Effects of the egg incubation environment on turtle carapace development

Developing organisms are often exposed to fluctuating environments that destabilize tissue-scale processes and induce abnormal phenotypes. This might be common in species that lay eggs in the external environment and with little parental care, such as many reptiles. In turtles, morphological development has provided striking examples of abnormal phenotypic patterns, though the influence of the environment remains unclear. To this end, we compared fluctuating asymmetry, as a proxy for developmental instability, in turtle hatchlings incubated in controlled laboratory and unstable natural conditions. Wild and laboratory hatchlings featured similar proportions of supernumerary scales (scutes) on the dorsal shell (carapace). Such abnormal scutes likely elevated shape asymmetry, which was highest in natural nests. Moreover, we tested the hypothesis that hot and dry environments cause abnormal scute formation by subjecting eggs to a range of hydric and thermal laboratory incubation regimes. Shape asymmetry was similar in hatchlings incubated at five constant temperatures (26–30°C). A hot (30°C) and severely Dry substrate yielded smaller hatchlings but scutes were not overtly affected. Our study suggests that changing nest environments contribute to fluctuating asymmetry in egg-laying reptiles, while clarifying the conditions at which turtle shell development remains buffered from the external environment.     

Spatial mapping of phosphorus influx in bean root systems using digital autoradiography

A novel technique was developed to spatially map the phosphorus net influx capacity in intact root systems. The method is based on digital autoradiography and permits the quantification of phosphorus influx at high spatial resolution (2 mm). Roots of 18-d-old common bean plants were exposed to (32)P-labelled orthophosphate, quickly frozen, excised, lyophilized, scanned, and exposed to a storage phosphor screen. Plots of (32)P content versus root length (distance from the root tip or from the base of the root) were obtained for three different root classes: basal, basal laterals, and taproot laterals. Radioactivity detected by filmless autoradiography correlated well (r(2)=0.99) with measurements made by scintillation counting. Basal roots absorbed 2.5 times and 1.9 times more phosphorus than the taproot lateral and basal lateral root classes, respectively, in the first 20 mm from the root apex. External phosphorus markedly affected influx: roots averaged 5, 16, and 34 pmol P min(-1) in the apical 20 mm when exposed to 1, 5, and 10 microM P solutions, respectively. The spatial pattern of phosphorus influx along the root axes of the different root classes was rather homogeneous when measured on a root surface area basis. Phosphorus influx in the older segments of basal roots (those next to the hypocotyl) did not differ from the newer segments close to the root apex. However, a heterogeneous pattern was detected for basal roots when measured on a length basis, indicating that both root class and diameter constitute main factors controlling the spatial pattern of net influx.     

Kluyveromyces lactis sexual pheromones. Gene structures and cellular responses to alpha-factor

The Kluyveromyces lactis genes for sexual pheromones have been analyzed. The alpha-factor gene encodes a predicted polypeptide of 187 amino acid residues containing four tridecapeptide repeats (WSWITLRPGQPIF). A nucleotide blast search of the entire K. lactis genome sequence allowed the identification of the nonannotated putative a-pheromone gene that encodes a predicted protein of 33 residues containing one copy of the dodecapeptide a-factor (WIIPGFVWVPQC). The role of the K. lactis structural genes KlMFalpha1 and KlMFA1 in mating has been investigated by the construction of disruption mutations that totally eliminate gene functions. Mutants of both alleles showed sex-dependent sterility, indicating that these are single-copy genes and essential for mating. MATalpha, Klsst2 mutants, which, by analogy to Saccharomyces cerevisiae, are defective in Galpha-GTPase activity, showed increased sensitivity to synthetic alpha-factor and increased capacity to mate. Additionally, Klbar1 mutants (putatively defective in alpha-pheromone proteolysis) showed delay in mating but sensitivity to alpha-pheromone. From these results, it can be deduced that the K. lactis MATa cell produces the homolog of the S. cerevisiaealpha-pheromone, whereas the MATalpha cell produces the a-pheromone.     

Reduced IgG anti-small nuclear ribonucleoprotein autoantibody production in systemic lupus erythematosus patients with positive IgM anti-cytomegalovirus antibodies

Introduction

In rheumatoid arthritis (RA), synovial fluid (SF) contains a large number of neutrophils that contribute to the inflammation and destruction of the joints. The SF also contains granulocyte-macrophage colony-stimulating factor (GM-CSF), which sustains viability of neutrophils and activates their functions. Using proteomic surveillance, we here tried to elucidate the effects of GM-CSF on neutrophils.

Methods

Neutrophils stimulated by GM-CSF were divided into four subcellular fractions: cytosol, membrane/organelle, nuclei, and cytoskeleton. Then, proteins were extracted from each fraction and digested by trypsin. The produced peptides were detected using matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS).

Results

We detected 33 peptide peaks whose expression was upregulated by more than 2.5-fold in GM-CSF stimulated neutrophils and identified 11 proteins out of the 33 peptides using MALDI-TOF/TOF MS analysis and protein database searches. One of the identified proteins was neutrophil gelatinase-associated lipocalin (NGAL). We confirmed that the level of NGAL in SF was significantly higher in patients with RA than in those with osteoarthritis. We next addressed possible roles of the increased NGAL in RA. We analysed proteome alteration of synoviocytes from patients with RA by treatment with NGAL in vitro. We found that, out of the detected protein spots (approximately 3,600 protein spots), the intensity of 21 protein spots increased by more than 1.5-fold and the intensity of 10 protein spots decreased by less than 1 to 1.5-fold as a result of the NGAL treatment. Among the 21 increased protein spots, we identified 9 proteins including transitional endoplasmic reticulum ATPase (TERA), cathepsin D, and transglutaminase 2 (TG2), which increased to 4.8-fold, 1.5-fold and 1.6-fold, respectively. Two-dimensional electrophoresis followed by western blot analysis confirmed the upregulation of TERA by the NGAL treatment and, moreover, the western blot analysis showed that the NGAL treatment changed the protein spots caused by post-translational modification of TERA. Furthermore, NGAL cancelled out the proliferative effects of fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) on chondrocytes from a patient with RA and proliferative effect of FGF-2 on chondrosarcoma cells.

Conclusions

Our results indicate that GM-CSF contributes to the pathogenesis of RA through upregulation of NGAL in neutrophils, followed by induction of TERA, cathepsin D and TG2 in synoviocytes. NGAL and the upregulated enzymes may therefore play an important role in RA.     

Carpology,germination and seedling performance in Digitalis thapsi L. (Scrophulariaceae)

Summary

We analyse the fruiting phase of the life cycleof Digitalis thapsi L. We studied the production and size of seeds and capsules, and the interpopulational variability and inter-relationships of these parameters. The capsule volume, number of seeds per capsule, of seeds per individual, and of capsules per individual were inter-related and fairly affected by the medium, whereas seed weight was found to bea more independent character. A germination study showed that the species has agreat germinative capacity and has positive photoblasty. It is non-dormant especially if incubated under experimental conditions close to the climatic conditions of the site of provenance of the samples. An increase in temperature speeds up the process without increasing germinability. The morphological and biometric characteristics of the first stages of development of the species following germination were analysed. It was found that seed weight is not positively correlated with cotyledon length nor with any of the other parameters con-sidered, and that the length of the fifth leaf is a useful parameter to express inter-populational genetic variability. Seedling production in spring seems to be influenced more by physical than by chemical factors.     

Novel carbohydrate specificity of the 16-kDa galectin from Caenorhabditis elegans: binding to blood group precursor oligosaccharides (type 1, type 2, Talpha,and Tbeta) and gangliosides

Galectins, a family of soluble beta-galactosyl-binding lectins, are believed to mediate cell-cell and cell-extracellular matrix interactions during development, inflammation, apoptosis, and tumor metastasis. However, neither the detailed mechanisms of their function(s) nor the identities of their natural ligands have been unequivocally elucidated. Of the several galectins present in the nematode Caenorhabditis elegans, the 16-kDa "proto" type and the 32-kDa "tandem-repeat" type are the best characterized so far, but their carbohydrate specificities have not been examined in detail. Here, we report the carbohydrate-binding specificity of the recombinant C. elegans 16-kDa galectin and the structural analysis of its binding site by homology modeling. Our results indicate that unlike the galectins characterized so far, the C. elegans 16-kDa galectin interacts with most blood group precursor oligosaccharides (type 1, Galbeta1,3GlcNAc, and type 2, Galbeta1,4GlcNAc; Talpha, Galbeta1,3GalNAcalpha; Tbeta, Galbeta1,3GalNAcbeta) and gangliosides containing the Tbeta structure. Homology modeling of the C. elegans 16-kDa galectin CRD revealed that a shorter loop containing residues 66-69, which enables interactions of Glu(67) with both axial and equatorial -OH at C-3 of GlcNAc (in Galbeta1,4GlcNAc) or at C-4 of GalNAc (in Galbeta1,3GalNAc), provides the structural basis for this novel carbohydrate specificity.     

Large-Scale Patterns of Quercus ilex, Quercus suber, and Quercus pyrenaica Regeneration in Central-Western Spain

In Central-Western Spain, forests and woodlands composed of Quercus sp. support outstanding levels of biodiversity, but there is increasing concern about their long-term persistence due to a lack of regeneration. We hypothesize that this regenerative lack is operating on a large geographic scale; that there are differences in the abundance of regeneration between three oak species; that oak regeneration is governed mainly by forest management and structure; and that shrubs act as important physical protectors of seedlings and saplings. We analyzed whether densities of oak seedlings and saplings in several size classes were related to stand-structure, understory, and physiographic variables potentially affecting regeneration. Data collected at a regional level (1 km × 1 km grid) by the Spanish Forest Inventory were evaluated from 2,816 plots. Results revealed that regeneration failure was common for all size categories, from small seedlings to large saplings, and for the three oak species studied, especially the evergreens. Of the Quercus ilex, Q. suber, and Q. pyrenaica plots studied, 49%, 62%, and 20% were lacking any small seedlings, and 82%, 96%, and 56% did not have any large saplings, respectively. Regeneration was positively correlated with tree cover and density, especially of small and medium-sized trees, and negatively correlated with the presence of large trees, indicating that regeneration failure is mostly associated with more open, uniform, and/or aged woodlands. Regeneration densities of Q. ilex and Q. suber were positively correlated with all understory variables, suggesting that the presence of pioneer shrubs represent a major safe site for early tree recruitment, independent from specific shrub species.     

Murine cytomegalovirus with a transposon insertional mutation at open reading frame m155 is deficient in growth and virulence in mice

A pool of murine cytomegalovirus (MCMV) mutants was previously generated by using a Tn3-based transposon mutagenesis approach (X. Zhan, M. Lee, J. Xiao, and F. Liu, J. Virol. 74:7411-7421, 2000). In this study, one of the MCMV mutants, Rvm155, which contained the transposon insertion in open reading frame m155, was characterized in vitro for its replication in tissue culture and in vivo for its growth and virulence in immunodeficient SCID mice. Compared to the wild-type strain and a rescued virus that restored the m155 region, the mutant is significantly deficient in growth in many organs of the infected animals. At 21 days postinfection the titers of Rvm155 in the salivary glands, lungs, spleens, livers, and kidneys of the intraperitoneally infected SCID mice were lower than the titers of the wild-type virus and the rescued virus by 50-, 1,000-, 500-, 100-, and 500-fold, respectively. Moreover, the viral mutant was attenuated in killing the SCID mice, as none of the SCID mice that were intraperitoneally infected with Rvm155 died until 38 days postinfection while all the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results provide the first direct evidence that a disruption of m155 expression leads to attenuation of viral virulence and growth in animals. Moreover, these results suggest that m155 is a viral determinant for optimal MCMV growth and virulence in vivo.     

Instability of plasmid DNA sequences: Macro and micro evolution of the antibiotic resistance plasmid R6-5

Summary Detailed examination of the structure of cloned DNA fragments of the R6-5 antibiotic resistance plasmid has revealed a substantial degree of polynucleotide sequence heterogeneity and indicates that sequence rearrangements in plasmids and possibly other replicons occur more frequently than has hitherto been appreciated. The sequence changes in cloned R6-5 fragments were shown in some instances to have occurred prior to cloning, i.e. existed in the original population of R6-5 molecules that was obtained from a single bacterial clone and by several different criteria judged to be homogeneous,and in others to have occurred either during the cloning procedure or during subsequent propagation of hybrid molecules. The molecular changes that are described involved insertion/deletion of the previously characterized IS2 insertion element, formation of a new inverted repeat structure probably by duplication of a preexisting R6-5 DNA sequence, sequence inversion, and loss and gain of restriction endonuclease cleavage sites.