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     

Role of NAD+ and ADP-Ribosylation in the Maintenance of the Golgi Structure

We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114–1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065–7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD⁺ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD⁺-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD⁺ triggered Golgi disassembly by BFA. This effect of NAD⁺ was mimicked by the use of pre–ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200–14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre–ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50–enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.     

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.     

Silencing of Mammalian Sar1 Isoforms Reveals COPII‐Independent Protein Sorting and Transport

The Sar1 GTPase coordinates the assembly of coat protein complex‐II (COPII) at specific sites of the endoplasmic reticulum (ER). COPII is required for ER‐to‐Golgi transport, as it provides a structural and functional framework to ship out protein cargoes produced in the ER. To investigate the requirement of COPII‐mediated transport in mammalian cells, we used small interfering RNA (siRNA)‐mediated depletion of Sar1A and Sar1B. We report that depletion of these two mammalian forms of Sar1 disrupts COPII assembly and the cells fail to organize transitional elements that coordinate classical ER‐to‐Golgi protein transfer. Under these conditions, minimal Golgi stacks are seen in proximity to juxtanuclear ER membranes that contain elements of the intermediate compartment, and from which these stacks coordinate biosynthetic transport of protein cargo, such as the vesicular stomatitis virus G protein and albumin. Here, transport of procollagen‐I is inhibited. These data provide proof‐of‐principle for the contribution of alternative mechanisms that support biosynthetic trafficking in mammalian cells, providing evidence of a functional boundary associated with a bypass of COPII .     

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.     

Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments

The organization of secretory traffic remains unclear, mainly because of the complex structure and dynamics of the secretory pathway. We have thus studied a simplified system, a single synchronized traffic wave crossing an individual Golgi stack, using electron tomography. Endoplasmic-reticulum-to-Golgi carriers join the stack by fusing with cis cisternae and induce the formation of intercisternal tubules, through which they redistribute their contents throughout the stack. These tubules seem to be pervious to Golgi enzymes, whereas Golgi vesicles are depleted of both enzymes and cargo. Cargo then traverses the stack without leaving the cisternal lumen. When cargo exits the stack, intercisternal connections disappear. These findings provide a new view of secretory traffic that includes dynamic intercompartment continuities as key players.     

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.