Vicariance patterns in the Mediterranean Sea: east–west cleavage and low dispersal in the endemic seagrass Posidonia oceanica

Aim The seagrass, Posidonia oceanica is a clonal angiosperm endemic to the Mediterranean Sea. Previous studies have suggested that clonal growth is far greater than sexual recruitment and thus leads to low clonal diversity within meadows. However, recently developed microsatellite markers indicate that there are many different genotypes, and therefore many distinct clones present. The low resolution of markers used in the past limited our ability to estimate clonality and assess the individual level. New high‐resolution dinucleotide microsatellites now allow genetically distinct individuals to be identified, enabling more reliable estimation of population genetic parameters across the Mediterranean Basin. We investigated the biogeography and dispersal of P. oceanica at various spatial scales in order to assess the influence of different evolutionary factors shaping the distribution of genetic diversity in this species. Location The Mediterranean. Methods We used seven hypervariable microsatellite markers, in addition to the five previously existing markers, to describe the spatial distribution of genetic variability in 34 meadows spread throughout the Mediterranean, on the basis of an average of 35.6 (± 6.3) ramets sampled. Results At the scale of the Mediterranean Sea as a whole, a strong east–west cleavage was detected (amova) . These results are in line with those obtained using previous markers. The new results showed the presence of a putative secondary contact zone at the Siculo‐Tunisian Strait, which exhibited high allelic richness and shared alleles absent from the eastern and western basins. F statistics (pairwise θ ranges between 0.09 and 0.71) revealed high genetic structure between meadows, both at a small scale (about 2 to 200 km) and at a medium scale within the eastern and western basins, independent of geographical distance. At the intrameadow scale, significant spatial autocorrelation in six out of 15 locations revealed that dispersal can be restricted to the scale of a few metres. Main conclusions A stochastic pattern of effective migration due to low population size, turnover and seed survival is the most likely explanation for this pattern of highly restricted gene flow, despite the importance of an a priori seed dispersal potential. The east–west cleavage probably represents the outline of vicariance caused by the last Pleistocene ice age and maintained to this day by low gene flow. These results emphasize the diversity of evolutionary processes shaping the genetic structure at different spatial scales.     

Fruiting at High Temperature and Its Genetic Control in the Basidiomycete Flammulina velutipes

Of 10 geographic strains of Flammulina velutipes, 4 were found capable of fruiting at 22°C (Fr_H) rather than at the typical 15°C (Fr_L). Crosses made between Fr_H and Fr_L monokaryons were never observed to fruit at 22°C. However, some hybrids did fruit at the intermediate temperature of 18°C when grown on appropriate substrates, indicating incomplete dominance of the low-temperature requirement. Analysis of progeny of five Fr_H × Fr_L crosses indicated that a minimum of two genes appears to control the requirement for fruiting at ≤15°C. The genes are not closely linked to either incompatibility locus.     

Initial stages in the course of adventitious bud formation on embryos of Picea abies

Adventitious buds on embryos of Picea abies (L.) Karst. developed after a pulse treatment with 250 μ M benzyladenine (BA) of pH 5.5 for 2 h. Light and temperature regimes were not critical during the initial stages. Adventitious buds developed faster after a pulse treatment and the variation among different experiments was lower compared to when the embryos were cultured on media supplemented with BA. Various stages of the differentiation of adventitious buds were identified: stage 1 - appearance of meristematic centres (approximately the first two weeks); stage 2 - development of adventitious bud primordia (approximately the third week); stage 3 - adventitious bud development (from approximately the 4th to the 8th week). This system may be used for further studies on bud differentiation.     

Transient enhancement of serum somatomedin levels prior to weaning of growth-impaired vasopressin-deficient Brattleboro rats

Somatomedin serum levels of congenitally vasopressin-deficient Brattleboro rats were determined postnatally between day 1 and 55, and compared with heterozygous control values. Assays were performed with a radioimmunoassay of insulin-like growth factor 1 (IGF-1). A transient enhancement of immunoreactive IGF-1 levels between day 8 and 21 of age and a reduction in adulthood was found. This observation shows that the early growth impairments of the Brattleboro mutant are not due to a deficiency of IGF-1.     

Antibodies to spiroperidol and their anti-idiotypes as probes for studying dopamine receptors

Spiroperidol was covalently conjugated to bovine serum albumin (BSA). Conjugated spiroperidol was almost as efficient as free spiroperidol in its binding capacity to dopamine receptor. Antibodies to spiroperidol were produced in rabbits following repeated immunizations with the conjugate of spiroperidol and BSA. The obtained antibodies have an apparent K_D of 0.02 nM for [³H]-spiroperidol. These antibodies bind also to other butyrophenones with IC₅₀ values three to four orders of magnitude higher than the IC₅₀ obtained with unlabeled spiroperidol. Antibodies were purified from anti-spiroperidol sera by affinity chromatography. Anti-idiotypic antibodies were raised in rabbits by immunization with the purified anti-spiroperidol antibodies. Some rabbits produced anti-idiotypic antibodies which bind to rat and calf striatum.     

The effect of osmotic `shock'' on the swelling pattern and respiratory control of rat-liver mitochondria

1. Rat-liver mitochondria suspended in 0.25m-sucrose were exposed for a few seconds to strongly hypo-osmotic conditions, and then the osmolarity of the medium was raised again to 0.25 with the aid of tris chloride (osmotic ;shock'). 2. Mitochondria after hypo-osmotic pretreatment lost their capacity for slow energy-dependent swelling in iso-osmotic tris buffer and showed no respiratory control. 3. Swelling could be induced in the ;shocked' mitochondria by ATP but not by addition of respiratory substrates. 4. It was shown that cytochrome c is lost from ;shocked' mitochondria when they come into contact with the tris buffer present in the assay medium, and that the changes observed in the pattern of swelling, as well as in respiratory control, are directly connected with this loss of cytochrome c. 5. The results of the investigation are discussed with regard to the role of cytochrome c in swelling and respiratory control.     

Differential Effect of Polyamines on T4 Morphogenesis

The 5-fluorouracil (5 FU) technique for the phenotypic reversion of amber mutants was used to demonstrate that under certain circumstances, in the presence of putrescine or spermidine, early mutants have an enhanced response to 5 FU, whereas late mutants have a delayed response. Bacteria infected by T4D wild-type bacteriophage did not produce phage in the presence of high putrescine concentrations. Pulse treatments with putrescine showed that the production of lysozyme depends on a putrescine-sensitive process that begins immediately after infection at 26 C and ends at 36 min or even later. The addition of putrescine at any time during the critical period between 0 and 36 min led to a corresponding delay in lysozyme synthesis after the inhibitor was removed. Intracellular phage maturation was delayed by the addition of 100 mumoles of putrescine per ml. Early enzymes were not affected by the diamine, but the level of phage deoxyribonucleic acid was considerably decreased by the inhibitor. The putrescine-sensitive process that affects the timing of maturation is suggested to be the natural process controlling the T4 "clock."     

Relation between optical configuration and immunogenicity of synthetic polypeptides

1. Three random linear copolymers composed of two or three of the amino acids d-tyrosine, d-glutamic acid, d-alanine and d-lysine, and a branched multichain copolymer with a poly-d-lysine backbone and polymeric side chains of d-tyrosine and d-glutamic acid, were found to be non-antigenic in rabbits, by precipitin and passive cutaneous anaphylaxis, and in guinea pigs, by delayed hypersensitivity tests. The corresponding four copolymers of l-amino acids were shown to be antigenic by all the three criteria. 2. No immunological cross-reactions were observed between the polypeptides composed of d-amino acids and the corresponding l-amino acid copolymers. 3. Similarly, an azobenzenearsonic acid conjugate of poly-d-tyrosine was shown to be non-antigenic in guinea pigs, in contrast with an analogous conjugate of poly-l-tyrosine. Animals sensitized with the conjugate of poly-l-tyrosine did not exhibit delayed skin reactions, when cross-tested with the d-conjugate. 4. A linear polymer composed of d-tyrosine, l-glutamic acid and l-alanine was found to be immunogenic and to cross-react with the corresponding polymer composed exclusively of d-amino acids.     

Phytochrome-mediated effects on extracellular peroxidase activity, lignin content and bending resistance in etiolated Vicia faba epicotyls

Etiolated Vicia faba seedlings were exposed to continuous red light to investigate whether changes in extracellular peroxidase activity were correlated in time and localization with changes in extension growth and/or lignin content in the subapical region of the epicotyl. Continuous red light: (a) increased extracellular peroxidase activity after a lag of ca 0.5 h, followed by a maximum peak after 2.5 h due to slightly acidic isoforms (pI = 6–6.5, according to isoelectrofocusing gels), a minimum after 4 h and a second maximum after 8 h due to acidic isoforms (pI=4–5), (b) increased lignin content and epicotyl resistance to bending after a lag of ca 4 h, i.e. simultaneously with changes in acidic extracellular peroxidase activity, and (c) reduced extension growth to a stable rate after a lag of ca 1 h, not coinciding with the kinetics of any of the extracellular peroxidase isoforms. These effects of continuous red light were at least partially mediated by phytochrome. Tissue printing and anatomical studies revealed red light effects on extracellular peroxidase activity and lignin content mainly in the outer cortical parenchyma. The results are consistent with the involvement of phyto-chrome-mediated effects on extracellular peroxidases (acidic isoforms) in the transduction chain leading to lignin responses to red light.     

The making of the minibody: An engineered β-protein for the display of conformationally constrained peptides

Conformationally constraining selectable peptides onto a suitable scaffold that enables their conformation to be predicted or readily determined by experimental techniques would considerably boost drug discovery process by reducing the gap between the discovery of a peptide lead and the design of a peptidomimetic with a more desirable pharmacological profile. With this in mind, we designed the minibody, a 61-residue β-protein aimed at retaining some desirable features of immunogloblin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of main chain conformation of the same regions, based on the ‘canonical structures’ model. To test the ability of the minibody scaffold to support functional sites we also designed a metal binding version of the protein by suitably choosing the sequences of its loops. The minibody was produced both by chemical syntyhesis and expression in E. coli and charactgerized by size exclusion chromatography, UV CD (circular dichroism) spectroscopy and metal binding activity. All our data supported the model, but a more detailed structural characterization of the molecule was impaired by its low soubility. We were able to overcome this problem both by further; mutagenesis of the framework and by addition of a solublizing motif. The minibody is being used to select constrained human IL-6 peptidic ligands from a library displayed on the surface of the f1 bacteriophage.