Free Phosphate Groups Research Articles

Effect of phytic acid/rust conversion layer on corrosion protection of rusty mild steel

In this study, a rust/phytic acid (rust/PA) conversion layer was prepared on rusty mild steel by dipping rusty steel panels into a phytic acid solution. After treating with phytic acid, the free phosphate group in the conversion layer chelated with Fe3+ and generated insoluble salt, which could form a new dense conversion layer. The dense rust/PA conversion layer effectively strengthened the interfacial bonding between the coatings and rusty metal substrate and exhibited good corrosion protection and defect healing abilities.

Ultra-acceleration of Photochemical Cytosine Deamination by Using a 5'-Phosphate-Substituted Oligodeoxyribonucleotide Probe Containing a 3-Cyanovinylcarbazole Nucleotide at Its 5'-End.

Genes are the blueprints for the architectures of living organisms, providing the backbone of the information required for formation of proteins. Changes in genes lead to disorders, and these disorders could be rectified by reversing the mutations that caused them. Photochemical methods currently in use for site-directed mutagenesis employ the photoactive 3-cyanovinylcarbazole (CNV K) nucleotide incorporated in the oligodeoxyribonucleotide (ODN) backbone. The major drawback of this method, the requirement for high temperature, has been addressed, and deamination has previously been achieved at 37 °C but with low efficiency. Here, efficient deamination has been accomplished under physiological conditions by using a short complementary photoactive ODN with a 5'-phosphate group in the -1 position with respect to the target cytosine. It is hypothesized that the free phosphate group affects the microenvironment around the target cytosine by activating the incoming nucleophile through hydrogen bonding with the water molecule, thus facilitating nucleophilic attack on the cytosine C-4 carbon. The degree of deamination observed in this technique is high and the effect of the phosphate group is to accelerate the deamination reaction.

Computational studies of the binding profile of phosphoinositide PtdIns (3,4,5) P₃ with the pleckstrin homology domain of an oomycete cellulose synthase.

Saprolegnia monoica is a model organism to investigate Saprolegnia parasitica, an important oomycete which causes considerable loss in aquaculture every year. S. monoica contains cellulose synthases vital for oomycete growth. However, the molecular mechanism of the cellulose biosynthesis process in the oomycete growth is still poorly understood. Some cellulose synthases of S. monoica, such as SmCesA2, are found to contain a plecsktrin homology (PH) domain, which is a protein module widely found in nature and known to bind to phosphoinositides, a class of signaling compounds involved in many biological processes. Understanding the molecular interactions between the PH domain and phosphoinositides would help to unravel the cellulose biosynthesis process of oomycetes. In this work, the binding profile of PtdIns (3,4,5) P3, a typical phosphoinositide, with SmCesA2-PH was studied by molecular docking, molecular dynamics and metadynamics simulations. PtdIns (3,4,5) P3 is found to bind at a specific site located at β1, β2 and β1-β2 loop of SmCesA2-PH. The high affinity of PtdIns (3,4,5) P3 to SmCesA2-PH is contributed by the free phosphate groups, which have electrostatic and hydrogen-bond interactions with Lys88, Lys100 and Arg102 in the binding site.

Modification of the 1-Phosphate Group during Biosynthesis of Capnocytophaga canimorsus Lipid A.

Capnocytophaga canimorsus, a commensal bacterium of dog's mouth flora causing severe infections in humans after dog bites or scratches, has a lipopolysaccharide (LPS) (endotoxin) with low-inflammatory lipid A. In particular, it contains a phosphoethanolamine (P-Etn) instead of a free phosphate group at the C-1 position of the lipid A backbone, usually present in highly toxic enterobacterial Gram-negative lipid A. Here we show that the C. canimorsus genome comprises a single operon encoding a lipid A 1-phosphatase (LpxE) and a lipid A 1 P-Etn transferase (EptA). This suggests that lipid A is modified during biosynthesis after completing acylation of the backbone by removal of the 1-phosphate and subsequent addition of an P-Etn group. As endotoxicity of lipid A is known to depend largely on the degree of unsubstituted or unmodified phosphate residues, deletion of lpxE or eptA led to mutants lacking the P-Etn group, with consequently increased endotoxicity and decreased resistance to cationic antimicrobial peptides (CAMP). Consistent with the proposed sequential biosynthetic mechanism, the endotoxicity and CAMP resistance of a double deletion mutant of lpxE-eptA was similar to that of a single lpxE mutant. Finally, the proposed enzymatic activities of LpxE and EptA based on sequence similarity could be successfully validated by mass spectrometry (MS)-based analysis of lipid A isolated from the corresponding deletion mutant strains.

Isolation and Characterization of Phytase from Chicken Manure Bacteria

Cereals in animal feed contain anti-nutrients of phytic acid that has capability of chelating proteins and cations. Phytasecan be employed to reduce phytic acid through hydrolyzing phytic acid into free phosphate group and lower derivate ofinositol phosphate. The aim of the study was to isolate and characterize phytase obtained from chicken manure bacteria.The study included the screening of phytase-producing bacteria from chicken manure, the homology analysis of bacterium,and the determination of phytase activity. Phytase activity was measured from concentration of free phosphate. The resultsshowed that one phytase-producing isolate obtained from chicken manure grew in the medium containing 5% rice branextract at 37°C for 5 d. Based on a phylogeny is tree analysis of the genes related to 16S rRNA, the isolate was identified asAcinetobacter sp. TZ1. The extracellular phytase expressed by Acinetobacter sp. TZ1 exhibited optimum reactions at pH5 and 50°C. The enzyme showed activity of 64,6 nmol mL–1min–1 and specific activity of 236 nmol min–1mg–1. Relativemolecular mass of phytase TZ1 was ~35 kDa. Phytase obtained is potential to improve animal feed quality by hydrolyzingphytic acid.

One-step conjugation of aminoferrocene to phosphate groups as electroactive probes for electrochemical detection of sequence-specific DNA

A straightforward electrochemical DNA biosensing approach based on exploiting organometallic compound, aminoferrocene (AFC), as electroactive probes was firstly demonstrated, where the probes could be directly labeled to the free phosphate groups of the hybridized PNA/DNA heteroduplexes merely through one-step conjugation in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and imidazole. Briefly, mercapto-terminated peptide nucleic acid (PNA) was firstly immobilized onto gold electrode and used as the capture probes for the specific recognition of target single-stranded DNA (ssDNA). After hybridization, AFC probes were directly labeled to the free 5′-terminal phosphate groups, which were activated by EDC and imidazole, of the hybridized PNA/DNA heteroduplexes, and then they were exploited as the electroactive probes to monitor the hybridization. As the captured ssDNA was labeled with AFC in the stoichiometric ratio of 1:1, thus the electrochemical analysis of the proportionally labeled AFC based on differential pulse voltammetry (DPV) enabled a quantitative determination of sequence-specific DNA. Under optimal conditions, the approach presented a good linear relationship between the current intensities and logarithm of ssDNA concentrations in the range from 0.1nM to 100nM with a detection limit of 93pM, and it rendered satisfactory analytical performance in serum samples. Furthermore, it exhibited excellent specificity toward single-nucleotide polymorphism (SNP) and precluded complicated protocols. More importantly, the simplicity of this approach together with its compatibility with standard micro-fabrication techniques makes it great potential in practical applications, especially in microarray areas where simple procedures are preferred.

Cloning and expression of apyrase gene from Ancylostoma caninum in Escherechia coli.

Apyrase encoding metal-ions activated plasma membrane protease is present in animal and plant tissues. This enzyme can hydrolyze ADP and ATP pyrophosphate bond, resulting in AMP and free phosphate groups, and plays an important role for insects and parasites to evade host immune system. However localization and function of apyrase in the canine hookworm, Ancylostoma caninum, remains unknown. To analyze apyrase gene in A. caninum (a eukaryotic parasitic hookworm), a pair of primers was designed according to the previous EST data. The full-length cDNA of apyrase gene was amplified from A. caninum by RT-PCR. The partial cDNA of apyrase encodes 249 amino acid protein was expressed in Escherechia coli. The recombinant protein was induced to express under proper conditions and the molecular size was as expected. The recombinant protein was purified. The transcripts of apyrase in different stages of A. caninum were analyzed by the Real-time PCR assay, and Immuno-localization assays were used to research the protein expression in different stages of A. caninum.

Paracrine stimulation of vascular smooth muscle proliferation by diadenosine polyphosphates released from proximal tubule epithelial cells

The purinergic receptor system plays an important role in the regulation of both vascular and tubular functions within the kidney; however, the release of purinergic agonists other than ATP by renal tissue is not known. In this investigation, we determine if kidney tissue is a source of diadenosine polyphosphates, which have high affinity for the P(2X) and P(2Y) receptors. Both diadenosine pentaphosphate and hexaphosphate were identified by matrix-assisted laser desorption ionization-mass spectrometry in extracts purified from both whole porcine kidney and from cloned cells of the LLC-PK1 cell line. Both polyphosphates in nanomolar concentrations were found to significantly stimulate the proliferation of vascular smooth muscle cells derived from rat thoracic aortas. The purinergic-receptor antagonist, suramin, did not significantly affect the growth-stimulatory properties of the polyphosphates. The growth stimulation of vascular smooth muscle cells by platelet-derived growth factor was potentiated by both diadenosine polyphosphates. We conclude that diadenosine polyphosphates are endogenous purinergic agonists of the kidney that have physiologic and pathophysiologic relevance. These epithelial cell metabolic products have vasoregulatory properties while linking the energy supply and tubular function.

Phosphopeptide-selective column-switching RP-HPLC with a titania precolumn.

A methodology of phosphopeptide-selective analysis coupled with column-switching HPLC utilizing titania as precolumn media is presented. Phosphopeptides were selectively enriched on titania packing within a protein/peptide mixture without any additional procedure, and analyzed by column-switching high-performance liquid chromatography. First, phospho-compounds were separated from complex mixtures by trapping them under acidic conditions on a titania packing, where non-phosphorylated compounds were effused out of the precolumn. Subsequently, phospho-compounds were desorbed from the titania column under a specific condition and analyzed. The behavior of phospho-compounds on a titania surface, especially adsorption/desorption, was precisely examined and optimized. A phosphoric buffer was successively employed for the elution of phosphopeptides on a titania surface by competition with the free phosphate group. From the successes of a selective concentration/analysis of phosphopeptides with column-switching HPLC with a titania precolumn, a novel phosphopeptide-selective RP-HPLC analysis has been shown to have an application possibility as a tool for phosphoproteomics.

Synthesis of adenosine derivatives as transcription initiators and preparation of 5' fluorescein- and biotin-labeled RNA through one-step in vitro transcription.

Expanding our previous finding of an adenosine-initiated transcription system, we now demonstrate that either the 5' site or the N6 site of adenosine nucleotides can be modified extensively without abolishing their ability to initiate transcription under the T7 phi2.5 promoter. Two series of amino derivatives of adenosine nucleotides were synthesized. Fluorescein and biotin groups were coupled to AMP derivatives through linkers of different sizes and hydrophobicities. Both fluorescein- and biotin-conjugated (at either the 5' or N6 site) adenosine nucleotides can act as efficient transcription initiators, producing fluorescein- and biotin-labeled RNA at the specific 5' end by a one-step transcription procedure, eliminating posttranscriptional modification. Furthermore, N6-modified adenosine derivative-initiated transcription synthesizes 5' end modified RNA with a free phosphate group, providing the possibility for further derivatization. The current finding makes easily available a variety of site-specifically functionalized RNA, which may be used in nucleic acid detection, RNA structural and functional investigation, and generation and isolation of novel functional RNA.

Activation of Phospholipase D by Phosphatidic Acid: ENHANCED VESICLE BINDING, PHOSPHATIDIC ACID-Ca2+ INTERACTION, OR AN ALLOSTERIC EFFECT?

The activity of bacterial phospholipase D (PLD), a Ca2+-dependent enzyme, toward phosphatidylcholine bilayers was enhanced 7-fold by incorporation of 10 mol % phosphatidic acid (PA) in the vesicle bilayer. Addition of other negatively charged lipids such as phosphatidylinositol, phosphatidylmethanol, and oleic acid either inhibited or had no effect on enzyme activity. Only negatively charged lipids with a free phosphate group, phosphatidylinositol 4-phosphate and lyso-PA, had the same effect as PA on enzyme activity. Changes in vesicle curvature and fusion were not the reason for PA activation; rather, a metal ion-induced lateral segregation of PA in the vesicle bilayer correlated with PLD activation. Significant PA activation was also observed with monomer phosphatidylcholine substrate upon the addition of PA vesicles. The PA activation was caused by Ca2+.PA interacting with PLD at an allosteric site other than active site.

Activation of a cyclic nucleotide phosphodiesterase 4 (PDE4) from rat thymocytes by phosphatidic acid

The cytosolic cyclic nucleotide phosphodiesterase (PDE) activity from rat thymocytes was resolved into five peaks by HPLC. Only two forms of the cAMP-specific PDE4 were found to be sensitive to physiologically relevant phosphatidic acid (PA) concentrations. PA activated the PDE4-peak 3 form, the fatty acid composition and unsaturation degree determining the efficiency of PA. The PDE4-peak 2 form was inhibited only by PA with saturated fatty acyl groups. PDE4 activation was specific of anionic phospholipids, a free phosphate group in the phospholipid molecule being required for maximum activation. These results suggest that PA may contribute to the lowering of cAMP level required in the early steps of a lympho-proliferative response, thus regulating immune functions through PDE4 activation.

Electrospray tandem mass spectrometry of nucleotides.

The tandem mass spectra (MS/MS) following electrospray ionization of nucleotides (from 2- to 6-mers) have been examined under a range of collision and ionization conditions. In general, abundant peaks due to the free bases and phosphate groups are observed, along with a limited number of sequence ions. A number of strategies for increasing the extent of the observed fragmentation were investigated. Increasing the skimmer potential of the ion source, thereby increasing the initial internal energy of the incident ions, did not significantly enhance the sequence-specific fragmentation, although the relative intensities of fragment ions increased. In MS/MS spectra of different adducts (i.e. [M + H]+, [M-H]-) of dinucleotides the intensity of the ions due to the bases (BH2+ or B-) is dependent on both the base position (i.e. either 3' or 5') and the nature of the adduct ion. These data show that MS/MS spectra of [M-H]- ions of dinucleotides generated by electrospray ionization may be equally as diagnostic as MS/MS spectra obtained following fast-atom bombardment. With [M + Li]+ ions, more sequence ions were observed compared to the corresponding protonated and deprotonated species. For oligonucleotide hexamers, MS/MS spectra of the [M-2H]2- ions show fragment ions due to loss of bases followed by cleavage of the 3' C--O bond in the sugar from which the base is lost (i.e. w ions); however, a strong preference for loss of any particular base is not observed. Finally, MS/MS spectra of 5'-ApCpC-3' and 5'-d(AGGCCT)-3' obtained on a magnetic sector orthogonal acceleration time-of-flight mass spectrometer demonstrate the potential utility of this instrumental configuration for MS/MS analysis of oligonucleotides.

Preparation of ferric adsorbent paper and its interaction with phosphate-containing biomolecules.

A procedure for the synthesis of a selective adsorbent for phosphate-containing biomolecules is described. The sorbent is based on Whatman chromatography paper, which is activated with epichlorohydrine, followed by the coupling of iminodiacetic acid to the active surface of the sorbent. The immobilized complex-forming chelating groups are saturated with ferric ions. The synthesized adsorbent is a counterpart to Chelating Sepharose and makes it possible to extend the use of immobilized ferric chelating groups for analytical purposes. It displays a high affinity towards compounds containing free terminal phosphate groups (phosphopeptides, nucleotides). The results of the binding experiments are compared to the corresponding data obtained with Chelating Sepharose gels.

An endonuclease activity in human polymorphonuclear neutrophils that removes 8-hydroxyguanine residues from DNA

An endonuclease that specifically removes 8-hydroxyguanine (oh 8Gua) from DNA has been isolated from Escherichia coli. As the amount of oh 8Gua produced in DNA of X-ray-irradiated mice is known to decrease with time after irradiation, an attempt was made to find a similar activity in human polymorphonuclear neutrophils (PMNs) using a synthetic dsDNA containing oh 8Gua as a substrate. The PMN enzyme was isolated free of other DNases, and found to cleave the substrate DNA simultaneously at 2 sites, the phosphodiester bonds 5′ and 3′ to oh 8Gua, producing free hydroxyl and phosphate groups, respectively. The enzyme showed almost no activity on DNAs containing other kinds of modified base tested or mismatched DNA. Thus human cells also contain an endonuclease that specifically removes oh 8Gua residues from DNA.

Model studies on iron(III) ion affinity chromatography : Interaction of immobilized metal ions with nucleotides

Mononucleotides are able to bind with immobilized iron(III) ions at low pH in the presence of 1 M sodium chloride and can be desorbed by increasing the pH. All the mononucleotides studied, bound at pH 5.5 to gel-chelated iron(III) ions, were eluted from the adsorbent in the pH range 7.0-7.4. No significant difference was observed in the elution profiles of mono-, di-, tri- and tetraphosphate nucleotides or their deoxy forms. Nucleosides, cyclic mononucleotides and dinucleotides containing all phosphate groups in the internal position do not bind to the immobilized metal ion under these conditions. The results obtained indicate that for interaction of nucleotides with immobilized iron(III) ions, one free terminal phosphate group is responsible.

Structure of human sperm chromatin: a study on the accessibility of DNA to macromolecules.

The structure of human sperm chromatin compared with somatic chromatin (liver) was studied by titration of the exposed DNA-phosphate groups with poly-1-lysine (3000 and 28,100 MW) and by their susceptibility to the hydrolytic action of micrococcal nuclease and DNase I. With both sizes of polylysine used, the binding values were significantly lower for sperm chromatin (0.31 +/- 0.05) than for liver chromatin (0.52 +/- 0.05), indicating the presence of about 30% and 52% of free phosphate groups, respectively. Interaction with liver chromatin left no polylysine molecules partially unbound ("wastage") even when 28,100 MW polylysine was used; on the contrary, sperm chromatin showed 26% of "wasted" polylysine even when the smaller polymer was used, indicating that in sperm chromatin the accessible DNA zones are usually no longer than 42 A, that is, 12 base pair. Sperm chromatin was notably more susceptible to both micrococcal nuclease and DNase I action than liver chromatin. However, in the presence of saturating concentrations of polylysine they were similarly protected. Micrococcal nuclease and DNase I hydrolysis products of sperm fractions when submitted to electrophoresis produced a polydisperse smearing pattern along the gel that was difficult to correlate with the presence of nucleosomal structure.

The involvement of nucleosomes in Giemsa staining of chromosomes. A new hypothesis on the banding mechanism.

A new hypothesis is proposed on the involvement of nucleosomes in Giemsa banding of chromosomes. Giemsa staining as well as the concomitant swelling can be explained as an insertion of the triple charged hydrophobic dye complex between the negatively-charged super-coiled helical DNA and the denatured histone cores of the nucleosomes still present in the fixed chromosomes. New cytochemical data and recent results from biochemical literature on nucleosomes are presented in support of this hypothesis. Chromosomes are stained by the Giemsa procedure in a purple (magenta) colour. Giemsa staining of DNA and histone (isolated or in a simple mixture) in model experiments results in different colours, indicating that a higher order configuration of these chromosomal components lies at the basis of the Giemsa method. Cytophotometry of Giemsa dye absorbance of chromosomes shows that the banding in the case of saline pretreatment is due to a relative absence of the complex in the faintly coloured bands (interbands). Pretreatment with trypsin results in an increase in Giemsa dye uptake in the stained bands. Cytophotometric measurements of free phosphate groups before and after pretreatment with saline, reveal a blocking of about half of the free phosphate groups indicating that a substantial number of free amino groups is still present in the fixed chromosomes. Glutaraldehyde treatment inhibited Giemsa-banding irreversibly while the formaldehyde-induced disappearance of the bands could be restored by a washing procedure. These results correlate with those of biochemical nucleosome studies using the same aldehydes. Based on these findings and on the known properties of nucleosomes, a mechanism is proposed that explains the collapse of the chromosome structure when fixed chromosomes are transferred to aqueous buffer solutions. During homogeneous Giemsa staining reswelling of the unpretreated chromosome is explained by insertion of the hydrophobic Giemsa complex between the hydrophobic nucleosome cores and the superhelix DNA. Selective Giemsa staining of the AT-enriched bands after saline pretreatment is thought to be due to the, biochemically well-documented, higher affinity of arginine-rich proteins present in the core histones for GC-enriched DNA, which prevents the insertion of the Giemsa complex in the interbands. Production of Giemsa bands by trypsin pretreatment can be related to the action of this enzyme on the H1 histones and subsequent charge rearrangements.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochemical demonstration of DNAse in pollen.

Pollen DNAse of Chlorophytum comosum was demonstrated cytochemically by coupling the breakdown of DNA (due to the endogenous DNAse) with acid phosphatase supplied to the reaction mixture. Precipitation of the free phosphate groups (from acid phosphatase reaction on the freed nucleotides) as lead phosphate results in a electron dense deposit, visible with the electron microscope. Enzyme activity using this technique was localized in vesicles along the intine (principally along the aperture region) and around the generative cell.

Anisotropic staining of apurinic acid with toluidine blue.

Using normal rat liver imprints, studies were carried out on the effects of histone extraction and the formation of aldehyde groups from deoxyribose on anisotropic toluidine blue staining of depurinized DNA after sodium bisulfite treatment. The anisotropic effect of bisulfite was found to be determined by binding of bisulfite ions to the aldehyde groups of apurinic acid which, together with free phosphate groups of DNA ensure coparallel attachment of the dye molecules. It was also shown that at pH 5.0 toluidine blue binds with both the phosphate and aldehyde groups of apurinic acid, to give anisotropic staining.