Transforming growth factor β1 induces mitogenesis in fetal rat brown adipocytes

The presence of transforming growth factor β₁ (TGF-β₁) for 24 or 48 h stimulated DNA synthesis, the percentage of cells in the S + G₂/M phases of the cell cycle, and cell number, as compared to quiescent cells. The mitogenic capacity of TGF-β₁ (1 pM) was similar to that shown by 10% fetal calf serum (FCS). TGF-β₁ for 48 h increased by 5-fold the percentage of cells containing (³H)thymidine-labeled nuclei as compared to quiescent cells. In addition, single fetal brown adipocytes, showing their typical multilocular fat droplets phenotype, become positive for (³H)thymidine-labeled nuclei in response to TGF-β₁. Moreover, TGF-β₁ induced the mRNA expression of a complete set of proliferation-related genes, such as c-fos (30 min), c-myc and β-actin (2 h), and H-ras, cdc2 kinase, and glucose 6-phosphate dehydrogenase (G6PD) at 4 and 8 h, as compared to quiescent cells. Concurrently, TGF-β₁ for 12 h increased the protein content of proliferating cellular nuclear antigen (PCNA) by 6-fold and p21-ras by 2-fold. Although our results demonstrate that TGF-β₁ induces the expression of very early genes related to cell proliferation, TGF-β₁ could be acting either as a mitogen or as a survival factor to induce proliferation in fetal brown adipocytes. © 1996 Wiley-Liss, Inc.     

Energy and speleogenesis: Key determinants of terrestrial species richness in caves

The aim of this study was to unravel the relative role played by speleogenesis (i.e., the process in which a cave is formed), landscape‐scale variables, and geophysical factors in the determination of species richness in caves. Biological inventories from 21 caves located in the southeastern Iberian Peninsula along with partial least square (PLS) regression analysis were used to assess the relative importance of the different explanatory variables. The caves were grouped according to the similarity in their species composition; the effect that spatial distance could have on similarity was also studied using correlation between matrices. The energy and speleogenesis of caves accounted for 44.3% of the variation in species richness. The trophic level of each cave was the most significant factor in PLS regression analysis, and epigenic caves (i.e., those formed by the action of percolating water) had significantly more species than hypogenic ones (i.e., those formed by the action of upward flows in confined aquifers). Dissimilarity among the caves was very high (multiple‐site β_sim = 0.92). Two main groups of caves were revealed through the cluster analysis, one formed by the western caves and the other by the eastern ones. The significant—but low—correlation found between faunistic dissimilarity and geographical distance (r = .16) disappeared once the caves were split into the two groups. The extreme beta‐diversity suggests a very low connection among the caves and/or a very low dispersal capacity of the species. In the region under study, two main factors are intimately related to the richness of terrestrial subterranean species in caves: the amount of organic material (trophic level) and the formation process (genesis). This is the first time that the history of a cave genesis has been quantitatively considered to assess its importance in explaining richness patterns in comparison with other factors more widely recognized.     

A cryptic invasion within an invasion and widespread introgression in the European water frog complex: consequences of uncontrolled commercial trade and weak international legislation

In Western Europe, many pond owners introduce amphibians for ornamental purposes. Although indigenous amphibians are legally protected in most European countries, retailers are circumventing national and international legislation by selling exotic nonprotected sibling species. We investigated to what extent non‐native species of the European water frog complex (genus Pelophylax) have become established in Belgium, using morphological, mitochondrial and nuclear genetic markers. A survey of 87 sampling sites showed the presence of non‐native water frogs at 47 locations, mostly Marsh frogs (Pelophylax ridibundus). Surprisingly, at least 19% of all these locations also harboured individuals with mitochondrial haplotypes characteristic of Anatolian water frogs (Pelophylax cf. bedriagae). Nuclear genotyping indicated widespread hybridization and introgression between P. ridibundus and P. cf. bedriagae. In addition, water frogs of Turkish origin obtained through a licensed retailer, also contained P. ridibundus and P. cf. bedriagae, with identical haplotypes to the wild Belgian populations. Although P. ridibundus might have invaded Belgium by natural range expansion from neighbouring countries, our results suggest that its invasion was at least partly enhanced by commercial trade, with origins as far as the Middle East. Also the invasion and rapid spread of Anatolian lineages, masked by their high morphological similarity to P. ridibundus, is likely the result of unregulated commercial trade. We expect that Anatolian frogs will further invade the exotic as well as the native range of P. ridibundus and other Pelophylax species elsewhere in Western and Central Europe, with risks of large‐scale hybridization and introgression.     

Effects of calcium in the nitrogen-fixing symbiosis between actinorhizal <Emphasis Type="Italic">Discaria trinervis</Emphasis> (Rhamnaceae) and <Emphasis Type="Italic">Frankia</Emphasis>

We have analysed the growth and symbiotic performance of actinorhizal Discaria trinervis at various Ca supply regimes. We aimed at discriminating between specific, if any, effects on nodulation and general growth stimulation by Ca. The hypothesis that a high Ca supply would interfere with nodulation by Frankia was also tested. Results showed that plant growth increased with Ca supply. Nodulation was stimulated by moderate levels of Ca, but inhibited by Ca higher than 0.77 mM. Growth of nodules was less affected by Ca than shoot and root growth. Ca concentration of symbiotic plants increased with Ca supply, but nitrogen concentration was independent of Ca at concentrations which did not impair plant growth. All together, these results show that Ca has a positive effect on the establishment and functioning of the symbiosis between Discaria trinervis and Frankia. However, the positive influence of Ca was more likely due to a promotion of plant growth rather than a direct effect on nodule growth and nitrogen fixation itself. At high levels of Ca supply nodulation was impaired. Given the intercellular infection pathway in Discaria trinervis, we suggest that the increment of Ca availability would strengthen its root cell walls, thus decreasing Frankia penetration of the root.     

Redesign of a protein–peptide interaction: Characterization and applications

The design of protein–peptide interactions has a wide array of practical applications and also reveals insight into the basis for molecular recognition. Here, we present the redesign of a tetratricopeptide repeat (TPR) protein scaffold, along with its corresponding peptide ligand. We show that the binding properties of these protein–peptide pairs can be understood, quantitatively, using straightforward chemical considerations. The recognition pairs we have developed are also practically useful for the specific identification of tagged proteins. We demonstrate the facile replacement of these proteins, which we have termed T‐Mods (TPR‐based recognition module), for antibodies in both detection and purification applications. The new protein–peptide pair has a dissociation constant that is weaker than typical antibody–antigen interactions, yet the recognition pair is highly specific and we have shown that this affinity is sufficient for both Western blotting and affinity purification. Moreover, we demonstrate that this more moderate affinity is actually advantageous for purification applications, because extremely harsh conditions are not required to dissociate the T‐Mod‐peptide interaction. The results we present are important, not only because they represent a successful application of protein design but also because they help define the properties that should be sought in other scaffolds that are being developed as antibody replacements.     

Evidence of an American origin for symbiosis-related genes in Rhizobium lusitanum

Randomly amplified polymorphic DNA (RAPD) analysis was used to investigate the diversity of 179 bean isolates recovered from six field sites in the Arcos de Valdevez region of northwestern Portugal. The isolates were divided into 6 groups based on the fingerprint patterns that were obtained. Representatives for each group were selected for sequence analysis of 4 chromosomal DNA regions. Five of the groups were placed within Rhizobium lusitanum, and the other group was placed within R. tropici type IIA. Therefore, the collection of Portuguese bean isolates was shown to include the two species R. lusitanum and R. tropici. In plant tests, the strains P1-7, P1-1, P1-2, and P1-16 of R. lusitanum nodulated and formed nitrogen-fixing symbioses both with Phaseolus vulgaris and Leucaena leucocephala. A methyltransferase-encoding nodS gene identical with the R. tropici locus that confers wide host range was detected in the strain P1-7 as well as 24 others identified as R. lusitanum. A methyltransferase-encoding nodS gene also was detected in the remaining isolates of R. lusitanum, but in this case the locus was that identified with the narrow-host-range R. etli. Representatives of isolates with the nodS of R. etli formed effective nitrogen-fixing symbioses with P. vulgaris and did not nodulate L. leucocephala. From sequence data of nodS, the R. lusitanum genes for symbiosis were placed within those of either R. tropici or R. etli. These results would support the suggestion that R. lusitanum was the recipient of the genes for symbiosis with beans from both R. tropici and R. etli.