Toxic effects of Ag(I) and Hg(II) on Candida albicans and C. maltosa: a flow cytometric evaluation

The effects of Ag(I) and Hg(II) on membrane potential and integrity of cells of Candida albicans and C. maltosa were determined with a flow cytometric procedure that employed an anionic membrane potential-sensitive dye, bis-(1,3-dibutylbarbituric acid) trimethine oxonol, and a membrane integrity indicator, propidium iodide. The membrane potentials of cells of both species were reduced rapidly within 15 min of exposure to Ag(I). No threshold dose for Hg(II) existed, and cells of both species lost membrane potential gradually in Hg(II) solutions. Cells of both species lost membrane integrity more rapidly in Ag(I) solutions than in Hg(II) solutions. In Ag(I) solutions, the decrease in the numbers of cells recoverable in culture occurred at a rate similar to the rate of cell depolarization and membrane permeabilization. In Hg(II) solutions, loss of cell recoverability preceded the loss of membrane potential and membrane integrity. C. albicans, in contrast to C. maltosa, showed no loss of membrane integrity after exposure to Hg(II) solutions for 1 h. Different rates of binding of Ag(I) and Hg(II) between the two species suggest that the two ions target different primary sites.     

In vitro and in vivo anticandidal efficacy of green synthesized gold nanoparticles using Spirulina maxima polysaccharide

This study, for the first time, demonstrated an unprecedented approach for the green synthesis of gold (Au) nanoparticles (NPs) using the polysaccharide of Spirulina maxima as a reducing agent. Time-kill kinetic analysis was used to evaluate the antifungal activity of the green synthesized Au NPs against the pathogenic Candida albicans (C. albicans). The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were found to be 32 μg/mL and 64 μg/mL, respectively. Ultra-structural analysis indicated prominent damage on cell wall of the C. albicans after Au NPs treatment, and suggested that the treatment could increase the membrane permeability and disintegration of cells leading to cellular death. The results of propidium iodide (PI) uptake assay showed the higher level of cell death in Au NPs treated C. albicans cells, further confirming the loss of plasma membrane integrity. Cytotoxicity analysis of Au NPs on HEK293T and A549 cells showed no cytotoxic effect up to 64 μg/mL of Au NPs concentration, indicating the potential use in in vivo studies. Also, the recovery of C. albicans infected zebrafish after Au NPs therapy suggest green synthesized Au NPs from S. maxima polysaccharide as a prospective anticandidal agent.     

Synthesis and X-ray crystal structure of [Ag(phendio)<Subscript>2</Subscript>]ClO<Subscript>4</Subscript> (phendio = 1,10-phenanthroline-5,6-dione) and its effects on fungal and mammalian cells

The Cu(II) and Ag(I) complexes, [Cu(phendio)₃](ClO₄)₂4H₂O and [Ag(phendio)₂]ClO₄ (phendio = 1,10-phenanthroline-5,6-dione), are prepared in good yield by reacting phendio with the appropriate metal perchlorate salt. The X-ray crystal structure of the Ag(I) complex shows it to have a pseudo tetrahedral structure. `Metal-free' phendio and the Cu(II) and Ag(I) phendio complexes strongly inhibit the growth of the fungal pathogen Candida albicans, and are more active than their 1,10-phenanthroline analogues. The simple Ag(I) salts, AgCH<sub>3</sub>CO<sub>2</sub>, AgNO<sub>3</sub> and AgClO<sub>4</sub>.H<sub>2</sub>O display superior anti-fungal properties compared to analogous simple Cu(II) and Mn(II) salts, suggesting that the nature of the metal ion strongly influences activity. Exposing C. albicans to `metal-free' phendio, simple Ag(I) salts and [Ag(phendio)₂]ClO₄ causes extensive, non-specific DNA cleavage. `Metal-free' phendio and [Ag(phendio)₂]ClO₄ induce gross distortions in fungal cell morphology and there is evidence for disruption of cell division. Both drugs also exhibit high anti-cancer activity when tested against cultured mammalian cells.     

Effect of miconazole on diO-C6-(3) accumulation in mitochondria ofCandida albicans

A flow cytometric method was used to investigate the effect of miconazole (MCZ) on yeast-form cells ofCandida albicans. Relative changes in electric potential of mitochondrial and cytoplasmic membranes were assessed by 3,3′-dihexyloxacarbocyanine iodide (diO-C₆-(3)) and bis-(1,3-dibutyl-barbituric acid) trimethine oxonol (diBA-C₄-(3)) stainings, respectively. WhenC. albicans was exposed to MCZ at 10 μg/ml (a fungistatic concentration) for 2 h, no change appeared in cytoplasmic membrane potential, which was revealed by constant fluorescence intensity of diBA-C₄-(3)-stained cells. On the other hand, the cells lost the ability to accumulate diO-C₆-(3) in mitochondria by MCZ treatment. Time- and dose-responses in fluorescence intensity reflected that MCZ affected the mitochondrial activity ofC. albicans.     

Synthesis, characterization and antimicrobial activity of a series of substituted coumarin-3-carboxylatosilver(I) complexes

A series of new coumarin-derived carboxylate ligands and their silver(I) complexes have been synthesized, characterized and screened for their in vitro antibacterial activity against a range of Gram-positive and Gram-negative bacteria as well as for their antifungal activity against a clinical isolate of Candida albicans. The ligands were synthesised by either acid or base hydrolysis of their corresponding esters, which in turn were synthesised via the Knoevenegal reaction. The reaction of silver(I) nitrate with the coumarin carboxylate ligands in either aqueous or aqueous/ethanol solutions allowed the isolation of a series of novel Ag(I) carboxylate complexes. Whilst none of the ligands showed any antimicrobial activity, a number of the Ag(I) complexes exhibited potent activity. In particular, Ag(I) complexes of hydroxy-substituted coumarin carboxylates demonstrated potent activity against the clinically important methicillin-resistant Staphylococcus aureus (MRSA) bacterium (MIC₈₀ = 0.63 μM).     

Anticandidal activity of hetero-dinuclear copper(II) Mn(II) Schiff base and its potential action of the mechanism

Invasive fungal infections are one of the major challenges especially for immunosuppressed patients since they are drug resistant and pathogen to patients. Therefore, developing new, efficient and nonresistant antifungal agents have been a primary focus of international research. In the current study, a novel Schiff base [hetero-dinuclear copper(II) Mn(II) complex] (SB) derivative was investigated for its anticandidal activity against Candida albicans and possible mechanisms inducing cell death. The results revealed that SB treatment induces apoptotic and necrotic pathways in C. albicans ATCC10231 strain. Intracellular reactive oxygen species production determined by 2′,7′-dichlorofluorescein diacetate staining was triggered by SB and amphotericin B administrations in a dose-dependent manner. Gene expression analysis demonstrated that SB exposure resulted in regulation of critical development and stress related gene expressions. SB treatment directly upregulated expression of stress related genes, DDR48 and RIM101, while suppressed important cell signaling and antibiotic resistance acquiring related genes such as HSP90, ERG11 and EFG1. Furthermore, CaMCA1 mRNA levels were found to be significantly high in SB-treated yeast cells, indicating possible caspase-like mechanism activation. Scanning electron microscopy analysis confirmed that SB treatment led to severe cell wall integrity disruption and wrinkling. The study will encourage development of SB-based anticandidal regimens but further studies are highly warranted to understand limitations and the extended use in the routine.     

Inhibition of Zn(II) Binding Type IA Topoisomerases by Organomercury Compounds and Hg(II)

Type IA topoisomerase activities are essential for resolving DNA topological barriers via an enzyme-mediated transient single strand DNA break. Accumulation of topoisomerase DNA cleavage product can lead to cell death or genomic rearrangement. Many antibacterial and anticancer drugs act as topoisomerase poison inhibitors that form stabilized ternary complexes with the topoisomerase covalent intermediate, so it is desirable to identify such inhibitors for type IA topoisomerases. Here we report that organomercury compounds were identified during a fluorescence based screening of the NIH diversity set of small molecules for topoisomerase inhibitors that can increase the DNA cleavage product of Yersinia pestis topoisomerase I. Inhibition of relaxation activity and accumulation of DNA cleavage product were confirmed for these organomercury compounds in gel based assays of Escherichia coli topoisomerase I. Hg(II), but not As(III), could also target the cysteines that form the multiple Zn(II) binding tetra-cysteine motifs found in the C-terminal domains of these bacterial topoisomerase I for relaxation activity inhibition. Mycobacterium tuberculosis topoisomerase I activity is not sensitive to Hg(II) or the organomercury compounds due to the absence of the Zn(II) binding cysteines. It is significant that the type IA topoisomerases with Zn(II) binding domains can still cleave DNA when interfered by Hg(II) or organomercury compounds. The Zn(II) binding domains found in human Top3α and Top3β may be potential targets of toxic metals and organometallic complexes, with potential consequence on genomic stability and development.     

Circular dichroism, kinetic and mass spectrometric studies of copper(I) and mercury(II) binding to metallothionein

The metalloprotein metallothionein (MT) is remarkable in its metal binding properties: for the mammalian protein, well-characterized species exist for metal to sulfur ratios of M7S20, M12S20, and M18S20, where M = Cd(II), Zn(II), Hg(II), Ag(I), Au(I), and Cu(I). Optical spectra in general, and circular dichroism (CD) and luminescence spectra in particular, provide rich detail of a complicated metal binding chemistry when metals are added directly to the metal-free or zinc-containing protein. CD spectral data unambiguously identify key metal to protein stoichiometric ratios that result in well-defined structures. Electrospray ionization-mass spectrometry data are reported for reactions in which Hg(II) binds to apo-MT 2A as previously described from CD data. Emission spectra in the 450-750 nm region have been reported for metallothioneins containing Ag(I), Au(I), and Cu(I). The luminescence of Cu-MT can also be detected directly from mammalian and yeast cells. We report both steady-state and new dynamic data for titrations of Zn-MT with Cu(I). Analysis of kinetic data for the addition of the first two Cu(I) atoms to Zn-MT indicates a first-order mechanism over a concentration range of 5-50 microM. Three-dimensional modeling was carried out using the results of the CD and EXAFS studies, model calculations for Zn7-MT, Hg7-MT, and Cu12-MT are described.     

Reduction of Hg(II) to Hg(0) by Biogenic Magnetite from two Magnetotactic Bacteria

Understanding the biogeochemical cycle of the highly toxic element mercury (Hg) is necessary to predict its fate and transport. In this study, we determined that biogenic magnetite isolated from Magnetospirillum gryphiswaldense MSR-1 and Magnetospirillum magnetotacticum MS-1 was capable of reducing inorganic mercury [Hg(II)] to elemental mercury [Hg(0)]. These two magnetotactic bacteria (MTB) lacked mercuric resistance operons in the genomes. However, they revealed high resistance to Hg(II) under atmospheric conditions and an even higher resistance under microaerobic conditions (1% O₂ and 99% N₂). Neither strain reduced Hg(II) to Hg(0) under atmospheric conditions. However, a slow rate (0.05–0.21 µM·d^?1) of Hg(II) loss occurred from late log phase to stationary phase in two MTBs' culture media under microaerobic conditions. Increased Hg(II) entered both cells under microaerobic conditions relative to atmospheric conditions. The majority of Hg(II) was still blocked by the cell membrane. Hg(II) reduction was more effective when biogenic magnetite was extracted out, with or without the magnetosome membrane envelope. When magnetosome membrane was present, 8.55–13.53% of 250 nM Hg(II) was reduced to Hg(0) by 250 mg/L biogenic magnetite suspension within 2 hours. This ratio increased to 55.07–64.70% while magnetosome membrane was removed. We concluded that two MTBs contributed to the reduction of Hg(II) to Hg(0) at a slow rate in vivo. Such reduction was more favorable to occur when biogenic magnetite is released from dead cells. It proposed a new biotic pathway for the formation of Hg(0) in aquatic systems.     

Nerol triggers mitochondrial dysfunction and disruption via elevation of Ca2+ and ROS in Candida albicans

The antifungal activity of Nerol (NEL) against Candida albicans, a pathogenic fungus, has a minimum inhibitory concentration (MIC) of 4.4 mM that causes noteworthy candidacidal activity through an apoptosis-like mechanism. Calcium (Ca²⁺) levels and reactive oxygen species (ROS) production, which are the major causes of apoptosis, were determined in C. albicans cells treated with NEL and were found to increase, which related to mitochondrial dysfunction and disruption. A series of characteristic changes of apoptosis caused by NEL, including mitochondrial membrane depolarization, cytochrome c (cyt c) release, and metacaspase activation were examined using a flow cytometer and Western blot. The results showed that an increase in intracellular Ca²⁺ and ROS led to dramatically decreased mitochondrial membrane potential (MMP); cyt c was also released from the mitochondria to the cytosol. Other early apoptotic features were also observed with the metacaspase activation. Finally, the morphological changes of the cells were observed, including phosphatidylserine (PS) externalization, nuclear condensation, and DNA fragmentation through Annexin V-FITC and PI double staining, TUNEL assay, and DAPI staining. The results supported the hypothesis that NEL was involved in the apoptosis of C. albicans cells not only at the early stages, but also at the late stages. In summary, NEL can trigger mitochondrial dysfunction and disruption via elevation of Ca²⁺ and ROS leading to apoptosis in C. albicans. This research on the mechanism of cell death triggered by NEL against C. albicans has important significance for providing a novel treatment of C. albicans infections.     

Green synthesis and structural classification of Acacia nilotica mediated-silver doped titanium oxide (Ag/TiO2) spherical nanoparticles: Assessment of its antimicrobial and anticancer activity

Current exanimation reports, green fabrication of silver doped TiO₂ nanoparticles (Ag/TiO₂) using aqueous extract of Acacia nilotica as bio-reductant and assess its potential as antimicrobial and anticancer agent. The obtained spherical Ag/TiO₂ were characterized by various analytical techniques including FTIR, (XRD), (FE-SEM EDS), and (TEM). Synthesized Ag/TiO₂ demonstrated broad spectrum antibacterial and anticandidal activity. The order of antimicrobial activity was found to be E. coli > C. albicans > MRSA > P. aeruginosa. In addition, cytotoxicity and oxidative stress of Ag/TiO₂ nanoparticles in (MCF-7) cells was also investigated. Outcomes of MTT assay showed concentration dependent reduction in cell viability. Further, synthesized NPs reduced the level of glutathione, induced ROS generation and lipid peroxidation in the treated cells. Therefore, it is envisaged that these spherical nanoparticles may be exploited in drug delivery, pharmaceutical, and food industry.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

Physiological and DNA characterization of Candida maltosa,a hydrocarbon-utilizing yeast

Selected yeasts classified as Candida sake van Uden et Buckley were examined for their physiological, morphological and immunological properties and their DNA relatedness. Candida maltosa Komagata, Nakase et Katsuya is herein recognized as a species separate from C. sake. Candida maltosa was distinguished from C. sake and from C. tropicalis by insignificant DNA reassociation. In addition, C. maltosa was distinguished from C. sake by its higher maximal growth temperature and lower guanine plus cytosine content of its DNA and from C. tropicalis by its failure to utilize soluble starch for growth and its resistance to cycloheximide. The species C. cloacae and C. subtropicalis are placed in synonymy with C. maltosa.     

The influence of Zn(II) and Mn(II) on catalytic activity of C. albicans aspartic proteinases

The interaction of secreted aspartic proteinases from C. albicans (C. albicans SAP) with ZnCl2 and MnCl2 has been studied. Logarithms of stability constant from the data of electronic spectroscopy were calculated for the complexes of SAP with Zn (II) (SAP-ZnII, logβ = 4.73 ± 0.20) and with Mn(II) (SAP-MnII, logβ = 7.02 ± 0.20). The composition and maximum accumulation of complexes in solution were calculated. The optimal conditions of hydrolysis of the substrate, HAS (human serum albumin) in the presence of C. albicans SAP-MnII and SAP-ZnII proteinases were determined. These were: [HSA] = 1 mg/ml, [SAP] = 2.33 μM, pH = 4.5, incubation time of 25 min. The activity of C. albicans SAP in the presence of different concentrations of ZnCl₂ and MnCl₂ was evaluated under optimal conditions of enzymatic hydrolysis. For the first time the activating action of 0.5 μM ZnCl₂ on catalytic activity of C. albicans SAP proteinase has been demonstrated. The maximal rate of enzymatic reaction (V _max), the Michaelis constant (K _m ) and constants of effects in the presence and in the absence of the effector, ZnCl₂, were calculated. The albuminase activity of C. albicans pathogenic strains of different localization was evaluated in the presence and the absence of the effector of ZnCl₂.     

Membrane damage as first and DNA as the secondary target for anti‐candidal activity of antimicrobial peptide P7 derived from cell‐penetrating peptide ppTG20 against Candida albicans

P7, a peptide analogue derived from cell‐penetrating peptide ppTG20, possesses antibacterial and antitumor activities without significant hemolytic activity. In this study, we investigated the antifungal effect of P7 and its anti‐Candida acting mode in Candida albicans. P7 displayed antifungal activity against the reference C. albicans (MIC = 4 μM), Aspergilla niger (MIC = 32 μM), Aspergillus flavus (MIC = 8 μM), and Trichopyton rubrum (MIC = 16 μM). The effect of P7 on the C. albicans cell membrane was examined by investigating the calcein leakage from fungal membrane models made of egg yolk l ‐phosphatidylcholine/ergosterol (10 : 1, w/w) liposomes. P7 showed potent leakage effects against fungal liposomes similar to Melittin‐treated cells. C. albicans protoplast regeneration assay demonstrated that P7 interacted with the C. albicans plasma membrane. Flow cytometry of the plasma membrane potential and integrity of C. albicans showed that P7 caused 60.9 ± 1.8% depolarization of the membrane potential of intact C. albicans cells and caused 58.1 ± 3.2% C. albicans cell membrane damage. Confocal laser scanning microscopy demonstrated that part of FITC‐P7 accumulated in the cytoplasm. DNA retardation analysis was also performed, which showed that P7 interacted with C. albicans genomic DNA after penetrating the cell membrane, completely inhibiting the migration of genomic DNA above the weight ratio (peptide : DNA) of 6. Our results indicated that the plasma membrane was the primary target, and DNA was the secondary intracellular target of the mode of action of P7 against C. albicans. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

The removal of mercury (II) from water by Ag supported on nanomesoporous silica

In this study, the synthesis of SBA-15/Ag nanocomposite materials with different amounts of silver (2.5, 5, and 10 %) has been investigated under acidic conditions by using P123 as a template via the direct method. The nanocomposites of SBA-15 were synthesized by the same method and by the addition of silver salt. Finally, the nanocomposite materials were examined for the removal of mercury ions from wastewater as an adsorbent by the reverse titration method. Characterization was carried out through x-ray diffraction analysis (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and N₂ adsorption-desorption (Brunauer–Emmett–Teller). XRD spectra confirmed the presence of silver nanoparticles within the amorphous silica matrix of SBA-15. The Barrett–Joyner–Halenda analysis showed that SBA-15 and SBA-15/Ag have a narrow pore size distribution. SEM images demonstrated that the morphology of the matrix of SBA-15 is in spherical state. Furthermore, wavelength dispersive x-ray spectroscopy identified the presence and distribution of silver nanoparticles inside the pore channels and outside of them. Typical TEM images of SBA-15 and SBA-15/Ag (5 wt.%) indicated a regular hexagonal pore structure with long-range order and long channels. In SBA-15/Ag (5 wt.%) sample, the nanoparticles of silver was found into the pores and outside of them. The removal of mercury ions from wastewater using mesoporous silica nanocomposite containing silver nanoparticles was studied by the reverse titration analysis. The best capacity of adsorption of mercury ions from wastewater was obtained for SBA-15/Ag (5 wt.%) sample, which was equal to 42.26 mg/g in 20 min at pH of 7. The Freundlich model was used to explain the adsorption characteristics for the heterogeneous surface, and \( {K}_{\mathrm{f}} \) (adsorption capacity) and n (adsorption intensity) were determined for Hg (II) ion adsorption on SBA-15/Ag nanocomposite materials with different amounts of silver (2.5, 5, and 10 %). The value of R ² was about 0.99, 0.99, 0.98, and 0.98 and K _f was about 42, 48, 58, and 58 mg/g for SBA-15/Ag, SBA-15/Ag (2.5 %), SBA-15/Ag (5 %), and SBA-15/Ag (10 %), respectively. Furthermore, the values of n >1 show a favorable adsorption process for Hg (II) ion adsorption on SBA-15/Ag nanocomposite materials. Moreover, the Langmuir isotherm model evaluation showed that the correlation coefficients for all concentrations were R ² >0.99, indicating that Hg (II) ions were adsorbed on the surface of SBA-15/Ag via chemical and physical interaction. Additionally, the analytic hierarchy process (AHP) and Technique of Order Preference Similarity to the Ideal Solution (TOPSIS) methods that depend on the criteria of the surface area, amount of adsorbent, pore volume, and cost of synthesis were used. The evaluation of results showed that the best sample was SBA-15/Ag (5 wt.%). Furthermore, the research work highlighted the antibacterial nanocomposite with suitable adsorption of Hg (II) ions from water solutions and supported its potential for environmental applications. This nanocomposite can be used in the absorption domain of Hg (II) ions from water solutions.     

The temperature and humidity preferences of Haemaphysalis longicornis, Ixodes Holocyclus and Rhipicephalus sanguineus (Ixodidae): studies on eggs

The eggs of Haemaphysalis longicornis, Ixodes holocydus and Rhipicephalus sanguineus were exposed to a range of temperatures and humidities to compare the temperature and humidity requirements of ticks from climatically dissimilar habitats. The response to changes in humidity was also studied with H. longicornis. Hatching in I. holocydus eggs occurred from 18 to 28°C and at a saturation deficit of 2 mm Hg or less. The eggs of R. sanguineus hatched between 18 and 38°C and tolerated saturation deficits up to 20 mm Hg. The eggs of H. longicornis hatched between 18 and 35°C and at saturation deficits up to 8 mm Hg. When eggs of H. longicornis were exposed to moderately dry conditions for different periods of time and then transferred to moist conditions, incubation periods and mortality were reduced in comparison with eggs kept continuously under dry conditions. On the other hand, mortality of H. longicornis eggs was reduced in moderately dry conditions, provided that they had been exposed to a moist environment for about 10 days previously. Weight loss of eggs in dry air and the rate of development of eggs were examined and are discussed. The eggs of I. holocylus lost weight very rapidly in dry air, whereas weight loss from R. sanguineus eggs was much slower. Weight loss from H. longicornis eggs fell between these two rates. The rate of development was fastest in R. sanguineus and slowest in I. holocydus.     

The temperature and humidity preferences of Haemaphysalis longicornis,ixodes holocyclus and Rhipicephalus sanguineus (ixodidae): Studies on engorged larvae

The engorged larvae of Haemaphysalis longicornis, Ixodes holocyclus and Rhipicephalus sanguineus were exposed to a range of temperatures and humidities to see whether the nature of their requirements in the laboratory were similar to the climate within the geographic range of each species. The response of H. longicornis and I. holocyclus to changes in humidity was also studied. Moulting of I. holocyclus larvae occurred from 18 to 28°C and at a saturation deficit of 4 mm Hg or less. The larvae of R. sanguineus moulted between 18 and 38°C and tolerated saturation deficits up to 35 mm Hg. The larvae of H. longicornis moulted between 15 and 38°C at saturation deficits up to 8 mm Hg. When engorged larvae of H. longicornis and I. holocyclus were exposed to very dry conditions for different periods of time and then transferred to moist conditions, the minimum pre-moult period and mortality was increased in comparison with larvae kept continuously under moist conditions. On the other hand, mortality of H. longicornis larvae was reduced in very dry conditions provided that they had been exposed to a moist environment for about 5 days previously. The larvae of I. holocyclus required at least 12 days exposure to a moist environment before any survived to moult in moderately dry conditions. The engorged larvae of both H. longicornis and I. holocyclus lost weight rapidly in dry air, whereas weight loss from R. sanguineus larvae was much slower. The rate of development was fastest in R. sanguineus and slowest in I. holocyclus.     

Optimization of cellular uptake of zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine for maximal photoinactivation of Candida albicans

Cellular uptake and photodynamic action of zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc⁴⁺) was examined in Candida albicans. In vitro investigations showed that ZnPPc⁴⁺ was rapidly bound to C. albicans cells. The binding of phthalocyanine to cells was dependent on ZnPPc⁴⁺ concentrations (1–10 μM) and cells densities (10⁶–10⁸ cells mL^?1). A high amount of ZnPPc⁴⁺ retained in the cells after two washing steps, indicating a strong interaction between the photosensitizer and C. albicans. The uptake was temperature dependent, although the difference between 37 °C and 4 °C was about 10 %. Also, the amount of ZnPPc⁴⁺ bound to C. albicans was affected when the cells were incubated for a longer time with azide and 2,4-dinitrophenol (DNP) prior to treatment with ZnPPc⁴⁺. Cell survival after irradiation was dependent on the irradiation period, ZnPPc⁴⁺ concentration and cells density. Photoinactivation of C. albicans cells was elevated even after two washing steps. The strong dependence of uptake on cell density reveals the strength and avidity of the binding of ZnPPc⁴⁺ to C. albicans cells. The accumulation behaviour of ZnPPc⁴⁺ suggests that mainly an affinity-mediated binding mechanism can be involved. Therefore, ZnPPc⁴⁺ is an interesting phthalocyanine for photodynamic inactivation (PDI) of yeasts in liquid suspensions.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.