The immediate-early growth response in regenerating liver and insulin-stimulated H-35 cells: comparison with serum-stimulated 3T3 cells and identification of 41 novel immediate-early genes.

Liver regeneration provides a unique system for analysis of mitogenesis in intact, fully developed animals. Cellular immediate-early genes likely play an important role in cell cycle regulation and have been extensively studied in mitogen-stimulated fibroblasts lymphocytes but not in liver. We have begun to characterize the immediate-early growth response genes of mitogen-stimulated liver cells, specifically, regenerating liver and insulin-stimulated Reuber H-35 hepatoma cells, and to address differences in growth response between different cell types. Through subtraction and differential screening of cDNA libraries from regenerating liver and insulin-treated H-35 cells, we have extensively characterized 341 differentially expressed clones and identified 52 immediate-early genes. These genes have been partially sequenced and subjected to Northern (RNA) blot analysis, and 41 appear to be novel. Surprisingly, two-thirds of these genes are also expressed in BALB/c 3T3 cells, but only 10 were identified in previous studies of 3T3 cells, and of these, 6 include well-known genes like jun and fos, and only 4 are novel. Approximately one-third of the immediate-early genes identified in mitogen-stimulated liver cells or serum-stimulated NIH 3T3 cells are expressed in a tissue-specific fashion, indicating that cell type-specific regulation of the proliferative response occurs during the immediate-early period. Our findings indicate that the immediate-early response is unusually complex for the first step in a regulatory cascade, suggesting that multiple pathways must be activated. The abundance of immediate-early genes and the highly varied pattern of their expression in different cell types suggest that the tissue specificity of the proliferative response arises from the particular set of these genes expressed in a given tissue.     

Effect of cold acclimation on the photosynthetic performance of two ecotypes of Colobanthus quitensis (Kunth) Bartl

The effects of cold acclimation of two ecotypes (Antarctic and Andes) of Colobanthus quitensis (Kunth) Bartl. Caryophyllaceae on their photosynthetic characteristics and performance under high light (HL) were compared. Non-acclimated plants of the Antarctic ecotype exhibited a higher (34%) maximal rate of photosynthesis than the Andes ecotype. In cold-acclimated plants the light compensation point was increased. Dark respiration was significantly increased during the exposure to 4 degrees C in both ecotypes. Cold-acclimated Antarctic plants showed higher Phi(PSII) and qP compared with the Andes ecotype. In addition, the Antarctic ecotype exhibited higher heat dissipation (NPQ), especially in the cold-acclimated state, which was mainly associated with the fast relaxing component of non-photochemical quenching (NPQ(F)). By contrast, the Andes ecotype exhibited a lower NPQ(F) and a significant increase in the slowly relaxing component (NPQ(s)) at low temperature and HL, indicating higher sensitivity to low temperature-induced photoinhibition. Although the xanthophyll cycle was fully operational in both ecotypes, cold-acclimated Antarctic plants exposed to HL exhibited higher epoxidation state of the xanthophyll cycle pigments (EPS) compared with the cold-acclimated Andes ecotype. Thus, the photosynthetic apparatus of the Antarctic ecotype operates more efficiently than that of the Andes one, under a combination of low temperature and HL. The ecotype differences are discussed in relation to the different climatic conditions of the two Colobanthus.     

Adaptive processes drive ecomorphological convergent evolution in antwrens (Thamnophilidae)

Phylogenetic niche conservatism (PNC) and convergence are contrasting evolutionary patterns that describe phenotypic similarity across independent lineages. Assessing whether and how adaptive processes give origin to these patterns represent a fundamental step toward understanding phenotypic evolution. Phylogenetic model‐based approaches offer the opportunity not only to distinguish between PNC and convergence, but also to determine the extent that adaptive processes explain phenotypic similarity. The Myrmotherula complex in the Neotropical family Thamnophilidae is a polyphyletic group of sexually dimorphic small insectivorous forest birds that are relatively homogeneous in size and shape. Here, we integrate a comprehensive species‐level molecular phylogeny of the Myrmotherula complex with morphometric and ecological data within a comparative framework to test whether phenotypic similarity is described by a pattern of PNC or convergence, and to identify evolutionary mechanisms underlying body size and shape evolution. We show that antwrens in the Myrmotherula complex represent distantly related clades that exhibit adaptive convergent evolution in body size and divergent evolution in body shape. Phenotypic similarity in the group is primarily driven by their tendency to converge toward smaller body sizes. Differences in body size and shape across lineages are associated to ecological and behavioral factors.     

Binding of sulphonated indigo derivatives to RepA-WH1 inhibits DNA-induced protein amyloidogenesis

The quest for inducers and inhibitors of protein amyloidogenesis is of utmost interest, since they are key tools to understand the molecular bases of proteinopathies such as Alzheimer, Parkinson, Huntington and Creutzfeldt–Jakob diseases. It is also expected that such molecules could lead to valid therapeutic agents. In common with the mammalian prion protein (PrP), the N-terminal Winged-Helix (WH1) domain of the pPS10 plasmid replication protein (RepA) assembles in vitro into a variety of amyloid nanostructures upon binding to different specific dsDNA sequences. Here we show that di- (S2) and tetra-sulphonated (S4) derivatives of indigo stain dock at the DNA recognition interface in the RepA-WH1 dimer. They compete binding of RepA to its natural target dsDNA repeats, found at the repA operator and at the origin of replication of the plasmid. Calorimetry points to the existence of a major site, with micromolar affinity, for S4-indigo in RepA-WH1 dimers. As revealed by electron microscopy, in the presence of inducer dsDNA, both S2/S4 stains inhibit the assembly of RepA-WH1 into fibres. These results validate the concept that DNA can promote protein assembly into amyloids and reveal that the binding sites of effector molecules can be targeted to inhibit amyloidogenesis.     

The unusual intersubunit ferroxidase center of Listeria innocua Dps is required for hydrogen peroxide detoxification but not for iron uptake. A study with site-specific mutants

The role of the ferroxidase center in iron uptake and hydrogen peroxide detoxification was investigated in Listeria innocua Dps by substituting the iron ligands His31, His43, and Asp58 with glycine or alanine residues either individually or in combination. The X-ray crystal structures of the variants reveal only small alterations in the ferroxidase center region compared to the native protein. Quenching of the protein fluorescence was exploited to assess stoichiometry and affinity of metal binding. Substitution of either His31 or His43 decreases Fe(II) affinity significantly with respect to wt L. innocua Dps (K approximately 10(5) vs approximately 10(7) M(-)(1)) but does not alter the binding stoichiometry [12 Fe(II)/dodecamer]. In the H31G-H43G and H31G-H43G-D58A variants, binding of Fe(II) does not take place with measurable affinity. Oxidation of protein-bound Fe(II) increases the binding stoichiometry to 24 Fe(III)/dodecamer. However, the extent of fluorescence quenching upon Fe(III) binding decreases, and the end point near 24 Fe(III)/dodecamer becomes less distinct with increase in the number of mutated residues. In the presence of dioxygen, the mutations have little or no effect on the kinetics of iron uptake and in the formation of micelles inside the protein shell. In contrast, in the presence of hydrogen peroxide, with increase in the number of substitutions the rate of iron oxidation and the capacity to inhibit Fenton chemistry, thereby protecting DNA from oxidative damage, appear increasingly compromised, a further indication of the role of ferroxidation in conferring peroxide tolerance to the bacterium.     

Exit,Punishment and Rewards in Commons Dilemmas: An Experimental Study

Commons dilemmas are interaction situations where a common good is provided or exploited by a group of individuals so that optimal collective outcomes clash with private interests. Although in these situations, social norms and institutions exist that might help individuals to cooperate, little is known about the interaction effects between positive and negative incentives and exit options by individuals. We performed a modified public good game experiment to examine the effect of exit, rewards and punishment, as well as the interplay between exit and rewards and punishment. We found that punishment had a stronger effect than rewards on cooperation if considered by itself, whereas rewards had a stronger effect when combined with voluntary participation. This can be explained in terms of the ‘framing effect’, i.e., as the combination of exit and rewards might induce people to attach higher expected payoffs to cooperative strategies and expect better behaviour from others.     

A uracil-DNA glycosylase inhibitor encoded by a non-uracil containing viral DNA

Uracil-DNA glycosylase (UDG) is an enzyme involved in the base excision repair pathway. It specifically removes uracil from both single-stranded and double-stranded DNA. The genome of the Bacillus subtilis phage 29 is a linear double-stranded DNA with a terminal protein covalently linked at each 5'-end. Replication of 29 DNA starts by a protein-priming mechanism and generates intermediates that have long stretches of single-stranded DNA. By using in vivo chemical cross-linking and affinity chromatography techniques, we found that UDG is a cellular target for the early viral protein p56. Addition of purified protein p56 to B. subtilis extracts inhibited the endogenous UDG activity. Moreover, extracts from 29-infected cells were deficient in UDG activity. We suggested that inhibition of the cellular UDG is a defense mechanism developed by 29 to prevent the action of the base excision repair pathway if uracil residues arise in their replicative intermediates. Protein p56 is the first example of a UDG inhibitor encoded by a non-uracil-containing viral DNA.