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.     

Cr(VI) reduction in a chromate-resistant strain of Candida maltosa isolated from the leather industry

A Cr(VI)-resistant yeast was isolated from tanning liquors from a leather factory in Leon, Guanajuato, Mexico. Based on morphological and physiological analyses and the D1/D2 domain sequence of the 26S rDNA, the yeast was identified as Candida maltosa. Resistance of the strain to high Cr(VI) concentrations and its ability to chemically reduce chromium was studied. When compared to the three laboratory yeasts Candida albicans, Saccharomyces cerevisiae and Yarrowia lipolytica, the C. maltosa strain was found to tolerate chromate concentrations as high as 100 micro g/ml. In addition to this phenotypic trait, the C. maltosa strain showed ability to reduce Cr(VI). Chromate reduction occurred both in intact cells (grown in culture medium or in soil containing chromate) as well as in cell-free extracts. NADH-dependent chromate reductase activity was found associated with soluble protein and, to a lesser extent, with the membrane fraction.     

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.     

Antimicrobial and anticancer activity of tetrahedral, chelated, diphosphine silver(I) complexes: comparison with copper and gold

Tetrahedral, bischelated Ag(I) diphosphine complexes [Ag(P-P)2]NO3, where P-P is Ph2P(CH2)2PPh2 (dppe), Et2P(CH2)2PPh2 (depe), and cis-Ph2P(CH = CH)PPh2 (dppey), are potently cytotoxic to B16 melanoma cells in vitro (IC50 4 microM) and exhibit good activity against ip P388 leukemia in mice. The complex [Ag(dppe)2]NO3 is active against M5076 reticulum cell sarcoma. The antibacterial and antifungal activities of Ag(I) diphosphine and related Cu(I) and Au(I) complexes were assessed. The complexes [Au(dppey)2]Cl, [Au(dppp)2]Cl and (CuCl)2(dppe)3 show modest activity against three of the 12 bacterial strains tested, but all complexes exhibit antifungal activity against three strains of C. albicans in a "defined" medium, [Ag(depe)2]NO3 and [Au(dppp)2]Cl having comparable activity to fungizone. Antifungal activity of the complexes is reduced in Sabouraud's broth medium, and lost altogether for the Ag(I) complexes. Reactions of some of the Ag(I) complexes with glutathione and blood plasma were studied by 31P NMR.     

Use of the coagglutination reaction of yeast-like Candida maltosa fungi for detecting fimbriae in intestinal bacteria

The capacity of nonpathogenic yeast-like C. maltosa strains to coagglutinate Escherichia coli has been studied. C. maltosa cells have also been shown to coagglutinate E. coli possessing mannose-sensitive adhesins in a wide range of their concentrations (5-140 bacterial cells per C. maltosa cell). Strains belonging to types CFA/I and CFA/II with fimbriae, similarly to their corresponding paired genetically related strains without these adhesins, are practically incapable of agglutinating C. maltosa cells, while strains K88 and B41 react with them. The reaction occurs at a concentration of 9.5-37.0 and 38.0-55.5 bacteria respectively per C. maltosa cell and is not inhibited by 1% d-mannose. The suggestion that C. maltosa cell surface glycoproteins contain not only receptors for E. coli fimbriae, type I, but also components similar in their structure to receptors specific to the mannose-resistant adhesins of strains K88, K99 and 41, has been confirmed by hemagglutination inhibition with C. maltosa surface antigens as inhibiting agents.     

Further evidence of species variation in mechanisms of epithelial cell loss in mammalian small intestine: ultrastructural studies on the reindeer (Rangifer tarandus) and seal (Phoca groenlandica)

Ultrastructural studies were conducted on mechanisms of epithelial cell loss in the small intestine of seal and reindeer. Mechanisms maintaining epithelial integrity were distinguished from those that did not and the non-epithelial cell types involved were identified. Three types of cell extrusion were noted. In two, tight junctional integrity was preserved and anucleate apical cell fragments (rather than complete cells) were lost into the lumen. In reindeer (type 1), this involved creating large intercellular spaces extending from the preserved apical cap to the lamina propria and containing enterocyte debris probably phagocytosed by subepithelial macrophages. A variant of this process (type 2) involved the gradual shrinkage of individual cells, which became more electron-dense, and the in situ degeneration of their nucleated subapical portions. Degenerated cell fragments and membrane whorls were confined to narrow intercellular spaces between approximating adjacent healthy enterocytes. The mechanism of removal of these fragments was unclear. In both cases, the proximity of intraepithelial lymphocytes suggested that they were involved in cell targetting and killing. Evidence of apoptotic nuclei was not found but nucleated cell fragments could have been washed out of the lumen during tissue preparation. Type 2 cell loss was seen in both species, as was another mechanism (type 3) reminiscent of necrosis. In contrast to other mechanisms, this was accompanied by breaks in epithelial continuity following gradual loss of cell electron density and total or subtotal degradation of organelles and membranes. In seal, this terminated in the loss of an abnormal cell apex and exposure of the contents of the cell remnant to the lumen. In reindeer, all the cell remnants may have been extruded before total membrane degeneration but, in both species, the otherwise tight epithelial barrier was clearly breached. Again, intraepithelial lymphocytes were associated with sites of necrosis. These findings provide evidence for further species differences in mechanisms of epithelial cell extrusion and suggest that necrotic cell loss may be more common than previously admitted.     

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.     

Death by apoptosis in salivary glands of females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

The salivary glands of females of the tick Rhipicephalus sanguineus at three feeding stages: unfed, engorged, and at day three post-engorgement, were subjected to cytochemical methods of enzymatic analysis and cell viability. Comparing glands at these stages, was observed distinct staining patterns in cells of different types of acini, specially in degenerating types III, II, I, which were affected in this sequence by cell death. This study also revealed changes in: nuclei, staining intensity for acid phosphatase and ATPase activities, and permeability of the plasma membrane. Acid phosphatase activity was inversely proportional to that of ATPase, while ATPase activity was always proportional to membrane integrity. The glands of unfed females exhibited high metabolic activity and cells with intact nucleus and plasma membrane, suggesting that the presence of acid phosphatase detected in these individuals may participate in the normal physiology of some acini, as they were not undergoing degeneration. In acini I and II of engorged females, we observed cells with intact membranes, as well as changes characterized by nuclear changes, decrease in ATPase activity, and stronger acid phosphatase activity. At day three post-engorgement, degeneration progressed to more advanced stages, loss of membrane integrity was observed in most cells (of some type I acini, most type II acini, and all type III acini), as well as prominent nuclear changes, decrease in ATPase activity, and intense acid phosphatase activity, resulting in apoptotic bodies. During the death of cells nuclear changes preceded cytoplasmic ones in the following sequence: nuclear changes, loss of ATPase activity, loss of integrity of the plasma membrane, increase in acid phosphatase activity, and formation of apoptotic bodies. The presence of acid phosphatase with a secondary role (late) during cell death, degrading final cell remnants, characterized this process in the glands of R. sanguineus females as atypical or non-classic apoptosis.     

Phagocytosis of nonapoptotic cells dying by caspase-independent mechanisms

Caspase activation, exposure of phosphatidylserine (PS) on the outer surface of the plasma membrane, and rapid phagocytic removal of dying cells are key features of apoptosis. Nonapoptotic/necrotic modes of death occur independent of caspase activation, but the role of phagocytosis is largely unknown. To address this issue, we studied phagocytosis by human monocyte-derived macrophages (HMDM) and rat microglial cells. Target cells (Jurkat) were stimulated by several different methods that all caused caspase-independent death. First, we induced necrosis by combining toxins with ATP-depleting agents. Under these conditions, neither PS was exposed nor were such cells phagocytosed before their death. However, once the plasma membrane integrity was lost, the dead cells were rapidly and efficiently engulfed by HMDM. Next, we triggered Jurkat cell death with staurosporine in the presence of the pan-caspase inhibitor zVAD-fmk. Under these conditions, death occurred by delayed necrosis and without exposure of PS. Nevertheless, such lethally challenged cells were phagocytosed before the loss of membrane integrity. Finally, we triggered Ca2+ influx in Jurkat cells with an ionophore, or in neurons by glutamate receptor stimulation, respectively. In both models, PS was exposed on the cell surface. Ca2+-stressed cells were phagocytosed starting at 30 min after stimulation. Protein kinase C inhibitors prevented Ca2+-mediated PS exposure and phagocytosis. Essentially, similar phagocytosis data were obtained for all models with HMDM and microglia. We conclude that also cells dying nonapoptotically and independent of caspase activation may be recognized and removed before, or very quickly after, membrane lysis.     

Analysis of the genome of plants. II. Characterization of repetitive DNA in barley (Hordeum vulgare) and wheat (Triticum aestivum).

Barley and wheat DNAs have been characterized by studying their kinetics of reassociation, melting properties and sedimentation behaviour in neutral CsCl gradients as well as in Cs2SO4 gradients containing Ag+ or Hg2+. In both species, reassociation kinetics have revealed the presence of approx. 76% redundant nucleotide sequences which have been grouped into very rapidly reassociating (Cot 0-0.01), rapidly reassociating (Cot 0.01-1.0) and slowly reassociating (Cot 1-100) fractions. The barley Cot 0-0.01 and Cot 0.01-1.0 fractions as well as the wheat Cot 0.01-1.0 fraction form narrow bands upon centrifugation in CsCl gradients. Under similar experimental conditions both Cot 0.01 and Cot 1.0-100 wheat fractions and the barley Cot 1.0-100 fraction form broad bands each having several shoulders. Thermal denaturation studies of most of the above reassociated fractions have shown a considerable degree of order in their duplexes with an average hyperchromicity of 21.5%. When native, high molecular weight barley DNA is centrifuged in Ag+/CS2SO4 density gradients (RF = 0.2), two satellites appear on the heavier side of the main band, as against one in the case of wheat. The two minor peaks, designated as satellites I and II, have buoyant densities of 1.702 and 1.698 g/cm3, respectively, in neutral CsCl gradients and together represent about 8-9% of total barley DNA. Upon centrifugation in Hg2+/CS2SO4 density gradients, one satellite is observed in both barley and wheat and it accounts for 1-2% of their genomes.     

Evolution of codon usage patterns: the extent and nature of divergence between Candida albicans and Saccharomyces cerevisiae.

Codon usage in a sample of 28 genes from the pathogenic yeast Candida albicans has been analysed using multivariate statistical analysis. A major trend among genes, correlated with gene expression level, was identified. We have focussed on the extent and nature of divergence between C.albicans and the closely related yeast Saccharomyces cerevisiae. It was recently suggested that significant differences exist between the subsets of preferred codons in these two species [Brown et al. (1991) Nucleic Acids Res. 19, 4293]. Overall, the genes of C.albicans are more A + T-rich, reflecting the lower genomic G + C content of that species, and presumably resulting from a different pattern of mutational bias. However, in both species highly expressed genes preferentially use the same subset of 'optimal' codons. A suggestion that the low frequency of NCG codons in both yeast species results from selection against the presence of codons that are potentially highly mutable is discounted. Codon usage in C.albicans, as in other unicellular species, can be interpreted as the result of a balance between the processes of mutational bias and translational selection. Codon usage in two related Candida species, C.maltosa and C.tropicalis, is briefly discussed.     

Thermal difference circular dichroism of Pf1 filamentous virus and effects of mercury(II), silver(I), and copper(II)

The circular dichroism (CD) of Pfl filamentous virus has been examined over the temperature range 0-40 degrees C, in the absence and presence of Hg(II), Ag(I), and Cu(II). Thermal difference CD spectra were obtained by subtraction of spectra recorded above and below a thermally induced structure transition near 12 degrees C. The thermal difference spectra look like they arise from shifts in two exciton bands, one centered at 230 nm and the other at 290 nm. The amplitudes on either side of a crossover at 230 nm are 10 times those of a crossover at 290 nm. It is proposed that the difference spectra result from thermally induced shifts in coupled oscillator interactions between Tyr40 residues of the coat protein and the guanine and cytosine bases of the DNA. Metal ions can reduce or block these shifts. The changes in ellipticities at 220, 237, and 270 nm induced by changing the temperature have inflections near 12 degrees C. Ag(I) and Hg(II), which are known to bind to the DNA bases in Pfl, reduce or eliminate the inflections in the thermal profiles, depending on the metal ion type and concentration. Cu(II) ions do not affect the profiles. The spectral changes and the effects of the metal ions indicate intimate contact between the DNA bases and the protein subunits in the virion.     

Cytological response of hemocytes in the European flat oyster, Ostrea edulis, experimentally exposed to mercury

Molluscs bivalves have been widely used as bioindicators to monitor contamination levels in coastal waters. In addition, many studies have attempted to analyze bivalve organs, considered pollutant-targets, to understand the bio-accumulation process and to characterize the effects of pollutants on the organisms. Here we analyzed the effects of mercury exposure on flat oyster hemocytes. Optical and electronic microscope procedures were used to characterize hemocyte morphology. In addition, cell solutions treated with acridine orange were analyzed by flow cytometry and laser scanning cytometry in order to evaluate the variations of cytoplasmic granules (red fluorescence, ARF) and cell size (green fluorescence, AGF) of hemocyte populations over time. Light and electron microscopical studies enabled us to differentiate four hemocyte subpopulations, agranulocytes (Types I and II) and granulocytes (Types I and II). Slight morphological differences were observed between control and Hg-exposed cells only in granulocytes exposed to Hg for 30 days, where condensed chromatin and partially lysed cytoplasmic regions were detected. Flow and laser scanning cytometry studies allowed us to differentiate three hemocyte populations, agranulocytes (R1) and granulocytes (R2 and R3). The exposure time to Hg increased the average red fluorescence (ARF) of agranulocytes and small granulocytes, while there was no change in large granulocytes, which showed a loss of membrane integrity. In control oysters, the three hemocyte populations showed an increase of ARF after 19 days of exposure although initial values were restored after 30 days. The average green fluorescence (AGF) was more stable than the ARF throughout the experiment. In Hg-exposed oysters, the values of AGF of agranulocytes showed an increase at half Hg-exposure period while the AGF values of large granulocytes decreased throughout the experiment, confirming the instability of these types of cells. The relative percentage of small granulocytes and granulocytes showed time variations in both control and exposed oysters. However, the values of small granulocytes remained constant during the whole experiment. The fact that there were only changes in agranulocytes and large granulocytes suggested a possible relationship between these two types of cells. In a quantitative study, we found a significant linear relationship between the agranulocytes and large granulocytes.     

Inactivation of Escherichia coli and Lactobacillus plantarum in relation to membrane permeabilization due to rapid chilling followed by cold storage

The relationship between membrane permeabilization and loss of viability by chilling depending on the chilling rate was investigated in two bacterial models: one Gram-positive bacterium, Lactobacillus plantarum, and one Gram-negative bacterium, Escherichia coli. Cells were cold shocked slowly (2°C/min) or rapidly (2,000°C/min) from physiological temperature to 0°C and maintained at this temperature for up to 1 week. Loss of membrane integrity was assessed by the uptake of the fluorescent dye propidium iodide (PI). Cell death was found to be strongly dependent on the rate of temperature downshift to 0°C. Prolonged incubation of cells after the chilling emphasized the effect of treatment on the cells, as the amount of cell death increased with the length of exposure to low temperature, particularly when cells were rapidly chilled. More than 5 and 3-log reductions in cell population were obtained with L. plantarum and E. coli after the rapid cold shock followed by 7-day storage, respectively. A correlation between cell inactivation and membrane permeabilization was demonstrated with both bacterial strains. Thus, loss of membrane integrity due to the chilling treatments was directly involved in the inactivation of vegetative bacterial cells.     

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.     

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.     

In vitro selection of SV40 T antigen epitope loss variants by site-specific cytotoxic T lymphocyte clones

SV40-transformed cells of C57BL/6 (B6) mouse origin (H-2b) express four distinct predominant antigenic sites, I, II, III, and IV, on SV40 large tumor (T) Ag that are recognized by SV40 T Ag-specific CTL clones. In this study, we selected SV40 T Ag-positive cell lines which had lost one or more of the antigenic sites, by in vitro cocultivation of a SV40-transformed B6 mouse kidney cell line (K-0) with SV40 T Ag site-specific CTL clones, Y-1 (site I specific), Y-2 (site II specific), Y-3 (site III specific), and Y-4 (site IV specific). All of the CTL-resistant cell lines expressed large quantities of cell surface H-2 class I Ag. K-1 cells selected by CTL clone Y-1 lost the expression of antigenic sites I, II, and III, but not site IV. K-2 and K-3 cells selected by CTL clones Y-2 and Y-3, respectively, were found to be negative for sites II and III but expressed sites I and IV. K-4 cells selected by CTL clone Y-4 lost the expression of only site IV. K-1,4 cells (sites I-, II-, III-, IV-) were selected from K-1 cells by cocultivation with CTL clone Y-4, K-2,4 cells (sites I+, II-, III-, IV-) were selected from K-2 cells by CTL clone Y-4. K-3,1 cells (sites I-, II-, III-, IV+) were selected from K-3 cells by CTL clone Y-1, and K-3,1,4 cells (sites I-, II-, III-, IV-) were selected from K-3,1 cells by CTL clone Y-4. From K-4 cells, K-4,1 cells (sites I-, II-, III-, IV-) and K-4,3 cells (sites I+, II-, III-, IV-) were selected by CTL clone Y-1 and Y-3, respectively. The antigenic site loss variant cell lines K-1, K-1,4, K-3,1 K-3,1,4, K-4,1, and K-4,3 synthesized SV40 T Ag molecules of 75, 75, 78, 78, 81, and 88 kDa, respectively. Expression of wild-type SV40 T Ag in the antigenic site loss variants by infection with SV40 or transfection with cloned SV40 DNA restored the CTL recognition sites on the variant cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Processing of the Man(10)GlcNAc (M(10)) oligosaccharide by alpha-mannosidases from Candida albicans

The hydrolysis of Man(10)GlcNAc (M(10)) by purified alpha-mannosidases and its further processing by a mixed membrane preparation from Candida albicans were studied. Incubation of the oligosaccharide with purified alpha-mannosidases I (E-I) or II (E-II) from C. albicans released 1 and 2 mol of mannose per mol of M(10), respectively. This treatment converted M(10) into an acceptor substrate of further mannose residues from GDP-Man as catalyzed by membrane-bound mannosyltransferases. Elongation of E-I- or E-II-trimmed M(10) yielded a low molecular mass product (14-17 mannose residues added), and in the case of E-II, a minor amount of an additional product of a higher molecular mass. Our results indicate that purified alpha-mannosidases participate in N-glycan processing in C. albicans.