Kanyawim Kirtikara

Sulforhodamine B colorimetric assay for cytotoxicity screening

The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method described here has been optimized for the toxicity screening of compounds to adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for OD determination at 510 nm using a microplate reader. The results are linear over a 20-fold range of cell numbers and the sensitivity is comparable to those of fluorometric methods. The method not only allows a large number of samples to be tested within a few days, but also requires only simple equipment and inexpensive reagents. The SRB assay is therefore an efficient and highly cost-effective method for screening.

Development of antibody array for simultaneous detection of foodborne pathogens.

Pathogenic bacterial contaminations present serious problems for food industry and public health. Rapid, accurate and affordable assays are needed. In this study, antibody arrays to simultaneously detect two foodborne pathogenic bacteria (Escherichia coli O157:H7 and Salmonella spp.) have been developed using chemiluminescent detecting system. Solid supports using nitrocellulose membrane and poly-l-lysine (PLL) glass slide were compared and optimized for antibody array construction. Many parameters including optimal concentrations of antibodies, blocking reagents, assay time, storage time, sensitivity and cross-reactivity were considered during optimization. This study revealed that the PLL slide was a more suitable support due to highly accurate results and the absence of non-specific background. Phosphate-buffered saline (PBS, pH 7.2) and 3% skim milk in PBS buffer were optimal spotting and blocking reagents, respectively. With the same sensitivity for bacterial detection as in a conventional ELISA (10(5)-10(6)CFU/ml for the E. coli O157:H7 and 10(6)-10(7)CFU/ml for Salmonella detections), this antibody array has advantages of a much shorter assay time of 1h and much lower required amounts of antibodies. Moreover, there was no cross-reactivity in the detection among bacteria tested in this study. Bacteria detection in food sample was feasible as demonstrated using bacteria-added milk.

Planotetraspora thailandica sp. nov., isolated from soil in Thailand.

A Gram-positive-staining, filamentous bacterial strain that developed cylindrical sporangia containing four oval- to rod-shaped spores at the ends of short sporangiophores on branched aerial mycelium was isolated from tropical rainforest soil near a hot spring. The cell-wall peptidoglycan contained meso-diaminopimelic acid, glutamic acid and alanine as cell-wall amino acids; the whole-cell hydrolysate contained rhamnose, madurose, glucose, galactose and 3-O-methylmannose as whole-cell sugars. The predominant menaquinone was MK-9(H(4)). Mycolic acids were not detected. The diagnostic phospholipid was phosphatidylethanolamine. The predominant cellular fatty acids were iso-C(16 : 0) and 10-methylated C(17 : 0). The G+C content of the DNA was 71.1 mol%. The phenotypic and chemotaxonomic analyses showed that the isolate had characteristics typical of members of the genus Planotetraspora. Furthermore, 16S rRNA gene sequence analysis also indicated that this strain belongs to the genus Planotetraspora, but as a putative novel species. Following phenotypic, chemotaxonomic and genotypic studies, the isolate is proposed to be a representative of a novel species, to be named Planotetraspora thailandica sp. nov. The type strain is BCC 21825(T) (=NBRC 104271(T)). An emended description of the genus Planotetraspora is also presented.

Cyclohexene, diketopiperazine, lactone and phenol derivatives from the sea fan-derived fungi Nigrospora sp. PSU-F11 and PSU-F12

Nigrosporanenes A (1) and B (2), two new cylohexene derivatives, and tyrosol (3) were isolated from the sea fan-derived fungus Nigrospora sp. PSU-F11, whereas five known compounds: 4-hydroxybenzoic acid (4), aplysiopsene D (5), 3-isochromanone (6), (−)-drimenin (7) and diketopiperazine derivative (8), were obtained from the fungus Nigrospora sp. PSU-F12. Their structures were established by spectroscopic evidence. We also tested their cytotoxic (on African green monkey kidney fibroblast and breast cancer cells), antioxidant (in the DPPH assay), and antibacterial (against the standard Staphylococcus aureus ATCC 25923 and methicillinresistant S. aureus) activities.

Antimalarial preracemosols A and B, possible biogenetic precursors of racemosol from Bauhinia malabarica Roxb.

Racemosol and demethylracemosol, together with their possible biogenetic precursors, preracemosol A and preracemosol B, were isolated from the roots of Bauhinia malabarica Roxb. While only racemosol and demethylracemosol exhibited cytotoxicity against KB and BC cell lines, all four compounds exhibited moderate antimalarial activity.

Insights into the prostanoid pathway in the ovary development of the penaeid shrimp Penaeus monodon.

The prostanoid pathway converts polyunsaturated fatty acids (PUFAs) into bioactive lipid mediators, including prostaglandins, thromboxanes and prostacyclins, all of which play vital roles in the immune and reproductive systems in most animal phyla. In crustaceans, PUFAs and prostaglandins have been detected and often associated with female reproductive maturation. However, the presence of prostanoid biosynthesis genes remained in question in these species. In this study, we outlined the prostanoid pathway in the black tiger shrimp Penaeus monodon based on the amplification of nine prostanoid biosynthesis genes: cytosolic phospholipase A2, hematopoietic prostaglandin D synthase, glutathione-dependent prostaglandin D synthase, prostaglandin E synthase 1, prostaglandin E synthase 2, prostaglandin E synthase 3, prostaglandin F synthase, thromboxane A synthase and cyclooxygenase. TBLASTX analysis confirmed the identities of these genes with 51-99% sequence identities to their closest homologs. In addition, prostaglandin F2α (PGF2α), which is a product of the prostaglandin F synthase enzyme, was detected for the first time in P. monodon ovaries along with the previously identified PUFAs and prostaglandin E2 (PGE2) using RP-HPLC and mass-spectrometry. The prostaglandin synthase activity was also observed in shrimp ovary homogenates using in vitro activity assay. When prostaglandin biosynthesis was examined in different stages of shrimp ovaries, we found that the amounts of prostaglandin F synthase gene transcripts and PGF2α decreased as the ovaries matured. These findings not only indicate the presence of a functional prostanoid pathway in penaeid shrimp, but also suggest a possible role of the PGF2α biosynthesis in shrimp ovarian development.

ReproArrayGTS: a cDNA microarray for identification of reproduction-related genes in the giant tiger shrimp Penaeus monodon and characterization of a novel nuclear autoantigenic sperm protein (NASP) gene.

Expressed sequence tags (ESTs) were established from various tissues of the giant tiger shrimp (Penaeus monodon). To simultaneously examine expression patterns of a large number of transcripts in ovaries and testes of P. monodon, a cDNA microarray (ReproArray(GTS)) containing 4992 features amplified from cDNAs of ovary (1920) and testis (3072) EST libraries was constructed and subjected to high-throughput gene expression analysis in four different stages of ovarian development (previtellogenesis, vitellogenesis, early cortical rod and late cortical rod stages). Several transcripts were found to be differentially expressed during P. monodon ovarian development. Among many important reproduction-related genes with differential expression from microarray data, nuclear autoantigenic sperm protein (NASP) was further characterized by RACE-PCR. The full-length cDNA of P. monodon NASP (PmNASP) was 2126 bp in length containing an open reading frame (ORF) of 1812 bp corresponding to a deduced protein of 603 amino acids with 5? and 3?UTRs of 93 and 202 bp (excluding the poly A tail), respectively. Higher PmNASP transcript levels at later stages of ovarian development was consistently confirmed by quantitative real-time PCR. This study indicated that ReproArray(GTS) is effective for high-throughput screening of genes that play important roles in ovarian development of P. monodon.

A Chemiluminescent Antibody Array System for Detection of Foodborne Pathogens in Milk

A foodborne antibody array (FAbA) was developed as a rapid, inexpensive, and accurate detection to simultaneously detect Escherichia coli O157:H7 and Salmonella spp. using a custom-made chemiluminescent detector. We have optimized fabrication processes, selected the antibody concentrations, calibrated the signals to the pathogen amount, and minimized assay time. In comparison with ∼4 hours for ELISA, FAbA takes only 75 minutes to achieve relatively the same sensitivities (8 × 104 and 5 × 107 CFU/ml for E. coli and Salmonella, respectively). The FAbA was able to detect the pathogens in milk at the same detection limits as in the buffer.

Up-regulation of microsomal prostaglandin E synthase-1 in COX-1 and COX-2 knock-out mouse fibroblast cell lines

In this paper we investigated the possible involvement of prostaglandin E synthases (PGESs) in compensatory mechanism. Our findings showed that microsomal (m)PGES-1 expression was significantly up-regulated in COX knock-out (K/O) cells whereas the expression of cytosolic PGES was not changed indicating that the induction of mPGES-1 may, at least in part, contribute to the substantial increase of PGE(2) production in COX K/O cell lines. The selective up-regulation of mPGES-1 in COX-2 K/O cells suggests that mPGES-1 may be metabolically coupled with COX-1 for PGE(2) formation. Addition of arachidonic acid caused significant induction of mPGES-1 and COX-2 in WT cells, whereas COX-1 and cPGES were not affected. Our earlier and the current studies demonstrate the coregulation of cPLA(2), COX, and mPGES-1, in PGE(2) synthesis pathway, and that these enzymes contribute to the elevation of PGE(2) level when one COX isoform is absent.

A Sensitive Direct ELISA for Detection of Prostaglandin E2

Abstract In order to improve the indirect ELISA for detection of PGE2, a modified direct ELISA technique was developed to measure PGE2 in cell culture supernatants. An evaluation of three types of coating buffer showed that PGE2 was adsorbed efficiently to the solid phase using the gelatin phosphate buffer. The sensitivity of the assay was increased by employing polyclonal rabbit anti‐PGE2 antibody dilution of 1/100 and 1% skimmed milk as a blocking solution, with the detection limit of 7.8–500 ng/well. The within‐run and between‐run coefficients of variation (CV) ranges were 3.2–3.7% and 3.4–3.8%, respectively. A linear standard curve was observed over the range of 0.078–5 µg/mL with a coefficient of determination (r2) of 0.99. Our results indicated that the developed direct ELISA was sensitive and suitable for a quick determination of PGE2 levels from cell culture supernatants.