Challenging level of rigid-body approach involving numerical elements (CHLORAINE) applied to repeated elastin peptides.

Elastic proteins and derived biomaterials contain numerous tandemly repeated peptides along their sequences, ranging from a few copies to hundreds. These repetitions are responsible for their biochemical, biological and biomechanical properties. These sequences are considered to be intrinsically disordered, and the variations in their behavior are actually mainly due to their high flexibility and lack of stable secondary structures originating from their unique amino acid sequences. Consequently, the simulation of elastic proteins and large elastomeric biomaterials using classical molecular dynamics is an important challenge. Here, we propose a novel approach that allows the application of the DURABIN protocol to repeated elastin-like peptides (r-ELPs) in a simple way. Four large r-ELPs were studied to evaluate our method, which was developed for simulating extracellular matrix proteins at the mesoscopic scale. After structure clustering applied on molecular dynamic trajectories of constitutive peptides (5-mers and 6-mers), the main conformations were used as starting points to define the corresponding primitives, further used as rigid body fragments in our program. Contributions derived from electrostatic and molecular hydrophobicity potentials were tested to evaluate their influence on the interactions during simple mesoscopic simulations. The CHLORAINE approach, despite the thinner granularity due to the size of the patterns used, was included in the DURABIN protocol and emerges as a promising way to simulate elastic macromolecular systems.

Molecular and biochemical characterization of the sunflower (Helianthus annuus L.) cytosolic and plastidial enolases in relation to seed development.

In the present study, we describe the molecular and biochemical characterization of sunflower (Helianthus annuus L.) enolase (ENO, EC 4.2.1.11) proteins, which catalyze the formation of phosphoenolpyruvate, the penultimate intermediate in the glycolytic pathway. We cloned and characterized three cDNAs encoding different ENO isoforms from developing sunflower seeds. Studies using fluorescently tagged ENOs confirmed the predicted subcellular localization of ENO isoforms: HaENO1 in the plastid while HaENO2 and HaENO3 were found in the cytosol. The cDNAs were used to express the corresponding 6(His)-tagged proteins in Escherichia coli. The proteins were purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Recombinant HaENO1 and HaENO2, but not HaENO3 were shown to have enolase activity, in agreement with data obtained with the Arabidopsis homolog proteins. Site directed mutagenesis of several critical amino acids was used to attempt to recover enolase activity in recombinant HaENO3, resulting in very small increases that were not additive. A kinetic characterization of the two active isoforms showed that pH had similar effect on their velocity, that they had similar affinity for 2-phosphoglycerate, but that the kcat/Km of the plastidial enzyme was higher than that of the cytosolic isoform. Even though HaENO2 was always the most highly expressed transcript, the levels of expression of the three ENO genes were remarkably distinct in all the vegetative and reproductive tissues studied. This indicates that in seeds the conversion of 2-phosphoglycerate to phosphoenolpyruvate takes place through the cytosolic and the plastidial pathways therefore both routes could contribute to the supply of carbon for lipid synthesis. The identity of the main source of carbon during the period of stored products synthesis is discussed.

Molecular and epigenetic regulations and functions of the LAFL transcriptional regulators that control seed development.

The LAFL (i.e. LEC1, ABI3, FUS3, and LEC2) master transcriptional regulators interact to form different complexes that induce embryo development and maturation, and inhibit seed germination and vegetative growth in Arabidopsis. Orthologous genes involved in similar regulatory processes have been described in various angiosperms including important crop species. Consistent with a prominent role of the LAFL regulators in triggering and maintaining embryonic cell fate, their expression appears finely tuned in different tissues during seed development and tightly repressed in vegetative tissues by a surprisingly high number of genetic and epigenetic factors. Partial functional redundancies and intricate feedback regulations of the LAFL have hampered the elucidation of the underpinning molecular mechanisms. Nevertheless, genetic, genomic, cellular, molecular, and biochemical analyses implemented during the last years have greatly improved our knowledge of the LALF network. Here we summarize and discuss recent progress, together with current issues required to gain a comprehensive insight into the network, including the emerging function of LEC1 and possibly LEC2 as pioneer transcription factors.

Plasmin releases the anti-tumor peptide from the NC1 domain of collagen XIX.

During tumor invasion, tumor cells degrade the extracellular matrix. Basement membrane degradation is responsible for the production of peptides with anti-tumor properties. Type XIX collagen is associated with basement membranes in vascular, neuronal, mesenchymal and epithelial tissues. Previously, we demonstrated that the non-collagenous NC1, C-terminal, domain of collagen XIX [NC1(XIX)] inhibits the migration capacities of tumor cells and exerts a strong inhibition of tumor growth. Here, we demonstrate that plasmin, one of the most important enzyme involved in tumor invasion, was able to release a fragment of NC1(XIX), which retained the anti-tumor activity. Molecular modeling studies showed that NC1(XIX) and the anti-tumor fragment released by plasmin (F4) adopted locally the same type I ß-turn conformation. This suggests that the anti-tumor effect is conformation-dependent. This study demonstrates that collagen XIX is a novel proteolytic substrate for plasmin. Such release may constitute a defense of the organism against tumor invasion.

Short communication: The water footprint of dairy products: case study involving skim milk powder.

In the context of global water scarcity and food security concerns, water footprints are emerging as an important sustainability indicator in the agriculture and food sectors. Using a recently developed life cycle assessment-based methodology that takes into account local water stress where operations occur, the normalized water footprints of milk products from South Gippsland, one of Australia's major dairy regions, were 14.4 L/kg of total milk solids in whole milk (at farm gate) and 15.8 L/kg of total milk solids in skim milk powder (delivered to export destination). These results demonstrate that dairy products can be produced with minimal potential to contribute to freshwater scarcity. However, not all dairy production systems are alike and the variability in water footprints between systems and products should be explored to obtain strategic insights that will enable the dairy sector to minimize its burden on freshwater systems from consumptive water use.

The role of trehalose-6-phosphate synthase in Arabidopsis embryo development.

We previously showed that trehalose-6-phosphate synthase 1 (TPS1), which catalyses the first step in trehalose synthesis, is essential for embryo maturation in Arabidopsis. The tps1 mutant embryos develop more slowly than wild type. Patterning in the tps1 embryos appears normal but they do not progress past the torpedo stage to cotyledon stage, which is when storage reserves start to accumulate in the expanding cotyledons. Our initial data led to the hypothesis that trehalose metabolism plays a key role in regulating storage reserve accumulation by allowing the embryo to respond to the dramatic increase in sucrose levels that occurs at the torpedo stage of embryo development. More recent data demonstrate that while the tps1 mutant is blocked in the developmental progression of embryos from torpedo to cotyledon stage the expression of genes involved in the accumulation of storage reserves proceeds in a similar fashion to wild type. Thus it appears that induction of metabolic processes required for accumulation of storage reserves in tps1 occurs independently of the developmental stage and instead follows a temporal programme similar to wild-type seeds in the same silique.

Application of constant current, low voltage electrical stimulation systems to pig carcasses and its effects on pork quality.

This study examined the effectiveness of a constant current, low voltage electrical stimulation system on improving pork quality when applied to pigs at 2 min post-exsanguination. A total of 48 female Duroc×Large White/Landrace pigs of 85-90 kg liveweight were randomly allocated immediately prior to slaughter to one of four constant current electrical stimulation treatments: control (no electrical stimulation), 50, 200 and 400 mA. Stimulation was applied to pig carcasses at 2 min post-exsanguination for 30 s. No differences (P>0.05) in WB shear force values, muscle lightness or PSE incidence of pork M. longissimus lumborum (LL) was found due to electrical stimulation treatment. Muscle pH of the LL muscle was lower (P<0.001) in carcasses in the 200 and 400 mA treatments compared to those from carcasses in both the 50 mA and control treatment groups, when measured at the various time points from 40 min to 8 h post-slaughter. Although carcasses stimulated with 200 and 400 mA had higher percentage drip loss (P<0.05) and purge (P<0.001), this was not found to impact WB shear force values, muscle lightness or PSE incidence.

Effect of low voltage electrical stimulation of pig carcasses and ageing on sensory attributes of fresh pork.

The effect of electrically stimulating pig carcasses and ageing on sensory attributes of pork was evaluated in this study. A total of 48 female pigs [Duroc×Large White/Landrace (A; n=24) and Large White/Landrace (B; n=24)] were randomly allocated immediately prior to slaughter to one of two low voltage stimulation treatments; no stimulation or 150 mA applied for 30 s at 2 min post-exsanguination. Each side of the carcass was then randomly allocated to an ageing treatment of either 2 or 7 days post-slaughter. Muscle pH of the M. longissimus lumborum was lower (P<0.001) in electrically stimulated carcasses when measured from 40 min to 8 h post-slaughter compared with non-stimulated carcasses. Percentage drip loss, muscle lightness and PSE incidence were not influenced (P>0.05) by electrical stimulation. Electrical stimulation of pig carcasses and ageing pork for 7 days post-slaughter both improved (P<0.001) consumer scores for tenderness, juiciness, overall liking and quality category, however the interaction term of electrical stimulation and ageing was not significant for any of the sensory attributes. Pork from non-stimulated carcasses that was aged for 2 day post-slaughter was less tender (P<0.01) compared with pork in all other treatments. These results indicate that electrical stimulation (150 mA applied for 30 sec at 2 min post-exsanguination) was effective in improving eating quality attributes of pork, particularly when pork was aged for only 2 days post-slaughter, without detrimentally affecting colour or drip loss.

Delayed infection after immunization with a peptide from the transmembrane glycoprotein of the feline immunodeficiency virus.

Recent advances in the quantitative assessment of viral burden, by permitting the extension of criteria applied to assess the efficacy of vaccines from all-or-none protection to diminution of the viral burden, may allow the identification of original immunogens of value in combined vaccines. Peptides corresponding to three domains of the envelope glycoproteins of feline immunodeficiency virus that are recognized during natural infection were used to immunize cats. After challenge with a primary isolate of feline immunodeficiency virus, the development of acute infection was monitored by quantitative assessment of the viral burden in plasma and tissues by competitive reverse transcription-PCR, by measurement of the humoral response developed to viral components, and by lymphocyte subset analysis. Whereas immunization with two peptides derived from the surface glycoprotein had no effect on the early course of infection, immunization with a peptide derived from the transmembrane glycoprotein delayed infection, as reflected by a diminished viral burden in the early phase of primary infection and delayed seroconversion. This peptide, located in the membrane-proximal region of the extracellular domain, has homology to an epitope of human immunodeficiency virus type 1 recognized by a broadly neutralizing monoclonal antibody. These results suggest that lentivirus transmembrane glycoproteins share a determinant in the juxtamembrane ectodomain which could be of importance in the design of vaccines against AIDS.

Enhancement of feline immunodeficiency virus (FIV) infection after DNA vaccination with the FIV envelope.

Despite intensive experimentation to develop effective and safe vaccines against the human immunodeficiency viruses and other pathogenic lentiviruses, it remains unclear whether an immune response that does not afford protection may, on the contrary, produce adverse effects. In the present study, the effect of genetic immunization with the env gene was examined in a natural animal model of lentivirus pathogenesis, infection of cats by the feline immunodeficiency virus (FIV). Three groups of seven cats were immunized by intramuscular transfer of plasmid DNAs expressing either the wild-type envelope or two envelopes bearing mutations in the principal immunodominant domain of the transmembrane glycoprotein. Upon homologous challenge, determination of plasma virus load showed that the acute phase of viral infection occurred earlier in the three groups of cats immunized with FIV envelopes than in the control cats. Genetic immunization, however, elicited low or undetectable levels of antibodies directed against envelope glycoproteins. These results suggest that immunization with the FIV env gene may result in enhancement of infection and that mechanisms unrelated to enhancing antibodies underlay the observed acceleration.

Genotypic variation of quantitative trait loci controlling in vitro androgenesis in maize.

A quantitative trait loci (QTL) analysis for androgenetic capability has been conducted on three different crosses in maize, including very high and nonresponding lines for androgenesis. The doubled haploid lines derived by anther culture from the crosses DH5 x DH7, A188 x DH7, and R6 x DH99 showed a range of 0-70%, 0-40%, and 0-50% androgenetic responding anthers, respectively. The genotypic heritability of means for this trait is close to 0.90 for A188 x DH7 and 0.78 for R6 x DH99. The QTL analysis involved in each population the mapping of more than 100 loci covering a large part of the genome with reasonably spaced markers averaging 12 cM. Different measurements describing the androgenetic process were studied: AC, percentage of responding anthers; ELS, number of androgenetic embryos produced per 100 plated anthers; PLE, number of plantlets regenerated per 100 embryos; PLA, number of plantlets per 100 plated anthers. In each cross, three to four QTLs were found for AC, explaining 30-40% of the phenotypic variation. The QTL detected for PLA was also strong QTL for AC or ELS. This agrees with the observation that these last two traits are good predictors for final plantlet yield. The QTLs found were specific, although the same line DH7 was used in two crosses and DH99 derived from DH5 and DH7 in the third cross. These results suggest that the transfer of the androgenetic capabilities in elite germplasm will still involve a phenotypic evaluation of the androgenetic performances. A backcross-assisted selection based only on the genotype at the QTL is probably possible but only within the crosses used for this QTL analysis.

Molecular and morphological evaluation of doubled-haploid lines in maize. 2. Comparison with single-seed-descent lines.

Doubled-haploid (DH) and single-seed-descent (SSD) lines in maize have been compared for quantitatively inherited traits and for RFLP markers. The comparisons of the distributions for agromorphological traits do not allow definite conclusions to be drawn on the similarity of the two reproductive systems. We have used more than 100 RFLP markers to provide a precise description of the parental allele frequency and the recombination fractions. A comparison of two DH populations shows that non-random meiotic reassortment is influenced by differences in the anther culture capacities of the two parental lines. For the DH lines derived from the cross DH5 x DH7, involving two responsive lines in anther culture, the distortion in segregation (P < 0.05) affected less than 20% of the genome with half of the deviations towards each parent. DH lines derived from the cross A188 x DH7, where A188 is a non-responsive line, showed more than twice this level of distortion and an excess of DH7 alleles was found for almost all of the skewed loci. The recombination fractions were homogeneous between the two DH populations for most of the genome. The genome sizes calculated with the DH and the SSD lines derived from the same cross, A188 x DH7, were also similar, which suggests that no selection against recombinant gametes occurs during anther culture. The observed recombination fraction after five meioses (SSD) is on average twice as large as after one meiosis (DH). No difference is observed for recombination fractions greater than 20%. Despite a precise description of the material at the molecular level, it has not been possible to make a definite conclusion as to whether or not the differences in some morphological characters are the consequences of differences in the segregation ratio and/or the recombination frequency. However, the agromorphological evaluation shows a narrow range in differences between the two types of lines and suggests that the use of DH lines is possible in breeding programmes.

[Monitoring cervical dilatation by impedance]. / Partogramme enregistré par impédancemétrie.

Several different physics procedures have been tried to mechanize the recording of partograms. Can a measure of impedance of tissue Z using potential difference V, according to Ohm's law V = Z1, and 1 is a constant, be correlated with a measure of cervical dilatation using vaginal examination? This was our hypothesis. The tissue impedance meter was made to our design and applied according to a bipolar procedure. Our work was carried out on 28 patients. 10 patients were registered before labour started in order to test the apparatus and to record the impedance variations without labour taking place, and 18 patients were registered in labour to see whether there was any correlation. The level of impedance in the cervix without labour was 302.7 Ohms with a deviation of 8.2. Using student's t tests it was found that there was a significant correlation (p less than 0.001) in four measurements between the impedance measure and measures obtained by extrapolating the degrees of dilatation calculated from vaginal examination. This is a preliminary study in which we have defined the conditions that are necessary to confirm these first results and to further develop the method.