Purification and cDNA cloning of the antimicrobial peptide apMolluscidin from the pen shell, Atrina pectinata.

A 5.6 kDa antimicrobial peptide (AMP) was purified from acidified gill extract of the pen shell, Atrina pectinata, by cation exchange and C18 reversed-phase high performance liquid chromatography. Comparison of the amino acid sequences and molecular weight of this peptide with those of other known AMPs revealed that it had high sequence homology with that of cgMolluscidin or hdMolluscidin; it was designated apMolluscidin. apMolluscidin comprises 59 amino acid residues containing several dibasic residue repeats and sequence repeats such as Lys-Lys and Lys-Gly. apMolluscidin exhibited potent antimicrobial activity against both Gram-positive bacteria including Bacillus subtilis (minimal effective concentration [MEC], 2.1 µg/mL), and Gram-negative bacteria including E. coli D31 (MEC, 0.5 µg/mL), without hemolytic activity. However, it did not show any activity against fungi such as Candida albicans. Secondary structure prediction suggested that it might form two helical regions and have an amphipathic structure. Full-length apMolluscidin cDNA contained 812 base pairs (bp), including a 5'-untranslated region (UTR) of 82 bp, a 3'-UTR of 547 bp, and a coding sequence of 183 bp encoding 60 amino acids (containing Met). Furthermore, qPCR analyses revealed that the mature peptide translated from apMolluscidin mRNA is expressed in a tissue-specific manner in locations such as the gill and siphon. These results indicate that apMolluscidin might be related to the innate immune defense system of abalone and may not act directly on the bacterial membrane. This is the first report of an AMP from the pen shell with a fully identified amino acid sequence.

The Edible Red Alga Porphyra yezoensis Promotes Neuronal Survival and Cytoarchitecture in Primary Hippocampal Neurons.

The edible red alga Porphyra yezoensis is among the most popular marine algae and is of economic and medicinal importance. In the present study, the neurotrophic and neuroprotective activities of the ethanol extract of P. yezoensis (PYE) were investigated in primary cultures of hippocampal neurons. Results revealed that PYE significantly increased neurite outgrowth at an optimal concentration of 15 µg/mL. PYE dose-dependently increased viable cells, significantly accelerated the rate of neuronal differentiation in cultures, promoted axodendritic arborization, and eventually induced synaptogenesis. In addition to morphological development, PYE also promoted functional maturation as indicated by the staining of live cultures with FM 1-43. Moreover, PYE increased neuronal survivability, which was attributed to reduced apoptosis and its ROS scavenging activity. Taurine, a major organic acid in PYE (2.584/100 mg of dry PYE) promoted neurite outgrowth in a dose-dependent manner, and this promotion was suppressed by the taurine antagonist isethionic acid. The study indicates that PYE and its active component, taurine, facilitate neuronal development and maturation and have a neuroprotective effect.

Conversion of red-algae Gracilaria verrucosa to sugars, levulinic acid and 5-hydroxymethylfurfural.

This study employed a statistical methodology to investigate the optimization of conversion conditions and evaluate the reciprocal interaction of reaction factors related to the process of red-algae Gracilaria verrucosa conversion to sugars (glucose, galactose), levulinic acid and 5-hydroxymethylfurfural (5-HMF) by acidic hydrolysis. Overall, the conditions optimized for glucose formation included a higher catalyst concentration than did those for galactose, and these conditions for galactose were similar to those for 5-HMF. Levulinic acid production, meanwhile, was optimized at a higher reaction temperature, a higher catalyst concentration, and a longer reaction time than was glucose, galactose or 5-HMF production. By this approach, the optimal yields (and reaction conditions) for glucose, galactose, levulinic acid, and 5-HMF were as follows: glucose 5.29 g/L (8.46 wt%) (reaction temperature 160 °C, catalyst concentration 1.92%, reaction time 20 min), galactose 18.38 g/L (29.4 wt%) (160 °C, 1.03%, 20 min), levulinic acid 14.65 g/L (18.64 wt%) (180.9 °C, 2.85%, 50 min), and 5-HMF 3.74 g/L (5.98 wt%) (160.5 °C, 1%, 20 min).

Comparison of Ecklonia cava, Ecklonia stolonifera and Eisenia bicyclis for phlorotannin extraction.

Phlorotannins are polyphenols of marine algae, particularly brown seaweed, having multiple biological activities. A reverse phase-high performance liquid chromatography method was developed for rapid and routine quantification of two major phlorotannins, dieckol and phlorofucofuroeckol-A (PFE-A), from boiling water- and organic solvent-extracts of brown seaweeds Ecklonia cava, E. stolonifera and Eisenia bicyclis. The regression equations for dieckol and PFE-A were as follows: the concentration (mg ml(-1)) = 16.56 x peak height (cm) + 0.44, and the concentration = 20.60 x peak height (cm) + 0.11, with correlation coefficients of 0.996 and 0.999, respectively. Compared to organic solvent extraction, the recovery yield of dieckol from boiling water extracts of E. cava, E. stolonifera and E. bicyclis was 86%, 93%, and 98%, respectively. The recovery yield of PFE-A was 74%, 86% and 62%, respectively. Antioxidant activity was detected in each E. bicyclis water extract (91%), followed by E. stolonifera (90%) and E. cava (74%). Dieckol and PFE-A showed almost 9- and 7-fold stronger antioxidant activity than the standard butylhydroxytoluene, and 6-and 4-fold greater than L-ascorbic acid in molar concentration, respectively.

The marine alga Gelidium amansii promotes the development and complexity of neuronal cytoarchitecture.

Neurotrophic factors are vital not only to support neuronal development but also to protect mature neurons from atrophy in neurodegenerative diseases. As an effort to explore natural sources that possess neurotrophic activity, we screened common marine algae for their neuritogenic activity in the developing rat hippocampal neurons in culture. Of the 22 seaweed species examined, ethanol extracts of Gelidium amansii (GAE) exhibited potent neuritogenic activity, followed by Undaria pinnatifida and Sargassum fulvellum extracts. The effects of GAE were dose dependent with an optimum concentration of 15 µg/mL. The GAE significantly promoted the initial neuronal differentiation from the stage I into the stage II and increased the indices of axonal and dendritic development such as the length, the numbers of primary processes, and branching frequencies by a minimum of twofold compared with the vehicle control. These results show that marine algae are promising candidates for neurotrophic potentials.

Isolation and structural determination of the antifouling diketopiperazines from marine-derived Streptomyces praecox 291-11.

Marine derived actinomycetes constituting 185 strains were screened for their antifouling activity against the marine seaweed, Ulva pertusa, and fouling diatom, Navicula annexa. Strain 291-11 isolated from the seaweed, Undaria pinnatifida, rhizosphere showed the highest antifouling activity and was identified as Streptomyces praecox based on a 16S rDNA sequence analysis. Strain 291-11 was therefore named S. praecox 291-11. The antifouling compounds from S. praecox 291-11 were isolated, and their structures were analyzed. The chemical constituents representing the antifouling activity were identified as (6S,3S)-6-benzyl-3-methyl-2,5-diketopiperazine (bmDKP) and (6S,3S)-6-isobutyl-3-methyl-2,5-diketopiperazine (imDKP) by interpreting the nuclear magnetic resonance and high-resolution mass spectroscopy data. Approximately 4.8 mg of bmDKP and 3.1 mg of imDKP were isolated from 1.2 g of the S. praecox 291-11 crude extract. Eight different compositions of culture media were investigated for culture, the TBFeC medium being best for bmDKP and TCGC being the optimum for imDKP production. Two compounds respectively showed a 17.7 and 21 therapeutic ratio (LC50/EC50) to inhibit zoospores, and two compounds respectively showed a 263 and 120.2 therapeutic ratio to inhibit diatoms.

Detoxification of acidic catalyzed hydrolysate of Kappaphycus alvarezii (cottonii).

Red seaweed, Kappaphycus alvarezii, holds great promise for use in biofuel production due to its high carbohydrate content. In this study, we investigated the effect of fermentation inhibitors to the K. alvarezii hydrolysate on cell growth and ethanol fermentation. In addition, detoxification of fermentation inhibitors was performed to decrease the fermentation inhibitory effect. 5-Hydroxymethylfurfural and levulinic acid, which are liberated from acidic hydrolysis, was also observed in the hydrolysate of K. alvarezii. These compounds inhibited ethanol fermentation. In order to remove these inhibitors, activated charcoal and calcium hydroxide were introduced. The efficiency of activated charcoals was examined and over-liming was used to remove the inhibitors. Activated charcoal was found to be more effective than calcium hydroxide to remove the inhibitors. Detoxification by activated charcoal strongly improved the fermentability of dilute acid hydrolysate in the production of bioethanol from K. alvarezii with Saccharomyces cerevisiae. The optimal detoxifying conditions were found to be below an activated charcoal concentration of 5%.

Comparison of sulfuric and hydrochloric acids as catalysts in hydrolysis of Kappaphycus alvarezii (cottonii).

In this study, hydrolysis of marine algal biomass Kappaphhycus alvarezii using two different acid catalysts was examined with the goal of identifying optimal reaction conditions for the formation of sugars and by-products. K. alvarezii were hydrolyzed by autoclave using sulfuric acid or hydrochloric acid as catalyst with different acid concentrations (0.1-1.0 M), substrate concentrations (1.0-13.5%), hydrolysis time (10-90 min) and hydrolysis temperatures (100-130 (°)C). A difference in galactose, glucose, reducing sugar and total sugar content was observed under the different hydrolysis conditions. Different by-product compounds such as 5-hydroxymethylfurfural and levulinic acid were also observed under the different reaction conditions. The optimal conditions for hydrolysis were achieved at a sulfuric acid concentration, temperature and reaction time of 0.2 M, 130 °C and 15 min, respectively. These results may provide useful information for the development of more efficient systems for biofuel production from marine biomass.

The Edible Seaweed Gelidium amansii Promotes Structural Plasticity of Hippocampal Neurons and Improves Scopolamine-Induced Learning and Memory Impairment in Mice.

BACKGROUND: Gelidium amansii has been gaining profound interest in East Asian countries due to its enormous commercial value for agar production and its extensive pharmacological properties. Previous studies have shown that the ethanol extract of Gelidium amansii (GAE) has promising neurotrophic effects in in vitro conditions. OBJECTIVES: The present study aimed at investigating the protective effects of GAE against scopolamine-induced cognitive deficits and its modulatory effects on hippocampal plasticity in mice. METHODS: For memory-related behavioral studies, the passive avoidance test and radial arm maze paradigm were conducted. The brain slices of the hippocampus CA1 neurons of experimental mice were then prepared to perform Golgi staining for analyzing spine density and its characteristic shape, and immunohistochemistry for assessing the expression of different pre- and postsynaptic proteins. RESULTS: Following oral administration of GAE (0.5 mg/g body weight), mice with memory deficits exhibited a significant increase in the latency time on the passive avoidance test and a decrease in the number of working and reference memory errors and latency time on the radial arm maze test. Microscopic observations of Golgi-impregnated tissue sections and immunohistochemistry of hippocampal slices showed that neurons from GAE-treated mice displayed higher spine density and spine dynamics, increased synaptic contact, and the recruitment of memory-associated proteins such as N-methyl-D-aspartate receptors (NR2A and NR2B) and postsynaptic density-95 (PSD-95) when compared with the control group. CONCLUSION: With these memory-protective functions and a modulatory role in underlying memory-related events, GAE could be a potential functional food and a promising source of pharmacological agents for the prevention and treatment of memory-related brain disorders.

The complete mitochondrial genome of Liparis ochotensis and a preliminary phylogenetic analysis.

Liparis ochotensis is a snailfish commonly confused with similar fish species because of unclear morphological characteristics. Moreover, molecular genetic studies have not been conducted for snailfish in Korea. Here, we report the complete mitogenome sequence of L. ochotensis, obtained via long PCR using universal primers for the fish mitogenome. The L. ochotensis mitogenome is 17,522 bp long, comprising 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and one control region. A neighbour-joining phylogenetic tree based on CO1 sequences depicted a close relationship with Liparis gibbus. The complete mitogenome is a valuable resource to classify and conserve L. ochotensis.

Gelidium amansii Attenuates Hypoxia/Reoxygenation-Induced Oxidative Injury in Primary Hippocampal Neurons through Suppressing GluN2B Expression.

Oxidative stress is known to be critically implicated in the pathophysiology of several neurological disorders, including Alzheimer's disease and ischemic stroke. The remarkable neurotrophic activity of Gelidium amansii, which has been reported consistently in a series of our previous studies, inspired us to investigate whether this popular agarophyte could protect against hypoxia/reoxygenation (H/R)-induced oxidative injury in hippocampal neurons. The primary culture of hippocampal neurons challenged with H/R suffered from a significant loss of cell survival, accompanied by apoptosis and necrosis, DNA damage, generation of reactive oxygen species (ROS), and dissipation of mitochondrial membrane potential (ΔΨm), which were successfully attenuated when the neuronal cultures were preconditioned with ethanolic extract of G. amansii (GAE). GAE also attenuated an H/R-mediated increase of BAX and caspase 3 expressions while promoting Bcl-2 expression. Moreover, the expression of N-methyl-d-acetate receptor subunit 2B (GluN2B), an extrasynaptic glutamate receptor, was significantly repressed, while synaptic GluN2A expression was preserved in GAE-treated neurons as compared to those without GAE intervention. Together, this study demonstrates that GAE attenuated H/R-induced oxidative injury in hippocampal neurons through, at least in part, a potential neuroprotective mechanism that involves inhibition of GluN2B-mediated excitotoxicity and suppression of ROS production, and suggests that this edible seaweed could be a potential source of bioactive metabolites with therapeutic significance against oxidative stress-related neurodegeneration, including ischemic stroke and neurodegenerative diseases.

The ethanol extract from the rhodophyta Gloiopeltis furcata and its active ingredient docosahexaenoic acid improve exercise performance in mice.

The effectiveness of natural bioresources at enhancing exercise performance is of interest to those in sports and health care. The use of 29 common seaweed species as supplements to enhance exercise performance and the recovery from physical fatigue was evaluated. The ethanol extract of the red seaweed Gloiopeltis furcata (GFE) had the greatest effect on forelimb grip strength and swimming endurance in mice. The optimal daily dose of GFE was 0.1 mg per 10 µl per g of body weight. GFE significantly increased muscle mass but had little effect on body weight and fatty deposits. The extract also significantly raised the blood superoxide dismutase and high-density lipoprotein cholesterol levels, while reducing the lactate and urea levels (p < 0.05). Docosahexaenoic acid (DHA) from GFE made the greatest contribution to improving physical exercise performance. These results support the use of GFE and DHA in health food products for enhancing physical performance. PRACTICAL APPLICATIONS: The study shows the exercise enhancement and anti-fatigue activities of GFE using the forelimb grip strength test, forced swimming endurance test, muscle mass measurement, and blood biochemical parameters. These results support the use of GFE and its active constituent DHA in functional foods or nutraceuticals for enhancing physical performance.

The hot water extract and active components nicotinamide and guanosine of the leather carp Cyprinus carpio nudis improve exercise performance in mice.

We identified the main active, exercise performance-enhancing compounds in a hot water extract of the leather carp, Cyprinus carpio nudus, as nicotinamide and guanosine. Mice were fed casein (30 mg/ml) enriched with nicotinamide (0.1 mg/ml) and guanosine (0.05 mg/ml) once daily for a week at 10 µl/g body weight. Swimming endurance (57%) and forelimb grip strength (21%) were increased significantly. The diet had little effect on body weight. After the swimming exercise, the blood glucose and superoxide dismutase levels were significantly higher (137% and 131%, respectively) than in the saline controls. The blood lactate level was 90% of that in the controls. The estimated amount of nicotinamide in the carp fillet was 26.2 mg/kg. These results suggest that the triple combination of casein with nicotinamide and guanosine improves exercise performance and delays the onset of fatigue, supporting the traditional use of carp extract in healthcare as a tonic soup. PRACTICAL APPLICATIONS: The triple-combination of casein (30 mg/ml) + nicotinamide (0.1 mg/ml) + guanosine (0.05 mg/ml) significantly enhanced the exercise performance and anti-fatigue in mice, supporting the traditional use of carp extract in healthcare as a tonic soup.

Protective effects of a mixed plant extracts derived from Astragalus membranaceus and Laminaria japonica on PTU-induced hypothyroidism and liver damages.

Protective effects of a mixed hot water extracts of Astragalus membranaceus (AWE) and Laminaria japonica (LWE), AWE: LWE 85:15 (g/g; AL mix), were investigated against propylthiouracil (PTU)-induced hypothyroidism in rats. Rats were challenged with PTU, resulting in, increased thyroid gland weight, decreased liver weight and antioxidant activities, reduced serum tri-iodothyronine and thyroxine levels with increased thyroid stimulating hormone levels, and elevated serum aspartate aminotransferase level. However, orally administered AL mix with 100, 200, and 400 mg kg-1  day-1 , significantly inhibited such abnormalities, dose-dependently. Moreover, PTU-induced abnormal histological architecture of the rat thyroid gland and liver were also significantly ameliorated by an AL mix. The results suggested that, therapeutic use of AL mix for treating hypothyroidism can be characterized by its diversified active ingredients particularly iodine and ferulic acid as confirmed by phytochemical analyses. PRACTICAL APPLICATIONS: The AL mix has synergistic effects in modulating thyroid hormone synthesis and preventing liver damages in PTU-induced hypothyroid rats. These effects of AL mix are mainly related to its richness specifically in iodine and ferulic acid. The growing interests of iodine and ferulic acid in AL mix are principally due to their beneficial effects in releasing sufficient thyroid hormones in hypothyroid conditions and promoting liver-protective functions through its antioxidant and anti-inflammatory potentials, respectively. Moreover, the results of AL mix are well-matched with the effects of standard drug levothyroxine in the present study. Therefore, appropriate dosage of AL mix will be promising as new medicinal food for preventing thyroid dysfunctions and its related liver damages.

Identification of the minimum region of flatfish myostatin propeptide (Pep45-65) for myostatin inhibition and its potential to enhance muscle growth and performance in animals.

Myostatin (MSTN) negatively regulates skeletal muscle growth, and its activity is inhibited by the binding of MSTN propeptide (MSTNpro), the N-terminal domain of proMSTN that is proteolytically cleaved from the proMSTN. Partial sequences from the N-terminal side of MSTNpro have shown to be sufficient to inhibit MSTN activity. In this study, to determine the minimum size of flatfish MSTNpro for MSTN inhibition, various truncated forms of flatfish MSTNpro with N-terminal maltose binding protein (MBP) fusion were expressed in E. coli and purified. MSTNpro regions consisting of residues 45-68, -69, and -70 with MBP fusion suppressed MSTN activity with a potency comparable to that of full-sequence flatfish MSTNpro in a pGL3-(CAGA)12-luciferase reporter assay. Even though the MSTN-inhibitory potency was about 1,000-fold lower, the flatfish MSTNpro region containing residues 45-65 (MBP-Pro45-65) showed MSTN-inhibitory capacity but not the MBP-Pro45-64, indicating that the region 45-65 is the minimum domain required for MSTN binding and suppression of its activity. To examine the in vivo effect of MBP-fused, truncated flatfish MSTNpro, MBP-Pro45-70-His6 (20 mg/kg body wt) was subcutaneously injected 5 times for 14 days in mice. Body wt gain and bone mass were not affected by the administration. Grip strength and swimming time were significantly enhanced at 7 d after the administration. At 14 d, the effect on grip strength disappeared, and the extent of the effect on swimming time significantly diminished. The presence of antibody against MBP-Pro45-70-His6 was observed at both 7 and 14 d after the administration with the titer value at 14 d being much greater than that at 7 d, suggesting that antibodies against MBP-Pro45-70-His6 neutralized the MSTN-inhibitory effect of MBP-Pro45-70-His6. We, thus, examined the MSTN-inhibitory capacity and in vivo effect of flatfish MSTNpro region 45-65 peptide (Pep45-65-NH2), which was predicted to have no immunogenicity in silico analysis. Pep45-65-NH2 suppressed MSTN activity with a potency similar to that of MBP-Pro45-65 but did not suppress GDF11, or activin A. Pep45-65-NH2 blocked MSTN-induced Smad2 phosphorylation in HepG2 cells. The administration of Pep45-65 (20 mg/kg body wt, 5 times for 2 weeks) increased the body wt gain with a greater gain at 14 d than at 7 d and muscle wt. Grip strength and swimming time were also significantly enhanced by the administration. Antibody titer against Pep45-65 was not detected. In conclusion, current results indicate that MSTN-inhibitory proteins with heterologous fusion partner may not be effective in suppressing MSTN activity in vivo due to an immune response against the proteins. Current results also show that the region of flatfish MSTNpro consisting of 45-65 (Pep45-65) can suppress mouse MSTN activity and increase muscle mass and function without invoking an immune response, implying that Pep45-65 would be a potential agent to enhance skeletal muscle growth and function in animals or to treat muscle atrophy caused by various clinical conditions.

Spinogenesis and Synaptogenesis Effects of the Red Seaweed Kappaphycus alvarezii and Its Isolated Cholesterol on Hippocampal Neuron Cultures.

Neurotrophic factors promote the formation of spines and synapses in neuron development and maintenance. Synaptic connections enhance memory in the brain. In this study, the effects of Kappaphycus alvarezii ethanolic extract (EKA) and its isolated cholesterol (iCHOL) on spinogenesis and synaptogenesis of hippocampal neurons were evaluated. Compared with the vehicle, both EKA and iCHOL significantly promoted generation of dendritic filopodia (2.4- and 2.2-fold, respectively) and spine (1.7- and 1.4-fold) formations in spinogenesis; they also increased presynaptic (3.6- and 2.6-fold), postsynaptic (2.5- and 2.9-fold), and cocolonized (3.8- and 3.0-fold) puncta, which enhances synaptic function (P< 0.05). Further, EKA- and iCHOL-treated neurons showed significantly improved functional presynaptic plasticity (1.6- and 1.4-fold, respectively, at 17 days in vitro; P<0.05). These results indicate that K. alverezii facilitates neuronal development, and support its use as a functional food to reduce neurological disorders and prevent brain aging via helping to reconstruct partially damaged neural networks.

Effects of the brown seaweed Undaria pinnatifida on erythematous inflammation assessed using digital photo analysis.

The brown seaweed Undaria pinnatifida (Harvey) Suringar produced potent inhibition of erythematous inflammation assessed using digital photo analysis. The analysis technique was validated by laser speckle flowgraphy and blood vessel contraction. The methanol extract suppressed erythema by 50% when applied within 1 h before or 15 min after application of phorbol myristate acetate. Erythema reduction to half-maximal values took 12 h with the extract, compared with 25 h with the vehicle. The blade part of the thallus showed the highest activity, while the northern type of U. pinnatifida had slightly higher activity than the southern type. The active constituents were stearidonic acid and eicosapentaenoic acid. These findings reinforce the claims of the health care industry and indigenous medicine that U. pinnatifida can be used as a health food and remedy for inflammation-related symptoms.

A methoxylated fatty acid isolated from the brown seaweed Ishige okamurae inhibits bacterial phospholipase A2.

A methoxylated fatty acid that inhibits phospholipase A(2) (PLA(2); EC 3.1.1.4) was purified from the brown seaweed Ishige okamurae. Approximately 8.1 mg of the inhibitory compound, 7-methoxy-9-methylhexadeca-4,8-dienoic acid, was isolated from 1 kg of I. okamurae powder. Recombinant PLA(2) derived from the pathogenic bacterium Vibrio mimicus was used as the target enzyme. The methoxylated fatty acid compound competitively inhibited PLA(2) with a Ki value of 3.9 microg/mL. The concentrations required for 50% inhibition of PLA(2), oedema and erythema were 1.0 microg/mL, 3.6 mg/mL and 4.6 mg/mL, respectively. The compound strongly inhibited PLA(2) activity in vitro and had potent antiinflammatory activity in vivo.

Application of the rpoS gene for species-specific detection of Vibrio vulnificus by real-time PCR.

Vibrio vulnificus is a causative agent of serious diseases in humans, resulting from the contact of wound with seawater or consumption of raw seafood. Several studies aimed at detecting V. vulnificus have targeted vvh as a representative virulence toxin gene belonging to the bacterium. In this study, we targeted the rpoS gene, a general stress regulator, to detect V. vulnificus. PCR specificity was identified by amplification of 8 V. vulnificus templates and by the loss of a PCR product with 36 non-V. vulnificus strains. The PCR assay had the 273-bp fragment and the sensitivity of 10 pg DNA from V. vulnificus. SYBR Green I-based real-time PCR assay targeting the rpoS gene showed a melting temperature of approximately 84 degrees for the V. vulnificus strains. The minimum level of detection by real-time PCR was 2 pg of purified genomic DNA, or 103 V. vulnificus cells from pure cultured broth and 103 cells in 1 g of oyster tissue homogenates. These data indicate that real-time PCR is a sensitive, species-specific, and rapid method for detecting this bacterium, using the rpoS gene in pure cultures and in infected oyster tissues.

Identification of a Taq DNA polymerase inhibitor from the red seaweed Symphyocladia latiuscula.

Two inhibitors of Taq DNA polymerase were isolated from the marine red alga Symphyocladia latiuscula. The inhibitors were purified by methanol extraction, molecular fractionation below 3000 MW and reverse-phase HPLC. The purified compound SL-1 containing three bromines was identified as 2,3,6-tribromo-4,5-dihydroxybenzyl alcohol (C7H5Br3O3: MW374) by NMR and MS analyses. The purified compound SL-2 was identified as 2,3, 6-tribromo-4,5-dihydroxybenzyl methyl ether(C8H7Br3O3: MW388). In a 25-microl reaction mixture containing 1.5 units of Taq DNA polymerase, the enzyme was completely inhibited by 0.5 microg SL-1 or 5 microg SL-2.