Stress integrated tests and cytological analyses reveal Brassica villosa subsp. drepanensis seed quality decrease upon long-term storage

Under stress integrated germination test (SIGT), seeds undergo osmo-saline stresses, which enable to detect differences in vigour of long-term stored seeds with high germination percentage (G%). The quality of Brassica villosa subsp. drepanensis seeds stored in a genebank (at ? 20°C for 16 years) was compared with seeds at harvest by standard germination tests (GT), SIGT and cytogenetic analysis. No differences were detected in G% and mean germination time under GT. Conversely, SIGT performed with NaCl ? 0.9 MPa osmotic potential did not influence G% at harvest but reduced that of stored seeds, SIGT at ? 1.4 MPa reduced G% of both. Cytogenetic analysis showed reduction of mitotic index, appearance of chromosomal aberrations and smaller nucleoli in stored seeds compared with harvest seeds germinated in water. SIGT at ? 0.9 MPa had no effect on mitotic index, but increased chromosome aberrations and nucleoli number. SIGT at ? 1.4 MPa inhibited G% of harvest and stored seeds, reduced mitoses in harvest and completely prevented it in stored seeds. The results indicate that GT does not faithfully reflect the quality of stored seeds, with misinterpretation of their vigour, whereas SIGT and cytogenetical parameters are sensitive, reliable and inexpensive methods for early prediction of genetic erosion in germplasm banks.     

Ploidy reduction and genome segregation in cultured carrot cell lines. I. Prophase chromosome reduction

Cytological analysis of different carrot cell lines in culture has shown various cytogenetic anomalies generating new levels of ploidy and novel chromosome numbers. Polyploidy may be considered a reservoir of variability that can be released in the form of distinct new segregants of different ploidy. Mechanisms alternative to mitosis (reductional grouping, prophase chromosome reduction) operate from a polyploid state (possibly reached by means of endopolyploidy, endomitosis, nuclear fusion, or restitution nuclei) to generate new levels of ploidy and novel chromosome numbers necessary for selection to operate in vitro. The segregational phenomena require chromosome recognition in haploid set complements and abnormal behaviour of mitoses; the resulting chromosome variability suggests that chromosomes are arranged, in the resting nuclei, in an orderly and predictable manner.The knowledge of the molecular events governing these mechanisms, and how to control them, would be of great help for future applications of plant cell culture.     

Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry: αB-CRYSTALLIN DISSOCIATES β2-MICROGLOBULIN OLIGOMERS*

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β₂-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m. From analysis of the NMR spectra collected at various R3Aβ2m to αB-crystallin molar subunit ratios, it is concluded that the structured β-sheet core and the apical loops of R3Aβ2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type β2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type β2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated β2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing β2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.     

Introduction of sheep meat breeds in extensive systems: Lamb carcass characteristics

Genotype effects on lamb carcass traits were investigated in a 4-year study aimed at assessing potential benefits from introducing meat breeds into the wool-oriented extensive sheep systems of northeastern Patagonia, Argentina. Five ram [Corriedale: CO; Border Leicester: BL; Île de France: IF; Texel: TX; and synthetic CRIII (25% Merino, 37.5% IF, 37.5% TX)] and 5 dam (CO; synthetic CRIII; BLCO: BL × CO; IFCO: IF × CO; and TXCO: TX × CO) genotypes were represented in the study. Data were collected from 436 male lambs of 9 genotypes (CO × CO, BL × CO, IF × CO, TX × CO, CRIII × CO, CRIII × BLCO, CRIII × IFCO, CRIII × TXCO, and CRIII × CRIII). Hot carcass weights and dressing yields were determined after slaughtering. Carcasses were given conformation and subcutaneous fat scores using the EUROP system [scale varying from E (best) to P (poorest) for conformation, and from 1 (lean) to 5 (overfat) for subcutaneous fat]. Linear measurements of carcass length and width were recorded and carcass compactness indices were calculated from those. Purebred CO acted as a standard for comparisons. On a constant liveweight basis, genotypes CRIII × IFCO and CRIII × CRIII presented higher (P < 0.05) carcass weight and dressing yield than CO × CO and BL × CO. Crossbred and synthetic genotypes showed higher (P < 0.05) carcass width than CO × CO. With the exception of BL × CO the remaining genotypes showed higher (P < 0.05) carcass width/length ratio than CO × CO. The probability that carcasses of crossbred and synthetic lambs presented better conformation than CO × CO was higher than 84%. Carcasses of CRIII × IFCO lambs were given the best conformation scores. The probability that BL × CO carcasses presented higher subcutaneous fat than the remaining genotypes exceeded 79%. Our results indicate significant improvements in carcass conformation arising from crossing. Sheep farmers in extensive systems could take advantage of the higher fatness of BL crossbred lambs to produce light carcasses with adequate fat cover, a crucial industry requirement. Terminal crossbreeding with Île de France, Texel, and CRIII rams could be implemented to improve carcass conformation thus matching market demand for heavy carcasses with limited fat content. Second cross schemes did not improve carcass commercial traits over the best terminal cross or the synthetic CRIII breed.     

Molecular insights into cell toxicity of a novel familial amyloidogenic variant of β2‐microglobulin

The first genetic variant of β₂‐microglobulin (b2M) associated with a familial form of systemic amyloidosis has been recently described. The mutated protein, carrying a substitution of Asp at position 76 with an Asn (D76N b2M), exhibits a strongly enhanced amyloidogenic tendency to aggregate with respect to the wild‐type protein. In this study, we characterized the D76N b2M aggregation path and performed an unprecedented analysis of the biochemical mechanisms underlying aggregate cytotoxicity. We showed that, contrarily to what expected from other amyloid studies, early aggregates of the mutant are not the most toxic species, despite their higher surface hydrophobicity. By modulating ganglioside GM1 content in cell membrane or synthetic lipid bilayers, we confirmed the pivotal role of this lipid as aggregate recruiter favouring their cytotoxicity. We finally observed that the aggregates bind to the cell membrane inducing an alteration of its elasticity (with possible functional unbalance and cytotoxicity) in GM1‐enriched domains only, thus establishing a link between aggregate‐membrane contact and cell damage.     

Structural model and ligand interactions of the Xanthomonas axonopodis pv. citri oligopeptide-binding protein

The oligopeptide-binding protein, OppA, ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides by several bacterial species. In the present study, we report a structural model and an oligopeptide docking analysis of the OppA protein expressed by Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The X. citri OppA structural model showed a conserved three-dimensional structure, irrespective of the low amino acid identities with previously defined structures of Bacillus subtilis and Salmonella typhimurium orthologs. Oligopeptide docking analysis carried out with the proposed model indicated that the X. citri OppA preferentially binds tri- and tetrapeptides. The present study represents the first structural analysis of an OppA ortholog expressed by a phytopathogen and contributes to the understanding of the physiology and nutritional strategies of X. citri.     

A partially structured species of beta 2-microglobulin is significantly populated under physiological conditions and involved in fibrillogenesis

The folding of beta(2)-microglobulin (beta(2)-m), the protein forming amyloid deposits in dialysis-related amyloidosis, involves formation of a partially folded conformation named I(2), which slowly converts into the native fold, N. Here we show that the partially folded species I(2) can be separated from N by capillary electrophoresis. Data obtained with this technique and analysis of kinetic data obtained with intrinsic fluorescence indicate that the I(2) conformation is populated to approximately 14 +/- 8% at equilibrium under conditions of pH and temperature close to physiological. In the presence of fibrils extracted from patients, the I(2) conformer has a 5-fold higher propensity to aggregate than N, as indicated by the thioflavine T test and light scattering measurements. A mechanism of aggregation of beta(2)-m in vivo involving the association of the preformed fibrils with the fraction of I(2) existing at equilibrium is proposed from these results. The possibility of isolating and quantifying a partially folded conformer of beta(2)-m involved in the amyloidogenesis process provides new opportunities to monitor hemodialytic procedures aimed at the reduction of such species from the pool of circulating beta(2)-m but also to design new pharmaceutical approaches that consider such species as a putative molecular target.     

Detection of two partially structured species in the folding process of the amyloidogenic protein beta 2-microglobulin

beta 2-Microglobulin is a small, major histocompatibility complex class I-associated protein that undergoes aggregation and accumulates as amyloid deposits in human tissues as a consequence of long-term haemodialysis. The folding process of this amyloidogenic protein has been studied in vitro by diluting the guanidine hydrochloride-denatured protein in refolding buffer at pH 7.4 and monitoring the folding process by means of a number of spectroscopic probes that allow the native structure of the protein to be detected as it develops. These techniques include fluorescence spectroscopy, far and near-UV circular dichroism, 8-anilino-1-naphthalenesulfonic acid binding and double jump assays. All spectroscopic probes indicate that a significant amount of structure forms within the dead-time of stopped-flow measurements (<5 ms). The folding reaction goes to completion through a fast phase followed by a slow phase, whose rate constants are ca 5.1 and 0.0030 s(-1) in water, respectively. Unfolding-folding double jump experiments, together with the use of peptidyl prolyl isomerase, reveal that the slow phase of folding of beta 2-microglobulin is not fundamentally determined by cis/trans isomerisation of X-Pro peptide bonds. Other folding-unfolding double jump experiments also suggest that the fast and slow phases of folding are not related to independent folding of different populations of protein molecules. Rather, we provide evidence for a sequential mechanism of folding where denatured beta 2-microglobulin collapses to an ensemble of partially folded conformations (I(1)) which fold subsequently to a more highly structured species (I(2)) and, finally, attain the native state. The partially folded species I(2) appears to be closely similar to previously studied amyloidogenic forms of beta 2-microglobulin, such as those adopted by the protein at mildly acid pH values and by a variant with six residues deleted at the N terminus. Since amyloid formation in vivo originates from partial denaturation of beta 2-microglobulin under conditions favouring the folding process, the long-lived, partially structured species detected here might be significantly populated under some physiological conditions and hence might play an important role in the process of amyloid formation.     

Root proliferation in perennial grasses of low and high palatability

Root proliferation of desirable (Stipa clarazii andS. tenuis) and undesirable (S.ambigua)perennial grasses was studied in semiarid rangelands of Central Argentina(40°39S, 62°54W) in 1998. On 17 September, soil coreswereremoved from the edge of the plant, metal structures lined with screen mesh(hereafter called bags) were buried in the holes, and root-free soil was placedinto these structures. Numbers of green tillers and circumference per plant hadpreviously been determined. Since plants were of unequal size among species,root length and root dry weight data are reported on a per green tiller basis.Half of the plants was defoliated to 5 cm stubble height on 17September and/or 12 October, while the other half remained undefoliated(controls). Bags were destructively harvested either 20 days after the firstdefoliation (first sampling) or 56 days after the second defoliation (secondsampling) by digging out soil very carefully around each bag. Roots were washedfrom soil, root length estimated by the line intercept method, root dry weightdetermined after oven-drying, and root length per unit root dry weightcalculated from the two measured variables. Root length and dry weight weremorethan 96% greater on defoliated and undefoliated plants ofS. clarazii than on those of S.tenuisor S. ambigua for both sampling dates. Root length perunitroot dry weight, however, was more than 43% greater (p < 0.05) inS. tenuis than in S. clarazii andS. ambigua during the second sampling. Defoliated plantshada similar root length and root dry weight than undefoliated plants in all threespecies, although plants of S. tenuis defoliated twiceshowed a greater (p < 0.05) root length than undefoliated controls. Rootlength and root dry weight were similar between sampling periods, except onundefoliated plants of S. tenuis which had a greater (p<0.05) root length and root dry weight at the first than at the second sampling.Although root length per unit root dry weight may be greater inS. tenuis than in S. clarazii andS. ambigua, greater root length and dry weight increasesinS. clarazii after defoliation appear determinant incontributing to explain its greater competitive ability and defoliationtolerance when compared with the other two species.Nomenclature of taxa followed.