Conditional tamoxifen Cre induced mutagenesis in the embryonic kidney in organ culture

Developmental control genes are often sequentially and repeatedly functional during embryogenesis, and for this reason conditional mutagenesis tools are often required to study their roles in detail. Cre recombinase fused to the modified estrogen hormone-binding domain (ER(Tm)) generates a Cre in which the recombination activity of the LoxP-containing gene can be regulated by the nonsteroidal estrogen analogue 4-hydroxytamoxifen (4OH-TM). ER(Tm) may provide a useful way of achieving conditional mutagenesis in conjunction with the classic organ culture methods of experimental embryology. We used embryonic kidneys separated from the Cre-ER(Tm); R26R embryos to assay whether efficient 4OH-TM-inducible genomic recombination can be achieved in organ culture and in experimentally induced kidney mesenchymes. Our results indicate that the inducible ER(Tm) Cre/loxP system indeed provides an effective way of conditionally mutagenizing genes in kidney organ culture and tissue conjugates.     

Anatomical basis of variation in mesophyll resistance in eastern Australian sclerophylls: news of a long and winding path

In sclerophylls, photosynthesis is particularly strongly limited by mesophyll diffusion resistance from substomatal cavities to chloroplasts (r (m)), but the controls on diffusion limits by integral leaf variables such as leaf thickness, density, and dry mass per unit area and by the individual steps along the diffusion pathway are imperfectly understood. To gain insight into the determinants of r (m) in leaves with varying structure, the full CO(2) physical diffusion pathway was analysed in 32 Australian species sampled from sites contrasting in soil nutrients and rainfall, and having leaf structures from mesophytic to strongly sclerophyllous. r (m) was estimated based on combined measurements of gas exchange and chlorophyll fluorescence. In addition, r (m) was modelled on the basis of detailed anatomical measurements to separate the importance of different serial resistances affecting CO(2) diffusion into chloroplasts. The strongest sources of variation in r (m) were S (c)/S, the exposed surface area of chloroplasts per unit leaf area, and mesophyll cell wall thickness, t (cw). The strong correlation of r (m) with t (cw) could not be explained by cell wall thickness alone, and most likely arose from a further effect of cell wall porosity. The CO(2) drawdown from intercellular spaces to chloroplasts was positively correlated with t (cw), suggesting enhanced diffusional limitations in leaves with thicker cell walls. Leaf thickness and density were poorly correlated with S (c)/S, indicating that widely varying combinations of leaf anatomical traits occur at given values of leaf integrated traits, and suggesting that detailed anatomical studies are needed to predict r (m) for any given species.     

High-frequency metabolism study in a large and shallow temperate lake reveals seasonal switching between net autotrophy and net heterotrophy

Respiratory CO₂ release from inland waters is a major process in the global carbon cycle, retaining more than half of the carbon flux from terrestrial sources that otherwise would reach the sea. The strongly lake type-specific balance between primary production and respiration determines whether a lake acts regionally as a net sink or source of CO₂. This study presents two-year (2009, 2010) results of high-frequency metabolism measurements in the large and shallow polymictic eutrophic Lake V?rtsj?rv (area 270?km²; mean depth 2.8?m). We estimated the net ecosystem production (NEP), community respiration (R) and gross primary production (GPP) from continuous measurements of oxygen, irradiance, wind and water temperature. A sinusoidal model fitted to the calculated metabolic rates showed the prevalence of net autotrophy (mean GPP:R?>?1) from early spring until August/September, whereas during the rest of the year heterotrophy (mean GPP:R?2 neutral on an annual basis. Community respiration lagged behind GPP by approximately 2?weeks, which could be explained by the bulk of the phytoplankton biomass accounted for by filamentous cyanobacteria that are considered mostly inedible to zooplankton, and the seasonally increasing role of sediment resuspension. In the warmer year 2010, the seasonal peaks of GPP, R and NEP were synchronously shifted nearly 1?month earlier compared with 2009. The strong stimulating effect of temperature on both GPP and R and its negative effect on NEP revealed by the multiple regression analysis suggests increasing metabolic rates and increasing heterotrophy in this lake type in a warmer climate.     

Role of CD44 in the organization of keratinocyte pericellular hyaluronan

CD44 is a ubiquitous cell surface glycoprotein, involved in important cellular functions including cell adhesion, migration, and modulation of signals from cell surface receptors. While most of these CD44 functions are supposed to involve hyaluronan, relatively little is known about the contribution of CD44 to hyaluronan maintenance and organization on cell surface, and the role of CD44 in hyaluronan synthesis and catabolism. Blocking hyaluronan binding either by CD44 antibodies, CD44-siRNA or hyaluronan decasaccharides (but not hexasaccharides) removed most of the hyaluronan from the surfaces of both human (HaCaT) and mouse keratinocytes, resembling results on cells from CD44−/− animals. In vitro, compromising CD44 function led to reduced and increased amounts, respectively, of intracellular and culture medium hyaluronan, and specific accumulation below the cells. In vivo, CD44-deficiency caused no marked differences in hyaluronan staining intensity or localization in the fetal skin or in adult ear skin, while tail epidermis showed a slight reduction in epidermal hyaluronan staining intensity. However, CD44-deficient tail skin challenged with retinoic acid or tape stripping revealed diffuse accumulation of hyaluronan in the superficial epidermal layers, normally negative for hyaluronan. Our data indicate that CD44 retains hyaluronan in the keratinocyte pericellular matrix, a fact that has not been shown unambiguously before, and that hyaluronan abundance in the absence of CD44 can result in hyaluronan trapping in abnormal locations possibly interfering there with normal differentiation and epidermal barrier function.     

Relation of chronotype to sleep complaints in the general Finnish population

Individuals show variation in their preference for the daily timing of activities. In this study the authors analyzed whether chronotypes associate with sleep duration and sleep-related complaints. The authors used the National FINRISK Study 2007 Survey data on 3696 women and 3162 men, representative of the Finnish population aged 25 yrs and older, for the assessment of chronotype and self-reported sleep. Evening types experienced insomnia symptoms, had nightmares, and had used recently hypnotics significantly more often than other chronotypes among both men and women. In a multinominal logistic regression model predicting insufficient sleep, the association of eveningness with insufficient sleep was not abolished after adjustment for sex, age, and sleep duration. The prevalence of short sleepers was significantly higher in evening types among men than among women, whereas that of long sleepers was significantly higher in evening types among both men and women, as compared with the other chronotypes. These results indicate that eveningness predisposes individuals to a range of sleep complaints.     

Wnt-4 signaling is involved in the control of smooth muscle cell fate via Bmp-4 in the medullary stroma of the developing kidney

Wnt-4, a member of the Wnt family of secreted signaling molecules, is essential for nephrogenesis, but its expression in the presumptive medulla suggests additional developmental roles in kidney organogenesis. We demonstrate here that Wnt-4 signaling plays also a role in the determination of the fate of smooth muscle cells in the medullary stroma of the developing kidney, as a differentiation marker, smooth muscle alpha-actin (alpha-SMA), is markedly reduced in the absence of its signaling. Wnt-4 probably performs this function by activating the Bmp-4 gene encoding a known differentiation factor for smooth muscle cells, since Bmp-4 gene expression was lost in the absence of Wnt-4 while Wnt-4 signaling led to a rescue of Bmp-4 expression and induction of alpha-SMA-positive cells in vitro. Recombinant Bmp-4 similarly rescued the differentiation of alpha-SMA-expressing cells in cultured Wnt-4-deficient embryonic kidney. The lack of smooth muscle cell differentiation leads to an associated deficiency in the pericytes around the developing vessels of the Wnt-4-deficient kidney and apparently leads to a secondary defect in the maturation of the kidney vessels. Thus, besides being critical for regulating mesenchymal to epithelial transformation in the cortical region in nephrogenesis, Wnt-4 signaling regulates the fate of smooth muscle cells in the developing medullary region.     

Seasonality Pattern of Biomass Accumulation in a Drifting Furcellaria Lumbricalis Community in the Waters of the West Estonian Archipelago, Baltic Sea

A free-floating, loose form of Furcellaria lumbricalis (Huds.) Lamour is rare in the Baltic Sea area. Kassari Bay, situated in the West Estonian Archipelago Sea area contains the largest known community of this kind. Here the free-floating mixed Furcellaria lumbricalis-Coccotylus truncatus (Paela) M. J. Wynne et J. N. Heine community inhabits sandy bottom, covering up to 120 km². Commercial exploitation of the community started in 1966 and has led to regular monitoring surveys for the quantification of the commercial resource. The aim of the present study was to determine the potential growth rates of the two community-forming species as well as to test different environmental factors affecting their growth. Results showed that the highest growth rates were measured in shallower depths (4 m) for both species. The seasonal growth pattern was also very similar for both species, showing the highest growth rates during the beginning of summer. Incubation of both species in another sea area with apparently similar basic environmental conditions (the northern part of the Gulf of Riga, Kõiguste Bay) resulted in significantly lower growth rates during the whole incubation period.