Antitranspirant functions of stem periderms and their influence on corticular photosynthesis under drought stress

Transpiration and photosynthesis of current-year stems and adult leaves of different deciduous tree species were investigated to estimate their probable influence on carbon balance. Peridermal transpiration of young stems was found to be rather small as compared to the transpiration of leaves (stem/leaf like 1/5–1/20). A characteristic that was mainly attributable to the lower peridermal conductance to water and CO₂, which made up only 8–28% of stomatal conductance. Water vapour conductance was significantly lower in stems, but also non-responsive to PAR, which led to a comparatively higher water use efficiency (WUE, ratio assimilation/transpiration). Thus, although corticular photosynthesis reached only 11–37% of leaf photosynthesis, it may be a means of improving the carbon balance of stems under limited water availability. The influence of drought stress on primary photosynthetic reactions was also studied. Under simulated drought conditions the drying time needed to provoke a 50% reduction (t ₅₀) in dark- and light-adapted PSII efficiency (Fv/Fm, ΔF/Fm′) was up to ten times higher in stems than in leaves. Nevertheless, up to a relative water deficit (RWD) of around 40–50% dark-adapted PSII efficiency of leaves and stems was rather insensitive to dehydration, showing that the efficiency of open PS II reaction centres is not impaired. Thus, it may be concluded that in stems as well as in leaves the primary site of drought damage is at the level of dark enzyme reactions and not within PSII. However, enduring severe drought caused photoinhibitory damage to the photosynthetic apparatus of leaves and stems; thereby RWD₅₀ values (= RWD needed to provoke a 50% reduction in Fv/Fm ad ΔF/Fm′) were comparably lower in stems as compared to leaves, indicating a possibly higher drought sensitivity of the cortex chlorenchyma.     

Isozyme differentiation of aldolase and pyruvate kinase in fetal,regenerating, preneoplastic,and malignant rat hepatocytes during culture

Summary Aldolase and pyruvate kinase isozymes were investigated in cultured hepatocytes from fetal, regenerating, and 2-acetyl-aminofluorene-fed rat liver as well as in some epithelial liver cell lines. Our results show that: (a) cell proliferation and prolonged expression of specific isozymes were found only in cultured hepatocytes from 17-day old fetuses; (b) the fetal type of pyruvate kinase expressed in regenerating and carcinogen-treated liver was temporarily lost only in cultured hepatocytes from regenerating liver; (c) the adult type of aldolase and pyruvate kinase was absent in one epithelial cell line derived from a carcinogen-treated liver and in the hepatoma tissue cell (HTC) line but was found in the Faza clone of the Reuber H₃₅ cell line during the 50 first passages in vitro; and (d) the isozyme pattern of pyruvate kinase was always more strongly shifted than that of aldolase. The observations suggest that: (a) hepatocytes from carcinogen-treated liver exhibit the same lack of ability to proliferate in primary culture as normal adult hepatocytes; (b) adult hepatocytes can produce fetal isozymes without prior cell division; (c) pyruvate kinase is a stronger marker of dedifferentiation (retrodifferentiation) than aldolase; and (d) regulatory processes of isozyme expression are different during ontogenesis, regeneration, and hepatocarcinogenesis. This work was supported by the “Institut National de la Santé et de la Recherche Médicale” and the “Fondation pour la Recherche Medicale Fran?aise”     

M-CSF stimulated differentiation requires persistent MEK activity and MAPK phosphorylation independent of Grb2-Sos association and phosphatidylinositol 3-kinase activity

Macrophage colony-stimulating factor (M-CSF) is a physiological regulator of monocyte-macrophage lineage. Ectopic expression of the M-CSF receptor (M-CSFR, or Fms) in murine myeloid cell line FDC-P1 (FD/Fms cells) results in M-CSF-dependent macrophage differentiation. Previously, we observed that M-CSF induces two temporally distinct phases of mitogen-activated protein kinase (MAPK) phosphorylation. Here we show that levels of phosphorylated MAPK kinase MEK1 follow the same kinetics as MAPK phosphorylation, characterized by an early and transient phase (the first 30 min of M-CSF stimulation) and a late and persistent phase from 4 h of stimulation. The MEK inhibitor U0126 strongly inhibited both phases of MAPK phosphorylation as well as FD/Fms cell differentiation, indicating that MAPK may relay M-CSF differentiation signaling downstream of M-CSFR. Treatment of FD/Fms cells with U0126 during the first hour of M-CSF stimulation reversibly blocked the early phase of MAPK phosphorylation but did not affect differentiation. In contrast, U0126 still inhibited FD/Fms cell differentiation when its addition was delayed by 24 h. This demonstrated that late and persistent MEK activity is specifically required for macrophage differentiation to occur. Furthermore, disrupting Grb2-Sos complexes with a specific blocking peptide did not prevent FD/Fms cells differentiation in response to M-CSF, nor did it abolish MAPK phosphorylation. The role of phosphatidylinositol 3-kinase (PI 3-kinase), another potential regulator of the MAPK pathway, was examined using the specific inhibitor LY294002. This compound could not impede FD/Fms cell commitment to macrophage differentiation and did not significantly affect MAPK phosphorylation in response to M-CSF. Therefore, M-CSF differentiation signaling in myeloid progenitor cells is mediated through persistent MEK activity but it is not strictly dependent upon Grb2-Sos interaction or PI 3-kinase activity.     

Structural stability and dynamics of hydrogenated and perdeuterated cytochrome P450cam (CYP101)

Perdeuterated and hydrogenated cytochrome P450cam (P450cam), from Pseudomonas putida, has been characterized concerning thermal stability and structural dynamics. For the first time, Fourier transform infrared (FTIR) spectroscopy was used to characterize a perdeuterated protein. The secondary structure compositions were determined from the fitted amide I' spectral region, giving band populations at 10 degrees C for the perdeuterated protein of 22% between 1605 and 1624 cm(-1) (beta-sheets), 47% between 1633 and 1650 cm(-1) (alpha-helix (29%) plus unordered/3(10)-helix (18%)), and 28% between 1657 and 1677 cm(-1) (turns) and for the hydrogenated protein of 22% between 1610 and 1635 cm(-1) (beta-sheets), 52% between 1640 and 1658 cm(-1) (alpha-helix (41%) plus unordered/3(10)-helix (11%)), and 24% between 1665 and 1680 cm(-1) (turns).Thermal unfolding experiments revealed that perdeuterated P450cam was less stable than the hydrogenated protein. The midpoint transition temperatures were 60.8 and 64.4 degrees C for the perdeuterated and hydrogenated P450cam, respectively. Step-scan time-resolved FTIR was applied to the P450cam-CO complex to study the ligand-rebinding process after flash photolysis. Rebinding of the ligand occurred with the same kinetics and rate constants k(on), 8.9 x 10(4) and 8.3 x 10(4) M(-1) s(-1) for the perdeuterated and hydrogenated P450cam, respectively.Perdeuterated P450cam was expressed for a neutron crystallographic study to determine the specific hydration states and hydrogen-bonding networks at the active site. The analyses presented here show that perdeuterated P450cam is structurally similar to its hydrogenated counterpart, despite its reduced thermal stability, suggesting that information obtained from the neutron structure will be representative of the normal hydrogenated P450cam.     

Fostering responsible research with genome editing technologies: a European perspective

In this consensus paper resulting from a meeting that involved representatives from more than 20 European partners, we recommend the foundation of an expert group (European Steering Committee) to assess the potential benefits and draw-backs of genome editing (off-targets, mosaicisms, etc.), and to design risk matrices and scenarios for a responsible use of this promising technology. In addition, this European steering committee will contribute in promoting an open debate on societal aspects prior to a translation into national and international legislation.     

Establishment,Characterization and Chemosensitivity of Three Mismatch Repair Deficient Cell Lines from Sporadic and Inherited Colorectal Carcinomas

Background

Colorectal cancer (CRC) represents a morphologic and molecular heterogenic disease. This heterogeneity substantially impairs drug effectiveness and prognosis. The subtype of mismatch repair deficient (MMR-D) CRCs, accounting for about 15% of all cases, shows particular differential responses up to resistance towards currently approved cytostatic drugs. Pre-clinical in vitro models representing molecular features of MMR-D tumors are thus mandatory for identifying biomarkers that finally help to predict responses towards new cytostatic drugs. Here, we describe the successful establishment and characterization of three patient-derived MMR-D cell lines (HROC24, HROC87, and HROC113) along with their corresponding xenografts.

Methodology

MMR-D cell lines (HROC24, HROC87, and HROC113) were established from a total of ten clinicopathological well-defined MMR-D cases (120 CRC cases in total). Cells were comprehensively characterized by phenotype, morphology, growth kinetics, invasiveness, and molecular profile. Additionally, response to clinically relevant chemotherapeutics was examined in vitro and in vivo.

Principal Findings

Two MMR-D lines showing CIMP-H derived from sporadic CRC (HROC24: K-ras^wt, B-raf^mut, HROC87: K-ras^wt, B-raf^mut), whereas the HROC113 cell line (K-ras^mut, B-raf^wt) was HNPCC-associated. A diploid DNA-status could be verified by flow cytometry and SNP Array analysis. All cell lines were characterized as epithelial (EpCAM⁺) tumor cells, showing surface tumor marker expression (CEACAM⁺). MHC-class II was inducible by Interferon-γ stimulation. Growth kinetics as well as invasive potential was quite heterogeneous between individual lines. Besides, MMR-D cell lines exhibited distinct responsiveness towards chemotherapeutics, even when comparing in vitro and in vivo sensitivity.

Conclusions

These newly established and well-characterized, low-passage MMR-D cell lines provide a useful tool for future investigations on the biological characteristics of MMR-D CRCs, both of sporadic and hereditary origin. Additionally, matched patient-derived immune cells allow for comparative genetic studies.     

Different Assembly Processes Drive Shifts in Species and Functional Composition in Experimental Grasslands Varying in Sown Diversity and Community History

Background

The prevalence of different biotic processes (limiting similarity, weaker competitor exclusion) and historical contingency due to priority effects are in the focus of ongoing discussions about community assembly and non-random functional trait distributions.

Methodology/Principal Findings

We experimentally manipulated assembly history in a grassland biodiversity experiment (Jena Experiment) by applying two factorially crossed split-plot treatments to all communities: (i) duration of weeding (never weeded since sowing or cessation of weeding after 3 or 6 years); (ii) seed addition (control vs. seed addition 4 years after sowing). Spontaneous colonization of new species in the control treatment without seed addition increased realized species richness and functional richness (FRic), indicating continuously denser packing of niches. Seed addition resulted in forced colonization and increased realized species richness, FRic, functional evenness (FEve) and functional divergence (FDiv), i.e. higher abundances of species with extreme trait values. Furthermore, the colonization of new species led to a decline in FEve through time, suggesting that weaker competitors were reduced in abundance or excluded. Communities with higher initial species richness or with longer time since cessation of weeding were more restricted in the entry of new species and showed smaller increases in FRic after seed addition than other communities. The two assembly-history treatments caused a divergence of species compositions within communities originally established with the same species. Communities originally established with different species converged in species richness and functional trait composition over time, but remained more distinct in species composition.

Conclusions/Significance

Contrasting biotic processes (limiting similarity, weaker competitor exclusion) increase functional convergence between communities initially established with different species. Historical contingency with regard to realized species compositions could not be eradicated by cessation of weeding or forced colonization and was still detectable 5 years after application of these treatments, providing evidence for the role of priority effects in community assembly.     

In Silico Analysis of Usher Encoding Genes in Klebsiella pneumoniae and Characterization of Their Role in Adhesion and Colonization

Chaperone/usher (CU) assembly pathway is used by a wide range of Enterobacteriaceae to assemble adhesive surface structures called pili or fimbriae that play a role in bacteria-host cell interactions. In silico analysis revealed that the genome of Klebsiella pneumoniae LM21 harbors eight chromosomal CU loci belonging to γκп and ϭ clusters. Of these, only two correspond to previously described operons, namely type 1 and type 3-encoding operons. Isogenic usher deletion mutants of K. pneumoniae LM21 were constructed for each locus and their role in adhesion to animal (Intestine 407) and plant (Arabidopsis thaliana) cells, biofilm formation and murine intestinal colonization was investigated. Type 3 pili usher deleted mutant was impaired in all assays, whereas type 1 pili usher deleted mutant only showed attenuation in adhesion to plant cells and in intestinal colonization. The LM21ΔkpjC mutant was impaired in its capacity to adhere to Arabidopsis cells and to colonize the murine intestine, either alone or in co-inoculation experiments. Deletion of LM21kpgC induced a significant decrease in biofilm formation, in adhesion to animal cells and in colonization of the mice intestine. The LM21∆kpaC and LM21∆kpeC mutants were only attenuated in biofilm formation and the adhesion abilities to Arabidopsis cells, respectively. No clear in vitro or in vivo effect was observed for LM21∆kpbC and LM21∆kpdC mutants. The multiplicity of CU loci in K. pneumoniae genome and their specific adhesion pattern probably reflect the ability of the bacteria to adhere to different substrates in its diverse ecological niches.