Antimicrobial function of SHßAP, a novel hemoglobin ß chain-related antimicrobial peptide, isolated from the liver of skipjack tuna, Katsuwonus pelamis.

A 2.3 kDa of antimicrobial peptide was purified from an acidified liver extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase HPLC. A comparison of the amino acid sequence of the purified peptide with those of other known polypeptides revealed high homology with the C-terminus of hemoglobin ß-chain; thus, this peptide was designated as the Skipjack Hemoglobin ß chain-related Antimicrobial Peptide (SHßAP). SHßAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 6.5-57.0 µg/mL), Gram-negative bacteria, such as Escherichia coli D31, Pseudomonas aeruginosa, Salmonella enterica, Shigella sonnei, and two Vibrio parahaemolyticus species (MECs, 2.0-19.0 µg/mL), and against Candida albicans (MEC; 12.0 µg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide was heatstable and pH resistant but is sensitive to proteases and salt. SHßAP did not show membrane permeabilization and killing ability. The secondary structural prediction and the homology modeling expected that this peptide formed an amphipathic α-helical structure. This is the first report the purification of a novel antimicrobial peptide related to the C-terminus of hemoglobin ß-chain from marine fish.

Nano-sized silver patterning using nano-ink with an indirect patterning method of nanoimprint and lift-off processes.

Silver nanopatterns were fabricated using a nanoimprint and lift-off process using silver nano-ink. Particle-based silver nano-ink was used to fabricate metal patterns below 100 nm in a lift-off process without the undercut shape of polymer patterns typically required in a conventional lift-off process using evaporated metal. The silver film made by spin-coating the silver nano-ink and removing the solvent using PDMS, has a low density compared to evaporated metal film; hence, unnecessary nano-ink that is not part of the pattern area is easily eliminated during the lift-off process. Using the proposed patterning process, patterns defined on a stamp were successfully transferred to the nano-ink through nanoimprinted polymer patterns on a four-inch Si wafer. Compared to other metal patterning methods that involve lift-off processes, the proposed metal patterning process is a simple and cost-effective process capable of fabricating the micro- and nano-sized metal patterns due to its low operating temperature and one-step polymer patterning process by nanoimprinting.

Complete mitochondrial genome of the ectomycorrhizal fungus Tricholoma matsutake.

Tricholoma matsutake is an ectomycorrhizal fungus belonging to the class Agaricomycetes and the phylum Basidiomycota. Here, we report the complete mitochondrial genome sequence of T. matsutake. The mitochondrial DNA (mtDNA) of T. matsutake seems to be concatenated, and harbors 76,037 nucleotides, with an overall GC content of 20.69%. The mtDNA comprises 15 protein-coding genes, 28 tRNA genes, and 2 rRNA genes. A comparative mitogenomic analysis between Agaricomycetes and other classes (Exobasidiomycetes, Pucciniomycetes, Tremellomycetes, and Ustilaginomycetes) revealed that species belonging to the class Agaricomycetes preferably harbor the codon UUA (coding for leucine) in their mtDNAs, whereas other classes prefer the codon CUA. This bias in the usage of the leucine codon could be the result of evolutionary divergence between the Agaricomycetes and other classes. The T. matsutake mtDNA sequence thus provides insight into the evolutionary characteristics of the Agaricomycetes mtDNAs.

The complete mitochondrial genome of the javeline goby Acanthogobius hasta (Perciformes, Gobiidae) and phylogenetic considerations.

We isolated Acanthogobius hasta mitochondrial DNA by long-polymerase chain reaction (long-PCR) with conserved primers, and sequenced this mitogenome with primer walking. The resultant A. hasta mitochondrial DNA sequence was found to consist of 16,663 bp with a structural organization conserved relative to that of other fish. In this paper, we report the basic characteristics of the A. hasta mitochondrial genome including structural organization, base composition of rRNAs and the tRNAs and protein-encoding genes, and characteristics of mitochondrial tRNAs. These findings are applicable to molecular phylogenetics in the suborder Gobioidei.

The complete mitochondrial genome of the floating goby, Gymnogobius petschiliensis (Perciformes, Gobiidae).

We isolated floating goby Gymnogobius petschiliensis mitochondrial DNA by long-polymerase chain reaction (Long-PCR) with conserved primers, and sequenced the mitogenome by primer walking using flanking sequences. The G. petschiliensis mitochondrial DNA has 16,424 bp and its structural organization is similar to the mitochondrial DNAs of other fish, and mammals. We analyzed phylogenetic relationships derived from the mitochondrial cytochrome b gene. We report the basic characteristics of the G. petschiliensis mitochondrial genome including its structural organization and the base composition of the rRNAs, tRNAs and protein-coding genes as well as characteristics of tRNAs. These features are used to analyze phylogenetic relationship among the 60 species of the genus Gymnogobius.

Synthesis and characterization of tri(ethylene oxide)-attached poly(amidoamine) dendrimer layers on gold.

This paper describes the synthesis of a tri(ethylene oxide)-attached fourth-generation poly(amidoamine) dendrimer (EO3-dendrimer) and the characterization of its layers on gold. NMR analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that about 61 amine groups of a G4 PAMAM dendrimer were covalently conjugated with tri(ethylene oxide) units, accounting for a 95% modification level. Layers of the EO3-dendrimer were formed on gold, and the resulting surface was characterized by infrared reflection absorption spectroscopy, ellipsometry, and contact angle goniometry. The EO3-dendrimer resulted in more hydrophilic and less compact layers with no substantial deformation of the molecule during layer formation by virtue of the EO3 units, compared to a PAMAM dendrimer. Interestingly, the specific binding of avidin to the biotinylated layers of the EO3-dendrimer approached a surface density of 5.2 +/- 0.2 ngmm-2, showing about 92% of full surface coverage. The layers of the EO3-dendrimer were found to be more resistant to nonspecific adsorption of proteins than PAMAM dendrimer layers when bovine serum albumin and serum proteins were tested.

Cloning and sequence analysis of the self-fertilizing fish Rivulus marmoratus immediate early gene c-fos.

We have cloned the proto-oncogene c-fos from a self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) after screening of R. marmoratus lambdaGEM-11 genomic DNA library, and sequenced over 12 kb including all exons, introns and the promoter region. The R. marmoratus c-fos gene consisted of one noncoding exon and four exons with high similarity to those of fugu and mammals. We sequenced approximately 7 kb of the R. marmoratus c-fos gene promoter region to gain a better understanding of the molecular anatomy of the immediate response of this gene upon cellular damage. In the promoter region, R. marmoratus c-fos gene has seven xenobiotic response elements (XREs) and eight metal response elements (MREs) as well as two estradiol (E2), 4 NFkappaB, 2 CarG, 2 prolactin (PRL) motifs and one pit1 site, while the 3'-UTR of this gene contains the estrogen response element (ERE). The seven XRE and eight MRE motifs raise the possibility of its regulation by exposure to environmental pollutants. In this paper, we discuss the gene structure of R. marmoratus c-fos gene and compare its promoter region with those of other organisms' c-fos genes. We propose its potential use in ecotoxicology.

Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphrodite fish Rivulus marmoratus and the Japanese medaka Oryzias latipes.

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.

The intertidal harpacticoid copepod Tigriopus japonicus (Crustacea: Copepoda) beta-actin gene: cloning, sequence and intraspecies variation.

The Tigriopus japonicus beta-actin genes were amplified from genomic DNA by polymerase chain reaction (PCR) and cloned into pCRII vector. Several clones of the T. japonicus beta-actin gene spanned 1662-1676 bp with gains or losses of some bases in intron 3 or 4 but they consisted generally of 5 exons and 4 introns with very high homology, implying polymorphism of this gene. The exon and intron boundaries were matched with the GT/AG rule. The T. japonicus beta-actin gene showed high homology to the fish (Rivulus marmoratus) and human genes, 99.2 and 98.4%, respectively at the amino acid sequence level. The phylogenetic implications inferred from the T. japoncius beta-actin gene are discussed.

cDNA cloning of translationally controlled tumor protein/histamine releasing factor (TCTP/HRF) from the intertidal harpacticoid copepod Tigriopus japonicus.

We synthesized a cDNA library from the intertidal copepod Tigriopus japonicus, converted it to phagemids and sequenced expressed sequence tags (ESTs). Of these, Tigriopus translationally controlled tumor protein/histamine releasing factor (TCTP/HRF) was further characterized. The Tigriopus TCTP/HRF gene encoded 172 amino acid residues and showed high similarity to Drosophila but moderate similarity to other annelids (e.g. Brugia, Wuchereria and C. elegans). The Tigriopus TCTP/HRF gene appeared in the same clade as the annelids. Here, we describe the analysis of the Tigriopus TCTP/HRF gene.

Cloning and mRNA expression analysis of the gene encoding phenylalanine ammonia-lyase of the ectomycorrhizal fungus Tricholoma matsutake.

The ectomycorrhizal fungus Tricholoma matsutake grows symbiotically with Pinus densiflora. Phenylalanine ammonia-lyase (E.C. 4.3.1.24) catalyzes the conversion of L-phenylalanine to trans-cinnamic acid. The role of fungal phenylalanine ammonia-lyase, however, has not been clear until now. In this study, the gene encoding phenylalanine ammonia-lyase (PAL), which was isolated from T. matsutake, was cloned and characterized. The PAL gene (tmpal) consists of 2,160 nucleotides, coding for a polypeptide containing 719 amino acid residues. The deduced amino acid sequence of tmpal from T. matsutake shows high identity (70%) with that from Laccaria bicolor. Comparative analysis of the PAL genes among T. matsutake and other species of the class Agaricomycetes showed that both active sites and binding sites were significantly conserved among these genes. The transcriptional analysis of the PAL gene revealed a differential gene expression pattern depending on the developmental stages (mycelium, primordium, stipe, pileus, and gills) of T. matsutake. These results suggest that the PAL gene in T. matsutake plays an important role in multiple physiological functions.

Metagenomic analysis of fungal communities inhabiting the fairy ring zone of Tricholoma matsutake.

Tricholoma matsutake, an ectomycorrhiza that has mutual relationships with the rootlet of Pinus denisflora, forms a fruiting body that serves as a valuable food in Asia. However, the artificial culture of this fungus has not been successful. Soil fungi, including T. matsutake, coexist with many other microorganisms and plants; therefore, complex microbial communities have an influence on the fruiting body formation of T. matsutake. Here, we report on the structures of fungal communities associated with the fairy ring of T. matsutake through the pyrosequencing method. Soil samples were collected inside the fairy ring zone, in the fairy ring zone, and outside the fairy ring zone. A total of 37,125 sequencing reads were obtained and 728 to 1,962 operational taxonomic units (OTUs) were observed in the sampling zones. The fairy ring zone had the lowest OTUs and the lowest fungal diversity of all sampling zones. The number of OTUs and fungal taxa inside and outside the fairy ring zone was, respectively, about 2 times and 1.5 times higher than the fairy ring. Taxonomic analysis showed that each sampling zone has different fungal communities. In particular, out of 209 genera total, 6 genera in the fairy ring zone, such as Hemimycena, were uniquely present and 31 genera, such as Mycena, Boletopsis, and Repetophragma, were specifically absent. The results of metagenomic analysis based on the pyrosequencing indicate a decrease of fungal communities in the fairy ring zone and changes of fungal communities depending on the fairy ring growth of T. matsutake.

Mass spectrometric analysis of affinity-captured proteins on a dendrimer-based immunosensing surface: investigation of on-chip proteolytic digestion.

The monolayer of fourth-generation poly(amidoamine) dendrimers was adopted to construct the immunoaffinity surface of an antibody layer. The antibody layer as a bait on the dendrimer monolayer was found to result in high binding capacity of antigenic proteins and a reliable detection. The affinity-captured protein at the immunosensing surface was subjected to direct on-chip tryptic digestion, and the resulting proteolytic peptides were analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The performance of the on-chip digestion procedure was investigated with respect to the ratio of trypsin to protein, digestion time, composition of a reaction buffer, and the amount of affinity-captured protein on a surface. Addition of a water-miscible organic solvent to a reaction buffer had no significant effect on the digestion efficiency under the optimized digestion conditions. The on-chip digestion method identified the affinity-captured bovine serum albumin (BSA), lysozyme, and ferritin at the level of around 100 fmol. Interestingly, the detected number of peptide hits through the on-chip digestion was almost similar regardless of the amount of captured protein ranging from low- to high-femtomole levels, whereas the efficiency of in-solution digestion decreased significantly as the amount of protein decreased to low-femtomole levels. The structural alignment of the peptide fragments from on-chip-digested BSA revealed that the limited exterior of the captured protein is subjected to attack by trypsin. The established detection procedures enabled the identification of BSA in the biological mixtures at the level of 0.1 ng/mL. The use of antibodies against the proteins involved in the metabolic pathway of L-threonine in Escherichia coli also led to discrimination of the respective target proteins from cell lysates.

[A case of hyperinfection syndrome with Strongyloides stercoralis]

A case of Strongyloides stercoralis infection wss experienced in a 73-year old Korean female patient, was hospitalized with relapse of cholecystitis. The patient developed cough and dyspnea 17 days after the admission. On the 27th hospitalized day, diarrhoea, nausea, vomiting and abdominal pain started. A number of parasitic larvae were incubated at 25 degrees C for 2 days. Typical fork tailed filariform larvae of S. stercoralis (Bavay, 1876) Stiles and Hassall, 1902, were identified after cultivation. There was no improvement of diarrhoea after the medication with mebendazole. After the administration of thiabendazole, however, diarrhoea was stopped. On the 6th day of medication, S. stercoralis larvae were no more detected, and thereafter no larva was observed by repeated stool examinations upto 2 months after chemotherapy. The patient had the history of administration of steroid for articular rheumatism. Therefore this case seems to be a hyperinfection of S. stercoralis due to an autoinfection and to be the first report on the hyperinfected strongyloidiasis in Korea. Related literature was briefly reviewed.