In Vivo Metal Substitution in Bacteroides Fragilis Superoxide Dismutase

Bacteroides fragilis. an obligate anaerobe, synthesizes an azide-inhibitable iron-containing superoxide dismutase when grown in complex medium. Cells grown anaerobically in complex media containing dcsferrioxamine (Desferal^TM, Ciba-Geigy) and graded concentrations of Mn synthesize the azide-resistant manganese-containing SOD. The fraction of MnSOD activity in dialyzed cell extracts increased prograsively as the Mn concentration in the medium increased. The fraction of MnSOD activity also increased in extracts of cells grown in the medium with I mM Mn but with graded concentrations of desferrioxamine (0–10 micromolar). The SOD activity in the cells grown under the various conditions varied but not in a causal relationship with either Mn or desferrioxamine concentration. Electrophoresis reveaicd that the SOD activity in cells grown in the absence or presence of I mM Mn migrated with the same relative mobility and exhibited identical activity patterns when examined separately or as a mixture. These data are consistent with substitution of Mn for Fe in the B. fragilis apoprotein under anaerobic conditions and support the model of a single protein binding either Fe or Mn.     

In vitro studies indicate a quinone is involved in bacterial Mn(II) oxidation

Manganese(II)-oxidizing bacteria play an integral role in the cycling of Mn as well as other metals and organics. Prior work with Mn(II)-oxidizing bacteria suggested that Mn(II) oxidation involves a multicopper oxidase, but whether this enzyme directly catalyzes Mn(II) oxidation is unknown. For a clearer understanding of Mn(II) oxidation, we have undertaken biochemical studies in the model marine α-proteobacterium, Erythrobacter sp. strain SD21. The optimum pH for Mn(II)-oxidizing activity was 8.0 with a specific activity of 2.5 nmol × min⁻¹ × mg⁻¹ and a K _m = 204 μM. The activity was soluble suggesting a cytoplasmic or periplasmic protein. Mn(III) was an intermediate in the oxidation of Mn(II) and likely the primary product of enzymatic oxidation. The activity was stimulated by pyrroloquinoline quinone (PQQ), NAD⁺, and calcium but not by copper. In addition, PQQ rescued Pseudomonas putida MnB1 non Mn(II)-oxidizing mutants with insertions in the anthranilate synthase gene. The substrate and product of anthranilate synthase are intermediates in various quinone biosyntheses. Partially purified Mn(II) oxidase was enriched in quinones and had a UV/VIS absorption spectrum similar to a known quinone requiring enzyme but not to multicopper oxidases. These studies suggest that quinones may play an integral role in bacterial Mn(II) oxidation.     

Influence of Amino Acids Shiff Bases on Irradiated DNA Stability In Vivo

To reveal protective role of the new Mn(II) complexes with Nicotinyl-l-Tyrosinate and Nicotinyl-l-Tryptophanate Schiff Bases against ionizing radiation. The DNA of the rats liver was isolated on 7, 14, and 30 days after X-ray irradiation. The differences between the DNA of irradiated rats and rats pre-treated with Mn(II) complexes were studied using the melting, microcalorimetry, and electrophoresis methods. The melting parameters and the melting enthalpy of rats livers DNA were changed after the X-ray irradiation: melting temperature and melting enthalpy were decreased and melting interval was increased. These results can be explained by destruction of DNA molecules. It was shown that pre-treatment of rats with Mn(II) complexes approximates the melting parameters to norm. Agarose gel electrophoresis data confirmed the results of melting studies. The separate DNA fragments were revealed in DNA samples isolated from irradiated animals. The DNA isolated from animals pre-treated with the Mn(II) chelates had better electrophoretic characteristics, which correspond to healthy DNA. Pre-treatment of the irradiated rats with Mn(II)(Nicotinil-l-Tyrosinate) and Mn(II)(Nicotinil-l-Tryptophanate)₂ improves the DNA characteristics.     

In vitro digestion method to evaluate solubility of dietary zinc,selenium and manganese in salmonid diets

BackgroundThe determination of dietary mineral solubility is one of the main steps in the evaluation of their availability for a given species.MethodsThis study proposed an in vitro digestion method (acidic and alkaline hydrolysis). The method was applied to evaluate the solubility of inorganic and organic forms of zinc (Zn), selenium (Se) and manganese (Mn) in salmonid diets. An inorganic mineral (IM) diet was supplemented with zinc sulphate, sodium selenite and manganous sulphate and an organic mineral (OM) diet was supplemented with zinc chelate of glycine, l-selenomethionine and manganese chelate of glycine.ResultsThe solubility of Zn was similar in both diets tested. The amount of soluble Zn was low in the acidic hydrolysis (3–8%) and lower in the alkaline hydrolysis (0.4–2%). The solubility of Se was higher in the OM diet (7–34%) compared with the IM diet (3–12%). Regarding Mn, after the acidic hydrolysis the solubility was higher in the IM diet (6–25%) than the OM diet (4–17%). The in vitro solubility were compared with in vivo availability of Zn, Se and Mn. Data obtained for solubility (%) of Zn, Se and Mn was lower when compared with apparent availability (%) of Zn, Se and Mn.ConclusionData obtained demonstrated that solubility of Zn, Se and Mn was influenced by the mineral chemical form supplemented to the diet and by the gastrointestinal environment. The solubility of Zn, Se and Mn was not comparable with the apparent availability of Zn, Se and Mn. Nevertheless, the effect of the chemical form of the minerals was similar for the solubility of Zn, Se and Mn and the apparent availability of Zn, Se and Mn. Considering the overall results of this study, the in vitro method could replace some of the in vivo studies for a qualitative evaluation but not for a quantitative evaluation.     

In vivo manganese exposure modulates Erk, Akt and Darpp-32 in the striatum of developing rats, and impairs their motor function

Manganese (Mn) is an essential metal for development and metabolism. However, exposures to high Mn levels may be toxic, especially to the central nervous system (CNS). Neurotoxicity is commonly due to occupational or environmental exposures leading to Mn accumulation in the basal ganglia and a Parkinsonian-like disorder. Younger individuals are more susceptible to Mn toxicity. Moreover, early exposure may represent a risk factor for the development of neurodegenerative diseases later in life. The present study was undertaken to investigate the developmental neurotoxicity in an in vivo model of immature rats exposed to Mn (5, 10 and 20 mg/kg; i.p.) from postnatal day 8 (PN8) to PN12. Neurochemical analysis was carried out on PN14. We focused on striatal alterations in intracellular signaling pathways, oxidative stress and cell death. Moreover, motor alterations as a result of early Mn exposure (PN8-12) were evaluated later in life at 3-, 4- and 5-weeks-of-age. Mn altered in a dose-dependent manner the activity of key cell signaling elements. Specifically, Mn increased the phosphorylation of DARPP-32-Thr-34, ERK1/2 and AKT. Additionally, Mn increased reactive oxygen species (ROS) production and caspase activity, and altered mitochondrial respiratory chain complexes I and II activities. Mn (10 and 20 mg/kg) also impaired motor coordination in the 3^rd, 4^th and 5^th week of life. Trolox™, an antioxidant, reversed several of the Mn altered parameters, including the increased ROS production and ERK1/2 phosphorylation. However, Trolox™ failed to reverse the Mn (20 mg/kg)-induced increase in AKT phosphorylation and motor deficits. Additionally, Mn (20 mg/kg) decreased the distance, speed and grooming frequency in an open field test; Trolox™ blocked only the decrease of grooming frequency. Taken together, these results establish that short-term exposure to Mn during a specific developmental window (PN8-12) induces metabolic and neurochemical alterations in the striatum that may modulate later-life behavioral changes. Furthermore, some of the molecular and behavioral events, which are perturbed by early Mn exposure are not directly related to the production of oxidative stress.     

In vivo localization of manganese in the hyperaccumulator Gossia bidwillii (Benth.) N. Snow & Guymer (Myrtaceae) by cryo-SEM/EDAX

Gossia bidwillii (Myrtaceae) is a manganese (Mn)-hyperaccumulating tree native to subtropical eastern Australia. It typically contains foliar Mn levels in excess of 1% dry weight. However, in G. bidwillii and other Mn-hyperaccumulating species, the cellular and subcellular localization of Mn has not been measured. Quantitative in vivo cryo-scanning electron microscopy (SEM)/energy dispersive X-ray analysis (EDAX) was used to localize Mn and other elements in tissue collected from mature trees growing in a natural population. Cryo-SEM showed that the leaf mesophyll is differentiated as a double-layer palisade mesophyll above spongy mesophyll. Transmission electron microscopy (TEM) revealed that the palisade and epidermal cells are highly vacuolated. EDAX data were used to estimate in situ vacuolar Mn concentrations of all cell types in fresh cryo-fixed leaf tissues. The highest average vacuolar Mn concentration of over 500 mM was found in the upper-layer palisade mesophyll, while the lowest concentration of around 100 mM was found in the spongy mesophyll. Qualitative in vivo cryo-SEM/EDAX was employed to further investigate the spatial distribution of Mn in fresh leaf tissues and young bark tissue, which was also found to have a high Mn concentration. It is concluded that Mn distribution in G. bidwillii is quantitatively different to metal distribution in other hyperaccumulating species where the highest localized concentrations of these elements occur in non-photosynthmetic tissues such as epidermal cells and associated dermal structures including trichomes and leaf hairs.     

In vitro transactivation of Bacillus subtilis RNase P RNA

We have partially characterised an alpha4-fucosyltransferase (alpha4-FucT) from Vaccinium myrtillus, which catalysed the biosynthesis of the Lewis(a) adhesion determinant. The enzyme was stable up to 50 degrees C. The optimum pH was 7.0, both in the presence and in the absence of Mn(2+). The enzyme was inhibited by Mn(2+) and Co(2+), and showed resistance towards inhibition with N-ethylmaleimide. It transferred fucose to N-acetylglucosamine in the type I Galbeta3GlcNAc motif from oligosaccharides linked to a hydrophobic tail and glycoproteins (containing the type I motif). Sialylated oligosaccharides containing the type II Galbeta4GlcNAc motif were not acceptors. The catalytic mechanism of the plant alpha4-FucT possibly involves a His residue, and it must have arisen by convergent evolution relative to its mammalian counterparts.     

L-DOPA Inhibits In Vitro Phosphorylation of Melanoma Glycoproteins

L-DOPA had no effect on the endogenous phosphorylation of proteins after extraction with 1% Triton X-100 from hamster melanoma. When proteins were purified further by wheat germ-agglutinin chromatography, however, a dramatic and dose-dependent inhibitory effect of DOPA on glycoprotein phosphorylation was observed in the presence of Mn ⁺².     

In Situ Determination of Manganese(II) Speciation in Deinococcus radiodurans by High Magnetic Field EPR: DETECTION OF HIGH LEVELS OF Mn(II) BOUND TO PROTEINS*

High magnetic field high frequency electron paramagnetic resonance techniques were used to measure in situ Mn(II) speciation in Deinococcus radiodurans, a radiation-resistant bacteria capable of accumulating high concentrations of Mn(II). It was possible to identify and quantify the evolution of Mn(II) species in intact cells at various stages of growth. Aside from water, 95-GHz high field electron nuclear double resonance showed that the Mn(II) ions are bound to histidines and phosphate groups, mostly from fructose-1,6-bisphosphate but also inorganic phosphates and nucleotides. During stationary growth phase, 285-GHz continuous wave EPR measurements showed that histidine is the most common ligand to Mn(II) and that significant amounts of cellular Mn(II) in D. radiodurans are bound to peptides and proteins. As much as 40% of the total Mn(II) was in manganese superoxide dismutase, and it is this protein and not smaller manganese complexes, as has been suggested recently, that is probably the primary defense against superoxide.     

In vitro replication of bacteriophage PRD1 DNA. Metal activation of protein-primed initiation and DNA elongation.

Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism. Compared to Mg2+, the use of Mn2+ as the metal activator of the phage DNA polymerase results in a great stimulation of the initiation reaction. The molecular basis of the observed stimulatory effect is an increase in the velocity of the reaction. The phage DNA polymerase is also able to catalyze the formation of the initiation complex in the absence of DNA template. Although the presence of Mn2+ does not affect either the polymerization activity or the processivity of the DNA polymerase, this metal is unable to activate the overall replication of the phage genome. This can be explained by a deleterious effect of Mn2+ on the 3'-5'-exonucleolytic and/or the strand-displacement activity, the latter being an intrinsic function of the viral DNA polymerase required for protein-primed DNA replication.     

In vivo competition between iron and manganese for occupancy of the active site region of the manganese-superoxide dismutase of Escherichia coli

Three forms of the dimeric manganese superoxide dismutase (MnSOD) were isolated from aerobically grown Escherichia coli which contained 2 Mn, 1 Mn and 1 Fe, or 2 Fe, respectively. These are designated Mn2-MnSOD, Mn,Fe-MnSOD, and Fe2-MnSOD. Substitution of iron in place of manganese, eliminated catalytic activity, decreased the isoelectric point, and increased the native electrophoretic anodic mobility, although circular dichroism, high performance liquid chromatography gel exclusion chromatography, and sedimentation equilibrium revealed no gross changes in conformation. Moreover, replacement of iron by manganese restored enzymatic activity. Fe2-MnSOD and the iron-superoxide (FeSOD) of E. coli exhibit distinct optical absorption spectra. These data indicate that the active site environments of E. coli MnSOD and FeSOD must differ. They also indicate that competition between iron and manganese for nascent MnSOD polypeptide chains occurs in vivo, and copurification of these variably substituted MnSODs can explain the substoichiometric manganese contents and the variable specific activities which have been reported for this enzyme.     

In silico analyses of superoxide dismutases (SODs) of rice (Oryza sativa L.)

Superoxide dismutases (SODs), members of the metalloenzymes family are most effective intracellular enzymatic antioxidant in aerobic organisms. These enzymes provide the first line of defense in plants against the toxic effects of elevated levels of reactive oxygen species (ROS) generated during various environmental stresses. The availability of high-throughput computational tools has provided better opportunities to characterize the protein features and determine their function. In the present study an attempt was made to gain an insight into the structure and evolution of subunits of SODs (Cu-Zn, Mn and Fe SODs) of rice. The 3-Dimensional structures of SODs were modeled based on available X-ray crystal structures and further validated. The primary sequence, secondary and tertiary structure analysis revealed Mn and Fe SOD to be structurally homologous while Cu-Zn SOD is un-related to either of them. Comparative structural study also revealed former two were dominated by α-helices followed by β-strands in contrast; Cu-Zn SOD dominated by β-strands. Molecular phylogeny indicated a common evolutionary origin of Mn and Fe SOD while Cu-Zn SOD may have evolved separately.     

In vitro bioaccessibility of metals from tape tea – A low-cost emerging drug

BackgroundAn in vitro physiologically relevant test based on the standard Unified Bioaccessibility Method (UBM) combined with inductively coupled plasma mass spectrometry was performed in this study to ascertain the elemental bioaccessibility pools of tape tea as emerging low-cost abuse drug under fasted conditions.MethodsElemental quantification in tape tea and body fluid extracts was performed by an inductively coupled plasma quadrupole mass spectrometer - ICP-MS, and for sample preparation of the bioaccessibility extracts prior to ICP-MS analysis, a microwave-assisted acid decomposition was applied by using a microwave oven. The Unified Bioaccessibility Method (UBM) was considered for investigation of elemental bioaccessibility in tape tea, required a full set of organic compounds, salts, and enzymes.ResultsConsidering total element evaluation through ICP-MS, Co, Ni, Mn, and Zn are found at the highest concentrations in the sample, namely 415 ± 36, 202 ± 55, 1389 ± 225 and 2397 ± 197 μg L⁻¹, respectively. Regarding the oral bioaccessibility test, after both gastric and gastrointestinal extractions Co, Ni, and Mn are fully bioaccessible while for Zn the bioaccessibility is ca. 66 %.ConclusionAccording to the first results in the literature proposed for these samples, the bioaccessibility results indicate an increment in day-to-day total element concentration and depending on the concentration of each element that an individual consumes in its usual diet, the total concentration can exceed the TDI. There are several possible toxic effects caused by the excess of Co, Ni and Mn, which might be expected by their high total concentrations.     

In situ localization of galactosyltransferase in surface mucous cells of the rat stomach.

Cryostate sections of the rat stomach fundus were incubated in the presence of uridine 5'-diphosphate-[3H]-galactose. The radioactivity in the surface mucous epithelial cells was shown by autoradiography to be specifically incorporated into a supranuclear area, the area where in these cells the Glogi system is situated. The incorporation lasted only 5-6 min and was Mn++ dependent. Galactose was probably incorporated into a beta-glycosidic bond.     

In vitro RNA synthesis by intact rat brain nuclei

The characteristics of DNA-dependent RNA polymerase activity in intact rat cerebral cortex nuclei tested at low ionic strength are presented. The system was most dependent on the presence of spermidine and an ATP-generating system and to a lesser extent on Mg2+ and K+ for maximal incorporation. Substitution of Mg2+ by Mn2+ or of K+ by Na+ resulted in substantially less activity than under the optimum conditions described. Maximal incorporation was about 10 per cent that of brain nuclear systems of high ionic strength. In addition, the labelling patterns of the in vitro RNA products were shown to be very similar to those found in vivo. The stability of isolated nuclei toward degradation of RNA synthetic capacity and products formed was much greater than that of a similar liver system.     

In vitro study of cysteine oxidase in rat brain

Effects of some cysteine analogs and other compounds on in vitro cysteine oxidase were studied in rat brain microsomes. Among the tested compounds, maximum inhibition of microsomal cysteine oxidase was by alpha,alpha'-dipyridyl and the least inhibition by dithiothreitol. Kinetic and dialysis studies found L-homocysteine to be a competitive and reversible inhibitor of cysteine oxidase. Epinephrine was shown to inhibit cysteine oxidase, whereas pyridoxal HCl activated cysteine oxidase at the same concentration. Except for the Mg2+ ion, other metallic ions inhibited cysteine oxidase activity in the following order: Zn2+ greater than Cu2+ greater than Li+ greater than Ca2+ greater than Co2+ greater than K+ greater than Mn2+. A 12 mM concentration of Mg2+ ion was required to obtain maximum cysteine oxidase activity.     

In vivo analysis of metal distribution and expression of metal transporters in rice seed during germination process by microarray and X-ray Fluorescence Imaging of Fe, Zn, Mn, and Cu

To investigate the flow of the metal nutrients iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) during rice seed germination, we performed microarray analysis to examine the expression of genes involved in metal transport. Many kinds of metal transporter genes were strongly expressed and their expression levels changed during rice seed germination. We found that metal transporter genes such as ZIP family has tendency to decrease in their expressions during seed germination. Furthermore, imaging of the distribution of elements (Fe, Mn, Zn, and Cu) was carried out using Synchrotron-based X-ray microfluorescence at the Super Photon ring-8 GeV (SPring-8) facility. The change in the distribution of each element in the seeds following germination was observed by in vivo monitoring. Iron, Mn, Zn, and Cu accumulated in the endosperm and embryos of rice seeds, and their distribution changed during rice seed germination. The change in the patterns of mineral localization during germination was different among the elements observed.     

Catalytic properties of phenol carboxylase. In vitro study of CO2: 4-hydroxybenzoate isotope exchange reaction

Phenol is metabolized in a denitrifying bacterium in the absence of molecular oxygen via para-carboxylation to 4-hydroxybenzoate (biological Kolbe-Schmitt synthesis). The enzyme system catalyzing the presumptive carboxylation of phenol, tentatively named 'phenol carboxylase', catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate (specific activity 0.1 mumol 14CO2 incorporated into 4-hydroxybenzoate x min-1 x mg-1 cell protein) which is considered a partial reaction of the overall enzyme catalysis; 14C from [14C]phenol was not exchanged into 4-hydroxybenzoate ring positions to a significant extent. The 14CO2 isotope exchange reaction was studied in vitro. The reaction was dependent on the substrates CO2 and 4-hydroxybenzoate and required K+ and Mn2+. The actual substrate was CO2 rather than HCO3-. The apparent Km values were 1 mM dissolved CO2, 0.2 mM 4-hydroxybenzoate, 2 mM K+, and 0.1 mM Mn2+. The cationic cocatalysts could be substituted by ions of similar ionic radius: K+ could be replaced to some extent by Rb+, but not by Li+, Na+, Cs+, or NH4+; Mn2+ could be replaced to some extent by Fe2+ greater than Mg2+, Co2+, but not by Ni2+, Zn2+, Ca2+, or Cu2+. The exchange reaction was not strictly specific for 4-hydroxybenzoate, however it required a p-hydroxyl group; derivatives of 4-hydroxybenzoate with OH, CH3 or Cl substituents in m-position did react, whereas those with substitutions in the o-position were inactive or were inhibitory. The enzyme was induced when cells were grown on phenol, but not on 4-hydroxybenzoate. Comparison of SDS/PAGE protein patterns of cells grown on phenol or 4-hydroxybenzoate revealed several additional protein bands in phenol-grown cells. The possible role of similar enzymes in the anaerobic metabolism of phenolic compounds is discussed.