Maximin 9, a novel free thiol containing antimicrobial peptide with antimycoplasma activity from frog Bombina maxima

Amphibian skin is a rich resource of antimicrobial peptides, like maximins and maximin Hs from frog Bombina maxima. Novel cDNA clones encoding a precursor protein, which comprises a novel maximin peptide (maximin 9) and reported maximin H3, were isolated from two constructed skin cDNA libraries of B. maxima. The predicted primary structure of maximin 9 is GIGRKFLGGVKTTFRCGVKDFASKHLY-NH2. A surprising substitution is at position 16, with a free cysteine in maximin 9 rather than usual conserved glycine in other reported maximins. Maximin 9, the homodimer form and its Cys(16) to Gly(16) mutant were synthesized and their antimicrobial activities were evaluated. Unlike previously reported maximin 3, the tested bacterial and fungal strains were resistant to maximin 9, its homodimer and the Cys(16) to Gly(16) mutant (with MICs > 100 mu M). On the other hand, interestingly, while eight clinical Mollicutes strains were generally resistant to maximin 9 homodimer and its Cys(16) to Gly(16) mutant, most of them are sensitive to maximin 9 at a peptide concentration of 30 mu M, especially in the presence of dithiothreitol. These results indicate that the presence of a reactive Cys residue in maximin 9 is important for its antimycoplasma activity. The diversity of antimicrobial peptide cDNA structures encountered in B. maxima skin cDNA libraries and the antimicrobial specificity differences of the peptides may reflect well the species' adaptation to the unique microbial environments. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Cloning of bradykinin precursor cDNAs from skin of Bombina maxima reveals novel bombinakinin M antagonists and a bradykinin potential peptide

Bombinakinin M (DLPKINRKGP-bradykinin) is a bradykinin-related peptide purified from skin secretions of the frog Bombina maxima. As previously reported, its biosynthesis is characterized by a tandem repeats with various copy numbers of the peptide and sometimes co-expressed with other structure-function distinguishable peptides. At present study, two novel cDNAs encoding bombinakinin M and its variants were cloned from a cDNA library from the skin of the frog. The encoded two precursor proteins are common in that each contains three repeats of a novel 16-amino acid peptide unit and one copy of kinestatin at their N- and C-terminal parts, respectively. They differ in that the first precursor contains two copies of bombinakinin M and the second one contains one copy of a novel bombinakinin M variant. Bombinakinin M was found to elicit concentration-dependent contractile effects on guinea pig ileum, with an EC50 value of 4 nM that is four times higher than that of bradykinin (1 nM). Interestingly, the synthetic peptide (DYTIRTRLH-amide), as deduced from the 16-amino acid peptide repeats in the newly cloned cDNAs, possessed weak inhibitory activity on the contractile effects of bombinakinin M, but not on that of bradykinin. Furthermore, the newly identified bombinakinin M variant (DLSKMSFLHG-Ile(1)-bradykinin), did not show contractile activity on guinea pig ileum, but showed potentiation effect on the myotropic activity of bradykinin. In a molar raito of 1:58, it augmented the activity of bradykinin up to two-fold. (C) 2004 Elsevier B.V. All rights reserved.

Incidence of ulcer type of disease in wild fishes of Bangladesh

Disease occurred in wild fish species investigated in different water bodies like canals, ditches, beel, haor, flood plain in 17 districts of Dhaka division. Haemorrhagic lesions were observed on the body surface of affected fishes. Incidence of the disease in the investigated water bodies ranged from 0 to 100%. In total 19 fish species were found to be affected and prevalence of infection ranged from 0.0 to 100.0%. Channa punctatus and Puntius ticto were severely affected in all locations. Percentage of infection in these fishes ranged from 0.0 to 100.0. The highest infection was observed in Netrokona, Kishoreganj, and Mymensingh districts. Bacterial genera isolated from the lesions of these affected fishes were Aeromonas, Pseudomonas, Flavobacterium, Micrococcus, and Staphylococcus. Among these isolates Aeromonas was the dominant. Abundance of Aeromonas in the lesions among the investigated bacteria ranged from 75 to 90%. Five identified Aero monas lrydrophila were examined for their pathogenicity and were able to infect the experimental fish, silver barb (Puntius gonionotus). The pathogen Aeromonas hydrophila was thus considered to have an association with the outbreak of ulcer type of disease in the investigated fish species.

Low temperature (< 100 °c) deposited P-type cuprous oxide thin films: Importance of controlled oxygen and deposition energy

With the emergence of transparent electronics, there has been considerable advancement in n-type transparent semiconducting oxide (TSO) materials, such as ZnO, InGaZnO, and InSnO. Comparatively, the availability of p-type TSO materials is more scarce and the available materials are less mature. The development of p-type semiconductors is one of the key technologies needed to push transparent electronics and systems to the next frontier, particularly for implementing p-n junctions for solar cells and p-type transistors for complementary logic/circuits applications. Cuprous oxide (Cu2O) is one of the most promising candidates for p-type TSO materials. This paper reports the deposition of Cu2O thin films without substrate heating using a high deposition rate reactive sputtering technique, called high target utilisation sputtering (HiTUS). This technique allows independent control of the remote plasma density and the ion energy, thus providing finer control of the film properties and microstructure as well as reducing film stress. The effect of deposition parameters, including oxygen flow rate, plasma power and target power, on the properties of Cu2O films are reported. It is known from previously published work that the formation of pure Cu2O film is often difficult, due to the more ready formation or co-formation of cupric oxide (CuO). From our investigation, we established two key concurrent criteria needed for attaining Cu2O thin films (as opposed to CuO or mixed phase CuO/Cu2O films). First, the oxygen flow rate must be kept low to avoid over-oxidation of Cu2O to CuO and to ensure a non-oxidised/non-poisoned metallic copper target in the reactive sputtering environment. Secondly, the energy of the sputtered copper species must be kept low as higher reaction energy tends to favour the formation of CuO. The unique design of the HiTUS system enables the provision of a high density of low energy sputtered copper radicals/ions, and when combined with a controlled amount of oxygen, can produce good quality p-type transparent Cu2O films with electrical resistivity ranging from 102 to 104 Ω-cm, hole mobility of 1-10 cm2/V-s, and optical band-gap of 2.0-2.6 eV. These material properties make this low temperature deposited HiTUS Cu 2O film suitable for fabrication of p-type metal oxide thin film transistors. Furthermore, the capability to deposit Cu2O films with low film stress at low temperatures on plastic substrates renders this approach favourable for fabrication of flexible p-n junction solar cells. © 2011 Elsevier B.V. All rights reserved.

Localization and distribution of tissue type and urokinase type plasminogen activators and their inhibitors type 1 and 2 in human and rhesus monkey fetal membranes

Fetal membranes consist of 10 distinct layers including components of amnion, chorion and decidua, the latter being of maternal origin. They form mechanically integrated sheets capable of retaining amniotic fluid and play an essential role in protecting fetal growth and development in the pregnant uterus. The extracellular matrix, substrate for plasminogen activators (PAs), is an important supportive framework of the fetal membranes. :Fetal membranes from women with preterm premature rupture of membranes may differ in their protease activity compared with normal membranes. To identify the presence of PAs and their inhibitors (PAI) and their possible role in the process of fetal membrane rupture, this study in investigated the distribution and localization of both protein and mRNA for tissue (t) and urokinase (u) PA and their inhibitors type 1 (PAI-1) and type 2 (PAI-2) in amniochorion of human and rhesus monkey using conventional and. confocal immunofluorescence microscopy. In situ hybridization analysis showed that the distribution and localization of mRNAs for tPA, uPA, PAI-I and PAI-2 were similar in the fetal membranes of human and rhesus monkey; no obvious species difference was observed. Evidence of tPA mRNA was detected in amniotic epithelium, trophoblast cells and nearly all cells of the decidual layer. Strong expression of uPA mRNA was noted in the decidual cells which increased in intensity as the abscission point was approached. Weak staining in chorion laeve trophoblast was also detected. In situ hybridization experiments showed PAI-1 mRNA to be concentrated mainly in the decidual cells, some of which were interposed into the maternal-facing edge of the chorion laeve. Maximal labelling of the decidua occurred towards the zone of abscission. Weak expression of PAI-1 mRNA nas also noted in some cells of the chorion laeve. The distribution of PAI-2 mRNA in amniochorion was also concentrated in the cells of the decidual layer, maximum expression of the mRNA was in the level of abscission. No detectable amount of mRNAs for tPA, uPA, PAI-1 and PAI-2 was found in the fibroblast, reticular and spongy layers. Distribution of the proteins of tPA, uPA and PAI-1 in the fetal membranes of these two species was consistent with the distribution of their mRNA. Anti-PAI-2 immunofluorescence was found to be strongly concentrated in the amniotic epithelium, but PAI-2 mRNA was negative in this layer, suggesting that the epithelium-associated PAI-2 is not of epithelial origin. These findings suggest that a local fibrinolysis in fetal membranes generated by precisely balanced expression of PAs and their inhibitors via paracrine or autocrine mechanisms may play an essential role in fetal membrane development, maturation and in membrane rupture. Following an analysis of the distribution and synthesis of activators and inhibitors it was found that they may play a role in abscission during the third stage of labour. (C) 1998 W. B. Saunders Company Ltd.

LiMn2-xTixO4 spinel-type compounds (x ≤ 1): Structural, electrical and magnetic properties

LiMn2-xTixO4 compounds with 0 ≤ x ≤ 1 were prepared by solid state reaction and Pechini technique. Powder X-ray diffraction showed that all samples crystallize with the spinel crystal structure (S.G. Fd3-m). The cubic unit-cell parameter increases with the Ti content. The influence of the Ti content and cationic distribution on the magnetic properties of the compounds was studied by measuring the temperature and magnetic field dependences of the magnetization: substitution by non-magnetic d0 Ti4+ ions appeared to weaken the magnetic interactions between the manganese ions. The electrical properties of LiMnTiO4 were studied by AC impedance spectroscopy and DC polarisation measurements, which revealed the electronic character of the conduction process. © 2006 Elsevier B.V. All rights reserved.

Structural analysis of plate-type fuel assemblies and development of a non-destructive method to assess their integrity

This work is concerned with the structural behaviour and the integrity of parallel plate-type nuclear fuel assemblies. A plate-type assembly consists of several thin plates mounted in a box-like structure and is subjected to a coolant flow that can result in a considerable drag force. A finite element model of an assembly is presented to study the sensitivity of the natural frequencies to the stiffness of the plates' junctions. It is shown that the shift in the natural frequencies of the torsional modes can be used to check the global integrity of the fuel assembly while the local natural frequencies of the inner plates can be used to estimate the maximum drag force they can resist. Finally a non-destructive method is developed to assess the resistance of the inner plates to bear an applied load. Extensive computational and experimental results are presented to prove the applicability of the method presented. © 2013 Elsevier B.V. All rights reserved.

Expressional induction of Paralichthys olivaceus cathepsin B gene in response to virus, poly I:C and lipopolysaccharide

Cathepsin B is a lysosomal cysteine protease of the papain-like enzyme family with multiple biological functions. In this study, Paralichthys olivaceus cathepsin B (PoCatB) cDNA was isolated from flounder embryonic cells (FEC) treated with UV-inactivated grass carp hemorrhage virus (GCHV) and subsequently identified as a vitally induced gene. The full length cDNA of PoCatB is 1801 bp encoding 330-amino acids. The deduced protein has high homology to all known cathepsin B proteins, containing an N-terminal signal peptide, cysteine protease active sites, the occluding loop segment and a glycosylation site, all of which are conserved in the cathepsin B family. PoCatB transcription of FEC cells could be induced by turbot (Scophthalmus maximus) rhabdovirus (SMRV), UV-inactivated SMRV, UV-inactivated GCHV, poly I:C or lipopolysaccharide (LPS), and SMRV or poly I:C was revealed to be most effective among the five inducers. In normal flounder, PoCatB mRNA was detectable in all examined tissues. Moreover, SMRV infection could result in significant upregulation of PoCatB mRNA, predominantly in spleen, head kidney, posterior kidney, intestine, gill and muscle with 18.2,10.9, 24.7,12, 31.5 and 18 fold increases at 72 h post-infection respectively. These results provided the first evidence for the transcriptional induction of cathepsin B in fish by virus and LPS, indicating existence of a novel function in viral defense. (C) 2008 Elsevier Ltd. All rights reserved.

Gene structure of an antimicrobial peptide from mandarin fish, Siniperca chuatsi (Basilewsky), suggests that moronecidins and pleurocidins belong in one family: the piscidins

The gene of piscidin, an antimicrobial peptide, has been cloned from the mandarin fish, Siniperca chuatsi. From the first transcription initiation site, the mandarin fish piscidin gene extends 1693 nucleotides to the end of the 3' untranslated region and contains four exons and three introns. A predicted 79-residue prepropeptide consists of three domains: a signal peptide (22 aa), a mature peptide (22 aa) and a C-terminal prodomain (35 aa). The shortage of XQQ motif in the prodomain of mandarin fish piscidin and the similar gene structure between moronecidins (piscidins) and pleurocidins may indicate that they are derived from the same ancestor gene. We thus suggest that piscidin should be used as a terminology for these antimicrobial peptides in the future. The mandarin fish piscidin mRNA was abundant in intestine, spleen, pronephros and kidney analysed by real-time polymerase chain reaction. After stimulation with lipopoly saccharides (LPS), a marked increase in transcripts was observed in most tissues, indicating that piscidin is not only a constitutively expressed molecule, but also has an increased response to bacterial infection. The synthetic, amidated mandarin fish piscidin exhibited different antimicrobial activity against different fish bacterial pathogens, especially against species of Aeromonas, which may to certain extent reflect the pathogenicity of these bacteria.

Discovery and characterization of two types of liver-expressed antimicrobial peptide 2 (LEAP-2) genes in rainbow trout

The sequences and gene organisation of two LEAP-2 molecules (LEAP-2A and LEAP-2B) from rainbow trout, Oncorhynchus mykiss are presented. Both genes consist of a 3 exon/2 intron structure, with exon sizes comparable to known mammalian genes. LEAP-2A notably differs from LEAP-2B in having larger introns and a larger 3'UTR. The predicted proteins contain a signal peptide and prodomain, followed by a mature peptide of 41 aa containing four conserved cysteines. The RXXR cleavage site to release the mature peptide was also conserved. Both genes were found to be constitutively expressed in the liver, with expression in the intestine, and to a lesser extent the skin, evident after bacterial challenge. (C) 2004 Elsevier B.V. All rights reserved.

Cloning and expression analysis in mature individuals of two chicken type-II GnRH (cGnRH-II) genes in common carp (Cyprinus carpio)

Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide family, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II precursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplication hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the expression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.

CYCLIC PEPTIDE HEPATOTOXINS FROM FRESH-WATER CYANOBACTERIAL (BLUE-GREEN-ALGAE) WATERBLOOMS COLLECTED IN CENTRAL CHINA

Toxic cyanobacteria (blue-green algae) waterblooms have been found in several Chinese water bodies since studies began there in 1984. Waterbloom samples for this study contained Anabaena circinalis, Microcystis aeruginosa and Oscillatoria sp. Only those waterblooms dominated by Microcystis aeruginosa were toxic by the intraperitoneal (i.p.) mouse bioassay. Signs of poisoning were the same as with known hepatotoxic cyclic peptide microcystins. One toxic fraction was isolated from each Microcystis aeruginosa sample. Two hepatotoxic peptides were purified from each of the fractions by high-performance liquid chromatography and identified by amino acid analysis followed by low and high resolution fast-atom bombardment mass spectrometry (FAB-MS). LD50 i.p. mouse values for the two toxins were 245-mu-g/kg (Toxin A) and 53-mu-g/g (Toxin B). Toxin content in the cells was 0.03 to 3.95 mg/g (Toxin A) and 0.18 to 3.33 mg/kg (Toxin B). The amino acid composition of Toxin A was alanine [1], arginine [2], glutamic acid [1] and beta-methylaspartic acid [1]; for Toxin B it was the same, except one of the arginines was replaced with a leucine. Low- and high-resolution FAB-MS showed that the molecular weights were 1,037 m/z (Toxin A) and 994 m/z (Toxin B), with formulas of C49H76O12N13 (Toxin A) and C49H75O12N10 (Toxin B). It was concluded that Toxin A is microcystin-RR and Toxin B is microcystin-LR, both known cyclic heptapeptide hepatotoxins isolated from cyanobacteria in other parts of the world. Sodium borohydride reduction of microcystin-RR yielded dihydro-microcystin-RR (m/z = 1,039), an important intermediate in the preparation of tritium-labeled toxin for metabolism and fate studies.

Growth and Characterization of GaSb-Based Type-II InAs/GaSb Superlattice Photodiodes for Mid-Infrared Detection

InAs/GaSb superlattice (SL) midwave infrared photovoltaic detectors are grown by molecular beam epitaxy on GaSb(001) residual p-type substrates. A thick GaSb layer is grown under the optimized growth condition as a buffer layer. The detectors containing a 320-period 8ML/8ML InAs/GaSb SL active layer are fabricated with a series pixel area using anode sulfide passivation. Corresponding to 50% cutoff wavelengths of 5.0 mu m at 77 K, the peak directivity of the detectors is 1.6 x 10(10) cm.Hz(1/2) W-1 at 77 K.