Role of permanent dicentric systems in carrot somatic embryogenesis

Summary A permanent dicentric chromosome system was studied on carrot cultures and regenerated somatic embryos at different stages of development. The large chromosomal variability of the cultures and the presence of the breakage-fusion bridge cycle did not interfere with the initial developmental process up to the seedling stage but subsequent growth proceeded only if healing of the broken ends or dicentric loss had occurred. The behaviour of the dicentric chromosome in culture and during somatic embryogenesis is discussed in relation to chromosomal variability, abnormal development and the somaclonal variation that such mechanisms may generate in regenerated plants.     

Structure-function relationship in spinach ferredoxin-NADP+ reductase as studied by limited proteolysis

Studies of limited proteolysis on purified ferredoxin-NADP+ reductase with various proteases were performed in the presence and absence of the flavoprotein ligands. Both the diaphorase and the ferredoxin-dependent activities of the enzyme were followed as well as the proteolytic pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with further characterization of the polypeptides produced. These experiments revealed that only two regions of the flavoprotein are susceptible to the attack of the proteases used: (a) the N-terminal chain which can be cleaved only up to Lys35 and (b) the sequence segment 235-250. It can be inferred that these regions are on the surface of the protein molecule and presumably have a very flexible conformation adaptable to the protease active site. The deletion of the N-terminal region up to Thr36 of the native reductase (Mr 35,000) produced a truncated form (Mr about 31,000) which had full diaphorase activity but lost the capacity to catalyze the ferredoxin-dependent reaction. Proteolytic cleavage at the 235-250 segment of the sequence yielded a nicked protein (Mr about 30,000 by gel filtration; 23,000 plus 7,000 in denaturing electrophoresis) devoid of both activities. Protection by the flavoprotein ligands implies that the 23-35 region of the sequence is part of the binding site for ferredoxin and the 235-250 polypeptide segment is in the NADP(+)-binding site.     

DNA heterogeneity and genetic control of tumorigenesis in nicotiana tumorous and nontumorous genotypes

The possible relevance of changes in amounts of highly repetitive DNA sequences for plant differentiation and dedifferentiation processes has been suggested in several cases. Data are lacking however on (1) the genetic control of these phenomena and (2) cause-effect relationships between DNA amplification and specific ontogenetic patterns. The present study was carried out on a Nicotiana genetic system consisting of the tumorous amphidiploid N glauca X N langsdorffii, a nontumorous mutant of it, their F₁, and a backcross to the tumorous parent. Backcross segregation ratios were shown to be compatible with a “single gene” hypothesis, the F₁ plant being nontumorous but showing a low percentage of tumors induced by wounds, 6-azauracil or X-rays. In vitro studies of excised pith tissue grown on Linsmaier and Skoog medium for different periods of time showed the presence, confirmed by cytological analyses, of amplification of highly repetitive sequences only in the nontumorous stock, as judged by reassociation experiments in the first 24–96 hours of culture. CsCl analytical ultracentrifugation of those sequences showed the appearance in the same stock of a heavy DNA satellite (density = 1.721 gm/ml), whose presence was also confirmed by derivative melting curves. Amplification seemed to be essential for the initiation of cell division, which was completely inhibited in the nontumorous genotype and partially influenced in the F₁ by incorporation during the critical period (24–96 hours of the primary explant) of 5-bromo-2′-deoxy-uridine. The results are discussed in terms of an hypothesis of an integrated gene-controlled, hormone-mediated regulatory system of cell proliferation involving changes in target repetitive DNA sequences.     

ErbB2 Receptor Over-Expression Improves Post-Traumatic Peripheral Nerve Regeneration in Adult Mice

In a transgenic mice (BALB-neuT) over-expressing ErbB2 receptor, we investigated the adult mouse median nerve in physiological and pathological conditions. Results showed that, in physiological conditions, the grip function controlled by the median nerve in BALB-neuT mice was similar to wild-type (BALB/c). Stereological assessment of ErbB2-overexpressing intact nerves revealed no difference in number and size of myelinated fibers compared to wild-type mice. By contrast, after a nerve crush injury, the motor recovery was significantly faster in BALB-neuT compared to BALB/c mice. Moreover, stereological assessment revealed a significant higher number of regenerated myelinated fibers with a thinner axon and fiber diameter and myelin thickness in BALB-neuT mice. At day-2 post-injury, the level of the mRNAs coding for all the ErbB receptors and for the transmembrane (type III) Neuregulin 1 (NRG1) isoforms significantly decreased in both BALB/c and BALB-neuT mice, as shown by quantitative real time PCR. On the other hand, the level of the mRNAs coding for soluble NRG1 isoforms (type I/II, alpha and beta) increased at the same post-traumatic time point though, intriguingly, this response was significantly higher in BALB-neuT mice with respect to BALB/c mice. Altogether, these results suggest that constitutive ErbB2 receptor over-expression does not influence the physiological development of peripheral nerves, while it improves nerve regeneration following traumatic injury, possibly through the up-regulation of soluble NRG1 isoforms.     

Dynamics of the temperature-humidity index in the Mediterranean basin

The study was aimed at describing the temperature humidity index (THI) dynamics over the Mediterranean basin for the period 1951–2007. The THI combines temperature and humidity into a single value, and may help to predict the effects of environmental warmth in farm animals. In particular, on the basis of THI values, numerous studies have been performed to establish thresholds for heat stress in dairy cows. The THI was calculated by using monthly mean values of temperature and humidity obtained from the National Center for Environmental Prediction/National Center for Atmospheric Research reanalysis project. The analysis demonstrated a high degree of heterogeneity of THI patterns over the Mediterranean basin, a strong north–south gradient, and an overall warming during the study period, which was particularly marked during summer seasons. Results indicated that several areas of the basin present summer THI values which were unfavorable to cow welfare and productivity, and that risk of heat stress for cows is generally greater in the countries of the south coast of the basin. Furthermore, THI data from the summer 2003 revealed that severe positive anomalies may impact areas normally characterized by a favorable climate for animal production. In conclusion, THI dynamics should be taken into careful consideration by farmers and policy makers operating in Mediterranean countries when planning investments in the sector of animal production. The investments should at least partially be directed towards implementation of adaptation measures, which may help to alleviate the impact of hot on farm animals welfare, performance and health.     

FUS-dependent liquid–liquid phase separation is important for DNA repair initiation

RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid–liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, and 53BP1 and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using superresolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nanofoci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSB repair complexes.