A novel 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis: characterization,purification and kinetic properties

The enzyme, desacetoxyvindoline 4-hydroxylase, was purified to apparent homogeneity from Catharanthus roseus by ammonium sulfate precipitation and successive chromatography on Sephadex G-100, green 19-agarose, hydroxylapatite, -kg sepharose and Mono Q. The 4-hydroxylase was characterized by its strict specificity for position 4 of desacetoxyvindoline suggesting it to catalyze the second to last step in vindoline biosynthesis. The molecular mass of the native and denatured 4-hydroxylase was 45 kDa and 44.7 kDa, respectively, suggesting that the native enzyme is a monomer. Two-dimensional isoelectric focusing under denaturing conditions resolved the purified 4-hydroxylase into three charge isoforms of pIs 4.6, 4.7 and 4.8. The purified 4-hydroxylase exhibited no requirement for divalent cations, but inactive enzyme was reactivated in a time-dependent manner by incubation with ferrous ions. The enzyme was not inhibited by EDTA or SH-group reagents at concentrations up to 10 mM. The mechanism of action of desacetoxyvindoline 4-hydroxylase was investigated. The results of substrate interaction kinetics and product inhibition studies suggest an Ordered Ter Ter mechanism where -kg is the first substrate to bind followed by the binding of O₂ and desacetoxyvindoline. Their K _m values for -kg, O₂ and desacetoxyvindoline are 45 M, 45 M and 0.03 M, respectively. The first product to be released was deacetylvindoline followed by CO₂ and succinate, respectively.Abbreviations -kg -ketoglutarate or 2-oxoglutarate - NMT N-methyltransferase - SAM S-adenosyl-l-methionine - TLC thin layer chromatography - VBL vinblastine - VCR vincristine     

Lipid conjugates of antiretroviral agents. II. Disodium palmityl phosphonoformate: anti-HIV activity, physical properties, and interaction with plasma proteins

Disodium palmityl phosphonoformate, a novel lipid phosphoester of the anti HIV agent phosphonoformate (foscarnet), inhibits HIV replication in H9 cells and syncytia formation in MOLT-3 cells as effectively as foscarnet itself, as shown by dose-response data from assays for expression of p17 and p24 viral antigens and syncytia formation. Protein binding studies indicate that in serum, the derivative exists bound to albumin and the lipoproteins, and would therefore be likely to exhibit improved serum lifetime in vivo.     

IL-15Rα deficiency leads to mitochondrial and myofiber differences in fast mouse muscles

The purpose of this study was to determine mitochondrial changes in fast muscles from interleukin-15 receptor alpha knockout (IL-15RαKO) mice. We tested the hypothesis that fast muscles from IL-15RαKO mice would have a greater mitochondrial density and altered internal structure compared to muscles from control mice. In fast muscles from IL-15RαKO mice, mitochondrial density was 48% greater with a corresponding increase in mitochondrial DNA content. Although there were no differences in the relative size of isolated mitochondria, internal complexity was lower in mitochondria from IL-15RαKO mice. These data support an increase in mitochondrial biogenesis and provide direct evidence for a greater density and altered internal structure of mitochondria in EDL muscles deficient in IL-15Rα.     

Apomixis in plant reproduction: a novel perspective on an old dilemma

Seed is one of the key factors of crop productivity. Therefore, a comprehension of the mechanisms underlying seed formation in cultivated plants is crucial for the quantitative and qualitative progress of agricultural production. In angiosperms, two pathways of reproduction through seed exist: sexual or amphimictic, and asexual or apomictic; the former is largely exploited by seed companies for breeding new varieties, whereas the latter is receiving continuously increasing attention from both scientific and industrial sectors in basic research projects. If apomixis is engineered into sexual crops in a controlled manner, its impact on agriculture will be broad and profound. In fact, apomixis will allow clonal seed production and thus enable efficient and consistent yields of high-quality seeds, fruits, and vegetables at lower costs. The development of apomixis technology is expected to have a revolutionary impact on agricultural and food production by reducing cost and breeding time, and avoiding the complications that are typical of sexual reproduction (e.g., incompatibility barriers) and vegetative propagation (e.g., viral transfer). However, the development of apomixis technology in agriculture requires a deeper knowledge of the mechanisms that regulate reproductive development in plants. This knowledge is a necessary prerequisite to understanding the genetic control of the apomictic process and its deviations from the sexual process. Our molecular understanding of apomixis will be greatly advanced when genes that are specifically or differentially expressed during embryo and embryo sac formation are discovered. In our review, we report the main findings on this subject by examining two approaches: i) analysis of the apomictic process in natural apomictic species to search for genes controlling apomixis and ii) analysis of gene mutations resembling apomixis or its components in species that normally reproduce sexually. In fact, our opinion is that a novel perspective on this old dilemma pertaining to the molecular control of apomixis can emerge from a cross-check among candidate genes in natural apomicts and a high-throughput analysis of sexual mutants.     

Potential role of oxidative exoenzymes of the extremophilic fungus Pestalotiopsis palmarum BM-04 in biotransformation of extra-heavy crude oil

Large amount of drilling waste associated with the expansion of the Orinoco Oil Belt (OOB), the biggest proven reserve of extra-heavy crude oil (EHCO) worldwide, is usually impregnated with EHCO and highly salinized water-based drilling fluids. Oxidative exoenzymes (OE) of the lignin-degrading enzyme system (LDS) of fungi catalyse the oxidation of a wide range of toxic pollutants. However, very little evidences on fungal degradation or biotransformation of EHCO have been reported, which contain high amounts of asphaltenes and its biodegradation rate is very limited. The aims of this work were to study the ability of Pestalotiopsis palmarum BM-04 to synthesize OE, its potential to biotransform EHCO and to survive in extreme environmental conditions. Enzymatic studies of the LDS showed the ability of this fungus to overproduce high amounts of laccase (LACp) in presence of wheat bran or lignin peroxidase (LIPp) with EHCO as sole carbon and energy source (1300 U mgP⁻¹ in both cases). FT-IR spectroscopy with Attenuated Total Reflectance (ATR) analysis showed the enzymatic oxidation of carbon and sulfur atoms in both maltenes and asphaltenes fractions of biotreated EHCO catalysed by cell-free laccase-enriched OE using wheat bran as inducer. UV-visible spectrophotometry analysis revealed the oxidation of the petroporphyrins in the asphaltenes fraction of biotreated EHCO. Tolerance assays showed the ability of this fungus to grow up to 50 000 p.p.m. of EHCO and 2000 mM of NaCl. These results suggest that P. palmarum BM-04 is a hopeful alternative to be used in remediation processes in extreme environmental conditions of salinity and EHCO contamination, such as the drilling waste from the OOB.     

An Overview of Ten Italian Horse Breeds through Mitochondrial DNA

BackgroundThe climatic and cultural diversity of the Italian Peninsula triggered, over time, the development of a great variety of horse breeds, whose origin and history are still unclear. To clarify this issue, analyses on phenotypic traits and genealogical data were recently coupled with molecular screening.MethodologyTo provide a comprehensive overview of the horse genetic variability in Italy, we produced and phylogenetically analyzed 407 mitochondrial DNA (mtDNA) control-region sequences from ten of the most important Italian riding horse and pony breeds: Bardigiano, Esperia, Giara, Lipizzan, Maremmano, Monterufolino, Murgese, Sarcidano, Sardinian Anglo-Arab, and Tolfetano. A collection of 36 Arabian horses was also evaluated to assess the genetic consequences of their common use for the improvement of some local breeds.ConclusionsIn Italian horses, all previously described domestic mtDNA haplogroups were detected as well as a high haplotype diversity. These findings indicate that the ancestral local mares harbored an extensive genetic diversity. Moreover, the limited haplotype sharing (11%) with the Arabian horse reveals that its impact on the autochthonous mitochondrial gene pools during the final establishment of pure breeds was marginal, if any. The only significant signs of genetic structure and differentiation were detected in the geographically most isolated contexts (i.e. Monterufolino and Sardinian breeds). Such a geographic effect was also confirmed in a wider breed setting, where the Italian pool stands in an intermediate position together with most of the other Mediterranean stocks. However, some notable exceptions and peculiar genetic proximities lend genetic support to historical theories about the origin of specific Italian breeds.     

Understanding Genetic Diversity and Population Structure of a Poa pratensis Worldwide Collection through Morphological,Nuclear and Chloroplast Diversity Analysis

Poa pratensis L. is a forage and turf grass species well adapted to a wide range of mesic to moist habitats. Due to its genome complexity little is known regarding evolution, genome composition and intraspecific phylogenetic relationships of this species. In the present study we investigated the morphological and genetic diversity of 33 P. pratensis accessions from 23 different countries using both nuclear and chloroplast molecular markers as well as flow cytometry of somatic tissues. This with the aim of shedding light on the genetic diversity and phylogenetic relationships of the collection that includes both cultivated and wild materials. Morphological characterization showed that the most relevant traits able to distinguish cultivated from wild forms were spring growth habit and leaf colour. The genome size analysis revealed high variability both within and between accessions in both wild and cultivated materials. The sequence analysis of the trnL-F chloroplast region revealed a low polymorphism level that could be the result of the complex mode of reproduction of this species. In addition, a strong reduction of chloroplast SSR variability was detected in cultivated materials, where only two alleles were conserved out of the four present in wild accessions. Contrarily, at nuclear level, high variability exist in the collection where the analysis of 11 SSR loci allowed the detection of a total of 91 different alleles. A Bayesian analysis performed on nuclear SSR data revealed that studied materials belong to two main clusters. While wild materials are equally represented in both clusters, the domesticated forms are mostly belonging to cluster P2 which is characterized by lower genetic diversity compared to the cluster P1. In the Neighbour Joining tree no clear distinction was found between accessions with the exception of those from China and Mongolia that were clearly separated from all the others.     

PTX3 interacts with inter-alpha-trypsin inhibitor: implications for hyaluronan organization and cumulus oophorus expansion

Pentraxin 3 (PTX3) and heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI) are essential for hyaluronan (HA) organization within the extracellular matrix of the cumulus oophorus, which is critical for in vivo oocyte fertilization and female fertility. In this study, we examined the possibility that these molecules interact and cooperate in this function. We show that HCs and PTX3 colocalize in the cumulus matrix and coimmunoprecipitate from cumulus matrix extracts. Coimmunoprecipitation experiments and solid-phase binding assays performed with purified human IalphaI and recombinant PTX3 demonstrate that their interaction is direct and not mediated by other matrix components. PTX3 does not bind to IalphaI subcomponent bikunin and, accordingly, bikunin does not compete for the binding of PTX3 to IalphaI, indicating that PTX3 interacts with IalphaI subcomponent HC only. Recombinant PTX3-specific N-terminal region, but not the PTX3-pentraxin C-terminal domain, showed the same ability as full-length protein to bind to HCs and to enable HA organization and matrix formation by Ptx3(-/-) cumulus cell oocyte complexes cultured in vitro. Furthermore, a monoclonal antibody raised against PTX3 N terminus, which inhibits PTX3/IalphaI interaction, also prevents recombinant full-length PTX3 from restoring a normal phenotype to in vitro-cultured Ptx3(-/-) cumuli. These results indicate that PTX3 directly interacts with HCs of IalphaI and that such interaction is essential for organizing HA in the viscoelastic matrix of cumulus oophorus, highlighting a direct functional link between the two molecules.     

Stability of <Emphasis Type="Italic">Potato virus X</Emphasis> expression vectors is related to insert size: implications for replication models and risk assessment

We investigated the stability of expression constructs based on Potato virus X (PVX) as a function of insert length. Five different inserts ranging in length from 261 to 1,758 bp (human proinsulin, murine interleukin-10, HIV-1 nef, petunia expansin-1 and human gad65) were expressed using a PVX vector in Nicotiana benthamiana plants for three sequential passages. Using a competitive RT-PCR approach we demonstrated that insert–deletion could occur in the first infection cycle for all inserts, but that this was much more likely to be the case for longer ones. This suggested a negative correlation between insert length and vector stability. Sequence analysis of the deleted constructs suggested that recombination usually occurred at sites close to the duplicated sub-genomic promoter, but in a smaller number of cases the foreign gene itself was probably involved, resulting in partially deleted constructs containing transgene fragments. The implications of these results in the context of recombinant protein expression and its risks are discussed.     

A new disease-specific machine learning approach for the prediction of cancer-causing missense variants

High-throughput genotyping and sequencing techniques are rapidly and inexpensively providing large amounts of human genetic variation data. Single Nucleotide Polymorphisms (SNPs) are an important source of human genome variability and have been implicated in several human diseases, including cancer. Amino acid mutations resulting from non-synonymous SNPs in coding regions may generate protein functional changes that affect cell proliferation. In this study, we developed a machine learning approach to predict cancer-causing missense variants. We present a Support Vector Machine (SVM) classifier trained on a set of 3163 cancer-causing variants and an equal number of neutral polymorphisms. The method achieve 93% overall accuracy, a correlation coefficient of 0.86, and area under ROC curve of 0.98. When compared with other previously developed algorithms such as SIFT and CHASM our method results in higher prediction accuracy and correlation coefficient in identifying cancer-causing variants.