Variable inter and intraspecies alkaline phosphatase activity within single cells of revived dinoflagellates

Adaptation of cell populations to environmental changes is mediated by phenotypic variability at the single-cell level. Enzyme activity is a key factor in cell phenotype and the expression of the alkaline phosphatase activity (APA) is a fundamental phytoplankton strategy for maintaining growth under phosphate-limited conditions. Our aim was to compare the APA among cells and species revived from sediments of the Bay of Brest (Brittany, France), corresponding to a pre-eutrophication period (1940’s) and a beginning of a post-eutrophication period (1990’s) during which phosphate concentrations have undergone substantial variations. Both toxic marine dinoflagellate Alexandrium minutum and the non-toxic dinoflagellate Scrippsiella acuminata were revived from ancient sediments. Using microfluidics, we measured the kinetics of APA at the single-cell level. Our results indicate that all S. acuminata strains had significantly higher APA than A. minutum strains. For both species, the APA in the 1990’s decade was significantly lower than in the 1940’s. For the first time, our results reveal both inter and intraspecific variabilities of dinoflagellate APA and suggest that, at a half-century timescale, two different species of dinoflagellate may have undergone similar adaptative evolution to face environmental changes and acquire ecological advantages.Subject terms: Climate-change ecology, Microbial ecology     

A portable system for simultaneous measurement of transpiration and CO2 exchange

A portable field system for simultaneous measurement of transpiration and CO₂ exchange from leaves of fruit trees is described. CO₂ concentration is measured by means of infra-red gas analysis, using small gas samples collected in syringes. Methods for analysing small gas samples are compared. The leaf chamber described can also be used in a conventional laboratory open gas-exchange system, its small volume permitting measurement of very rapid changes in gas exchange in response to experimental stimuli.

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Resumen Este artículo describe un sistema portátil para medir simultaneamente transpiración e intercambio de CO₂ en hojas de árboles frutales en el campo. Para medir la velocidad de fotosíntesis se colectan pequeñas muestras de gas en jeringas y se inyectan al analizador de gases de luz infra-roja. Se comparan varios métodos para analizar las muestras. Se describe la cámara de absorción de CO₂ utilizada, la cual puede tambien incorporarse a un sistema abierto convencional, su pequeño volumen permite detectar cambios rápidos en el intercambio de gases cuando se producen alteraciones en el ambiente.

     

Chemical composition and antimicrobial activity of the essential oil of Cacalia tangutica (Maxim.) Hand.-Mazz

Essential oil of the subterranean part of Cacalia tangutica (Maxim.) Hand.-Mazz was analyzed by gas chromatography (GC)-mass spectrum (MS) tech-nique in two different capillary columns of different polar-ities. Thirty-one components were identified in the oil and the main compounds were a-zingiberene (13.49%), germa-crene D (10.76%), a-pinene (8.54%), caryophyllene(Z-) (6.36%), linalool (6.16%), β-myrcene (4.89%),β-ocimene (Z-) (4.40%)and ocimenone(Z-) (3.58%). The antimicro-bial activity of the oil was evaluated against 2 fungi and 12 bacteria including 6 clinically isolated strains using the agar disc diffusion and broth microdilution methods. The results show that the oil presented a broad antimicro-bial spectrum and had better antimicrobial activity against yeast and gram-positive bacteria. The minimum inhibitory concentration values were 0.16-5.00 g/L and minimum bactericidal concentration values were 0.16-5.00 g/L.     

Cytoskeletal changes and the system of regulation of alkaline phosphatase activity in human periodontal ligament cells induced by mechanical stress

The periodontal ligament (PDL) is a specialized, mechanically responsive tissue that adapts via cellular responses to equilibrate the effects of mechanical stress on teeth. However, the mechanism of remodelling by which individual cells in periodontal tissue detect and respond to mechanical stress is not well understood. To identify the cellular mechanisms induced by mechanical stress in the periodontal ligament, we examined the effects of cyclic stretching on periodontal ligament fibroblast-like cells (PDL cells). Furthermore, we investigated the effects of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and interaction with peripheral blood mononuclear cells (PBMCs) on mechanically-simulated PDL cells. PDL cells were cultured on type I collagen-coated silicon membranes with 10% FBS alpha-MEM, and then subjected to cyclic mechanical stimulation (1 s stretching/1 s relaxation, 15% maximum elongation). Alkaline phosphatase activity was monitored by cytochemical and spectrophotometric methods. Morphologically, the cells assumed a spindle shape, and the cytoskeletal components, including microtubules and F-actin filaments, were aligned perpendicular to the strain force vector. Cyclic stretching decreased ALPase activity in PDL cells. The anabolic systemic hormone 1,25(OH)(2)D(3) increased ALPase activity, but this effect was suppressed by cyclic stretching. ALPase activities were reduced by co-culture with PBMCs, including lymphocytes and monocytes. This PBMC-induced ALPase reduction was synergistically reduced by cyclic stretching. ALPase activity was decreased by co-culture with PBMCs, and ALPase activity was reduced synergistically by treatment with PBMCs and cyclic stretching. We conclude that PDL cells changed their shape and alignment in response to cyclic stretching. Furthermore, local factors, such as mechanical stress and PBMCs, showed synergistic suppressive effects on ALPase activity.     

Solubility properties of alkaline phosphatase from matrix vesicles

Alkaline phosphatase has been extracted from matrix vesicles of a calcifying cartilage with 0.15 M KCl, 0.4 M guanidinium chloride and 0.05 M deoxycholate/50% butanol mixture. The catalytic properties of the three extracts have been compared. Although the highest amount of enzyme activity is extracted with the latter reagent (55%), some of it is also extracted with KCl (11%) and guanidinium (7%). By submitting isolated matrix vesicles to a short time sonication the distribution pattern of the alkaline phosphatase activity in the extracts is clearly modified, as the amount extracted with KCl increases from 14 to 50% and the portion extracted with deoxycholate decreases from 55 to 27% of the total enzyme activity of matrix vesicles. The enzymatic preparations were comparable on the basis of specific activities, affinity for the substrates (p-nitrophenylphosphate, ATP), thermostability, sensitivity to inhibitors and activators. By electrofocusing a value of pI = 4.15 was found for the alkaline phosphatase of matrix vesicles independently of the extraction medium. These results contradict the concept that alkaline phosphatase is exclusively an intrinsic membrane protein.     

Chemical composition and antimicrobial activity of the essential oil of Cacalia tangutica (Maxim.) Hand.-Mazz

Essential oil of the subterranean part of Cacalia tangutica (Maxim.) Hand.-Mazz was analyzed by gas chromatography (GC)-mass spectrum (MS) technique in two different capillary columns of different polarities. Thirty-one components were identified in the oil and the main compounds were α-zingiberene (13.49%), germacrene D (10.76%), α-pinene (8.54%), caryophyllene(Z-) (6.36%), linalool (6.16%), β-myrcene (4.89%), β-ocimene (Z-) (4.40%)and ocimenone(Z-) (3.58%). The antimicrobial activity of the oil was evaluated against 2 fungi and 12 bacteria including 6 clinically isolated strains using the agar disc diffusion and broth microdilution methods. The results show that the oil presented a broad antimicrobial spectrum and had better antimicrobial activity against yeast and gram-positive bacteria. The minimum inhibitory concentration values were 0.16–5.00 g/L and minimum bactericidal concentration values were 0.16–5.00 g/L. __________ Translated from Journal of Wuhan University (Science Edition), 2007, 53 (2): 198–203 [译自: 武汉大学学报(理学版)]     

Phototactic responses of four marine dinoflagellates with different types of eyespot and chloroplast

Great structural variety is seen in the eyespot of dinoflagellates, a structure involved in phototaxis. Although there are several works on the phototactic responses in some species of dinoflagellates, none of the dinoflagellates used in these studies possessed an eyespot and, therefore, we have no knowledge of the relationship between eyespot type and phototactic response. In this study, we determined wavelength dependency curves for phototaxis in four marine dinoflagellates that possess a different type of either eyespot or chloroplast. These include: (i) a dinoflagellate possessing a peridinin-containing ohioroplast with an eyespot (Scrippsiella hexapraecingula Horiguchi et Chihara); (ii) a dinoflagellate containing a diatom endosymbiont and with the type B eyespot sensu Dodge (1984; (Peridinium foli-aceum (Stein) Biecheler); (iii) a dinoflagellate with peri-dinin-containing chloroplasts, but lacking an eyespot (Atexandrium hiranoi Kita et Fukuyo); and (iv) a dinoflagellate with fucoxanthin, 19′-hexanoyloxyfucoxanthin and 19′-butanoyloxyfucoxanthin, but lacking an eyespot (Gymnodinium mikimotoi Miyabe et Kominami ex Oda), Regardless of the eyespot or the chloroplast type, all four dinoflagellates showed similar wavelength dependency curves for phototaxis, with sensitivity between 380 and 520 nm, the highest peak at approximately 440 or 460 nm and smaller peaks or shoulders at 400–420 nm and 480–500 nm. Substantial peaks have also been noted in the ultraviolet range (260–280 nm). The ultrastructural study of the eye-spot of Scrippsiella hexapraecingula revealed that the eyespot consists of two layers of lipid globules and probably acts as a quarter-wave stack antenna.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.     

Chemical modification and composition of tetrameric isozyme K of alkaline phosphatase from harp seal intestinal mucosa

1. The carbohydrate content of isozyme K of alkaline phosphatase (EC 3.1.3.1) from harp seal intestinal mucosa was examined. The presence of N-acetylglucosamine, N-acetylgalactosamine and considerable amounts of mannose residues was shown. 2. The amino acid content of seal alkaline phosphatase was determined. A high extent of homology (85%) between bovine and seal alkaline phosphatases was demonstrated. 3. By chemical modification lysine, dicarboxylic acids, arginine and tyrosine residues of tetrameric seal alkaline phosphatase are located near or at the active site. By contrast, the modification of either thiol or imidazole groups resulted in no alterations of the enzyme activity. 4. It has been demonstrated that inorganic phosphate is an inhibitor of alkaline phosphatase and entirely prevents the enzyme inactivation with succinic anhydride.     

The effect of long wave ultraviolet radiation (UV-A) on the photosynthetic activity of natural population of freshwater phytoplankton

The effect of ultraviolet radiation on diel changes and depth profiles of phytoplankton photosynthesis was studied in four temperate freshwater lakes. Photosynthetic oxygen production was determined by incubating lake water in light and dark bottles under various weather conditions. Half the light bottles were wrapped with sheets of vinyl chloride film to exclude light with wavelengths shorter than 400 nm. The inhibition of photosynthesis due to UV-A (320–400 nm) was observed during most of the daytime and was very strong around noon on both sunny and cloudy days. On sunny days, when the surface waters of the highly eutrophic Lake Suwa and Senzoku Pond were dominated by denseMicrocystis populations, cumulative daily production at the surface, estimated from the incubation of bottles from which UV-A was excluded by the vinyl film, were about double the rates obtained from glass bottles in which UV-A was present. The UV-A inhibition was detected from the surface toca 20 cm depth in hypereutrophic lakes and at depths greater than 50 cm in mesotrophic lakes. Analysis of the photosynthesis-irradiance (P-I) relationship obtained in the present study shows β, a parameter that describes photo-inhibition, is higher in the presence of UV-A than in its absence. This indicates that UV-A is the major cause of photo-inhibition of phytoplankton photosynthesis.     

Diel changes in the alkaline phosphatase activity of bacteria and phytoplankton in the hypereutrophic Villerest reservoir (Roanne,France)

The diel changes of the size fractioned alkaline phosphatase activity (APA) were studied in relation to several abiotic and biotic factors in Villerest reservoir (located on the Loire river, near the city of Roanne, France), bihourly during two days in July 1992. The APA measured in this work exceeded considerably those reported in the literature, suggesting that dissolved mineral phosphorus was not available to microorganisms. At 1 m, the APA was primarily due to bacteria which actively assimilated organic P compounds released by photosynthetic algal metabolism. At 5, 10 and 20 m, the APA was predominantly algal. The high concentrations in SRP (soluble reactive phosphorus) would indicate that orthophosphates were not bioavailable. The reverse (i. e availability to phytoplankton) would have resulted in undetectable levels of P-PO _inf4 ^sup3– due to the massive proliferation of algae in Villerest reservoir.     

Reduction of alkaline phosphatase activity in aged human osteogenic periodontal ligament fibroblasts exhibiting short telomeres

The osteogenic cell type of human periodontal ligament fibroblasts (PDLF) undergoes senescence at finite population doubling numbers unrelated to donor ages. This study investigated telomere lengths of osteogenic PDLF from differently aged donors and alterations of the osteoblast-like properties in the aged PDLF with short telomeres. Telomere lengths of osteogenic PDLF were biased towards long or short among all 15- to 51-year-old individuals, and did not show a normal distribution by Pearsons test or a correlation to donor age by simple regression analysis. In osteogenic PDLF, senescence-associated -galactosidase was expressed in 78.5% of cells in the clones with short telomeres (mean 3.02 kbp), and in 9.4% of cells in the clones with long telomeres (mean 13.06 kbp). These results suggest that human periodontium comprises aged osteogenic PDLF without correlation to age. Osteogenic PDLF with long telomeres strongly expressed alkaline phosphatase (ALPase) activity whereas cells with short telomeres expressed ALPase activity to a weaker extent. Total activity of ALPase in the clones of osteogenic PDLF with long telomeres was significantly higher than that in the clones with short telomeres. The produced amounts of both osteopontin and osteocalcin in the clones of osteogenic PDLF with long telomeres were slightly but statistically significantly smaller than those in the clones with short telomeres. These findings suggest that aged osteogenic PDLF reduce the expression of ALPase activity but that there is not a critical alteration in bone-associated protein production. Aged osteogenic PDLF may impair the ability to induce ALPase-dependent calcification.This work was supported by a grant-in-aid for Scientific Research (B) (2) from the Ministry of Education, Science, Sports, and Culture of Japan (No. 12470379).     

Electron-cytochemical localization of alkaline phosphatase to G cells of Necturus maculosus antrum

Summary Electron-cytochemical localization of alkaline phosphatase activity was performed on G cells of Necturus maculosus antral mucosa. Alkaline phosphatase activity was localized to the nuclear membrane, the Golgi/endoplasmic reticulum, and the limiting membranes of G cell peptide-secretion vesicles. There was no specific localization of alkaline phosphatase activity to the plasma membrane. Treatment of the tissues with levamisole (an alkaline phosphatase inhibitor) did not markedly reduce the specific alkaline phosphatase activity. Specific lead deposition was reduced by removal of the substrate from the reaction mixture. The results from this study on N. maculosus G cells demonstrate that alkaline phosphatase activity can be found in a non-mammalian gastric endocrine cell and that specific activity was localized primarily to those intracellular structures involved with protein biosynthesis.     

Amplified luminometric assays of alkaline phosphatase using riboflavin phosphates

An asasy for alkaline phosphatase is described which is based on the hydrolysis of riboflavin phosphates (5′FMN or 4′FMN) to produce riboflavin. This is converted to 5′FMN using riboflavin kinase, and then asayed using the bacterial bioluminescent system from Vibrio harveyi or V. Fischeri. The most sensitive assay is obtained using 4′FMN, which can measure less than 20 amol after a 1-hour incubation.     

Biotechnological significance of toxic marine dinoflagellates

Dinoflagellates are microalgae that are associated with the production of many marine toxins. These toxins poison fish, other wildlife and humans. Dinoflagellate-associated human poisonings include paralytic shellfish poisoning, diarrhetic shellfish poisoning, neurotoxic shellfish poisoning, and ciguatera fish poisoning. Dinoflagellate toxins and bioactives are of increasing interest because of their commercial impact, influence on safety of seafood, and potential medical and other applications. This review discusses biotechnological methods of identifying toxic dinoflagellates and detecting their toxins. Potential applications of the toxins are discussed. A lack of sufficient quantities of toxins for investigational purposes remains a significant limitation. Producing quantities of dinoflagellate bioactives requires an ability to mass culture them. Considerations relating to bioreactor culture of generally fragile and slow-growing dinoflagellates are discussed. Production and processing of dinoflagellates to extract bioactives, require attention to biosafety considerations as outlined in this review.     

Stimulation of alkaline phosphatase activity in Ishikawa cells induced by various phytoestrogens and synthetic estrogens

Xenoestrogens, phytoestrogens and synthetic estrogens, are able to bind to estrogen receptors, and to mimic estrogenic activities in a cell and tissue specific manner. For the characterization of environmental estrogens mainly mammary derived and yeast based models have been used. The aim of this study was therefore to assess selected natural and synthetic compounds in an endometrial derived model. We measured the relative estrogenic potency of phytoestrogens (genistein, daidzein, coumestrol, some naringenins), synthetic estrogens (bisphenol A, octylphenol, nonylphenol, o,p′-DDT), mycoestrogen (zearalanone) as well as extracts of Cimicifuga racemosa on alkaline phosphatase (AlkP) activity in the endometrial derived adenocarcinoma cell line Ishikawa. We used a modified multiwell plate in vitro bioassay based on the estrogen-specific and dose-dependent enhancement of AlkP activity in this cell line. Estradiol, which induced AlkP at levels as low as 10⁻⁸ M, was used as positive control. Most of the compounds studied showed a clear dose-dependent estrogenic effect. Compared to the vehicle control (ethanol) all phyto- and mycoestrogens, stimulated the AlkP activity 2–4-fold at a concentration of 10⁻⁶ M. The synthetic chemicals bisphenol A and nonylphenol showed an effect at 10⁻⁶ M, octylphenol at 10⁻⁵ M. Effects of o,p′-DTT could not be measured. ICI 182,780, a pure estrogen receptor antagonist, significantly inhibited these effects. The latter result demonstrated the estrogen receptor dependency of this process. In summary, most of the phytoestrogens and industrial chemicals tested, behaved as estrogen receptor agonists in terms of the stimulation of AlkP activity.     

Induction of alkaline phosphatase in cultured human fibroblasts: Comparison of normal cells and those from patients with cystic fibrosis

The membrane glycoprotein enzyme, alkaline phosphatase was induced in cultured human fibroblasts by dibutyryl cyclic AMP, sodium butyrate, the serum glycoprotein fetuin, the Tamm-Horsfall urinary glycoprotein, and by a number of inhibitors of DNA synthesis. The uninduced basal enzyme activity increased at later stages of growth when the cells became confluent. Induction by dibutyryl cyclic AMP or fetuin was most effective when the agents were added after the cells had reached stationary phase and was maximal after at least two days of exposure. The levels of induction resulting from the addition of pairs of the agents, dibutyryl cyclic AMP, n-butyrate and fetuin were additive indicating that these have different modes of action. The inhibitors of DNA synthesis, cytosine arabinoside, hydroxyurea, and methothrexate were less effective inducers. Bromodeoxyuridine which also has non-DNA mediated effects induced to the same extent as dibutyryl cyclic AMP.Similar experiments with sex- and age-matched cell strains derived from patients with cystic fibrosis failed to detect differences in the levels of induction from those observed in normal cells. In addition, the combined inductive effects of Tamm-Horsfall glycoprotein, isoproterenol and theophylline, were similar with normal and cystic fibrosis cells.     

Distribution of activity of alkaline phosphatase and Mg-dependent adenosine triphosphatase in the cranial dura mater-arachnoid interface zone of the rat

Summary The distribution of the activity of alkaline phosphatase and Mg-dependent adenosine triphosphatase was studied in the encephalic dura mater-arachnoid borderline (interface) zone of albino Wistar rats. Intense clustering of electron-dense granules that indicated alkaline phosphatase activity was observed in the inner dural cells, the neurothelial cells, the outermost row of the outer arachnoidal cells and in the intercellular cleft between the latter two (the so-called electron-dense band). The remainder of the outer arachnoidal cells contained almost no reaction product. Mg-adenosine triphosphatase activity was distributed differently; a lack of reaction product was observed not only in the outer arachnoidal cells, but also in the zone occupied by the electron-dense band. The data confirm histochemically the barrier properties of the dura mater-arachnoid interface zone.