GAMBIERDISCUS YASUMOTOI SP. NOV. (DINOPHYCEAE), A TOXIC BENTHIC DINOFLAGELLATE FROM SOUTHEASTERN ASIA

A new, toxin-producing, benthic dinoflagellate named Gambierdiscus yasumotoi sp. nov. was isolated from macroalgae from the fringing coral reef surrounding the Singapore island of Pulau Hantu. The plate formula of G. yasumotoi is P_o , 3', 7", 6c, 6s, 5"', and 2"". Gambierdiscus yasumotoi has a globular shape and is not anterior-posteriorly compressed as are the two other known species in this genus, G. toxicus Adachi et Fukuyo 1979 and G. belizeanus Faust 1995. The girdle descends one to two girdle widths without overhang in contrast to the ascending girdles of G. toxicus and G. belizeanus. The dimensions of cultured G. yasumotoi were 45–63 μm in length, 38–50 μm in transdiameter and 43–61 μm in dorsoventral diameter. The thecae are smooth without areolae. The apical pore plate has the characteristic fishhook shape of Gambierdiscus, but it is significantly longer than G. toxicus. Cells from young cultures of G. yasumotoi are positively phototropic.     

Ecdysteroids influence the circadian system timing ecdysis in the insectRhodnius prolixus (Hemiptera)

Summary 20-hydroxyecdysone (20HE) injections induced transient delays in the time of ecdysis inRhodnius prolixus reared in L/D cycles. Sustained phase delays in the ecdysis rhythm were revealed by transfer to constant dark during the scotophase following 20HE injection. The magnitude of the phase delays depended on the time in the L/D cycle at which 20HE was injected with major delays occurring at times when the endogenous titre is declining. Therefore the increases and decreases in the endogenous titre which are themselves timed in a circadian fashion may be involved in phase setting the ecdysis rhythm to the environmental cycle. Populations maintained in LL which are arrhythmic with respect to both ecdysteroid titres and ecdysis, can be induced to display gated ecdysis by injection of either 20HE or antiserum to ecdysteroids. Multiple injections of 20HE or antiserum are capable of inducing an ecdysis rhythm whose period (22.3 h) and gate location are very similar to that produced by altering the environmental cycle. Therefore manipulations of the endogenous titre of ecdysteroids can mimic the effects of L/D cycles on the timing of ecdysis. Ecdysis inRhodnius may therefore be timed at least partially as a result of circadian timing of the ecdysteroid titre.Abbreviations AZT Arbitrary Zeitgeber Time - DD constant darkness - LL constant light - L/D 24 h light dark cycle - 12L/12D 12 h of light 12 h of dark - 20HE 20-hydroxyecdysone     

EXOGENOUS STEROIDS AND GROWTH OF NEOSPONGIOCOCCUM SP. (CHLOROPHYTA)1

Of the various types of steroids found in nature, only sterols (steroids whose molecules possess an 8- to 10-carbon atom hydrocarbon side chain at position 17 of the perhydrocyclopentanophenanthrene ring) are known to be common constituents of algae. Little is known of the effects of steroids in the environment upon the growth and survival of algae. This paper investigates the growth of the green alga Neospongiococcum sp. in medium containing steroids. Bile acids are not inhibitory, even at a concentration of 100 ppm. Some sterols inhibit growth when present in the medium at a concentration of 100 ppm, but not at 10 ppm. Testosterone and β-estradiol, which have no carbon atom side chain at ring position 17, inhibit growth at a concentration of only 10 ppm. Steroids whose molecules possess a 2-carbon atom side chain at ring position 17 and a keto group at the α-carbon of this side chain, such as pregnenolone, inhibit growth at a concentration of as little as 1 ppm. Respiration is also inhibited by pregnenolone at this level .     

SPORE RELEASE IN ACROCHAETIUM SP. (RHODOPHYTA) IS BACTERIALLY CONTROLLED1

The facultative red algal epiphyte Acrochaetium sp. liberated spores preferentially and recruited more successfully in laboratory cultures when its host Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira was present. The same effect was also induced by cell‐free medium from G. chilensis, suggesting it contained a molecular signal. Antibiotics prevented spore release in Acrochaetium sp., even when G. chilensis was present, suggesting a prokaryotic origin of the signal. Simultaneous application of N‐butyl‐homoserine‐lactone (BHL) restored the spore‐release capacity, which demonstrated that spore release was not directly inhibited by the antibiotics and indicated that bacterially generated N‐acyl‐homoserine‐lactones (AHLs) regulate spore release. An involvement of AHL was further indicated by the fact that two different halofuranone inhibitors of AHL receptors also inhibited spore release when they were applied at relatively low concentrations. Of seven different AHLs tested, only BHL induced the effect. However, BHL was only active at relatively high concentrations (100 μM), and it was not detected in spore‐release‐inducing medium of G. chilensis. Another water‐soluble AHL or an AHL structure analog is therefore probably the active compound in G. chilensis cultures. The data presented demonstrate that life cycle completion in Acrochaetium sp. strongly depends on bacteria, which are not always present in sufficient numbers on the alga itself. Exogenous bacteria that are associated with G. chilensis or with other potential substrates may therefore trigger timely spore liberation in Acrochaetium sp., provided that the necessary concentration of AHL is reached. This first finding of AHL perception in a red alga confirms that AHL signalling is more widespread among eukaryotes than was thought until recently. However, spore release of a second red alga, Sahlingia subintegra (Rosenv.) Kornmann, was unaffected by AHL, and the reaction observed is therefore not universal.     

Moulting of a deep-sea galatheid crab (Anomura, Galatheidae) at a depth of 3572 m

The moulting of the deep-sea galatheid crab, Mundopsissp. was observed using a seafloor observatory at a depth of 3572 m in Nankai Trough, western Japan. The duration of the ecdysis was 147 seconds. The crab rested on the substratum until ecdysis was complete. After ecdysis, it flicked and moved away from its exuviae. The newly moulted galatheid crab did not consume its cast exoskeleton.     

CELL DIVISION IN THE DINOFLAGELLATE GAMBIERDISCUS TOXICUS IS PHASED TO THE DIURNAL CYCLE AND ACCOMPANIED BY ACTIVATION OF THE CELL CYCLE REGULATORY PROTEIN,CDC2 KINASE1

The cell division cycle in several pelagic dinoflagellate species has been shown to be phased with the diurnal cycle, suggesting that their cell cycle may be regulated by a circadian clock. In this study, we examined the cell cycle of an epibenthic dinoflagellate, Gambierdiscus toxicus Adachi and Fukuyo (Dinophyceae), and found that cell division was similarly phased to the diurnal cycle. Cell division occurred during a 3-h window beginning 6 h after the onset of the dark phase. Cell cycle progression in higher eukaryotes is regulated by a cell cycle regulatory protein complex consisting of cyclin and the cyclin-dependent kinase CDC2. In this report, we identified a CDC2-like kinase in G. toxicus that displays activity in vitro against a known substrate of CDC2 kinase, histone H1. As in higher eukaryotes, CDC2 kinase was expressed constitutively in G. toxicus throughout the cell cycle, but it was activated only late in the dark phase, concurrent with the presence of mitotic cells. These results indicate that cell division in G. toxicus is regulated by molecular controls similar to those found in higher eukaryotes.     

Factors affecting vesicle formation and acetylene reduction (nitrogenase activity) in Frankia sp. CpI1

Vesicle formation and acetylene reduction (nitrogenase activity) were observed when washed hyphae from cultures of Frankia sp. CpI1 were transferred to a nitrogen-free medium containing ethylenediaminetetraacetic acid and succinate. Succinate could be replaced by malate or fumarate, but not other carbon sources. Maximum acetylene reduction and vesicle numbers were observed at a pH of 6.0-6.5, at 25-30 degrees Centigrade, and at atmos pheric Po2 or somewhat less (5-20 kPa). Addition of 1 mM NH4Cl almost completely inhibited vesicle formation and acetylene-reducing activity, but did not immediately inhibit such reducing activity by cultures with preexisting vesicles. Acetylene-reducing activity was never observed in the absence of vesicle formation.     

IMMUNOLOCALIZATION OF AN α-1,4-GLUCAN LYASE IN YOUNG AND MATURE PARTS OF THE RED ALGA GRACILARIOPSIS SP. (RHODOPHYTA)

The enzyme α-1,4-glucan lyase (EC 4.2.2.13) was studied in cells of young and mature parts of the red alga Gracilariopsis sp. by using immunogold labeling in ultrastructural studies. In young tissues, the α-1,4-glucan lyase was observed at two different sites: around the starch granules in the cytosol and in the stroma of the chloroplast. In mature tissues, the α-1,4-glucan lyase was present only in the chloroplasts. The possible role of this starch-degrading enzyme in red algae is discussed.     

On the stability of the ripple phase in the DPPC/PLPC/water ternary system

The effect of incorporation of 1-palmitoyl-sn-glycero-3-phosphocholine (PLPC) on the structure of the P_β′ ripple mesophase in aqueous dispersions of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) has been studied by differential scanning calorimetry (DSC) and scanning dilatometry (SD). For samples containing 34 wt. % ²H₂O and 0–15 wt. % PLPC, a pretransition was observed by DSC. The pretransition disappears at 15 wt. % PLPC. The behavior of thermodynamic functions at the pretransition and main transition gives new insights on the structural changes produced by PLPC on bilayers of DPPC.     

Associated bacterial flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus

The growth, toxicity, and associated bacterial flora of 10 clonal cultures of the toxic benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus isolated from the coastal waters of southwest Puerto Rico have been examined. Clonal cultures of O. lenticularis grew more rapidly and at broader temperature ranges than those of G. toxicus. All five Ostreopsis clones were toxic, while only one of the five Gambierdiscus clones was poisonous. The degree of toxicity among poisonous clones was highly variable. The number of associated bacterial genera and their frequency of occurrence were quite variable among clones of both dinoflagellate genera. Bacterial isolates represented six genera (Nocardia, Pseudomonas, Vibrio, Aeromonas, Flavobacterium, and Moraxella) in addition to coryneform bacteria. Extracts of dinoflagellate-associated bacteria grown in pure culture were not toxic. Gambierdiscus clones were characterized by the frequent presence of Pseudomonas spp. (four of five clones) and the absence of coryneforms. In O. lenticularis, only one of five clones showed the presence of Pseudomonas spp., and Moraxella sp. was absent altogether. Detailed analyses of toxicity and associated microflora in a selected Ostreopsis clone, repeatedly cultivated (four times) over a period of 160 days, showed that peak cell toxicities developed in the late static and early negative culture growth phases. Peak Ostreopsis cell toxicities in the stationary phase of culture growth were correlated with significant increases in the percent total bacteria directly associated with these cells. Changes in the quantity of bacteria directly associated with microalgal cell surfaces and extracellular matrices during culture growth may be related to variability and degree of toxicity in these laboratory-cultured benthic dinoflagellates.     

Combined effects of aphidicolin and retinoic acid on proliferation and differentiation of human leukaemic (HL-60) cells

The relationships between replicative DNA synthesis and retinoic acid (RA)-induced differentiation of human promyelocytic leukaemic (HL-60) cells are evaluated with the use of Aphidicolin, a specific and reversible inhibitor of DNA polymerase alpha (alpha). Addition of a sublethal concentration of Aphidicolin (0.4 microM) in culture for 3 days suppresses DNA synthesis to a similar level of the resting stage (day 8) in control cultures. DNA synthesis is reactivated to the level observed in the growing stage of control cultures once Aphidicolin is removed after 3 days in culture. The level of DNA synthesis at the early stage of RA-induction (day 3) is suppressed by only 17% when compared to control cultures. The inhibitory effect of Aphidicolin on DNA synthesis in both control cultures and RA-induced cell cultures is similar. However, no reactivation of DNA synthesis is observed after removal of Aphidicolin on day 3 from RA-induced cell cultures. Flow cytometric analysis of DNA content on day 3 reveals that cells accumulate in G1 and early S phases of the cell cycle after exposure to Aphidicolin with or without RA. Of interest is the fact that, while Aphidicolin alone did not induce cells to differentiate, neither did it interfere with RA-induced cell differentiation (the rate of RA-induced cell differentiation in the presence of Aphidicolin is similar to that of RA-treated cultures in the absence of Aphidicolin). These results suggest that the combined use of Aphidicolin and RA may inhibit leukaemic cell proliferation more effectively without causing severe cytotoxicity and without interfering with RA-induced cell differentiation.     

Synergistic Interactions Between α1- and α2-Adrenergic Receptors in Activating 3H-Inositol Phosphate Formation in Primary Glial Cell Cultures

Norepinephrine (NE)-stimulated 3H-inositol phosphate (3H-InsP) formation in primary glial cell cultures is thought to be due to alpha 1-adrenergic receptor activation. Surprisingly, the alpha 1-selective agonists phenylephrine and methoxamine showed only 12-21% of the intrinsic activity of NE in activating this response. Although the alpha 2-selective agonist UK 14,304 was itself inactive, inclusion of UK 14,304 increased the response to the alpha 1-selective agonists by about threefold. This increase was concentration-dependent and occurred at all time points examined. 6-Fluoro-NE and alpha-methyl-NE mimicked the effect of NE in glial cultures, although with lower potencies. However, several partial agonists were ineffective in activating this response, in both the presence and absence of UK 14,304. Synergistic interactions were not observed for alpha 1-mediated responses in slices of rat cerebral cortex, either for formation of 3H-InsPs or potentiation of isoproterenol- or adenosine-stimulated cyclic AMP accumulation. Both UK 14,304 and phenylephrine inhibited NE-stimulated 3H-InsP formation in concentrations similar to those necessary to activate this response directly. These results suggest that NE activates 3H-InsP formation in primary glial cultures by synergistic actions on both alpha 1- and alpha 2-adrenergic receptors. The agonists UK 14,304 and phenylephrine also can act to inhibit the response to NE competitively.     

Protein-Lipopolysaccharide Complexes Produced by Virulent and Non-Virulent Isolates of Verticillium albo-atrum and V. dahliae are Non-Specific Toxins to Tomato and Potato

Toxic protein-lipopolysaccharide complexes (PLPC) were isolated from culture filtrates of Verticillium albo-atrum (isolate WCS 800) and V. dahliae (WCS 070) isolates, both virulent to tomato and potato cultivars, and from an isolate of,V. albo-atrum (V22W) which was non-virulent to these hosts. The virulent isolates each produced one major PLPC in culture and the non-virulent isolate produced two (fractions 1 and 2, characterized by their elution pattern after gel filtration). Virulence in these three isolates was not related to quantity of PLPC produced in culture. However, PLPC from the virulent isolates were toxic to tomato in a leaf bioassay at 4μg ml^-1 (WCS 800) and 20μg ml^-1 (WCS 070) but the two PL, PC from the non-virulent isolate required concentrations of 100 and 1000 μg ml^-1 for toxicity. Production of a modified, less toxic PLPC in V22W may partly account for its non-virulence. Gel filtration of PLPC from the three isolates on a calibrated Sephacryl S-400 column, eluted in phosphate buffer plus NaCl, indicated a compound of heterogeneous molecular mass with an average of 126,000 daltons for the PLPC of WCS 800, WCS 070 and, fraction 1 of V22W, and 25,000 daltons for fraction 2 of V22W. Attempts to extract a low molecular weight toxin from the PLPC by extended dialysis were unsuccessful. The susceptibility of 12 tomato and 19 potato cultivars to the Verticillium isolates was compared with their sensitivity to PLPC. Susceptibility was not correlated with toxin sensitivity and the PLPC were concluded to be non-specific toxins in these hosts.     

Aromatic Polyketide Synthases (Purification,Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits)

p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response.     

中国蝼蛄属研究及一新种记述(直翅目,蝼蛄科)

给出了中国蝼蛄属的检索表,并描述了该属1新种G.mabiana sp.nov..新种与尼泊尔种类 G.pygmaea相似,但可以通过如下特征加以区分:径脉末端不分岔,翅室呈三角形,阳茎基片横向骨片的侧端尖锐;此新种还与河南蝼蛄G.henana相似,区别为:新种前翅超过第5节背板,后翅到达第4节背板后缘,而河南蝼蛄G.henana前后翅均未伸达腹部第4节背板后缘;新种前胸背板无心纹斑,河南蝼蛄G.henana有;新种阳茎侧突囊弯钩状,后者为弯月形.     

Associated bacterial flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus.

The growth, toxicity, and associated bacterial flora of 10 clonal cultures of the toxic benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus isolated from the coastal waters of southwest Puerto Rico have been examined. Clonal cultures of O. lenticularis grew more rapidly and at broader temperature ranges than those of G. toxicus. All five Ostreopsis clones were toxic, while only one of the five Gambierdiscus clones was poisonous. The degree of toxicity among poisonous clones was highly variable. The number of associated bacterial genera and their frequency of occurrence were quite variable among clones of both dinoflagellate genera. Bacterial isolates represented six genera (Nocardia, Pseudomonas, Vibrio, Aeromonas, Flavobacterium, and Moraxella) in addition to coryneform bacteria. Extracts of dinoflagellate-associated bacteria grown in pure culture were not toxic. Gambierdiscus clones were characterized by the frequent presence of Pseudomonas spp. (four of five clones) and the absence of coryneforms. In O. lenticularis, only one of five clones showed the presence of Pseudomonas spp., and Moraxella sp. was absent altogether. Detailed analyses of toxicity and associated microflora in a selected Ostreopsis clone, repeatedly cultivated (four times) over a period of 160 days, showed that peak cell toxicities developed in the late static and early negative culture growth phases. Peak Ostreopsis cell toxicities in the stationary phase of culture growth were correlated with significant increases in the percent total bacteria directly associated with these cells. Changes in the quantity of bacteria directly associated with microalgal cell surfaces and extracellular matrices during culture growth may be related to variability and degree of toxicity in these laboratory-cultured benthic dinoflagellates.     

Inhibition of p56(lck) tyrosine kinase by isothiazolones

Lck encodes a 56-kDa protein-tyrosine kinase, predominantly expressed in T lymphocytes, crucial for initiating T cell antigen receptor (TCR) signal transduction pathways, culminating in T cell cytokine gene expression and effector functions. As a consequence of a high-throughput screen for selective, novel inhibitors of p56(lck), an isothiazolone compound was identified, methyl-3-(N-isothiazolone)-2-thiophenecarboxylate(A-125800), which inhibits p56(lck) kinase activity with IC50 = 1-7 microM. Under similar assay conditions, the isothiazolone compound was equipotent in blocking the ZAP-70 tyrosine kinase activity but was 50 to 100 times less potent against the catalytic activities of p38 MAP kinase and c-Jun N-terminal kinase 2alpha. A-125800 blocked activation-dependent TCR tyrosine phosphorylation and intracellular calcium mobilization in Jurkat T cells (IC50 = 35 microM) and blocked T cell proliferation in response to alloantigen (IC50 = 14 microM) and CD3/CD28-induced IL-2 secretion (IC50 = 2.2 microM) in primary T cell cultures. Inhibition of p56(lck )by A-125800 was dose- and time-dependent and was irreversible. A substitution of methylene for the sulfur atom in the isothiazolone ring of the compound completely abrogated the ability to inhibit p56(lck) kinase activity and TCR-dependent signal transduction. Incubation with thiols such as beta-ME or DTT also blocked the ability of the isothiazolone to inhibit p56(lck) kinase activity. LC/MS analysis established the covalent modification of p56(lck) at cysteine residues 378, 465, and 476. Together these data support an inhibitory mechanism, whereby cysteine -SH groups within the p56(lck) catalytic domain react with the isothiazolone ring, leading to ring opening and disulfide bond formation with the p56(lck) enzyme. Loss of p56(lck) activity due to -SH oxidation has been suggested to play a role in the pathology of AIDS. Consequently, a similar mechanism of sulfhydryl oxidation leading to p56(lck) inhibition, described in this report, may occur in the intact T cell and may underlie certain T cell pathologies.     

Kinetics and mechanism of dissimilative Fe(III) reduction by Pseudomonas sp. 200

The kinetics and mechanism of Fe(III) reduction to Fe(II) were studied in pure batch cultures of Pseudomonas sp. 200. The rate of iron reduction has been mechanistically related to aqueous phase iron speciation. In the absence of microbial activity the iron reduction rate was negligible. Initial rates of microbial iron reduction were accelerated more than 20-fold by the addition of equimolar quantities of nitrilotriacetic acid (NTA) to media initially containing 1.86 x 10(-3)M total Fe(III). Numerical techniques were utilized to quantify relationships between the observed rate of Fe(II) production and the calculated (equilibrium) aqueous phase speciation. These results indicate that soluble ferric iron species are not equivalent in terms of their susceptibility to bacterial (dissimilative) iron reduction. The concentration of Fe(NTA)(OH)(2) (2-) correlated strongly with observed iron reduction rates. Ferrous iron species appeared to inhibit the reduction process.     

Plasma 1-palmitoyl-2-linoleoyl phosphatidylcholine. Evidence for extensive phospholipase A1 hydrolysis and hepatic metabolism of the products

1-Palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) labeled in either the choline, glycerol, palmitate, or linoleate component in reconstituted rat high density lipoprotein (rHDL), was administered by vein to rats with bile fistula and taurocholate infusion. PLPC disappeared from plasma in a monoexponential fashion with a half-life of 50 min. A small fraction, about 14%, of PLPC disappearance was due to removal of linoleate from the sn-2 ester bond to form plasma cholesterol esters, presumably by lecithin-cholesterol acyltransferase. Otherwise, nearly all of the PLPC components that disappeared from blood in 1 h were recovered in the liver. The choline, glycerol, and linoleate components appeared predominantly in hepatic phosphatidylcholine (PC). These three components remained together in the liver with similar fractions of each in individual PC molecular species, most notably 1-stearoyl-2-linoleoyl-PC and dilinoleoyl-PC as well as PLPC. However, the palmitate component was spread among hepatic triglyceride, free fatty acid, other phospholipids, and all palmitate-containing molecular species of PC. Less than 2% of any administered PLPC component appeared in 1-stearoyl-2-arachidonyl-PC, the major species by mass in the liver. The palmitate component from plasma PLPC appeared in biliary PC at a more rapid rate than glycerol and linoleate components; the latter components appeared in bile in identical fashion. The results show that about two-thirds of plasma PLPC disappearance is due to phospholipase A1 hydrolysis, probably hepatic lipase. The putative produce, 2-linoleoyl-lysoPC, is efficiently reacylated with a saturated fatty acid in the liver, conserving PC.