Normal growth rate in rats is recovered after a period of zinc deficiency by restoration of zinc supply by means of a zinc-fortified Petit Suisse cheese

Fortification of a Petit Suisse cheese with zinc sulfate and zinc gluconate stabilized with glycine was used as a tool to overcome zinc-deficiency effects on total-body growth and skeletal growth. Animals were divided in 4 groups of 10 rats: basal (B), control (C), depletion-repletion 1 (DR1), and depletion-repletion 2 (DR2). These four groups were fed with four diets: basal (2 ppm Zn), control (30 ppm Zn), DR1, and DR2; they received a basal diet for 14 d and a control diet for the other 14 d of the experiment, using zinc sulfate for DR1 and zinc gluconate stabilized with glycine for DR2. After 28 d of the experiment, total-body weight and weight gain of the control and DR1 and DR2 animals were not statistically different (p<0.05), Femur weight and femur zinc content of DR1 and DR2 did not achieve the values of control animals (p<0.05), but they were higher than that of basal animals. Our results show that restoration of dietary zinc levels by means of food fortification normalized weight gain, as an indicator of total-body growth, and presented a trend to normalize bone weight, as a marker of skeletal growth, in young rats and independently of the zinc source used.     

Redox characterization of Geobacter sulfurreducens cytochrome c7: physiological relevance of the conserved residue F15 probed by site-specific mutagenesis

The complete genome sequence of the delta-proteobacterium Geobacter sulfurreducens reveals a large abundance of multiheme cytochromes. Cytochrome c(7), isolated from this metal ion-reducing bacterium, is a triheme periplasmic electron-transfer protein with M(r) 9.6 kDa. This protein is involved in metal ion-reducing pathways and shares 56% sequence identity with a triheme cytochrome isolated from the closely related delta-proteobacterium Desulfuromonas acetoxidans (Dac(7)). In this work, two-dimensional NMR was used to monitor the heme core and the general folding in solution of the G. sulfurreducens triheme cytochrome c(7) (PpcA). NMR signals obtained for the three hemes of PpcA at different stages of oxidation were cross-assigned to the crystal structure [Pokkuluri, P. R., Londer, Y. Y., Duke, N. E. C., Long, W. C., and Schiffer, M. (2004) Biochemistry 43, 849-859] using the complete network of chemical exchange connectivities, and the order in which each heme becomes oxidized was determined at pH 6.0 and 8.2. Redox titrations followed by visible spectroscopy were also performed in order to monitor the macroscopic redox behavior of PpcA. The results obtained showed that PpcA and Dac(7) have different redox properties: (i) the order in which each heme becomes oxidized is different; (ii) the reduction potentials of the heme groups and the global redox behavior of PpcA are pH dependent (redox-Bohr effect) in the physiological pH range, which is not observed with Dac(7). The differences observed in the redox behavior of PpcA and Dac(7) may account for the different functions of these proteins and constitute an excellent example of how homologous proteins can perform different physiological functions. The redox titrations followed by visible spectroscopy of PpcA and two mutants of the conserved residue F15 (PpcAF15Y and PpcAF15W) lead to the conclusion that F15 modulates the redox behavior of PpcA, thus having an important physiological role.     

Redox behaviour of the haem domain of flavocytochrome c3 from Shewanella frigidimarina probed by NMR

Flavocytochrome c3 from Shewanella frigidimarina (fcc3) is a tetrahaem periplasmic protein of 64 kDa with fumarate reductase activity. This work reports the first example of NMR techniques applied to the assignment of the thermodynamic order of oxidation of the four individual haems for such large protein, expanding its applicability to a wide range of proteins. NMR data from partially and fully oxidised samples of fcc3 and a mutated protein with an axial ligand of haem IV replaced by alanine were compared with calculated chemical shifts, allowing the structural assignment of the signals and the unequivocal determination of the order of oxidation of the haems. As oxidation progresses the fcc3 haem domain is polarised, with haems I and II much more oxidised than haems III and IV, haem IV being the most reduced. Thus, during catalysis as an electron is taken by the flavin adenosine dinucleotide from haem IV, haem III is eager to re-reduce haem IV, allowing the transfer of two electrons to the active site.     

Site-directed mutagenesis of a phenylalanine residue strictly conserved in cytochromes c 3

 Reduction of the haems in tetrahaem cytochromes c ₃ is a cooperative process, i.e., reduction of each of the haems depends on the redox states of the other haems. Furthermore, electron transfer is coupled to proton transfer (redox-Bohr effect). Two of its haems and a strictly conserved nearby phenylalanine residue, F20, in Desulfovibrio vulgaris (Hildenborough) cytochrome c ₃ form a structural motif that is present in all cytochromes c ₃ and also in cytochrome c oxidase. A putative role for this phenylalanine residue in the cooperativity of haem reduction was investigated. Therefore, this phenylalanine was replaced, with genetic techniques, by isoleucine and tyrosine in D. vulgaris (Hildenborough) cytochrome c ₃. Cyclic voltammetry studies revealed a small increase (30 mV) in one of the macroscopic redox potentials in the mutated cytochromes. EPR showed that the main alterations occurred in the vicinity of haem I, the haem closest to residue 20 and one of the haems responsible for positive cooperativities in electron transfer of D. vulgaris cytochrome c ₃. NMR studies of F20I cytochrome c ₃ demonstrated that the haem core architecture is maintained and that the more affected haem proton groups are those near the mutation site. NMR redox titrations of this mutated protein gave evidence for only small changes in the relative redox potentials of the haems. However, electron/electron and proton/electron cooperativity are maintained, indicating that this aromatic residue has no essential role in these processes. Furthermore, chemical modification of the N-terminal amino group of cytochrome c ₃ backbone, which is also very close to haem I, had no effect on the network of cooperativities. Received: 25 June 1996 / Accepted: 26 August 1996     

Effect of carbon source on the formation of polyhydroxyalkanoates (PHA) by a phosphate-accumulating mixed culture

In the present work, attention was devoted to understand how different carbon substrates and their concentration can influence the production of PHA by polyphosphate-accumulating bacteria. Acetate, propionate, and butyrate were tested independently. The composition of the polymers formed was found to vary with the substrate used. Acetate leads to the production of a copolymer of hydroxybutyrate (HB) and hydroxyvalerate (HV) with the HB units being dominant. With propionate, HV units are mainly produced and only a small amount of HB is synthesized. When butyrate is used, the amount of polymer formed is much lower with the HB units being produced to a higher extent. The yield of polymer produced per carbon consumed (Y_P/S) was found to diminish from acetate (0.97) to propionate (0.61) to butyrate (0.21). Using a mixture of acetate, propionate, and butyrate and increasing the carbon concentration, although maintaining the relative concentration of each substrate, propionate is primarily consumed and consequently, PHA synthesized was enriched in HV units. The polymers obtained in all experiments were copolymers with the average molecular weight of the most representative fraction higher when hydroxybutyrate units were present in considerable amounts. All the polymers synthesized were found to be quite homogeneous and their average molecular weight is of the same order of magnitude as the ones commercially available.     

Phylogeography and ecological niche modelling in Eugenia uniflora (Myrtaceae) suggest distinct vegetational responses to climate change between the southern and the northern Atlantic Forest

In this study, we evaluate phylogeographic patterns and predictions of ecological niche modelling (ENM) for Eugenia uniflora (Myrtaceae), a widely distributed taxon in the Atlantic forest domain, to understand the effect of past climatic oscillations on the demographic history of this species. An analysis of phylogeographic population structure and demography was conducted on E. uniflora from 46 localities in natural environments across the distribution range of the species based on three plastid markers. ENM was also performed to predict suitable environments and areas of dramatic decrease in future suitability for the species under distinct representative concentration pathways (RCPs). Eugenia uniflora exhibited higher haplotype and nucleotide diversity in the southern part of its distribution than in the northern part. Two divergent lineages were revealed in the phylogenetic analysis of haplotypes, with an estimated divergence at c. 4.9 Mya. The populations in the northern and central regions of the range probably experienced population growth, whereas populations in the southern region are marked by historical demographic stability. ENM results indicate that the distribution of E. uniflora was fragmented in cool periods and was broader and more connected during warm periods during Pleistocene. The results suggest distinct evolutionary histories in southern to northern populations, indicating region‐specific responses to changes.     

mtDNA analysis of <Emphasis Type="Italic">Mytilopsis</Emphasis> (Bivalvia,Dreissenidae) invasion in Brazil reveals the existence of two species

The present study sought to identify general patterns of genetic variability and structure of fish stocks (migratory and non-migratory species) along one of the largest Brazilian rivers, the São Francisco. Given that genetic variability of populations of all organisms is governed by both present and past influences, herein we investigate both these aspects by evaluating the current genetic differences between populations of five species (Leporinus piau, Megaleporinus reinhardti, Pimelodus maculatus, Prochilodus argenteus, and Pygocentrus piraya) along the entire extension of the river, as well as their demographic history. Analyses were done through sequences of two mitochondrial fragments and microsatellite data. In general, the data showed no support for recent fragmentation of stocks by the dams present in this river, and that all species show signs of past population expansion. We discuss the possible reasons for the common patterns found between these species, including the influence of the river’s topography and history.     

Structural studies of Desulfovibrio vulgaris ferrocytochrome c3 by two-dimensional NMR.

Two-dimensional NMR has been used to make specific assignments for the four haems in Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c3 and to determine their haem core architecture. The NMR signals from the haem protons were assigned according to type using two-dimensional NMR experiments which led to four sets of signals, one for each of the haems. Specific assignments were obtained by calculating the ring current shifts which arise from other haems and aromatic residues. Observation of interhaem NOEs confirmed the assignments and established that the relative orientation of the haems is identical to that found in the crystal structure of D. vulgaris (Miyazaki F.) ferricytochrome c3. Assignments were also made for all the aromatic residues except for the haem ligands and F20, which is shifted under the main envelope of signals. The NOEs observed between these aromatic protons and haem protons confirm the similarity between the structures in solution and in the crystal. The assignments reported here are the basis for the cross-assignments of the four microscopic haem redox potentials to specific haems in the protein structure [Salgueiro, C. A., Turner, D. L., Santos, H., LeGall, J. and Xavier, A. V. (1992) FEBS Lett., in the press]     

Nonredundant Roles for Cytochrome c2 and Two High-Potential Iron-Sulfur Proteins in the Photoferrotroph Rhodopseudomonas palustris TIE-1

The purple bacterium Rhodopseudomonas palustris TIE-1 expresses multiple small high-potential redox proteins during photoautotrophic growth, including two high-potential iron-sulfur proteins (HiPIPs) (PioC and Rpal_4085) and a cytochrome c₂. We evaluated the role of these proteins in TIE-1 through genetic, physiological, and biochemical analyses. Deleting the gene encoding cytochrome c₂ resulted in a loss of photosynthetic ability by TIE-1, indicating that this protein cannot be replaced by either HiPIP in cyclic electron flow. PioC was previously implicated in photoferrotrophy, an unusual form of photosynthesis in which reducing power is provided through ferrous iron oxidation. Using cyclic voltammetry (CV), electron paramagnetic resonance (EPR) spectroscopy, and flash-induced spectrometry, we show that PioC has a midpoint potential of 450 mV, contains all the typical features of a HiPIP, and can reduce the reaction centers of membrane suspensions in a light-dependent manner at a much lower rate than cytochrome c₂. These data support the hypothesis that PioC linearly transfers electrons from iron, while cytochrome c₂ is required for cyclic electron flow. Rpal_4085, despite having spectroscopic characteristics and a reduction potential similar to those of PioC, is unable to reduce the reaction center. Rpal_4085 is upregulated by the divalent metals Fe(II), Ni(II), and Co(II), suggesting that it might play a role in sensing or oxidizing metals in the periplasm. Taken together, our results suggest that these three small electron transfer proteins perform different functions in the cell.