The expression and localization of urokinase-type plasminogen activator and its type 1 inhibitor are regulated by retinoic acid and fibroblast growth factor in human teratocarcinoma cells.

Human Tera 2 embryonal carcinoma cells switch gradually from rapidly growing undifferentiated cells to almost nonproliferating cells during retinoic acid (RA)-induced neuronal differentiation. This process is associated with the increased expression of type 1 plasminogen activator inhibitor (PAI 1) mRNA, and the secreted inhibitor is immobilized to the pericellular area. Furthermore, the differentiation is accompanied by a decrease in the amount of both the secreted tissue-type PA (tPA) and the mainly cell-associated urokinase-type PA (uPA) activity. In RA-differentiated cells, uPA becomes localized at the vinculin-rich cell-substratum adhesion sites. Fibroblast growth factor activity has been associated with various events during embryonal growth and with the regulation of proteolytic enzymes. A short-term treatment of the undifferentiated Tera 2 cells with basic fibroblast growth factor (bFGF) increases uPA mRNA levels and the cell-associated uPA activity, whereas the secretory tPA activity decreases. bFGF induces PAI 1 mRNA expression in the undifferentiated cells, but unlike PAI 1 protein after RA-treatment, the inhibitor does not accumulate around the cells but is released in the medium. A similar exposure to bFGF has less effect on the RA-differentiated Tera 2 cells. Under these conditions bFGF treatment leads to an increase in the amounts of PAI 1 and uPA mRNAs, but no changes in the localization of these components can be seen. Differentiation of human embryonal carcinoma cells is thus connected with an altered response to bFGF.     

Exogenous fibronectin is not required for organogenesis in vitro

The biological effect of plasma fibronectin on the differentiation of embryonic mouse kidney and tooth was studied in organ cultures. Transferrin (50 micrograms/ml) was a strong mitogen for kidney cells, whereas the addition of soluble fibronectin (50 to 250 micrograms/ml) had no detectable effect on differentiation or proliferation. The same serum-free, transferrin-containing medium did not support tooth differentiation. However, fibronectin was not a necessary serum component because fibronectin-free serum supported tooth development. It was demonstrated with antibodies specific for human fibronectin that the exogenously added human fibronectin at 50 micrograms/ml did not become incorporated to the cultured organs. Only minimal incorporation to the kidney basement membrane area was observed when fibronectin concentration was 250 micrograms/ml. The mesenchymal stroma and the basement membranes of the kidney and tooth rudiments cultured in fibronectin-free media stained intensely with conventional fibronectin antibodies, indicating endogenous production of fibronectin. Outgrowing epithelial cells from isolated kidney tubules produced fibronectin as well as laminin. The results suggest that the fibronectin found in the stroma and basement membranes is an endogenous product of the developing tissues and that plasma fibronectin is not required for in vitro organogenesis. The results also indicate that it is difficult to study the effect of fibronectin on morphogenetic processes because it may not penetrate the organ explants in vitro.     

Reorganization of intermediate filament cytoskeleton in induced metanephric mesenchyme cells is independent of tubule morphogenesis

The metanephric mesenchyme becomes converted into epithelial tubules if cultured in transfilter contact with an inductor tissue. The expression of intermediate filaments (IFs), used as cell-type-specific markers has been studied in this model system for differentiation and organogenesis. In immunofluorescence microscopy of frozen sections, the undifferentiated cells of isolated metanephric mesenchymes uniformly showed IFs of vimentin type only. Also, when cultured as a monolayer, cells from the uninduced mesenchymes showed only vimentin filaments. In frozen sections of transfilter explants, epithelial tubules apparently negative for vimentin could be seen after 3 days in culture, but expression of cytokeratin could not be demonstrated in the developing tubules until the fourth day of culture. Sections of explants cultured further showed tubule cells with distinct fibrillar cytokeratin positivity. The appearance of cytokeratin in the explants was also demonstrated with immunoblotting experiments, using two different cytokeratin antibodies. Expression of IFs was further examined in monolayer cultures of metanephric mesenchymes which had been initially exposed to a short transfilter induction pulse. In these experiments, cytokeratin-positive cells could be demonstrated after a total of 4 days in culture. Double immunofluorescence experiments showed varying amounts of vimentin in the cytokeratin-positive cells: after 4 days in culture, most cytokeratin-positive cells still showed vimentin-positivity although often in a nonfibrillar form. During further culture, gradual disappearance of vimentin-specific fluorescence was observed in cytokeratin-positive cells. The results suggest that the vimentin-positive metanephric mesenchyme cells lose their fibrillar vimentin organization upon induction that leads to kidney tubule formation. This change may be essential for the transformation from an undifferentiated mesenchymal cell into a specialized epithelial cell. Cytokeratin filaments, regarded as a marker for epithelial cells, seem to appear simultaneously with or soon after the change in vimentin organization. These changes in IF expression also occur in monolayer cultures of mesenchyme cells initially exposed to a short transfilter induction pulse. This suggests that epithelial differentiation, as revealed by the emergence of cytokeratin positivity, may occur even in the absence of a clear morphological differentiation and three-dimensional organization of the cells.     

Common structural elements in the architecture of the cofactor-binding domains in unrelated families of pyridoxal phosphate-dependent enzymes

A detailed comparison of the structures of aspartate aminotransferase, alanine race-mase, the beta subunit of tryptophan synthase, D-amino acid aminotransferase and glycogen phosphorylase has revealed more extensive structural similarities among pyridoxal phosphate (PLP)-binding domains in these enzymes than was observed previously. These similarities consist of seven common structural segments of the polypeptide chain, which form an extensive common structural organization of the backbone chain responsible for the appropriate disposition of key residues, some from the aligned fragments and some from variable loops joined to these fragments, interacting with PLPs in these enzymes. This common structural organization contains an analogous hydrophobic minicore formed from four amino acid side chains present in the two most conserved structural elements. In addition, equivalent alpha-beta-alpha-beta supersecondary structures are formed by these seven fragments in three of the five structures: alanine racemase, tryptophan synthase and glycogen phosphorylase. Despite these similarities, it is generally accepted that these proteins do not share a common heritage, but have arisen on five separate occasions. The common and contiguous alpha-beta-alpha-beta structure accounts for only 28 residues and all five enzymes differ greatly in both the orientation of the PLP pyridoxal rings and their contacts with residues close to the common structural elements.     

The effects of continuous illumination on southern and northern Micrasterias strains

Different strains of Micrasterias (Chlorophyta, Conjugatophyceae); M. rotata (Grev.) Ralfs ex. Ralfs and M. denticulata Breb. ex. Ralfs var. angulosa (Hantzsch) W. & G. S. West from northern and southern Finland were treated with continuous illumination in order to study the cellular effects of the treatment and whether the tolerance to continuous light of the northern Finnish strains is related to the different daylenght conditions in northern and southern areas. During the growing season the Finnish strains normally live in long-day conditions or even in continuous light (between 60 and 70°N), and they also tolerated continuous illumination in the laboratory. Ultrastructural changes were found especially in the chloroplasts, where formation of calcium precipitates of different forms and sizes and also formation of plastoglobuli containing lipids appeared. However, even in 4-week treatments the ultrastructure of cells of these northern strains was not totally disrupted, contrary to what was found in southern M. torreyi , studied earlier. Southern and northern strains tolerated continuous illumination in different ways. They seem to differ from each other physiologically, and the differences are possibly located in their ionic metabolism and regulation. The injuries sustained during continuous illumination of Micrasterias may largely be caused by the accumulation of Ca²⁺ in cytoplasm and organelles, especially in the chloroplasts.     

Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast

NOP1 is an essential nucleolar protein in yeast that is associated with small nucleolar RNA and required for ribosome biogenesis. We have cloned the human nucleolar protein, fibrillarin, from a HeLa cDNA library. Human fibrillarin is 70% identical to yeast NOP1 and is also the functional homologue since either human or Xenopus fibrillarin can complement a yeast nop1- mutant. Human fibrillarin is localized in the yeast nucleolus and associates with yeast small nucleolar RNAs. This shows that the signals within eucaryotic fibrillarin required for nucleolar association and nucleolar function are conserved from yeast to man. However, human fibrillarin only partially complements in yeast resulting in a temperature-sensitive growth, concomitantly altered rRNA processing and aberrant nuclear morphology. A suppressor of the human fibrillarin ts-mutant was isolated and found to map intragenically at a single amino acid position of the human nucleolar protein. The growth rate of yeast nop1- strains expressing Xenopus or human fibrillarin or the human fibrillarin suppressor correlates closely with their ability to efficiently and correctly process pre-rRNA. These findings demonstrate for the first time that vertebrate fibrillarin functions in ribosomal RNA processing in vivo.     

Climate-driven synchrony in seed production of masting deciduous and conifer tree species

Aims Understanding fluctuations in plant reproductive investment can constitute a key challenge in ecology, conservation and management. Masting events of trees (i.e. the intermittent and synchronous production of abundant seeding material) is an extreme example of such fluctuations. Our objective was to establish the degree of spatial and temporal synchrony in common four masting tree species in boreal Finland and account for potential causal drivers of these patterns.Methods We investigated the spatial intraspecific and temporal interspecific fluctuations in annual seed production of four tree species in Finland, silver birch Betula pendula Roth, downy birch Betula pubescens Ehrh., Norway spruce Picea abies (L.) H.Karst. and rowanberry Sorbus aucuparia L. We also tested to see whether variations in seed production were linked to annual weather conditions. Seeding abundance data were derived from tens of stands per species across large spatial scales within Finland during 1979 to 2014 (for rowanberries only 1986 to 2014).Important findings All species showed spatial synchrony in seed production at scales up to 1000 km. Annual estimates of seed production were strongly correlated between species. Spring and summer temperatures explained most variation in crop sizes of tree species with 0-to 2-year time lags, whereas rainfall had relatively little influence. Warm weather during flowering (May temperature) in the flowering year (Year t) and 2 years before (t ?2) were correlated with seed production. However, high May temperatures during the previous year (t^-1) adversely affected seed production. Summer temperatures in Year t^-1 was positively correlated with seed production, likely because this parameter enhances the development of flower primordials, but the effect was negative with a time lag of 2 years. The negative feedback in temperature coefficients is also likely due to patterns of resource allocation, as abundant flowering and seed production in these species is thought to reduce the subsequent initiation of potential new flower buds. Since the most important weather variables also showed spatial correlation up to 1000 km, weather parameters likely explain much of the spatial and temporal synchrony in seed production of these four studied tree species.     

Diversification of lindsaeoid ferns and phylogenetic uncertainty of early polypod relationships

We analysed one nuclear gene (18S) and seven plastid markers [five protein coding (atpA, atpB, rbcL, rpoC1, rps4) and two non‐coding (trnH‐psbA, trnL‐trnF] for 31 members of Polypodiales and four outgroup taxa. We focused our sampling on the lindsaeoids and associated ferns in order to obtain a better understanding of the diversification of the early polypods. However, the exact phylogenetic position of Saccoloma and Cystodium remained uncertain. Based on relaxed molecular clock analyses, it appears that the crown group lindsaeoids diversified in the Caenozoic, more or less simultaneously with the main radiation of other Polypodiales, even though the original divergence between the lindsaeoid and non‐lindsaeoid polypods occurred before the end of the Jurassic. The current pantropical distribution of lindsaeoids can be explained by either long‐distance dispersal across the oceans or vicariance caused by the retreat of previously widely distributed tropical forests from higher to lower latitudes. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170 , 489–503.     

The many costs of sex

Explaining the evolution of sex is challenging for biologists. A 'twofold cost' compared with asexual reproduction is often quoted. If a cost of this magnitude exists, the benefits of sex must be large for it to have evolved and be maintained. Focusing on benefits can be misleading, as this sidelines important questions about the cost of sex: what is the source of the twofold cost: males, genome dilution or both? Does the cost deviate from twofold? What other factors make sex costly? How should the costs of sex be empirically measured? The total cost of sex and how it varies in different contexts must be known to determine the benefits needed to account for the origin and maintenance of sex.     

Neph3 associates with regulation of glomerular and neural development in zebrafish

Neph3 (filtrin) is a membrane protein expressed in the glomerular epithelial cells (podocytes), but its role in the glomerulus is still largely unknown. To characterize the function of Neph3 in the glomerulus, we employed the zebrafish as a model system. Here we show that the expression of neph3 in pronephros starts before the onset of nephrin and podocin expression, peaks when the nephron primordium differentiates into glomerulus and tubulus, and is then downregulated upon glomerular maturation. By histology, we found that neph3 is specifically expressed in pronephric podocytes at 36 hpf. Furthermore, disruption of neph3 expression by antisense morpholino oligonucleotides results in distorted body curvature and transient pericardial edema, the latter likely reflecting perturbation of glomerular osmoregulatory function. Histological analysis of neph3 morphants reveals altered glomerular morphology and dilated pronephric tubules. The phenotype of neph3 morphants, curved body and pericardial edema, is rescued by wild-type zebrafish neph3 mRNA. In addition to glomerulus, neph3 is highly expressed in the developing brain and specific regions of mature midbrain and hindbrain. In line with this, neph3 morphants show aberrant brain morphology. Collectively, the expression of neph3 in glomerulus and brain together with the morphant phenotype imply that neph3 is a pleiotropic gene active during distinct stages of tissue differentiation and associates directly in the regulation of both glomerular and neural development.