Morphological diversification of fish as a consequence of the divergence of ontogenetic trajectories]

The possibilities of the investigation of ontogenetic changes in the morphological features of fish using multidimensional "ontogenetic channels," within which individual ontogenetic trajectories run, are demonstrated using African barbs. (Barbus intermedius complex) as an example. The position of ontogenetic channels allows one to judge how the differences of adult individuals in morphological characters arise during development and how their morphological diversity is formed.     

Expression of a releasable form of annexin II by human keratinocytes

Annexin II is a multifunctional calcium-dependent phospholipid binding protein whose presence in epidermis has previously been reported. However, like other members of annexin family, annexin II has been regarded as either an intracellular protein or associated with the cellular membrane. Here, we report the presence of a releasable annexin II and p11, two monomers of annexin II tetramer, in keratinocyte-conditioned medium (KCM). Proteins present in KCM were fractionated on a gel filtration column and following further evaluation, a releasable protein with apparent MW of 36 kDa was identified. Further characterization identified this protein as the p36 monomer of annexin II tetramer. The phospho-tyrosine antibody did not visualize this protein as the phosphorylated form of p36. Several experiments were conducted to examine whether this protein is soluble or associated with keratinocyte cell membranes in the conditioned medium. A centrifugation of conditioned medium was not able to bring this protein down into the pellet. Surprisingly, the results of Western analysis identified p36 and p11, two monomers of the annexin II tetramer, in conditioned medium derived from either keratinocytes cultured alone or keratinocytes co-cultured with fibroblasts. In contrast to the keratinocyte-conditioned medium in which annexin II was easily detectable, both monomers were barely detectable in conditioned medium collected from dermal fibroblasts. This finding was in contrast to the cell lysates in which p36 was detectable in both keratinocytes and fibroblasts. However, the amount of this protein was markedly higher in keratinocyte lysate relative to that of dermal fibroblasts. Conditioned medium derived from keratinocyte established from adult showed a higher level of annexin II compared to that of keratinocytes established from newborn babies. The expression of p11 seems to increase with differentiation of keratinocytes derived from either adult or newborn skin samples. When the site of annexin synthesis in human skin was examined by immunohistochemical staining, the antibody for p36 localized the annexin to the keratinocyte cell members in the basal and suprabasal keratinocytes. In conclusion, Western blot detection of both p36 and p11 in conditioned medium from skin cells revealed that human keratinocytes, but not fibroblasts, express a releasable monomer form of annexin II which is regulated by differentiation status of keratinocytes. This finding is consistent with the localization of annexin II detected by immunohistochemical staining.     

Viroid RNA systemic spread may depend on the interaction of a 71-nucleotide bulged hairpin with the host protein VirP1

Viroids are noncoding circular single-stranded RNAs that are propagated systemically in plants. VirP1 is a protein from tomato, which is an excellent host for potato spindle tuber viroid (PSTVd), and it has been isolated by virtue of its specific in vitro binding to PSTVd RNA. We report on the specific in vivo interaction of VirP1 with full-length viroid RNA as well as with subfragments in the three-hybrid system. The terminal right domain (TR) of PSTVd was identified as a strong interacting partner for VirP1. A weaker partner is provided by a right-hand subfragment of hop stunt viroid (HSVd), a viroid that infects tomato poorly. We present a sequence and structural motif of the VirP1-interacting subfragments. The motif is disturbed in the replicative but nonspreading R+ mutant of the TR. According to our in vivo and in vitro binding assays, the interaction of this mutant with VirP1 is compromised. We propose that the AGG/CCUUC motif bolsters recognition of the TR by VirP1 to achieve access of the viroid to pathways that propagate endogenous RNA systemic signals in plants. Systemic trafficking has been suggested for miRNA precursors, of which the TR, as a stable bulged hairpin 71 nt long, is quite reminiscent.     

Analysis of the odontogenic and osteogenic potentials of dental pulp in vivo using a Col1a1-2.3-GFP transgene

Recently, transgenic mice that carry a Green Fluorescent Protein (GFP) reporter gene fused to 2.3 kb fragment of rat Col1a1 regulatory sequences (pOBCol2.3GFPemd) were generated. In the present study, we have examined the patterns of expression of Col1a1-2.3-GFP during odontoblast differentiation in this transgenic line. We report that Col1a1-2.3-GFP is expressed in newly differentiated odontoblasts secreting predentin and fully differentiated odontoblasts. The pattern of expression of Col1a1-2.3-GFP in odontoblasts is correlated with that of dentin sialophosphoprotein (DSPP). Col1a1-2.3-GFP is also expressed in the osteoblasts and osteocytes of alveolar bone. The pattern of expression of Col1a1-2.3-GFP in osteocytes is correlated with the expression of Dmp1. These observations indicate the 2.3 kb rat Col1a1 promoter fragment has sufficient strength and specificity to monitor the stage-specific changes during both odontoblast and osteoblast differentiation. We also used coronal pulp tissues isolated from postnatal pOBCol2.3GFPemd transgenic animals to follow their differentiation after transplantation under the kidney capsule. Our observations provide direct evidence that the dental pulp contains competent progenitor cells capable of differentiating into new generations of odontoblast-like cells which express high levels of Col1a1-2.3-GFP and DSPP and secrete tubular containing reparative dentin. We also report that the dental pulp is capable of giving rise to atubular bone-like tissue containing osteocytes expressing high levels of Col1a1-2.3-GFP and Dmp1. Our studies indicate that pOBCol2.3GFPemd transgenic animals provide a powerful tool for direct examination of the underlying mechanisms and the signaling pathways involved in dentin regeneration and repair, stem cell properties and heterogeneity of the dental pulp.     

Quinolinate dehydrogenase and 6-hydroxyquinolinate decarboxylase involved in the conversion of quinolinic acid to 6-hydroxypicolinic acid by<Emphasis Type="Italic"> Alcaligenes</Emphasis> sp. strain UK21

In the conversion of quinolinic acid to 6-hydroxypicolinic acid by whole cells of Alcaligenes sp. strain UK21, the enzyme reactions involved in the hydroxylation and decarboxylation of quinolinic acid were examined. Quinolinate dehydrogenase, which catalyzes the first step, the hydroxylation of quinolinic acid, was solubilized from a membrane fraction, partially purified, and characterized. The enzyme catalyzed the incorporation of oxygen atoms of H₂O into the hydroxyl group. The dehydrogenase hydroxylated quinolinic acid and pyrazine-2,3-dicarboxylic acid to form 6-hydroxyquinolinic acid and 5-hydroxypyrazine-2,3-dicarboxylic acid, respectively. Phenazine methosulfate was the preferred electron acceptor for quinolinate dehydrogenase. 6-Hydroxyquinolinate decarboxylase, catalyzing the nonoxidative decarboxylation of 6-hydroxyquinolinic acid, was purified to homogeneity and characterized. The purified enzyme had a molecular mass of approximately 221 kDa and consisted of six identical subunits. The decarboxylase specifically catalyzed the decarboxylation of 6-hydroxyquinolinic acid to 6-hydroxypicolinic acid, without any co-factors. The N-terminal amino acid sequence was homologous with those of bacterial 4,5-dihydroxyphthalate decarboxylases.     

The dual functions of biphenyl-degrading ability of Pseudomonas sp. KKS102: energy acquisition and substrate detoxification

The bph operon of Pseudomonas sp. KKS102 is constituted of 11 bph genes which encode enzymes for biphenyl assimilation. Growth of a mutant in which a large part of the bph operon was deleted was inhibited by biphenyl in a concentration-dependent manner. We constructed a series of bph operon deletion mutants and tested for their biphenyl sensitivity. Growth inhibition by biphenyl was more prominent with the mutants defective in bphA1, bphB, bphC, and bphD, which were clustered in the bph operon and working in the early stage of the biphenyl degradation. The mutant defective in bphE, which was working at the late stage and forming a different cluster from the early stage genes, was not much inhibited by biphenyl. These indicate that biphenyl is detoxified by enzymes which function in the early stage of biphenyl assimilation and thus detoxification of substrates as well as energy acquisition could have played an important role in the evolution of the KKS102 bph operon.     

Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.     

Differences in Energy Balance-Related Behaviours in European Preschool Children: The ToyBox-Study

Background

The aim of the current study was to compare levels of energy balance-related behaviours (physical activity, sedentary behaviour, and dietary behaviours (more specifically water consumption, sugar-sweetened beverage consumption and unhealthy snacking)) in four- to six-year-old preschoolers from six European countries (Belgium, Bulgaria, Germany, Greece, Poland, and Spain) within the ToyBox cross-sectional study.

Methods

A sample of 4,045 preschoolers (4.77 ± 0.43 years; 52.2% boys) had valid physical activity data (steps per day), parents of 8,117 preschoolers (4.78 ± 0.46 years; 53.0% boys) completed a parental questionnaire with questions on sedentary behaviours (television viewing, computer use, and quiet play), and parents of 7,244 preschoolers (4.77 ± 0.44 years; 52.0% boys) completed a food frequency questionnaire with questions on water consumption, sugar-sweetened beverage consumption and unhealthy snacking.

Results

The highest levels of physical activity were found in Spain (12,669 steps/day on weekdays), while the lowest levels were found in Bulgaria and Greece (9,777 and 9,656 steps/day on weekdays, respectively). German preschoolers spent the least amount of time in television viewing (43.3 min/day on weekdays), while Greek preschoolers spent the most time in television viewing (88.5 min/day on weekdays). A considerable amount of time was spent in quiet play in all countries, with the highest levels in Poland (104.9 min/day on weekdays), and the lowest levels in Spain (60.4 min/day on weekdays). Belgian, German, and Polish preschoolers had the lowest intakes of water and the highest intakes of sugar-sweetened beverages. The intake of snacks was the highest in Belgian preschoolers (73.1 g/day) and the lowest in Greek preschoolers (53.3 g/day).

Conclusions

Across six European countries, differences in preschoolers’ energy balance-related behaviours were found. Future interventions should target European preschoolers’ energy balance-related behaviours simultaneously, but should apply country-specific adaptations.