Redistribution of phosphate deposits in the algaScenedesmus quadricauda deprived of exogenous phosphate—an ultra-cytochemical study

Summary The green algaScenedesmus quadricauda (Turp.) Bréb. was cultivated in the presence or absence of orthophosphate and synchronized daughter or mother cells were cytochemically stained. Forin situ capturing of water soluble phosphates Ca²⁺ and Mg²⁺ ions were added to the ice-cold glutaraldehyde fixative to form a polymeric metal-phosphate complex which was equivalent to the energy-rich condensed polyphosphates in staining by alkaline lead acetate. The X-ray microanalysis of the extensive stained deposits proved the presence of phosphorus. In orthophosphate-supplied daughter cells cytoplasmic vacuoles contained round stained bodies; a layer of phosphate-containing paracrystals encompassing some starch grains and a fine stained layer delineating the chloroplast envelope were also observed. In the equivalent mother cells only the material inside theloculi of stacked thylakoids was stained. In orthophosphate starved daughter cells filamentous phosphate-containing paracrystals filled extensive cytoplasmic vacuoles. A stained layer covered the chloroplast envelope and continuous stained layers appeared inside theloculi of stacked thylakoids. Mother cells that develop from these daughter cells were filled with starch grains and showed only peripheral stained deposits. The results are compared with the biochemical evidence of phosphate turnover in algal cells.Abbreviations ADP adenosine diphosphate - ATP adenosine triphosphate - ATPase adenosine triphosphatase - EDAX energy dispersive analysis of X-rays - Pi orthophosphate - PPi pyrophosphate - PP polyphosphate - PhAR photosynthetic active radiation - TCA trichloroacetic acid     

Time-courses of size-fractionated phosphate uptake: are larger cells better competitors for pulses of phosphate than smaller cells?

Summary Time-course experiments of phosphate uptake by size-fractionated phytoplankton were conducted in oligotrophic Kennedy and Sproat Lakes. The objective was to determine if large phytoplankton obtained more phosphate than smaller cells, when the nutrient was present at higher concentrations. Studies at Kennedy Lake revealed that uptake rates in the 0.2–3.0 m fraction were very sensitive to the time they were exposed to elevated concentrations; rates determined over the 60–120 min interval were less than 30% of those recorded over the 0–60 min interval. In contrast, there was little difference in uptake rates over these intervals for cells>3.0 m. At Sproat Lake phosphate incorporation into the two size fractions was followed after the aerial fertilization of the lake with inorganic nutrients. Following nutrient addition the proportion of phosphate entering the>3.0 m size fraction increased from ca. 35% to ca. 85%. Despite these observations, it is doubtful that larger cells are able to sequester enough phosphate from pulses to realize the same specific growth rates as their smaller counterparts.     

Molecular modeling of the intercalation of porphyrins into α-zirconium phosphate

In this work, n-alkylamines (number of carbon atoms ranging from 3 to 10) were investigated in detail by molecular modeling as spacers for intercalating porphyrins into α-zirconium phosphate (α-ZrP). Pre-intercalated n-alkylamines can form either a flat monolayer or a canted bilayer in the gallery of α-ZrP. Based on the interlayer state and intercalative potential of the two modes in α-ZrP, it is suggested that the flat monolayer is a better spacer than the bilayer and that n-propylamine (PA) and n-butylamine (BA) in mobile monolayers are the best spacers among the n-alkylamines studied, as is also found experimentally. The intercalation behavior of TMPyP [5,10,15,20-tetrakis (1-methylpyridinium-4-yl) porphyrin] and several other porphyrins was investigated by calculating the intercalative potential. The calculated results showed that the porphyrins were densely packed in a canted monolayer model, and an increase of polarity of the substituent would facilitate the intercalation of the porphyrins. Figure Schematic representation of platform of intercalated spacers and guests taking n-butylamine and TMPyP as an example, respectively: a a flat monolayer of n-bultylamine in α-ZrP; b a canted monolayer of TMPyP in α-ZrP; c the top layer of the canted bilayer n-bultylamine in α-ZrP (the gray area indicates the amphiphilic distribution on the interface between α-ZrP layers and n-alkylamine/porphyrin).      

Structure and properties of starch/α-zirconium phosphate nanocomposite films

Glycerol-plasticized pea starch/α-zirconium phosphate (PS/ZrP) nanocomposite films with different loading levels of α-zirconium phosphate (α-ZrP) were prepared by a casting and solvent evaporation method. The effects of the α-ZrP on the structure and properties of the PS/ZrP films were characterized by Fourier transform infrared (FT-IR) spectroscopy, wide-angle X-ray diffraction (XRD), scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and tensile testing. The results indicated that hydrogen bonds formed between pea starch (PS) and α-ZrP, which improved the compatibility between PS and α-ZrP. Compared with the neat PS, the tensile strength (σ_b) and elongation at break (ε_b) of the PS/ZrP nanocomposite films were significantly enhanced with an increase in α-ZrP content. The maximum values of σ_b and ε_b reached 9.44 MPa and 47.5%, respectively, at 0.3% α-ZrP and 25% glycerol as plasticizer. The moisture uptake of the nanocomposite films, measured in an environment with 92% relative humidity, was reduced by the addition of α-ZrP. The structure and properties of pea starch-based films were modified and improved by the incorporation of α-ZrP.     

Microscopic study on resorption of β-tricalcium phosphate materials by osteoclasts

Sintered compounds prepared with β-tricalcium phosphate (β-TCP) are commonly used as biocompatible materials for bone regenerative medicine. Although implanted β-TCP is gradually replaced with new bone after resorption by osteoclasts, exactly how osteoclasts resorb β-TCP is not well understood. To elucidate this mechanism, we analyzed the structure of β-TCP discs on which mouse mature osteoclasts were cultured using scanning electron microscopy. We found that β-TCP was resorbed by mature osteoclasts on one side of each disc, as evidenced by the formation of multiple spine-like crystals at the exposed areas. Because osteoclasts secrete acid to resorb bone minerals, we mimicked this acidification by dipping β-TCP slices into HCl solution (pH 2.0). However, no spine-like crystals appeared even though the size of each β-TCP particle was reduced. On dentin slices, osteoclasts formed clear actin rings, which are cytoskeletal structures characteristic of bone-resorbing osteoclasts. No clear actin rings were observed in osteoclasts cultured on β-TCP slices, although small actin dots were observed. Analysis by transmission electron microscopy showed that osteoclasts attached to β-TCP particles. These results suggest that osteoclasts resorb β-TCP particles independently of clear actin ring formation.     

Flavonoids: True or promiscuous inhibitors of enzyme? The case of deoxyxylulose phosphate reductoisomerase

Flavonoids, due to their physical and chemical properties (among them hydrophobicity and metal chelation abilities), are potential inhibitors of the 1-deoxyxylulose 5-phosphate reductoisomerase and most of the tested flavonoids effectively inhibited its activity with encouraging IC₅₀ values in the micromolar range. The addition of 0.01% Triton X100 in the assays led however, to a dramatic decrease of the inhibition revealing that a non-specific inhibition probably takes place. Our study highlights the possibility of erroneous conclusions regarding the inhibition of enzymes by flavonoids that are able to produce aggregates in micromolar range. Therefore, the addition of a detergent in the assays prevents possible false positive hits in high throughput screenings.     

Seven-membered cyclic phosphate: synthesis and properties of S-cycloadenosine 2′,5′-cyclic phosphate and cordycepin 2′,5′-cyclic phosphate. Studies of nucleosides and nucleotides. LXIV1). Purine cyclonucleosides 25

8,3′-Anhydro-8-mercapto-9-β-D-xylofuranosyladenine (8,3′-s-cycloadenosine) was phosphorylated with cyanoethyl phosphate and DCC to 5′-phosphate. After 6-amino group was benzoylated, the monophosphate was treated with DCC to give a cyclic phosphate (II). The structure of compound II was elucidated as 8,3′-s-cycloadenosine 2′,5′-cyclic phosphate by UV, NMR and CD spectra, as well as enzymatic hydrolyses. When compound II was desulfurized with Raney nickel, cordycepin 2′,5′-cyclic phosphate (III) was obtained. Although compound III could be obtained from cordycepin 5′-phosphate with DCC, the yield was extremely low.     

Degradation of phytate in the gut of pigs ‐ pathway of gastrointestinal inositol phosphate hydrolysis and enzymes involved

The present study gives an overview on the whole mechanism of phytate degradation in the gut and the enzymes involved. Based on the similarity of the human and pigs gut, the study was carried out in pigs as model for humans. To differentiate between intrinsic feed phytases and endogenous phytases hydrolysing phytate in the gut, two diets, one high (control diet) and the other one very low in intrinsic feed phytases (phytase inactivated diet) were applied. In the chyme of stomach, small intestine and colon inositol phosphate isomers and activities of phytases and alkaline phosphatases were determined. In parallel total tract phytate degradation and apparent phosphorus digestibility were assessed. In the stomach chyme of pigs fed the control diet, comparable high phytase activity and strong phytate degradation were observed. The predominant phytate hydrolysis products were inositol phosphates, typically formed by plant phytases. For the phytase inactivated diet, comparable very low phytase activity and almost no phytate degradation in the stomach were determined. In the small intestine and colon, high activity of alkaline phosphatases and low activity of phytases were observed, irrespective of the diet fed. In the colon, stronger phytate degradation for the phytase inactivated diet than for the control diet was detected. Phytate degradation throughout the whole gut was nearly complete and very similar for both diets while the apparent availability of total phosphorus was significantly higher for the pigs fed the control diet than the phytase inactivated diet. The pathway of inositol phosphate hydrolysis in the gut has been elucidated.     

Inorganic phosphate homeostasis in sodium-dependent phosphate cotransporter Npt2b⁺/⁻ mice

An inorganic phosphate (P(i))-restricted diet is important for patients with chronic kidney disease and patients on hemodialysis. Phosphate binders are essential for preventing hyperphosphatemia and ectopic calcification. The sodium-dependent P(i) (Na/P(i)) transport system is involved in intestinal P(i) absorption and is regulated by several factors. The type II sodium-dependent P(i) transporter Npt2b is expressed in the brush-border membrane in intestinal epithelial cells and transports P(i). In the present study, we analyzed the phenotype of Npt2b(-/-) and hetero(+/-) mice. Npt2b(-/-) mice died in utero soon after implantation, indicating that Npt2b is essential for early embryonic development. At 4 wk of age, Npt2b(+/-) mice showed hypophosphatemia and low urinary P(i) excretion. Plasma fibroblast growth factor 23 levels were significantly decreased and 1,25(OH)(2)D(3) levels were significantly increased in Npt2b(+/-) mice compared with Npt2b(+/+) mice. Npt2b mRNA levels were reduced to 50% that in Npt2b(+/+) mice. In contrast, renal Npt2a and Npt2c transporter protein levels were significantly increased in Npt2b(+/-) mice. At 20 wk of age, Npt2b(+/-) mice showed hypophosphaturia and reduced Na/P(i) cotransport activity in the distal intestine. Npt2b(+/+) mice with adenine-induced renal failure had hyperphosphatemia and high plasma creatinine levels. Npt2b(+/-) mice treated with adenine had significantly reduced plasma P(i) levels compared with Npt2b(+/+) mice. Intestinal Npt2b protein and Na(+)/P(i) transport activity levels were significantly lower in Npt2b(+/-) mice than in the Npt2b(+/+) mice. The findings of the present studies suggest that Npt2b is an important target for the prevention of hyperphosphatemia.     

Teichoic acids possessing phosphate–sugar linkages in strains of Lactobacillus plantarum

Cell walls of strains of Lactobacillus plantarum lacking the group D precipitinogen (a glucosylribitol teichoic acid) contain glucosylglycerol teichoic acid in which the glycosidic substituents are attached to the primary hydroxyl group of glycerol. Three distinct repeating units have been isolated from the teichoic acid preparation of strain C106, indicating either that the polymer is complex or that the wall contains a mixture of teichoic acids. Walls of streptobacteria differ from those of L. plantarum and contain neither teichoic acid nor diaminopimelic acid.     

Binding of reduced nicotinamide adenine dinucleotide phosphate destabilizes the iron−sulfur clusters of human mitoNEET

The outer mitochondrial membrane protein mitoNEET is a cellular target of the antidiabetic drug pioglitazone. Binding of pioglitazone stabilizes the protein against [2Fe-2S] cluster release. Here, we report that reduced nicotinamide adenine dinucleotide phosphate (NADPH) can bind to homodimeric mitoNEET, influencing the stability of the [2Fe-2S] cluster that is bound within a loop region (Y71?H87) in each subunit. Nuclear magnetic resonance (NMR) and isothermal titration calorimetry experiments demonstrated that NADPH binds weakly to mitoNEET(44?108), a soluble domain of mitoNEET containing residues 44?108. Visible?UV absorption measurements revealed the destabilizing effect of NADP binding on the [2Fe-2S] clusters. Disruption of the three-dimensional structure of mitoNEET(44?108) as a result of decomposition of the iron?sulfur clusters was observed by NMR and circular dichroism experiments. Binding of NADPH facilitated release of the iron?sulfur clusters from the protein at pH≤7.0. Residues K55 and H58 of each subunit of mitoNEET were shown to be involved in NADPH binding. NADPH binding may perturb the interactions of K55 and H58 from one subunit with H87′ and R73′, respectively, from the other subunit, thereby interfering with [2Fe-2S] cluster binding. This may account for the destabilization effect of NADPH binding on the [2Fe-2S] clusters.     

Mutational analysis of the 5′-OH oligonucleotide phosphate acceptor site of T4 polynucleotide kinase

T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5′-kinase and 3′-phosphatase activities that function in nucleic acid repair. The N-terminal kinase domain belongs to the P-loop phosphotransferase superfamily. The kinase is distinguished by a tunnel-like active site with separate entrances on opposite sides of the protein for the NTP phosphate donor and a 5′-OH single-stranded oligonucleotide phosphate acceptor. Here, we probed by mutagenesis the roles of individual amino acids that comprise the acceptor binding site. We thereby identified Glu57 as an important residue, by virtue of its participation in a salt bridge network with two catalytic residues identified previously: Arg38, which binds the 3′-phosphate of the terminal 5′-OH nucleotide, and the putative general base Asp35 that contacts the nucleophilic 5′-OH group. The 5′-OH nucleoside fits into a pocket lined by aliphatic amino acids (Val131, Pro132 and Val135) that make van der Waals contacts to the nucleobase. Whereas subtraction of these contacts by single alanine substitutions for Val131 or Val135 and glycine for Pro132 had modest effects on kinase activity, the introduction of bulkier phenylalanines for Val131 and Val135 were deleterious, especially V131F, which severely impeded both substrate binding (increasing K_m by 15-fold) and catalysis (decreasing k_cat by 300-fold).     

The redox state of free nicotinamide–adenine dinucleotide phosphate in the cytoplasm of rat liver

1. The concentrations of the oxidized and reduced substrates of the ;malic' enzyme (EC 1.1.1.40) and isocitrate dehydrogenase (EC 1.1.1.42) were measured in freeze-clamped rat livers. By assuming that the reactants of these dehydrogenase systems are at equilibrium in the cytoplasm the [free NADP(+)]/[free NADPH] ratio was calculated. The justification of the assumption is discussed. 2. The values of this ratio obtained under different nutritional conditions (well-fed, 48hr.-starved, fed with a low-carbohydrate diet, fed with a high-sucrose diet) were all of the same order of magnitude although characteristic changes occurred on varying the diet. The value of the ratio fell on starvation and on feeding with the low-carbohydrate diet and rose slightly on feeding with the high-sucrose diet. 3. The mean values of the ratio were calculated to be between 0.001 and 0.015, which is about 100000 times lower than the values of the cytoplasmic [free NAD(+)]/[free NADH] ratio. 4. The differences in the redox state of the two nicotinamide-adenine dinucleotide couples can be explained on a simple physicochemical basis. The differences are the result of equilibria that are determined by the equilibrium constants of a number of highly active readily reversible dehydrogenases and transaminases and the concentrations of the substrates and products of these enzymes. 5. The decisive feature is the fact that the NAD and NADP couples share substrates. This sharing provides a link between the redox states of the two couples. 6. The application of the method of calculation to data published by Kraupp, Adler-Kastner, Niessner & Plank (1967), Goldberg, Passonneau & Lowry (1966) and Kauffman, Brown, Passonneau & Lowry (1968) shows that the redox states of the NAD and NADP couples in cardiac-muscle cytoplasm and in mouse-brain cytoplasm are of the same order as those in rat liver. 7. The determination of the equilibrium constant at 38 degrees , pH7.0 and I 0.25 (required for the calculation of the [free NADP(+)]/[free NADPH] ratio), gave a value of 3.44x10(-2)m for the ;malic' enzyme (with CO(2) rather than HCO(3) (-) as the reactant) and a value of 1.98x10(-2)m(-1) for glutathione reductase.     

Partitioning of α-lactalbumin and β-lactoglobulin in aqueous two-phase systems of polyvinylpyrrolidone and potassium phosphate

In the present study, the partitioning of α-lactalbumin, β-lactoglobulin, and cheese whey proteins in aqueous two-phase system of polyvinylpyrrolidone-potassium phosphate is investigated. The partitioning of proteins in this system depends on the polymer and salt weight percents in feed, temperature, and pH. The orthogonal central composite design is used to study the effects of different parameters on partitioning of α-lactalbumin and β-lactoglobulin. A second order model is proposed to determine the impact of these parameters. The results of the model show that the weight percent of the salt in feed has a large effect on the protein partitioning. The weight percent of polyvinylpyrrolidone in the feed increases the partitioning coefficients. By increasing the temperature, the viscosity of polyvinylpyrrolidone is reduced and the protein can easily be transferred from one phase to the other phase. The pH of the aqueous two phase system can alter the protein partitioning coefficient through the variation of the protein net charge.     

The biocompatibility of calcium phosphate cements containing alendronate-loaded PLGA microparticles in?vitro

The composite of poly-lactic-co-glycolic acid (PLGA) and calcium phosphate cements (CPC) are currently widely used in bone tissue engineering. However, the properties and biocompatibility of the alendronate-loaded PLGA/CPC (APC) porous scaffolds have not been characterized. APC scaffolds were prepared by a solid/oil/water emulsion solvent evaporation method. The morphology, porosity, and mechanical strength of the scaffolds were characterized. Bone marrow mesenchymal stem cells (BMSCs) from rabbit were cultured, expanded and seeded on the scaffolds, and the cell morphology, adhesion, proliferation, cell cycle and osteogenic differentiation of BMSCs were determined. The results showed that the APC scaffolds had a porosity of 67.43 ± 4.2% and pore size of 213 ± 95 µm. The compressive strength for APC was 5.79 ± 1.21 MPa, which was close to human cancellous bone. The scanning electron microscopy, cell counting kit-8 assay, flow cytometry and ALP activity revealed that the APC scaffolds had osteogenic potential on the BMSCs in vitro and exhibited excellent biocompatibility with engineered bone tissue. APC scaffolds exhibited excellent biocompatibility and osteogenesis potential and can potentially be used for bone tissue engineering.     

Is the isotopically exchangeable phosphate of a loamy soil the plant-available P?

Carduus nutans L. is an invasive pasture/grassland species which may undergo rapid population growth through positive feedback. Plants ofC. nutans produce a vegetative rosette, and after several months produce stems containing flower-heads, during which time the rosette leaves die and decompose. We investigated the influence ofC. nutans on the nitrogen-fixation ability ofTrifolium repens L. in three experiments. The first experiment was set up in a mixture design, and demonstrated that seedlings ofT. repens were more susceptible to competition with otherT. repens seedlings than toC. nutans seedlings. Nodule numbers and acetylene reduction per unit root, and acetylene reduction per unit nodules were adversely affected by increasingT. repens, but notC. nutans densities. The second experiment was of an additive design, with separate partitions to isolate above-ground and below-ground interference. FloweringC. nutans plants strongly inhibitedT. repens root growth, nodulation and acetylene reduction, but usually only when shoot interference was permitted. This appears to be due to decomposition of rosette leaves, which was maximal at this stage. The third experiment involved monitoring effects of taggedC. nutans individuals againstT. repens in the field. This experiment showed that acetylene reduction was severely influenced by floweringC. nutans (when rosette leaves were decomposing), even when only mild reduction ofT. repens growth was observed, and these effects persisted for some months after theC. nutans plants had died. The results of these experiments in combination suggest that decomposing rosette leaves have a strong potential to inhibitT. repens nitrogen fixation. It appears that allelopathy is involved, since alternative explanations (e.g. root competition byC. nutans; effects ofC. nutans on soil moisture, microbial nutrient immobilisation and light availability; facilitation of herbivores byC. nutans) can be effectively discounted. Although invasive species are often assumed to be associated with soil nitrogen build-up, we believe that some invasive species such asC. nutans have the potential to induce long-term decline of soil nitrogen input.