Influence of the culture medium on the expression of surface polypeptides of Yersinia enterocolitica

Three polypeptides (200, 46, and 25 kDal) encoded by the virulence plasmid were detected by SDS-PAGE in the outer membrane of Yersinia enterocolitica 09 grown at 37°C in brain-heart infusion medium. Bacteria grown at the same temperature in the tissue culture medium RPMI 1640 expressed five additional polypeptides (170, 135, 118, 100, and 98 kDal), but the 25-kDal band was not seen. The protein profile in RPMI 1640 resembles the expression pattern displayed by yersiniae when grown in vivo. The immunoblot of total membrane proteins of bacteria grown in brain-heart infusion medium revealed eight plasmid-encoded polypeptides, four of which were also in the outer membrane preparations, including a 28-kDal polypeptide. These peptides do not coincide with known plasmid-encoded outer membrane proteins.     

Cloning and expression pattern of a gene encoding an alpha-xylosidase active against xyloglucan oligosaccharides from Arabidopsis

An alpha-xylosidase active against xyloglucan oligosaccharides was purified from cabbage (Brassica oleracea var. capitata) leaves. Two peptide sequences were obtained from this protein, the N-terminal and an internal one, and these were used to identify an Arabidopsis gene coding for an alpha-xylosidase that we propose to call AtXYL1. It has been mapped to a region of chromosome I between markers at 100.44 and 107.48 cM. AtXYL1 comprised three exons and encoded a peptide that was 915 amino acids long, with a potential signal peptide of 22 amino acids and eight possible N-glycosylation sites. The protein encoded by AtXYL1 showed the signature regions of family 31 glycosyl hydrolases, which comprises not only alpha-xylosidases, but also alpha-glucosidases. The alpha-xylosidase activity is present in apoplastic extractions from Arabidopsis seedlings, as suggested by the deduced signal peptide. The first eight leaves from Arabidopsis plants were harvested to analyze alpha-xylosidase activity and AtXYL1 expression levels. Both increased from older to younger leaves, where xyloglucan turnover is expected to be higher. When this gene was introduced in a suitable expression vector and used to transform Saccharomyces cerevisiae, significantly higher alpha-xylosidase activity was detected in the yeast cells. alpha-Glucosidase activity was also increased in the transformed cells, although to a lesser extent. These results show that AtXYL1 encodes for an apoplastic alpha-xylosidase active against xyloglucan oligosaccharides that probably also has activity against p-nitrophenyl-alpha-D-glucoside.     

Cell cycle flow cytometric analysis in the diagnosis and management of colorectal carcinoma

OBJECTIVE: To establish prognostic models and protocols for individualized management in colorectal carcinoma patients based on both clinical and DNA flow cytometric parameters. STUDY DESIGN: Prospective study of 88 colon carcinoma patients with a minimum follow-up of 12 months, operated on with the intent to cure and not treated with radiotherapy or chemotherapy. All the cases were subjected to a clinical evaluation: age, sex, tumor localization and size, histologic grade, tumor stage, disease-free interval, survival and flow cytometric study (ploidy, DNA index and S-phase fraction [SPF]). RESULTS: From the total of 88 neoplasms studied, 56 (63.6%) were from males and 32 (36.4%) from females; 30 (34%) were located in the right side of the colon, 7 (8%) in the transverse colon and 51 (58%) in the left side of the colon. Eleven (12.5%) were stage I, 52 (59.1%) stage II and 25 (28%) stage III. Forty-two (47.7%) were diploid and 46 (52.3%) aneuploid. The S-phase mean was 14.6% (12% for diploids and 16.9% for aneuploids). During the follow-up period, 26.1% of diploid tumors recurred, whereas aneuploid tumors recurred in 36.9% (P < .05). SPF from diploid and aneuploid tumors was analyzed separately. CONCLUSION: Regarding relapse-free interval, the behavior of diploid tumors with a high SPF was similar to that of aneuploid ones. Two kinetic profiles were established, favorable (diploid tumors with low S phase) and unfavorable (diploid with high S phase and all aneuploid tumors), that had significant prognostic value for progression and survival and that allowed identification of patients at high risk of recurrence. We formulated a prognostic index according to SPF and tumor stage that has discriminatory capacity for biologic behavior in colorectal tumors.     

Changes in alpha-xylosidase during intact and auxin-induced growth of pine hypocotyls

A complete cDNA from Pinus pinaster Aiton, potentially coding for an alpha-xylosidase able to remove the xylose residue from xyloglucan oligosaccharides, has been cloned. Its sequence was homologous to previously published alpha-xylosidase genes from Arabidopsis and nasturtium. The protein also showed the two signature regions of family 31 of glycosyl hydrolases. The gene expression level was quantified by competitive RT-PCR, under different growth conditions, throughout seedling development, in different regions along the hypocotyls and in auxin-treated hypocotyl segments, and related with growth capacity and alpha-xylosidase activity. A role of alpha-xylosidase in regulating the level of xyloglucan oligosaccharides within the apoplast is proposed. The action of an alpha-xylosidase removing the xylose residue, would make possible the action of a beta-glucosidase deblocking the xyloglucan oligosaccharide degradation and it could serve as a control point for the regulation of the apoplastic levels of xyloglucan oligosaccharides.     

Evolutionary divergence of β–expansin structure and function in grasses parallels emergence of distinctive primary cell wall traits

Expansins are wall‐loosening proteins that promote the extension of primary cell walls without the hydrolysis of major structural components. Previously, proteins from the EXPA (α–expansin) family were found to loosen eudicot cell walls but to be less effective on grass cell walls, whereas the reverse pattern was found for EXPB (β–expansin) proteins obtained from grass pollen. To understand the evolutionary and structural bases for the selectivity of EXPB action, we assessed the extension (creep) response of cell walls from diverse monocot families to EXPA and EXPB treatments. Cell walls from Cyperaceae and Juncaceae (families closely related to grasses) displayed a typical grass response (‘β–response’). Walls from more distant monocots, including some species that share with grasses high levels of arabinoxylan, responded preferentially to α–expansins (‘α–response’), behaving in this regard like eudicots. An expansin with selective activity for grass cell walls was detected in Cyperaceae pollen, coinciding with the expression of genes from the divergent EXPB–I branch that includes grass pollen β–expansins. The evolutionary origin of this branch was located within Poales on the basis of phylogenetic analyses and its association with the ‘sigma’ whole‐genome duplication. Accelerated evolution in this branch has remodeled the protein surface in contact with the substrate, potentially for binding highly substituted arabinoxylan. We propose that the evolution of the divergent EXPB–I group made a fundamental change in the target and mechanism of wall loosening in the grass lineage possible, involving a new structural role for xylans and the expansins that target them.     

Heritability of seed weight in Maritime pine,a relevant trait in the transmission of environmental maternal effects

Quantitative seed provisioning is an important life-history trait with strong effects on offspring phenotype and fitness. As for any other trait, heritability estimates are vital for understanding its evolutionary dynamics. However, being a trait in between two generations, estimating additive genetic variation of seed provisioning requires complex quantitative genetic approaches for distinguishing between true genetic and environmental maternal effects. Here, using Maritime pine as a long-lived plant model, we quantified additive genetic variation of cone and seed weight (SW) mean and SW within-individual variation. We used a powerful approach combining both half-sib analysis and parent–offspring regression using several common garden tests established in contrasting environments to separate G, E and G × E effects. Both cone weight and SW mean showed significant genetic variation but were also influenced by the maternal environment. Most of the large variation in SW mean was attributable to additive genetic effects (h²=0.55–0.74). SW showed no apparent G × E interaction, particularly when accounting for cone weight covariation, suggesting that the maternal genotypes actively control the SW mean irrespective of the amount of resources allocated to cones. Within-individual variation in SW was low (12%) relative to between-individual variation (88%), and showed no genetic variation but was largely affected by the maternal environment, with greater variation in the less favourable sites for pine growth. In summary, results were very consistent between the parental and the offspring common garden tests, and clearly indicated heritable genetic variation for SW mean but not for within-individual variation in SW.     

Genetics, phosphorus availability, and herbivore-derived induction as sources of phenotypic variation of leaf volatile terpenes in a pine species

Oleoresin produced and stored in pine tree leaves provides direct resistance to herbivores, while leaf volatile terpenes (LVT) in the resin are also powerful airborne infochemicals. Resin concentration and profile show considerable spatial and temporal phenotypic variation within and among pine populations. LVT biochemistry is known to be under genetic control, and although LVT should be plastic to diverse abiotic and biotic environmental factors such as nutrient availability and herbivore attack, little is known about their relative contributions and interactive effects. The aim of this paper was to clarify whether reduced phosphorus availability could increase the LVT concentration and affect the expression of herbivore-derived induced defences, and how plasticity would contribute to the phenotypic variation of LVT. The constitutive and methyl-jasmonate (MeJa) induced LVT concentration and profile were analysed in 17 half-sib Pinus pinaster families growing under two levels of P-availability (complete and P-limited fertilization). Individual terpene concentrations showed large additive genetic variation, which was more pronounced in the control than in MeJa-induced pines. MeJa application did not affect the LVT concentration, but significantly modified the LVT profile by depleting the α-pinene content and reducing the sesquiterpene fraction. Low P-availability strongly reduced plant growth and foliar nutrient concentrations, but did not affect LVT concentration and profile, and did not interact with MeJa-induction. Results indicate a strong homeostasis of LVT concentration to P-availability, and minor changes in the LVT profile due to MeJa-induction. Genetic variation appears to be the main source of phenotypic variation affecting the LVT concentration in this pine species.     

Methyl Jasmonate Increases the Tropane Alkaloid Scopolamine and Reduces Natural Herbivory in Brugmansia suaveolens: Is Scopolamine Responsible for Plant Resistance?

The tropane alkaloid (TA) scopolamine is suggested to protect Brugmansia suaveolens (Solanaceae) against herbivorous insects. To test this prediction in a natural environment, scopolamine was induced by methyl jasmonate (MJ) in potted plants which were left 10?days in the field. MJ-treated plants increased their scopolamine concentration in leaves and herbivory decreased. These findings suggest a cause?Ceffect relationship. However, experiments in laboratory showed that scopolamine affect differently the performance of the specialist larvae of the ithomiine butterfly Placidina euryanassa (C. Felder & R. Felder) and the generalist fall armyworm Spodoptera frugiperda (J. E. Smith): the specialist that sequester this TA from B. suaveolens leaves was not negatively affected, but the generalist was. Therefore, scopolamine probably acts only against insects that are not adapted to TAs. Other compounds that are MJ elicited may also play a role in plant resistance against herbivory by generalist and specialist insects, and deserve future investigations.