Manganese(II) adsorption and oxidation by whole cells and a membrane fraction of Pedomicrobium sp. ACM 3067

Heat treatment of Pedomicrobium sp. ACM 3067 enhanced the adsorption of Mn(II) to whole cells but abolished Mn(II)-oxidising activity. In whole cells, optimal Mn(II)-oxidising activity occurred at pH 7 and 25 °C. The apparent K _m of the Mn(II)-oxidising system for Mn(II) was 26 μM. These data confirm that Mn(II) oxidation is an enzymic process in Pedomicrobium sp. ACM 3067. Measurement of Mn(II) oxidation during the growth cycle demonstrated that the highest activity occurred during early- to mid-exponential phase and was independent of the presence of Mn in the growth medium. Mn(II)-oxidising activity was localised to the membrane fraction. Transmission electron microscopy showed that this fraction consisted of double-layered membrane vesicles. Positively charged molecules such as poly-l-lysine interfered with the adsorption and oxidation of Mn(II) by whole cells and membranes. Similarly, aminoglycoside antibiotics such as gentamicin sulfate proved to be potent inhibitors of Mn(II) oxidation. Treatment of cells with the copper chelator diethyldithiocarbamate inhibited Mn(II) oxidation. Enzyme activity was restored by the addition of Cu(II) ions, but not by Co(II) nor Zn(II). We conclude that Mn(II) oxidation in Pedomicrobium sp. ACM 3067 is catalysed by a Cu-dependent enzyme. Received: 14 September 1998 / Accepted: 4 January 1999     

Reconstitution of the water-oxidizing complex in manganese-depleted photosystem II preparations using synthetic binuclear Mn(II) and Mn(IV) complexes: production of hydrogen peroxide

Reconstitution of Mn-depleted PSII particles with synthetic binuclear Mn complexes (one Mn(II)₂ complex and one Mn(IV)₂ complex) was examined. In both cases the electron-transfer rates in the reconstituted systems were found to be up to 75–82% of that measured in native PSII but the oxygen evolution activity remained lower (<5–40%). However, hydrogen peroxide was also produced by the reconstituted samples. These samples therefore represent a new type of reconstituted PSII that generates hydrogen peroxide as the final product in reconstituted PSII centers.     

Highly Responsive Bioinspired AgNPs Probe for the Precise Colorimetric Detection of the Mn(II) in Aqueous Systems

A green, rapid, and cost-effective probe for the precise colorimetric detection of Mn(II) ions has been investigated. The AgNPs were prepared via heating method by utilizing the extract obtained from Bhilwa (Semecarpus anacardium Linn) nuts (B−AgNPs). The Mn(II) ions induce the aggregation of B−AgNPs, resulting in color changes from yellowish brown to dark red along with the red shift in the surface plasmon resonance (SPR) peak from 404 to 432 nm. The aggregation of B−AgNPs was further confirmed by FTIR, HRTEM, and DLS techniques. The developed probe displayed an excellent linear response towards Mn(II) ions in the linear range from 20 to 0.001 ppm. In comparison to the currently available methods for the detection of Mn(II), the proposed probe provides an enhanced detection limit, i.e., 0.001 ppm. The developed colorimetric probe can be successfully applied for the detection of Mn(II) ions in water samples (pond water), thus demonstrating its potential use in field applications.

     

A capillary electrophoresis method for the characterization of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and the analysis of inhibitors by in-capillary enzymatic microreaction

A capillary electrophoresis (CE) method for the characterization of recombinant NTPDases 1, 2, and 3, and for assaying NTPDase inhibitors has been developed performing the enzymatic reaction within the capillary. After hydrodynamic injection of plugs of substrate solution with or without inhibitor in reaction buffer, followed by a suspension of an enzyme-containing membrane preparation, and subsequent injection of another plug of substrate solution with or without inhibitor, the reaction took place close to the capillary inlet. After 5 min, the electrophoretic separation of the reaction products was initiated by applying a constant current of −60 μA. The method employing a polyacrylamide-coated capillary and reverse polarity mode provided baseline resolution of substrates and products within a short separation time of less than 7 min. A 50 mM phosphate buffer (pH 6.5) was used for the separations and the products were detected by their UV absorbance at 210 nm. The Michaelis–Menten constants (K_m) for the recombinant rat NTPDases 1, 2, and 3 obtained with this method were consistent with previously reported data. The inhibition studies revealed pronounced differences in the potency of reactive blue 2, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), suramin, and N⁶-diethyl-β,γ-dibromomethylene-ATP (ARL67156) towards the NTPDase isoforms. Notably, ARL67156 does not inhibit all NTPDases, having only a minor inhibitory effect on NTPDase2. Dipyridamole is not an inhibitor of the NTPDase isoforms investigated. The new method is fast and accurate, it requires only tiny amounts of material (nanoliter scale), no sample pretreatment and can be fully automated; thus it is clearly superior to the current standard methods.     

Theoretical study of the hydroxylation of phenolates by the Cu2O2(N,N′-dimethylethylenediamine)2 2+ complex

Tyrosinase catalyzes the ortho hydroxylation of monophenols and the subsequent oxidation of the diphenolic products to the resulting quinones. In efforts to create biomimetic copper complexes that can oxidize C–H bonds, Stack and coworkers recently reported a synthetic μ-η²:η²-peroxodicopper(II)(DBED)₂ complex (DBED is N,N′-di-tert-butylethylenediamine), which rapidly hydroxylates phenolates. A reactive intermediate consistent with a bis-μ-oxo-dicopper(III)-phenolate complex, with the O–O bond fully cleaved, is observed experimentally. Overall, the evidence for sequential O–O bond cleavage and C–O bond formation in this synthetic complex suggests an alternative mechanism to the concerted or late-stage O–O bond scission generally accepted for the phenol hydroxylation reaction performed by tyrosinase. In this work, the reaction mechanism of this peroxodicopper(II) complex was studied with hybrid density functional methods by replacing DBED in the μ-η²:η²-peroxodicopper(II)(DBED)₂ complex by N,N′-dimethylethylenediamine ligands to reduce the computational costs. The reaction mechanism obtained is compared with the existing proposals for the catalytic ortho hydroxylation of monophenol and the subsequent oxidation of the diphenolic product to the resulting quinone with the aim of gaining some understanding about the copper-promoted oxidation processes mediated by 2:1 Cu(I)O₂-derived species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis and characterization of new optically active poly(azo-ester-imide)s via interfacial polycondensation

N,N′-Pyromelliticdiimido-di-l-amino acids (1a–1d) were prepared from the reaction of pyromellitic dianhydride with the corresponding l-amino acids in a solution of glacial acetic acid/pyridine (3:2) at refluxing temperature. 4,4′-sulfonyl bis(4,1-phenylene) bis(diazene-2,1-diyl) diphenol, 4,4′-oxy bis(4,1-phenylene) bis(diazene-2,1-diyl) diphenol and 4,4′-methylene bis(4,1-phenylene) bis(diazene-2,1-diyl) diphenol, were prepared from 4,4′-diamino diphenyl sulfone, 4,4′-diamino diphenyl ether, 4,4′-diamino diphenyl methane, sodium nitrite and phenol following the general procedure of diazo coupling. Interfacial polycondensation method was used to prepare the corresponding poly(azo-ester-imid)s (PAEI _1–12 ) in biphasic solution of water/dichloromethane. The resulting polymers (PAEIs) have been obtained in high yields having good inherent viscosities (0.32–0.57 dl g⁻¹), optical activities and thermal stabilities.     

Significance of Stereochemistry of 3′-Terminal Phosphorothioate-modified Primer in DNA Polymerase-mediated Chain Extension

Influence of stereochemistry of the 3′-terminal phosphorothioate (PS)-modified primers was studied in a single base extension (SBE) assay to evaluate any improvements in specificity. SBE reactions were catalyzed by members of the high fidelity Pfu family of DNA polymerases with (exo+) or without (exo−) 3′ → 5′ exonucleolytic activity. The diastereomerically pure PS-labeled primers used in these studies were obtained either by the stereospecific chemical synthesis invented in our laboratory or by the more conventional ion-exchange chromatographic method for separation of a mixture of diastereomers (R_P and S_P). When the SBE reaction was performed in the presence of mispaired 2′-deoxyribonucleoside triphosphates (dNTPs), the “racemic” 3′-phosphorothioate primer mixture resulted in a lower level of 3′ → 5′ exonuclease-mediated cleavage products in comparison to the SBE reactions carried out with the corresponding unmodified primer. When the diastereomerically pure R_P 3′-phosphorothioate primer was examined, the results were largely the same as for the racemic 3′-phosphorothioate primer mixture. In contrast, a 3′-PS primer of S_P configuration displayed significantly improved performance in the SBE reaction. This included the lack of 3′ → 5′ proofreading products, less mispriming, and improved yield of incorporation of the correct nucleotide.     

Initial step of B12-dependent enzymatic catalysis: energetic implications regarding involvement of the one-electron-reduced form of adenosylcobalamin cofactor

Density functional theory analysis was performed to elucidate the impact of one-electron reduction upon the initial step of adenosylcobalamin-dependent enzymatic catalysis. The transition state (TS) corresponding to the Co–C bond cleavage and subsequent hydrogen abstraction from the substrate was located. The intrinsic reaction coordinate calculations predicted that the reaction consisting of Co–C5′ bond cleavage in [Co^III(corrin^•)]–Rib (where Rib is ribosyl) and hydrogen-atom abstraction from the CH₃–CH₂–CHO substrate occurs in a concerted fashion. The computed activation energy barrier of the reaction (15.0 kcal/mol) was lowered by approximately 54.5% in comparison with the reaction involving the positively charged cofactor model (Im–[Co^III(corrin)]–Rib⁺, where Im is imidazole; energy barrier = 33.0 kcal/mol). The Im base was detached during the TS search in the reaction involving the one-electron-reduced analogue. Thus, to compare the energetics of the two reactions, the axial Im ligand detachment energy for the Im–[Co^III(corrin^•)]–Rib model was computed [7.6 kcal/mol (gas phase); 4.6 kcal/mol (water)]. Consequently, the effective activation energy barrier for the reaction mediated by the Im-off [Co^III(corrin^•)]–Rib was estimated to be 22.6 kcal/mol, which implied an overall 31.5% reduction in the energetic demands of the reaction. Considering that the lengthened Co–N_axial bond has been observed in X-ray crystal structure studies of B₁₂-dependent mutases, the catalytic impact induced by one-electron reduction of the cofactor is expected to be higher in the presence of the enzymatic environment.     

Highly sensitive field test lateral flow immunodiagnostics of PVX infection

A test system is described and expanded upon for mass field immunochromatography assay on porous membrane carriers for rapid diagnostics of potato virus X (PVX) in potato leaf tissue and sprout extracts using colloidal gold nanoparticles as a marker. Sensitivity of the assay developed for PVX identification is found to be comparable to the sensitivity of solid-phase sandwich-ELISA. Complete assay time does not exceed 15 min, and the lower limit of the PVX detection in non-clarified leaf extract is 2 ng/ml. A single measurement requires 0.1–0.2 ml (3–5 drops) of tested solution only (extracted from 10–20 mg of potato leaf tissue or sprouts). The simplicity and reliability of the method makes it especially efficient in direct rapid monitoring of many infected potato specimens in the field, as verified by field trials of 360 clones of 28 domestic and foreign cultivars of potato. A diagnostic kit for routine analyses of potato viral infections both in the laboratory and in the field is described and expanded upon.     

Direct colorimetric assay of microcystin using protein phosphatase

A new direct colorimetric assay of microcystin in water and algal samples is proposed consisting of two procedures as follows: 1) the elimination of phosphorus in the sample and concentration of microcystin using a C₁₈ cartridge, 2) the detection of the released phosphorus by the ascorbic acid method and determination of protein phosphatase (PP) inhibition by microcystin. The optimum amounts of phosphorylase α and PP-1 in 50 μL concentrated sample were 50 μg/50 μL buffer and 1.0 unit/50 μL buffer, respectively, for the best assay. The pH for the maximum activity of PP-1 was 8. The minimum detectable concentration for this method was about 0.02 μg/L, which is sufficient to meet the proposed guideline level of 1 μg microcystin/L in drinking water. Consequently, it would seem that the proposed direct colorimetric assay using PP is a rapid, easy, and convenient method for the detection of microcystin in water and algal samples.     

A rapid method for liquid β-galactosidase reporter assay in Saccharomyces cerevisiae

Although β-galactosidase assay is widely used for various studies in yeast a quantitative estimation of low enzyme activities with standard reactives remains hampered. It requires long reaction time and large amounts of cells. To overcome existing limitations we developed protocol, which incorporates realization reaction in miniaturized format, cell lysis in reaction buffer and simplification the normalization of β-galactosidase activity. These features allow faster reaction kinetics, accurate and simple quantification of low enzyme activities. To perform studies in vivo conditions we constructed a reporter plasmids based on the low copy yeast vector Ycp50. We adapted our assay on the yeast protein Rpn4 which is highly unstable with a half-life of only 2 min. We demonstrated that detection of Rpn4–LacZ fusion is achieved in 40 min in our method, whereas in standard assay it requires 4–5 h. Moreover, we implemented our approach for promoter dissection investigation. Thus, we present rapid, convenient and less labor-intensive method for assessment β-galactosidase activity.     

Manganese tolerance in subterranean clover (Trifolium subterraneum L.) genotypes grown with ammonium nitrate or symbiotic nitrogen

Summary A wide range of clover accessions were screened for reaction to manganese (Mn) in solution culture. Growth was supported with ammonium nitrate (NH₄NO₃) or symbiotic nitrogen to assess Mn effects on symbiosis and the suitability of NH₄NO₃ dependent growth for assessing Mn tolerance in clover. Reduction of dry matter at Mn 45 ppm varied 0–70%, at Mn 90 ppm, 38–92%, the extent depending on genotype. Tolerant clovers tended to restrict the movement of Mn from roots to shoots. Several previously untested lines were the most tolerant while some commercial lines possessed poor tolerance. Ranks of tolerance for the two nitrogen (N) sources at Mn 45 ppm were correlated suggesting no dominant, discriminatory effects of N source on Mn tolerance; but inclusion of symbiotic effectiveness in a multiple correlation improved the relation between relative tolerances of genotypes under different N sources. Mn affected some aspects of symbiosis. Total nodule nitrogenase activity mainly reflected effects of Mn on nodule number but nitrogenase activity per nodule also contributed. To establish relative tolerances of subterranean clover to Mn growth with NH₄NO₃ is suitable and useful when symbiotic effectiveness is unknown.     

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.     

Catalysis and Selectivity in Prebiotic Synthesis: Initiation of the Formation of Oligo(U)s on Montmorillonite Clay by Adenosine-5′-methylphosphate

Adenosine-5′-methylphosphate (MepA) initiates the oligomerization of the 5′-phosphorimidazolide of uridine (ImpU) in the presence of montmorillonite clay. Longer oligomers form because the 5′-phosphate is blocked with a methyl group that prevents the formation of cyclic- and pyrophosphate-containing compounds. The MepA initiates 69–84% of the 5–9 charge oligomers, respectively. The montmorillonite catalyst also provides selectivity in the oligomerization reactions so that the main reaction pathway is MepA → MepA³′pU → MepA³′pU²′pU → MepA³′pU²′pU³′pU. MepA did not enhance the oligomerization of ImpA. The relative rates of the reactions were determined from an investigation of the products in competitive reactions. Selectivity was observed in the reaction of ImpU with equimolar amounts of MepA³′pU and MepA²′pU where the relative reaction rates are 10.3:1, respectively. In the reaction of ImpA with MepA³′pA and MepA²′pA the ImpA reacts 5.2 times faster with MepA³′pA. In the competitive reaction of ImpU and ImpA with MepA³′pA and MepA³′pU the elongation proceeds on MepA³′pA 5.6 times more rapidly than with MepA³′pU. There is no correlation between the extent of binding to the montmorillonite and reaction rates in the formation of longer oligomers. The formation of more than two sequential 2′,5′-linkages in the oligomer chain proceeds more slowly than the addition to a single 2′,5′-link or a 3′,5′-link and either chain termination or elongation by a 3′,5′-linage occurs. The central role that catalysis may have had in the prebiotic formation of biopolymers is discussed. Note added in proof: There are errors in the high resolution mass spectral data given in Section 4.2.1. The high resolution mass spectrum found for the cyclic dimer of UpUp (C-UpUp) was 657.02260. C₁₈H₂₁N₄O₁₆P₂Na₂ requires 657.02232. The high resolution mass spectrum found for the cyclic dimer of ApAp (C-ApAp) was 725.05850. C₂₀H₂₂N₁₀O₁₂P₂Na₃ requires 725.05839.     

Highly efficient photoactivation of Mn-depleted photosystem II by imidazole-liganded manganese complexes

The oxygen-evolving complex (OEC) of Mn-depleted photosystem II (PSII) can be reconstituted in the presence of exogenous Mn or a Mn complex under weak illumination, a process called photoactivation. Synthetic Mn complexes could provide a powerful system to analyze the assembly of the OEC. In this work, four mononuclear Mn complexes, [(terpy)₂Mn^II(OOCH₃)]·2H₂O (where terpy is 2,2′:6′,2″-terpyridine), Mn^II(bzimpy)₂, Mn^II(bp)₂(CH₃CH₂OH)₂ [where bzimpy is 2,6-bis(2-benzimidazol-2-yl)pyridine] and [Mn^III(HL)(L)(py)(CH₃OH)]CH₃OH (where py is pyridine) were used in photoactivation experiments. Measurements of the photoreduction of 2,6-dichorophenolindophenol and oxygen evolution demonstrate that photoactivation is more efficient when Mn complexes are used instead of MnCl₂ in reconstructed PSII preparations. The most efficient recoveries of oxygen evolution and electron transport activities are obtained from a complex, [Mn^III(HL)(L)(py)(CH₃OH)]CH₃OH, that contains both imidazole and phenol groups. Its recovery of the rate of oxygen evolution is as high as 79% even in the absence of the 33-kDa peptide. The imidazole ligands of the Mn complex probably accelerate P ₆₈₀ ^•+ reduction and consequently facilitate the process of photoactivation. Also, the strong intermolecular hydrogen bond probably facilitates interaction with the Mn-depleted PSII via reorganization of the hydrogen-bonding network, and therefore promotes the recovery of oxygen evolution and electron transport activities.     

Metabolization of [Ru(η6-C6H5CF3)(pta)Cl2]: a cytotoxic RAPTA-type complex with a strongly electron withdrawing arene ligand

Abstract  

The anticancer ruthenium–arene compound [Ru(η⁶-C₆H₅CF₃)(pta)Cl₂] (where pta is 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane), termed RAPTA-CF3, with the electron-withdrawing α,α,α-trifluorotoluene ligand, is one of the most cytotoxic RAPTA compounds known. To rationalize the high observed cytotoxicity, the hydrolysis of RAPTA-CF3 in water and brine (100 mM sodium chloride) and its reactions with the protein ubiquitin and a double-stranded oligonucleotide (5′-GTATTGGCACGTA-3′) were studied using NMR spectroscopy, high-resolution Fourier transform ion cyclotron resonance mass spectrometry, and gel electrophoresis. The aquation of the ruthenium–chlorido complex was accompanied by a loss of the arene ligand, independent of the chloride concentration, which is a special property of the compound not observed for other ruthenium–arene complexes with relatively stable ruthenium–arene bonds. Accordingly, the mass spectra of the biomolecule reaction mixtures contained mostly [Ru(pta)]–biomolecule adducts, whereas [Ru(pta)(arene)] adducts typical of other RAPTA compounds were not observed in the protein or DNA binding studies. Gel electrophoresis experiments revealed a significant degree of decomposition of the oligonucleotide, which was more pronounced in the case of RAPTA-CF3 compared with RAPTA-C. Consequently, facile arene loss appears to be responsible for the increased cytotoxicity of RAPTA-CF3.     

Real-time kinetics of discontinuous and highly conformational metal-ion binding sites of prion protein

The prion protein (PrP) is a metalloprotein with an unstructured region covering residues 60–91 that bind two to six Cu(II) ions cooperatively. Cu can bind to PrP regions C-terminally to the octarepeat region involving residues His111 and/or His96. In addition to Cu(II), PrP binds Zn(II), Mn(II) and Ni(II) with binding constants several orders of magnitudes lower than those determined for Cu. We used for the first time surface plasmon resonance (SPR) analysis to dissect metal binding to specific sites of PrP domains and to determine binding kinetics in real time. A biosensor assay was established to measure the binding of PrP-derived synthetic peptides and recombinant PrP to nitrilotriacetic acid chelated divalent metal ions. We have identified two separate binding regions for binding of Cu to PrP by SPR, one in the octarepeat region and the second provided by His96 and His111, of which His96 is more essential for Cu coordination. The octarepeat region at the N-terminus of PrP increases the affinity for Cu of the full-length protein by a factor of 2, indicating a cooperative effect. Since none of the synthetic peptides covering the octarepeat region bound to Mn and recombinant PrP lacking this sequence were able to bind Mn, we propose a conformational binding site for Mn involving residues 91–230. A novel low-affinity binding site for Co(II) was discovered between PrP residues 104 and 114, with residue His111 being the key amino acid for coordinating Co(II). His111 is essential for Co(II) binding, whereas His96 is more important than His111 for binding of Cu(II).     

Sequence preference in DNA binding: de novo designed helix–turn–helix metallopeptides recognize a family of DNA target sites

The DNA-binding behavior and target sequences of two designed metallopeptides have been investigated with an iterative electrophoresis mobility shift assay followed by PCR amplification, and by circular dichroism spectroscopy. Peptides P3W and P5b were designed based on the structural similarity of the helix–turn–helix motif of homeodomains and the EF-hand motifs of calmodulin, as previously described for P3W. Like P3W, P5b binds both Eu(III) (K _d=12.6±1.9 μM) and Ca(II) (K _d=70±8 μM) with reasonable affinity. Binding selection from a library of randomized 8-mer DNA oligonucleotide sequences identified one target family for CaP5b [5′-pur-T-pur-G-(G/C)-3′], and two target sites for CaP3W [5′-(A/T)-G-G-G-(T/C)-3′ and 5′-A-T-(G/T)-T-G-3′]. Circular dichroism studies indicate that unlike EuP3W, EuP5b is poorly folded in the absence of DNA. In the presence of DNA containing target-binding sites for both peptides, both EuP3W and EuP5b increase in helical content, in the latter case significantly. These results suggest that EuP5b binding to target DNA involves an induced-fit mechanism. These small chimeric metallopeptides have been found to bind selectively to DNA targets, analogous to natural protein–DNA interactions. This corroborates our earlier conclusions (J. Am. Chem. Soc. 125:6656, 2003) that sequence-preferential DNA cleavage by Ce(IV)P3W was due to sequence recognition. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Effect of Supplemental Inorganic Zn and Mn and their Interactions on the Performance of Broiler Chicken,Mineral Bioavailability,and Immune Response

The purpose of this study was to investigate the interaction and main effects of supplemental Zn and Mn levels on growth, tissue mineral uptake, and immune response in broiler chicken. A basal diet of corn–soybean meal was supplemented with Zn at 40, 80, or 160 ppm and Mn at 60, 120, or 240 ppm in a factorial pattern to constitute nine experimental diets. Each diet was offered to nine replicates of six chicks in stainless steel battery brooders. At 35 days of age, body weight gain, feed conversion efficiency, hock joint scores, tibia weight, tibia strength, and percent ash were not influenced by Zn and Mn levels and their interactions. The concentration of Zn (207–238 ppm) and Mn (11.8–16.3 ppm) in tibia increased linearly with progressive raise of mineral inclusion in diets. Mn at 240 ppm level caused higher retention of Zn in tibia, but not vice versa. Manganese either alone or in combination with Zn (Zn160/Mn120 ppm) significantly reduced Cu retention (10.1–7.2 ppm) in tibia. Even in the hepatic tissue, Zn (93.6–98.4 ppm) and Mn (9.3–10.2 ppm) concentration increased linearly with their levels of inclusion in diets. When Zn and Mn levels were maintained at 4:3 ratio (80:60 or 160:120 ppm), the concentration of Zn (100–106 ppm) in liver was higher, while that of Mn was significantly more with low level of Zn (40 ppm) in diet. However, Mn supplementation at 120 ppm level and above significantly decreased Cu accumulation (19.5–17.1 ppm) in liver, but Mn × Zn interaction had no effect on Cu retention. The immune response measured as antibody titers to sheep RBC increased (5.9–7.9 log₂) significantly with higher Zn (80 ppm) supplementation and cell-mediated immune response to phytohemagglutinin (0.57–0.78) with Mn level at 120 ppm. In summary, Zn (40 ppm) and Mn (60 ppm) as recommended by NRC was sufficient for broiler performance and bone parameters. Mn complimented Zn retention in tibia and antagonized Cu in tibia and liver tissues. Higher levels of Zn (80 ppm) and Mn (120 ppm) than those recommended by NRC were needed for improved immune response in broilers at 35 days of age.     

Involvement of manganese peroxidase in the transformation of macromolecules from low-rank coal by Phanerochaete chrysosporium

Manganese peroxidase (Mn peroxidase) catalyses the oxidation of Mn(II) to Mn(III), a diffusible non-specific oxidant likely to be involved in the transformation of polyphenolic macromolecules from brown coal by the white-rot fungus Phanerochaete chrysosporium. We report here that solubilised macromolecules from Morwell brown coal were depolymerised by Mn(III) ions when incubated under hyperbaric O₂. However, under N₂ or air they were polymerised, suggesting that net depolymerisation by Mn(III) requires molecular oxygen to inhibit coupling of coal radicals. Coal macromolecules were also polymerised when separated by a semipermeable membrane from a culture of P. chrysosporium or from a solution of Mn peroxidase, Mn(II) and H₂O₂, probably by Mn(III) crossing the membrane. In oxygenated cultures in which Mn peroxidase␣was up-regulated by Mn(II), the extent of depolymerisation correlated with cumulative Mn peroxidase activity suggesting that Mn-peroxidase-generated Mn(III) has a central role in initial depolymerisation of coal molecules in vivo. However, mutant ME446-B17-1, which produces Mn peroxidase but not lignin peroxidase, polymerised coal macromolecules in oxygenated cultures. In sum, it appears Mn peroxidase can both polymerise and depolymerise brown coal macromolecules and that, in vivo, both hyperbaric O₂ and lignin peroxidase are also required to force net depolymerisation to products assimilable by cells. Received: 4 September 1997 / Received revision: 29 January 1998 / Accepted: 30 January 1998