Fibronectin‐binding proteins of Clostridium perfringens recognize the III1‐C fragment of fibronectin

The Clostridium perfringens strain 13 genome contains two genes (fbpA, fbpB) that encode putative Fbp. Both rFbpA and rFbpB were purified and their reactivity with human serum Fn was analyzed. To determine the region of the Fn molecule recognized by rFbp, a plate binding assay using N‐terminal 70‐kDa peptide, III₁‐C peptide, and 110‐kDa peptide containing III_2–10 of Fn was performed. Both rFbp bound to the III₁‐C peptide of Fn but not to the other peptides. However, the III₁‐C fragment of Fn is known to be cryptic in serum Fn. Then, rFbp‐BP from Fn were purified by rFbp‐affinity chromatography. The yield of purified proteins was approximately 1% of the applied Fn on a protein basis. Western blotting analysis of the rFbp‐BP, using four different anti‐Fn monoclonal antibodies, revealed that the rFbp‐BP carried partial Fn antigenicity. Bindings of rFbp to rFbp‐BP were inhibited by the presence of the III₁‐C peptide, suggesting that rFbp‐BP express the III₁‐C fragment. The binding of Fn to III₁‐C was inhibited by the presence of either rFbpA or rFbpB. This result that suggests C. perfringens Fbps may inhibit the formation of Fn‐matrix in vivo.     

Voltammetric studies of Zn and Fe complexes of EDTA: Evidence for the push mechanism

The `push' hypothesis for the antioxidant action of Zn²⁺ is based on its displacement of iron from a low molecular weight pro-oxidant complex. In this study, the chemical plausibility of that proposed function is investigated by cyclic voltammetry. As a model for a pro-oxidative low molecular weight iron complex the Fe^II/IIIEDTA couple was examined. This complex was selected for its well-defined electrochemical, iron stability constants, and similarity to other low molecular weight chelates in physiological fluids in terms of logical binding sites, i.e. amino, and carboxylate groups. Also investigated were iron complexes of nitrilotriacetic acid and DL-glutamic acid. Results demonstrate that approximately 90% of the cyclic voltammetric peak current for Fe^IIIEDTA reduction and the EC′ current for the mediated reduction of H₂O₂ by Fe^II/IIIEDTA (Fenton Reaction) are lost when Zn²⁺ is introduced to a 1:1 molar ratio relative to iron. All experiments were conducted in HEPES buffered solutions at pH 7.4. Iron (II/III) complexes of nitrilotriacetic acid and DL-glutamic acid followed the same trends. Cyclic voltammetric experiments indicate that Zn²⁺ displaces Fe^III from EDTA despite the much larger stability constant for the iron complex (10^25.1) versus zinc (10^16.50). The hydrolysis aided displacement of Fe^III from EDTA by Zn²⁺ is considered by the equilibria modeling program, HySS. With Fe^III hydrolysis products included, Zn²⁺ is able to achieve 90% displacement of iron from EDTA, a result consistent with cyclic voltammetric observations. Published online December 2004     

Growth of Pseudomonas fluorescens in modified atmosphere packaged tofu

Aims: The objective was to study the growth of Pseudomonas in a food product (tofu) where it typically occurs as a spoilage organism, and when this product is stored under modified atmosphere. Methods and Results: A Pseudomonas strain was isolated from the endogenous microflora of tofu. Tofu was inoculated with the strain, packaged in different gas conditions (air, 100% N₂, 30% CO₂/70% N₂ or 100% CO₂) and stored under refrigerated conditions. Microbial loads and the headspace gas composition were monitored during storage. Conclusions: The strain was capable of growing in atmospheres containing no or limited amounts of oxygen and increased amounts of carbon dioxide. Even when 100% CO₂ was used, growth could not be inhibited completely. Significance and Impact of Study: In contrast to the general characteristics of the genus Pseudomonas (strictly aerobic, highly sensitive to CO₂), it should not be expected in the food industry that removing oxygen from the food package and increasing the carbon dioxide content, combined with cold storage, will easily avoid spoilage by Pseudomonas species. Guarantee of hygienic standards and combination of strategies with other microbial growth inhibiting measures should be implemented.     

Kinetic studies on the reduction of the R2 subunit of mouse ribonucleotide reductase with hydroxyurea, hydrazine, phenylhydrazine and hydroxylamine

 Four reductions of the R2 subunit of mouse ribonucleotide reductase have been studied and found to exhibit different behaviour from that of Escherichia coli R2. An important difference is that there is no stable met-R2 (Fe₂ ^II I) form of mouse R2. With hydroxyurea, hydrazine and hydroxylamine uniphasic kinetics are observed for the combined reduction of radical Tyr ^˙ and Fe₂ ^II I components to tyrosine and Fe₂ ^II respectively. The rate constants, determined at 370 nm (emphasising Fe^III decay) and 417 nm (emphasising Tyr ^˙ decay), differ by factors of 2–3, allowing some mechanistic features to be defined. The studies with hydrazine are particularly important. In the case of E. coli R2, a first phase corresponding to two-equivalent reduction of the met-R2 component has been observed [18]. It is likely that the four times slower second phase reaction of active E. coli R2 also corresponds to the Fe₂ ^II I → Fe₂ ^II change and is followed by fast intramolecular Fe₂ ^II reduction of the higher potential Tyr ^˙. The latter changes are believed to hold also for (active) mouse R2. The Fe^IIFe^III semi forms have been detected at low levels by EPR for mouse R2 (9%) and E. coli (∼5%) in previous studies. Further substrate reduction of Fe^IIFe^III occurs at a comparable rate to account for the transient behaviour of Fe^IIFe^III. For mouse R2 the combined Fe^III decay processes (which we are unable to separate) give smaller uniphasic rate constants at 370 nm than at 417 nm. A fitted-base-line (FBL) treatment of absorbance changes at 417 nm targets more closely the Tyr ^˙ decay as a means of monitoring the rate-determining step. The FBL method gives rate constants k (M^–1 s^–1) at 25  °C and pH 7.5 for hydroxyurea (1.46), hydrazine (0.163) and hydroxylamine (4.4). Surprisingly, phenylhydrazine, with a less strong reduction potential (0.25 V), gives a substantially faster reduction of the Tyr ^˙ as the only redox step (rate constant 27 M^–1 s^–1). In this case a slower second phase at 370 nm is independent of reductant and corresponds to rate-controlling release of Fe^III. Overall the results indicate a more reactive redox centre for mouse R2 and help develop further an understanding of factors affecting the reactivity of R2. Received: 11 October 1996 / Accepted: 11 February 1997     

Trans-pyridine tetraammine complexes of RuII and RuIII with N7-coordninated purine nucleosides

 The synthesis, spectroscopic, and electrochemical properties of trans-[L(Pyr)(NH₃)₄Ru^II/III] (Pyr=py, 3-phpy, 4-phpy, 3-bnpy, or 4-bnpy; L=H₂O, Guo, dGuo, 1MeGuo, Gua, Ino, or G⁷-DNA) are reported. As expected, the Pyr ligand slows DNA binding by trans-[(H₂O)(Pyr)(NH₃)₄Ru^II]²⁺ relative to [(H₂O)(NH₃)₅Ru^II]²⁺ and favors reduction of Ru^III by about 150 mV. The pyridine ligand also promotes the disproportionation of Ru^III to afford the corresponding complexes of Ru^II and, presumably, Ru^IV. For L=Ino, disproportionation follows the rate law: d[Ru^II]/dt=k ₀[Ru^III]+k ₁[OH^–][Ru^III], k ₀=(2.7±0.7)×10^–4s^–1 and k ₁=70±1 M^–1s^–1. Received: 11 May 1998 / Accepted: 3 March 1999     

Cis-diamminedichloroplatinum(II) Induced Distortion of a Single and Double Stranded Deoxydecanucleosidenona-phosphate Studied by Nuclear Magnetic Resonance

Abstract

The structural distortion of a single- and a double-stranded decadeoxynucleotide upon binding of cis-PtCI₂(NH₃)₂ was studied by ¹H-NMR. After selective platination of d(T-C-T- C-G-G-T-C-T-C) (I) at the central d(-GpG-) site (resulting in I-Pt), several non-exchangeable base protons as well as H1′, H2′ H2″ and H3′ protons could be assigned by means of conventional NMR double-resonance techniques. Addition of the complementary decamer strand to I and I-Pt yielded the double-stranded III and III-Pt, respectively. All non-exchangeable base, H1′, and most of the H2′ and H2″ protons in the two double stranded compounds could be assigned using 2D-chemical shift correlation (COSY) and nuclear Overhauser enhancement (NOESY) techniques. The double stranded compound III appears to adopt a B-DNA like structure. Comparison of NOEs and proton-proton coupling constants in the d(-GpG-)·cisPt part in I-Pt and III-Pt reveals that their structure displays large similarity. Significant chemical shift changes (i.e, larger than 0.1 ppm) between III and III-Pt are restricted to the central four base pairs. It follows that the outer three base pairs, located on either side of the central four base pairs in III-Pt are likely to adopt a regular B-DNA type helix. The observed large upfield and downfield chemical shifts in the d(-CpGpG-) part of III with respect to III-Pt can be rationalized by describing the distortion of the double helix as a kink. A discussion of the observed physical effects upon platination of a double-stranded oligonucleotide is presented.     

Mechanism of Short Term Fe Reduction by Roots : Evidence against the Role of Secreted Reductants

The hypothesized role of secreted reducing compounds in Fe^III reduction has been examined with Fe-deficient peanuts (Arachis hypogaea L. cv A124B). Experiments involved the exposure of roots to (a) different gas mixtures, (b) carbonyl cyanide m-chlorophenylhydrazone (CCCP), and (c) agents which impair membrane integrity.

Removing roots from solution and exposing them to air or N₂ for 10 minutes did not result in any accumulation in the free space of compounds capable of increasing rates of Fe^III reduction when roots were returned to solutions. On the contrary, exposing roots to N₂ decreased rates of Fe^III reduction. CCCP also decreased rates of Fe^III reduction.

Acetic acid and ethylenediaminetetraacetic acid (disodium salt) (EDTA) impaired the integrity and function of the plasma membranes of roots of Fe-deficient peanuts. That is, in the presence of acetic acid or EDTA, there was an efflux of K⁺ from the roots; K⁺ (⁸⁶Rb) uptake was also impaired. Acetic acid increased the efflux from the roots of compounds capable of reducing Fe^III. However, both acetic acid and EDTA caused rapid decreases in rates of Fe^III reduction by the roots. In addition to peanuts, acetic acid also decreased rates of Fe^III reduction by roots of Fe-deficient sunflowers (Helianthus annuus L. cv Sobrid) but not maize (Zea mays L. cv Garbo).

These results suggest that, at least in the short term, the enhanced Fe^III reduction by roots of Fe-deficient plants is not due to the secretion of reducing compounds.

     

Positive selection for resistance to 2-deoxyglucose gives rise, in Streptococcus salivarius, to seven classes of pleiotropic mutants, including ptsH and ptsl missense mutants

We have used the toxic non-metabolizabie glucose/ mannose analogue 2-deoxygiucose to isolate a comprehensive collection of mutants of the phosphoenoipyruvate:sugar phosphotransferase system from Streptococcus salivarius. To increase the range of possible mutations, we isolated spontaneous mutants on different media containing 2-deoxyglucose and various metabolizable sugars, either lactose, meli-biose, galactose or fructose. We found that the frequency at which 2-deoxygiucose-resistant mutants Were isolated varied according to the growth substrate. The highest frequency was obtained with the combination galactose and 2-deoxygiucose and was 15-fold higher than the rate observed with the mixture melibiose and 2-deoxygiucose, the combination that gave the lowest frequency. By combining results from: (i) Western biol analysis of III^Man, a specific component of the phosphoenolpyruvate:mannose phosphotransferase system in S. salivarius; (ii) rocket immunoelectrophoresis of HPr and EI, the two general energy-coupling proteins of the phosphotransferase system; and (iii) from gene sequencing, mutants could be assigned to seven classes. Class 1 was composed of strains devoid of III^Man_L, a low-molecular-weight form of III^Man_L (35200), class 2 was composed of strains exhibiting a reduced level of III^Man_L, class 3 was composed of strains devoid of both forms of III^Man (III^Man_L as well as III^Man_H, the high-molecular-weight form of III^Man (38900)), class 4 was composed of mutants bearing a mutation in ptsH, the gene encoding HPr, class 5 was composed of mutants bearing a mutation in ptsl, the gene encoding EI, class 6 was composed of 2-deoxygiucose-resistant strains without any apparent defect in PTS components, and class 7 was composed of strains possessing both forms of III^Man but abnormal levels of HPr and/or EI without any mutation in the ptsH and/or the ptsI genes. Preliminary characterization of representative strains of each class is reported.     

Metal-metal bonding and charge localisation in [Ru2X9], X=Cl or Br; n=1, 2, 3, 4. A spectroelectrochemical study

A spectroelectrochemical study of [Ru₂X₉]ⁿ⁻, X=Cl, Br; n=1, 2, 3, 4 has been undertaken. Stable solutions of n=4, 2, 1 can be formed by electrolysis at low temperatures. Analysis of the Vis-NIR spectra of the complexes indicate that the Ru^II---R^III dimers (n=4) have delocalised mixed valence and that the Ru^III---R^III (n=3) dimers have a strong Ru---Ru bond. The more oxidised materials do not form a Ru---Ru bond; the spectroscopic data indicates the Ru^III---R^IV dimers have localised valency.     

Mechanism of dithionite reduction of the R2 protein of E. coli and mouse ribonucleotide reductase: evidence for reduction of FeIII 2 prior to the tyrosyl radical

 Dithionite has been found to reduce directly (without mediators) the Escherichia coli R2 subunit of ribonucleotide reductase. With dithionite (∼10 mM) in large excess, the reaction at 25  °C is complete in ∼10 h. Preparations of E. coli R2 have an Fe^III ₂ (met-R2) component in this work at ∼40% levels, alongside the fully active enzyme Fe^III ₂ . . . Tyr*, which has a tyrosyl radical at Tyr-122. In the pH range studied (7–8) the kinetics are biphasic. Rate laws for both phases give [S₂O₄ ^2–] and not [S₂O₄ ^2–]^1/2 dependencies, and saturation kinetics are observed for the first time in R2 studies. No dependence on pH was detected. The kinetics (25  °C) of the first phase are reproduced in separate experiments using only met-R2, with association of S₂O₄ ^2– to met-R2, K=330 M^–1, occurring prior to electron transfer, k _et=4.8×10^–4 s^–1, I=0.100 M (NaCl). The second phase assigned to the reaction of Fe^III ₂ . . . Tyr* with S₂O₄ ^2– gives K=800 M^–1 and k _et=5.6×10^–5 s^–1. Bearing in mind the substantially smaller reduction potential for Fe^III ₂ compared to Tyr*, this is a quite remarkable finding, with implications similar to those already reported for the reaction of R2 with hydrazine, but with additional information provided by the saturation kinetics. The similarity in rates for the two phases (∼fourfold difference) suggests that reduction of Fe^III ₂ is occurring in both cases, and since S₂O₄ ^2– is involved a two-equivalent change is proposed with the formation of Fe^II ₂ . . . Tyr* in the case of active R2. As a sequel to the second phase, intramolecular reduction of the strongly oxidising Tyr* by the Fe^II ₂ is rapid, and further decay of Fe^IIFe^III is also fast. There is no stable mouse met-R2 form, and the single-phase reaction with dithionite gives saturation kinetics with K=208 M^–1 and k _et=1.7±10^–3 s^–1. Mechanistic implications, including the applicability of a pathway for electron transfer via Fe_A, are considered. Received: 25 February 1998 / Received: 20 August 1998     

Spectrophotometric kinetic study and analytical implications of the glucose oxidase-catalyzed reduction of [MIII(LL)2Cl2]+ complexes by d-glucose (M=Os and Ru, LL=2,2′-bipyridine and 1,10-phenanthroline type ligands)

 Glucose oxidase-catalyzed reduction of cis[M^III (LL)₂Cl₂]⁺ (M=Os and Ru) complexes to cis[M^II (LL)₂Cl₂] (LL=2,2′-bipyridine and 1,10-phenanthroline type ligands) by d-glucose is a first-order process in the complex and the enzyme in aqueous buffered solution. The reaction follows MichaelisMenten kinetics in d-glucose and the rate is independent of d-glucose concentration above 0.03 M. The reactivity decreases in the series [Ru(bpy)₂Cl₂]⁺ > [Os(phen)₂Cl₂]⁺ > [Os(4,4′-Me₂bpy)₂Cl₂]⁺ > [Os(4,7Me₂phen)₂Cl₂]⁺. The measured second-order rate constant for the oxidation of reduced glucose oxidase by [Os(phen)₂Cl₂]⁺ in air equals 1.2×10⁵ M^–1 s^–1 at pH 6.7, [d-glucose] 0.05 M, and 25  °C, which is ca. 20% less than that when the reaction solutions are purged with argon. In the case of [Ru(bpy)₂Cl₂]⁺ the rate constant equals 1.8×10⁵ M^–1 s^–1 under similar conditions in air, showing higher reactivity of Ru complexes compared with Os ones. The reduction is pH-dependent with a maximum around 7. Added for solubilization of poorly soluble metal complexes, surfactants decrease the rates of the enzymatic reaction. The retardation effect increases in the series: cetyltrimethylammonium bromide < Triton X-100 < sodium dodecyl sulfate, i.e. on going from positively charged to neutral and then to negatively charged surfactants. The behavior of the Os^III and Ru^III complexes toward reduced glucose oxidase contrasts to that of recently studied ferricenium cations. As opposed to the latter, the former do not show kinetically meaningful binding with the enzyme, and the Michaelis kinetics typical of the ferricenium case is not realized for the Os^III, and Ru^III species. The systems Os^III- or Ru^III-glucose oxidase are convenient for routine "one pot" spectrophotometric monitoring of the d-glucose content in samples, since the metal reduction to M^II is accompanied by a strong increase in absorbance in the visible spectral region. Received: 1 July 1998 / Accepted: 13 January 1999     

Cellulose-binding domain of endoglucanase III from Trichoderma reesei disrupting the structure of cellulose

The DNA fragment encoding the cellulose binding domain of endoglucanase III (CBD_{EG III}) from Trichoderma reesei was subcloned and expressed in E. coli. The CBD_{EG III} had a high affinity for cellulose. The morphological and structural changes of cellulose after treatment with CBD_{EG III} indicated a 17% decrease in number of hydrogen bonds and a 16.5% decrease in crystalline index. X-ray diffraction and IR spectra analyses indicated that the destabilization and breakage of the hydrogen bonds in crystalline cellulose accounted for the non-hydrolytic disruption of the structure of cellulose.     

Magnetite as a precursor for green rust through the hydrogenotrophic activity of the iron‐reducing bacteria Shewanella putrefaciens

Magnetite (Fe^IIFe^III₂O₄) is often considered as a stable end product of the bioreduction of Fe^III minerals (e.g., ferrihydrite, lepidocrocite, hematite) or of the biological oxidation of Fe^II compounds (e.g., siderite), with green rust (GR) as a mixed Fe^II‐Fe^III hydroxide intermediate. Until now, the biotic transformation of magnetite to GR has not been evidenced. In this study, we investigated the capability of an iron‐reducing bacterium, Shewanella putrefaciens, to reduce magnetite at circumneutral pH in the presence of dihydrogen as sole inorganic electron donor. During incubation, GR and/or siderite (Fe^IICO₃) formation occurred as secondary iron minerals, resulting from the precipitation of Fe^II species produced via the bacterial reduction of Fe^III species present in magnetite. Taking into account the exact nature of the secondary iron minerals and the electron donor source is necessary to understand the exergonic character of the biotic transformation of magnetite to GR, which had been considered to date as thermodynamically unfavorable at circumneutral pH. This finding reinforces the hypothesis that GR would be the cornerstone of the microbial transformations of iron‐bearing minerals in the anoxic biogeochemical cycle of iron and opens up new possibilities for the interpretation of the evolution of Earth's history and for the understanding of biocorrosion processes in the field of applied science.     

Electronic structural investigations of ruthenium compounds and anticancer prodrugs

Several Ru^III compounds are propitious anticancer agents although the precise mechanisms of action remain unknown. With this paper we start to establish an experimental library of X-ray absorption spectroscopy (XAS) data for ten Ru compounds wherein the ligands [Cl⁻, dimethyl sulfoxide, imidazole, and indazole] were varied systematically to provide electronic structural information for future use in correlating spectroscopic signatures with chemical properties. Despite the considerable difference in the coordination environments of the complexes studied, the overall differences in spectral features and electronic structures calculated using density functional theory are unexpectedly small. However, the differences in the electronic structure of the Ru^III prodrugs KP1019 ([IndH][trans-RuCl₄(Ind)₂], Ind is indazole) and ICR ([ImH][trans-RuCl₄(Im)₂], Im is imidazole) observed in the XAS data show correlation with known chemical and biological activities in addition to the donor abilities of imidazole compared with indazole and reduction potentials of the complexes. These semiquantitative results lay the groundwork for future biochemical studies into the structure–function relationships of Ru-based anticancer drugs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Accounting for cryptic population substructure enhances detection of inbreeding depression with genomic inbreeding coefficients: an example from a critically endangered marsupial

Characterizing inbreeding depression in wildlife populations can be critical to their conservation. Coefficients of individual inbreeding can be estimated from genome‐wide marker data. The degree to which sensitivity of inbreeding coefficients to population genetic substructure alters estimates of inbreeding depression in wild populations is not well understood. Using generalized linear models, we tested the power of two frequently used inbreeding coefficients that are calculated from genome‐wide SNP markers, F_H and F^^III, to predict four fitness traits estimated over two decades in an isolated population of the critically endangered Leadbeater's possum. F_H estimates inbreeding as excess observed homozygotes relative to equilibrium expectations, whereas F^^III quantifies allelic similarity between the gametes that formed an individual, and upweights rare homozygotes. We estimated F_H and F^^III from 1,575 genome‐wide SNP loci in individuals with fitness trait data (N = 179–237 per trait), and computed revised coefficients, F_H^{by group} and F^^{IIIby group}, adjusted for population genetic substructure by calculating them separately within two different genetic groups of individuals identified in the population. Using F_H or F^^III in the models, inbreeding depression was detected for survival to sexual maturity, longevity and whether individuals bred during their lifetime. F^^{IIIby group} (but not F_H^{by group}) additionally revealed significant inbreeding depression for lifetime reproductive output (total offspring assigned to each individual). Estimates of numbers of lethal equivalents indicated substantial inbreeding load, but differing between inbreeding estimators. Inbreeding depression, declining population size, and low and declining genetic diversity suggest that genetic rescue may assist in preventing extinction of this unique Leadbeater's possum population.     

Insolubility of Cr2O3 in Bioaccessibility Tests Points to Requirement for a New Human Oral Reference Dose for Trivalent Chromium

Bioaccessibility measurements have the potential to improve the accuracy of risk assessments and reduce the potential costs of remediation when they reveal that the solubility of chemicals in a matrix (e.g., soil) differs markedly from that in the critical toxicity study (i.e., the key study from which a toxicological or toxicity reference value is derived). We aimed to apply this approach to a brownfield site contaminated with chromium, and found that the speciation was Cr^III, using a combination of alkaline digestion/diphenylcarbazide complexation and X-ray absorption near edge structure analysis. The bioaccessibility of Cr₂O₃, the compound on which a reference dose for Cr^III is based, was substantially lower (<0.1%) than that of the Cr^III in the soils, which was a maximum of 9%, giving relative bioaccessibility values of 13,000% in soil. This shows that the reference dose is based on essentially an insoluble compound, and thus we suggest that other compounds be considered for toxicity testing and derivation of reference dose. Two possibilities are CrCl₃·6H₂O and KCr(SO₄)₂·12H₂O, which have been used for derivation of ecological toxicity reference values and are soluble at a range of dosing levels in our bioaccessibility tests.     

A new class of clear mutants from coliphage 434 hy not complementing CII and C3 mutants for lysogenization

Summary Clear mutants which differ from regular C _I , C _II , CIIIand y mutants have been isolated from phage 434 hy. These mutants resemble C _I mutants in plaque and spot phenotype but efficiently complement C _I mutants for lysogenization. Like C _II mutants, they do not complement authentic C _II mutants for lysogenization but in contrast to C _II mutants they also fail to complement C _III mutants. They map between the lambda-434 non-homology region and Co ₁ (aC _II mutant). On account of this map position adjacent to C _II the mutants of the new type are called C _IIa . They arise from phage 434 hy with a frequency comparable to that of C _I and C _II mutants. Such mutants are also obtained from phage lambda but apparently not from phage b₅. C _IIa mutants would not fit into a picture of three independently acting cistrons C _I , C_II, and C _III . The hypothesis is presented that C _IIa and C _II mutants are in the same structural gene. Two possibilities are discussed that would account for the complementation patterns: 1. C _IIa mutants may block the expression of gene C _III in cis position; or 2. the products of genes C _II and C _III function through an oligomeric complex they form.     

Mechanism of Fe uptake by the leaf symplast: Is Fe inactivation in leaf a cause of Fe deficiency chlorosis?

The mechanism of iron (Fe) uptake from the leaf apoplast into leaf mesophyll cells was studied to evaluate the putative Fe inactivation as a possible cause of Fe deficiency chlorosis. For this purpose, sunflower (Helianthus annuus L.) and faba bean plants (Vicia faba L.) were precultured with varied Fe and bicarbonate (HCO ₃ ^- ) supply in nutrient solution. After 2–3 weeks preculture, Fe^III reduction and ⁵⁹Fe uptake by leaf discs were measured in solutions with Fe supplied as citrate or synthetic chelates in darkness. The data clearly indicate that Fe^III reduction is a prerequisite for Fe uptake into leaf cells and that the Fe nutritional status of plants does not affect either process. In addition, varied supply of Fe and HCO ₃ ^- to the root medium during preculture had no effect on pH of the xylem sap and leaf apoplastic fluid. A varied pH of the incubation solution had no significant effect on Fe^III reduction and Fe uptake by leaf discs in the physiologically relevant pH range of 5.0–6.0 as measured in the apoplastic leaf fluid. It is concluded that Fe inactivation in the leaf apoplast is not a primary cause of Fe deficiency chlorosis induced by bicarbonate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Improvement of Metabolic and Histological Changes of Adiposity in Rats by Synthetic Oleoyl Chalcones

We previously reported that synthetic oleoyl chalcones had a favorable effect to alleviate metabolic consequences of obesity in male SD rats. In this work, we prepared and characterized by spectroscopic tools, a set of six oleoyl chalcones ( 5a–c , 10 and 11a,b ). The comparative effects of the previously prepared oleoyl chalcones and their new synthetic analogs on metabolic and histological changes in obese male SD rats were studied. It was found that the oleoyl chalcones III_a and IV were the best in improving many metabolic parameters, e. g., FBG, FI, ISI, TG, and total cholesterol. They cured systemic inflammation, through inhibition of the TNF-α and induction of adiponectin production. Moreover, chalcones III_a and IV alleviated the oxidative stress accompanying obesity through the induction of the antioxidant enzymes GPX, SOD and CAT besides, GSH. Interestingly, chalcones III_a and IV exerted hepatoprotective potency and ameliorated the manifestations of NAFLD via inhibition of apoptosis and induction of autophagy of hepatic cells. In conclusion, the oleoyl chalcones III_a and IV were the most effective candidates among the series of synthetic chalcones in correcting body weight and the consequent metabolic and histological changes in adiposity.     

Kinetic, structural and electrostatic aspects of the reduction of pentacyanoferrate(III) complexes by myoglobin

 The mechanism of the reduction of pentacyanoferrate(III) complexes by oxymyoglobin has been studied by conventional and high-pressure kinetic methods, and also by structural modelling. The results of this and an earlier study show that an outer-sphere mechanism is operating for electron transfer between oxymyoglobin and Fe^III(CN)₅L ^{n –}, independent of the lability of the ligand L. The electron transfer process is preceded by precursor formation at a specific site on the protein close to the protein heme pocket. Received: 10 November 1998 / Accepted: 25 February 1999