to ACE via interactions with His383, His387, and Glu411 residues. Particularly, similar to the thiol group of captopril, the cysteine thiol group of the most potent peptide VVCVPW may play a key role in the binding of this peptide to the ACE active site.     

Characterization of an Alginate Lyase,FlAlyA, from Flavobacterium sp. Strain UMI-01 and Its Expression in Escherichia coli

A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I—Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.     

野生二粒小麦PYL基因家族的鉴定及其在逆境胁迫下的表达特性分析

脱落酸(ABA)是一类重要的植物内源激素,在调控植物生长发育和胁迫响应等方面发挥着关键作用。基因作为ABA的受体,在ABA信号转导过程中发挥着重要作用。为深入发掘野生二粒小麦PYL基因,本研究利用生物信息学对野生二粒小麦的PYL家族进行全基因组鉴定与分析。结果在野生二粒小麦基因组中共鉴定到24个PYL基因(TdPYLs),分布在1A、1B、2A、2B、3A、3B、4A、4B、7A和7B染色体上,且所有成员均含有PYR-PYL-RCAR-like功能域;系统进化分析发现,24个TdPYL基因可分为3个亚家族,同一亚家族成员间具有相似的基因结构和保守结构域;对这些PYL基因的启动子顺式元件进行预测,发现它们含有大量与逆境胁迫、光调节和ABA响应相关的元件;基于RNA-seq数据的表达特性分析发现,它们在不同组织中以及逆境胁迫下表达差异明显,并鉴定到多个与组织特异性以及胁迫响应相关的PYL基因;进一步对PYL基因的单倍型组成进行分析,发现在野生二粒小麦、栽培二粒小麦和硬粒小麦中PYL基因的遗传变异和单倍型组成具有明显的差异,发生了显著的遗传分化。本研究为深入揭示野生二粒小麦PYL基因的调控功能及其进化机制研究提供了有益信息。