Characteristics of Triose Phosphate／Phosphate Translocator from Wheat and Its Role in the Distribution of Assimilates

In plants, triose phosphate/phosphate translocator (TPT) is the first regulation point forpartitioning of photosynthate between source and sink. Studies on the characteristic of TPT and itsregulation on the distribution of assimilates are critical for improving the utilization rate of photosyntheticassimilates. Chloroplasts with intactness of more than 91% and high purity were isolated from wheat( Triticurn aestivurn L. cv. Jing 411) leaves. Analysis of SDS-PAGE and labeling with an irreversible specificinhibitor, [H3]2^-DIDS (4, 4‘-diisothiocyano-2, 2‘-stilbenedisulfonate, DIDS) demonstrated that wheat TPTwas a chloroplast membrane protein with a 35 kD molecular weight, which comprised about 15% of the totalmembrane proteins of chloroplasts. Western blotting analysis showed that wheat TPT is uniquelydistributed in the envelope membrane of chloroplasts, but not detected in the membranes of vacuoles andmitochondria. The silicone-oil-layer centrifugation system was employed to study the kinetic properties ofTPT. The results showed that the maximal transport activity of TPT was the highest for dihydroxyacetonephosphate (DHAP)/inorganic phosphate (Pi), then for phosphoenolpyruvate (PEP)/Pi and glucose-6-phosphate (G6P)/Pi. The Km value of TPT was the lowest for DHAP, followed by Pi, PEP and G6P,therefore the most preferred substrate of TPT is DHAP. The transport of wheat TPT to DHAP was stronglyinhibited by DIDS with a degree of 95%. Inhibition of TPT transport activity led to an obvious accumulationof starch in chloroplasts, therefore the TPT protein of wheat controls the export of TP out of chloroplastsinto cytosol. Except for the need of participating in the Calvin cycle, the ratio of TP exported out ofchloroplast to the one used for synthesizing starch was at least 93.6:6.4. The TPT protein from wheat hasmuch high transport efficiency, which plays an important role in the regulation of the distribution ofassimilates in wheat chloroplasts.     

Phosphate effects in the fermentation of α-amylase by Bacillus amyloliquefaciens

Summary The effects of phosphate on -amylase fermentation byBacillus amyloliquefaciens were investigated. It was observed through batch culture that optimal phosphate level which maximizes -amylase biosynthesis exists. High concentration of phosphate level promotes maltose uptake and growth of the microorganism, while high maltose uptake rate in the microorganism at the same time represses the enzyme biosynthesis presumably due to catabolite repression inside the microorganism. In continuous cultivation, a steady state of -amylase biosynthesis was obtained by maintaining phosphate level at a certain level. In fed-batch culture, by intermittant feeding of phosphate as well as maltose, higher activity of -amylase in the broth was obtained compared to the result from single nutrient feeding.     

Isolation of Phosphate Solubilizing Bacteria from Maize Rhizosphere and Their Potential for Rock Phosphate Solubilization–Mineralization and Plant Growth Promotion

Phosphate solubilizing bacteria (PSB) play a significant role in plant P nutrition by their effect on soil P dynamics and their subsequent ability to make P available to plants via solubilization and mineralization processes. This study aimed to evaluate the effect of separate and combined use of indigenous PSB, poultry manure (PM) and compost on solubilization and mineralization of rock phosphate (RP) and their subsequent effect on growth and P accumulation of maize (Zea mays L.). A group of fifty seven bacteria were isolated from the rhizosphere/rhizoplane of maize that had been grown in soils collected from varying altitudes (655–2,576 m) of the mountain region of Rawalakot, Azad Jammu and Kashmir, Pakistan. After screening, the capacity of eleven isolates to solubilize mineral phosphate was quantitatively evaluated using insoluble Ca₃(PO₄)₂ in culture medium as a time course study through spectrometer. The growth hormone producing (IAA) capacity of the isolates was also determined. Furthermore, five potential isolates were tested for their ability to increase P release capacity (mineralization) of insoluble RP in an incubation study. The effect of PSB inoculation on maize was determined in a completely randomized greenhouse experiment where root and shoot biomass and P accumulation in plants were assessed. The P solubilization index of selected isolates varied from 1.94 to 3.69, while the P solubilization efficiency ranged between 94.1% and 269.0%. The isolates MRS18 and MRS27 displayed the highest values. The P solubilization in the liquid medium was maximum at 6 and 9 days of incubation ranging between 9.91 and 44.04 µgmL^?1 and the isolates MRS27 and MRS34 exhibited the highest solubilization. Six isolates showed additional capability of producing IAA ranging between 2.66 and 28.41 µgmL^?1. Results of the incubation study indicated that P release capacity (P mineralization) of RP-amended soil varied between 6.0 and 11.8 µgPg^?1 that had been significantly increased to 30.6–36.3 µgPg^?1 (maximum value) when PSB were combined with RP. The combined application of PSB and organic amendments (PM, compost) with RP further increased P mineralization by releasing a maximum of 37.7 µgPg^?1 compared with separate application of RP (11.8 µgPg^?1) and organic amendments (21.5 and 16.5 µgPg^?1). The overall effect of PSB (as a group) with RP over RP alone on maize growth showing a relative increase in shoot length 21%, shoot fresh weight 42%, shoot dry weight 24%, root length 11%, root fresh weight 59%, root dry weight 35% and chlorophyll content 32%. This study clearly indicates that use of PSB, and organic amendments with insoluble RP could be a promising management strategy to enhance P availability in soil pool and improve plant growth in intensive cropping systems.     

Phosphate carrier of liver mitochondria: The reaction of its SH groups with mersalyl, 5,5′-dithio-bis-nitrobenzoate,andN-ethylmaleimide and the modulation of reactivity by the energy state of the mitochondria

The inhibitory effect of three SH reagents, mersalyl, 5,5-dithio-bis-nitrobenzoate, andN-ethylmaleimide, on P_i transport in rat liver mitochondria was investigated under a variety of conditions. Mersalyl binds at room temperature with both high (K _d<10 µM) and low affinity to mitochondria. Inhibition of P_i transport by mersalyl goes in parallel with titration of the high-affinity sites, inhibition being complete when 3.5–4.5 nmol/mg protein is bound to the mitochondria. At concentrations of mersalyl equal to or higher than 10 µM, inhibition of P_i transport occurs in less than 10 sec. At concentrations of mersalyl lower than 10 µM, the rate of reaction with the P_i carrier is considerably decreased. At a concentration of 100 µM, 5,5-dithio-bisnitrobenzoate fully inhibits P_i transport in about 1 min at room temperature. Nearly total inhibition is attained when as little as 40–50 pmol/mg is bound to mitochondria. Upon incubation longer than 1 min, additional SH groups, not belonging to the P_i carrier, begin to react. The uncoupler carbonyl cyanidep-trifluoromethoxyphenylhydrazone decreases the rate of reaction of mersalyl, 5,5-dithio-bis-nitrobenzoate, andN-ethylmaleimide with the P_i carrier. Preincubation with P_i has a similar effect. We propose that both carbonyl cyanidep-trifluoromethoxyphenylhydrazone and P_i act by increasing the acidity of the mitochondrial matrix. Protonation of the P_i carrier at the matrix side would change the accessibility of its SH groups at the outer surface of the inner membrane. This might correspond to a membrane-Bohr effect, possibly related to the opening of a gating pore in the P_i carrier.     

Ligustrazine Phosphate Ethosomes for Treatment of Alzheimer’s Disease, In Vitro and in Animal Model Studies

In the present study, we have investigated transdermal administration of ligustrazine phosphate (LP), as an antioxidant, for the treatment of Alzheimer's disease (AD). The LP transdermal ethosomal system was designed and characterized. Franz-type diffusion cells and confocal laser scanning microscopy were used for the in vitro permeation studies. Furthermore, the effect of LP transdermal ethosomal system on AD was evaluated in the scopolamine-induced amnesia rats by evaluating the behavioral performance in the Morris water maze test. The activities of the antioxidant enzymes and the levels of the lipid peroxidation product malondialdehyde (MDA) in the brain of rats were also determined. The results showed that both the penetration ability and the drug deposition in skin of the LP ethosomal system were significantly higher than the aqueous one. The LP transdermal ethosomal system could recover the activities of the antioxidant enzymes and the levels of MDA in the brain of the amnesic rats to the similar status of the normal rats, which was also indirectly reflected by the improvement in the behavioral performance. In conclusion, LP might offer a potential alternative therapeutic drug in the fight against AD, and ethosomes could be vesicles of choice for transdermal delivery of LP.     

Effects of Detergent β-Octylglucoside and Phosphate Salt Solutions on Phase Behavior of Monoolein Mesophases

Using small-angle x-ray scattering (SAXS), we investigated the phase behavior of mesophases of monoolein (MO) mixed with additives commonly used for the crystallization of membrane proteins from lipidic mesophases. In particular, we examined the effect of sodium and potassium phosphate salts and the detergent β-octylglucoside (βOG) over a wide range of compositions relevant for the crystallization of membrane proteins in lipidic mesophases. We studied two types of systems: 1), ternary mixtures of MO with salt solutions above the hydration boundary; and 2), quaternary mixtures of MO with βOG and salt solutions over a wide range of hydration conditions. All quaternary mixtures showed highly regular lyotropic phase behavior with the same sequence of phases (Lα, Ia3d, and Pn3m) as MO/water mixtures at similar temperatures. The effects of additives in quaternary systems agreed qualitatively with those found in ternary mixtures in which only one additive is present. However, quantitative differences in the effects of additives on the lattice parameters of fully hydrated mesophases were found between ternary and quaternary mixtures. We discuss the implications of these findings for mechanistic investigations of membrane protein crystallization in lipidic mesophases and for studies of the suitability of precipitants for mesophase-based crystallization methods.     

Lack of Control in Inorganic Phosphate Uptake by Catharanthus roseus (L.) G. Don Cells (Cytoplasmic Inorganic Phosphate Homeostasis Depends on the Tonoplast Inorganic Phosphate Transport System?)

Inorganic phosphate (Pi) uptake by Catharanthus roseus (L.) G. Don cells was studied in relation to its apparent uncontrolled uptake using 31P-nuclear magnetic resonance spectroscopy. Kinetics of Pi uptake by the cells indicated that apparent Km and Vm were about 7 [mu]M and 20 [mu]mol g-1 fresh weight h-1, respectively. Pi uptake in Murashige-Skoog medium under different Pi concentrations and different initial cell densities followed basically the same kinetics. When supplied with abundant Pi, cells absorbed Pi at a constant rate (Vm) for the first hours and accumulated it in the vacuole. As the endogenous pool expanded, the rate of Pi uptake gradually decreased to nil. Maximum Pi accumulation was 100 to 120 [mu]mol g-1 fresh weight if cell swelling during Pi uptake (about 2-fold in cell volume) was not considered. Results indicated that (a) the rate of Pi uptake by Catharanthus cells was independent of initial cell density and was constant over a wide range of Pi concentrations (2 mM to about 10 [mu]M) unless the cells were preloaded with excess Pi, and (b) there was no apparent feedback control over the Pi uptake process in the plasma membrane to avoid Pi toxicity. The importance of the tonoplast Pi transport system in cytoplasmic Pi homeostasis is discussed.     

Selectivty Assessments of a Sequential Extraction Procedure for Potential Trace Metals’ Mobility and Bioavailability in Phosphate Rocks from Jordan Phosphate Mines

A modified six-step sequential extraction procedure was used to fractionate and determine the following trace metals: U⁴⁺, As⁵⁺, Cd²⁺, Cr⁺², Cu²⁺, Ni²⁺, Pb²⁺, Zn²⁺, and V⁵⁺ in three different phosphate rocks from mines in Jordan. The mean values of uranium in the samples investigated were 98 ± 6 mgkg^?1, 92 ± 3 mgkg^?1, 215 ± 6 mgkg^?1, and 159 ± 13 mgkg^?1, respectively. The sequential extraction results obtained showed that most of the U⁴⁺ in these samples was strongly bound with 87%, 93%, 97%, and 93% of the total content, respectively, remaining in the samples after the sequential extraction steps were performed. Hence, 13%–7% and 3%–7%, respectively, of the U⁴⁺ is distributed in the most labile form, indicating that the majority of the U⁴⁺ in these samples was highly incorporated within the apatite present in the samples. The aforementioned was in agreement with the XRD and SEM-EDX results obtained. The apparent mobility of U⁴⁺, As⁵⁺, Cd²⁺, Cr⁺², Cu²⁺, Ni²⁺, Pb²⁺, Zn²⁺, and V⁵⁺ (using all six extraction steps) from the Al-Abied and Al-Hasa samples was as follows: As⁵⁺ (30.17%)> Cu²⁺ (6.55%)> Zn²⁺(4.34%)> Cd²⁺ (3.84%) Cr⁺² (3.66%)> Pb²⁺ (2.57%)> V⁵⁺ (53%)> Ni²⁺ (1.71%)> U⁴⁺ (0.99%). The mobility of As⁵⁺, Cd²⁺, Cu²⁺, Cr⁺², Ni²⁺, Pb²⁺, U⁴⁺, Zn²⁺, and V⁵⁺ (using all six extraction steps) from Eshidiya samples was as follows: As⁵⁺ (17.32%)> Cr⁺² (4.84%)> Zn²⁺ (4.25%)> Pb²⁺ (4.19%)> Cu²⁺ (3.49%)> V⁵⁺ (1.42%)> Cd²⁺ (0.78) U⁴⁺ (0.09%)> Ni²⁺ (0%).     

Modulation of Brain Glutathione Reductase and Peroxiredoxin 2 by α-Tocopheryl Phosphate

α-Tocopheryl phosphate (αTP) is a phosphorylated form of α-tocopherol. Since it is phosphorylated in the hydroxyl group that is essential for the antioxidant property of α-tocopherol, we hypothesized that αTP would modulate the antioxidant system, rather than being an antioxidant agent per se. α-TP demonstrated antioxidant activity in vitro against iron-induced oxidative stress in a mitochondria-enriched fraction preparation treated with 30 or 100 µM α-TP. However, this effect was not observed ex vivo with mitochondrial-enriched fraction from mice treated with an intracerebroventricular injection of 0.1 or 1 nmol/site of αTP. Two days after treatment (1 nmol/site αTP), peroxiredoxin 2 (Prx2) and glutathione reductase (GR) expression and GR activity were decreased in cerebral cortex and hippocampus. Glutathione content, glutathione peroxidase, and thioredoxin reductase activities were not affected by αTP. In conclusion, the persistent decrease in GR and Prx2 protein content is the first report of an in vivo effect of αTP on protein expression in the mouse brain, potentially associated to a novel and biologically relevant function of this naturally occurring compound.     

Purification of 5′-O-Trityl-on Oligoribonucleotides. Investigation of Phosphate Migration During Purification and Detritylation.

Abstract

Synthetic oligoribonucleotides (RNA) are efficiently prepared with 2′-O-tert-butyldimethylsilyl nucleoside 3′-O-phosphoramidites with labile base-protection; A_dmf or A_Pac, G_dmf, C_ibu, U. After cleavage from the polystyrene support, the exocyclic amine protecting groups are removed with conc. NH₄OH: ethanol/3:1 by heating at 55°C for 3–5 h. The 2′-O- silyl protecting groups are removed with tetra-n-butylammonium fluoride in THF or more conveniently with neat triethylamine trihydrofluoride. To gain the advantages of increased capacity on reverse phase HPLC and the convenience of cartridge based purification (OPC, Oligonucleotide Purification Cartridge), the 5′ trityl was left on the RNA as the final protecting group to be removed. The mild conditions which are effective for trityl removal are shown to preserve 3′-5′ phosphate linkage integrity in RNA. The absence of phosphate migration is demonstrated by model studies, utilizing N⁴ -isobutyryl-5′-O-DMT-3′-O-TBDMS-2′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) as a control monomer and digestion by 3′-5′ selective P1 nuclease and alkaline phosphatase and HPLC analysis. Oligoribonucleotides were analyzed by Microgel capillary electrophoresis, anion-exchange HPLC, and the enzymatic digest/HPLC method.

     

Influence of Architecture of β-Tricalcium Phosphate Scaffolds on Biological Performance in Repairing Segmental Bone Defects

Background

Although three-dimensional (3D) β-tricalcium phosphate (β-TCP) scaffolds serve as promising bone graft substitutes for the segmental bone defect treatment, no consensus has been achieved regarding their optimal 3D architecture.

Methods

In this study, we has systematically compared four types of β-TCP bone graft substitutes with different 3D architectures, including two types of porous scaffolds, one type of tubular scaffolds and one type of solid scaffolds, for their efficacy in treating segmental bone defect in a rabbit model.

Results

Our study has demonstrated that when compared to the traditional porous and solid scaffolds, tubular scaffolds promoted significantly higher amount of new bone formation in the defect regions as shown by X-ray, micro CT examinations and histological analysis, restored much greater mechanical properties of the damaged bone evidenced by the biomechanical testing, and eventually achieved the complete union of segmental defect. Moreover, the implantation of tubular scaffolds enhanced the neo-vascularization at the defect region with higher bone metabolic activities than others, as indicated by the bone scintigraphy assay.

Conclusions

This study has further the current knowledge regarding the profound influence of overall 3D architecture of β-TCP scaffolds on their in vivo defect healing performance and illuminated the promising potential use of tubular scaffolds as effective bone graft substitute in treating large segmental bone defects.     

Role of Calcium and Adenosine Cyclic 3′-5′ Phosphate in Action of Adrenocorticotropin

ALTHOUGH adenosine cyclic monophosphate (cyclic AMP) has been proposed as a mediator through which many hormones exert their physiological effects¹, it is also well established that calcium plays a crucial role in hormone release². Both calcium^3,4 and cyclic AMP^1,5 have been implicated in the action of adrenocorticotropin (ACTH) on the adrenal cortex and although various hypotheses have been advanced concerning their roles in steroid production and release, elucidation of their functions in the adrenal gland is hindered because most studies have been carried out on in vitro systems where the physiological release response cannot be studied. The isolated cat adrenal gland perfused in situ ⁶ approximates the situation in vivo, yet eliminates the influence of several factors, including the anterior pituitary. In the intact adrenal preparation one can also measure both steroid synthesis and release and can better evaluate the respective effects of cyclic AMP and calcium on these processes.     

Oxyanion-Mediated Protein Stabilization: Differential Roles of Phosphate for Preventing Inactivation of Bacterial α-Glucan Phosphorylases

Maltodextrin phosphorylase (MalP) from Escherichia coli and starch phosphorylase (StP) from Corynebacterium callunae are significantly stabilized in the presence of phosphate against inactivation by elevated temperature or urea. The stabilizing effect of phosphate was observed at ion concentrations below 50 mM. Therefore, it is probably due to preferential binding of phosphate to the folded conformations of the phosphorylases. For StP, phosphate binding inhibited the dissociation of the active-site cofactor pyridoxal 5′-phosphate. Phosphate-liganded StP was at least 500-fold more stable at 60d`C than the free enzyme at the same temperature. It showed an apparent transition midpoint of 5.2 M for irreversible denaturation by urea, and this midpoint was increased by a denaturant concentration of 4M relative to the corresponding transition midpoint of free StP in urea. The mechanisms of inactivation and denaturation of MalP at 45d`C and by urea involve formation of a cofactor-containing, insoluble protein aggregate. Under denaturing conditions, phosphate was shown to inhibit aggregation of the reversibly inactivated MalP dimer.     

Regulation of Acid Phosphatase Synthesis in Baker’s Yeast Protoplast by Phosphate Compounds

The regulation of acid phosphatase synthesis by various phosphate compounds was examined in Baker’s yeast protoplasts. Synthesis was repressed by inorganic phosphate and phosphomonoesters. Phosphomonoesters were hydrolysed by a small amount of non-specific acid phosphatase present in the protoplast membrane. The inorganic phosphate that was liberated and incorporated into protoplasts probably repressed acid phosphatase synthesis. Phosphodiesters, such as 3′, 5′-cyclic AMP, 3′, 5′-cyclic CMP and 3′, 5′-cyclic GMP, promoted acid phosphatase synthesis. The effect of 3′, 5′-cyclic AMP was not to overcome hexose repression, because high hexose did not repress acid phosphatase synthesis. 3′, 5′-cyclic AMP did not overcome repression of the enzyme synthesis by inorganic phosphate. From these observations 3′, 5′-cyclic nucleotides probably had some effect on the yeast acid phosphatase-synthesizing system but the exact role of the nucleotides is obscure.     

Lipid Phosphate Phosphatase 3 Stabilization of β-Catenin Induces Endothelial Cell Migration and Formation of Branching Point Structures

Endothelial cell (EC) migration, cell-cell adhesion, and the formation of branching point structures are considered hallmarks of angiogenesis; however, the underlying mechanisms of these processes are not well understood. Lipid phosphate phosphatase 3 (LPP3) is a recently described p120-catenin-associated integrin ligand localized in adherens junctions (AJs) of ECs. Here, we tested the hypothesis that LPP3 stimulates β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) to induce EC migration and formation of branching point structures. In subconfluent ECs, LPP3 induced expression of fibronectin via β-catenin/LEF-1 signaling in a phosphatase and tensin homologue (PTEN)-dependent manner. In confluent ECs, depletion of p120-catenin restored LPP3-mediated β-catenin/LEF-1 signaling. Depletion of LPP3 resulted in destabilization of β-catenin, which in turn reduced fibronectin synthesis and deposition, which resulted in inhibition of EC migration. Accordingly, reexpression of β-catenin but not p120-catenin in LPP3-depleted ECs restored de novo synthesis of fibronectin, which mediated EC migration and formation of branching point structures. In confluent ECs, however, a fraction of p120-catenin associated and colocalized with LPP3 at the plasma membrane, via the C-terminal cytoplasmic domain, thereby limiting the ability of LPP3 to stimulate β-catenin/LEF-1 signaling. Thus, our study identified a key role for LPP3 in orchestrating PTEN-mediated β-catenin/LEF-1 signaling in EC migration, cell-cell adhesion, and formation of branching point structures.Angiogenesis, the formation of new blood vessels, involves several well-coordinated cellular processes, including endothelial cell (EC) migration, synthesis and deposition of extracellular matrix proteins, such as fibronectin, cell-cell adhesion, and formation of branching point structures (1-3, 19, 33); however, less is known about the underlying mechanisms of these processes (6, 8, 12, 14, 16, 17). For example, adherens junctions (AJs), which mediate cell-cell adhesion between ECs, may be involved in limiting the extent of cell migration (2, 14, 38, 40). VE-cadherin, a protein found in AJs, is a single-pass transmembrane polypeptide responsible for calcium-dependent homophilic interactions through its extracellular domains (2, 38, 40). The VE-cadherin cytoplasmic domain interacts with the Armadillo domain-containing proteins, β-catenin, γ-catenin (plakoglobin), and p120-catenin (p120ctn) (2, 15, 38, 40, 43). Genetic and biochemical evidence documents a crucial role of β-catenin in regulating cell adhesion as well as proliferation secondary to the central position of β-catenin in the Wnt signaling pathway (13, 16, 25, 31, 44). In addition, the juxtamembrane protein p120ctn regulates AJ stability via binding to VE-cadherin (2, 7, 9, 15, 21, 28, 32, 43). The absence of regulation or inappropriate regulation of β-catenin and VE-cadherin functions is linked to cardiovascular disease and tumor progression (2, 6).We previously identified lipid phosphate phosphatase 3 (LPP3), also known as phosphatidic acid phosphatase 2b (PAP2b), in a functional assay of angiogenesis (18, 19, 41, 42). LPP3 not only exhibits lipid phosphatase activity but also functions as a cell-associated integrin ligand (18, 19, 35, 41, 42). The known LPPs (LPP1, LPP2, and LPP3) (20-23) are six transmembrane domain-containing plasma membrane-bound enzymes that dephosphorylate sphingosine-1-phosphate (S1P) and its structural homologues, and thus, these phosphatases generate lipid mediators (4, 5, 23, 35, 39). All LPPs, which contain a single N-glycosylation site and a putative lipid phosphatase motif, are situated such that their N and C termini are within the cell (4, 5, 22, 23, 35, 39). Only the LPP3 isoform contains an Arg-Gly-Asp (RGD) sequence in the second extracellular loop, and this RGD sequence enables LPP3 to bind integrins (18, 19, 22). Transfection experiments with green fluorescent protein (GFP)-tagged LPP1 and LPP3 showed that LPP1 is apically sorted, whereas LPP3 colocalized with E-cadherin at cell-cell contact sites with other Madin-Darby canine kidney (MDCK) cells (22). Mutagenesis and domain swapping experiments established that LPP1 contains an apical targeting signal sequence (FDKTRL) in its N-terminal segment. In contrast, LPP3 contains a dityrosine (109Y/110Y) basolateral sorting motif (22). Interestingly, conventional deletion of Lpp3 is embryonic lethal, since the Lpp3 gene plays a critical role in extraembryonic vasculogenesis independent of its lipid phosphatase activity (11). In addition, an LPP3-neutralizing antibody was shown to prevent cell-cell interactions (19, 42) and angiogenesis (42). Here, we addressed the hypothesis that LPP3 plays a key role in EC migration, cell-cell adhesion, and formation of branching point structures by stimulating β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) signaling.     

Hydration of Cytidine, 2′-Deoxycytidine and their Phosphate Salts in the Aqueous Solutions. A Molecular Dynamics Computer Simulation Study

Abstract

To compare the hydration pattern of the cytidine (Cyd) and 2′-deoxycytidine (dCyd) in the aqueous solutions at the level of microscopic interactions, Molecular Dynamics (MD) computer simulations have been undertaken. The results indicate that the hydration of the heterocyclic base moiety in cytidine and 2′-deoxycytidine has a hydrophobic character. None of the three potential Watson—;Crick base pair centres hydrogen bonds with the water molecules and the formation of something akin to a clathrate cage structure of water around base moieties of nucleosides in the aqueous solution is suggested. In contrast, the hydration of Cyd and dCyd sugar moieties shows a hydrophilic character and the three-dimensional networks of H-bonds involving all hydrophilic centres are formed differently around the ribose and 2′-deoxyribose. The sugar hydroxyl groups participate in the hydrogen bonding with water both as H-donor and as H-acceptor. Their donor-acceptor abilities have been evaluated and compared. The coordination numbers, the geometrical data of the first hydration shell, and the number of hydrogen bonds have been calculated. The changes in the pattern of hydration with the increased concentration of nucleosides and upon nucleoside protonation are discussed. The analysis of the pairwise interaction energies are also presented.     

Blockade by Polyphloretin Phosphate of the Prostaglandin F<Subscript>2α</Subscript>, Action on Isolated Human Bronchi

POLYPHLORETIN phosphate (PPP), a polymer with an inhibitory effect on some enzyme systems, such as alkaline phosphatase and hyaluronidase, has been synthesized by Diczfalusy et al.¹ by phosphorylating phloretin with phosphorus oxychloride. PPP has a molecular weight of about 15,000 and is readily soluble in water at pH 7. It has been tried in the treatment of oedema^2,3 and in vitro it inhibits the aggregation of red blood corpuscles⁴ and selectively antagonizes the effect of prostaglandin E₂ (PGE₂) on the intraocular pressure of the rabbit eye⁵. In the light of this finding we have examined whether PPP (supplied by Dr B. Högberg) could also modify the action of prostaglandins, when tested on some other preparation— in this case isolated human bronchi. During this study an investigation of the inhibition by PPP of the PGE₂ and PGF_2α action on isolated bird colon, rabbit jejunum and uterus has been reported⁶. It has been shown that PGE₁ and usually also PGE₂ have a bronchodilating effect in guinea-pig^7,8 and man⁹, while PGF_2α constricts bronchial smooth muscle in vivo and invitro^8,10,11. PGF_2α and PGE₂ have been isolated from human lungs^12,13 and both seem to be released in the anaphylactic reaction of isolated guinea-pig lung.     

Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer's Aβ peptide

The oligomerization of Aβ peptide into amyloid fibrils is a hallmark of Alzheimer’s disease. Due to its biological relevance, phosphate is the most commonly used buffer system for studying the formation of Aβ and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers. Herein, we examine the effects on the fibrillation and oligomerization mechanism of Aβ peptide that occur due solely to the influence of phosphate buffer. We reveal that significant differences in amyloid fibrillation are observed due to fibrillation being initiated in phosphate or HEPES buffer (at physiological pH and temperature). Except for the differing buffer ions, all experimental parameters were kept constant. Fibril formation was assessed using fluorescently monitored kinetic studies, microscopy, X-ray fiber diffraction and infrared and nuclear magnetic resonance spectroscopies. Based on this set up, we herein reveal profound effects on the mechanism and speed of Aβ fibrillation. The three histidine residues at positions 6, 13 and 14 of Aβ(1–40) are instrumental in these mechanistic changes. We conclude that buffer plays a more significant role in fibril formation than has been generally acknowledged.     

A Unique β-1,2-Mannosyltransferase of Thermotoga maritima That Uses Di-myo-Inositol Phosphate as the Mannosyl Acceptor

In addition to di-myo-inositol-1,3′-phosphate (DIP), a compatible solute widespread in hyperthermophiles, the organic solute pool of Thermotoga maritima comprises 2-(O-β-d-mannosyl)-di-myo-inositol-1,3′-phosphate (MDIP) and 2-(O-β-d-mannosyl-1,2-O-β-d-mannosyl)-di-myo-inositol-1,3′-phosphate (MMDIP), two newly identified β-1,2-mannosides. In cells grown under heat stress, MDIP was the major solute, accounting for 43% of the total pool; MMDIP and DIP accumulated to similar levels, each corresponding to 11.5% of the total pool. The synthesis of MDIP involved the transfer of the mannosyl group from GDP-mannose to DIP in a single-step reaction catalyzed by MDIP synthase. This enzyme used MDIP as an acceptor of a second mannose residue, yielding the di-mannosylated compound. Minor amounts of the tri-mannosylated form were also detected. With a genomic approach, putative genes for MDIP synthase were identified in the genome of T. maritima, and the assignment was confirmed by functional expression in Escherichia coli. Genes with significant sequence identity were found only in the genomes of Thermotoga spp., Aquifex aeolicus, and Archaeoglobus profundus. MDIP synthase of T. maritima had maximal activity at 95°C and apparent K_m values of 16 mM and 0.7 mM for DIP and GDP-mannose, respectively. The stereochemistry of MDIP was characterized by isotopic labeling and nuclear magnetic resonance (NMR): DIP selectively labeled with carbon 13 at position C1 of the l-inositol moiety was synthesized and used as a substrate for MDIP synthase. This β-1,2-mannosyltransferase is unrelated to known glycosyltransferases, and within the domain Bacteria, it is restricted to members of the two deepest lineages, i.e., the Thermotogales and the Aquificales. To our knowledge, this is the first β-1,2-mannosyltransferase characterized thus far.Thermotoga maritima was first isolated from hot marine sediments on Vulcano Island, Italy, being able to grow between 55°C and 90°C (14). This strictly anaerobic bacterium ferments a variety of simple and complex carbohydrates to acetate, hydrogen, and CO₂ (10). In line with these metabolic traits, a substantial percentage of the genes annotated in the genome of this hyperthermophile are allocated to the metabolism of mono- and polysaccharides (8, 23). Therefore, T. maritima has been pointed out as a source of glycoside hydrolases with potential industrial relevance, namely, in processes of conversion of biomass into biofuels (3, 34).Like many other hyperthermophiles isolated from marine environments, Thermotoga maritima is slightly halophilic (optimum NaCl concentration of 2.7%, wt/vol) and has developed biochemical strategies to counterbalance the external osmotic pressure. The accumulation of low-molecular-mass organic compounds in the cytoplasm is the most common osmoadaptation mechanism, which enables a rapid response to fluctuations in the salinity of the external medium. Interestingly, the organic solutes encountered in organisms adapted to thrive in hot environments are clearly different from those used by mesophiles, leading to the view that osmolytes of (hyper)thermophiles could play an additional role as protectors of macromolecules and other cellular components against heat damage. This notion is further fuelled by the finding that the total pool of organic solutes of (hyper)thermophiles increases notably not only at supraoptimal salinity but also in response to heat stress conditions (30).Over the last decade, our team has directed considerable effort to assess the role of osmolytes in the thermo-adaptation strategies of hyperthermophiles. Despite the scarcity of genetic tools for manipulation of marine hyperthermophiles, a number of novel organic solutes were identified and the corresponding biosynthetic pathways characterized at the genetic and biochemical levels (15, 17, 30), providing critical knowledge for engaging in elucidation of the molecular basis of the whole process, from the sensing of stress to the synthesis of specific osmolytes. In this context, we recently reported the characterization of the pathway for synthesis of di-myo-inositol-1,3′-phosphate, the most common solute within hyperthermophiles (5). Additionally, the genes and enzymes involved in the relevant reaction steps were disclosed. The synthesis proceeds via a phosphorylated form of DIP, and the respective synthase is a membrane-associated enzyme that catalyzes the condensation of CDP-inositol with inositol-1-phosphate (26, 27).The solute pool in members of the order Thermotogales was investigated a few years ago (19). Thermotoga neapolitana responded to heat stress with a strong accumulation of DIP and DIP derivatives. One of the solutes was assigned to a mannosylated form of DIP, at that time designated di-mannosyl-di-myo-inositol phosphate; moreover, the presence of a second DIP derivative was proposed, but its structure remained elusive. Therefore, we set out to fully characterize the solute pool of Thermotoga spp. and to identify the genes and the enzyme(s) involved in the synthesis of the DIP derivatives. Members of the genus Thermotoga accumulated DIP and two mannosylated forms of this compound, herein fully characterized using isotopic labeling, NMR, and mass spectrometry. Moreover, the pathway for the synthesis of these novel solutes was identified, leading to the discovery of a unique β-1,2-mannosyltransferase that catalyzes the transfer of the mannosyl group from GDP-mannose to DIP.