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A simple and facile processing strategy to enhance the thermomechanical properties of sulfur-rich polymers has been reported.

The crystal structures of SdgB and SdgA from Staphylococcus aureus provide functional and structural insights into the glycosylation mechanism in staphylococcal adhesion., The initiation of infection of host tissues by Staphylococcus aureus requires a family of staphylococcal adhesive proteins containing serine–aspartate repeat (SDR) domains, such as ClfA. The O-linked glycosylation of the long-chain SDR domain mediated by SdgB and SdgA is a key virulence factor that protects the adhesive SDR proteins against host proteolytic attack in order to promote successful tissue colonization, and has also been implicated in staphylococcal agglutination, which leads to sepsis and an immunodominant epitope for a strong antibody response. Despite the biological significance of these two glycosyltransferases involved in pathogenicity and avoidance of the host innate immune response, their structures and the molecular basis of their activity have not been investigated. This study reports the crystal structures of SdgB and SdgA from S. aureus as well as multiple structures of SdgB in complex with its substrates (for example UDP, N-acetylglucosamine or SDR peptides), products (glycosylated SDR peptides) or phosphate ions. Together with biophysical and biochemical analyses, this structural work uncovered the novel mechanism by which SdgB and SdgA carry out the glycosyl-transfer process to the long SDR region in SDR proteins. SdgB undergoes dynamic changes in its structure such as a transition from an open to a closed conformation upon ligand binding and takes diverse forms, both as a homodimer and as a heterodimer with SdgA. Overall, these findings not only elucidate the putative role of the three domains of SdgB in recognizing donor and acceptor substrates, but also provide new mechanistic insights into glycosylation of the SDR domain, which can serve as a starting point for the development of antibacterial drugs against staphylococcal infections.

The metallo-β-lactamase fold is the most abundant metal-binding domain found in two major kingdoms: bacteria and archaea. Despite the rapid growth in genomic information, most of these enzymes, which may play critical roles in cellular metabolism, remain uncharacterized in terms of structure and function. In this study, X-ray crystal structures of SAV1707, a hypothetical metalloenzyme from Staphylococcus aureus, and its complex with cAMP are reported at high resolutions of 2.05 and 1.55 Å, respectively, with a detailed atomic description. Through a functional study, it was verified that SAV1707 has Ni2+-dependent phosphodiesterase activity and Mn2+-dependent endonuclease activity, revealing a different metal selectivity depending on the reaction. In addition, the crystal structure of cAMP-bound SAV1707 shows a unique snapshot of cAMP that reveals the binding mode of the intermediate, and a key residue Phe511 that forms π–π interactions with cAMP was verified as contributing to substrate recognition by functional studies of its mutant. Overall, these findings characterized the relationship between the structure and function of SAV1707 and may provide further understanding of metalloenzymes possessing the metallo-β-lactamase fold.

Toxin-antitoxin (TA) systems are ubiquitously found in bacteria and are related to cell maintenance and survival under environmental stresses such as heat shock, nutrient starvation, and antibiotic treatment. Here, we report for the first time the crystal structure of the Staphylococcus aureus TA complex YoeBSa1-YefMSa1 at a resolution of 1.7 A. This structure reveals a heterotetramer with a 2:2 stoichiometry between YoeBSa1 and YefMSa1. The N-terminal regions of the YefMSa1 antitoxin form a homodimer characteristic of a hydrophobic core, and the C-terminal extended region of each YefMSa1 protomer makes contact with each YoeBSa1 monomer. The binding stoichiometry of YoeBSa1 and YefMSa1 is different from that of YoeB and YefM of E. coli (YoeBEc and YefMEc), which is the only structural homologue among YoeB-YefM families; however, the structures of individual YoeBSa1 and YefMSa1 subunits in the complex are highly similar to the corresponding structures in E. coli. In addition, docking simulation with a minimal RNA substrate provides structural insight into the guanosine specificity of YoeBSa1 for cleavage in the active site, which is distinct from the specificity of YoeBEc for adenosine rather than guanosine. Given the previous finding that YoeBSa1 exhibits fatal toxicity without inducing persister cells, the structure of the YoeBSa1-YefMSa1 complex will contribute to the design of a new category of anti-staphylococcal agents that disrupt the YoeBSa1-YefMSa1 complex and increase YoeBSa1 toxicity.

The crystal structure of SAV0491, a TatD-related DNase from the Gram-positive bacterium Staphylococcus aureus, was determined at 1.85 Å resolution, providing functional and structural insights into a putative DNA-binding mode of SAV0491., TatD has been thoroughly investigated as a DNA-repair enzyme and an apoptotic nuclease, and still-unknown TatD-related DNases are considered to play crucial cellular roles. However, studies of TatD from Gram-positive bacteria have been hindered by an absence of atomic detail and the resulting inability to determine function from structure. In this study, an X-ray crystal structure of SAV0491, which is the TatD enzyme from the Gram-positive bacterium Staphylococcus aureus (SaTatD), is reported at a high resolution of 1.85 Å with a detailed atomic description. Although SaTatD has the common TIM-barrel fold shared by most TatD-related homologs, and PDB entry 2gzx shares 100% sequence identity with SAV0491, the crystal structure of SaTatD revealed a unique binding mode of two phosphates interacting with two Ni2+ ions. Through a functional study, it was verified that SaTatD has Mg2+-dependent nuclease activity as a DNase and an RNase. In addition, structural comparison with TatD homologs and the identification of key residues contributing to the binding mode of Ni2+ ions and phosphates allowed mutational studies to be performed that revealed the catalytic mechanism of SaTatD. Among the key residues composing the active site, the acidic residues Glu92 and Glu202 had a critical impact on catalysis by SaTatD. Furthermore, based on the binding mode of the two phosphates and structural insights, a putative DNA-binding mode of SaTatD was proposed using in silico docking. Overall, these findings may serve as a good basis for understanding the relationship between the structure and function of TatD proteins from Gram-positive bacteria and may provide critical insights into the DNA-binding mode of SaTatD.

We isolated and purified an antimicrobial peptide (AMP) from the mantle of the hard-shelled mussel, Mytilus coruscus. The peptide was purified through C18 reversed-phase high-performance liquid chromatography, and displayed antibacterial activity. Total molecular mass of 11,182 Da was determined using matrix-assisted laser desorption ionization time-of-flight mass spectrophotometry. The N-terminal 23-amino acid sequence of its purified peak was obtained through Edman degradation, revealing 82% identity with myticusin-1 of M. coruscus. Complete sequence of the target peptide was determined through cDNA cloning and rapid amplification of cDNA ends. The complete sequence comprised 574 bp with a 387-bp open reading frame (ORF) encoding 24 amino acids of a signal peptide and 104 amino acids of a mature peptide, which was named myticusin-beta. Furthermore, we discovered two novel isoforms of myticusin-beta. We constructed and expressed recombinant myticusin-beta, which displayed antimicrobial activity against gram-positive (Bacillus cereus, Bacillus subtilis, Clostridium perfringens, Staphylococcus aureus, Streptococcus iniae, Streptococcus mutans) and gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Vibrio alginolyticus, Klebsiella pneumoniae). Purified recombinant myticusin-beta also showed anti-parasitic activity at various concentrations. A short AMP analog was designed and synthesized based on the sequence of myticusin-beta, with markedly improved antimicrobial activity. Expression of myticusin-beta was detected in the mantle at the highest level, followed by hemocytes. The results obtained in this work suggest that myticusin-beta is an immune-related AMP of M. coruscus and an effective alternative to antibiotics.

American oyster defensin (AOD) was previously purified from acidified gill extract of the American oyster, Crassostrea virginica. AOD is composed of 38 amino acids with three disulfide bonds and exhibits strong antimicrobial activity against Gram-positive bacteria as well as significant activity against Gram-negative bacteria. Here, to develop promising peptides into antibiotic candidates, we designed five arginine-rich analogs (A0, A1, A2, A3, and A4), predicted their loop and extended strand/random structures—including nine amino acids and a disulfide bond derived from the C-terminus of AOD—and described their antimicrobial and cytotoxic effects, as well as their modes of action. In our experimental results, the A3 and A4 analogs exhibited potent antimicrobial activity against all test organisms—including four Gram-positive bacteria, six Gram-negative bacteria, and Candida albicans—without cell toxicity. A sequence of experiments, including a membrane permeabilization assay, DNA binding study, and DNA polymerization inhibition test, indicated that the two analogs (A3 and A4) possibly&nbsp, did not act directly on the bacterial membrane but instead interacted with intracellular components such as DNA or DNA amplification reactions. AOD analogs also showed strong bacterial inhibition activity in the plasma environment. In addition, analog-treated microbial cells clearly exhibited membrane disruption, damage, and leakage of cytoplasmic contents. Collectively, our results suggest that two analogs, A3 and A4, have potent antimicrobial activity via DNA interaction and have the potential for development into novel antimicrobial agents.

This paper presents an on-chip implementation of an analog processor-in-memory (PIM)-based convolutional neural network (CNN) in a biosensor. The operator was designed with low power to implement CNN as an on-chip device on the biosensor, which consists of plates of 32 × 32 material. In this paper, 10T SRAM-based analog PIM, which performs multiple and average (MAV) operations with multiplication and accumulation (MAC), is used as a filter to implement CNN at low power. PIM proceeds with MAV operations, with feature extraction as a filter, using an analog method. To prepare the input feature, an input matrix is formed by scanning a 32 × 32 biosensor based on a digital controller operating at 32 MHz frequency. Memory reuse techniques were applied to the analog SRAM filter, which is the core of low power implementation, and in order to accurately grasp the MAC operational efficiency and classification, we modeled and trained numerous input features based on biosignal data, confirming the classification. When the learned weight data was input, 19 mW of power was consumed during analog-based MAC operation. The implementation showed an energy efficiency of 5.38 TOPS/W and was differentiated through the implementation of 8 bits of high resolution in the 180 nm CMOS process.

Acylphosphatase is the smallest enzyme that is widely distributed in many diverse organisms ranging from archaebacteria to higher-eukaryotes including the humans. The enzyme hydrolyzes the carboxyl-phosphate bonds of the acyl phosphates which are important intermediates in glycolysis, membrane pumps, tricarboxylic acid cycle, and urea biosynthesis. Despite its biological importance in critical cellular functions, very limited structural investigations have been conducted on bacterial acylphosphatases. Here, we first unveiled the crystal structure of SaAcP, an acylphosphatase from gram-positive S. aureus at the atomic level. Structural insights on the active site together with mutation study provided greater understanding of the catalytic mechanism of SaAcP as a bacterial acylphosphatase and as a putative apyrase. Furthermore, through NMR titration experiment of SaAcP in its solution state, the dynamics and the alterations of residues affected by the phosphate ion were validated. Our findings elucidate the structure-function relationship of acylphosphatases in gram-positive bacteria and will provide a valuable basis for researchers in the field related to bacterial acylphosphatases.

Two species of brown seaweeds, Saccharina japonica and Undaria pinnatifida were fermented by the red molds; Monascus purpureus and Monascus kaoliang to increase their bio-functional properties. The phenolic contents of S. japonica fermented by M. purpureus (SjMp) and M. kaoliang (SjMk) were the highest 71.53 ± 2.25 and 66.50 ± 4.64 mg gallic acid equivalent/g extract, respectively, whereas the highest flavonoid content was evident in S. japonica fermented by M. purpureus (SjMp) and U. pinnatifida fermented by M. purpureus (UpMp) (27.93 ± 0.28 and 26.88 ± 1.24 mg quercetin equivalent/g extract, respectively). Reducing sugar, protein and essential fatty acids levels also increased in fermented seaweeds. The antioxidant activities of fermented seaweed extracts exhibited significantly (p

Graphene oxides coated by tannic acid (GOT) can be obtained easily by the self-polymerization of tannic acid in basic buffer solution on a graphene oxide surface. Polyamide reverse osmosis nanocomposite membranes containing GOT in the active layer were prepared by the interfacial polymerization using an organic solution containing trimesoyl chloride and an aqueous solution containing m -phenylene diamine and GOT. The polyamide membrane containing GOT (PA-GOT) showed significantly improved performances such as water flux, chlorine resistance, and antimicrobial properties, compared to the polyamide membrane without any additives and the polyamide membranes containing only tannic acid and/or graphene oxide. These high performances of PA-GOT membrane could be ascribed to a various of advantageous properties of GOT such as improved hydrophilicity, oxidative stress capability, barrier property, and compatibility with the polymer matrix.

We isolated and characterised a cDNA encoding the aspartic protease cathepsin E (CTSE) in Korean rose bitterling, Rhodeus uyekii. The full-length Rhodeus uyekii CTSE (RuCTSE) cDNA (1396 bp) contains an open reading frame of 1218 bp, encoding 405 amino acids. Alignment of multiple CTSE protein sequences revealed that two of the aspartyl protease active site residues and a disulphide bond were well-conserved among the other CTSE sequences. Phylogenetic analysis revealed that RuCTSE is most closely related to freshwater fish cathepsin E. RuCTSE is widely expressed in the liver, spleen, ovary, testis, brain, eye, intestine, muscle, fin, stomach, and kidney. This first report of teleost CTSE will provide important information related to the identification of other cathepsin E genes in various fish species and will serve as a useful molecular tool to help clarify biological activities in other teleosts.

An antimicrobial peptide with 55 amino acid residues was purified by C18 reversed-phase high-performance liquid chromatography (HPLC) from foot extract of the hard-shelled mussel, Mytilus coruscus. This peptide showed strong antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as fungi. The purified peptide was determined to have a molecular mass of 6202 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrophotometry (MALDI-TOF/MS). The identified 20-amino acid sequence of the purified peak by Edman degradation shared 100% identity with the N-terminal regions of mytichitin-1, mytichitin-2, mytichitin-3, mytichitin-4, mytichitin-5, and chitinase-like protein-1, and so was named mytichitin-CBD. The cDNA of mytichitin-CBD was cloned and sequenced by rapid amplification of cDNA ends (RACE). The mRNA transcripts were mainly detected in foot tissue, and they were up-regulated and peaked at 4 h after bacterial infection. We constructed and expressed recombinant mytichitin-CBD protein which displayed antimicrobial activity against Gram-negative bacteria Gram-positive bacteria and the fungus as well as anti-parasitic activity against scuticociliates. The results of this study demonstrate that the peptide isolated from M. coruscus is related to the innate immune system of this marine invertebrate and is a possible alternative to antibiotics.

A 5.6 kDa antimicrobial peptide (AMP) was purified from acidified gill extract of the pen shell, Atrina pectinata, by cation exchange and C18 reversed-phase high performance liquid chromatography. Comparison of the amino acid sequences and molecular weight of this peptide with those of other known AMPs revealed that it had high sequence homology with that of cgMolluscidin or hdMolluscidin; it was designated apMolluscidin. apMolluscidin comprises 59 amino acid residues containing several dibasic residue repeats and sequence repeats such as Lys-Lys and Lys-Gly. apMolluscidin exhibited potent antimicrobial activity against both Gram-positive bacteria including Bacillus subtilis (minimal effective concentration [MEC], 2.1 μg/mL), and Gram-negative bacteria including E. coli D31 (MEC, 0.5 μg/mL), without hemolytic activity. However, it did not show any activity against fungi such as Candida albicans. Secondary structure prediction suggested that it might form two helical regions and have an amphipathic structure. Full-length apMolluscidin cDNA contained 812 base pairs (bp), including a 5′-untranslated region (UTR) of 82 bp, a 3′-UTR of 547 bp, and a coding sequence of 183 bp encoding 60 amino acids (containing Met). Furthermore, qPCR analyses revealed that the mature peptide translated from apMolluscidin mRNA is expressed in a tissue-specific manner in locations such as the gill and siphon. These results indicate that apMolluscidin might be related to the innate immune defense system of abalone and may not act directly on the bacterial membrane. This is the first report of an AMP from the pen shell with a fully identified amino acid sequence.

The ywpF gene (SAV2097) of the Staphylococcus aureus strain Mu50 encodes the YwpF protein, which may play a role in antibiotic resistance. Here, we report the first crystal structure of the YwpF superfamily from S. aureus at 2.5-A resolution. The YwpF structure consists of two regions: an N-terminal core β-barrel domain that shows structural similarity to type VI secretion system (T6SS) proteins (e.g., Hcp1, Hcp3, and EvpC) and a C-terminal two-helix pair. Although the monomer structure of S. aureus YwpF resembles those of T6SS proteins, the dimer/tetramer model of S. aureus YwpF is distinct from the functionally important hexameric ring of T6SS proteins. We therefore suggest that the S. aureus YwpF may have a different function compared to T6SS proteins. Proteins 2015; 83:781–788. © 2015 Wiley Periodicals, Inc.

Poly-γ-glutamic acid (γ-PGA) is an unusual anionic homopolyamide that is biodegradable, edible, and nontoxic. It has a wide variety of industrial applications depending on its combined cations, and molecular weight. In this study, extracellular viscous γ-PGA produced by halotolerant Bacillus sp. SJ-10 isolated from a traditional Korean salted-fermented seafood was purified and characterized. The physicochemical analysis indicated that the γ-PGA produced by Bacillus sp. SJ-10 consists primarily of d-glutamic acid residues combined with sodium cations. When batch fermentation was performed with 8% NaCl for 3 d, Bacillus sp. SJ-10 produced approximately 24.7g/L γ-PGA with a molecular weight of approximately 400 kilodaltons (kDa). Under fermentation conditions with 6% NaCl, the maximum yield was 26.2g/L regardless of the molecular weight. The γ-PGA sodium salt with a molecular weight of 400kDa exhibited antioxidant activity by scavenging 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radicals and reducing molybdenum, with maximal scavenging activity at 0.5mg/mL and reducing activity at 1mg/mL (20μg ascorbic acid-equivalent), respectively. These results suggest the potential use of γ-PGA in the food, cosmetic, and biomedical industries for its antioxidant qualities. Our results also provide an economical method for controlling the molecular weight of the γ-PGA produced.

We describe the antimicrobial function of peptides derived from the C-terminus of the olive flounder LBP BPI precursor protein. The investigated peptides, namely, ofLBP1N, ofLBP2A, ofLBP4N, ofLBP5A, and ofLBP6A, formed α-helical structures, showing significant antimicrobial activity against several Gram-negative bacteria, Gram-positive bacteria, and the yeast Candida albicans, but very limited hemolytic activities. The biological activities of these five analogs were evaluated against biomembranes or artificial membranes for the development of candidate therapeutic agents. Gel retardation studies revealed that peptides bound to DNA and inhibited migration on an agarose gel. In addition, we demonstrated that ofLBP6A inhibited polymerase chain reaction. These results suggested that the ofLBP-derived peptide bactericidal mechanism may be related to the interaction with intracellular components such as DNA or polymerase.

Screening of a gene library from Paenibacillus sp. PBS-2 generated in Escherichia coli led to the identification of a clone with lipolytic activity. Sequence analysis showed an open reading frame encoding a polypeptide of 378 amino acid residues with a predicted molecular mass of 42 kDa. The esterase displayed 69% and 42% identity with the putative β-lactamases from Paenibacillus sp. JDR-2 and Clostridium sp. BNL1100, respectively. The esterase contained a Serx- x-Lys motif that is conserved among all β-lactamases found to date. The protein PBS-2 was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at 18°C. The enzyme is a serine protein and was active against p-nitrophenyl esters of C2, C4, C8, and C10. The optimum pH and temperature for enzyme activity were pH 9.0 and 30°C, respectively. Relative activity of 55% remained at up to 5°C with an activation energy of 5.84 kcal/mol, which indicates that the enzyme is cold-adapted. Enzyme activity was inhibited by Cd(2+), Cu(2+), and Hg(2+) ions. As expected for a serine esterase, activity was inhibited by phenylmethylsulfonyl fluoride. The enzyme was remarkably active and stable in the presence of commercial detergents and organic solvents. This cold-adapted esterase has potential as a biocatalyst and detergent additive for use at low temperatures.

The selected isolate, Bacillus sp. SW1-1 showed antibacterial activity against both Gram-positive and Gram-negative bacteria involved in fish diseases, including Edwardsiella tarda, Streptococcus iniae, S. parauberis, Vibrio anguillarum, and V. harveyi. The Maximum bacteriocin production was observed at 30°C after 24 h with brain heart infusion medium (pH 7.0). The bacteriocin SW1-1 was purified by 50% ammonium sulfate precipitation, followed by HiPrep diethylaminoethyl 16/10 FF and Sephacryl S-100 High resolution column chromatography. The substance was characterized as a bacteriocin-like inhibitory substance with a molecular mass of 38 kDa. Bacteriocin SW1-1 was sensitive to the proteolytic action of pepsin, trypsin, chymotrypsin, and protease types I and XIV, and relatively heat labile, despite the fact that bacteriocin activity was still detected after heating at 100°C for 30 min. The activity of bacteriocin SW1-1 was stable in the pH range of 2.0–11.0, and relatively unaffected by organic chemicals. The bacteriocin SW1-1 had a bacteriolytic mechanism, resulting in cell wall degradation of E. tarda. These characteristics indicate that this bacteriocin may be a potential candidate for alternative agent to control important pathogens of fish diseases in aquaculture.

Vibrio anguillarum, a causative agent of vibriosis in finfish, crustaceans, and bivalves, is a Gram-negative, motile marine bacterium. Most bacteria have developed survival strategies in various environments. The aim of this study was to investigate the changes in protein expression of V. anguillarum O1 incubated under different conditions using two dimensional electrophoresis and MALDI-TOF MS/MS analysis. Result indicated that peptidyl-prolyl cis/trans isomerase (PPIase) expression was increasingly appeared when incubated at low temperature (15°C) and alkaline conditions (pH 10). Subsequently, the ppi gene from V. anguillarum O1 was isolated and overexpressed in Escherichia coli to characterize the biochemical properties. The cloned ppi gene encoded 206 amino acids containing the conserved regions identified in FK506 binding pocket. To determine the optimal conditions of the purified recombinant PPIase protein (VaFKBP22), we used Succinyl-Ala-Phe-Pro-Phe-p nitroanilide as substrate and the highest enzymatic activity was found at 5°C and pH 6. VaFKBP22 was detected in the cytoplasm and periplasm of V. anguillarum O1. In addition, VaFKBP22 also showed chaperone activity and did not show cytotoxic activity.

A series of organic/inorganic hybrid branched (BCPs) and linear (LCPs)-graft copolymers comprising poly(ethylene glycol) methyl ether methacrylate (PEGMA), 3-(3,5,7,9,11,13,15-heptaisobutyl-pentacyclo[9.5.1.13,9.15,15.17,13]octasiloxane-1-yl)propyl methacrylate (MA-POSS), and ethylene glycol dimethacrylate (EGDMA) were synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization for application to solid polymer electrolyte (SPE) materials in high-temperature lithium-ion batteries. Dimensionally stable free-standing films were obtained when the MA-POSS contents in BCPs and LCPs are larger than 21 mol% and maintained their original shape and storage modulus even if the temperature increases up to 90 °C. The maximum ionic conductivity value of the BCP electrolyte containing 21 mol% of MA-POSS was 1.6 × 10−4 S cm−1 at 60 °C and that of the LCP electrolyte containing 21 mol% of MA-POSS was 5.6 × 10−5 S cm−1 at 60 °C, indicating that the branched-graft copolymer electrolyte has higher ionic conductivity than the linear-graft counterpart, due to the increased chain mobility, as estimated by a smaller Tg value. All-solid-state batteries prepared using the BCP electrolyte showed a reasonable cell performance at 60 °C without causing safety problems, demonstrating great potential of BCPs as SPE materials for high-temperature battery systems.

Antimicrobial peptides are a pivotal component of the invertebrate innate immune system. In this study, we identified a lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) gene from the pacific abalone Haliotis discus hannai (HDH), which is involved in the pattern recognition mechanism and plays avital role in the defense mechanism of invertebrates immune system. The HDH-LGBP cDNA consisted of a 1263-bp open reading frame (ORF) encoding a polypeptide of 420 amino acids, with a 20-amino-acid signal sequence. The molecular mass of the protein portion was 45.5 kDa, and the predicted isoelectric point of the mature protein was 4.93. Characteristic potential polysaccharide binding motif, glucanase motif, and β-glucan recognition motif were identified in the LGBP of HDH. We used its polysaccharide-binding motif sequence to design two novel antimicrobial peptide analogs (HDH-LGBP-A1 and HDH-LGBP-A2). By substituting a positively charged amino acid and amidation at the C-terminus, the pI and net charge of the HDH-LGBP increased, and the proteins formed an α-helical structure. The HDH-LGBP analogs exhibited antibacterial and antifungal activity, with minimal effective concentrations ranging from 0.008 to 2.2 μg/mL. Additionally, both were toxic against human cervix (HeLa), lung (A549), and colon (HCT 116) carcinoma cell lines but not much on human umbilical vein cell (HUVEC). Fluorescence-activated cell sorter (FACS) analysis showed that HDH-LGBP analogs disturb the cancer cell membrane and cause apoptotic cell death. These results suggest the use of HDH-LGBP analogs as multifunctional drugs.

The gene encoding an esterase from Photobacterium sp. MA1-3 was cloned in Escherichia coli using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (948 bp) corresponded to a protein of 315 amino acid residues with a molecular weight of 35 kDa and a pI of 6.06. The deduced protein showed 74% and 68% amino acid sequence identities with the putative esterases from Photobacterium profundum SS9 and Photobacterium damselae, respectively. Absence of a signal peptide indicated that it was a cell-bound protein. Sequence analysis showed that the protein contained the signature G-X-S-X-G included in most serine-esterases and lipases. The MA1-3 esterase was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at 18°C. The enzyme was a serine-esterase and was active against C2, C4, C8 and C10 p-nitrophenyl esters. The optimum pH and temperature for enzyme activity were pH 8.0 and 30°C, respectively. Relative activity remained up to 45% even at 5°C with an activation energy of 7.69 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was inhibited by Cd 2+ , Cu 2+ , Zn 2+ , and Hg 2+ ions.

A bacterial strain that produces a cold-adapted esterase was isolated from tidal flats and identified as Shewanella sp. Ke75. In the present study, the corresponding gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (957 bp) corresponded to a protein of 318 amino acid residues with a calculated molecular weight of 34875 Da. The esterase showed 68 and 57% identities with the putative esterases of Shewanella amazonensis SB2B and Colwellia psychrerythraea 34H, respectively. The esterase contained a putative leader sequence, as well as the conserved catalytic triad (Ser, His, Asp), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein Ke75 was produced in both soluble and insoluble forms when Escherichia coli cells harboring the gene were cultured at 30°C. The enzyme showed specificity for C4 (butyrate) as a substrate, with little activity toward the other p-nitrophenyl esters tested. The optimum pH and temperature for enzyme activity were pH 9.0 and 30°C, respectively. Relative activity remained up to 60% even at 5°C with an activation energy of 6.29 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was enhanced in the presence of Mn2+ ions, but inhibited by Cd2+, Cu2+, Hg2+, and Zn2+ ions.

We identified full-length cDNAs encoding pituitary adenylyl cyclase-activating polypeptide (PACAP), PACAP-related peptide (PRP), and PACAP-specific receptor (PAC1R) from olive flounder, Paralichthys olivaceus. Two variant mRNA forms were created by alternative splicing. Comparison of genomic and cDNA sequences of the PRP-PACAP precursor revealed that skipping of exon 4 within PRP resulted in two variant transcripts: a long form encoding both PRP and PACAP and a short form encoding PACAP only. Both transcripts were constitutively observed only in the brain, whereas the short form appeared in gut tissues, such as the intestine and pyloric cecum in fish challenged with a pathogen, but not in healthy fish. Furthermore, expression of the long PRP/PACAP transcript gradually increased in the intestine of flounder challenged with bacteria, suggesting that PRP and/or PACAP may serve as a regulator(s) of the immune system, especially in the gastrointestinal tract of olive flounder. The biological functions of PACAP and PRP were investigated by exogenous treatment of flounder embryogenic cells (hirame natural embryonic cells, HINAE cells) with synthetic peptides of fPACAP-38 and/or fPRP-45. Intracellular cyclic adenosine monophosphate (cAMP) production in PAC1R-overexpressing HINAE cells was regulated by fPACAP-38 in a concentration-dependent manner, but was not regulated by fPRP-45. Results from real-time quantitative polymerase chain reaction revealed that PAC1R mRNA was specifically induced by fPACAP-38 but not by fPRP-45; PACAP significantly increased TNF-α mRNA but not growth hormone (GH) mRNA in HINAE cells; however, PRP affected GH but not TNF-α mRNA expression. These results suggest that the expression ratio of PRP and PACAP is regulated at the transcriptional level depending on the tissues and conditions, and that the unique biological roles of PRP and PACAP differ from that of mammalian PRP.

For bacteria, cysteine thiol groups in proteins are commonly used as thiol-based switches for redox sensing to activate specific detoxification pathways and restore the redox balance. Among the known thiol-based regulatory systems, the MarR/DUF24 family regulators have been reported to sense and respond to reactive electrophilic species, including diamide, quinones, and aldehydes, with high specificity. Here, we report that the prototypical regulator YodB of the MarR/DUF24 family from Bacillus subtilis uses two distinct pathways to regulate transcription in response to two reactive electrophilic species (diamide or methyl-p-benzoquinone), as revealed by X-ray crystallography, NMR spectroscopy, and biochemical experiments. Diamide induces structural changes in the YodB dimer by promoting the formation of disulfide bonds, whereas methyl-p-benzoquinone allows the YodB dimer to be dissociated from DNA, with little effect on the YodB dimer. The results indicate that B. subtilis may discriminate toxic quinones, such as methyl-p-benzoquinone, from diamide to efficiently manage multiple oxidative signals. These results also provide evidence that different thiol-reactive compounds induce dissimilar conformational changes in the regulator to trigger the separate regulation of target DNA. This specific control of YodB is dependent upon the type of thiol-reactive compound present, is linked to its direct transcriptional activity, and is important for the survival of B. subtilis. This study of B. subtilis YodB also provides a structural basis for the relationship that exists between the ligand-induced conformational changes adopted by the protein and its functional switch.

Salinisphaera sp. P7-4 was isolated from the intestine of silver whiting, Sillago japonicas caught in the Pacific Ocean, and the esterase gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (951 bp) corresponded to a protein of 316 amino acid residues with a molecular weight of 34,443. The esterase had 46 and 44% identities with the esterase enzymes of Ralstonia eutropha JMP134 and Rhodopseudomonas palustris HaA2, respectively. The primary structure of P7-4 esterase showed the conserved catalytic triad (Ser, Asp, His), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein P7-4 was successfully expressed in Escherichia coli in a biologically active form. The enzyme showed high catalytic activity at low temperatures (5-25° C) with an activation energy of 2.18 kcal/mol. This result indicated that the esterase from Salinisphaera sp. P7-4 is a new cold-adapted enzyme. The enzyme preferentially hydrolyzed acyl-group chains with short chain lengths of ≤10 carbon. Metal ions such as Cd2(+), Co2(+), Cu2(+), Hg2(+), Ni2(+) and Zn2(+) inhibited enzymatic activity. Additionally, EDTA has no effect on its activity, whereas inhibition was observed with PMSF, a serine hydrolase inhibitor.

The brown alga Saccharina japonica is abundant on rocky coasts of Far East Asia, including Korea, Japan, and China. S. japonica produces high levels of compounds used in the food, cosmetic, and pharmaceutical industries. Thus, many studies have focused on the biosynthesis, extraction, purification, and application of carbohydrates, as well as biochemical features that yield cellular proteins. However, total protein isolation has proved difficult, due to viscous polysaccharides on the surface of S. japonica. To extract total proteins cleanly from S. japonica, we examined various lysis buffers and detergents for effective cell lysis and removal of polysaccharide. Lysis solution D (7 M urea, 4% [3-(3-cholami-dopropyl dimethylammonio) propanesulfonate], 2 M thio-urea, 100 mM dithiothreitol, 4% pharmalyte, 4% polyvinylpyrrolidone) achieved a comparatively high yield of protein extraction, with 12 mg of proteins purified per 1 g of dry weight of S. japonica. Proteins isolated using lysis solution D and subjected to two-dimension polyacrylamide gel electrophoresis generated more than 200 protein spots. Of these, 60 spots were analyzed by matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) and MALDI-TOF/MS/MS. A database search revealed that these proteins include glyceraldehyde-3-phosphate dehydrogenase, tryptophan synthase α chain, 6-phosphogluconate dehydrogenase (6PGD), actin, phosphoglycerate kinase, elongation factor Tu, kinesin, fucoxanthin-chlorophyll a–c binding protein F precursor and ATP synthase subunit β. Many protein spots were unidentified. When S. japonica was incubated at different pH, tryptophan synthase α chain and variant surface glycoprotein 7 precursor were highly expressed at pH 7.5 and 9.5, respectively, whereas 6PGD and kinesin showed low expression at pH 9.5.

The untranscribed DNA sequences of the intergenic spacer regions (ISRs) in the groESL gene of 23 Vibrio species were determined and compared. ISR sequence length (41–85 bp) was variable. Vibrio species could be divided into three groups according to the length and homology of their ISR sequences. DNA array hybridization using ISR-specific probes accurately distinguished Vibrio parahaemolyticus and Vibrio vulnificus from other species.

We isolated and characterized two cDNA clone encoding warm-temperature acclimation-associated 65-kDa proteins (PoWap65-1 and PoWap65-2) from the olive flounder, Paralichthys olivaceus. The deduced amino acid sequences of PoWap65s showed overall identities of 33-73% with other fish Wap65 and mammalian hemopexin-like proteins. The 5'-flanking regions of both PoWap65-encoding genes contained various putative transcriptional elements. While PoWap65-1 and PoWap65-2 were structurally similar, they exhibited highly differential patterns of expression. PoWap65-1 was expressed only in the liver, whereas PoWap65-2 transcripts were detected in a wide range of tissues. The accumulation of PoWap65s mRNA was expressed differentially during development. Expression of them in warm temperatures also differed in flounder embryonic cells. PoWap65-1 was upregulated by temperature stimulation whereas PoWap65-2 was not detected. PoWap65s were highly regulated by Edwardsiella tarda infection and hypoxia. Pathogen challenge induced PoWap65-2 expression in the liver whereas PoWap65-1 was downregulated. Hypoxia induced the expression of both PoWap65s in the liver of juvenile fish.

Peroxiredoxins (Prxs), also known as natural killer cell enhancing factors in fish, role as antioxidant proteins and participate in a variety of biological processes, including H2O2-mediated cell signaling, molecular chaperoning, and mitochondrial function. In this study, we isolated and characterized a Prx 1 cDNA from the Korean rose bitterling Rhodeus uyekii, and designated it RuPrx 1. The RuPrx 1 cDNA encodes a 197-amino-acid polypeptide that belongs to the class of typical 2-Cys Prxs that contain peroxidatic and resolving cysteines. The deduced RuPrx 1 protein shows strong homology (77.38–92.89 %) with Prx 1 proteins from other species, including fish, amphibians, and mammals, and it is most closely related to rainbow smelt Prx 1. RuPrx 1 mRNA was ubiquitously detected in all tested tissues and its expression was comparatively high in the brain, intestine, kidney, liver, ovary, stomach, and testis. Expression of RuPrx 1 mRNA in liver peaked 3 h post-infection with Aeromonas hydrophila and decreased 24 h post-infection while the expression in intestine decreased 24 h post-infection. These results suggest that RuPrx 1 is conserved through evolution and may play roles similar to its mammalian counterparts.

Acinetobacter venetians V28 was isolated from the intestine of righteye flounder, Poecilopsetta plinthus caught in Vietnam seawater, and the esterase gene was cloned using a shotgun method. The amino acid sequence deduced from the nucleotide sequence (1,017 bp) corresponded to a protein of 338 amino acid residues with a molecular weight of 37,186. The esterase had 87% and 72% identities with the lipases of A. junii SH205 and A. calcoaceticus RUH2202, respectively. The esterase contained a putative leader sequence, as well as the conserved catalytic triad (Ser, His, Asp), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein from the strain V28 was produced in both a soluble and an insoluble form when the Escherichia coli cells harboring the gene were cultured at 18 degrees C. The maximal activity of the purified enzyme was observed at a temperature of 40 degrees C and pH 9.0 using p-NP-caprylate as substrate; however, relative activity still reached to 70% even at 5 degrees C with an activation energy of 3.36 kcal/mol, which indicated that it was a cold-adapted enzyme. The enzyme was a nonmetalloprotein and was active against p-nitrophenyl esters of C4, C8, and C14. Remarkably, this enzyme retained much of its activity in the presence of commercial detergents and organic solvents. This cold-adapted esterase will be applicable as catalysts for reaction in the presence of organic solvents and detergents.

We identified outer membrane vesicle (OMV) production in Vibrio anguillarum O1, a major fish pathogen that causes vibriosis, and characterized the OMVs. They were produced during normal growth, and were appeared as spherical vesicle fractions. The protein profile of the OMVs was similar to that of the outer membrane proteins, and the 38-kDa major protein band of OMV was identified as OmpU. The OMVs had enzyme activity of metalloprotease, hemolysin, and phospholipase, and stimulated the production of proinflammatory cytokines, such as TNF-alpha, IL-1beta, and IL-6 when injected into the flounder.

Human epidermal growth factor (hEGF) is a polypeptide of 53 amino acids, is an important autocrine/paracrine factor in the human body, and is used in the pharmaceutical and cosmetics industries. We constructed a fusion hEGF protein with a collagen-binding domain (CBD) composed of 33 amino acids from Vibrio mimicus metalloprotease (VMCBD). The CBD segment of the metalloprotease was fused at the C terminus of the hEGF protein. The recombinant fusion protein was expressed in Escherichia coli and purified. The purified hEGF protein promoted greater growth of human/A-431 cells than did the control hEGF. The fusion EGF protein also showed collagen-binding activity with type I collagen. In contrast, hEGF did not bind to type I collagen. These results suggest that recombinant hEGF protein fused to VMCBD may be able to remain for a long period at injured epidermal tissue acting as a healing agent.

Star-shaped polymers (SPCs) containing poly(ethylene glycol) (PEG) and renewable cardanol side groups were synthesized by atom transfer radical polymerization (ATRP). Water-soluble SPCs were turned into water-insoluble materials by self-cross-linking reaction of the unsaturated hydrocarbon chains of cardanol moieties upon UV irradiation. The SPC-coated membranes with UV curing exhibited noticeably higher bio- and oil-fouling resistance than the bare polysulfone (PSf) membrane during filtration experiments, whereas the SPC-coated membranes without UV curing showed a large flux-decline caused by fouling compared to that of the bare membrane, because SPCs were washed out during filtration. The enhanced antifouling properties of the SPC-coated membranes with UV curing were ascribed to a large quantity of PEG moieties on the surfaces stabilized by the cross-linked polymeric structure, leading to decreased interactions with proteins and oils.