Synthesis of Hydroxyapatite with Antibacterial Properties Using a Microwave-Assisted Combustion Method.

The prevention of implant-associated infections has been increasing clinically in orthopedic surgery. Hydroxyapatite with antibacterial properties was synthesized using a microwave-assisted combustion method. High crystallinity at low temperature can be achieved using this method. The synthesized hydroxyapatite exhibited a superior clear zone for both Gram-positive and Gram-negative bacteria. Electron spin resonance (ESR) and X-ray photoelectron spectroscopy (XPS) were used for the radical investigation. The application of intelligent ink testing and an antioxidant assay using DPPH reduction were also used to confirm the existence of radicals. These techniques provided data confirming that radicals are responsible for the antibacterial properties. The synthesized antibacterial hydroxyapatite would be a good candidate for the prevention any infection with medical implants and injection materials causing failure in bone repair.

Molecular cloning and characterization of a novel bi-functional α-amylase/subtilisin inhibitor from Hevea brasiliensis.

A novel cDNA encoding a bi-functional α-amylase/subtilisin inhibitor (HbASI) was isolated from rubber (Hevea brasiliensis) leaves cultivar RRIM600. The HbASI had strong homology with the soybean trypsin inhibitor (Kunitz) family of protease inhibitors. Its putative amino acid sequence was similar to that of the α-amylase/subtilisin inhibitor from Ricinus communis (72% identity). Genomic sequencing indicated that the HbASI gene contained no introns. The messenger RNA of HbASI was detected in leaf, hypocotyl and root. The recombinant HbASI expressed extracellularly in Pichia pastoris exhibited inhibitory activity against α-amylase from Aspergillus oryzae, trypsin and subtilisin A. The HbASI gene was induced in the rubber leaves infected with a rubber tree pathogen, Phytophthora palmivora. It was also enhanced by salicylic acid (SA) treatment and mechanical wounding. In addition, the biological activity of the HbASI protein involving in the plant defence responses was also investigated. The HbASI at a concentration of 0.16 mg mL(-1) could inhibit the mycelium growth of P. palmivora. These data suggested that the HbASI protein might play a crucial role in defence against pathogen of rubber trees.

RNA Polymerase II Second Largest Subunit Molecular Identification of Boletus griseipurpureus Corner From Thailand and Antibacterial Activity of Basidiocarp Extracts.

BACKGROUND: Boletus griseipurpureus Corner, an edible mushroom, is a putative ectomycorrhizal fungus. Currently, the taxonomic boundary of this mushroom is unclear and its bitter taste makes it interesting for evaluating its antibacterial properties. OBJECTIVES: The purpose of this study was to identify the genetic variation of this mushroom and also to evaluate any antibacterial activities. MATERIALS AND METHODS: Basidiocarps were collected from 2 north-eastern provinces, Roi Et and Ubon Ratchathani, and from 2 southern provinces, Songkhla and Surat Thani, in Thailand. Genomic DNA was extracted and molecular structure was examined using the RNA polymerase II (RPB2) analysis. Antibacterial activities of basidiocarp extracts were conducted with Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29523 and methicillin-resistant Staphylococcus aureus (MRSA) 189 using the agar-well diffusion method. RESULTS: All the samples collected for this study constituted a monophyletic clade, which was closely related with the Boletus group of polypore fungi. For the antibacterial study, it was found that the crude methanol extract of basidiomes inhibited the growth of all bacteria in vitro more than the crude ethyl acetate extract. CONCLUSIONS: Basidomes collected from four locations in Thailand had low genetic variation and their extracts inhibited the growth of all tested bacteria. The health benefits of this edible species should be evaluated further.

Trichoharzianol, a new antifungal from Trichoderma harzianum F031.

A new decalin derivative, trichoharzianol (1), together with three known compounds, eujavanicol A (2), 5-hydroxy-3-hydroxymethyl-2-methyl-7-methoxychromone (3), and 4,6-dihydroxy-5-methylphthalide (4), were isolated from Trichoderma harzianum F031. For the first time, compounds 2-4 were reported from the Trichoderma species. Their structures were characterized by spectroscopic methods. Trichoharzianol (1) showed the highest antifungal activity against Colletotrichum gloeosporioides, with a minimum inhibitory concentration (MIC) of 128 µg/mL.

Molecular cloning and biochemical characterization of a novel cystatin from Hevea rubber latex.

A novel cDNA encoding a cysteine proteinase inhibitor or phytocystatin was isolated from Hevea brasiliensis RRIM600 rubber latex cDNA library. The full-length HbCPI obtained from rapid amplification of cDNA ends contains 588 bp. An open reading frame of 306 bp encodes for a protein of 101 amino acids with the typical inhibitory motifs of phytocystatin superfamily, namely the central signature motif QXVXG, a GG doublet and LARFAV-like motifs in the N-terminal part, and conserved A/PW residues in the C-terminal region. Sequence comparison showed that the deduced amino acid sequence was similar to that of cysteine protease inhibitor from Manihot esculenta (84% identity). The HbCPI was subcloned into expression vector pQE-40 and then overexpressed in Escherichia coli M15 strain (pREP4) as a His-tagged recombinant protein with molecular mass approximately 13 kDa. The purified HbCPI showed thermal stable property and efficiently inhibited the protease activity of papain by non-competitive inhibition with K(i) value of 15.4 nM. Beside latex, HbCPI also transcripted in leaf and young seed. The HbCPI message accumulation was induced by phytopathogenic fungi Phytophthora palmivora infection. These data suggest that HbCPI might play crucial roles in defense mechanism against biotic stimuli.

Myxobolus supamattayai n. sp. (Myxosporea: Myxobolidae) from Thailand parasitizing the scale pellicle of wild mullet (Valamugil seheli).

A new myxosporean species, Myxobolus supamattayai n. sp., was isolated from wild mullet (Valamugil seheli) from the Andaman Sea, Thailand and described based on its morphology and molecular data. The myxosporean produced black plasmodia-like unique clinical sign on the skin with sporogonic stages and mature spores. Polysporous plasmodia, up to 2.5 mm in diameter, were found in epithelium tissue in the scale pocket. The spores measured 6.6 (6.2-7.0) µm in length, 6.5 (6.2-6.7) µm in width, smooth, and round board to ellipsoidal in valvular view. Spores were enclosed with intracapsular process which represents 5-7 and 11-12 in amount revealed in light microscopy and ultrastructure, respectively. The polar capsules were pyriform and of equal size, measuring 3.5 (3.4-3.6) µm in length and 2.0 (1.9-2.2) µm in width, with four to five turns of polar filament arranged perpendicularly to longitudinal axis of the polar capsule. In conclusion, this new species is entirely different from those previously described; however, this finding was assured by the partial sequence of SSU rRNA gene (1,666 bp) analysis that differed from all known species of Myxobolus Bütschli, 1882. The phylogenetic tree of the sequence data sets including those of freshwater and marine of Myxobolus spp. and the sister group (Henneguya spp.) was constructed to establish the relationship of this new species in Myxobolus clade and to explore its relations between their sister groups. Phylogenetic analysis indicated that a monophyletic group with Myxobolus spp. which infected mullet represents the newly formed species. These results suggested the presumably nearby evolution prospecting of Myxobolus species that were found in the same host.

Molecular cloning and expression of a mammalian homologue of a translationally controlled tumor protein (TCTP) gene from Penaeus monodon shrimp.

White spot syndrome, caused by white spot syndrome virus (WSSV), is a deadly disease of shrimps, causing a catastrophic loss in shrimp industries worldwide. In order to investigate molecular response of shrimp haemocyte to WSSV infection, we performed subtraction hybridization of mRNAs from healthy and WSSV-infected haemocyte. One of the genes that were severely down-regulated in moribund WSSV-infected-haemocyte was translationally controlled tumor protein (TCTP) (or fortilin). Strikingly, while there was a slight difference in the amount of TCTP message between normal and early WSSV-infected shrimps, shrimps that exhibited severe symptoms uniformly had very little TCTP in their haemocyte. Taken together with the fact that TCTP functions as an anti-apoptotic protein in mammals, our data suggest that TCTP in shrimp protects WSSV-infected shrimps from death.

A syntenin-like protein with postsynaptic density protein (PDZ) domains produced by black tiger shrimp Penaeus monodon in response to white spot syndrome virus infection.

We report the isolation and characterization of products from a subtractive cDNA library from the haemolymph of Penaeus monodon experimentally infected with white spot syndrome virus (WSSV). One cDNA derived from up-regulated mRNA was identified. A homology search indicated similarity to the putative protein syntenin (TE8). The nearly complete nucleotide sequence of TE8 was obtained by rapid amplification of cDNA (RACE). Its putative protein product contained a tandem repeat of PDZ domains (postsynaptic density protein or PSD-95, DlgA and ZO-1). We propose that TE8 may function as an adapter that couples PDZ-binding protein(s) in a signaling pathway involved in the shrimp response to WSSV.