Hepatic stellate cells function as regulatory bystanders

Regulatory T cells (Tregs) contribute significantly to the tolerogenic nature of the liver. The mechanisms, however, underlying liver-associated Treg induction are still elusive. We recently identified the vitamin A metabolite, retinoic acid (RA), as a key controller that promotes TGF-β-dependent Foxp3(+) Treg induction but inhibits TGF-β-driven Th17 differentiation. To investigate whether the RA producing hepatic stellate cells (HSC) are part of the liver tolerance mechanism, we investigated the ability of HSC to function as regulatory APC. Different from previous reports, we found that highly purified HSC did not express costimulatory molecules and only upregulated MHC class II after in vitro culture in the presence of exogenous IFN-γ. Consistent with an insufficient APC function, HSC failed to stimulate naive OT-II TCR transgenic CD4(+) T cells and only moderately stimulated α-galactosylceramide-primed invariant NKT cells. In contrast, HSC functioned as regulatory bystanders and promoted enhanced Foxp3 induction by OT-II TCR transgenic T cells primed by spleen dendritic cells, whereas they greatly inhibited the Th17 differentiation. Furthermore, the regulatory bystander capacity of the HSC was completely dependent on their ability to produce RA. Our data thus suggest that HSC can function as regulatory bystanders, and therefore, by promoting Tregs and suppressing Th17 differentiation, they might represent key players in the mechanism that drives liver-induced tolerance.     

The 5S ribosomal RNA sequences of a red algal rhodoplast and a gymnosperm chloroplast. Implications for the evolution of plastids and cyanobacteria

Summary The 5S ribosomal RNA sequences have been determined for the rhodoplast of the red algaPorphyra umbilicalis and the chloroplast of the coniferJuniperus media. The 5S RNA sequence of theVicia faba chloroplast is corrected with respect to a previous report. A survey of the known sequences and secondary structures of 5S RNAs from plastids and cyanobacteria shows a close structural similarity between all 5S RNAs from land plant chloroplasts. The algal plastid 5S RNAs on the other hand show much more structural diversity and have certain structural features in common with bacterial 5S RNAs. A dendrogram constructed from the aligned sequences by a clustering algorithm points to a common ancestor for the present-living cyanobacteria and the land plant plastids. However, the algal plastids branch off at an early stage within the plastid-cyanobacteria cluster, before the divergence between cyanobacteria and land plant chloroplasts. This evolutionary picture points to the occurrence of multiple endosymbiotic events, with the ancestors of the present algal plastids already established as photosynthetic endosymbionts at a time when the ancestors of the present land plant chloroplasts were still free-living cells.     

Pollen viability assessments in blackberries (Rubus subgen.Rubus)

Pollen viability has been investigated in 20 blackberry species using 3 methods, (1) cotton blue, (2) TTC, and (3) germination in a sucrose solution. Significant differences were found between species. Correlations between high pollen viability and high seed set were also obtained.     

Photostimulation effects on chicken egg development: Perspectives on human newborn treatment

It is well known that, under exposure to bright light, eggs tend to hatch earlier than control, without any damage to the birds. This report aims to systematically show the effect and establishes a proposal for a possible application to accelerate chicken egg formation, which could be extrapolated or adapted as a great advance in premature human newborns. Comparing several protocols, the experiments show that lower doses of light slowly delivered for 24 h promote higher efficiency in embryo development, increasing on average 25% of its size and more than 70% in weight when compared to the control. This weight difference shows promising results compared to rates of up to 17% found in the literature. These results can be a first step to reduce the stay of premature human infants in hospitals because light, when applied in very low doses, can accelerate the natural biological processes without risks.      

Growth rate rather than growth duration drives growth heterosis in maize B104 hybrids

Research in maize is often performed using inbred lines that can be readily transformed, such as B104. However, because the B104 line flowers late, the kernels do not always mature before the end of the growing season, hampering routine seed yield evaluations of biotech traits introduced in B104 at many geographical locations. Therefore, we generated five hybrids by crossing B104 with the early‐flowering inbred lines CML91, F7, H99, Mo17, and W153R and showed in three consecutive years that the hybrid lines proved to be suitable to evaluate seed yield under field conditions in a temperate climate. By assessing the two main processes driving maize leaf growth, being rate of growth (leaf elongation rate or LER) and the duration of growth (leaf elongation duration or LED) in this panel of hybrids, we showed that leaf growth heterosis was mainly the result of increased LER and not or to a lesser extent of LED. Ectopic expression of the transgenes GA20‐oxidase (GA20‐OX) and PLASTOCHRON1 (PLA1), known to stimulate the LER and LED, respectively, in the hybrids showed that leaf length heterosis can be stimulated by increased LER, but not by LED, indicating that LER rather than LED is the target for enhancing leaf growth heterosis.     

Chromosomal distribution of the hamster intracisternal A-particle (IAP) retrotransposons

The retrotransposon-like elements of the intracisternal A-particle (IAP) sequences occur in about 900 copies per haploid hamster cell genome. By applying the fluorescent in situ hybridization (FISH) technique and four different, cloned segments of the IAP element as hybridization probes, these elements were found to be distributed in specific patterns over many of the 44 hamster chromosomes. The hybridization patterns were very similar regardless of whether all four probes or only the IAPI probe carrying the long terminal repeat (LTR) region were used. The IAP elements were found most abundantly, though not exclusively, on the short arms of at least 12 of the autosomes. Of the sex chromosomes, the shorter Y chromosome was stained on both arms, and the X chromosome on one arm by the IAP probes. Primary Syrian hamster cells, the established Syrian hamster cell line BHK21, and the adenovirus type 12 (Ad12)-transformed BHK21 cell line T637 yielded very similar results. In Chinese hamster ovary (CHO) or 3T3 mouse cells, signals could not be elicited by FISH using the Syrian hamster IAP probes. On Southern blots, the DNAs from these cell lines hybridized very weakly, if at all, to the IAP sequences. Thus, IAP sequences were retroposed after Syrian hamster and mouse or Syrian and Chinese hamsters had diverged in evolution.     

Growth and growth substrate levels in spinach under non-steady state conditions of nitrogen nutrition and light

Spinach plants ( Spinacia oleracea L. cv. Subito) were grown in a complete nutrient solution under ample light intensity (14 h day⁻¹ at 660 μmol m⁻² s⁻¹) before being transferred either to a minus-N solution (experiment 1), or to limiting light conditions (6 h day⁻¹ at 220 μmol m⁻² s⁻¹; experiment 2). Shoot growth in experiment 1 decreased significantly from 0.24 day⁻¹ to 0.07 day⁻¹ after the fourth day of transfer. Root relative growth rate increased after 1 day from 0.25 to 0.31 day⁻¹, but decreased on the fifth day after transfer to 0.11 day⁻¹. Shoot growth in experiment 2 decreased significantly from 0.25 to 0.17 day⁻¹ after the fourth day of transfer, while root growth decreased to half of its original level (0.25 day⁻¹) already on the second day. Growth substrate levels in the plants (free sugars, free amino acids) and starch levels depended on the plant age, the moment in the diurnal cycle, and the imposed treatment. Fluctuations in shoot growth or root growth resulting from the light or N limitation could not be explained by a correspondent increase or decrease in the levels of growth substrates. The hypotheses underlying the functional equilibrium theory, assuming shoot and root growth to be controlled by N- and C-containing substrates respectively, and several other growth and partitioning models are therefore questioned. A neglect of the osmotic role of the free sugars in these models might be the explanation for this.     

Interaction between a Domain of the Negative Regulator of the Ras-ERK Pathway,SPRED1 Protein,and the GTPase-activating Protein-related Domain of Neurofibromin Is Implicated in Legius Syndrome and Neurofibromatosis Type 1

Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple café-au-lait macules, axillary freckling, learning disabilities, and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the Ras-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids and the C-terminal 20 amino acids of the GTPase-activating protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing α-helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120^GAP. Several mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome.     

Real-time PCR for detection and quantification,and histological characterization of <Emphasis Type="Italic">Neonectria ditissima</Emphasis> in apple trees

Key message

We designed a pair of primers from a region of the β-tubulin gene to detect and quantify Neonectria ditissima in wood of some infected apple cultivars, and optimized light microscopy to study fungal-plant interactions.

Abstract

Neone ctria ditissima, the causal pathogen of fruit tree canker, is a sordariomycete fungus that affects apple orchards, especially in north-western Europe. To prevent serious disease epidemics, an accurate, rapid, and sensitive method for detection of N. ditissima is needed for pathogen identification. A quantitative real-time PCR (qPCR) assay was developed for both detection and quantification of this pathogen in infected apple cultivars. Several primer sets were designed from regions of the β-tubulin gene. One primer set passed several validation tests, and the melting curve confirmed species-specific amplification of the correct product. In addition, the N. ditissima biomass could be detected at variable amounts in samples from the infection sites of six different cultivars, with ‘Aroma’ having the lowest amount of N. ditissima biomass and ‘Elise’ the highest. To complement the qPCR results, tissue from detached shoots and 1-year-old trees of ‘Cox’s Orange Pippin’ (susceptible) and ‘Santana’ (partially resistant) was used in a histopathology study. In both detached shoots and trees, fungal hyphae were found in cells of all tissues. No qualitative differences in the anatomy of the infected samples were observed between the cultivars. In the detached shoot experiment, both cultivars were affected but differences in the rate of disease progression suggest that the partially resistant cultivar could resist the fungus longer. The qPCR assay developed in our study produced reproducible results and can be used for detection of N. ditissima in infected trees.