Extraction and detection of guanosine 5'-diphosphate-3'-diphosphate in amino acid starvation cells of Clavibacter michiganensis.

Guanosine 5'-diphosphate-3'-diphosphate (ppGpp) is a small molecule nucleotide alarmone that can accumulate under the amino acid starvation state and trigger the stringent response. This study reported the extraction of ppGpp from the Gram-positive bacteria Clavibacter michiganensis through methods using formic acid, lysozyme, or methanol. Following extraction, ppGpp was detected through ultra-high-performance liquid chromatography (UHPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The methanol method showed the highest extraction efficiency for ppGpp among the three methods tested. C. michiganensis cells in exponential growth phase was induced in amino acid starvation by serine hydroxamate (SHX) and used for ppGpp extraction and detection. When using the methanol extraction method, the results showed that ppGpp concentrations in SHX-treated samples were 15.645 nM, 17.656 nM, 20.372 nM, and 19.280 nM at 0 min, 15 min, 30 min and 1 h, respectively, when detected using LC-MS/MS. This is the first report on ppGpp extraction and detection in Clavibacter providing a new idea and approach for nucleotide detection and extraction in bacteria.

Selective sensing of ATP by hydroxide-bridged dizinc(ii) complexes offering a hydrogen bonding cavity.

This work illustrates the highly selective fluorescence detection of ATP in the presence of other competing anions, such as AMP, ADP, PPi and other phosphates by using a set of hydroxide-bridged dizinc(ii) complexes offering a cavity lined with hydrogen bonds and other interactive forces. ATP, as a whole, was recognized by the synergic combination of Zn-phosphate bonding, ππ stacking between the adenine ring of ATP and the pyridine ring of the dizinc complex and hydrogen bonding interactions that modulate the cavity structure of the dizinc complexes.

Removal behaviors and mechanisms of orthophosphate and pyrophosphate by calcined dolomite with ferric chloride assistance.

Phosphate is one of the main contaminations in water, so an effective method of decreasing or removing phosphate is needed. The main purpose of this paper is to synthesize CaFe-LDHs and MgFe-LDHs from the mixture of calcined dolomite and ferric chloride to remove orthophosphate and pyrophosphate. The study showed that removal of orthophosphate was attributed to the precipitation by Ca2+ and adsorption by MgFe-LDHs, where the former played a main role. As for pyrophosphate, it was mainly removed by precipitation at the initial pyrophosphate concentration ranging from 3.228 to 17.04 mmol/L. When the initial concentrations became relatively higher, the removal efficiency of pyrophosphate decreased because the complexation effects by Fe3+, Ca2+ and Mg2+ took place.

Scalable Chemoenzymatic Synthesis of Inositol Pyrophosphates.

The inositol pyrophosphates (PP-InsPs) are an important group of cellular messengers that influence a broad range of biological processes. To elucidate the functions of these high-energy metabolites at the biochemical level, access to the purified molecules is required. Here, a robust and scalable strategy for the synthesis of various PP-InsPs [5PP-InsP5, 1PP-InsP5, and 1,5(PP)2-InsP4] is reported, relying on the highly active inositol hexakisphosphate kinase A from Entamoeba histolytica and the kinase domain of human diphosphoinositol pentakisphosphate kinase 2. A facile purification procedure using precipitation with Mg2+ ions and an optional strong anion exchange chromatography on an FPLC system afforded PP-InsPs in high purity. Furthermore, the newly developed protocol could be applied to simplify the synthesis of radiolabeled 5PP-InsP5-ß32P, which is a valuable tool for studying protein pyrophosphorylation. The chemoenzymatic method for obtaining PP-InsPs is readily amenable to both chemists and biologists and will thus foster future research on the multiple signaling functions of PP-InsP molecules.

Label-free upconversion nanoparticles-based fluorescent probes for sequential sensing of Cu2+, pyrophosphate and alkaline phosphatase activity.

An efficient near-infrared fluorescence probe has been developed for the sequential detection of Cu2+, pyrophosphate (P2O74-, PPi), and alkaline phosphatase (ALP), which is based on the "off-on-off" fluorescence switch of branched polyethyleneimine (PEI)-capped NaGdF4:Yb/Tm upconversion nanoparticles (UCNPs). The fluorescence is quenched via energy transfer from UCNPs to Cu2+ for the coordination of PEI with Cu2+. The strong affinity between Cu2+ and PPi leads to the formation of Cu2+-PPi complex and results in the detachment of Cu2+ from the surface of UCNPs, thus the fluorescence is triggered on. ALP-directed hydrolysis of PPi causes the disassembly of Cu2+-PPi complex and re-conjugation between Cu2+ with PEI, which leads to the switch-off fluorescence of UCNPs. The system allows sequential analysis of Cu2+, PPi, and ALP by modulating the switch of the fluorescence of UCNPs with detection limits of 57.8nM, 184nM, and 0.019U/mL for Cu2+, PPi, and ALP, respectively. By virtue of the NIR feature and excellent biocompatibility, the UCNPs-based probes are suitable for bioimaging. Taking Cu2+ visualization as a model, the nanoprobes have been successfully applied for intracellular imaging of Cu2+ in living cells.

Fast determination of bioactive phytic acid and pyrophosphate in walnuts using microwave accelerated extraction.

Bioactive compounds phytic acid (IP6) and pyrophosphate (PPi) are minor components of walnuts with the ability of being inhibitors of urolithiasis, among others. Since simultaneous analysis of IP6 and PPi have known drawbacks, a new method to determine their content in walnuts has been developed with emphasis on their extraction from walnuts by microwave-assisted extraction (MAE). Acid content of extracting solvent, extraction time and temperature were optimized. After extraction, compounds were purified by selective adsorption/desorption on an anion exchange solid phase extraction and analyzed by inductive coupled plasma/mass spectrometry. A mixture of H2SO4 and HCl as solvent to extract both, IP6 and PPi, provided results slightly higher than those determined by conventional extraction with no statistical difference. The possible hydrolysis of phytic acid by MAE was analyzed. Compared with the conventional acid extraction method, significant improvement is achieved by the MAE method reducing extraction time from 3h to 10min.

Phosphocalyculin C as a pyrophosphate protoxin of calyculin C in the marine sponge Discodermia calyx.

Calyculin C, a minor derivative of the calyculins, has an additional methyl group on C32 of calyculin A. A recent biosynthetic study of calyculins revealed that an end product of calyculin biosynthesis is the pyrophosphate form, phosphocalyculin A. However, the pyrophosphate counterpart derived from calyculin C had not been reported. We isolated phosphocalyculin C as a minor pyrophosphate derivative, by a detailed investigation of an extract from the sponge Discodermia calyx. The treatment of phosphocalyculin C with the D. calyx cell-free extract significantly enhanced its cytotoxicity, providing molecular evidence for its role as the protoxin of calyculin C.

Identification of bacteria directly from positive blood culture samples by DNA pyrosequencing of the 16S rRNA gene.

Rapid identification of the causative bacteria of sepsis in patients can contribute to the selection of appropriate antibiotics and improvement of patients' prognosis. Genotypic identification is an emerging technology that may provide an alternative method to, or complement, established phenotypic identification procedures. We evaluated a rapid protocol for bacterial identification based on PCR and pyrosequencing of the V1 and V3 regions of the 16S rRNA gene using DNA extracted directly from positive blood culture samples. One hundred and two positive blood culture bottles from 68 patients were randomly selected and the bacteria were identified by phenotyping and pyrosequencing. The results of pyrosequencing identification displayed 84.3 and 64.7 % concordance with the results of phenotypic identification at the genus and species levels, respectively. In the monomicrobial samples, the concordance between the results of pyrosequencing and phenotypic identification at the genus level was 87.0 %. Pyrosequencing identified one isolate in 60 % of polymicrobial samples, which were confirmed by culture analysis. Of the samples identified by pyrosequencing, 55.7 % showed consistent results in V1 and V3 targeted sequencing; other samples were identified based on the results of V1 (12.5 %) or V3 (31.8 %) sequencing alone. One isolate was erroneously identified by pyrosequencing due to high sequence similarity with another isolate. Pyrosequencing identified one isolate that was not detected by phenotypic identification. The process of pyrosequencing identification can be completed within ~4 h. The information provided by DNA-pyrosequencing for the identification of micro-organisms in positive blood culture bottles is accurate and could prove to be a rapid and useful tool in standard laboratory practice.

Determination of nucleoside analog mono-, di-, and tri-phosphates in cellular matrix by solid phase extraction and ultra-sensitive LC-MS/MS detection.

An ultra-sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated to facilitate the assessment of clinical pharmacokinetics of nucleotide analogs from lysed intracellular matrix. The method utilized a strong anion exchange isolation of mono-(MP), di-(DP), and tri-phosphates (TP) from intracellular matrix. Each fraction was then dephosphorylated to the parent moiety yielding a molar equivalent to the original nucleotide analog intracellular concentration. The analytical portion of the methodology was optimized in specific nucleoside analog centric modes (i.e. tenofovir (TFV) centric, zidovudine (ZDV) centric), which included desalting/concentration by solid phase extraction and detection by LC-MS/MS. Nucleotide analog MP-, DP-, and TP-determined on the TFV centric mode of analysis include TFV, lamivudine (3TC), and emtricitibine (FTC). The quantifiable linear range for TFV was 2.5-2000 fmol/sample, and that for 3TC/FTC was 0.1 200 pmol/sample. Nucleoside analog MP-, DP-, and TP-determined on the ZDV centric mode of analysis included 3TC and ZDV. The quantifiable linear range for 3TC was 0.1 100 pmol/sample, and 5-2000 fmol/sample for ZDV. Stable labeled isotopic internal standards facilitated accuracy and precision in alternative cell matrices, which supported the intended use of the method for MP, DP, and TP determinations in various cell types. The method was successfully applied to clinical research samples generating novel intracellular information for TFV, FTC, ZDV, and 3TC nucleotides. This document outlines method development, validation, and application to clinical research.

Expression, crystallization and preliminary crystallographic study of octaprenyl pyrophosphate synthase from Helicobacter pylori.

Octaprenyl pyrophosphate synthase (OPPs) is involved in the synthesis of the side chains of ubiquinone and menaquinone and catalyzes consecutive condensation reactions of farnesyl pyrophosphate with isopentenyl pyrophosphate to generate polyprenyl pyrophosphate and pyrophosphate. In order to investigate the roles played by OPPs in the metabolism of ubiquinone and menaquinone and the enzymatic mechanisms of these enzymes, analysis of the structure-function relationship of OPPs from Helicobacter pylori was initiated. The gene for OPPs was cloned, the protein was expressed, purified and crystallized and a diffraction data set was collected to 2.00 Šresolution. The crystals belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 109.33, c = 103.41 Å.

Molecular manipulation and analysis of inositol phosphate and pyrophosphate levels in Mammalian cells.

Lipid-derived inositol phosphates (InsPs) comprise a family of second messengers that arise through the action of six classes of InsP kinases, generally referred to as IPKs. Genetic studies have indicated that InsPs play critical roles in embryonic development, but the mechanisms of action for InsPs in mammalian cellular function are largely unknown. This chapter outlines a method for manipulating cellular InsP profiles through the coexpression of a constitutively active G protein and various IPKs. It provides a mechanism by which the metabolism of a variety of InsPs can be upregulated, enabling the evaluation of the effects of these InsPs on cellular functions.

Synthesis and nonradioactive micro-analysis of diphosphoinositol phosphates by HPLC with postcolumn complexometry.

A nonradioactive high-performance anion-exchange chromatographic method based on MDD-HPLC (Mayr Biochem. J. 254:585-591, 1988) was developed for the separation of inositol hexakisphosphate (InsP(6), phytic acid) and most isomers of pyrophosphorylated inositol phosphates, such as diphosphoinositol pentakisphosphate (PPInsP(5) or InsP(7)) and bis-diphosphoinositol tetrakisphosphate (bisPPInsP(4) or InsP(8)). With an acidic elution, the anion-exchange separation led to the resolution of four separable PPInsP(5) isomers (including pairs of enantiomers) into three peaks and of nine separable bisPPInsP(4) isomers into nine peaks. The whole separation procedure was completed within 20-36 min after optimization. Reference standards of all bisPPInsP(4) isomers were generated by a nonenzymatic shotgun synthesis from InsP(6). Hereby, the phosphorylation was brought about nonenzymatically when concentrated InsP(6) bound to the solid surface of anion-exchange beads was incubated with creatine phosphate under optimal pH conditions. From the mixture of pyrophosphorylated InsP(6) derivatives containing all theoretically possible isomers of PPInsP(5), bisPPInsP(4), and also some isomers of trisPPInsP(3), isomers were separated by anion-exchange chromatography and fractions served as reference standards of bisPPInsP(4) isomers for further investigation. Their isomeric nature could be partly assigned by comparison with position specifically synthesized or NMR-characterized purified protozoan reference compounds and partly by limited hydrolysis to PPInsP(5) isomers. By applying this nonradioactive analysis technique to cellular studies, the isomeric nature of the major bisPPInsP(4) in mammalian cells could be identified without the need to obtain sufficient material for NMR analysis.

Minimum handling method for the analysis of phosphorous inhibitors of urolithiasis (pyrophosphate and phytic acid) in urine by SPE-ICP techniques.

Pyrophosphate (PPi) and phytic acid (IP6) are natural phosphorous compounds with growing interest in the biomedical field due to their ability as potential inhibitors of urolithiasis among others. Existing methodologies for their evaluation show inconveniences mainly associated with sample treatment, matrix interferences and lack of resolution. The objective of the present work is the validation of a new method to determine both inhibitors in urine samples selectively and its application to the diagnosis of lithiasic patients. After urine purification by an off-line anion exchange solid phase extraction (SPE), based in an appropriate acidic elution gradient, the phosphorous compounds were analyzed by (31)P measurements by inductively coupled plasma mass spectrometry (ICP-MS) in the purified urine extracts. Linear range and limit of detection obtained were adequate for the analysis of the physiological amounts of the compounds in urine. The method was successfully applied to human urine samples, resulting in adequate accuracy and precision and allowing for the analysis of phosphorus inhibitors of urolithiasis in urine. The method simplicity and high sample throughput leads to a clear alternative to current determinations of the mentioned species in urine. Moreover, PPi and IP6 concentrations found in patients suffering from oxalocalcic urolithiasic were significantly lower than those for healthy controls, supporting the fact that the risk for oxalocalcic urolithiasis increases when urinary phosphorus inhibitors decrease. Thus, speciation of phosphorus inhibitors of urolithiasis in urine of stone formers can be performed, which is of unquestionable value in diagnostic, treatment and monitoring of urolithiasis.

PABLO analysis of RNA: 5'-phosphorylation state and 5'-end mapping.

Recent studies have revealed that 5'-end-dependent RNA degradation in prokaryotes is triggered by pyrophosphate removal from the 5'-terminus to generate a monophosphorylated intermediate that is readily degraded. This chapter describes how to examine the 5'-phosphorylation state of any specific bacterial RNA by PABLO analysis. The method is based on the ability of monophosphorylated, but not triphosphorylated, RNA 5'-ends to undergo splinted ligation to a DNA oligonucleotide when juxtaposed by base pairing to a bridging oligonucleotide. PABLO analysis not only makes it possible to quantify the proportion of a particular RNA that is monophosphorylated in bacterial cells but also provides a more reliable method than primer extension for high-resolution mapping of RNA 5'-termini.

Initiation of RNA decay in Escherichia coli by 5' pyrophosphate removal.

The common belief that endonucleolytic cleavage is the initial, rate-determining step of mRNA decay in Escherichia coli fails to explain the influence of 5' termini on the half-lives of primary transcripts. We have re-examined the initial events of RNA degradation in that organism by devising an assay to probe the 5' phosphorylation state of RNA and by employing a self-cleaving hammerhead ribozyme to investigate the degradative consequences of an unphosphorylated 5' end. These studies have identified a previously unrecognized prior step in decay that triggers subsequent internal cleavage by the endonuclease RNase E and thereby governs RNA longevity: the rate-determining conversion of a triphosphorylated to a monophosphorylated 5' terminus. Our findings redefine the role of RNase E in RNA degradation and explain how unpaired 5'-terminal nucleotides can facilitate access to internal cleavage sites within primary transcripts. Moreover, these results reveal a striking parallel between the mechanisms of mRNA decay in prokaryotic and eukaryotic organisms.

Preferential recognition of a microbial metabolite by human Vgamma2Vdelta2 T cells.

Human Vgamma2Vdelta2 T cells are stimulated by prenyl pyrophosphates, such as isopentenyl pyrophosphate (IPP), and play important roles in mediating immunity against microbial pathogens and have potent anti-tumor activity. (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) has been identified as a metabolite in the 2-C-methyl-D-erythritol-4 phosphate (MEP) pathway for isoprenoid biosynthesis that is used by many bacteria and protozoan parasites. We find that HMBPP is the major Vgamma2Vdelta2 T-cell antigen for many bacteria, including Mycobacterium tuberculosis, Yersinia enterocolitica and Escherichia coli. HMBPP was a 30 000-fold more potent antigen than IPP. Using mutant bacteria, we show that bacterial antigen levels for Vgamma2Vdelta2 T cells are controlled by MEP pathway enzymes and find no evidence for the production of 3-formyl-1-butyl pyrophosphate. Moreover, HMBPP reactivity required only germ line-encoded Vgamma2Vdelta2 TCR elements and is present at birth. Importantly, we show that bacterial HMBPP levels correlated with their ability to expand Vgamma2Vdelta2 T cells in vivo upon engraftment into severe combined immunodeficiency-beige mice. Thus, the production of HMBPP by a microbial-specific isoprenoid pathway plays a major role in determining whether bacteria will stimulate Vgamma2Vdelta2 T cells in vivo. This preferential stimulation by a common microbial isoprenoid metabolite allows Vgamma2Vdelta2 T cells to respond to a broad array of pathogens using this pathway.

Identification, purification and genetic deficiencies of the glucose-6-phosphatase system transport proteins.

Hepatic microsomal glucose-6-phosphatase (Glc-6-P'ase) is a complex multicomponent system containing at least three transport proteins, in addition to the catalytic subunit and a Ca2+ binding regulatory protein. The transport proteins have been designated T1 the glucose-6-phosphate transport protein, T2 a phosphate/pyrophosphate transport protein and T3 a glucose transport protein. Diagnosis of the genetic deficiencies of these transport proteins at present requires a complex kinetic analysis of the Glc-6-P'ase system as a whole. Here we describe the progress to date in our attempts to identify, purify and clone each transport protein with the ultimate aim of isolating specific cDNA probes for each transport protein which can be used for the diagnosis of types 1b, 1c and the putative 1d glycogen storage diseases.

A new cyclopyrophosphate as a bacterial antistressor?

In a number of bacteria an unusual glycosyl pyrophosphate (31P NMR signal chemical shift at about -15 ppm) was detected when the cells were subjected to oxidative stress. This substance from Brevibacterium ammoniagenes has now been identified as 2-methyl-butan-1,2,3,4,-tetraol-2,4-cyclopyrophosphate, which is accumulated in the cell under certain conditions in concentrations of of about 50 mM. It is now suggested that this compound is the long sought after bacterial antistressor.

High-performance liquid chromatographic determination of pyrophosphate in the presence of a 20,000-fold excess of orthophosphate.

An HPLC method was based on anion-exchange separation of pyrophosphate (diphosphate) and orthophosphate and postcolumn spectrophotometric detection at 140 degrees C with a molybdenum(V)-molybdenum(VI) reagent. The reagent was easy to prepare, stable for at least 6 months at room temperature, and ready for the determination of pyrophosphate and orthophosphate by the so-called heteropoly blue method without use of any reducing agent. A photodiode-array detector for HPLC indicated the spectral characteristics of the heteropoply blue complex that was detectable at 330-800 nm. The HPLC method had a wide dynamic range from 3 x 10(-7) to 5 x 10(-4) M for both pyrophosphate and orthophosphate with a relative standard deviation of measurement of 10 approximately 2%. Pyrophosphate of 5 x 10(-7) and 5 x 10(-6) M, respectively, could be determined in the presence of a 20,000-fold excess of orthophosphate; 0.01 and 0.1 M.

Structures of the monohydrate and dihydrate of (bidentate pyrophosphato) trans-diammine cis-diaqua chromium (III).

(OC-6-32)-Diamminediaqua [pyrophosphato(3-)]chromium(III) monohydrate, [Cr(NH3)2-(H2O)2(HP2O7)]. H2O, Mr = 315.1, triclinic, P1, a = 7.127 (2), b = 8.390 (2), c = 9.619 (2) A, alpha = 72.14 (2), beta = 98.86 (2), gamma = 76.98 (3) degrees, V = 517.7 (3) A3, Z = 2, Dx = 2.02 g cm-3, lambda(Cu K alpha) = 1.5418 A, mu = 129 cm-1, F(000) = 322, T = 293 K. (OC-6-32)-Diamminediaqua[pyrophosphato(3-)]-chromium(III) dihydrate, 0.5( [Cr(NH3)2(H2O)2-(HP2O7)].2H2O), Mr = 0.5(333.1), monoclinic, C2/m, a = 13.118 (3), b = 12.101 (3), c = 7.436 (2) A, beta = 105.09 (2) degrees, V = 1139.7 (3) A3, Z = 8, Dx = 1.94 g cm-3, lambda(Cu K alpha) = 1.5418 A, mu = 118 cm-1, F(000) = 684, T = 293 K. The structures were solved by the multi-solution technique and refined by the method of least squares to yield a final R index of 0.057 for 986 reflections in the monohydrate and a final R index of 0.043 for 1030 reflections in the dihydrate. The six-membered chromium pyrophosphate chelate ring is in a boat conformation for the monohydrate with an intramolecular hydrogen bond between an ammonia proton and a pyrophosphate O atom. In the dihydrate, the chelate ring is bisected by the mirror plane resulting in an unusual planar chelate ring conformation which does not permit intramolecular hydrogen bonding.