Intracellular Phosphate Concentration Research Articles

PitA Controls the H2- and H3-T6SSs through PhoB in Pseudomonas aeruginosa.

Pseudomonas aeruginosa possesses three type VI secretion systems (T6SSs) that are involved in interspecies competition, internalization into epithelial cells, and virulence. Host-derived mucin glycans regulate the T6SSs through RetS, and attacks from other species activate the H1-T6SS. However, other environmental signals that control the T6SSs remain to be explored. Previously, we determined PitA to be a constitutive phosphate transporter, whose mutation reduces the intracellular phosphate concentration. Here, we demonstrate that mutation in the pitA gene increases the expression of the H2- and H3-T6SS genes and enhances bacterial uptake by A549 cells. We further found that mutation of pitA results in activation of the quorum sensing (QS) systems, which contributes to the upregulation of the H2- and H3-T6SS genes. Overexpression of the phosphate transporter complex genes pstSCAB or knockdown of the phosphate starvation response regulator gene phoB in the ΔpitA mutant reduces the expression of the QS genes and subsequently the H2- and H3-T6SS genes and bacterial internalization. Furthermore, growth of wild-type PA14 in a low-phosphate medium results in upregulation of the QS and H2- and H3-T6SS genes and bacterial internalization compared to those in cells grown in a high-phosphate medium. Deletion of the phoB gene abolished the differences in the expression of the QS and T6SS genes as well as bacterial internalization in the low- and high- phosphate media. Overall, our results elucidate the mechanism of PitA-mediated regulation on the QS system and H2- and H3-T6SSs and reveal a novel pathway that regulates the T6SSs in response to phosphate starvation. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogenic bacterium that causes acute and chronic infections in humans. The type VI secretion systems (T6SSs) have been shown to associate with chronic infections. Understanding the mechanism used by the bacteria to sense environmental signals and regulate virulence factors will provide clues for developing novel effective treatment strategies. Here, we demonstrate a relationship between a phosphate transporter and the T6SSs and reveal a novel regulatory pathway that senses phosphate limitation and controls bacterial virulence factors in P. aeruginosa.

Pseudomonas aeruginosa Phosphate Transporter PitA (PA4292) Controls Susceptibility to Aminoglycoside Antibiotics by Regulating the Proton Motive Force.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that causes nosocomial infections in immunocompromised patients. β-lactam and aminoglycoside antibiotics are commonly used in the treatment of P. aeruginosa infections. Previously, we found that mutation in a PA4292 gene increases bacterial resistance to β-lactam antibiotics. In this study, we demonstrated that mutation in PA4292 increases bacterial susceptibility to aminoglycoside antibiotics. We further found enhanced uptake of tobramycin by the ΔPA4292 mutant, which might be due to an increase of proton motive force (PMF). Sequence analysis revealed PA4292 is homologous to the Escherichia coli phosphate transporter PitA. Mutation of PA4292 indeed reduces intracellular phosphate concentration. We thus named PA4292 as pitA. Although the PMF is enhanced in the ΔpitA mutant, the intracellular ATP concentration is lower than that in the isogenic wild-type strain PA14, which might be due to lack of the ATP synthesis substrate phosphate. Overexpression of the phosphate transporter complex genes pstSCAB in the ΔpitA mutant restores the intracellular phosphate concentration, PMF, ATP synthesis, and aminoglycosides resistance. In addition, growth of wild-type PA14 in a low-phosphate medium resulted in higher PMF and aminoglycoside susceptibility compared to cells grown in a high-phosphate medium. Overall, our results demonstrate the roles of PitA in phosphate transportation and reveal the relationship between intracellular phosphate and aminoglycoside susceptibility.

Rice ACID PHOSPHATASE 1 regulates Pi stress adaptation by maintaining intracellular Pi homeostasis.

The concentration and homeostasis of intracellular phosphate (Pi) are crucial for sustaining cell metabolism and growth. During short-term Pi starvation, intracellular Pi is maintained relatively constant at the expense of vacuolar Pi. After the vacuolar stored Pi is exhausted, the plant cells induce the synthesis of intracellular acid phosphatase (APase) to recycle Pi from expendable organic phosphate (Po). In this study, the expression, enzymatic activity and subcellular localization of ACID PHOSPHATASE 1 (OsACP1) were determined. OsACP1 expression is specifically induced in almost all cell types of leaves and roots under Pi stress conditions. OsACP1 encodes an acid phosphatase with broad Po substrates and localizes in the endoplasmic reticulum (ER) and Golgi apparatus (GA). The phylogenic analysis demonstrates that OsACP1 has a similar structure with human acid phosphatase PHOSPHO1. Overexpression or mutation of OsACP1 affected Po degradation and utilization, which further influenced plant growth and productivity under both Pi-sufficient and Pi-deficient conditions. Moreover, overexpression of OsACP1 significantly affected intracellular Pi homeostasis and Pi starvation signalling. We concluded that OsACP1 is an active acid phosphatase that regulates rice growth under Pi stress conditions by recycling Pi from Po in the ER and GA.

Cortisol enhances citrulline synthesis from proline in enterocytes of suckling piglets.

There are marked decreases in plasma concentrations of cortisol and arginine (an essential amino acid for neonates) as well as intestinal citrulline synthesis in piglets during the first 14days of life. The objective of this study was to test the hypothesis that increasing plasma cortisol concentrations by cortisol administration may prevent the decline in intestinal citrulline and arginine synthesis from proline, thereby possibly increasing plasma arginine concentration in suckling piglets and their growth. Seven-day-old pigs reared by sows received daily intramuscular injections of hydrocortisone 21-acetate (25mg/kg) or vehicle solution (saline) (n = 10/group). At 14days of age, piglets were used to prepare jejunal enterocytes. Cells were incubated at 37°C for 30min in oxygenated Krebs buffer containing 5mM glucose, 2mM [U-14C]proline, and 2mM glutamine. Cortisol treatment increased plasma cortisol concentration, mitochondrial proline oxidase and N-acetylglutamate synthase activities, cytosolic argininosuccinate lyase activity, and the intracellular concentrations of N-acetylglutamate and carbamoyl phosphate for citrulline and arginine synthesis. However, cortisol treatment induced the expression of intestinal arginase-II for arginine hydrolysis, resulting in no change in plasma arginine concentration. Administration of cortisol had no effect on milk consumption or the whole-body growth rate of piglets, but increased villus height in the jejunum and ileum. Collectively, these results suggest an important role for proline oxidase and N-acetylglutamate in regulating citrulline and arginine synthesis from proline in pig enterocytes. Because proline catabolism plays an important role in modulating protein synthesis, cell proliferation, and arginine production, our findings may have important implications for understanding the role of proline oxidase in the growth and health of the mammalian small intestine.

Intracellular Phosphate and ATP Depletion Measured by Magnetic Resonance Spectroscopy in Patients Receiving Maintenance Hemodialysis.

The precise origin of phosphate that is removed during hemodialysis remains unclear; only a minority comes from the extracellular space. One possibility is that the remaining phosphate originates from the intracellular compartment, but there have been no available data from direct assessment of intracellular phosphate in patients undergoing hemodialysis. We used phosphorus magnetic resonance spectroscopy to quantify intracellular inorganic phosphate (Pi), phosphocreatine (PCr), and βATP. In our pilot, single-center, prospective study, 11 patients with ESKD underwent phosphorus (31P) magnetic resonance spectroscopy examination during a 4-hour hemodialysis treatment. Spectra were acquired every 152 seconds during the hemodialysis session. The primary outcome was a change in the PCr-Pi ratio during the session. During the first hour of hemodialysis, mean phosphatemia decreased significantly (-41%; P<0.001); thereafter, it decreased more slowly until the end of the session. We found a significant increase in the PCr-Pi ratio (+23%; P=0.001) during dialysis, indicating a reduction in intracellular Pi concentration. The PCr-βATP ratio increased significantly (+31%; P=0.001) over a similar time period, indicating a reduction in βATP. The change of the PCr-βATP ratio was significantly correlated to the change of depurated Pi. Phosphorus magnetic resonance spectroscopy examination of patients with ESKD during hemodialysis treatment confirmed that depurated Pi originates from the intracellular compartment. This finding raises the possibility that excessive dialytic depuration of phosphate might adversely affect the intracellular availability of high-energy phosphates and ultimately, cellular metabolism. Further studies are needed to investigate the relationship between objective and subjective effects of hemodialysis and decreases of intracellular Pi and βATP content. Intracellular Phosphate Concentration Evolution During Hemodialysis by MR Spectroscopy (CIPHEMO), NCT03119818.

Phosphate Assay Kit in One Cell for Electrochemical Detection of Intracellular Phosphate Ions at Single Cells

In this paper, phosphate assay kit in one cell is realized for the electrochemical detection of intracellular phosphate ions at single cells. The components of the phosphate assay kit, including maltose phosphorylase, maltose, mutarotase, and glucose oxidase, are electrochemically injected into a living cell through a nanometer-sized capillary with the ring electrode at the tip. These components react with phosphate ions inside the cell to generate hydrogen peroxide that is electrochemically oxidized at the ring electrode for the qualification of intracellular phosphate ions. An average 1.7 nA charge was collected from eight individual cells, suggesting an intracellular phosphate concentration of 2.1 mM. The establishment in the electrochemical measurement of phosphate ions provides a special strategy to monitor the fluctuation of intracellular phosphate at single cells, which is significant for the future investigation of phosphate signal transduction pathway.

Phosphate and carbohydrate facilitate the formation of filamentous Salmonella enterica during osmotic stress.

Salmonella enterica is a human pathogen that can produce filamentous cells in response to environmental stress. The molecular mediators and biosynthetic pathways that contribute to the formation of filamentous cells (>10 µm in length) during osmotic stress are mostly unknown. The comparison of filamentous and non-filamentous cells in this study was aided by the use of a filtration step to separate cell types. Osmotic stress caused an efflux of phosphate from cells, and the addition of phosphate and a carbohydrate to Luria broth with 7 % NaCl (LB-7NaCl) significantly increased the proportion of filamentous cells in the population (58 %). In addition to direct measurements of intracellular and extracellular phosphate concentrations, the relative abundance of the iraP transcript that is induced by phosphate limitation was monitored. Non-filamentous cells had a greater relative abundance of iraP transcript than filamentous cells. IraP also affects the stability of RpoS, which regulates the general stress regulon, and was detected in non-filamentous cells but not filamentous cells. Markers of metabolic pathways for the production of acetyl-CoA (pflB, encoding for pyruvate formate lyase) and fatty acids (fabH) that are essential to membrane biosynthesis were found in greater abundance in filamentous cells than non-filamentous cells. There were no differences in the DNA, protein and biomass levels in filamentous and non-filamentous cells after 48 h of incubation, although the filamentous cells produced significantly (P<0.05) more acetate. This study found that phosphate and carbohydrate enhanced the formation of filamentous cells during osmotic stress, and there were differences in key regulatory elements and markers of metabolic pathways in filamentous and non-filamentous S. enterica.

TDAG51 is a key modulator of vascular calcification and osteogenic transdifferentiation of arterial smooth muscle cells

Cardiovascular disease (CVD) is the primary cause of morbidity and mortality in patients with chronic kidney disease (CKD). More than half of all deaths from patients with end-stage renal disease (ESRD) requiring dialysis can be attributed to CVD. Although traditional risk factors for CVD are prevalent in ESRD, they cannot fully explain the high mortality rates. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced CKD and is now recognized as an important predictor and an independent risk factor for cardiovascular and all-cause and mortality in these patients. VC in CKD is triggered by vascular smooth muscle cells (VSMCs) transforming into osteoblasts, a consequence of elevated circulating phosphate levels due to poor kidney function. Understanding and targeting this SMC trans-differentiation and subsequent VC represents a viable therapeutic avenue for managing CKD complications. Similar to bone formation, VC is a highly regulated process that draws many parallels with osteogenic differentiation. We have recently observed that T-cell Death Associated Gene 51 (TDAG51), a protein upregulated by oxidative and endoplasmic reticulum stress, is induced in SMCs under conditions of hyperphosphatemia and is expressed in the medial layer of calcified human vessels. We have found that ablation of TDAG51 reduces SMC trans-differentiation and VC, both in human SMCs treated with TDAG51 siRNA and in mouse VSMCs isolated from TDAG51−/− aortas. To explain these findings, we observed that TDAG−/− VSMCs express reduced levels of the of the Pit-1, a solute transporter responsible for cellular phosphate uptake. Consistent with this observation, reduced Pit-1 expression was associated with reduced intracellular phosphate levels and reduced phosphate-induced apoptosis. Further, TDAG−/− VSMCs exhibit significantly reduced transcriptional activity of a phosphate-inducible transcription factor, and well-established driver of VC, known as Runx2. Support or Funding Information This work was supported in part by research grants to Richard C. Austin from the Heart and Stroke Foundation of Ontario (T-6146), the Heart and Stroke Foundation of Canada (G-13-0003064 and G-15-0009389) and the Canadian Institutes of Health Research (74477). TDAG51 is involved in the development and progression of VC through the regulation of intracellular phosphate content and Runx2 transcriptional activity. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of a core Clostridium thermocellum kinetic metabolic model consistent with multiple genetic perturbations

BackgroundClostridium thermocellum is a Gram-positive anaerobe with the ability to hydrolyze and metabolize cellulose into biofuels such as ethanol, making it an attractive candidate for consolidated bioprocessing (CBP). At present, metabolic engineering in C. thermocellum is hindered due to the incomplete description of its metabolic repertoire and regulation within a predictive metabolic model. Genome-scale metabolic (GSM) models augmented with kinetic models of metabolism have been shown to be effective at recapitulating perturbed metabolic phenotypes.ResultsIn this effort, we first update a second-generation genome-scale metabolic model (iCth446) for C. thermocellum by correcting cofactor dependencies, restoring elemental and charge balances, and updating GAM and NGAM values to improve phenotype predictions. The iCth446 model is next used as a scaffold to develop a core kinetic model (k-ctherm118) of the C. thermocellum central metabolism using the Ensemble Modeling (EM) paradigm. Model parameterization is carried out by simultaneously imposing fermentation yield data in lactate, malate, acetate, and hydrogen production pathways for 19 measured metabolites spanning a library of 19 distinct single and multiple gene knockout mutants along with 18 intracellular metabolite concentration data for a Δgldh mutant and ten experimentally measured Michaelis–Menten kinetic parameters.ConclusionsThe k-ctherm118 model captures significant metabolic changes caused by (1) nitrogen limitation leading to increased yields for lactate, pyruvate, and amino acids, and (2) ethanol stress causing an increase in intracellular sugar phosphate concentrations (~1.5-fold) due to upregulation of cofactor pools. Robustness analysis of k-ctherm118 alludes to the presence of a secondary activity of ketol-acid reductoisomerase and possible regulation by valine and/or leucine pool levels. In addition, cross-validation and robustness analysis allude to missing elements in k-ctherm118 and suggest additional experiments to improve kinetic model prediction fidelity. Overall, the study quantitatively assesses the advantages of EM-based kinetic modeling towards improved prediction of C. thermocellum metabolism and develops a predictive kinetic model which can be used to design biofuel-overproducing strains.

Modélisation des transferts de phosphore pendant l’hémodialyse

Chronic kidney disease causes hyperphosphatemia, which is associated with increased cardiovascular risk and mortality. In patients with end-stage renal disease, haemodialysis allows the control of hyperphosphatemia. During a 4-h haemodialysis session, between 600 and 700mg of phosphate are extracted from the plasma, whereas the latter contains only 90mg of inorganic phosphate. The precise origin of phosphates remains unknown. The modelling of phosphorus transfers allows to predict the outcome after changes in dialysis prescription (duration, frequency) with simple two-compartment models and to describe the transfers between the different body compartments with more complex models. Work using 31P nuclear magnetic resonance spectroscopy performed in animals showed an increase in intracellular phosphate concentration and a decrease in intracellular ATP during a haemodialysis session suggesting an intracellular origin of phosphates.

Polyphosphate is involved in cell cycle progression and genomic stability in Saccharomyces cerevisiae.

Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4Δ) or hydrolyse polyP (ppn1Δ, ppx1Δ) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA.

Validation and Application of a Liquid Chromatography-Tandem Mass Spectrometry Method To Determine the Concentrations of Sofosbuvir Anabolites in Cells.

Sofosbuvir (SOF) is a highly efficacious and well-tolerated uridine nucleotide analog that inhibits the hepatitis C virus (HCV) NS5B polymerase enzyme. SOF is administered as a prodrug, which undergoes intracellular phosphorylation by host enzymes to a monophosphate, diphosphate, and finally a pharmacologically active triphosphate. In order to fully understand the clinical pharmacology of SOF, there is a great need to determine the intracellular phosphate concentrations of the drug. We describe the validation and utilization of a method to characterize SOF's disposition into various in vivo cell types, including hepatocytes, peripheral blood mononuclear cells (PBMC), and red blood cells (RBC). Standard bioanalytical validation criteria were applied to lysed cellular matrices, with a validated linear range of 50 to 50,000 fmol/sample for each phosphate moiety. The assay was utilized to collect the first data demonstrating concentrations of phosphorylated anabolites formed in PBMC, hepatocytes, and RBC in vivo during SOF therapy. Median concentrations in PBMC were 220 (range, 51.5 to 846), 70.2 (range, 25.8 to 275), and 859 (range, 54.5 to 6,756) fmol/10(6) cells in the monophosphate, diphosphate, and triphosphate fractions, respectively. In contrast, RBC triphosphate concentrations were much lower than those of PBMC, as the median concentration was 2.91 (range, 1.14 to 10.4) fmol/10(6) cells. The PBMC triphosphate half-life was estimated at 26 h using noncompartmental approaches, while nonlinear mixed-effect modeling was used to estimate a 69 h half-life for this moiety in RBC. The validated method and the data it generates provide novel insight into the cellular disposition of SOF and its phosphorylated anabolites in vivo.

Interactions of vitamin D and the proximal tubule.

Severe vitamin D deficiency (reduction in serum 25(OH)D concentration) in infants and children can cause features of the Fanconi syndrome, including phosphaturia, glycosuria, aminoaciduria, and renal tubular acidosis. This indicates that vitamin D and its metabolites influence proximal tubule function. Filtered 25(OH)D bound to vitamin D binding protein (DBP) is endocytosed by megalin-cubilin in the apical membrane. Intracellular 25(OH)D is metabolized to 1,25(OH)2D or calcitroic acid by 1-α-hydroxylase or 24-hydroxylase in tubule cell mitochondria. Bone-produced fibroblast growth factor 23 (FGF23) bound to Klotho in tubule cells and intracellular phosphate concentrations are regulators of 1-α-hydroxylase activity and cause proximal tubule phosphaturia. Aminoaciduria occurs when amino acid transporter synthesis is deficient, and 1,25(OH)2D along with retinoic acid up-regulate transporter synthesis by a vitamin D response element in the promoter region of the transporter gene. This review discusses evidence gained from studies in animals or cell lines, as well as from human disorders, that provide insight into vitamin D-proximal tubule interactions.

English

Himalayas are the natural reservoir of complex and diversified gene pool. Three Shiwalik Himalayan intracellular phytase producing budding yeasts were assayed for in vitro degradation of natural animal feed substrates. Phosphorus availability was found to enhance upto 70% yeast cultures during in vitro biodegradation of natural animal feed substrates. A direct correlation between intracellular phosphate concentration and phytase activity suggested the use of&nbsp; whole cell preparations in place of purified enzymes. Zymogram analysis revealed the presence of single high molecular weight isoform of the enzyme phytase. Based on 5.8S-ITS-rDNA sequencing, using ITS1 and ITS4 primers, the cultures were identified as Candida tropicalis (B4), Issatchenkia orientalis (PA4) and Pichia gluermondii (SS1). Indigenous I. orientalis strain PA4 was found superior&nbsp; among all the yeasts strains and therefore can be developed as successful inoculant for animal nutrition as well as environmental management under Himalayan ecosystems. &nbsp; &nbsp; Key words: Phytase, Shiwalik Himalaya, phytase biodegradation, 5.8S-ITS rDNA, Yeast identification.

Cell cycle regulation of purine synthesis by phosphoribosyl pyrophosphate and inorganic phosphate

Cells must increase synthesis of purine nucleotides/deoxynucleotides before or during S-phase. We found that rates of purine synthesis via the de novo and salvage pathways increased 5.0- and 3.3-fold respectively, as cells progressed from mid-G1-phase to early S-phase. The increased purine synthesis could be attributed to a 3.2-fold increase in intracellular PRPP (5-phosphoribosyl-α-1-pyrophosphate), a rate-limiting substrate for de novo and salvage purine synthesis. PRPP can be produced by the oxidative and non-oxidative pentose phosphate pathways, and we found a 3.1-fold increase in flow through the non-oxidative pathway, with no change in oxidative pathway activity. Non-oxidative pentose phosphate pathway enzymes showed no change in activity, but PRPP synthetase is regulated by phosphate, and we found that phosphate uptake and total intracellular phosphate concentration increased significantly between mid-G1-phase and early S-phase. Over the same time period, PRPP synthetase activity increased 2.5-fold when assayed in the absence of added phosphate, making enzyme activity dependent on cellular phosphate at the time of extraction. We conclude that purine synthesis increases as cells progress from G1- to S-phase, and that the increase is from heightened PRPP synthetase activity due to increased intracellular phosphate.

Utilization of multifrequency permittivity measurements in addition to biomass monitoring

In recent years measurement of permittivity signal has been increasingly used for online biomass monitoring of cell cultures. Breweries use it as an established method for fermenter inoculation and bioprocess control for instance [1]. In the case of animal cell cultures the correlation between permittivity and viable cell densities determined offline varies along cultivation time. Hence, several authors have used the permittivity signal as an indirect method for measuring oxygen and glutamine consumption as well as intracellular nucleotide phosphate concentrations [2,3]. The latest generation of biomass monitoring devices allows parallel measurement of permittivity at a range of frequencies leading to an improvement in the correlation between biomass and permittivity and providing a tool to further explore other aspects related to the physiological state of the cells.

The ferroxidase center is essential for ferritin iron loading in the presence of phosphate and minimizes side reactions that form Fe(III)-phosphate colloids

Ferritin iron loading was studied in the presence of physiological serum phosphate concentrations (1 mM), elevated serum concentrations (2-5 mM), and intracellular phosphate concentrations (10 mM). Experiments compared iron loading into homopolymers of H and L ferritin with horse spleen ferritin. Prior to studying the reactions with ferritin, a series of control reactions were performed to study the solution chemistry of Fe(2+) and phosphate. In the absence of ferritin, phosphate catalyzed Fe(2+) oxidation and formed soluble polymeric Fe(III)-phosphate complexes. The Fe(III)-phosphate complexes were characterized by electron microscopy and atomic force microscopy, which revealed spherical nanoparticles with diameters of 10-20 nm. The soluble Fe(III)-phosphate complexes also formed as competing reactions during iron loading into ferritin. Elemental analysis on ferritin samples separated from the Fe(III)-phosphate complexes showed that as the phosphate concentration increased, the iron loading into horse ferritin decreased. The composition of the mineral that does form inside horse ferritin has a higher iron/phosphate ratio (~1:1) than ferritin purified from tissue (~10:1). Phosphate significantly inhibited iron loading into L ferritin, due to the lack of the ferroxidase center in this homopolymer. Spectrophotometric assays of iron loading into H ferritin showed identical iron loading curves in the presence of phosphate, indicating that the ferroxidase center of H ferritin efficiently competes with phosphate for the binding and oxidation of Fe(2+). Additional studies demonstrated that H ferritin ferroxidase activity could be used to oxidize Fe(2+) and facilitate the transfer of the Fe(3+) into apo transferrin in the presence of phosphate.

Inorganic phosphate as an important regulator of phosphatases.

Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate (Pi). Pi starvation-responsive genes appear to be involved in multiple metabolic pathways, implying a complex Pi regulation system in microorganisms and plants. A group of enzymes is required for absorption and maintenance of adequate phosphate levels, which is released from phosphate esters and anhydrides. The phosphatase system is particularly suited for the study of regulatory mechanisms because phosphatase activity is easily measured using specific methods and the difference between the repressed and derepressed levels of phosphatase activity is easily detected. This paper analyzes the protein phosphatase system induced during phosphate starvation in different organisms.

New insights on sarcoplasmic reticulum calcium regulation in muscle fatigue

a consistent observation with fatigue in skeletal muscle is a decline in the amplitude of the myoplasmic Ca2+ transient, which is thought to result primarily from a reduced Ca2+ flux through the ryanodine receptor (RyR1) of the sarcoplasmic reticulum (SR) ([Fig. 1][1]). This in turn is thought to

Decreased Intracellular Phosphate and Magnesium Concentrations in Vascular Smooth Muscle Cells from Spontaneously Hypertensive Rats.

A decrease in total magnesium content is not a direct proof of a decreased magnesium ion concentration. It could reflect a phosphate alteration or an ATP metabolism disorder. Plasma phosphate levels are lower in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY), and defects in membrane regulation or mitochondrial ATP synthase occur. Only sparse data exist concerning cellular magnesium and phosphate concentrations in hypertensive cells. In aortic smooth muscle cells from 10 SHR of the Münster strain and 10 age-matched normotensive WKY, the intracellular phosphate and magnesium content was measured by electron probe X-ray microanalysis (Camscan CS 24 apparatus, Cambridge, U.K.). The Mg(++) content was 0.90 +/- 0.15 g/kg dry weight in SHR versus 1.15 +/- 0.10 g/kg dry weight in WKY (p < 0.05). Vascular smooth muscle phosphate content was 23.6 +/- 0.79 g/kg dry weight in WKY versus 15.81 +/- 1.22 g/kg dry weight in SHR (p < 0.01). Aortic smooth muscle cells from SHR are characterized by markedly lowered cellular phosphate and magnesium concentrations and an altered ATP metabolism. </hea