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Candida albicans is a major human pathogenic fungus that is distinguished by its capability to switch from a yeast to a hyphal morphology under different conditions. Here, we analyze the cellular effects of high concentrations of the iron chelator bathophenanthroline disulfonate (BPS). BPS inhibits cellular growth by withholding iron, but when iron chelation is overcome by the addition of hemoglobin as an iron source, the cells resume growth as hyphae. The BPS hyphal induction pathway was characterized by identifying the hyphal-specific transcription factors that it requires and by a forward genetic screen for mutants that fail to form hyphae in BPS using a transposon library generated in a haploid strain. Among the mutants identified are the DYRK1-like kinase Yak1 and Orf19.384, a homolog of the DYRK1-associated protein WDR68/DCAF7. Orf19.384 nuclear localization depends on Yak1, similar to their mammalian counterparts. We identified the hyphal suppressor transcription factor Sfl1 as a candidate target of Yak1-Orf19.384 and show that Sfl1 modification is similarly affected in the yak1 and orf19.384 mutant strains. These results suggest that DYRK1/Yak1 and WDR68/Orf19.384 represent a conserved protein pair that regulates cell differentiation from fungi to animals. [ABSTRACT FROM AUTHOR]

Candida albicans is a major human pathogenic fungus that is distinguished by its capability to switch from a yeast to a hyphal morphology under different conditions. Here, we analyze the cellular effects of high concentrations of the iron chelator bathophenanthroline disulfonate (BPS). BPS inhibits cellular growth by withholding iron, but when iron chelation is overcome by the addition of hemoglobin as an iron source, the cells resume growth as hyphae. The BPS hyphal induction pathway was characterized by identifying the hyphal-specific transcription factors that it requires and by a forward genetic screen for mutants that fail to form hyphae in BPS using a transposon library generated in a haploid strain. Among the mutants identified are the DYRK1-like kinase Yak1 and Orf19.384, a homolog of the DYRK1-associated protein WDR68/DCAF7. Orf19.384 nuclear localization depends on Yak1, similar to their mammalian counterparts. We identified the hyphal suppressor transcription factor Sfl1 as a candidate target of Yak1-Orf19.384 and show that Sfl1 modification is similarly affected in the yak1 and orf19.384 mutant strains. These results suggest that DYRK1/Yak1 and WDR68/Orf19.384 represent a conserved protein pair that regulates cell differentiation from fungi to animals.

Main conclusion: Fe uptake machinery of chloroplasts prefers to utilise Fe(III)-citrate over Fe-nicotianamine complexes.Iron uptake in chloroplasts is a process of prime importance. Although a few members of their iron transport machinery were identified, the substrate preference of the system is still unknown. Intact chloroplasts of oilseed rape (Brassica napus) were purified and subjected to iron uptake studies using natural and artificial iron complexes. Fe-nicotianamine (NA) complexes were characterised by 5 K, 5 T Mössbauer spectrometry. Expression of components of the chloroplast Fe uptake machinery was also studied. Fe(III)-NA contained a minor paramagnetic Fe(II) component (ca. 9%), a paramagnetic Fe(III) component exhibiting dimeric or oligomeric structure (ca. 20%), and a Fe(III) complex, likely being a monomeric structure, which undergoes slow electronic relaxation at 5 K (ca. 61%). Fe(II)-NA contained more than one similar chemical Fe(II) environment with no sign of Fe(III) components. Chloroplasts preferred Fe(III)-citrate compared to Fe(III)-NA and Fe(II)-NA, but also to Fe(III)-EDTA and Fe(III)-o,o′EDDHA, and the Km value was lower for Fe(III)-citrate than for the Fe-NA complexes. Only the uptake of Fe(III)-citrate was light-dependent. Regarding the components of the chloroplast Fe uptake system, only genes of the reduction-based Fe uptake system showed high expression. Chloroplasts more effectively utilize Fe(III)-citrate, but hardly Fe-NA complexes in Fe uptake. [ABSTRACT FROM AUTHOR]

Inflammatory Bowel Disease (IBD) is associated with systemic iron deficiency and has been managed with iron supplements which cause adverse side effects. Conversely, some reports highlight iron depletion to ameliorate IBD. The underlying intestinal response and comparative benefit of iron depletion and supplementation in IBD is unknown. The aims of this work were to characterize and compare the effects of iron supplementation and iron depletion in IBD. IBD was induced in Drosophila melanogaster using 3 % dextran sodium sulfate (DSS) in diet for 7 days. Using this model, we investigated the impacts of acute iron depletion (using bathophenanthroline disulfonate, BPS) and supplementation (using ferrous sulphate, FS), before and after IBD induction, on gut iron homeostasis, cell death, gut permeability, inflammation, antioxidant defence, antimicrobial response and several fly phenotypes. DSS decreased fly mass (p < 0.001), increased gut permeability (p < 0.001) and shortened lifespan (p = 0.035) compared to control. The DSS-fed flies also showed significantly elevated lipid peroxidation (p < 0.001), and the upregulated expression of apoptotic marker- drice (p < 0.001), tight junction protein – bbg (p < 0.001), antimicrobial peptide – dpta (p = 0.002) and proinflammatory cytokine – upd2 (p < 0.001). BPS significantly (p < 0.05) increased fly mass and lifespan, decreased gut permeability, decreased lipid peroxidation and decreased levels of drice , bbg , dpta and upd2 in IBD flies. This iron chelation (using BPS) showed better protection from DSS-induced IBD than iron supplementation (using FS). Preventive and curative interventions, by BPS or FS, also differed in outcomes. This may inform precise management strategies aimed at tackling IBD and its recurrence. [Display omitted] [ABSTRACT FROM AUTHOR]

Alkaline phosphatase (ALP) plays a vital role in dephosphorylation- and phosphorylation-related cellular regulation and signaling processes. Accordingly, the development of efficient methods for ALP activity assay is of significant importance in clinical diagnosis. In this work, a simple and practical method is reported for the first time for the sensitive and selective colorimetric assay of ALP activity by exploiting a water-soluble Cu(II)-phenanthroline complex as the probe, on the basis of the distinctive metal-to-ligand charge-transfer (MLCT) properties. This method is simply built on a two-step chromogenic reaction: the enzymatic hydrolysis of the substrate ascorbic acid 2-phosphate to ascorbic acid (AA), followed by the reduction of the colorimetric probe Cu(BPDS) 2 2− (BPDS=bathophenanthroline disulfonate) by AA to its cuprous form. The latter process triggers a turn-on spectral absorption at 424 nm and a striking color change of the solution from colorless to blackish-green. Needless of complicated protocols and instrumentation, this method allows a sensitive readout of ALP activity within a wide linear range of 0–200 mU mL − 1 , with a detection limit down to 1.25 mU mL −1 . Results also reveal that it is highly selective and holds great potential in ALP inhibitor efficiency evaluation. In addition, quantitative analysis of ALP activity in spiked serum samples has been realized successfully in the linear range of 0–200 mU mL −1 , with a detection limit of 1.75 mU mL −1 . Advantages of simplicity, wide linear range, high sensitivity and selectivity, low cost, and little background interference render this method great potential in practical applications. [ABSTRACT FROM AUTHOR]

We report a versatile approach for the colorimetric assay of alkaline phosphatase (ALP) activity based on the distinctive metal-to-ligand charge-transfer (MLCT) absorption properties of Fe(II)-phenanthroline reporter. In the presence of ALP, the applied substrate ascorbic acid 2-phosphate is enzymatically hydrolyzed to produce ascorbic acid, which then reduces Fe 3+ to Fe 2+ . The complexation of Fe 2+ with the bathophenanthroline disulfonate (BPS) ligand generates a blood-red Fe(BPS) 3 4− reporter, which is characterized by an intense MLCT absorption band at 535 nm in the visible range. Under optimal conditions, the spectral output exhibits a good quantitative relationship with ALP activity over the range of 0–220 mU mL −1 with a detection limit of 0.94 mU mL −1 . Moreover, the activity of ALP can also be conveniently judged through naked-eye observations. Results indicate that it is highly selective and can be applied to the screening of ALP inhibitors. In addition, it has been successfully employed to detect the endogenous ALP level of undiluted human serum samples, with a detection limit of 1.05 mU mL −1 being achieved. This approach avoids any elaborately designed substrates and holds considerable simplicity and flexibility for reporter design. This study broadens the horizon of the applications of phenanthroline-based transition metal complexes. Furthermore, an efficient and practical method like this has the potential to be widely used in clinical applications and in the point-of-care testing. [ABSTRACT FROM AUTHOR]

Addition of the impermeable iron II chelator bathophenanthroline disulfonate (BPS) to cultured Chinese hamster lung fibroblast (CCL 39 cells) inhibits DNA synthesis but not protein synthesis or cytoplasmic alkalinization, when cell growth is initiated with growth factors such as EGF plus insulin, thrombin, or ceruloplasmin. The BPS inhibition is reversed by addition of stoichiometric ferrous iron at stoichiometric concentration. BPS does not inhibit cell growth stimulated by fetal calf serum. The effect of the BPS differs from the inhibition of growth by hydroxyurea which acts on the ribonucleotide reductase. The BPS treatment leads to release of iron from the cells as determined by BPS iron II complex formation over 90 min. Cells treated with BPS just during starvation period cannot re-initiate DNA synthesis after mitogen stimulation even if BPS is removed from the medium and cells are previously washed. BPS treatment also inhibits transplasma membrane electron which is restored by incubation of cells with 10 μM ferric ammonium citrate. Growth factor stimulation of DNA synthesis is restored by addition of 1 μM ferrous ammonium sulfate or ferric ammonium citrate, or 0.1 μM diferric transferrin. Copper, cobalt, nickel, zinc, gallium, aluminum, or apotransferrin cannot restore the activity. The BPS effect is consistent with removal of iron from a site on the cell surface which controls electron transport and DNA synthesis. [ABSTRACT FROM AUTHOR]

• The procedure allows determination of free Fe(II) from in vivo microdialysis samples. • It leaves sufficient material for neurotransmitter determinations in the same samples. • Iron release and hROS production evoked by 6-OHDA administration were demonstrated. • Applicable to investigating conditions involving Fe(II) release and oxidative stress. Free, non-protein bound, Fe(II), which can catalyse the formation of the toxic highly-reactive oxygen species (hROS), has been implicated in several neurodegenerative conditions. The determination of free Fe(II) and Fe(III) in samples obtained from microdialysis experiments has been limited by the small amounts of sample available. This work describes the development of a HPLC, with absorbance detection, method, based on the complexation of Fe(II) with bathophenanthroline disulfonate (BS), which allows a complete extracellular iron analysis with the small sample amounts that are available from in vivo microdialysis in rat brain. Microdialysis experiments using 6-hydroxydopamine stimulation, showed that basal-as well as evoked levels of extracellular Fe(II) and total iron could be determined in parallel with measurements of hROS formation. Although a spectrophotometric BS-based assay has been reported for use in microdialysis samples from large animals, the present procedure is applicable to the small sample sizes available from studies in rat brain. It is simpler than the alternative, involving inductively-coupled plasma mass spectrometry. The procedure described is simple and sensitive, giving a linear response in the Fe(II) concentration range of 50 −2000 nM. A 20 min microdialysis sample (flow-rate 3 μl/min) yields sufficient material for triplicate determinations of the evoked release of Fe(II) and total iron whilst leaving sufficient sample volume for determining hROS and amine or amino-acid neurotransmitter release. [ABSTRACT FROM AUTHOR]

PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multidomain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of the Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9- and 10-nitro-octadeca-9-cis-enoic acid; OA-NO2) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e., Cys and His), modulating protein function and subcellular distribution in a reversible manner. Here, we show that OA-NO2 inhibits kinase activity by covalently adducting PknG remote from the catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO2-mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO2 induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. These results affirm the relevance of the Rbx domain as a target for PknG inhibition and support that electrophilic lipid reactions of Rbx-Cys may represent a new drug strategy for specific PknG inhibition., (Copyright © 2013 Elsevier Inc. All rights reserved.)