ACE Inhibitory Activity and Molecular Docking of Gac Seed Protein Hydrolysate Purified by HILIC and RP-HPLC.

Gac (Momordica cochinchinensis Spreng.) seed proteins (GSPs) hydrolysate was investigated for angiotensin I-converting enzyme (ACE) inhibitory activities. GSPs were hydrolyzed under simulated gastrointestinal digestion using a combination of enzymes, including pepsin, trypsin, and chymotrypsin. The screening of ACE inhibitory peptides from GSPs hydrolysate was performed using two sequential bioassay-guided fractionations, namely hydrophilic interaction liquid chromatography (HILIC) and reversed-phase high-performance liquid chromatography (RP-HPLC). Then, the peptides in the fraction with the highest ACE inhibitory activity were identified by LC-MS/MS. The flow-through (FT) fraction showed the most potent ACE inhibitory activity when HILIC fractionation was performed. This fraction was further separated using RP-HPLC, and the result indicated that fraction 8 (RP-F8) showed the highest ACE inhibitory activity. In the HILIC-FT/RP-F8 fraction, 14 peptides were identified using LC-MS/MS analysis coupled with de novo sequencing. These amino acid chains had not been recorded previously and their ACE inhibitory activities were analyzed in silico using the BIOPEP database. One fragment with the amino acid sequence of ALVY showed a significant ACE inhibitory activity (7.03 ± 0.09 µM). The Lineweaver-Burk plot indicated that ALVY is a competitive inhibitor. The inhibition mechanism of ALVY against ACE was further rationalized through the molecular docking simulation, which revealed that the ACE inhibitory activities of ALVY is due to interaction with the S1 (Ala354, Tyr523) and the S2 (His353, His513) pockets of ACE. Bibliographic survey allowed the identification of similarities between peptides reported as in gac fruit and other proteins. These results suggest that gac seed proteins hydrolysate can be used as a potential nutraceutical with inhibitory activity against ACE.

Change in Renal Glomerular Collagens and Glomerular Filtration Barrier-Related Proteins in a Dextran Sulfate Sodium-Induced Colitis Mouse Model.

Renal disease is not rare among patients with inflammatory bowel disease (IBD) and is gaining interest as a target of research. However, related changes in glomerular structural have rarely been investigated. This study was aimed at clarifying the changes in collagens and glomerular filtration barrier (GFB)-related proteins of glomeruli in a dextran sulfate sodium (DSS)-induced colitis mouse model. Acute colitis was induced by administering 3.5% DSS in Slc:ICR strain mice for eight days. Histological changes to glomeruli were examined by periodic acid-Schiff (PAS) and Masson's trichrome staining. Expressions of glomerular collagens and GFB-related proteins were analyzed by immunofluorescent staining and Western blot analysis. DSS-colitis mice showed an elevated disease activity index (DAI), colon shortening, massive cellular infiltration and colon damage, confirming that DSS-colitis mice can be used as an IBD animal model. DSS-colitis mice showed increased glycoprotein and collagen deposition in glomeruli. Interestingly, we observed significant changes in glomerular collagens, including a decrease in type IV collagen, and an increment in type I and type V collagens. Moreover, declined GFB-related proteins expressions were detected, including synaptopodin, podocalyxin, nephrin and VE-cadherin. These results suggest that renal disease in DSS-colitis mice might be associated with changes in glomerular collagens and GFB-related proteins. These findings are important for further elucidation of the clinical pathological mechanisms underlying IBD-associated renal disease.

Volatile and Nonvolatile Constituents and Antioxidant Capacity of Oleoresins in Three Taiwan Citrus Varieties as Determined by Supercritical Fluid Extraction.

As local varieties of citrus fruit in Taiwan, Ponkan (Citrus reticulata Blanco), Tankan (C. tankan Hayata), and Murcott (C. reticulate × C. sinensis) face substantial competition on the market. In this study, we used carbon dioxide supercritical technology to extract oleoresin from the peels of the three citrus varieties, adding alcohol as a solvent assistant to enhance the extraction rate. The supercritical fluid extraction was fractionated with lower terpene compounds in order to improve the oxygenated amounts of the volatile resins. The contents of oleoresin from the three varieties of citrus peels were then analyzed with GC/MS in order to identify 33 volatile compounds. In addition, the analysis results indicated that the non-volatile oleoresin extracted from the samples contains polymethoxyflavones (86.2~259.5 mg/g), limonoids (111.7~406.2 mg/g), and phytosterols (686.1~1316.4 µg/g). The DPPH (1,1-Diphenyl-2-picrylhydrazyl), ABTS [2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] scavenging and inhibition of lipid oxidation, which test the oleoresin from the three kinds of citrus, exhibited significant antioxidant capacity. The component polymethoxyflavones contributed the greatest share of the overall antioxidant capacity, while the limonoid and phytosterol components effectively coordinated with its effects.

Gallic Acid Content in Taiwanese Teas at Different Degrees of Fermentation and Its Antioxidant Activity by Inhibiting PKCδ Activation: In Vitro and in Silico Studies.

Teas can be classified according to their degree of fermentation, which has been reported to affect both the bioactive components in the teas and their antioxidative activity. In this study, four kinds of commercial Taiwanese tea at different degrees of fermentation, which include green (non-fermented), oolong (semi-fermented), black (fully fermented), and Pu-erh (post-fermented) tea, were profiled for catechin levels by using high performance liquid chromatography (HPLC). The result indicated that the gallic acid content in tea was directly proportional to the degree of fermentation in which the lowest and highest gallic acid content were 1.67 and 21.98 mg/g from green and Pu-erh tea, respectively. The antioxidative mechanism of the gallic acid was further determined by in vitro and in silico analyses. In vitro assays included the use of phorbol ester-induced macrophage RAW264.7 cell model for determining the inhibition of reactive oxygen species (ROS) production, and PKCδ and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit (p47) activations. The results showed that only at a concentration of 5.00 µM could gallic acid significantly (p < 0.05) reduce ROS levels in phorbol ester-activated macrophages. Moreover, protein immunoblotting expressed similar results in which activations of PKCδ and p47 were only significantly (p < 0.05) attenuated by 5.00 µM treatment. Lastly, in silico experiments further revealed that gallic acid could block PKCδ activation by occupying the phorbol ester binding sites of the protein.

Application of highly purified electrolyzed chlorine dioxide for tilapia fillet disinfection.

This research aimed to develop an electrolysis method to generate high-concentration chlorine dioxide (ClO2) for tilapia fillet disinfection. The designed generator produced up to 3500 ppm of ClO2 at up to 99% purity. Tilapia fillets were soaked in a 400 ppm ClO2 solution for 5, 10, and 25 min. Results show that total plate counts of tilapia, respectively, decreased by 5.72 to 3.23, 2.10, and 1.09 log CFU/g. In addition, a 200 ppm ClO2 solution eliminated coliform bacteria and Escherichia coli in 5 min with shaking treatment. Furthermore, ClO2 and trihalomethanes (THMs) residuals on tilapia fillets were analyzed by GC/MS and were nondetectable (GC-MS detection limit was 0.12 ppb). The results conform to Taiwan's environmental protection regulations and act governing food sanitation.

An innovative cell model revealed the inhibitory effect of flavanone structure on peroxynitrite production through interaction with the IKKß kinase domain at ATP binding site.

It is hypothesized that the oxidative/nitrosative stress inhibitory effect of a flavanone is governed by its chemical structure. However, the existing cell-based antioxidant assays primarily focus on single chemical to initiate toxic species production. In this study, a novel cell model using macrophage treated with a combination of PMA and LPS leading to generation of peroxynitrite was proposed to provide a more real physiological condition. Three flavanones (eriodictyol, naringenin, and pinocembrin) with different number of ortho-dihydroxyl groups on B-ring were used to provide a more comprehensive evaluation of the role of chemical structure in the new model. Dihydrorhodamine123 assay, protein immunoblotting, immunofluorescence assay, and in silico analysis by molecular docking between the flavanones and IKKß catalytic kinase domain at the ATP binding site were employed. Results indicated that the generation of peroxynitrite was decreased at 10 µM of flavanones; eriodictyol was the most effective inhibitor. Western blot analysis and confocal fluorescence image also showed that eriodictyol could inhibit iNOS and p47 protein expressions through the inhibition of NF-kB translocation and performed the maximal inhibition compared to that of the other groups. In addition, the highest CDOCKER energy values of eriodictyol (38.6703 kcal/mol) confirmed that the 3',4'-ortho-dihydroxylation on the B-ring played a crucial role in binding with IKKß kinase domain at ATP binding site. Finally, we propose that the ortho-dihydroxyl groups on B-ring of flavanone may influence directly the occupation of the ATP binding site of IKKß kinase domain leading to the abrogation of peroxynitrite formation in the innovative cell model.

Identification of a novel umami peptide in tempeh (Indonesian fermented soybean) and its binding mechanism to the umami receptor T1R.

Tempeh, a traditional Indonesian soybean product produced by fermentation, is especially popular because of its umami taste. In this study, a novel umami peptide GENEEEDSGAIVTVK (GK-15) was identified in the small peptide (<3 kDa) fraction of the water extract of tempeh using LC-MS/MS analysis and database-assisted identification. The umami taste of GK-15 was further validated using sensory evaluation, which suggested that GK-15 may be one of the key components contributing to the umami taste in tempeh. To rationalize the biological effect of GK-15, molecular docking of GK-15 into the N-terminal extracellular ligand-binding domain of the umami (T1R) receptor was performed. ZDOCK data showed that GK-15 could perfectly bind either to the open or closed conformation of T1R3. To the best of our knowledge, the present work is the first study to focus on the screening of umami peptides from tempeh.

Determination of Phenolic Compounds, Procyanidins, and Antioxidant Activity in Processed Coffea arabica L. Leaves.

The effects of dry processing and maturity on antioxidant activity, total phenolic content, total procyanidins, and identity of phenolic compounds in coffee leaves were evaluated. Fresh coffee leaves were tray-dried at 40 °C for 8 h before total phenolic content, total procyanidins, and antioxidant activity were analyzed. The drying process significantly (p < 0.05) improved the release of total phenolic content and total procyanidins compared with the fresh leaves. The results showed that the young leaves exposed to drying processes had the highest total phenolic content, total procyanidins, and DPPH radical scavenging activity. Therefore, the effect of different drying temperatures (30, 40, and 50 °C) in the young leaves were further analyzed. The results indicated that DPPH radical scavenging activity, total phenolic content, and total procyanidins were increasingly generated when exposed to an increase in drying temperatures, whereby the highest bioactivity was evident at 50 °C. The DPPH radical scavenging activity of the coffee leaf teas was significantly correlated with total phenolic content and total procyanidins. Identification of Coffea arabica L. bioactive compounds by LC-MS showed the presence of catechin or epicatechin, mangiferin or isomangiferin, procyanidin B, caffeoylquinic acids (CQA), caffeine, quercetin-3-O-glucoside, procyanidin C, rutin, and 3,4-diCQA. Coffea arabica L. leaf tea was confirmed to be a potential functional food rich in phenolic compounds with strong antioxidant activity.

Effects of Saccharomyces cerevisiae in association with Torulaspora delbrueckii on the aroma and amino acids in longan wines.

This study investigated the effects of monocultures of Saccharomyces cerevisiae and Torulaspora delbrueckii as well as simultaneous and sequential cultures of S. cerevisiae and T. delbrueckii on the nonvolatile and volatile compounds in longan wines. The four cultures had similar characteristics in longan wines. The main amino acids in all the fermentations were glutamic acid, arginine, alanine, leucine, proline, and GABA. The main volatile compounds in longan wines were ethanol, isoamyl alcohol, isobutanol, 2-phenylethanol, isoamyl acetate, ethyl decanoate, ethyl octanoate, ethyl hexanoate, and ethyl acetate, which can contribute more desired aroma compounds in wines. Among the four treatments, the longan wine fermented with the simultaneous culture produced the highest total volatile aroma content (345.26 mg/L). The simultaneous culture also had a better ability to generate a high level of the main volatile compounds in longan wines and also could achieve a noticeable intensity of floral and fruity aromas of wine as evaluated by calculation of the odor activity values.

Retrogradation and Digestibility of Rice Starch Gels: The Joint Effect of Degree of Gelatinization and Storage.

Retrogradation affects acceptability of starchy foods; however, it is preferred in some products such as rice noodles. Amylose content, gelatinization temperature, and storage condition were reported to affect retrogradation but with disputed data. The joint effects of these parameters were interested in this study. Rice starch was gelatinized using different temperatures (77, 81, 95, and 121 °C) and stored isothermally with temperature cycles for 10 days. Results showed that the most important parameter that affected retrogradation was storage time followed by storage condition and gelatinization temperature. The recrystallization rate constant (k) and Avrami exponent (n) of retrograded starches stored under temperature cycle were higher than isothermal storage. All samples showed similar polymorphs of a mixture of B and V types. High-temperature gelatinized starch gel stored under temperature cycle condition produced higher yield of resistant starch. The study provided useful information on how to apply these parameters to control the retrogradation of starchy foods, especially rice noodle. PRACTICAL APPLICATION: Retrogradation is found to be more prominent at higher gelatinization temperature and longer storage time. Resistant starch produced from retrograded starch depended largely on storage time than storage condition. This finding can be applied to improve rice noodle qualities (by increasing retrogradation) with lower digestibility (by producing higher resistant starch).

Induction of apoptosis by vitamin D2, ergocalciferol, via reactive oxygen species generation, glutathione depletion, and caspase activation in human leukemia Cells.

This study demonstrated that ergocalciferol was able to inhibit leukemia cell growth in a concentration-dependent manner. Exploration of the acting mechanisms involved this event revealed that ergocalciferol induced DNA fragmentation and increased sub-G1 DNA contents in HL-60 cells, both of which are hallmarks of apoptosis. Analysis of the integrity of mitochondria demonstrated that ergocalciferol caused loss of mitochondrial membrane potential with release cytochrome c to cytosol, generation of reactive oxygen species (ROS), and depletion of glutathione (GSH), suggesting that ergocalciferol may induce apoptosis in HL-60 cells through a ROS-dependent pathway. Further results show that caspases-2, -3, -6, and -9 were all activated by ergocalciferol, together with cleavage of the downstream caspase-3 targets, DNA fragmentation factor (DFF-45), and poly(ADP-ribose) polymerase. In addition, ergocalciferol led to the increase in pro-apoptotic factor Bax accompanied with the decrease in anti-apoptotic member Mcl-1, and the reduced Mcl-1 to Bax ratio may be a critical event concerning mitochondrial decay by ergocalciferol. Furthermore, ergocalciferol also led to induction of Fas death receptor closely linked to caspase-2 activation, suggesting the involvement of a Fas-mediated pathway in ergocalciferol-induced apoptosis. Totally, these findings suggest that ergocalciferol causes HL-60 apoptosis via a modulation of mitochondria involving ROS production, GSH depletion, caspase activation, and Fas induction. On the basis of anticancer activity of ergocalciferol, it may be feasible to develop chemopreventive agents from edible mushrooms or hop.

Synthesis of 1-piperideine-6-carboxylic acid produced by L-lysine-epsilon-aminotransferase from the Streptomyces clavuligerus gene expressed in Escherichia coli.

The gene (lat) encoding L-lysine epsilon-aminotransferase (LAT) in Streptomyces clavuligerus was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of lat predicted a single open reading frame (ORF) of 1371 bp, encoding a polypeptide of 457 amino acids with calculated molecular mass of 49.89 kDa. S. clavuligerus LAT was grouped into aminotransferase subfamily II of alpha family on the basis of sequence homology. A model system composed of the recombinant LAT in phosphate buffer was set up to study the biosynthesis of 2-acetyltetrahydropyridine. Lysine was found to be transformed to 1-piperideine-6-carboxylic acid. 2-Acetyltetrahydropyridine was characterized from the mixture of 1-piperideine-6-carboxylic acid and methylglyoxal. For the first time, we demonstrated that the L-lysine epsilon-aminotransferase is responsible for the formation of 1-piperideine-6-carboxylic acid, which may react with methylglyoxal to generate the acylated N-heterocyclic odorant 2-acetyltetrahydropyridine.

Delta1-pyrroline-5-carboxylic acid formed by proline dehydrogenase from the Bacillus subtilis ssp. natto expressed in Escherichia coli as a precursor for 2-acetyl-1-pyrroline.

Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Delta1-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of l-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.

Studies on the expression of liver detoxifying enzymes in rats fed seaweed (Monostroma nitidum).

The expression level of phase I (CYP1A1 and CYP1A2) and phase II (GST, and UGT) enzyme-coded genes were measured in liver microsomes of 30 Sprague-Dawley rats fed sea weed (Monostroma nitidum). Quantitative and qualitative analysis of the detoxifying enzymes were investigated using reverse transcription polymerase reaction (RT-PCR) and real time polymerase reaction (Real-time PCR) techniques. The antioxidative properties of seaweed were screened and investigated for its hepatoprotective activity in rat. There was no significant induction of GSTYa1, GSTYa2, and CYP1A2. However, an M. nitidum diet was found to significantly increase UGT1A1 and UGT1A6 mRNA levels and to decrease CYP1A1 mRNA levels in rat liver. Structural studies confirmed the presence of sulfated polysaccharides in the seaweed samples. The results demonstrate the potential of seaweed as a natural source of sulfated polysaccharide substances with potential use in chemoprevention medicine.

Traditional Small-Size Citrus from Taiwan: Essential Oils, Bioactive Compounds and Antioxidant Capacity.

Background: The calamondin (Citrus microcarpa Bunge) and the kumquat (Fortunella crassifolia Swingle) are two small-size citrus fruits that have traditionally been consumed in Taiwan; however, there has been a lack of scientific research regarding the active compounds and functionalities of these fruits. Methods: Analysis of volatile composition of essential oil and phytosterol was carried out using Gas Chromatography-Mass Spectrometry (GC-MS). Flavonoid and limonoid were analyzed by High Performance Liquid Chromatography (HPLC). Moreover, antioxidant capacity from their essential oils and extracts were assessed in vitro. Results: The compositions of the essential oils of both fruits were identified, with the results showing that the calamondin and kumquat contain identified 43 and 44 volatile compounds, respectively. In addition, oxygenated compounds of volatiles accounted for 4.25% and 2.04%, respectively, consistent with the fact that oxygenated compounds are generally found in high content in citrus fruits. In terms of flavonoids, the calamondin exhibited higher content than the kumquat, with disomin-based flavonoids being predominant; on the other hand, phytosterol content of kumquat was higher than that of calamondin, with amyrin being the dominant phytosterol. Both of them contain high amounts of limonoids. The ethanol extracts and essential oils of small-sized citrus fruits have been shown to have antioxidant effects, with those effects being closely related to the flavonoid content of the fruit in question. Conclusions: The present study also reviewed antioxidant activity in terms of specific bioactive compounds in order to find the underlying biological activity of both fruits. The calamondin and kumquat have antioxidant effects, which are in turn very important for the prevention of chronic diseases.

Macrophages in oxidative stress and models to evaluate the antioxidant function of dietary natural compounds.

Antioxidant testing of natural products has attracted increasing interest in recent years, mainly due to the fact that an antioxidant-rich diet might provide health benefits. Activated macrophages are a major source of reactive oxygen species, reactive nitrogen species, and peroxynitrite generated through the so-called respiratory burst. Constitutively released proinflammatory cytokine, especially tumor necrosis factor-α, triggers nuclear factor-κB, and activator protein-1 translocation leading to the over production of reactive oxygen species and reactive nitrogen species in macrophages. Activation of transcription factors in the long-lived tissue-resident macrophages and/or monocyte-derived macrophages, trigger epigenetic modifications leading to the pathogenesis of chronic diseases. Nutraceuticals including lipid raft structure disruption agent, cholesterol depletion agent, farnesyltransferase inhibitor, nuclear factor-κB blocker (α,ß-unsaturated carbonyl compounds), glucocorticoid receptor agonist, and peroxisome proliferator-activated receptor-γ agonist have long been used to inactive macrophage. The inhibition effects on the formation of nitric oxide, superoxide, and nitrite peroxide may be responsible for the anti-inflammatory functionalities. Activated macrophage models could be used to identify the active components for functional diets development through a multiple targets strategy.

Pyrrolidine dithiocarbamate inhibition of luteolin-induced apoptosis through up-regulated phosphorylation of Akt and caspase-9 in human leukemia HL-60 cells.

Previously, we observed that luteolin effectively inhibited cell growth and induced apoptosis in HL-60 cells. In that study, we also explored the modulatory effects and molecular mechanisms of pyrrolidine dithiocarbamate (PDTC) on the cytotoxicity of luteolin to HL-60 cells. In this study, we found that PDTC was able to inhibit luteolin-induced cell apoptosis in a dose-dependent manner. When HL-60 cells were treated with PDTC for 0.5 h before 60 microM luteolin treatment, the DNA ladder disappeared. Moreover, flow cytometry showed that PDTC had dose dependently decreased the percentage of apoptotic HL-60 cells and had not interfered with luteolin's ability to change the mitochondrial membrane potential or its ability to trigger the release of cytochrome c to cytosol. Detection by Western blotting, however, did show that PDTC had interfered with luteolin's ability to cleave poly(ADP-ribose)polymerase and DNA fragmentation of factor-45. Three hours after the PDTC-pretreated HL-60 cells were treated with 60 microM luteolin, the product cleaved from Akt started to appear. Therefore, not only was PDTC able to stop the apoptosis of HL-60 cells treated with luteolin, it was also found to increase phosphorylation of Akt and caspase-9. These results suggest that in the luteolin-induced apoptotic pathway, phosphorylation of procaspase-9 by survival signals might play an important role in the ultimate fate of HL-60 cells.

Reduction of histamine and biogenic amines during salted fish fermentation by Bacillus polymyxa as a starter culture.

Bacillus polymyxa D05-1, isolated from salted fish product and possessing amine degrading activity, was used as a starter culture in salted fish fermentation in this study. Fermentation was held at 35°C for 120 days. The water activity in control samples (without starter culture) and inoculated samples (inoculated with B. polymyxa D05-1) remained constant throughout fermentation, whereas the pH value rose slightly during fermentation. Salt contents in both samples were constant in the range of 17.5-17.8% during the first 60 days of fermentation and thereafter increased slowly. The inoculated samples had considerably lower levels of total volatile basic nitrogen (p < 0.05) than control samples at each sampling time during 120 days of fermentation. Aerobic bacterial counts in inoculated samples were retarded during the first 60 days of fermentation and thereafter increased slowly, whereas those of control samples increased rapidly with increased fermentation time. However, the aerobic bacterial counts of control samples were significantly higher (p < 0.05) than those of inoculated samples after 40 days of fermentation. In general, overall biogenic amine contents (including histamine, putrescine, cadaverine, and tyramine) in the control samples were markedly higher (p < 0.05) than those of the inoculated samples throughout fermentation. After 120 days of fermentation, the histamine and overall biogenic amine contents in the inoculated samples were reduced by 34.0% and 30.0%, respectively, compared to control samples. These results emphasize that the application of starter culture with amines degrading activity in salted fish products was effective in reducing biogenic amine accumulation.

Determination of histamine in milkfish stick implicated in food-borne poisoning.

An incident of food-borne poisoning causing illness in 37 victims due to ingestion of fried fish sticks occurred in September 2014, in Tainan city, southern Taiwan. Leftovers of the victims' fried fish sticks and 16 other raw fish stick samples from retail stores were collected and tested to determine the occurrence of histamine and histamine-forming bacteria. Two suspected fried fish samples contained 86.6 mg/100 g and 235.0 mg/100 g histamine; levels that are greater than the potential hazard action level (50 mg/100 g) in most illness cases. Given the allergy-like symptoms of the victims and the high histamine content in the suspected fried fish samples, this food-borne poisoning was strongly suspected to be caused by histamine intoxication. Moreover, the fish species of suspected samples was identified as milkfish (Chanos chanos), using polymerase chain reaction direct sequence analysis. In addition, four of the 16 commercial raw milkfish stick samples (25%) had histamine levels greater than the US Food & Drug Administration guideline of 5.0 mg/100 g for scombroid fish and/or products. Ten histamine-producing bacterial strains, capable of producing 373-1261 ppm of histamine in trypticase soy broth supplemented with 1.0% L-histidine, were identified as Enterobacter aerogenes (4 strains), Enterobacter cloacae (1 strain), Morganella morganii (2 strains), Serratia marcescens (1 strain), Hafnia alvei (1 strain), and Raoultella orithinolytica (1 strain), by 16S ribosomal DNA sequencing with polymerase chain reaction amplification.

Organization and promoter analysis of the zebrafish (Danio rerio) interferon gene.

Interferon plays important roles in confronting viral infections as the first line of defense. For the purpose of understanding the molecular mechanism which controls transcription of the interferon gene, we cloned and sequenced the interferon promoter region of the zebrafish interferon gene and characterized its activity using firefly luciferase transient transfection expression assays. Different fragments of the zebrafish interferon 5'-flanking region were transfected into ZFL cells. In these cell lines, maximum promoter activity was located in 2.2 kb of the zebrafish interferon 5' flanking region of the ZFL cell line. In this study, we investigated whether the viral replicative intermediate double-stranded RNA (herein we used synthetic polyinosinic-polycytidylic acid [poly(I):poly(C)] modifies the effects of interferon on gene expression. For this purpose, all zebrafish interferon promoter fragments were treated with either 1, 10, or 100 microg/ml poly(I):poly(C). The results showed that after treatment with 10 microg/ml poly(I):poly(C), high promoter activity was observed in the -2.2-kb interferon promoter fragment. Several putative transcription factors were shown in the promoter region, including IRF-1, C/EBP, NFkappaB, and GATA-1. Further study of the in vivo expression of the interferon promoter during development was carried out in transgenic zebrafish expressing an interferon promoter-driven green fluorescent protein (GFP) encoding the GFP cDNA transgene. Morphological studies of transgenic zebrafish indicated that the interferon promoter-driven GFP transcripts appeared in the yolk, head, and lymphoid organs. These results indicate that the interferon promoter is active in a tissue-specific manner, and suggest that the interferon promoter plays an important role in virus resistance during teleost growth.