Distinguishing between metabolically active and inactive roots by combined staining with 2,3,5-triphenyltetrazolium chloride and image colour analysis

The objective of the present work was to develop a method to distinguish between metabolically inactive and active parts of plant roots. White clover (Trifolium repens L.) roots were stained with 2,3,5-triphenyltetrazolium chloride (TTC) followed by root colour classification with an interactive scanner-based image analysis programme (WinRHIZO). Roots inactivated by boiling were unstained and pale brown, whereas fresh samples with predominantly metabolically active roots turned dark red, red or pale red after staining. A small amount of very young, presumable active roots (0.8% of total active root length) failed to stain red with TTC. The colour analysis of inactive and active roots was based on four colour classes for boiled roots and seven classes for fresh roots, respectively, as defined upon visual examination of images. Pixel colours falling outside the defined classes were allocated to the nearest defined class – an option that increased objectivity and stability and reduced the required number of colour classes. For the fresh white clover roots, 75–86% of the total root length was determined as active, while 3–7% of the boiled roots fell into the same category. The percentage of total root length measured by WinRHIZO that was identified as metabolically active was linearly correlated with the percentage of fresh roots in mixtures of fresh and boiled roots (R²=0.99). Colour classes chosen à priori from one experiment could be used to distinguish fairly satisfactorily between active and inactive roots of another white clover cultivar grown under other conditions, but failed to classify activity in ryegrass (Lolium multiflorum Lam.) root samples. In the latter case, colour classes needed to be re-defined in order to produce reliable data. Our work shows that WinRHIZOs colour identification sub-module provides a new promising tool to classify root activity as identified after staining with TTC, but colour classes must be carefully evaluated on every new occasion.     

p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and co-immunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.     

FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end–directed vesicle transport

Autophagy is the main eukaryotic degradation pathway for long-lived proteins, protein aggregates, and cytosolic organelles. Although the protein machinery involved in the biogenesis of autophagic vesicles is well described, very little is known about the mechanism of cytosolic transport of autophagosomes. In this study, we have identified an adaptor protein complex, formed by the two autophagic membrane-associated proteins LC3 and Rab7 and the novel FYVE and coiled-coil (CC) domain–containing protein FYCO1, that promotes microtubule (MT) plus end–directed transport of autophagic vesicles. We have characterized the LC3-, Rab7-, and phosphatidylinositol-3-phosphate–binding domains in FYCO1 and mapped part of the CC region essential for MT plus end–directed transport. We also propose a mechanism for selective autophagosomal membrane recruitment of FYCO1.     

Nucleocytoplasmic Shuttling of p62/SQSTM1 and Its Role in Recruitment of Nuclear Polyubiquitinated Proteins to Promyelocytic Leukemia Bodies

p62, also known as sequestosome1 (SQSTM1), A170, or ZIP, is a multifunctional protein implicated in several signal transduction pathways. p62 is induced by various forms of cellular stress, is degraded by autophagy, and acts as a cargo receptor for autophagic degradation of ubiquitinated targets. It is also suggested to shuttle ubiquitinated proteins for proteasomal degradation. p62 is commonly found in cytosolic protein inclusions in patients with protein aggregopathies, it is up-regulated in several forms of human tumors, and mutations in the gene are linked to classical adult onset Paget disease of the bone. To this end, p62 has generally been considered to be a cytosolic protein, and little attention has been paid to possible nuclear roles of this protein. Here, we present evidence that p62 shuttles continuously between nuclear and cytosolic compartments at a high rate. The protein is also found in nuclear promyelocytic leukemia bodies. We show that p62 contains two nuclear localization signals and a nuclear export signal. Our data suggest that the nucleocytoplasmic shuttling of p62 is modulated by phosphorylations at or near the most important nuclear localization signal, NLS2. The aggregation of p62 in cytosolic bodies also regulates the transport of p62 between the compartments. We found p62 to be essential for accumulation of polyubiquitinated proteins in promyelocytic leukemia bodies upon inhibition of nuclear protein export. Furthermore, p62 contributed to the assembly of proteasome-containing degradative compartments in the vicinity of nuclear aggregates containing polyglutamine-expanded Ataxin1Q84 and to the degradation of Ataxin1Q84.     

Viral and bacterial diseases of Atlantic cod Gadus morhua, their prophylaxis and treatment: a review

This review summarises the state of knowledge of both viral and bacterial diseases of Atlantic cod Gadus morhua, and their diagnosis, prophylaxis and treatment. The most important losses have been at the larval and juvenile stages, and vibriosis has long been the most important bacterial disease in cod, with Listonella (Vibrio) anguillarum dominant among pathogenic isolates. Vaccination of cod against pathogens such as L. anguillarum and Aeromonas salmonicida clearly demonstrates that the cod immune system possesses an effective memory and appropriate mechanisms sufficient for protection, at least against some diseases. Well-known viruses such as the nodavirus that causes viral encephalopathy and retinopathy (VER), infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) have been isolated from Atlantic cod and can be a potential problem under intensive rearing conditions. No commercial vaccines against nodavirus are currently available, whereas vaccines against IPNV infections based upon inactivated virus as well as IPNV recombinant antigens are available. A number of investigations of the pharmacokinetic properties of antibacterial agents in cod and their efficacy in treating bacterial infections have been reviewed.     

A branched beta-D-(1-->3,1-->6)-glucan from the marine diatom Chaetoceros debilis (Bacillariophyceae) characterized by NMR

The chrysolaminaran from the marine diatom Chaetoceros debilis was isolated and characterized by NMR spectroscopy. Cells were harvested in the stationary phase of growth after the medium had been depleted of nitrate when the chrysolaminaran content was expected to be at its highest. The chrysolaminaran was isolated with an yield of 17.5 mg/L, which corresponds to 15.8 pg/cell. 1H NMR indicated that the structure was similar to that of a beta-(1-->3) main chain with beta-(1-->6)-linked side chains. The degree of polymerization was found to be 30, corresponding to a molecular weight of approximately 4900. Thirty-three percent of the residues were found to be beta-(1-->6)-linked branches. The characteristics of the beta-(1-->6) branching were examined by NOESY NMR, which suggested pustulan-like branches, being beta-(1-->6) linked chains connected to the main chain with few branch points. Confirmation of the 1H NMR data was done by 13C-DEPT, TOCSY, COSY and HMQC NMR spectroscopy. The assignment of the resonances of the main beta-(1-->3) and beta-(1-->6) chains is presented. The structure proposed from our analyses is compared against other chrysolaminaran structures.     

Fourier Transform Infrared and Raman Spectroscopy for Characterization of Listeria monocytogenes Strains

The purpose of this study was to characterize the variation in biochemical composition of 89 strains of Listeria monocytogenes with different susceptibilities towards sakacin P, using Fourier transform infrared (FTIR) spectroscopy and Raman spectroscopy. The strains were also analyzed using amplified fragment length polymorphism (AFLP) analysis. Based on their susceptibilities to sakacin P, the 89 strains have previously been divided into two groups. Using the FTIR spectra and AFLP data, the strains were basically differentiated into the same two groups. Analyses of the FTIR and Raman spectra revealed that the strains in the two groups contained differences in the compositions of carbohydrates and fatty acids. The relevance of the variation in the composition of carbohydrates with respect to the variation in the susceptibility towards sakacin P for the L. monocytogenes strains is discussed.     

Genetics of self/nonself recognition in Serpula lacrymans

This study provides an analysis of the vegetative incompatibility system in Serpula lacrymans (Basidiomycota), a genetic system used to recognize nonself in fungi. Seventy-five worldwide isolates could be grouped into eight vegetative compatibility (VC) types, some of them distributed on different continents. Mating studies combined with vegetative incompatibility analyses revealed that the vegetative incompatibility response between isolates mainly could be explained by two biallelic vegetative incompatibility (vic) loci. The frequency distributions of the interpreted vic alleles do not seem to support the idea of frequency-dependent or balancing selection acting on the vic loci. We find little genetic variation at the vic loci and in one of the loci there was a significant heterozyote deficiency among strains in the overall material. The results may be explained by a recent worldwide dispersal of a few S. lacrymans isolates and, correspondingly, only a few vic alleles are being maintained in these populations.     

Emerging roles of DP and CRTH2 in allergic inflammation

The lipid mediator prostaglandin D(2) (PGD(2)) has long been implicated in various inflammatory diseases including asthma. PGD(2) elicits biological responses by activating two seven-transmembrane (7TM) G-protein-coupled receptors, the D-prostanoid receptor DP and the chemoattractant receptor homologous-molecule expressed on T-helper-type-2 cells (CRTH2), which are linked to different signaling pathways. Understanding how immune cells integrate and coordinate signals that are triggered by the same ligand is crucial for the development of novel anti-inflammatory therapies. Here, we examine the roles of DP and CRTH2 in the orchestration of complex inflammatory processes, and discuss their importance as emerging targets for the treatment of asthma and inflammatory diseases.