Synthesis, X-ray crystal structure and NMR characterisation of mononuclear Pd(II) and Pt(II) complexes of didentate ligands with NN′-donor set

Two new 3,5-dimethylpyrazolic derived ligands that are N1-substituted by diamine chains, 1-[2-(diethylamino)ethyl]-3,5-dimethylpyrazole (L1) and 1-[2-(dioctylamino)ethyl]-3,5-dimethylpyrazole (L2) were synthesised. Reaction of the ligands, L1 and L2, with [MCl₂(CH₃CN)₂] yielded [MCl₂(L)] (M = Pd(II), Pt(II)) complexes. These complexes were characterised by elemental analyses, conductivity measurements, IR, ¹H, ¹³C{¹H} and ¹⁹⁵Pt{¹H} NMR spectroscopies. The crystal structure of [PdCl₂(L1)] was determined by single-crystal X-ray diffraction methods. The structure consists of mononuclear units. The Pd(II) atom is coordinated by a pyrazolic nitrogen, an amine nitrogen and two chlorine atoms in a cis disposition. In this structure, C-H?Cl, C-H?H-C and C-H?C-H intermolecular interactions have been identified.     

Frataxin depletion in yeast triggers up-regulation of iron transport systems before affecting iron-sulfur enzyme activities

The primary function of frataxin, a mitochondrial protein involved in iron homeostasis, remains controversial. Using a yeast model of conditional expression of the frataxin homologue YFH1, we analyzed the primary effects of YFH1 depletion. The main conclusion unambiguously points to the up-regulation of iron transport systems as a primary effect of YFH1 down-regulation. We observed that inactivation of aconitase, an iron-sulfur enzyme, occurs long after the iron uptake system has been activated. Decreased aconitase activity should be considered part of a group of secondary events promoted by iron overloading, which includes decreased superoxide dismutase activity and increased protein carbonyl formation. Impaired manganese uptake, which contributes to superoxide dismutase deficiency, has also been observed in YFH1-deficient cells. This low manganese content can be attributed to the down-regulation of the metal ion transporter Smf2. Low Smf2 levels were not observed in AFT1/YFH1 double mutants, indicating that high iron levels could be responsible for the Smf2 decline. In summary, the results presented here indicate that decreased iron-sulfur enzyme activities in YFH1-deficient cells are the consequence of the oxidative stress conditions suffered by these cells.     

Presence of Novel,Potentially Homoacetogenic Bacteria in the Rumen as Determined by Analysis of Formyltetrahydrofolate Synthetase Sequences from Ruminants

Homoacetogens produce acetate from H₂ and CO₂ via the Wood-Ljungdahl pathway. Some homoacetogens have been isolated from the rumen, but these organisms are expected to be only part of the full diversity present. To survey the presence of rumen homoacetogens, we analyzed sequences of formyltetrahydrofolate synthetase (FTHFS), a key enzyme of the Wood-Ljungdahl pathway. A total of 275 partial sequences of genes encoding FTHFS were PCR amplified from rumen contents of a cow, two sheep, and a deer. Phylogenetic trees were constructed using these FTHFS gene sequences and the translated amino acid sequences, together with other sequences from public databases and from novel nonhomoacetogenic bacteria isolated from the rumen. Over 90% of the FTHFS sequences fell into 34 clusters defined with good bootstrap support. Few rumen-derived FTHFS sequences clustered with sequences of known homoacetogens. Conserved residues were identified in the deduced FTHFS amino acid sequences from known homoacetogens, and their presence in the other sequences was used to determine a “homoacetogen similarity” (HS) score. A homoacetogen FTHFS profile hidden Markov model (HoF-HMM) was used to assess the homology of rumen and homoacetogen FTHFS sequences. Many clusters had low HS scores and HoF-HMM matches, raising doubts about whether the sequences originated from homoacetogens. In keeping with these findings, FTHFS sequences from nonhomoacetogenic bacterial isolates grouped in these clusters with low scores. However, sequences that formed 10 clusters containing no known isolates but representing 15% of our FTHFS sequences from rumen samples had high HS scores and HoF-HMM matches and so could represent novel homoacetogens.Feed ingested by ruminant animals is fermented in the rumen by a complex community of microbes. This community produces, among other products, the volatile fatty acids acetate, propionate, and butyrate, which are absorbed across the rumen wall and satisfy a large part of the animals'' carbon and energy requirements. Hydrogen gas (H₂) is also formed and is the major precursor of the methane (CH₄) formed in ruminant animals. This ruminant-derived CH₄ is a contributor to global greenhouse gas emissions (46) and also represents an energy loss for the animals (34). Proposed ruminant greenhouse gas mitigation strategies include using feeds that produce less CH₄ and more volatile fatty acids (31). Alternative strategies include interventions that slow or halt methanogenesis by vaccination, using natural inhibitors found in plants, and supplementing feed with fats and oils or small-molecule inhibitors (31, 32). In the absence of methanogenesis, accumulation of H₂ could lead to a decrease in the rate of feed fermentation (31, 53) and hence a decrease in animal productivity. Other microbes that use H₂ without producing methane could be valuable in conjunction with intervention strategies that inhibit methanogens. This possibility has sparked interest in possible inoculation of ruminants with alternative H₂ users.Bacteria that use the Wood-Ljungdahl pathway to produce acetate from CO₂ are metabolically (6) and phylogenetically (48) diverse and are designated “homoacetogens.” Homoacetogens grow with H₂ or other suitable electron donors, such as formate or sugars, plus CO₂ as a terminal electron acceptor, heterotrophically with organic substrates such as sugars and methoxylated compounds, or mixotrophically with, e.g., H₂ and organic substrates. Homoacetogens have been reported to occur in a normally functioning rumen, but they are unlikely to compete with methanogens for H₂ (24, 25, 34). However, homoacetogens could play an important role in the disposal of H₂ if methanogens are not established in or are eliminated from the rumen (11, 17). At present, it is not clear whether resident rumen homoacetogens could fulfill the H₂ disposal role or whether homoacetogens would have to be added to the rumen to take over this role from the methanogens.Cultivation-based enumeration techniques have shown that the sizes of rumen acetogen populations range from undetectable to 1.2 × 10⁹ per g of rumen contents and that the prevalence of these acetogens depends on diet, animal age, and time of sampling (5, 7, 23, 24). Several homoacetogens, including Acetitomaculum ruminis (15), Eubacterium limosum (14, 17), Blautia schinkii, and Blautia producta (11), have been isolated from ruminants. Homoacetogens have also been isolated from the kangaroo forestomach, whose function is analogous to that of the rumen, which suggests that homoacetogenesis may play a role in hydrogen removal in the low-methane-emission forestomach (37).Because homoacetogens occur in different lineages of bacteria (48), traditional 16S rRNA gene-based surveys provide little information on their prevalence. The formyltetrahydrofolate synthetase (FTHFS) gene (fhs) has been used as a functional marker for homoacetogens, as the enzyme that it encodes catalyzes a key step in the reductive acetogenesis pathway (26). The structure of the enzyme of the homoacetogen Moorella thermoacetica has been reported, and putative functional features have been identified (27, 41, 42). FTHFS sequences from true homoacetogens differ from their homologs in sulfate-reducing bacteria and in other bacteria that degrade purines and amino acids via the glycine synthase-glycine reductase pathway (12, 21, 22, 26). At present, only a limited number of FTHFS sequences have been deposited in databases, and the vast majority of them are partial sequences retrieved from complex microbial communities. FTHFS sequences have been surveyed in sludge (39, 43, 54), termites (40, 44), salt marsh plant roots (21), horse manure (22), cow manure, freshwater sediment, rice field soil, and sewage (54), but so far only one study has investigated bovine ruminal FTHFS sequences (30). The rumen FTHFS sequences had low levels of similarity to the FTHFS sequences of known homoacetogens and could be sequences of novel homoacetogens. To our knowledge, no bacteria with these unique FTHFS sequences have been identified.The aims of this study were to assess the diversity of FTHFS gene sequences retrieved from rumen samples and to screen novel rumen isolates for the presence of FTHFS genes and test their ability to grow as homoacetogens. We used alignments of FTHFS sequences to define a homoacetogen similarity score based on the presence of diagnostic amino acids and developed a hidden Markov model to assess the likelihood that FTHFS sequences of unknown origin are sequences from true homoacetogens that are able to use H₂ or alternative electron donors for reductive acetogenesis.     

GTP-dependent structural rearrangement of the eRF1:eRF3 complex and eRF3 sequence motifs essential for PABP binding

Translation termination in eukaryotes is governed by the concerted action of eRF1 and eRF3 factors. eRF1 recognizes the stop codon in the A site of the ribosome and promotes nascent peptide chain release, and the GTPase eRF3 facilitates this peptide release via its interaction with eRF1. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay through its association with cytoplasmic poly(A)-binding protein (PABP) via PAM2-1 and PAM2-2 motifs in the N-terminal domain of eRF3. We have studied complex formation between full-length eRF3 and its ligands (GDP, GTP, eRF1 and PABP) using isothermal titration calorimetry, demonstrating formation of the eRF1:eRF3:PABP:GTP complex. Analysis of the temperature dependence of eRF3 interactions with G nucleotides reveals major structural rearrangements accompanying formation of the eRF1:eRF3:GTP complex. This is in contrast to eRF1:eRF3:GDP complex formation, where no such rearrangements were detected. Thus, our results agree with the established active role of GTP in promoting translation termination. Through point mutagenesis of PAM2-1 and PAM2-2 motifs in eRF3, we demonstrate that PAM2-2, but not PAM2-1 is indispensible for eRF3:PABP complex formation.     

Viability determination of Helicobacter pylori using propidium monoazide quantitative PCR

Background: While Helicobacter pylori exists in a bacillary form in both the natural habitat and the human host, detrimental environmental circumstances have been observed to lead to the conversion of H. pylori from the bacillary to the coccoid form. However, the viability or nonviability of coccoid forms remains to be established in H. pylori. The aim of this study was to determine whether the quantitative PCR combined with propidium monoazide could be an alternative and good technique to determine H. pylori viability in environmental samples and, to contribute to understanding of the role of the H. pylori forms. Materials and Methods: Viability, morphological distribution, and the number of live H. pylori cells were determined using a propidium monoazide‐based quantitative PCR method, at various time points. Results: Under adverse environmental conditions was observed the conversion of H. pylori from the bacillary to the coccoid form, and the decrease in amplification signal, in samples that were treated with propidium monoazide, over the time. Conclusions: Incorporation of propidium monoazide indicates that there is an increase in H. pylori cells with the damaged membrane over the study, leading to the manifestation of cellular degeneration and death. Consequently, quantitative PCR combined with propidium monoazide contributes to our understanding of the role of H. pylori cells, under adverse environmental conditions.     

Molecular characterization of ochratoxin A producing strains of the genus Penicillium

Sixty-six strains classified as P. verrucosum based on morphological criteria were characterized by molecular methods like RAPD, AFLP and ITS sequencing. Two groups could be identified by RAPD and AFLP analyses. The two RAPD as well as the two AFLP groups were completely coincidental. Strains in the two groups differed in their ability to produce ochratoxin A, with group I containing mainly high producing strains, and group II containing moderate to non-producing strains. The strains from group I originate from foods, such as cheeses and meat products, while the strains from group II originate from plants. The ribosomal ITS1-5.8S-ITS2 sequences were similar, except for two single nucleotide exchanges in several strains of each group. A chemotaxonomical analysis of some of the strains identified differences between the groups in secondary metabolite production. Strains from group I possessed the chemotype of P. nordicum and strains from group II that of P. verrucosum. The differences at the RAPD and AFLP level, which parallel the chemotypic differences, are consistent with the recent reclassification of ochratoxin A producing penicillia to be either P. verrucosum or P. nordicum. The homolgy between the ITS sequences however indicates phylogenetic relationship between the two species.     

Analysis of yeast populations during alcoholic fermentation: a six year follow-up study

Wine yeasts were isolated from fermenting Garnatxa and Xarel.lo musts fermented in a newly built and operated winery between 1995 and 2000. The species of non-Saccharomyces yeasts and the Saccharomyces cerevisiae strains were identified by ribosomal DNA and mitochondrial DNA RFLP analysis respectively. Non-Saccharomyces yeasts, particularly Hanseniaspora uvarum and Candida stellata, dominated the first stages of fermentation. However Saccharomyces cerevisiae was present at the beginning of the fermentation and was the main yeast in the musts in one vintage (1999). In all the cases, S. cerevisiae took over the process in the middle and final stages of fermentation. The analysis of the S. cerevisiae strains showed that indigenous strains competed with commercial strains inoculated in other fermentation tanks of the cellar. The continuous use of commercial yeasts reduced the diversity and importance of the indigenous S. cerevisiae strains.     

Statins inhibit the dimerization of beta-secretase via both isoprenoid- and cholesterol-mediated mechanisms

We have previously reported that protein lipidation in the form of palmitoylation and farnesylation is critical for the production of Abeta (amyloid beta-peptide), the dimerization of beta-secretase and its trafficking into cholesterol-rich microdomains. As statins influence these lipid modifications in addition to their effects on cholesterol biosynthesis, we have investigated the effects of lovastatin and SIMVA (simvastatin) at a range of concentrations chosen to distinguish different cellular effects on Abeta production and beta-secretase structure and its localization in bHEK cells [HEK-293 cells (human embryonic kidney cells) transfected with the Asp-2 gene plus a polyhistidine coding tag] cells. We have compared the changes brought about by statins with those brought about by the palmitoylation inhibitor cerulenin and the farnesyltransferase inhibitor CVFM (Cys-Val-Phe-Met). The statin-mediated reduction in Abeta production correlated with an inhibition of beta-secretase dimerization into its more active form at all concentrations of statin investigated. These effects were reversed by the administration of mevalonate, showing that these effects were mediated via 3-hydroxy-3-methylglutaryl-CoA-dependent pathways. At low (1 microM) statin concentrations, reduction in Abeta production and inhibition of beta-secretase dimerization were mediated by inhibition of isoprenoid synthesis. At high (>10 microM) concentrations of statins, inhibition of beta-secretase palmitoylation occurred, which we demonstrated to be regulated by intracellular cholesterol levels. There was also a concomitant concentration-dependent change in beta-secretase subcellular trafficking. Significantly, Abeta release from cells was markedly higher at 50 microM SIMVA than at 1 microM, whereas these concentrations resulted in similar reductions in total Abeta production, suggesting that low-dose statins may be more beneficial than high doses for the therapeutic treatment of Alzheimer's disease.     

Phytochemistry and antimycobacterial activity of Chlorophytum inornatum

In a project to investigate plant derived natural products from the Liliaceae with activity against fast-growing strains of mycobacteria, we have identified two new metabolites from Chlorophytum inornatum. The active principle, a new homoisoflavanone (1) was identified as 3-(4'-methoxybenzyl)-7,8-methylenedioxy-chroman-4-one. The metabolite assigned as 7-(1'-hydroxyethyl)-2-(2'-hydroxyethyl)-3,4-dihydrobenzopyran (2) was characterised by extensive 1- and 2D NMR spectroscopy. The antimycobacterial activity of this plant was mainly due to the homoisoflavonoid which exhibited minimum inhibitory values ranging from 16-256 microg/ml against four strains of fast-growing mycobacteria.     

Microsatellite segregation analysis and cytogenetic evidence for tetrasomic inheritance in the American yam Dioscorea trifida and a new basic chromosome number in the Dioscoreae

Despite the economic and cultural importance of the indigenous “Amerindian” yam Dioscorea trifida, very little is known about their origin, phylogeny, diversity and genetics. Consequently, conventional breeding efforts for the selection of D. trifida genotypes resistant to potyviruses which are directly involved in the regression of this species have been seriously limited. Our objective of this paper is to contribute to the clarification of the cytogenetic status, i.e., inheritance and chromosome number. Our results provide genetic evidence supporting tetrasomic behaviour of the genome of D. trifida based on chromosomal segregation pattern analysis using eight SSRs markers in three different crosses. This is the first reliable evidence of an autopolyploid species in the genus Dioscorea. The second major result in this study is the revealing of a new base chromosome number in the botanical section Macrogynodium to which D. trifida belongs. To date, our assumptions about the ploidy level of yams are based on the observations that the basic chromosome number is 10 or 9, and D. trifida was described as octoploid. The chromosome number of D. trifida accessions was also assessed using somatic chromosomic count techniques. Flow cytometry did not show significant variation of 2C DNA content among 80 accessions indicating homogeneity of the ploidy level of the cultivated D. trifida. This suggests that autotetraploidy is well established as well as the rule for the cultivated pool of D. trifida, even if the direct diploid ancestor remains to be identified. The data presented in this paper are significant and important for the effective breeding and conservation of the species and for elucidating the phylogeny and the origins of the yam and the evolution of the genus Dioscorea.