Exogenous Phosphate Research Articles

H+-dependent inorganic phosphate uptake in Trypanosoma brucei is influenced by myo-inositol transporter.

Trypanosoma brucei is an extracellular protozoan parasite that causes human African trypanosomiasis or "sleeping sickness". During the different phases of its life cycle, T. brucei depends on exogenous inorganic phosphate (Pi), but little is known about the transport of Pi in this organism. In the present study, we showed that the transport of 32Pi across the plasma membrane follows Michaelis-Menten kinetics and is modulated by pH variation, with higher activity at acidic pH. Bloodstream forms presented lower Pi transport in comparison to procyclic forms, that displayed an apparent K0.5=0.093±0.008mM. Additionally, FCCP (H+-ionophore), valinomycin (K+-ionophore) and SCH28080 (H+, K+-ATPase inhibitor) inhibited the Pi transport. Gene Tb11.02.3020, previously described to encode the parasite H+:myo-inositol transporter (TbHMIT), was hypothesized to be potentially involved in the H+:Pi cotransport because of its similarity with the Pho84 transporter described in S. cerevisiae and other trypanosomatids. Indeed, the RNAi mediated knockdown remarkably reduced TbHMIT gene expression, compromised cell growth and decreased Pi transport by half. In addition, Pi transport was inhibited when parasites were incubated in the presence of concentrations of myo-inositol that are above 300μM. However, when expressed in Xenopus laevis oocytes, two-electrode voltage clamp experiments provided direct electrophysiological evidence that the protein encoded by TbHMIT is definitely a myo-inositol transporter that may be only marginally affected by the presence of Pi. These results confirmed the presence of a Pi carrier in T. brucei, similar to the H+-dependent inorganic phosphate system described in S. cerevisiae and other trypanosomatids. This transport system contributes to the acquisition of Pi and may be involved in the growth and survival of procyclic forms. In summary, this work presents the first description of a Pi transport system in T. brucei.

Endojen ve Eksojen Adenozin Tri-Fosfatʼın Eritrosit Deformabilitesine Etkisi

Endojen ve Eksojen Adenozin Tri-Fosfatʼın Eritrosit Deformabilitesine Etkisi

Myocardial Protective Effect of Exogenous Creatine Phosphate in Children Undergoing Open Heart Surgery

Objective: To explore the protective role of exogenous Creatine Phosphate (PCr) in the postoperative cardiac muscle recovery in children undergoing open heart surgery. Material and methods: 63 cases of congenital heart disease were randomly divided into 3 groups; A, B and C; 21 cases in each group. Myocardial perfusion fluids were made of the same amount of blood in the St, Thomas Hospital cardioplegic solution type 2 (STH 2). In B and C group, intraoperative myocardial perfusion solution added with exogenous CP. All the groups were postoperatively treated with general fluid therapy, while group C was given postoperative intravenous exogenous CP. Trends of myocardial enzyme at different time points were monitored. Results: At the end of surgery (T2), enzyme levels in the plasma were significantly increased, no difference between the groups B and C but were significantly lower than that in group A (P<0.05); after surgery myocardial enzyme levels recovered fastest in Group C, slowest in A group, the differences between each group were significant (P<0.05). Conclusions: Perioperative and postoperative exogenous PCr have myocardial protective effect and accelerate the repair of injured myocardium due to ischemia during open heart surgery in children.

Transcriptional profiling of Neurospora crassa Δmak-2 reveals that mitogen-activated protein kinase MAK-2 participates in the phosphate signaling pathway

The filamentous fungus Neurospora crassa is an excellent model system for examining molecular responses to ambient signals in eukaryotic microorganisms. Inorganic phosphate (Pi) is an essential growth-limiting nutrient in nature and is crucial for the synthesis of nucleic acids and the flow of genetic information. The genetic and molecular mechanisms controlling the response to Pi starvation in N. crassa include at least four genes (nuc-2, preg, pogv, and nuc-1), which are involved in a hierarchical regulatory activation network. In a previous work, we identified a number of genes modulated by NUC-2 protein, including the mak-2 gene, which codes for a mitogen-activated protein kinase (MAPK), suggesting its participation in the phosphate signaling pathway. Thus, to identify other genes involved in metabolic responses to exogenous phosphate sensing and the functioning of the MAPK MAK-2, we performed microarray experiments using a mak-2 knockout strain (Δmak-2) grown under phosphate-shortage conditions by comparing its transcription profile to that of a control strain grown in low- and high-phosphate cultures. These experiments revealed 912 unique differentially expressed genes involved in a number of physiological processes related to phosphate transport, metabolism, and regulation as well as posttranslational modification of proteins, and MAPK signaling pathways. Quantitative Real-time PCR gene expression analysis of 18 selected genes, using independent RNA samples, validated our microarray results. A high Pearson correlation between microarray and quantitative Real-time PCR data was observed. The analysis of these differentially expressed genes in the Δmak-2 strain provide evidence that the mak-2 gene participates in the hierarchical phosphate-signaling pathway in N. crassa in addition to its involvement in other metabolic routes such as the isoprenylation pathway, thus revealing novel aspects of the N. crassa phosphorus-sensing network.

A Case of Pseudohyperphosphatemia in a Patient with Multiple Myeloma

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Transport of inorganic phosphate in Leishmania infantum and compensatory regulation at low inorganic phosphate concentration

BackgroundProliferation of Leishmania infantum depends on exogenous inorganic phosphate (Pi) but little is known about energy metabolism and transport of Pi across the plasma membrane in Leishmania sp. MethodsWe investigated the kinetics of 32Pi transport, the influence of H+ and K+ ionophores and inhibitors, and expression of the genes for the Na+:Pi and H+:Pi cotransporters. ResultsThe proton ionophore FCCP, bafilomycin A1 (vacuolar ATPase inhibitor), nigericin (K+ ionophore) and SCH28080 (an inhibitor of H+, K+–ATPase) all inhibited the transport of Pi. This transport showed Michaelis–Menten kinetics with K0.5 and Vmax values of 0.016±0.002mM and 564.9±18.06pmol×h−1×10−7cells, respectively. These values classify the Pi transporter of L. infantum among the high-affinity transporters, a group that includes Pho84 of Saccharomyces cerevisiae. Two sequences were identified in the L. infantum genome that code for phosphate transporters. However, transcription of the PHO84 transporter was 10-fold higher than the PHO89 transporter in this parasite. Accordingly, Pi transport and LiPho84 gene expression were modulated by environmental Pi variations. ConclusionsThese findings confirm the presence of a Pi transporter in L. infantum, similar to PHO84 in S. cerevisiae, that contributes to the acquisition of inorganic phosphate and could be involved in growth and survival of the promastigote forms of L. infantum. General significanceThis work provides the first description of a PHO84-like Pi transporter in a Trypanosomatide parasite of the genus Leishmania, responsible for many infections worldwide.

Buyer beware! Doctor be aware!—Answers

Acute hyperphosphatemia with hypocalcemic tetany from exogenous phosphate load was diagnosed. The serum phosphorous level was 45.2 mg/dl, total calcium 5.1 mg/dl and ionized calcium 1.83mg/dl (normal range 4.40–5.40 mg/dl). An adult Fleet® enema (C.B. Fleet Co., Lynchburg, VA) has a volume of 118 ml and contains 19 g of monobasic sodium phosphate monohydrate, 7 g of dibasic sodium phosphate heptahydrate and 4.4 g of sodium [1]. Hypocalcemia is induced by calcium complexing with phosphate, and the magnitude of hypocalcemia is proportional to the degree by which the solubility product of calcium phosphate is exceeded. Although the exact threshold is impossible to determine, a product as low as 60 mg/dl has been associated with increased visceral calcification [2, 3]. Phosphorous is the sixth most abundant element in the human body, and under normal conditions most total body phosphorous exists in the bone in the divalent form (HPO4 ). Of the remaining phosphorous 10 % exists intracellularly, and only 1 % is found in the extracellular fluid of which 15 % is protein bound and another 5 % is complexed as salts with cations such as sodium, calcium and magnesium [4, 5]. Active absorption of dietary phosphorous occurs in the small intestine (duodenum and jejunum) via sodium phosphate cotransporter 2b (NaPi2b). Passive absorption takes place throughout the intestine, including the colon. As a reference, the typical adult diet contains 1 g of phosphorous daily. The recommended elemental phosphorous intake for a 6-year-old child is 500 mg per day. Our patient received 15.8 g (488.5 mmol) of elemental phosphate within 12 h. Control of plasma phosphate is normally maintained by altering renal excretion or redistribution within the body compartments. Phosphate is freely filtered at the glomerulus, and 85 % is reabsorbed into the blood at the level of the proximal tubule. Phosphate enters the proximal tubule cell against an electrical gradient via the NaPi2a and NaPi2c transporters, both of which are located along the brush border membrane [5]. Inhibition of proximal tubule reabsorption produces phosphaturia, which not uncommonly can cause hypophosphatemia. In our case, the large exogenous phosphorous load overwhelmed the limited ability of the gut and kidney to regulate phosphorous absorption and excretion. Hyperphosphatemia occurs from exogenous sources, as in our case, or from endogenous sources, such as tumor lysis syndrome or rhabdomyolysis. In clinical medicine, chronic kidney disease is the most common cause. Severe This article refers to the article that can be found at http://dx/doi.org/ 10.1007/s00467-012-2338-y E. Anyaegbu : T. Keefe Davis (*) :K. Hruska :A. Beck (*) Division of Nephrology, Department of Pediatrics, Washington University School of Medicine, Box 8208, 660 S. Euclid Ave, St. Louis, MO 63110, USA e-mail: davis_tk@kids.wustl.edu e-mail: beck_a@kids.wustl.edu

Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum

BackgroundPhaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome has been sequenced (<30 Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis has been reported under different growth conditions. To elucidate P. tricornutum gene expression profiles during nutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P) and whole-genome transcripts were monitored over time via RNA-sequence determination.ResultsThe specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, the increase in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphate was depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be an early trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genes associated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10 were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulation after growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbon reduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbon assimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon (DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed (2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth. Alternative pathways that could utilize HCO3- were also suggested by the gene expression profiles (e.g., putative propionyl-CoA and methylmalonyl-CoA decarboxylases).ConclusionsThe results indicate that P. tricornutum continued carbon dioxide reduction when population growth was arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels. Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up of precursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actual enzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellular responses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.

Correction of Hypophosphatasia‐Associated Mineralization Deficiencies In Vitro by Phosphate/Pyrophosphate Modulation in Periodontal Ligament Cells

Mutations in the liver/bone/kidney alkaline phosphatase (ALPL) gene in hypophosphatasia (HPP) reduce the function of tissue non-specific alkaline phosphatase (ALP), resulting in increased pyrophosphate (PP(i)) and a severe deficiency in acellular cementum. We hypothesize that exogenous phosphate (P(i)) would rescue the in vitro mineralization capacity of periodontal ligament (PDL) cells harvested from HPP-diagnosed patients, by correcting the P(i)/PP(i) ratio and modulating expression of genes involved with P(i)/PP(i) metabolism. Ex vivo and in vitro analyses were used to identify mechanisms involved in HPP-associated PDL/tooth root deficiencies. Constitutive expression of PP(i)-associated genes was contrasted in PDL versus pulp tissues obtained from healthy individuals. Primary PDL cell cultures from patients with HPP (monozygotic twin males) were established to assay ALP activity, in vitro mineralization, and gene expression. Exogenous P(i) was provided to correct the P(i)/PP(i) ratio. PDL tissues obtained from healthy individuals featured higher basal expression of key PP(i) regulators, genes ALPL, progressive ankylosis protein (ANKH), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), versus paired pulp tissues. A novel ALPL mutation was identified in the twin patients with HPP enrolled in this study. Compared to controls, HPP-PDL cells exhibited significantly reduced ALP and mineralizing capacity, which were rescued by addition of 1 mM P(i). Dysregulated expression of PP(i) regulatory genes ALPL, ANKH, and ENPP1 was also corrected by adding P(i), although other matrix markers evaluated in our study remained downregulated. These findings underscore the importance of controlling the P(i)/PP(i) ratio toward development of a functional periodontal apparatus and support P(i)/PP(i) imbalance as the etiology of HPP-associated cementum defects.

Na+-dependent and Na+-independent mechanisms for inorganic phosphate uptake in Trypanosoma rangeli

BackgroundTrypanosoma rangeli is dependent on the presence of exogenous orthophosphate (Pi) for maximal growth and ecto-phosphatase activity is responsible for Pi supply under low Pi. Here we investigated the mechanisms of Pi uptake. MethodsWe investigated the kinetics of 32Pi transport, its Na+ and H+ dependence, its correlation with the Na+-ATPase and H+-ATPase, and gene expression of the Na+:Pi cotransporter and Na+-ATPase. ResultsT. rangeli grown under limiting Pi transports this anion to the cytosol in the absence and presence of Na+, suggesting that influx is mediated by both Na+-independent and Na+-dependent transporters. Cloning studies demonstrated that this parasite expresses a Pi transporter not previously studied in trypanosomatids. The H+ ionophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, decreased both components of 32Pi influx by 80–95%. The H+-ATPase inhibitor, bafilomycin A1, inhibited the Na+-independent mechanism. Furosemide, an inhibitor of ouabain-insensitive Na+-ATPase, decreased both uptake mechanisms of 32Pi to the same extent, whereas ouabain had no effect, indicating that the former is the pump responsible for inwardly directed Na+ and the electric gradients required by the transporters. Parasite growth in high Pi had a lower Pi influx than that found in those grown in low Pi, without alteration in TrPho89 expression, showing that turnover of the transporters is stimulated by Pi starvation. ConclusionsTwo modes of Pi transport, one coupled to Na+-ATPase and other coupled to H+-ATPase seem to be responsible for Pi acquisition during development of T. rangeli. General significanceThis study provides the first description of the mechanism of Pi transport across the plasma membrane of trypanosomatids.

Arabidopsis thaliana PECP1 — Enzymatic characterization and structural organization of the first plant phosphoethanolamine/phosphocholine phosphatase

Maintenance of cellular phosphate homeostasis is crucial for primary and energy metabolism. In plants, low exogenous phosphate availability activates adaptive responses that include the immediate liberation of Pi from phosphorylated metabolites by yet uncharacterized intracellular phosphatases. Based on transcriptional analyses, the Arabidopsis thaliana gene At1g17710, a member of the HAD (Haloacid Dehalogenase) superfamily, was one of the most promising candidates. Here, we show by recombinant protein production and analysis of purified protein that the gene At1g17710 encodes a phosphoethanolamine/phosphocholine phosphatase (EC 3.1.3.75). Thus, the gene product was termed AtPECP1. The present study demonstrates that the Mg2+-dependent enzyme exhibits pronounced specificity for both substrates. The enzyme displays a broad pH optimum ranging from pH 6 to pH 8. Comparison of Km values indicates a slightly higher affinity for phosphocholine (0.44mM) than for phosphoethanolamine (1.16mM). The catalytic efficiency, however, is markedly higher for phosphoethanolamine than for phosphocholine being 1.06×104M−1s−1 and 2.34×103M−1s−1, respectively. Size exclusion chromatography, native gel electrophoresis and SAXS experiments with recombinant protein clearly point to a rapid monomer–dimer equilibrium of protein subunits. Given its established substrate specificity the enzyme is likely to be involved in the liberation of inorganic phosphate from intracellular sources and is especially in demand under phosphate-deprived conditions.

Exogenous NADP increases the level of histone H2AX phosphorylation in mouse heart cells after ionizing radiation

Phosphorylation of replaceable histone H2AX occurs in megabase chromatin domains around DNA double-strand breaks (DSBs), and this modification called gamma-H2AX can be used as an effective marker for DSBs repair and DNA damage response. Using Western blotting and immunohistochemistry techniques we have studied here the influence of exogenous nicotinamide adenine dinucleotide phosphate (NADP) which could potentially increase the intracellular level of NAD+ and on the level of gamma-H2AX formation in mouse heart cells after ionizing radiation (IR). We have found that injection of NAD+ in different doses immediately after IR causes an increased level of gamma-H2AX in mouse heart cells 20 min after IR at the dose of 3 Gy compared to control mice after IR exposure. It indicates that it could be a relationship between intracellular NAD+ content and DNA damage response in vivo.

Phosphate systemically inhibits development of arbuscular mycorrhiza in Petunia hybrida and represses genes involved in mycorrhizal functioning

Most terrestrial plants form arbuscular mycorrhiza (AM), mutualistic associations with soil fungi of the order Glomeromycota. The obligate biotrophic fungi trade mineral nutrients, mainly phosphate (P(i) ), for carbohydrates from the plants. Under conditions of high exogenous phosphate supply, when the plant can meet its own P requirements without the fungus, AM are suppressed, an effect which could be interpreted as an active strategy of the plant to limit carbohydrate consumption of the fungus by inhibiting its proliferation in the roots. However, the mechanisms involved in fungal inhibition are poorly understood. Here, we employ a transcriptomic approach to get insight into potential shifts in metabolic activity and symbiotic signalling, and in the defence status of plants exposed to high P(i) levels. We show that in mycorrhizal roots of petunia, a similar set of symbiosis-related genes is expressed as in mycorrhizal roots of Medicago, Lotus and rice. P(i) acts systemically to repress symbiotic gene expression and AM colonization in the root. In established mycorrhizal roots, P(i) repressed symbiotic gene expression rapidly, whereas the inhibition of colonization followed with a lag of more than a week. Taken together, these results suggest that P(i) acts by repressing essential symbiotic genes, in particular genes encoding enzymes of carotenoid and strigolactone biosynthesis, and symbiosis-associated phosphate transporters. The role of these effects in the suppression of symbiosis under high P(i) conditions is discussed.

Effects of exogenous phosphorus and silicon on osteoblast differentiation at the interface with bioactive ceramics

In this study, we have investigated the effects of dissolved phosphorus and silicon on osteoblast differentiation in vitro. Neonatal rat calvarial osteoblasts were seeded on silica-calcium phosphate composites (SCPCS), hydroxyapatite (HA-200), and tissue culture polystyrene (TCPS) and incubated over 4 days in media containing 0 {minimal essential medium [MEM] (-)} or 3 mM β-glycerophosphate [MEM (+)]. Inductively coupled plasma analysis showed that P-content in original MEM (+) was 225% higher than that in MEM (-). Moreover, P-content in MEM (+) significantly increased to 3.4-4.4 mM and 3.6-4.7 mM after 2 and 4 days incubation with SCPC, respectively, owing to material dissolution and exogenous phosphate supplementation. In contrast, P-content in MEM (+) showed no change upon incubation with HA or TCPS. The P-content in MEM (-) incubated with SCPC was considerably lower than that in MEM (+). SCPC exhibited controlled Si-release in cell culture media [MEM (-) or MEM (+)], with Si-rich SCPC showing a significantly greater dissolution than Si-poor SCPC. Moreover, SCPC, unlike HA, demonstrated a cell- and solution-mediated dissolution over 4 days. Quantitative real-time PCR showed that in MEM (-), osteocalcin and osteopontin mRNA expression on Si-rich SCPC was significantly greater than that on HA, suggesting that Si plays an important role in enhancing bone-cell differentiation. However, osteoblast phenotypic expression on SCPC was significantly decreased after 4 days incubation in MEM (+), indicating that sustained exposure to elevated P-levels in the media can downregulate osteoblast function. Our results demonstrate that the controlled dissolution of SCPC provides a natural stimulus for bone-cell differentiation in vitro and could obviate the need of exogenous phosphate supplementation.

24R,25-Dihydroxyvitamin D3 [24R,25(OH) 2D 3] controls growth plate development by inhibiting apoptosis in the reserve zone and stimulating response to 1α,25(OH) 2D 3 in hypertrophic cells

Previously we showed that costochondral growth plate resting zone (RC) chondrocytes response primarily to 24R,25(OH) 2D 3 whereas prehypertrophic and hypertrophic (GC) cells respond to 1α,25(OH) 2D 3. 24R,25(OH) 2D 3 increases RC cell proliferation and inhibits activity of matrix processing enzymes, suggesting it stabilizes cells in the reserve zone, possibly by inhibiting the matrix degradation characteristic of apoptotic hypertrophic GC cells. To test this, apoptosis was induced in rat RC cells by treatment with exogenous inorganic phosphate (Pi). 24R,25(OH) 2D 3 blocked apoptotic effects in a dose-dependent manner. Similarly, apoptosis was induced in ATDC5 cell cultures and 24R,25(OH) 2D 3 blocked this effect. Further studies indicated that 24R,25(OH) 2D 3 acts via at least two independent pathways. 24R,25(OH) 2D 3 increases LPA receptor-1 (LPA R1) expression and production of lysophosphatidic acid (LPA), and subsequent LPA R1/3-dependent signaling, thereby decreasing p53 abundance. LPA also increases the Bcl-2/Bax ratio. In addition, 24R,25(OH) 2D 3 acts by increasing PKC activity. 24R,25(OH) 2D 3 stimulates 1-hydroxylase activity, resulting in increased levels of 1,25(OH) 2D 3, and it increases levels of phospholipase A2 activating protein, which is required for rapid 1α,25(OH) 2D 3-dependent activation of PKC in GC cells. These results suggest that 24R,25(OH) 2D 3 modulates growth plate development by controlling the rate and extent of RC chondrocyte transition to a GC chondrocyte phenotype.

PhoB Regulates Motility, Biofilms, and Cyclic di-GMP inVibrio cholerae

Signaling through the second messenger cyclic di-GMP (c-di-GMP) is central to the life cycle of Vibrio cholerae. However, relatively little is known about the signaling mechanism, including the specific external stimuli that regulate c-di-GMP concentration. Here, we show that the phosphate responsive regulator PhoB regulates an operon, acgAB, which encodes c-di-GMP metabolic enzymes. We show that induction of acgAB by PhoB positively regulates V. cholerae motility in vitro and that PhoB regulates expression of acgAB at late stages during V. cholerae infection in the infant mouse small intestine. These data support a model whereby PhoB becomes activated at a late stage of infection in preparation for dissemination of V. cholerae to the aquatic environment and suggest that the concentration of exogenous phosphate may become limited at late stages of infection.

Loss of mannosylphosphate from Candida albicans cell wall proteins results in enhanced resistance to the inhibitory effect of a cationic antimicrobial peptide via reduced peptide binding to the cell surface

The outermost layer of the Candida albicans cell wall is enriched with mannosylated glycoproteins. We have used a range of isogenic glycosylation mutants of C. albicans, which are defective to varying degrees in cell wall protein mannosylation, to investigate the role of the outermost layer of the yeast cell wall in mediating the fungicidal action of the cationic, alpha-helical antimicrobial peptide dermaseptin S3(1-16) [DsS3(1-16)]. The degree of phosphomannan loss, and concomitant reduction in surface negative charge, from the series of glycosylation mutants correlated with reduced levels of peptide binding to the cells. In turn, the reduced peptide binding correlated with enhanced resistance to DsS3(1-16). To ascertain whether DsS3(1-16) binds to negatively charged phosphate, we studied the effect of exogenous glucosamine 6-phosphate, and glucosamine hydrochloride as a negative control, on the antifungal efficacy of DsS3(1-16). Glucosamine 6-phosphate retarded the efficacy of DsS3(1-16), and this was attributed to the presence of phosphate, because addition of identical concentrations of glucosamine hydrochloride had little detrimental effect on peptide efficacy. Fluorescence microscopy with DsS3(1-16) tagged with fluorescein revealed that the peptide binds to the outer surface of the yeast cell, supporting our previous conclusion that the presence of exterior phosphomannan is a major determinant of the antifungal potency of DsS3(1-16). The binding of the peptide to the cell surface was a transient event that was followed by apparent localization of DsS3(1-16) in the vacuole or dissemination throughout the entire cytosol. The presence of glucosamine 6-phosphate clearly reduced the proportion of cells in the population that showed complete cytosolic staining, implying that the binding and entry of the peptide into the cytosol is significantly reduced due to the exogenous phosphate sequestering the peptide and reducing the amount of peptide able to bind to the surface phosphomannan. In conclusion, we present evidence that an antimicrobial peptide, similar to those employed by cells of the human immune system, has evolved to recognize molecular patterns on the surface of pathogens in order to maximize efficacy.

Phosphorus Deprivation Responses and Phosphonate Utilization in a Thermophilic Synechococcus sp. from Microbial Mats

The genomes of two closely related thermophilic cyanobacterial isolates, designated Synechococcus isolate OS-A and Synechococcus isolate OS-B', from the microbial mats of Octopus Spring (Yellowstone National Park) have been sequenced. An extensive suite of genes that are controlled by phosphate levels constitute the putative Pho regulon in these cyanobacteria. We examined physiological responses of an axenic OS-B' isolate as well as transcript abundances of Pho regulon genes as the cells acclimated to phosphorus-limiting conditions. Upon imposition of phosphorus deprivation, OS-B' stopped dividing after three to four doublings, and absorbance spectra measurements indicated that the cells had lost most of their phycobiliproteins and chlorophyll a. Alkaline phosphatase activity peaked and remained high after 48 h of phosphorus starvation, and there was an accumulation of transcripts from putative Pho regulon genes. Interestingly, the genome of Synechococcus isolate OS-B' harbors a cluster of phn genes that are not present in OS-A isolates. The proteins encoded by the phn genes function in the transport and metabolism of phosphonates, which could serve as an alternative phosphorus source when exogenous phosphate is low. The phn genes were upregulated within a day of eliminating the source of phosphate from the medium. However, the ability of OS-B' to utilize methylphosphonate as a sole phosphorus source occurred only after an extensive period of exposure to the substrate. Once acclimated, the cells grew rapidly in fresh medium with methylphosphonate as the only source of phosphorus. The possible implications of these results are discussed with respect to the ecophysiology of the microbial mats.

A Mg 2+-dependent ecto-phosphatase activity on the external surface of Trypanosoma rangeli modulated by exogenous inorganic phosphate

In this work, we characterized a Mg 2+-dependent ecto-phosphatase activity present in live Trypanosoma rangeli epimastigotes. This enzyme showed capacity to hydrolyze the artificial substrate for phosphatases, p-nitrophenylphosphate ( p-NPP). At saturating concentration of p-NPP, half-maximal p-NPP hydrolysis was obtained with 0.23 mM Mg 2+. Ca 2+ had no effect on the basal phosphatase activity, could not substitute Mg 2+ as an activator and in contrast inhibited the p-NPP hydrolysis stimulated by Mg 2+. The dependence on p-NPP concentration showed a normal Michaelis–Menten kinetics for this phosphatase activity with values of V max of 8.94 ± 0.36 nmol p-NP × h −1 × 10 −7 cells and apparent K m of 1.04 ± 0.16 mM p-NPP. Mg 2+-dependent ecto-phosphatase activity was stimulated by the alkaline pH range. Experiments using inhibitors, such as, sodium fluoride, sodium orthovanadate and ammonium molybdate, inhibited the Mg 2+-dependent ecto-phosphatase activity. Inorganic phosphate (Pi), a product of phosphatases, inhibited reversibly in 50% this activity. Okadaic acid and microcystin-LR, specific phosphoserine/threonine phosphatase inhibitors, inhibited significantly the Mg 2+-dependent ecto-phosphatase activity. In addition, this phosphatase activity was able to recognize as substrates only o-phosphoserine and o-phosphothreonine, while o-phosphotyrosine was not a good substrate for this phosphatase. Epimastigote forms of T. rangeli exhibit a typical growth curve, achieving the stationary phase around fifth or sixth day and the Mg 2+-dependent ecto-phosphatase activity decreased around 10-fold with the cell growth progression. Cells maintained at Pi-deprived medium (2 mM Pi) present Mg 2+-dependent ecto-phosphatase activity approximately threefold higher than that maintained at Pi-supplemented medium (50 mM Pi).

Sites of phytase and xylanase activities in the gastrointestinal tract of broiler chickens

Because digesta vary in pH in different gastrointestinal segments of poultry, exogenous phytase or xylanase may exhibit differences in activity along the gastrointestinal tract. Previous reports indicated that the stomach is the major site of exogenous microbial phytase activity, with no further activity found in the small intestine of piglets. Information regarding exogenous phytase or xylanase activity in the gastrointestinal tract of poultry is largely unavailable. Because exogenous phytase or xylanase activity in the digesta is extremely low, normal phytase or xylanase activity measurements are prone to errors resulting from background interference contributed by the exogenous inorganic phosphate or xylose in the digesta (Walsh et al., 1995). The aim of this study was to utilize electrophoresis activity stain to detect the activity of phytase or xylanase in different gastrointestinal segments of broiler chickens fed diets containing exogenous enzymes.