Development and application of fluorescence in situ hybridization (FISH) method for simple and rapid identification of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella in cultured and natural seawater

ABSTRACT:   The toxic dinoflagellates Alexandrium tamarense (Lebour) Balech and A. catenella (Whedon and Kofoid) Balech produce potent neurotoxins, such as saxitoxin and gonyautoxin and have been mainly responsible for paralytic shellfish poisoning (PSP) in Japan. To prevent a negative effect on the fishery industry, it is necessary to identify these toxic species precisely and rapidly before and during the bloom. In this paper, a rapid and simple protocol of a fluorescence in situ hybridization (FISH) method using ribosomal RNA (rRNA)-targeted probes has been established for identifying the cultured strains and natural cells of A. tamarense and A. catenella . Using the FISH method established in this study, it was possible to identify these toxic species species-specifically and rapidly, within 30 min. The procedure of detection constituted three steps: (i) fixation/dehydration; (ii) hybridization; and (iii) washing; this made the identification simple. Moreover, this method did not require either special techniques or equipment, and the cost for detection was low. The specificity, rapidity, and simplicity of the developed method suggest that it might be useful for routine monitoring of these toxic microalgae.     

Proportions of melanocyte stimulating hormone-immunoreactive cells in the adenohypophysis of Silky fowl and hyperpigmentation-free cockerels

Alpha‐melanocyte stimulating hormone (α‐MSH) is one of the main regulators for melanocytes, and the adenohypophysis is one of the major tissues for the synthesis of this hormone. The Silky fowl is a characteristic breed of chicken with hyperpigmentation throughout the body. The involvement of the adenohypophysis in the hyperpigmentation of this breed is not known. In the present study, the proportion of melanocyte stimulating hormone‐immunopositive cells (MSH cells) in the adenohypophysis was immunocytochemically compared between Silky, Red Cornish × New Hampshire (RN) crossbred and Japanese bantam cockerels at 15 weeks of age. After the body and gland were weighed, the adenohypophyseal cells were enzymatically dispersed and immunostained for α‐MSH, and the immunopositive MSH cells were counted. The weights of the body and adenohypophysis were heaviest in the RN crossbred, followed by the Silky and lightest in the bantam cockerels. In contrast, the ratio between adenohypophysis and bodyweight was much larger in the Silky and the bantam than in the RN crossbreed (P < 0.05). The population of MSH cells in the adenohypophysis was larger in the Silky (14.3%) than in the RN crossbreed (8.0%) and the bantam (8.1%) cockerels (P < 0.05). From these results, it was concluded that prepubertal Silky cockerel have numerous MSH cells in the adenohypophysis suggesting a relationship to hyperpigmentation.     

Taurine transporter from the giant Pacific oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>: function and expression in response to hyper- and hypo-osmotic stress

ABSTRACT:   Taurine is the primary osmolyte in marine molluscs, whose cellular osmo-conforming process is vital for environmental adaptation because of a lack of osmotic homeostasis. Here, cDNA cloning and expression, and functional analyses of taurine transporter (TAUT) from the giant Pacific oyster are reported on. The deduced amino-acid sequence of oyster TAUT (oyTAUT) showed 47–51% identity to those of vertebrate TAUT, whereas identity among the vertebrates is 78–95%. Functional analysis of oyTAUT expressed in Xenopus oocytes revealed that oyTAUT has a lower affinity and specificity for taurine and a requirement for higher NaCl concentration, compared with vertebrate TAUT. Taken together with similar functional properties of TAUT from mussel, indicated by our previous study, it is possible that these functional features reflect the internal environment of the molluscs (i.e. higher taurine and NaCl concentrations). Oyster taurine transporter mRNA expression was induced by not only hyper-osmotic stress, similar to other TAUT, but also hypo-osmotic stress. It is speculated that the expression in response to hypo-osmotic stress was induced by a substantial decrease in tissue taurine content following the decrease in the internal osmolality.     

Numbers of nitrate-reducing bacteria in the rumen as estimated by competitive polymerase chain reaction

Cell numbers of known species of nitrate- and nitrite-reducing bacteria, Selenomonas ruminantium, Veillonella parvula and Wollinella succinogenes , in the rumen of goats (25–30 kg) were estimated by competitive polymerase chain reaction (PCR). The number of S. ruminantium was the largest of the three species examined, and tended to be greater in goats fed a high-concentrate diet (5.6 × 10⁷ cells/mL rumen fluid) than in goats fed a high-roughage diet (1.3 × 10⁷ cells/mL). The number of V. parvula tended to be greater when goats were fed a high-roughage diet (6.7 × 10³/mL) than when fed a high-concentrate diet (3.2 × 10³/mL). The number of W. succinogenes was below the detectable level (< 1.0 × 10²/mL) when a high-concentrate diet was fed, but was significantly increased by feeding a high-roughage diet (1.6 × 10³/mL). Addition of potassium nitrate (6 g/day) to the high-concentrate diet tended to increase V. parvula , and significantly increased W. succinogenes , indicating that these two bacteria can be increased by feeding a diet containing nitrate.     

Screening of extracts of Japanese woods for melanin biosynthesis inhibition

The effects of 120 methanol extracts prepared from bark and heartwood of 69 types of Japanese wood on the melanin production of B16 melanoma cells were examined. The melanin content of B16 melanoma cells was determined spectrophotometrically at 405nm. The extracts were also examined for their effects on cell viability. We found that the methanol extracts of Fagus crenata (buna, wood, 100μg/ml), Sapium sebiferum (Nankinhaze, wood, bark, 10μg/ml), and Zelkova serrata (keyaki, wood, 10μg/ml) greatly inhibited the melanin production of B16 melanoma cells without significant cytotoxicity. However, these extracts did not inhibit tyrosinase activity at the concentration of 100μg/ml. These findings indicate that the depigmenting mechanism of these extracts involves the suppression of some pigmenting signals in stimulating melanogenesis rather than the inhibition of tyrosinase activity. Part of this study was presented at the 53rd Annual Meeting of the Japan Wood Research Society, Fukuoka, Japan, March 2003     

Effects of chronic acid fog exposure with soil acidification or nitrogen loading on nutrient status in Abies firma seedlings

To clarify the nutrient status in momi fir (Abies firma Sieb. et Zucc.) seedlings under complex stress conditions of acid fog with soil acidification or nitrogen loading, we exposed seedling shoots to simulated acid fog (pH 3) and simultaneously loaded the soil with acid or excess nitrogen for 17 months. Proton and nitrogen loading reduced K concentration in soil, but these treatments had little effect on the nutrient status of fine roots in momi fir seedlings. Acid fog exposure resulted in reduced concentrations of Mg, Al, and Fe in current-year needles and Mg, Al, Cu, Fe, and Zn in 1-year-old needles. The complex effects of acid fog exposure with proton or nitrogen loading on nutrient status were relatively slight. However, elements such as Al and Cu notably reflect the effects of proton loading. These results show that the nutrient status of trace elements, rather than major elements, in needles of momi fir seedlings is sensitive to exposure to acid fog or proton loading.     

Changes in the predicted function of the rumen bacterial community of Japanese Black beef cattle during the fattening stages according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses

We investigated changes in the predicted functions of the rumen bacterial community in Japanese Black beef cattle during fattening. Nine cattle were fed a high-concentrate diet during the early, middle, and late fattening stages consecutively (10–14, 15–22, and 23–30 months of age, respectively). The rumen fluid and solid samples collected at each stage were subjected to sequencing analyses. The sequencing results were clustered and classified into operational taxonomic units (OTUs). Representative sequences and a raw counting table for each OTU were submitted to the Piphillin website. The predicted functions were revealed by the Kyoto Encyclopedia of Genes and Genomes database as the ratio of the total sequence. In the early stage, “Biosynthesis of secondary metabolites” was significantly higher in the fluid fraction than in the solid fraction. “Two-component system” in the middle stage was significantly lower and “Purine metabolism” in the late stage was significantly higher in the fluid fraction than those in the solid fraction. The fluid fraction was significantly correlated with acetic acid, propionic acid, and bacterial metabolism, such as “Biosynthesis of secondary metabolites” and “Sugar metabolism.” Moreover, the solid fraction was correlated with “Purine metabolism” and “Biosynthesis of secondary metabolism”. These results suggest that the rumen bacterial community in Japanese Black beef cattle adapts to changes in rumen conditions by altering their functions in response to a long-term high-grain diet.     

Very low vitamin C activity of orally administered L-dehydroascorbic acid.

The biological activity of L-dehydroascorbic acid (DHA), which is easily formed from L-ascorbic acid (ASC) during storage and cooking processes, has been considered to be equivalent to that of ASC on the basis of studies made several decades ago, when a specific method to determine ASC was not available. The nutritional activity of orally ingested DHA has now been evaluated by comparing ASC concentrations in 12 tissues of rats administered four different doses of ASC. Determinations were made by using the specific and sensitive method, which had been developed by us. Here it is shown that the efficiency of DHA was almost 10% of that of ASC on a molar basis, based on animal experiments using the inherently scorbutic ODS rat, which is a convenient human model animal to investigate the metabolism of vitamin C. On the basis of these findings, it is proposed that it is necessary to reevaluate the nutritional requirement of vitamin C based on both ASC and DHA contents of foods.     

Evaluation of biological activities of extracts from the fruiting body of <Emphasis Type="Italic">Pleurotus citrinopileatus</Emphasis> for skin cosmetics

Pleurotus citrinopileatus Singer has recently become a popular delicacy in East Asian countries. We prepared a methanol extract, soluble fractions from the methanol extract, and a hot water extract of the fruiting bodies of P. citrinopileatus. The biological activities such as melanin biosynthesis inhibition, tyrosinase inhibition, antioxidant activities [1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, oxygen radical absorbance capacity (ORAC), and superoxide dismutase (SOD)-like activity], antibacterial activities, and antihyaluronidase activities of these extracts were evaluated. We found that the n-hexanesoluble, diethyl ether-soluble, and ethyl acetate-soluble fractions exhibited melanin biosynthesis inhibition in B16 melanoma cells, as well as antioxidant (ORAC) and antibacterial activities. However, the n-butanol-soluble and water-soluble fractions and the methanol and hot water extracts exhibited antioxidant (DPPH radical scavenging, SOD-like activity) and antihyaluronidase activities. These results indicate that the fruiting bodies of P. citrinopileatus have the potential to be used as an ingredient in skin cosmetics.     

Specialized inhibitory synaptic actions between nearby neocortical pyramidal neurons

We found that, in the mouse visual cortex, action potentials generated in a single layer-2/3 pyramidal (excitatory) neuron can reliably evoke large, constant-latency inhibitory postsynaptic currents in other nearby pyramidal cells. This effect is mediated by axo-axonic ionotropic glutamate receptor-mediated excitation of the nerve terminals of inhibitory interneurons, which connect to the target pyramidal cells. Therefore, individual cortical excitatory neurons can generate inhibition independently from the somatic firing of inhibitory interneurons.