Staphylococcus aureus transporters Hts, Sir, and Sst capture iron liberated from human transferrin by Staphyloferrin A, Staphyloferrin B, and catecholamine stress hormones, respectively, and contribute to virulence.

Staphylococcus aureus is a frequent cause of bloodstream, respiratory tract, and skin and soft tissue infections. In the bloodstream, the iron-binding glycoprotein transferrin circulates to provide iron to cells throughout the body, but its iron-binding properties make it an important component of innate immunity. It is well established that siderophores, with their high affinity for iron, in many instances can remove iron from transferrin as a means to promote proliferation of bacterial pathogens. It is also established that catecholamine hormones can interfere with the iron-binding properties of transferrin, thus allowing infectious bacteria access to this iron pool. The present study demonstrates that S. aureus can use either of two carboxylate-type siderophores, staphyloferrin A and staphyloferrin B, via the transporters Hts and Sir, respectively, to access the transferrin iron pool. Growth of staphyloferrin-producing S. aureus in serum or in the presence of holotransferrin was not enhanced in the presence of catecholamines. However, catecholamines significantly enhanced the growth of staphyloferrin-deficient S. aureus in human serum or in the presence of human holotransferrin. It was further demonstrated that the Sst transporter was essential for this activity as well as for the utilization of bacterial catechol siderophores. The substrate binding protein SstD was shown to interact with ferrated catecholamines and catechol siderophores, with low to submicromolar affinities. Experiments involving mice challenged intravenously with wild-type S. aureus and isogenic mutants demonstrated that the combination of Hts, Sir, and Sst transport systems was required for full virulence of S. aureus.

Critical role for cataplerosis via citrate in glucose-regulated insulin release.

The molecular mechanisms mediating acute regulation of insulin release by glucose are partially known. The process involves at least two pathways that can be discriminated on basis of their (in)dependence of closure of ATP-sensitive potassium (K+(ATP)) channels. The mechanism of the K+(ATP) channel-independent pathway was proposed to involve cataplerosis, the export of mitochondrial intermediates into the cytosol and in the induction of fatty acid-derived signaling molecules. In the present article, we have explored in fluorescence-activated cell sorter (FACS)-purified rat beta-cells the molecular steps involved in chronic glucose regulation of the insulin secretory response. When compared with culture in 10 mmol/l glucose, 24 h culture in 3 mmol/l glucose shifts the phenotype of the cells into a state with low further secretory responsiveness to glucose, lower rates of glucose oxidation, and lower rates of cataplerosis. Microarray mRNA analysis indicates that this shift can be attributed to differences in expression of genes involved in the K+(ATP) channel-dependent pathway, in cataplerosis and in fatty acid/cholesterol biosynthesis. This response was paralleled by glucose upregulation of the transcription factor sterol regulatory element binding protein 1c (SREBP1c) (ADD1) and downregulation of peroxisome proliferator-activated receptor (PPAR)-alpha and PPAR-beta (PPARdelta). The functional importance of cataplerosis via citrate for glucose-induced insulin release was further supported by the observation that two ATP-citrate lyase inhibitors, radicicol and (-)-hydroxycitrate, block part of glucose-stimulated release in beta-cells. In conclusion, chronic glucose regulation of the glucose-responsive secretory phenotype is associated with coordinated changes in gene expression involved in the K+(ATP) channel-dependent pathway, in cataplerosis via citrate and in acyl CoA/cholesterol biosynthesis.

(-)-Hydroxycitrate ingestion and endurance exercise performance.

We have been interested in the ergogenic aid effects of food components and supplements for enhancing endurance exercise performance. For this purpose, acute or chronic (-)-hydroxycitrate (HCA) ingestion might be effective because it promotes utilization of fatty acid as an energy source. HCA is a competitive inhibitor of the enzyme ATP: citrate lyase, thereby increasing inhibition of lipogenesis in the body. Many researchers have reported that less body fat accumulation and sustained satiety cause less food intake. After focusing on exercise performance with HCA ingestion, we came up with different results that show positive effects or not. However, our previously reported data showed increased use of fatty acids during moderate intensity exercise. For future research, HCA and co-ingestion of other supplements, such as carnitine or caffeine, might have greater effect on glycogen-sparing than HCA alone.

Ca2+ transport in isolated mouse liver mitochondria; role of reductive carboxylation and citrate?

The uptake of Ca2+ in isolated mouse liver mitochondria respiring on succinate in the presence of rotenone and added Pi, was inhibited by dibucaine, fluorocitrate, p-hydroxymercuribenzoate (PMB), malonate, palmitoyl-CoA, succinyl-CoA and trifluoroperazine. The release of accumulated Ca2+ was stimulated by arsenite, malonate, PMB, palmitoyl-CoA and succinyl-CoA, whereas the release was inhibited by dibucaine, fluorocitrate, trifluoroperazine, and by oligomycin, especially in the presence of ADP. The pyridine nucleotides were oxidized in mitochondria incubated with PMB. The observations suggest a possible contributory role of reductive carboxylation for the uptake of Ca2+, and a possible role of citrate for the retention of Ca2+ in isolated mouse liver mitochondria.

Development and de novo protein synthetic activity of bovine embryos produced in vitro in different culture systems.

In vitro matured (IVM) and fertilized (IVF) putative Day 1 zygotes (Day 0 = IVF) were allocated randomly to culture in formulations based on Synthetic Oviduct Fluid (SOF) medium and identified on the basis of their contrasting principal supplements, which were 10% v/v steer serum (SS; n = 558) or 4 mg/mL crystalline BSA (SBSA; n = 531) or 3 mg/mL polyvinyl alcohol (SPVA; n = 607) in 9 replicates. SBSA and SPVA also contained 10 microg/mL non-essential amino acids, while the former was further supplemented with 20 microL/mL essential amino acids and the latter with 0.5 mmol/L sodium citrate and 5 ng/mL epidermal growth factor. Zygotes were cultured in 20 microL drops (4 zygotes per drop) until Day 8 in an atmosphere of 5% CO2, 5% O2 and 90% N2 at 39 degrees C and droplets were renewed every 48 hours. The incidence of zygote cleavage was lower (P < 0.05) in SS (mean +/- SEM = 61 +/- 3%) than in SBSA (76 +/- 3%) but not in SPVA (72 +/- 4%) up to Day 3. The SPVA generated a lower yield of blastocysts on Day 7 (12 +/- 2%; P < 0.001) and by Day 8 (21 +/- 4%; P < 0.01) than did SS (33 +/- 3%; 40 +/- 3%) and SBSA (30 +/- 3%; 37 +/- 4%). Cell numbers (n) and diameters (d) of blastocysts on Day 8 were greater (P < 0.001; Replicates 1 to 5) in embryos from SBSA (n, 156 +/- 9; d, 203 +/- 4 microm) than in those from SS (n, 81 +/- 4; d, 177 +/- 3 microm) and SPVA (n, 76 +/- 5; d, 167 +/- 3 microm). Embryos produced in SS incorporated less 3H-phenylalanine into PCA-precipitable protein (replicates 6 to 9; log10 dpm = 3.03 +/- 0.04) than did embryos cultured in SBSA (3.21 +/- 0.03; P < 0.001) or in SPVA (3.14 +/- 0.03; NS). In conclusion, blastocyst yield was poor in SPVA, but the embryos had metabolic activities similar to those of embryos produced in SBSA. Blastocyst yields from SS were not compromised but their capacity for de novo protein synthesis was reduced significantly.

Citrate, not phosphate, can dissolve calcium oxalate monohydrate crystals and detach these crystals from renal tubular cells.

Dissolution therapy of calcium oxalate monohydrate (COM) kidney stone disease has not yet been implemented due to a lack of well characterized COM dissolution agents. The present study therefore aimed to identify potential COM crystal dissolution compounds. COM crystals were treated with deionized water (negative control), 5 mM EDTA (positive control), 5 mM sodium citrate, or 5mM sodium phosphate. COM crystal dissolution activities of these compounds were evaluated by phase-contrast and video-assisted microscopic examinations, semi-quantitative analysis of crystal size, number and total mass, and spectrophotometric oxalate-dissolution assay. In addition, effects of these compounds on detachment of COM crystals, which adhered tightly onto renal tubular cell surface, were also investigated. The results showed that citrate, not phosphate, had a significant dissolution effect on COM crystals as demonstrated by significant reduction of crystal size (approximately 37% decrease), crystal number (approximately 53% decrease) and total crystal mass (approximately 72% decrease) compared to blank and negative controls. Spectrophotometric oxalate-dissolution assay successfully confirmed the COM crystal dissolution property of citrate. Moreover, citrate could detach up to 85% of the adherent COM crystals from renal tubular cell surface. These data indicate that citrate is better than phosphate for dissolution and detachment of COM crystals.

Chemistry, physiological properties, and microbial production of hydroxycitric acid.

The tropical plants Garcinia cambogia and Hibiscus subdariffa produce hydroxycitric acid (HCA), of which the absolute configurations are (2S,3S) and (2S,3R), respectively. (2S,3S)-HCA is an inhibitor of ATP-citrate lyase, which is involved in fatty acid synthesis. (2S,3R)-HCA inhibits pancreatic alpha-amylase and intestinal alpha-glucosidase, leading to a reduction in carbohydrate metabolism. In this study, we review current knowledge on the structure, biological occurrence, and physiological properties of HCA. The availability of HCA is limited by the restricted habitat of its source plants and the difficulty of stereoselective organic synthesis. Hence, in our recent study, thousands of microbial strains were screened and finally two bacterial strains were, for the first time, found to produce trace amounts of HCA. The HCA variants produced were the Hibiscus-type (2S,3R) enantiomer. Subsequent genome shuffling rapidly generated a mutant population with improved HCA yield relative to the parent strain of bacteria. These bacteria are a potential alternative source of natural HCA.

Erwinia chrysanthemi requires a second iron transport route dependent of the siderophore achromobactin for extracellular growth and plant infection.

Full virulence of the pectinolytic enterobacterium Erwinia chrysanthemi strain 3937 depends on the production in planta of the catechol-type siderophore chrysobactin. Under iron-limited conditions, E. chrysanthemi synthesizes a second siderophore called achromobactin belonging to the hydroxy/carboxylate class of siderophore. In this study, we cloned and functionally characterized a 13 kb long operon comprising seven genes required for the biosynthesis (acs) and extracellular release (yhcA) of achromobactin, as well as the gene encoding the specific outer membrane receptor for its ferric complex (acr). The promoter of this operon was negatively regulated by iron. In a fur null mutant, transcriptional fusions to the acsD and acsA genes were constitutively expressed. Band shift assays showed that the purified E. chrysanthemi Fur repressor protein specifically binds in vitro to the promoter region of the acsF gene confirming that the metalloregulation of the achromobactin operon is achieved directly by Fur. The temporal production of achromobactin in iron-depleted bacterial cultures was determined: achromobactin is produced before chrysobactin and its production decreases as that of chrysobactin increases. Pathogenicity tests performed on African violets showed that achromobactin production contributes to the virulence of E. chrysanthemi. Thus, during infection, synthesis of these two different siderophores allows E. chrysanthemi cells to cope with the fluctuations of iron availability encountered within plant tissues. Interestingly, iron transport mediated by achromobactin or a closely related siderophore probably exists in other phytopathogenic bacterial species such as Pseudomonas syringae.

Concepts of citrate production and secretion by prostate: 2. Hormonal relationships in normal and neoplastic prostate.

A unique and major function of prostate secretory epithelial cells is to synthesize, accumulate, and secrete extraordinarily high levels of citrate. This function is regulated by testosterone and by prolactin. Concepts of the mechanisms of hormonal regulation are presented. The relationship of testosterone and prolactin to the origin and homologies of different prostate cell lines is described. The metabolic differentiation of citrate and non-citrate producing prostate secretory epithelial cells is discussed. Concepts of the pathogenesis of prostatic neoplasms are presented based on hormonal, metabolic, and homologous relationships associated with citrate production. Characterization of normal and neoplastic secretory epithelial cells by their citrate function is emphasized. The urgency and necessity for research relating to all aspects of prostate citrate production in normal and pathological prostate are emphasized.

Ralstonia solanacearum iron scavenging by the siderophore staphyloferrin B is controlled by PhcA, the global virulence regulator.

PhcA is a transcriptional regulator that activates expression of multiple virulence genes in the plant pathogen Ralstonia solanacearum. Relative to their wild-type parents, phcA mutants overproduced iron-scavenging activity detected with chrome azurol S siderophore detection medium. Transposon mutagenesis of strain AW1-PC (phcA1) generated strain GB6, which was siderophore negative but retained weak iron-scavenging activity. The ssd gene inactivated in GB6 encodes a protein similar to group IV amino acid decarboxylases, and its transcription was repressed by iron(III) and PhcA. ssd is the terminal gene in a putative operon that also appears to encode three siderophore synthetase subunits, a integral membrane exporter, and three genes with no obvious role in siderophore production. A homologous operon was found in the genomes of Ralstonia metallidurans and Staphylococcus aureus, both of which produce the polycarboxylate siderophore staphyloferrin B. Comparison of the siderophores present in culture supernatants of R. solanacearum, R. metallidurans, and Bacillus megaterium using chemical tests, a siderophore utilization bioassay, thin-layer chromatography, and mass spectroscopy indicated that R. solanacearum produces staphyloferrin B rather than schizokinen as was reported previously. Inactivation of ssd in a wild-type AW1 background resulted in a mutant almost incapable of scavenging iron but normally virulent on tomato plants. AW1 did not produce siderophore activity when cultured in tomato xylem sap, suggesting that the main location in tomato for R. solanacearum during pathogenesis is iron replete.

Elevated muscle citrate does not reduce carbohydrate utilization during tetanic stimulation.

The purposes of this study were to determine whether enhanced free fatty acid delivery would result in increased muscle citrate levels and to establish whether the effects of this putative phosphofructokinase inhibitor would be manifested during intense stimulation demanding glycogen as a fuel. Hind-limb muscles were perfused with either no or high (0.93 +/- 0.03 mM) free fatty acids for 10 min at rest, and during 5 min of tetanic stimulation. Muscles sampled at the end of the rest perfusion or stimulation were soleus (slow oxidative), red gastrocnemius (fast oxidative glycolytic), and white gastrocnemius (fast glycolytic). Muscle citrate content was unaffected during rest perfusion with no free fatty acids, whereas high free fatty acids significantly elevated citrate above control in soleus, red gastrocnemius, and white gastrocnemius (by 0.39 +/- 0.13, 0.53 +/- 0.10, and 0.29 +/- 0.07 mumol.g-1 dry muscle, respectively). Following 1 min of stimulation, citrate content in soleus and red gastrocnemius was not different from control in the absence of free fatty acids but accumulated significantly with high free fatty acids (0.26 +/- 0.05 and 0.28 +/- 0.04 mumol.g-1 dry muscle, respectively). Following 5 min of stimulation, soleus and red gastrocnemius citrate content decreased with no free fatty acids but increased significantly with high free fatty acids (0.42 +/- 0.10 mumol.g-1 dry muscle) in soleus and remained unchanged in red gastrocnemius.(ABSTRACT TRUNCATED AT 250 WORDS)

Urine citrate and renal stone disease.

Calcium stone disease is attributable to supersaturation of the urine with calcium and other salts, the presence of substances that promote crystallization and a deficiency of inhibitors of crystallization. Citrate is a potent inhibitor of calcium oxalate and calcium phosphate stone formation whose excretion is diminished in some patients with stone disease owing to idiopathic causes or secondary factors such as bowel disease and use of thiazides. The pH within the proximal tubule cells is an important determinant of citrate excretion. Multivariate analysis has shown that the urine concentrations of calcium and citrate are the most important factors in stone formation. In uncontrolled studies potassium citrate, which increases urinary citrate excretion, appears to be promising as a therapeutic agent for patients with stone disease and hypocitraturia refractory to other treatment. On the other hand, there are potential drawbacks to sodium alkali therapy, such as the precipitation of calcium phosphates.

Citrate and renal calculi: new insights and future directions.

Citrate is pathogenetically important in stone formation, because it retards the crystallization of stone-forming calcium salts and because its level in urine is low in many patients with nephrolithiasis. Potassium citrate is useful therapeutically, because it can often restore normal urinary citrate. Hypocitraturia often results from dietary aberrations, including sodium excess, and exaggerated intake of animal proteins. Hypocitraturia is frequently accompanied by a low net gastrointestinal absorption of alkali. New drugs are under development as improvements or refinements of currently available potassium citrate. They are potassium citrate 10-mEq-tablet preparation, effervescent calcium citrate, and potassium-magnesium citrate.

The role of transferrin and citrate in cellular uptake of aluminium.

The ability of human erythroleukaemia K562 cells to take up aluminium from Al-transferrin and Al-citrate has been examined. Uptake from Al-transferrin was dose-dependent over the range 68-544 ng/ml of aluminium, and increased over a 12-day period. In contrast, uptake from Al-citrate was low even at an aluminium concentration of 6800 ng/ml and did not increase over time. Neither form of aluminium greatly affected cell growth. It is concluded that Al-transferrin, rather than Al-citrate, is the physiologically relevant form of this metal with respect to cellular uptake, but that any metabolic abnormalities induced by aluminium do not affect proliferation of this cell line.

Production of siderophore by coagulase-negative staphylococci and its relation to virulence.

The ability to produce siderophore is considered to be a virulence factor for many pathogenic bacteria. To determine if siderophore production by coagulase-negative staphylococci (CNS) was related to virulence, 40 clinical isolates of CNS cultured from peritoneal dialysis fluid were compared with 38 commensal skin isolates. Siderophore activity was detected using the chrome azurol S liquid assay. Using precursor studies, Staphylococcus epidermidis isolates were shown to be more likely to produce the siderophore staphyloferrin A. Production of staphyloferrin B amongst non-Staphylococcus epidermidis species was associated with clinical isolates rather than commensal isolates, and therefore may play a role in pathogenicity.

Oxidative metabolism of the normal rat glomerulus.

Glomeruli from adult normal male Wistar rats were obtained by teasing a cortex slice with stainless steel needles. The enzyme content and the morphologic aspect of these glomeruli were assessed as a preliminary step to further metabolic studies. Robinson's medium appeared to be the most suitable medium. There was no loss of glutamic dehydrogenase, glucose-6-phosphate dehydrogenase or acid phosphatase. Lactate dehydrogenase was lost to about 50%. Electron microscopy showed morphologic signs of damage in the podocytes. The glomerular oxygen uptake was measured with the help of the Cartesian diver technique, using approximately 20 glomeruli per assay. The endogenous respiratory rate was linear for at least three hours. The endogenous respiratory rate was linear for at least three hours. The mean dry wt of lyophilized glomeruli was determined for 13 rats for which the glomerular oxygen uptake had been measured, and these data showed a glomerular Q-02 of 4 mul/hr/mg of dry wt. The following substances were tested for their influence on the oxygen uptake: acetate, alpha-oxoglutarate, citrate, oxalacetate, glutamate, alanine, all 10 mM; succinate, 2.5, 5 and 10 mM; glucose, 5, 10 and 20 mM; fructose 10 and 20 mM; and palmitate. Citrate increases the O-2 uptake/hr/glomerulus by 30%; glucose, 20 mM, by 30%; and succinate, 2.5 mM by 50% and 10 mM by 190%. In a Robinson's medium containing 35 mg of albumin/ml, the endogenous respiration is not different from that obtained in the inorganic medium but the oxygen uptake is increased 26% by glucose, 10 mM. From these data, it can be concluded that the oxygen uptake of the glomerulus is small. This fact explains its resistance to anoxia. The systematic investigation of possible substrates indicate that glucose, citrate and succinate may play a role in supporting this small oxidative metabolism.

Superoxide-dependent redox cycling of citrate-Fe3+: evidence for a superoxide dismutaselike activity.

Citrate-Fe3+, reportedly a physiological chelate, exhibits superoxide dismutaselike activity, as evidenced by the inhibition of xanthine oxidase-dependent cytochrome c reduction; the dismutation of xanthine oxidase-generated superoxide to hydrogen peroxide and oxygen, and the enhanced disproportionation of potassium superoxide. The catalytic activity of citrate-Fe3+ corresponds, on a molar basis, to 0.03% of that of copper- and zinc-containing superoxide dismutase. Although weak, this activity enables citrate-Fe3+ to inhibit superoxide and ADP-Fe3+ -dependent peroxidation of extracted microsomal lipids. Also, the dismutase activity of citrate-Fe3+ interferes with its ability to promote lipid peroxidation. It is proposed that chelation of Fe3+ by citrate may represent a protective mechanism against the deleterious consequences of superoxide generation.

Generation and degradation of fibrinopeptide A in stored platelet concentrate.

The levels of fibrinopeptide A (FPA) were measured in samples from stored platelet concentrates (PC) by radioimmunoassay. In 27 standard, citrated PC, the mean FPA was 13.6 ng/ml, which is elevated 5-6X over background levels. This value did not change significantly over a 7-day storage period. Addition of PGE-1 and theophylline resulted in higher initial levels of FPA (18.0 ng/ml) and a pronounced rise during the storage period (to 43.4 ng/ml by day 10). In contrast, addition of a thrombin inhibitor, D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-2 HCl (PPACK) or hirudin, resulted in lower initial levels of FPA relative to standard, citrated PC and a slow increase over time. Introduction of exogenous FPA into citrated PC resulted in a predicted elevation of FPA levels followed by a rapid loss of immunoreactivity (t1/2 = 18 h). Addition of PPACK did not affect this fall-off. However, PC prepared and stored in the presence of PGE-1 and theophylline showed a much slower fall-off of exogenous FPA (t1/2 = 38 h). These data indicate that FPA levels in samples from citrated PC represent a dynamic balance between generation and degradation processes and, thus, the data above underestimate the amount of thrombin activity present in stored, citrated PC.

Role of reversible phosphorylation of acetyl-CoA carboxylase in long-chain fatty acid synthesis.

Acetyl-CoA carboxylase, the rate-limiting enzyme in the biogenesis of long-chain fatty acids, is regulated by phosphorylation and dephosphorylation. The major phosphorylation sites that affect carboxylase activity and the specific protein kinases responsible for phosphorylation of different sites have been identified. A form of acetyl-CoA carboxylase that is independent of citrate for activity occurs in vivo. This active form of carboxylase becomes citrate-dependent upon phosphorylation under conditions of reduced lipogenesis. Therefore, phosphorylation-dephosphorylation of acetyl-CoA carboxylase is the enzyme's primary short-term regulatory mechanism; this control mechanism together with cellular metabolites such as CoA, citrate, and palmitoyl-CoA serves to fine-tune the synthesis of long-chain fatty acids under different physiological conditions.