Extracellular Alkaline Phosphatase Research Articles

Essential Role of Nuclear Factor of Activated T Cells (NFAT)-mediated Wnt Signaling in Osteoblast Differentiation Induced by Strontium Ranelate

The antiosteoporotic treatment strontium ranelate (SrRan) was shown to increase bone mass and strength by dissociating bone resorption and bone formation. To identify the molecular mechanisms of action of SrRan on osteoblasts, we investigated its effects on calcineurin-NFAT (nuclear factor of activated T cells) signaling, an important calcium sensitive pathway controlling bone formation. Using murine MC3T3-E1 and primary murine osteoblasts, we demonstrate that SrRan induces NFATc1 nuclear translocation, as shown by immunocytochemical and Western blot analyses. Molecular analysis showed that SrRan increased NFATc1 transactivation in osteoblasts, an effect that was fully abrogated by the calcineurin inhibitors cyclosporin A or FK506, confirming that SrRan activates NFATc1 signaling in osteoblasts. This has functional implications because calcineurin inhibitors blunted the enhanced osteoblast replication and expression of the osteoblast phenotypic markers Runx2, alkaline phosphatase, and type I collagen induced by SrRan. We further found that SrRan increased the expression of Wnt3a and Wnt5a as well as beta-catenin transcriptional activity in osteoblasts, and these effects were abolished by calcineurin inhibitors. The Wnt inhibitors sFRP1 and DKK1 abolished SrRan-induced osteoblast gene expression. Furthermore, blunting the Wnt5a receptor Ryk or RhoA that acts downstream of Ryk abrogated cell proliferation and osteoblast gene expression induced by SrRan. These results indicate that activation of NFATc1 and downstream canonical and non-canonical Wnt signaling pathways mediate SrRan-induced osteoblastic cell replication and differentiation, which provides novel insights into the mechanisms of action of this antiosteoporotic agent in osteoblastogenesis.

Prokaryotic activities and abundance in pelagic areas of the Ionian Sea

The Ionian Sea represents a suitable basin for studying the biogeochemical processes mediated by microbial activities. Because of its characteristics as a crossing region between the western and eastern Mediterranean Sea, it is one of the sites most affected by changes in water mass composition and dynamics, caused by the Eastern Mediterranean Transient (EMT). To date, relatively few data exist on microbial activities in pelagic areas of the Ionian Sea. From 1998 to 2004, during different research cruises, prokaryotic parameters (abundance, extracellular enzyme activities leucine aminopeptidase, β-glucosidase, alkaline phosphatase, bacterial production and respiration) were measured together with culturable bacteria and the main physical, chemical and trophic parameters (temperature, salinity, nutrients, particulated organic matter). The aim of the study was to describe the spatial and temporal variability in microbial activities involved in the carbon and phosphorus cycles, in different layers. Results showed that organic matter transformation mediated by the microbial community displayed a significant increase in autumn, highlighting the occurrence of significant changes at meso- and bathypelagic depths. Unlike the dark ocean, bacterial growth efficiency in the Ionian Sea, which increased with depth, seemed to vary from being a source of carbon in the epipelagic layer to a sink in the meso- and bathypelagic layers. The mechanism of phosphatase regulation showed a weak inverse correlation between specific phosphatase and inorganic P in all seasons except autumn. It is worth mentioning that the reported results constitute, to the best of our knowledge, one of the available datasets giving information about microbial activities in the Ionian Sea.

EXPRESSION AND DIVERSITY OF ALKALINE PHOSPHATASE EHAP1 IN EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler is a cosmopolitan coccolithophore species that forms massive blooms in low phosphorus seawater, partly due to its ability to utilize organic phosphate via extracellular alkaline phosphatase (AP). A novel AP gene, ehap1, was identified from the strain CCMP374. In this study, we examined the expression of ehap1 in various E. huxleyi strains and its genetic diversity in those strains and field populations. Two EHAP1 proteins (EHAP1a, 75 kDa and EHAP1b, 110 kDa) with virtually identical sequence were expressed under P limitation in all strains except one; a third protein (EHAP1c, 115 kDa) was expressed in a few strains. The correlation between AP activity and protein abundance suggests that EHAP1b is inactive and probably the precursor of EHAP1a. The transcript of ehap1 was induced by P depletion in all strains. The ehap1 gene sequence is highly conserved in these strains and field populations with <3% nucleic acid substitution. Most of the ehap1 sequences from one site in the English Channel and three sites in the Gulf of Alaska were essentially identical to one another. No EHAP1-like protein can be detected in other phytoplankton species tested via Western blot analysis. The rapid induction and high activity of EHAP1 in E. huxleyi suggest that it plays a significant role in P regeneration in the oligotrophic ocean where E. huxleyi is abundant. The EHAP1 antibody and gene-specific primers are well suited to study the dynamics of P limitation in field populations of E. huxleyi.

ALKALINE PHOSPHATASE IN FRESHWATER CLADOPHORA-EPIPHYTE ASSEMBLAGES: REGULATION IN RESPONSE TO PHOSPHORUS SUPPLY AND LOCALIZATION1

Extracellular alkaline phosphatase enzyme activity (APA) is important for algal phosphorus (P) acquisition in P-limited freshwater ecosystems and is often used as an indicator of P deficiency. APA allows access to organic P (monophosphate esters), but the regulation of APA in response to availability of both PO43− and organic P is poorly characterized. This study aimed to examine the regulation of APA in freshwater Cladophora-epiphyte assemblages in response to PO43− and a hydrolyzable organic P source, and for the first time to apply enzyme linked fluorescence (ELF) to localize APA within freshwater macroalgal-epiphyte assemblages. In response to elevated PO43− concentrations, a component of net APA was suppressed, but there was also a constitutive APA, which was maintained even after prolonged exposure to nearly 1,000 μM PO43− and saturation of internal P pools. When supplied with organic glycerol P as the sole P source, the algae maintained APA in excess of needs for supplying PO43− for uptake, resulting in PO43− release into the medium. Constitutive APA may be adaptive to growth under chronic P limitation in oligotrophic freshwater habitats. Excess APA and release of PO43− could benefit different algal and bacterial partners within assemblages. APA in both Cladophora sp. and epiphytic algae was localized with ELF only when ethanol fixation was omitted. In algal subsamples exposed to different P treatments, there was no correlation between bulk APA (using 4-methylumbelliferyl phosphate [MUP] substrate) and % cell labeling with ELF, suggesting that ELF labeling of APA was at best semiquantitative in the algal assemblages.

Phosphate solubilization potential and phosphatase activity of rhizospheric trichoderma spp.

Trichoderma sp., a well known biological control agent against several phytopathogens, was tested for its phosphate (P) solubilizing potential. Fourteen strains of Trichoderma sp. were isolated from the forest tree rhizospheres of pinus, deodar, bamboo, guava and oak on Trichoderma selective medium. The isolates were tested for their in-vitro P-solubilizing potential using National Botanical Research Institute Phosphate (NBRIP) broth containing tricalcium phosphate (TCP) as the sole P source, and compared with a standard culture of T. harzianum. All the cultures were found to solubilize TCP but with varying potential. The isolate DRT-1 showed maximum amount of soluble phosphate (404.07 εg.ml-1), followed by the standard culture of T. harzianum (386.42 εg.ml-1) after 96 h of incubation at 30±10C. Extra-cellular acid and alkaline phosphatases of the fungus were induced only in the presence of insoluble phosphorus source (TCP). High extra-cellular alkaline phosphatase activity was recorded for the isolate DRT-1 (14.50 U.ml-1) followed by the standard culture (13.41 U.ml-1) at 72h. The cultures showed much lesser acid phosphatase activities. Under glasshouse conditions, Trichoderma sp. inoculation increased chickpea (Cicer arietinum) growth parameters including shoot length, root length, fresh and dry weight of shoot as well as roots, in P-deficient soil containing only bound phosphate (TCP). Shoot weight was increased by 23% and 33% by inoculation with the isolate DRT-1 in the soil amended with 100 and 200 mg TCP kg-1 soil, respectively, after 60 d of sowing. The study explores high P-solubilizing potential of Trichoderma sp., which can be exploited for the solubilization of fixed phosphates present in the soil, thereby enhancing soil fertility and plant growth.

Purification and Properties of Alkaline Phosphatase from Bacillus Cereus

ABSTRACTExtracellular and membrane-bound alkaline phosphatases were produced at the middle stationary phase of growth by a strain Bacillus cereus. Twenty two percent of the enzyme activity was secreted into the culture media. An extracellular alkaline phosphatase (AP I) and a membrane-bound alkaline phosphatase (AP II) were purified 282-fold and 70-fold, respectively by a combination of chromatographic methods. Enzyme activity of alkaline phosphatase preparations was maximal at pH 9.5. Both enzymes were inhibited by EDTA and were reactivated by addition of Ca2+. The molecular weight of AP I was estimated to be 43 ± 1 kDa, and that of AP II was estimated to be 44 ± 1 kDa. Alkaline phosphatase activity of both enzyme preparation s was completely lost by heating at 80°C.

Efficient production of extracellular proteins with Escherichia coli by means of optimized coexpression of bacteriocin release proteins

Aiming to facilitate the accessibility of recombinant proteins produced with Escherichia coli, extracellular expression may be achieved by means of bacteriocin release protein (BRP) coexpression. Different types of BRPs were tested in order to optimize protein secretion into the culture medium. Those included the well-studied BRPs of the Colicin E1 and Cloacin DF13 bacteriocins and variants thereof. BRP expression was stringently controlled by means of the arabinose inducible P BAD promoter, which accounts for a broad-range adjustment of expression strength. Using appropriate arabinose concentrations, a concentration range was determined, that allowed efficient secretion of the model proteins alkaline phosphatase and β-lactamase, with 90–95% of the proteins released into the culture medium. Kinetic investigations into BRP expression and protein secretion revealed a rapid increase of extracellular protein concentration within 5–10 min past induction. Alternatively to fine-tuned BRP expression during cultivation, protein production and secretion could be decoupled by establishment of appropriate induction strategies and up to 90% of alkaline phosphatase was released into the culture medium within 3 h after reaching maximum biomasss concentrations. Both, fine-tuned and growth decoupled BRP expression accounted for extracellular alkaline phosphatase concentrations of roughly 500 mg l −1 of culture broth and selectivities of 50 mg of this enzyme per gram of cell dry mass, respectively.

Comparative studies on photosynthesis and phosphate metabolism of Cylindrospermopsis raciborskii with Microcystis aeruginosa and Aphanizomenon flos-aquae

The physiological differences for three bloom-forming cyanobacteria ( Cylindrospermopsis raciborskii, Microcystis aeruginosa, and Aphanizomenon flos-aquae) were investigated. In comparison with M. aeruginosa and A. flos-aquae, C. raciborskii exhibited a significantly higher concentration of carotenoids, higher values in maximum photosynthesis rate ( P m), apparent photosynthetic efficieny ( a), and maximum electron transport rate (ETR max) during the growth period. In addition, higher extracellular alkaline phosphatase activities and lower light compensation point ( I c) were also detected in C. raciborskii ( p < 0.05, ANOVA). Therefore, it is suggested that the higher photosynthetic activities, more effective uptake and utilization to phosphate, and low light requirements might play important roles in the occurrence and invasive behavior of C. raciborskii.

Limitations of using extracellular alkaline phosphatase activities as a general indicator for describing P deficiency of phytoplankton in Chinese shallow lakes

Extracellular phosphatase can be produced by phytoplankton to utilize organic phosphorus under phosphorus (P) deficiency. However, there is a controversy about its use as an indicator of P deficiency in natural phytoplankton community inferred by such an "induction-repression" mechanism. Size-fractionation of alkaline phosphatase activity (APA), soluble reactive phosphorus (SRP) concentration, algal density, and composition were determined in six Chinese shallow lakes ranking in gradient of trophic status, where a positive relationship between SRP concentration and algal density was observed. Enzyme-labeled fluorescence (ELF) method was used to localize phosphatase on cell membrane of algae. The so-called algal APA that associated with coarser particle (> 3.0 A mu m) accounted for the largest part of total APA. Within a lake with lower SRP concentration, the "induction-repression" mechanism held true. Contrastingly, both algal and total APA were positively related to SRP concentration based on the data across all study lakes with statistical significance, which may be explained firstly by algal composition. The lakes with higher SRP concentration were dominated by diatoms and green algae, while they easily produced extracellular phosphatases as evidenced by ELFA labeling. In parallel, the lakes with lower SRP concentration were dominated by cyanobacteria, while it was never ELFA-positive; secondly, ELFA-positive dots or structures suggested that, in lakes with higher trophic status, attached bacteria or heterotrophic microorganisms could substantially contribute to extracellular phosphatases for hydrolyzing organophosphoric compounds but probably utilizing the organic moiety as an organic carbon source. This process simultaneously produces inorganic P, leading to the co-occurrence of high phosphate concentration and APA. So, the contributor of APA are complex, which may produce extracellular phosphatase species-specific or not exclusively for P nutrient and consequently make it difficult to normalize APA with the exact biomass estimators. Therefore, it is not reasonable to use APA, normalized or not, as a general indicator for describing P deficiency of phytoplankton in shallow lakes especially eutrophic ones.

Microarray analysis of phosphate regulation in the marine cyanobacterium Synechococcus sp. WH8102

Primary productivity of open ocean environments, such as those inhabited by marine picocyanobacteria, is often limited by low inorganic phosphate (P). To observe how these organisms cope with P starvation, we constructed a full genome microarray for Synechococcus sp. WH8102 and compared differences in gene expression under P-replete and P-limited growth conditions, including both early P stress, during extracellular alkaline phosphatase induction, and late P stress. A total of 36 genes showed significant upregulation (>log(2) fold) whereas 23 genes were highly downregulated at the early time point; however, these changes in expression were maintained during late P stress for only 5 of the upregulated genes. Knockout mutants were constructed for genes SYNW0947 and SYNW0948, comprising a two-component regulator hypothesized to have a key function in regulating P metabolism. A high degree of overlap in the sets of genes affected by P stress conditions and in the knockout mutants supports this hypothesis; however, there is some indication that other regulators may be involved in this response in Synechococcus sp. WH8102. Consistent with what has been observed in many other cyanobacteria, the Pho regulon of this strain is comprised largely of genes for alkaline phosphatases, P transport or P metabolism. Interestingly, however, the exact composition and arrangement of the Pho regulon appears highly variable in marine cyanobacteria.

Differentiation of fetal osteoblasts and formation of mineralized bone nodules by 45S5 Bioglass ® conditioned medium in the absence of osteogenic supplements

Bioactive glasses bond strongly to bone in vivo and their ionic dissolution products have previously been shown to have stimulatory properties on adult and fetal osteoblasts and to induce the differentiation of embryonic stem cells towards the osteoblastic lineage in vitro. In the present study, the effect of 45S5 Bioglass ® conditioned medium with two different Si concentrations (15 μg/ml (BGCM/15) and 20 μg/ml (BGCM/20)) on human fetal osteoblast growth, differentiation and extracellular matrix production and mineralization was investigated. In the first instance, primary fetal osteoblasts were examined for the osteoblast phenotypic markers alkaline phosphatase (ALP), collagen type I (Col I) and OB Cadherin (Cadherin 11) (OB Cad) as well as for the mesenchymal stem cell markers CD105 and CD166. At passage 0 more than 50% of the population was positive for Col I and ALP, but at passage 2, the proportion of cells expressing ALP increased. In addition at passage 0 more than 50% of the fetal osteoblasts expressed the mesenchymal stem cell surface markers CD105 and CD166. Treatment with BGCM/15 and BGCM/20 in the absence of osteogenic supplements increased the gene expression of the bone extracellular matrix proteins alkaline phosphatase, osteonectin and bone sialoprotein as determined by quantitative real time reverse transcriptase-polymerase chain reaction (rt RT-PCR) analysis. Extracellular matrix production was also enhanced in the absence of osteogenic supplements by the 45S5 Bioglass ® conditioned medium as demonstrated by ALP enzymatic activity, osteocalcin and Col I protein synthesis. Furthermore, BGCM/15 and BGCM/20 significantly enhanced the formation of mineralized nodules, based on alizarin red histochemical staining, without necessitating the addition of β-glycerophosphate, l-ascorbate-2-phosphate or dexamethasone (commonly used osteogenic supplements).

Cold Stress Stimulates Intracellular Calcification by the Coccolithophore, Emiliania huxleyi (Haptophyceae) Under Phosphate-Deficient Conditions

Intracellular calcification by the coccolith-producing haptophyte Emiliania huxleyi (NIES 837) is regulated by various environmental factors. This study focused on the relationship between cold and phosphate-deficient stresses to elucidate how those factors control coccolith production. (45)Ca incorporation into coccoliths was more than 97% of the total (45)Ca incorporation by whole cells. In a batch culture, orthophosphate in the medium (final concentration, 28.7 muM) was rapidly depleted within 3 days, and then extracellular alkaline phosphatase (AP) activity, an indicator of phosphate deprivation, increased during the stationary growth phase. The increase in AP activity was slightly higher at 20 degrees C than at 12 degrees C. The calcification started to increase earlier than AP activity, and the increase was much higher at 12 degrees C than at 20 degrees C. Such enhancement of calcification was suppressed by the addition of phosphate, while AP activity was also suppressed after a transient increase. These results suggest that phosphate deprivation is a trigger for calcification and that a rather long induction period is needed for calcification compared to the increase in AP activity. While calcification was greatly stimulated by cold stress, other cellular activities such as growth, phosphate utilization, and the induction of AP activity were suppressed. The stimulation of coccolith production by cold stress was minimal under phosphate-sufficient conditions. The high calcification activity estimated by (45)Ca incorporation was confirmed by morphological observations of coccoliths on the cell surface under bright-field and polarization microscopy. These results indicate that phosphate deprivation is the primary factor for stimulating coccolith production, and cold stress is a secondary acceleration factor that stimulates calcification under conditions of phosphate deprivation.

Extracellular alkaline phosphatase is a sensitive marker for cellular stimulation and exocytosis in heterotroph cell cultures of Chenopodium rubrum

We investigated the response of extracellular phosphatase to heat shock in heterotrophic Chenopodium rubrum L. cell cultures. Surprisingly, in contrast to the generally used acid phosphatase, an extracellular alkaline phosphatase showed the most sensitive response. This phosphatase was characterized as a marker for cellular stimulation by its high correlations with induced changes of extracellular pH: 10microM nigericin (correlation coefficient r=0.91), 100microM salicylic acid (r=0.84), heat shock 5min 37 degrees C (r=0.79), and heat shock after pre-treatment with 5microM fusicoccin (r=0.92) or 0.5% ethanol (r=0.90). Cellular stimulation was estimated with concentrations of acids and bases, yielding similar levels of pH change (0.5 pH) in cell-free supernatant: salicylic acid (200microM), benzoic acid (600microM), HCl (140microM), NaOH (100microM), and KOH (100microM). The Golgi apparatus inhibitor Brefeldin A (200microM) reduced the heat-shock-induced phosphatase (-33%). The pH optimum of heat-shock-induced phosphatase was 3; however, there the proportion of constitutive phosphatase was higher than at pH 8-9.5, indicating different pH dependence of constitutive and induced activity. Thus, heat-shock-induced phosphatase was characterized by alkaline activity with inhibitors (10microM molybdate: -52%, 2.5mM phosphate: -64%, 10microM ZnCl(2): -82%), substrates (2.5mM, tyrosine phosphate: 255pkat g(-1), p-nitrophenyl phosphate: 92pkat g(-1), serine phosphate: 0, threonine phosphate: 0), Hill coefficient (nH=1.4) indicating two binding sites, and the extent of heat-shock stimulation (p-nitrophenyl phosphate: +190%, tyrosine phosphate: +180%). SDS-PAGE showed a correlation of alkaline phosphatase with the heat-shock-induced release of highly N-glycosylated 53kDa protein, detected by peroxidase-labeled concanavalin A affinoblotting after endoglycosidase H treatment. The 53kDa protein showed no in-gel phosphatase activity after SDS-PAGE and regeneration treatment, in contrast to a putative dimer (105kDa).

Microbial enzymatic activities in aerobic activated sludge model reactors

Communal, dairy and petroleum wastewaters were separately purified in three aerobic activated sludge model reactors to investigate selected enzymatic activities of microorganisms. Four extracellular enzymes, i.e. leucine-aminopeptidase (L-AMP), β-glucosidase (β-GLC), alkaline phosphatase (APA) and lipase (LIP), were studied. Experimental results show that enzymatic activities associated with microbial cells in the activated sludge flocs accounted for 54.5–97.4% of the total enzyme activities in model reactors, with LIP being the most active enzyme. Highest activities of L-AMP, β-GLC and APA were found in the dairy wastewater reactor. Kinetic parameters of the studied enzymes varied notably, high V max followed high a K m, suggesting that inhibition of enzymes took place in the activated sludge systems. There were no significant relationships between enzymatic activities and dissolved organic carbon (DOC) concentrations in the three aerobic reactors during the experiments.

Copper induction of phosphorus deficiency in Ulva fasciata (Ulvales, Chlorophyta)

T.-M. Lee, Y.-L. Huang and M.-H. Chen. 2005. Copper induction of phosphorus deficiency in Ulva fasciata (Ulvales, Chlorophyta). Phycologia 44: 620–628.The effects of Cu2+ on phosphorus (P) availability in Ulva fasciata Delile were studied by exposure to CuSO4 (0–100 µM) added in the nutrient-enriched artificial seawater media for 8 days. The growth rate decreased as medium Cu2+ contents increased, 50% growth inhibition was achieved at 9.8 µM Cu2+ (850 µg l−1). Tissue total Cu contents as well as cell wall–bound and intracellular Cu contents increased as medium Cu2+ contents increased. Normally, cell wall–bound Cu contents in Cu2+-treated thalli consisted of approximately 60% of total tissue Cu contents. When Cu2+ contents in the media were ≥ 50 µM, tissue P contents decreased in conjunction to tissue C : P and N : P molar ratio increased, but tissue C and N contents and tissue C : N molar ratio remained unchanged. Tissue soluble reactive phosphorus, soluble nonreactive phosphorus and total soluble phosphorus contents decreased, whereas Cu2+ contents in the medium were ≥ 50 µM. After 4 days of treatment, the inorganic oxyanion phosphate (Pi) uptake rate in 50 µM Cu2+-treated thalli (0.69 µmol h−1 g−1 d. wt. (dry weight)) decreased as compared with the control (2.25 to 0.69 µmol h−1 g−1 d. wt.). The apparent Km and Vmax values for Pi uptake in the control were 20.54 µM Pi and 2.79 µmol h−1 g−1 d. wt., respectively, and those in 50 µM Cu2+-treated thalli were 15.13 µM Pi and 0.81 µmol h−1 g−1 d. wt., respectively. There was an increase of medium Pi contents after 8 days of pre-exposure to 100 µM Cu2+, reflecting a leakage in response to extraordinarily high Cu2+ contents. The activities of extracellular alkaline phosphatase and intracellular acid phosphatase increased in both 50 and 100 µM Cu2+ treatments. Overall, Cu results in P deficiency in U. fasciata via a depression of Pi uptake, and a leakage of cellular P under exposure to excess high Cu contents. The role of Cu-adsorptive ability of cell walls in relation to Cu tolerance in U. fasciata is discussed.

THE EFFECTS OF PHOSPHITE ON PHOSPHATE STARVATION RESPONSES OF ULVA LACTUCA (ULVALES, CHLOROPHYTA)1

Effects of phosphite (Phi) on phosphate (Pi) starvation responses were determined in Ulva lactuca L. by incubation in Pi‐limited (1 μM NaH2PO4) or Pi‐sufficient (100 μM NaH2PO4) seawater containing 0–3 mM Phi. Exposure to 1 μM NaH2PO4 decreased the growth rate and the content of free Pi and esterified‐P but increased the activities of extracellular alkaline phosphatase (EC 3.1.2.1) and intracellular acid phosphatase (ACP; EC 3.1.2.2); two ACP isozymes observed by activity staining on isoelectric focussing (IEF) gel were induced. The Km value of Pi uptake rate was decreased by incubation with 1 μM NaH2PO4 and the decrease in Km value was inhibited by 2 mM Phi, reflecting the operation of a high‐affinity Pi uptake system at low Pi concentrations. In the presence of Phi, the growth rate of Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. As Phi concentrations were increased from 0 to 2 mM, the free Pi contents in both Pi‐sufficient and Pi‐starved thalli decreased, but the esterified‐P contents in Pi‐starved thalli increased, whereas those in Pi‐sufficient thalli increased at 1 mM Phi and decreased at 2 mM Phi. Cell wall localized AP activity in both Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. Intracellular ACP activity in Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM but was not affected in Pi‐sufficient thalli. The induction of ACP isozyme activity and high‐affinity Pi uptake system in Pi‐starved thalli was inhibited by Phi. The present investigation shows that Phi interrupts the sensing mechanisms of U. lactuca to Pi‐limiting conditions.

Fabrication of hydroxyapatite sponges by dextran sulphate/amino acid templating

We report a new template-directed method for the fabrication of hydroxyapatite (HAp) sponges by using amino-acid-coated HAp nanoparticles dispersed within a viscous polysaccharide (dextran sulfate) matrix, and describe the use of these materials for the viability and proliferation of human bone marrow stromal cells. The nanoparticles were prepared in the presence of excess amounts of aspartic acid, alanine or arginine, and subsequently organised into macroporous frameworks with typical pore sizes of 100–200 μm during thermal degradation of the dextran matrix. The sponge macrostructure was influenced by changes in the heating rate and sintering time, as well as the use of different amino acids or variations in dextran functional groups. Biocompatibility testing showed retention of cell viability, production of extracellular matrix and alkaline phosphatase expression, suggesting that it should be possible to exploit this novel fabrication method for potential applications in cartilage or soft tissue engineering.

TheLPB1Gene Is Important for Acclimation ofChlamydomonas reinhardtiito Phosphorus and Sulfur Deprivation

Organisms exhibit a diverse set of responses when exposed to low-phosphate conditions. Some of these responses are specific for phosphorus limitation, including responses that enable cells to efficiently scavenge phosphate from internal and external stores via the production of high-affinity phosphate transporters and the synthesis of intracellular and extracellular phosphatases. Other responses are general and occur under a number of different environmental stresses, helping coordinate cellular metabolism and cell division with the growth potential of the cell. In this article, we describe the isolation and characterization of a mutant of Chlamydomonas reinhardtii, low-phosphate bleaching (lpb1), which dies more rapidly than wild-type cells during phosphorus limitation. The responses of this mutant to nitrogen limitation appear normal, although the strain is also somewhat more sensitive than wild-type cells to sulfur deprivation. Interestingly, depriving the cells of both nutrients simultaneously allows for sustained survival that is similar to that observed with wild-type cells. Furthermore, upon phosphorus deprivation, the lpb1 mutant, like wild-type cells, exhibits increased levels of mRNA encoding the PHOX alkaline phosphatase, the PTB2 phosphate transporter, and the regulatory element PSR1. The mutant strain is also able to synthesize the extracellular alkaline phosphatase activity upon phosphorus deprivation and the arylsulfatase upon sulfur deprivation, suggesting that the specific responses to phosphorus and sulfur deprivation are normal. The LPB1 gene was tagged by insertion of the ARG7 gene, which facilitated its isolation and characterization. This gene encodes a protein with strong similarity to expressed proteins in Arabidopsis (Arabidopsis thaliana) and predicted proteins in Oryza sativa and Parachlamydia. A domain in the protein contains some similarity to the superfamily of nucleotide-diphospho-sugar transferases, and it is likely to be localized to the chloroplast or mitochondrion based on programs that predict subcellular localization. While the precise catalytic role and physiological function of the putative protein is not known, it may function in some aspect of polysaccharide metabolism and/or influence phosphorus metabolism (either structural or regulatory) in a way that is critical for allowing the cells to acclimate to nutrient limitation conditions.

Modulation of extracellular matrix synthesis and alkaline phosphatase activity of periodontal ligament cells by mechanical stress

Loss of occlusal function has been reported to induce atrophic changes in the periodontal ligament. It is likely that mechanical stress triggers the biological response of periodontal ligament. However, there have been few reports studying the correlation between mechanical stress of varying magnitude and periodontal ligament cell activities such as extracellular matrix (ECM) synthesis. The objective of this study is to clarify the influence of the mechanical stress on changes in mRNA expression levels of type I collagen and decorin genes, as well as alkaline phosphatase (ALP) activity in response to mechanical stress of varying magnitude. Bovine periodontal ligament cells were cultured on flexible-bottomed culture plates and placed on the BioFlex Loading Stations. Cells were elongated at 6 cycles/min (5 s on and 5 s off) at each of six levels of stretch (0.2, 1.0, 2.0, 3.0, 10, 18% increase in the surface area of the bottom) for 48 h. We measured mRNA expression levels of type I collagen and decorin genes using quantitative reverse transcription-polymerase chain reaction (RT-PCR), and ALP activity in periodontal ligament cell culture under cyclic mechanical stretching. Mechanical tensional stress of low magnitude induced the increase of both type I collagen and decorin mRNA expression without changing ALP activity in periodontal ligament cells. Mechanical tensional stress of high magnitude induced the increase of type I collagen and decorin mRNA expression while decreasing ALP activity. These results suggest that different magnitude of tensional force induces different responses from periodontal ligament cells, and that mechanical stress plays an important role in remodeling and functional regulation of periodontal ligament.

Dynamics of extracellular enzymatic activities in a shallow Mediterranean ecosystem (Tindari ponds, Sicily)

Three microbial extracellular enzymes, leucine aminopeptidase (LAP), β-glucosidase (β-glu) and alkaline phosphatase (AP), were studied in six small Mediterranean littoral ponds, to evaluate the diversity of microbial activities relative to prevailing environmental conditions. The marked diversification of the trophic states, ranging from oligotrophy to eutrophy, in the ponds was reflected in a range of enzyme patterns at different spatial and temporal scales. There were higher levels and greater variability of microbial activity in the oldest and most ‘confined’ ponds (ranges: 0.55–4360.00 nm h−1, 0.15–76.44 nm h−1, 1.29–1600.00 nm h−1 for LAP, β-glu and AP respectively) compared with the youngest and most seaward ponds (ranges: 22.64–612.0 nm h−1, 0.06–48.89 nm h−1, 0.32–744.0 nm h−1 for LAP, β-glu and AP respectively). The close relationship of the degradative potential with chlorophyll-a and particulate organic carbon could be a consequence of the stimulating effect of phytoplankton-released polymeric compounds (organic matter) and/or a response of the microbial community to warm temperatures, which were recorded from July to September. Within an area less than 1 km2, different aquatic ecosystems coexist and maintain their distinctive properties in terms of microbial biogeochemical processes.