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At least 685 records · Page 38Linked to original sources
PubMed1996

Underexpression of the 43 kDa inositol polyphosphate 5-phosphatase is associated with cellular transformation.

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the second messenger molecules inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. We have underexpressed the 43 kDa 5-phosphatase by stably transfecting normal rat kidney cells with the cDNA encoding the enzyme, cloned in the antisense orientation into the tetracycline-inducible expression vector pUHD10-3. Antisense-transfected cells demonstrated a 45% reduction in Ins(1,4,5)P3 5-phosphatase activity in the total cell homogenate upon withdrawal of tetracycline, and an approximately 80% reduction in the detergent-soluble membrane fraction of the cell, as compared with antisense-transfected cells in the presence of tetracycline. Unstimulated antisense-transfected cells showed a concomitant 2-fold increase in Ins(1,4,5)P3 and 4-fold increase in Ins(1,3,4,5)P4 levels. The basal intracellular calcium concentration of antisense-transfected cells (170 +/- 25 nM) was increased 1.9-fold, compared with cells transfected with vector alone (90 +/- 25 nM). Cells underexpressing the 43 kDa 5-phosphatase demonstrated a transformed phenotype. Antisense-transfected cells grew at a 1.7-fold faster rate, reached confluence at higher density and demonstrated increased [3H]thymidine incorporation compared with cells transfected with vector alone. Furthermore, antisense-transfected cells formed colonies in soft agar and tumours in nude mice. These studies support the contention that a decrease in Ins(1,4,5)P3 5-phosphatase activity is associated with cellular transformation.

3T3 Cells↗
PubMedDate not supplied

[Effect of cell wall polyphosphates on the sensitivity of the yeast Saccharomyces carlsbergensis to the damaging action of cetyltrimethylammonium bromide].

The effect of cetyltrimethylammonium bromide (CTAB) on Saccharomyces carlsbergensis cells grown in orthophosphate-containing medium and on cells starved of phosphorus was studied by electroorientation spectroscopy. CTAB (20 microM) was found to substantially damage the plasma membrane (PM)) of cells grown in the presence of orthophosphate, while cells starved of phosphorus were damaged to a lesser extent. Biochemical analysis of the content of inorganic polyphosphate (poly(P(i))) in the cells, as study of their capacity to adsorb 9-aminoacridine, a fluorescent cationic dye, suggested that the different sensitivity of the plasma membrane to CTAB was due to differences in the content of polyanionic molecules of poly(P(i)) in the cell wall and consequent differences in its net negative charge. Magnesium cations increased the time of CTAB penetration through the cell wall and reduced the damage inflicted on the PM.

Anti-Infective Agents, Local↗
PubMed1997

The generation of hydroxyl radicals in the reaction of molecular oxygen with polyphosphate complexes of ferrous ion.

The reaction of Fe2+ with molecular oxygen (autoxidation) was investigated in 20 mM phosphate buffer (pH 7.4) at 37 degrees C using a fluorescent OH probe, coumarin-3-carboxylic acid. The autoxidation of unchelated Fe2+ produces OH radicals. Polyphosphatic chelators (pyrophosphate and tri- and tetrapoly phosphate) enhanced the generation of radicals. This effect was explained by an alteration of the reaction mechanism. The two-electron reduction of the oxygen molecule and the generation of hydrogen peroxide intermediates are the major reactions during Fe2+ autoxidation. The polyphosphatic complexes of ferrous ion reduce molecular oxygen and reactive oxygen intermediates by a one-electron mechanism. The chelation of ferrous ion increases the generation of the superoxide radical and production of OH during ferrous ion autoxidation and in the Fenton reaction. The results consider the ferrous ion-polyphosphate system as a convenient model for the generation of hydroxyl radical in biological systems.

Ferrous Compounds↗
PubMed1998

Nickel sequestering by polyphosphate bodies in Staphylococcus aureus.

Metal incorporation and possible cellular compartmentalization of nickel by Staphylococcus aureus was investigated. Cells grown on nutrient agar were removed, washed and exposed to 10, 20, 50 or 100 ppm of nickel in distilled water. The cells were air-dried on Formvar coated grids and then examined in a transmission electron microscope operating in the scanning transmission mode. The spot setting was used in conjunction with an energy dispersive X-ray spectrometer to determine the location and relative amount of the nickel in different parts of the cells. The study showed that polyphosphate bodies bind large amounts of the metal. The peak height for nickel increased in the higher exposure amounts of nickel. No nickel was detected in the cell wall or cytoplasmic areas. The results are discussed in relation to nickel transport in cells and staphylococcal infections in humans.

Microscopy, Electron, Scanning Transmission↗
PubMed2000

Transcription of the inositol polyphosphate 1-phosphatase gene (INPP1) is upregulated in human colorectal cancer.

We have used suppression subtractive hybridization to demonstrate significant overexpression of the inositol polyphosphate 1-phosphatase gene (INPP1) in colorectal cancer compared with matched normal colon epithelium. Its gene product catalyses the hydrolysis of inositol 1,3,4-trisphosphate and inositol 1, 4-bisphosphate, a key molecule in the phosphoinositide metabolic and signaling pathways. Following confirmation of the differential expression by reverse Northern dot blot analysis, fully quantitative Taqman reverse transcriptase-polymerase chain reaction assays showed that its transcription was upregulated in 42/49 colorectal tumors. There was no significant difference in four tumors and reduced transcription was observed in three. This is the first study to report the upregulation of the INPP1 gene in a human cancer and should facilitate further studies looking at the role of phosphatidylinositol signaling reactions in human colorectal cancer.

Colorectal Neoplasms↗
PubMed1975

Incidence and sites of bone lesions detected by 99mTc-polyphosphate scans in patients with tumors.

The value of bone scanning with 99mTc-polyphosphate was assessed in 186 patients with various types of tumors. The sensitivity of this technique was greater than that of metastatic roentgenographic series and the reported results of 85-Sr-bone scans, in the detection of osseous involvement by tumors. Three cases with normal bone scans and abnormal roentgenographic studies illustrated the necessity and complementary value of comparing bone scan findings with radiographic studies. Patients with carcinoma of the breast, lung, or prostate displayed characteristic patterns of bone involvement by their tumors. The importance of clinical information, including bone symptoms, antecedent bone disease, and serum calcium and alkaline phosphatase, was stressed in the detection and interpretation of bone scan abnormalities.

Alkaline Phosphatase↗
PubMed1986

Analysis of inositol mono- and polyphosphates by gas chromatography/mass spectrometry and fast atom bombardment.

The electron ionization spectra of all of the positional isomers of myo-inositol monophosphate and of myo-inositol 1,2-cyclic phosphate were obtained by gas chromatography/mass spectrometry of the pertrimethylsilyl derivatives. The fragmentation pattern of pertrimethylsilyl myo-inositol-1-phosphate was studied using deuterium labeling. The phosphate moiety was found to direct fragmentation to produce fragment ions of useful intensity with specific carbon retention. The spectrum of pertrimethylsilyl myo-inositol-1,4-bisphosphate is also described. An electron impact gas chromatographic/mass spectrometric method for myo-inositol-1-phosphate has been developed, which has a sensitivity to a level of 0.1 pmol. The positive and negative ion fast atom bombardment spectra of myo-inositol hexakis(disodium phosphate) and myo-inositol hexakis(dihydrogen phosphate) are described. The lesser-phosphorylated inositol polyphosphates were also studied, including inositol pentakis and inositol tetrakis(dihydrogen phosphates) as well as D-myo-inositol-1,4,5-trisphosphate and D-myo-inositol-1,4-bisphosphate from human red blood cells. The sensitivity of fast atom bombardment for the measurement of the latter two substances allows their detection to a level of about 10 nmol. The fast atom bombardment spectrum of synthetic myo-inositol 1,2-cyclic phosphate revealed variable amounts of a dimer produced during its preparation.

Electrons↗
PubMed1990

Studies on the permethylation/dephosphorylation of inositol polyphosphates: an approach to a more sensitive assay.

Methods for the permethylation of inositol phosphates (i.e. the formation of the completely substituted C-O-methyl/P-O-methyl derivatives) have been studied as a precursor to preparing C-O-methyl inositols where the remaining inositol hydroxyl groups are at the positions originally occupied by the phosphomonoesters. Classical sodium-driven methylations, diazomethane methylations and methylation with methyl trifluoromethanesulfonate were studied and only the latter was found to produce completely alkylated inositol phosphates. Treatment of the permethylated substrates with methanolic HCl removed the dimethylphosphate groups to produce C-O-methyl inositols which are candidates for negative ion chemical ionization gas chromatographic/mass spectrometric analysis as heptafluorobutyryl C-O-methyl inositols. As an example, gas chromatographic/mass spectrometric analysis of myo-inositol 1,2,6-trisphosphate was carried out by methylation, dephosphorylation and conversion to the tris(heptafluorobutyryl) derivative. Detection at the low-femtomole level was achieved by this means. A limitation of the method may be that the methylation procedure appears to produce a variable degree of phosphate positional isomerization, with resulting loss of specificity. If stable isotope internal standards were available for the inositol polyphosphates of interest, this limitation could be compensated for.

Gas Chromatography-Mass Spectrometry↗
PubMed1988

Temporal relationship between inositol polyphosphate formation and increases in cytosolic Ca2+ in quiescent 3T3 cells stimulated by platelet-derived growth factor, bombesin and vasopressin.

We determined the temporal relationship between the formation of inositol phosphates and increase in cytosolic [Ca2+] elicited by bombesin, vasopressin and platelet-derived growth factor (PDGF) in quiescent Swiss 3T3 cells. These responses were measured under identical conditions. Bombesin caused a rapid increase in inositol 1,4,5-trisphosphate which coincided with the increase in cytosolic [Ca2+]. This was followed by a slower but marked increase in inositol 1,3,4-trisphosphate and inositol-bisphosphate. Vasopressin elicited a similar sequence of events. In sharp contrast, highly purified porcine PDGF induced increases in cytosolic [Ca2+] and inositol 1,4,5-trisphosphate that were temporally uncoupled: detectable inositol polyphosphate formation occurred after Ca2+ mobilization from intracellular stores. The same temporal dissociation was observed when a recombinant v-sis product was used instead of porcine PDGF. However, PDGF was as effective as bombesin in stimulating the formation of inositol phosphates after 5-10 min of incubation. The data suggest that PDGF increases cytosolic [Ca2+] via a different signal transduction pathway from that utilized by bombesin and vasopressin. These findings have important implications for understanding the signal transduction pathway activated by PDGF.

Animals↗
PubMed2002

Attachment, spreading, and matrix formation by human gingival fibroblasts on porous-structured titanium alloy and calcium polyphosphate substrates.

Porous calcium polyphosphate (CPP) structures represent promising resorbable implant systems that can promote anchorage to connective tissues. Previous studies focused on chondrocyte interactions with CPP, but there are limited data on interactions of soft connective tissue cells with these materials. We studied attachment, spreading, and matrix formation by human gingival fibroblasts when cultured on amorphous and crystalline CPP. Comparison with porous Ti6Al4V substrates of similar volume percent, porosity, and pore size distribution provided evaluations of fibroblast interactions with rapid, moderate, and nonbiodegradable systems, respectively. Cells were incubated on substrates in medium containing ascorbic acid and evaluated at 3, 24, 48, 72, and 96 h after plating. Attached cell counts, cytoplasmic actin filament area, and immunostained extracellular type 1 collagen or fibronectin were quantified by morphometric analyses using epifluorescence microscopy. Cell morphology and substrate interactions were evaluated by scanning electron microscopy. Spreading, attachment, and matrix production were similar for both CPP substrates. In contrast, titanium alloy substrates exhibited threefold more attachment and twofold more spreading than CPP substrates. The area per cell of immunostained extracellular collagen and fibronectin was similar for the three different substrates. The results indicate that the crystallinity and, hence, degradation rate of CPP substrates does not substantially affect the interactions of fibroblasts with CPP materials but that compared with titanium alloy substrates, spreading and attachment are inhibited.

Alloys↗
PubMed2002

Characterization of cartilagenous tissue formed on calcium polyphosphate substrates in vitro.

Successful joint resurfacing by tissue-engineered cartilage has been limited, in part, by an inability to secure the implant to bone. To overcome this, we have developed the methodology to form a cartilage implant in vitro consisting of a layer of cartilagenous tissue overlying a porous, biodegradable calcium polyphosphate (CPP) substrate. As bone will grow into the CPP after implantation, it will result in anchorage of the cartilage. In this study, the cartilagenous tissue formed in vitro after 8 weeks in culture was characterized and compared to native articular cartilage. Light microscopic examination of histological sections showed that there was a continuous layer of cartilagenous tissue on, and integrated with the subsurface of, the CPP substrate. The in vitro-formed tissue achieved a similar thickness to native articular cartilage (mean +/- SEM: in vitro = 0.94 +/- 0.03 mm; ex vivo = 1.03 +/- 0.01 mm). The cells in the in vitro-formed tissue synthesized large proteoglycans (Kav +/- SEM: in vitro = 0.27 +/- 0.01; ex vivo = 0.27 +/- 0.01) and type II collagen similar to the chondrocytes in the ex-vivo cartilage. The in vitro-formed tissue had a similar amount of proteoglycan (GAG microg/mg dry wt.: in vitro = 198 +/- 10; ex vivo = 201 +/- 13) but less collagen than the native cartilage (hydroxyproline microg/mg dry wt.: in vitro = 21 +/- 1; ex vivo = 70 +/- 8). The in vitro-formed tissue had only about 3% of the load-bearing capacity and stiffness of the native articular cartilage, determined from unconfined mechanical compression testing. Although low, this was within the range of properties reported by others for tissue-engineered cartilage. It is possible that the limited load-bearing capacity is the result of the low collagen content and further studies are required to identify the conditions that will increase collagen synthesis.

Animals↗
PubMed1988

Changes in inositol polyphosphate-sensitive calcium exchange in aortic smooth muscle cells in vitro.

Cells that expressed the muscle-specific intermediate filament protein desmin were cultured from the aorta of Fischer 344 rats. When the cultured cells were extracted with digitonin, they accumulated 45Ca2+ from the incubation medium in a manner that was stimulated by ATP and released subsequently by exposure to the Ca2+ ionophore A23187. Ca2+ bound in the presence of ATP was also released by exposure to inositol 1,4,5-trisphosphate (IP3). Like contraction in some kinds of smooth muscle, IP3 released Ca2+ in either the absence or the presence of the ATPase-inhibitor ruthenium red. When the responsiveness of digitonin-extracted cells cultured from 3-, 12-, and 24-month-old rats was compared, cells from the youngest group released only about one-half as much Ca2+ as cells from the 12- or 24-month-old rats. The results suggest that in the rat there are changes during maturation in the responsiveness to inositol polyphosphates of intracellular compartments that sequester Ca2+ for stimulus-contraction coupling in the aortic smooth muscle cell. These changes, characterized in smooth muscle cells in vitro, might contribute to the way vascular responsiveness is regulated in vivo.

Adenosine Triphosphate↗
PubMed2000

Lys-Ala mutations of type I adenylyl cyclase result in altered susceptibility to inhibition by adenine nucleoside 3'-polyphosphates.

Native and recombinant wild type and mutant forms of type I adenylyl cyclase, expressed in fall army worm ovarian cells (Sf9) cells, with mutations Lys-923-Ala, Lys-921-Ala, and Lys-350-Ala, retained the characteristic noncompetitive inhibition by adenine nucleoside 3'-polyphosphates, but exhibited substantially different sensitivities to inhibition by them. The type I K923A enzyme resulted in increased IC(50) values, e.g., >100-fold for 2'-deoxyadenosine-3'-monophosphate, but the shift diminished as the number of 3'-phosphates increased. The K921A mutation increased IC(50) values approximately 5-fold for all adenine nucleosides tested, whereas the K350A mutation increased IC(50) values approximately 6- to 8-fold for all adenine nucleosides tested except 2'-deoxyadenosine-3'-diphosphate, which was increased >/=2-fold. The data suggest that 3'-phosphates sufficiently increase binding affinity of these ligands to compensate for the reduced coordination of the adenine moiety induced by the K923A mutation. Moreover, the altered structures induced by both K350A and K921A mutations impair ligand binding in general, but paradoxically those resulting from the K350A change minimally affected nucleoside 3'-diphosphate binding, implying that selective changes in ligand binding can be induced by this site-specific mutation.

Adenine Nucleotides↗
PubMed1995

Use of fluorogenic substrates for detection and investigation of ectoenzymatic hydrolysis of diadenosine polyphosphates: a fluorometric study on chromaffin cells.

A set of procedures to assay and investigate ectoenzymatic hydrolysis of diadenosine polyphosphates (ApnA) in both intact cell or plasma membrane preparations is described. Procedures are based on the use of the fluorogenic ApnA analogs, epsilon-(ApnA), as artificial substrates. It is shown that these fluorogenic analogs behave as excellent substrates of the ectoenzyme present in cultured chromaffin cells. The ectoenzyme hydrolyzed all epsilon-(ApnA) tested (n = 2-6), always producing epsilon-AMP and epsilon-Ado 5'(n - 1) phosphate moieties. These released nucleotide moieties were then further catabolized up to epsilon-Ado by other ectonucleotidases. Epsilon-(Ap4A) hydrolysis by cultured cells displayed Km and Vmax values of 4.1 +/- 1.5 microM and 13.2 +/- 1.3 pmol/min x 10(6) cells, respectively, as measured by continuous fluorometric assays and 3.5 +/- 1.6 microM and 10.0 +/- 1.9 pmol/min x 10(6) cells by chromatographic-fluorometric assays. Using plasma membranes, values of 2.5 +/- 0.8 microM and 669 +/- 59 pmol/min x mg protein for Km and Vmax, respectively, were obtained through continuous fluorometric assays. ApnA and GpnG behaved as competitors and Ki values for these dinucleotides ranged between 0.7 and 3.5 microM. The ectoenzyme was activated by Mg2+ and Ca2+ and achieved maximal activity in the pH range 8.5-9.0.

Acid Anhydride Hydrolases↗
PubMed1994

Synaptotagmin is an inositol polyphosphate binding protein: isolation and characterization as an Ins 1,3,4,5-P4 binding protein.

We isolated a binding protein for inositol 1,3,4,5-tetrakisphosphate (InsP4) from detergent-solubilized mouse cerebellar membrane fractions by sequential column chromatographies. Partial amino acid sequencing of the purified sample revealed that the protein is essentially identical to rat synaptotagmin II, an integral membrane protein of synaptic vesicles. Immunoprecipitation experiment of [3H]InsP4 binding activity of the purified protein using polyclonal antibody against the C2A domain of rat synaptotagmin II also revealed that mouse synaptotagmin II is the InsP4 binding protein (IP4BP). Scatchard analysis of InsP4 binding to the IP4BP/synaptotagmin indicates a single binding site with a Kd of 30 nM. The present finding that InsP4 binds strongly to synaptotagmin II suggests an important role for inositol polyphosphates in the regulation of neurotransmitter release.

Amino Acid Sequence↗
PubMed1995

Oscillation of inositol polyphosphates in the embryonic cleavage cycle of the Xenopus laevis.

Evidence suggests that a transient increase in intracellular calcium ([Ca2+]i) is an important modulator during the cell division cycle in early embryos. We have recently shown that inhibition of Ins(1,4,5)P3-induced Ca2+ release in the cleaving Xenopus embryos greatly lengthens the cell cycle duration. In this report, we have directly measured the changes of Ins(1,4,5)P3 content during the first two cleavage cycles in the Xenopus embryos. HPLC profiles of cell extracts from dividing embryos show oscillations of inositol polyphosphates throughout the cleavage cycle with a transient production of Ins(1,4,5)P3 by the time of cleavage furrow completion. In addition, cyclic changes in inositol phospholipids, phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP) were detected during the cleavage cycle. These data strongly suggest the involvement of PIP2 turnover and periodic increase in Ins(1,4,5)P3 triggers [Ca2+]i transients during the early embryonic cell cycle in the Xenopus.

Animals↗