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Effects of infused glucose, sodium and potassium chlorides and polyphosphates on palatability of hot-boned pork.

Longissimus muscle sections were excised from eight pork carcasses 1 h postmortem and sectioned into six .5-kg roasts to determine the effects of glucose, salt and polyphosphates (aqueous solution to 110% of fresh weight) on palatability of hot-boned pork. Treatments were hot-boned control (HB) with no infusion or infusions of 2% KCl and 3% of a 1:1 mixture of sodium hexametaphosphate and sodium pyrophosphate (PP) plus either 8% NaCl; 2% glucose (G) plus 6% NaCl; 6% G plus 2% NaCl; or 8% G. Another muscle section was chilled at 0 degrees C for 24 h on each carcass as a cold-processed control (CP). The roasts were frozen until cooked and evaluated by a sensory panel. The infused groups were more tender, juicy and salty and higher in moisture and ash but lower in protein content than either the CP or HB controls (P less than .05). The fat content of the infused groups was lower than of the HB control but was not different from that of the CP control. Either 2% NaCl plus 6% G or equal amounts (4%) of NaCl and G produced the most tender and juicy product. The substitution of 4% glucose for NaCl not only reduced the NaCl content of the infusion solution, but also improved the palatability of the meat. This substitution allows production of a hot-boned, lower-sodium precooked pork that is tender and juicy.

Animals↗

Ammonium polyphosphate as a source of phosphorus and nonprotein nitrogen for monogastrics.

Ammonium polyphosphate (APP) was substituted for 0, 50 and 100% of the P supplied by defluorinated rock phosphate (DRP) in corn-soybean meal diets for growing rats and growing-finishing (G-F) pigs. The diets were formulated to contain 10% protein, .75% Ca and .60% P. Two-thirds of the P in the control diet was supplied by DRP. Replacement of 50 or 100% of the DRP with APP increased the dietary N by 7.25 and 14.5%, respectively. To evaluate nonprotein N (NPN) utilization, we used ammonium chloride (NH4Cl) to provide a level of N equivalent to that supplied by APP when it replaced 100% of the P supplied by DRP. These four treatments were repeated with supplementation of limiting amino acids (L-lysine, L-tryptophan and DL-methionine. Daily feed intake, rate of gain and feed:gain (F:G) of rats and G-F pigs were not influenced (P greater than .05) by the substitution of APP for DRP as a P source in corn-soybean meal diets with or without supplemental amino acids. F:G was improved (P less than .05) by the addition of limiting amino acids to diets for pigs, and a similar trend occurred in rats. The addition of NH4Cl to the 10% protein diets as a source of NPN resulted in no observable benefit for growing rats or G-F pigs. Percentages of ham and loin, percentages of bone ash and P content of the bone ash in pigs were not influenced by the dietary treatments.

Animal Feed↗

Two distinct waves of membrane-proximal B cell antigen receptor signaling differentially regulated by Src homology 2-containing inositol polyphosphate 5-phosphatase.

The phosphatidylinositol 3-kinase (PI3K) pathway plays a critical role in B cell activation and differentiation. Recruitment of pleckstrin homology (PH) domain-containing signal transduction proteins to the plasma membrane through binding to 3-phosphoinositide second messengers represents a major effector mechanism for PI3Ks. We have found that the PH domains of Bam32 and tandem PH domain-containing protein 2 (TAPP2) specify a temporally distinct wave of membrane recruitment compared with that of Bruton's tyrosine kinase (Btk), with recruitment of these two adaptors representing a later stage of the response. In this study we provide direct evidence that PH domain-dependent recruitment of Btk to the membrane is blocked by coligation of the inhibitory receptor FcgammaRII in human B lymphoma cells. In contrast, recruitment specified by the Bam32 or TAPP2 PH domains is completely insensitive to FcgammaRII inhibition. This differential regulation can be accounted for by Src homology 2-containing inositol polyphosphate 5-phosphatase (SHIP) activity alone, as expression of membrane-targeted SHIP completely abrogated Btk recruitment, but had no inhibitory effect on Bam32 or TAPP2 recruitment. Strikingly, kinetic analysis revealed that membrane recruitment of Bam32 and TAPP2 is actually more rapid under "inhibitory" signaling conditions. Analysis of 3-phosphoinositide generation under activating and inhibitory signaling conditions indicated that recruitment of Bam32 and TAPP2 is inversely correlated with the SHIP substrate/product ratio (phosphatidylinositol 3,4,5-trisphosphate/phosphatidylinositol 3,4-bisphosphate). Overexpression of TAPP2 in B cells led to an increase in the sustained phase of the calcium response and increased NF-AT-dependent transcriptional activation after B cell Ag receptor ligation. Together, these results suggest that Bam32 and TAPP2 adaptors define a novel group of SHIP-activated targets of PI3K that regulate B cell Ag receptor signaling.

Adaptor Proteins, Signal Transducing↗

Polyphosphate in bone.

Human bone-forming osteoblasts are an excellent model to investigate the multiple functions of inorganic polyphosphates (polyP) for the following reasons: 1) they contain relatively high amounts of polyP and polyP-dependent enzymes; 2) they allow the study of both general and specific functions of these polymers, and 3) medically relevant results can be expected from these studies.

Acid Anhydride Hydrolases↗

Application of polyphosphate metabolism to environmental and biotechnological problems.

The synthesis and degradation of polyphosphate (polyP) are influenced by the energy state of the cell and extracellular phosphate levels. The import of excess phosphate and its incorporation into polyP under phosphate- and energy-rich growth conditions allows organisms to survive when phosphate or energy are depleted. Under phosphate-starvation conditions, phosphate can be recovered from polyP by hydrolysis. When the organism is energy starved, energy can be recovered either by regenerating the high-energy phosphoanhydride bond donor (ATP in most cases) or by hydrolysis of polyP and subsequent secretion of orthophosphate to recharge the transmembrane proton gradient. Understanding how the energy state of the cell and environmental phosphate levels affect polyP metabolism is essential to improving such environmental processes as enhanced biological phosphorus removal, a treatment process that is widely used to remove excess phosphate from wastewater. Manipulation of the genes responsible for polyP metabolism can also be used to improve gene expression from phosphate-starvation promoters and to remove heavy metals from contaminated environments.

Biotechnology↗

Dependence of inorganic polyphosphate chain length on the orthophosphate content in the culture medium of the yeast Saccharomyces cerevisiae.

The content of inorganic linear polyphosphate (polyP) and the polymeric degree (n) of these compounds were determined in the process of growth of the yeast Saccharomyces cerevisiae VKM Y-1173 in a medium, which contained varying Pi amount with the constant level of all the necessary components. For this purpose, a combination of chemical methods of polyP extraction and 31P-NMR spectroscopy studies of their chain length were used. After 7 h of phosphate starvation, the yeast was shown to use almost completely the phosphate reserve in the form of polyP localized in various cell compartments to support their vitality. The polyP drop was followed by a considerable shortening of the polymer chain length of acid-soluble (polyP1) and two alkali-soluble (polyP3 and polyP4) fractions. Under the same conditions, the content of a salt-soluble fraction (polyP2) decreased almost 20-fold followed by a simultaneous increase of the chain length nearly 2-fold. As a result, fraction chain length ranged up to n = 40-45. Replacement of the yeast cells after phosphate starvation to a complete phosphate- and glucose-containing medium resulted in super-accumulation ("overcompensation") of polyP within 2 h mainly in polyP3 and, to a lesser degree, in polyP1, polyP2, and polyP5 fractions. In polyP4 fraction localized as polyP3 at the cell surface, the polyP super-accumulation was not detected. The increase of polyP amount in the fractions mentioned turned out not to be accompanied by simultaneous elongation of their chain length and occurred at the lowest level that is characteristic of a polymer level for each fraction. Further cultivation of the yeast on the complete medium during 2 h had little or no effect on polyP content in the cells but led to elongation of polyP chain length especially in the polyP3 and polyP4 fractions. A phenomenon of considerable elongation of polyP chain length against the background of their fixed content revealed in the yeast growing on the complete medium suggests that these organisms possess a previously unknown discrete way of polyP biosynthesis, which results first in the formation of comparatively low-molecular-mass chains followed by that of high-molecular-mass polymers.

Acid Anhydride Hydrolases↗

[Intracellular accumulation of monomer precursors of polyphosphates and polyhydroxyalkanoates from Acinetobacter calcoaceticus and Escherichia coli].

The formation of polyhydroxyalkanoates granules in anaerobically grown Escherichia coli cells was found to be preceded by the intracellular accumulation of carbonic acids (predominantly, acetic acid), amounting to 9% of the cytosol. The intracellular concentration of acidic metabolites increased after the lyophilization of the bacterial biomass and decreased after its long-term storage (3.5-13.5 years). The decrease in the concentration of acidic metabolites is likely due to the dehydration of dimeric carbonic acids in the viscoelastic cytosol of resting bacterial cells. The hydrophobic obligately aerobic cells of Acinetobacter calcoaceticus IEGM 549 are able to utilize a wide range of growth substrates (from acetate and citrate to hydrophobic hydrocarbons), which is considerably wider than the range of the growth substrates of E. coli (predominantly, carbohydrates). The minimal essential and optimal concentrations of orthophosphates in the growth medium of A. calcoaceticus were found to be tens of times lower than in the case of E. coli. The intracellular content of orthophosphates in A. calcoaceticus cells reached 35-77% of the total phosphorus content (Ptotal), providing for the intense synthesis of polyphosphates. The Ptotal of the A. calcoaceticus cells grown in media with different proportions between the concentrations of acetate and phosphorus varied from 0.7 to 3.3%, averaging 2%. This value of Ptotal is about two times higher than that observed for fermenting E. coli cells. Lowering the cultivation temperature of A. calcoaceticus from 37-32 to 4 degrees C augmented the accumulation of orthophosphates in the cytoplasm, presumably owing to a decreased requirement of growth processes for orthophosphate. In this case, if the concentration of phosphates in the cultivation medium was low, they were completely depleted.

Acinetobacter calcoaceticus↗

The significance of denitrifying polyphosphate accumulating organisms in biological nutrient removal activated sludge systems.

In this paper the advantages and disadvantages of denitrifying PAOs (polyphosphate accumulating organisms) in conventional BNRAS (biological nutrient removal activated sludge) and external nitrification BNRAS (ENBNRAS) systems are evaluated, with experimental data exhibiting a range of anoxic P uptake from low (<10%) to very high (>60%). The results indicate that the specific denitrification rate of the PAOs on internally stored PHB COD is about 1/5th of that of the "ordinary" heterotrophic organisms on SBCOD, and the PAOs contribute little (maximum 20%) to the denitrification in BNRAS systems even when the anoxic P uptake is high (60% of the total P uptake). Considering the unpredictable nature of anoxic P uptake and the reduction in BEPR it causes compared with aerobic P uptake BEPR, it is concluded that anoxic P uptake does not add a significant advantage to the BNR system.

Bacteria↗

Competition between polyphosphate- and glycogen-accumulating organisms in biological phosphorus removal systems--effect of temperature.

This study demonstrated that temperature is an important factor in determining the outcome of competition between polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating non-poly-P organisms (GAOs) and the resultant stability of enhanced biological phosphorus removal (EBPR) systems. At 20 degrees C and a 10-day sludge age, PAOs were dominant in the anaerobic/aerobic (A/O) SBR, however, at 30 degrees C and a 10-day sludge age, GAOs were dominant in the A/O SBR. For kinetic batch studies, the anaerobic specific acetate uptake rate of GAO-dominated sludge (1.34 x 10(-3) mg C/mg VSS x minute) was higher than the rate of PAO-dominated sludge (0.89 x 10(-3) mg C/mg VSS x minute) at 30 degrees C, leading to the eventual failure of EBPR processes at high temperatures.

Bioreactors↗

[Structure characterization of calcium polyphosphate bioceramics during sintering process].

Calcium polyphosphate (CPP) may be a promising bone substitute with controllably-degraded ability. In this investigation, the effects of sintering temperatures on its phase transformation and structure parameters, such as crystalline size distribution and micro-strain were investigated by X-ray diffraction (XRD). The phase composition was calculated with reference intensity ratio (RIR). The crystalline size distribution and micro-strain were calculated with Warren-Averbach Fourier transfer (W-A/FT) method. The results demonstrated that at the temperature of 585 degrees C-900 degrees C, the phase transformation of amorphous CPP into crystalline gamma-CPP and then into beta-CPP occurred,and the course of such transformation was accompanied with the significant change of the mean crystalline size (D) and the mean micro-strain (epsilon).

Biocompatible Materials↗

Short-term effects of carbon source on the competition of polyphosphate accumulating organisms and glycogen accumulating organisms.

The effectiveness of enhanced biological phosphorus removal (EBPR) systems is directly affected by the competition of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs). This study investigated the short-term effects of carbon source on PAO and GAO performance. The tests were designed to clearly determine the impact of volatile fatty acid (VFA) composition on the performance of two types of biomass, one enriched for PAOs and the other for GAOs. The two populations were enriched in separate reactors using identical operating conditions and very similar influent compositions with acetate as the sole carbon source. The only difference was that a very low level of phosphorus was present in the influent to the GAO reactor. The abundance of PAOs and GAOs was quantified using fluorescence in-situ hybridisation. The results clearly show that there are some very distinctive differences between PAOs and GAOs in their ability to utilise different carbon substrates. While both are able to take up acetate rapidly and completely, the GAOs are far slower at consuming propionate than the PAOs during short-term substrate changes. This provides a potentially highly valuable avenue to influence the competition between PAOs and GAOs. Other VFAs studied seem to be less usable in the short term by both PAOs and GAOs, as indicated by their much lower uptake rates.

Bacteria, Anaerobic↗

Inorganic polyphosphate and specific induction of apoptosis in human plasma cells.

BACKGROUND AND OBJECTIVES: Inorganic polyphosphate (polyP), a ubiquitous phosphate polymer with ATP-like bonds, has recently been related to a variety of functions including blood coagulation and cell proliferation. We investigated the effects of polyP in the biology of human plasma cells (PC), responsible for the production and maintenance of antibodies in response to antigens. DESIGN AND METHODS: The U266 myeloma cell line was used to study whether polyP affects immunoglobulin (Ig) secretion and survival. Different human cell lines were used to test the specificity of polyP on viability. We analyzed Ig secretion of PC from bone marrow and peripheral blood after polyP addition. A conventional tetanus toxoid booster immunization was used to increase the proportion of PC in order to examine the ex vivo effects of polyP. We also tested the effects of polyP on primary myeloma cells. Ig secretion and apoptosis were determined by ELISA and FACS respectively. RESULTS: Addition of polyP to human PC produced an unexpected inhibition of Ig secretion and stimulation of apoptosis. PolyP generated apoptosis specifically in PC, myeloma (malignant PC) cell lines, primary myeloma cells, and B lymphoid cell lines. Normal B cells, T cells, total blood mononuclear cells, and non-lymphoid cell lines were not affected by polyP. In the U266 myeloma cell line, polyP induced externalization of phosphatidylserine, activation of caspase-3, and arrest of the cell cycle. The protective effects of interleukin-6 did not overcome the polyP-induced apoptosis Interpretation and CONCLUSIONS: Taken together, our results suggest for the first time the relevance of the use of polyP to the humoral immune response and open prospects for polyP as a novel therapy for myeloma.

Apoptosis↗

Cloning and expression of human 75-kDa inositol polyphosphate-5-phosphatase.

Inositol polyphosphate-5-phosphatase (5-phosphatase) hydrolyzes inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thereby functions as a signal terminating enzyme in cellular calcium ion mobilization. A cDNA encoding human platelet 5-phosphatase has been isolated by screening for beta-galactosidase fusion proteins that bind to inositol 1,3,4,5-tetrakisphosphate. The sensitivity of the screening procedure was enhanced 50- to 100-fold by amplification of "sublibraries" prior to carrying out binding assays. The sequences derived from the "expression clone" were used to screen human erythroleukemia cell line and human megakaryocytic cell line cDNA libraries. We obtained two additional clones which together consist of 2381 base pairs. The amino-terminal amino acid sequence from the 75-kDa 5-phosphatase purified from platelets is identical to amino acids 38-56 predicted from the cDNA. This suggests that the platelet 5-phosphatase is formed by proteolytic processing of a larger precursor. The cDNA predicts that the mature enzyme contains 635 amino acids (Mr 72, 891). Antibodies directed against recombinant TrpE fusion proteins of either an amino-terminal region or a carboxyl-terminal region immunoprecipitate the enzyme activity from a preparation of the 75-kDa form of platelet 5-phosphatase (Type II) but do not precipitate the distinct 47-kDa 5-phosphatase (Type I) also found in platelets. In addition, the recombinant protein expressed in Cos-7 cells has the same 5-phosphatase activity as the platelet 5-phosphatase.

Amino Acid Sequence↗

Liquid chromatographic determination of L-ascorbate 2-polyphosphate in fish feeds by enzymatic release of L-ascorbate.

An accurate method was devised to assay L-ascorbic 2-polyphosphate esters (AsPP) in fish feed by phosphatase digestion followed by determination of the released L-ascorbic acid (AsA). Compressed yeast and dithiothreitol are added to the phosphatase reaction mixture to give 95-100% recovery of AsA, which is quantitated by reverse-phase liquid chromatography (LC) with electrochemical detection. Chromatograms of all feed digests showed baseline resolution of AsA. In 3 feeds, to which 75-125 ppm AsA equivalents in the form of AsPP were added, the assay procedure gave 98-100% recovery of AsA.

Animal Feed↗

Decreased bone uptake of technetium-99m polyphosphate in thalassemia major.

Bone scans were performed with Tc-99m stannous polyphosphate on four patients with thalassemia major. Three of the scans show generalized decrease in skeletal uptake of the radiopharmaceutical, associated with renal enlargement and markedly increased renal radioactivity. The skeletal findings are consistent with the known bone abnormalities in thalassemia major, which are secondary to the extensive marrow hyperplasia and include loss of trabeculae and cortical thinning with consequent loss of bone mass. The increased renal uptake is probably due in part to the increased renal excretion (secondary to the poor bone uptake) and in part to the tubular dilatation and renal enlargement associated with thalassemia major. In addition, the presence of excessive amounts of iron in these patients may play a role in both the skeletal and renal findings.

Adult↗

Characterization of a cDNA encoding the 43-kDa membrane-associated inositol-polyphosphate 5-phosphatase.

Agonist stimulation of cells results in phosphatidylinositol turnover and the generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which mobilizes intracellular calcium. The inositol-polyphosphate 5-phosphatase (5-phosphatase) enzymes hydrolyze Ins(1,4,5)P3 in a signal-terminating reaction. We have isolated a 2.7-kilobase (kb) composite cDNA, encoding the 43-kDa membrane-associated 5-phosphatase, by screening a human placental lambda gt11 library, using degenerate oligonucleotides. The 2.7-kb cDNA contains a 1.1-kb open reading frame, comprising 363 amino acids, which encodes a protein of a predicted molecular mass of 42 kDa. Amino acid sequence analysis demonstrates a number of potential sites for phosphorylation by protein kinase C and a CAAX motif in the COOH terminus, which may mediate membrane localization. The recombinant enzyme was expressed in COS-7 cells, resulting in a 50-fold increase in enzyme activity in the detergent-soluble membrane fraction of the cell (nanomole of Ins(1,4,5)P3 hydrolyzed per min/mg), but only a 2.5-fold increase in 5-phosphatase activity in the total cell homogenate. Sequence analysis demonstrated a 73-amino acid domain in the COOH terminus of the 43-kDa membrane-associated 5-phosphatase, which had 30% sequence identity and 67% similarity to a region in the 75-kDa 5-phosphatase and 34% identity and 70% similarity to a sequence in the protein that is encoded by the gene, defective in Lowe's oculocerebrorenal syndrome. As shown by RNA analysis the 43-kDa membrane-associated 5-phosphatase appears to be predominantly expressed in heart, brain, and skeletal muscle.

Amino Acid Sequence↗

Purification and characterization of a 43-kDa membrane-associated inositol polyphosphate 5-phosphatase from human placenta.

We have identified, isolated, and characterized a membrane-associated inositol polyphosphate 5-phosphatase (5-phosphatase) from the particulate fraction of human placenta. The enzyme was purified 3700-fold from a detergent extract of human placental membranes to apparent homogeneity, by chromatography on DEAE-Sepharose, S-Sepharose, hydroxylapatite, and Biosil SEC 250 HPLC gel filtration. The purified 5-phosphatase has a molecular mass of 43 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. The enzyme hydrolyzes inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) to inositol 1,4 bisphosphate (Ins(1,4)P2) with an apparent Km of 5 microM. The 43-kDa 5-phosphatase also hydrolyzes inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) with an apparent Km of 1.2 microM. The enzyme requires Mg2+ ions for activity and is inhibited by Ca2+ concentrations greater than 100 microM. Polyclonal antibodies developed against the membrane-associated enzyme immunoprecipitate the purified membrane-associated placental 5-phosphatase and the platelet Type I cytosolic enzyme, but not the 75-kDa platelet Type II 5-phosphatase. These results demonstrate that the purified membrane 5-phosphatase bears physical and immunological similarity with the Type I cytosolic platelet enzyme.

Chromatography, Liquid↗

Negative signaling in B lymphocytes induces tyrosine phosphorylation of the 145-kDa inositol polyphosphate 5-phosphatase, SHIP.

Stimulation of the B cell Ag receptor (BCR) has been reported to induce tyrosine phosphorylation of a 145-kDa protein and its association with the adapter protein Shc. We have identified this protein as the novel inositol polyphosphate 5-phosphatase (SHIP). Further analysis revealed that both maximal phosphorylation of SHIP and its association with Shc require co-clustering with the Fc receptor for IgG (Fc gamma RII) rather than stimulation of the BCR alone. Since co-clustering of the BCR and Fc gamma RII also down-regulates proliferation induced by Ag receptor stimulation, we hypothesize that tyrosine phosphorylation of SHIP and its association with Shc contribute to negative signaling through effects on inositol and phosphatidylinositol metabolism.

Animals↗