Sebacinales form ectendomycorrhizas with Cavendishia nobilis, a member of the Andean clade of Ericaceae, in the mountain rain forest of southern Ecuador

Cavendishia nobilis var. capitata is an endemic member of the Ericaceae growing as a hemiepiphyte in the tropical mountain rain forest of southern Ecuador. Mycorrhizas were collected from 20 individuals along an altitudinal gradient between 1850 and 2300 m. Transmission electron microscopy was used to study the symbiotic association in detail, and phylogenetic analyses based on nuclear rDNA coding for the ribosomal large subunit (nucLSU) were carried out to identify the associated mycorrhizal fungi. Microscopic and ultrastructural investigations showed the formation of a hyphal sheath, intercellular penetration of fine hyphae and colonization of the cortical cells by swollen hyphae of the same fungus. These structures were formed by hymenomycetes and ascomycetes. Molecular phylogenetic analysis detected seven groups of mycorrhizal fungi belonging to the Sebacinales. This is the first study to obtain evidence of ectendomycorrhizas in the Vaccinioideae. The ascomycetous nucLSU sequences belonged to members of the Leotiomycetes. The ectendomycorrhiza of C. nobilis with Sebacinales is discussed as a specific, hitherto undescribed mycorrhizal subcategory of ectomycorrhizas. We propose the term 'cavendishioid mycorrhiza'. This subcategory is most likely specific for the Andean clade of Ericaceae.     

A folded and functional protein domain in an amyloid-like fibril

The effect of the polypeptide environment on polyalanine-induced fibril formation was investigated with amyloidogenic fragments from PAPBN1, a nuclear protein controlling polyadenylation. Mutation-caused extensions of the natural 10 alanine sequence up to maximally 17 alanines result in fibril formation of PABPN1 and the development of the disease oculopharyngeal muscular dystrophy (OPMD). We explored the influence of fibril formation on the structure and function of a one-domain protein linked to the fibril-forming part of PABPN1. The well-characterized, stably folded, one-domain protein, cold-shock protein CspB from Bacillus subtilis, was fused either to the C terminus of the entire N-terminal domain of PABPN1 or directly to peptides consisting of 10 or 17 alanine residues. The fusion protein between the N-terminal domain of PABPN1 and CspB formed fibrils in which the structure and activity of CspB were retained. In the fibrils formed by fusions in which the polyalanine sequence was directly linked to CspB, CspB was unfolded. These results indicate that the folded conformation and the function of a protein domain can be maintained in amyloid-like fibrils, and that the distance between this domain and the fibril plays an important role.     

Characteristics of intracellular Ca2+ cycling in intact rat heart: a comparison of sex differences

Males and females show distinct differences in action potential waveform, ion channel expression patterns, and ECG characteristics. However, it is not known how sex-based differences in Ca2+ cycling might contribute to these differences in electrophysiological activity. The goal of this study was to investigate the differences in cellular Ca2+ transients in males and females and to examine how these might contribute to electrophysiological function. Ca2+ transients were measured in individual myocytes within microscopic regions of the fluo-4 AM-loaded left ventricular epicardium of intact rat heart of both sexes (3 to 5 mo old). Pacing protocols were used to measure transient characteristics at a basic cycle length of 500 ms and during 10-s trains of rapid pacing delivered to the left ventricular apex. Ca2+ transients were smaller in magnitude and longer in duration in females than in males. More importantly, the variability in Ca2+ transient characteristics between myocytes in a microscopic recording site (heterogeneity index) was greater for females than males for characteristics related to transient duration. The rate sensitivity of Ca2+ alternans development in individual myocytes was greater in females than in males, but there was also a greater heterogeneity in cellular responses to the rate dependence of alternans development in females. The longer Ca2+ transients in females were also associated with slower restitution, which was likely to be responsible for the development of Ca2+ and repolarization alternans at slower heart rates. These results demonstrate that there are distinct differences in cellular Ca2+ cycling in male and female rat hearts. Not only is there slower reuptake of Ca2+ in female rats, but there is greater local variability in Ca2+ cycling at the microscopic level. These sex-based differences in Ca2+ cycling could contribute to differences in ECG morphology and in arrhythmia sensitivity in males and females.     

Phenyl-tert-butylnitrone induces tumor regression and decreases angiogenesis in a C6 rat glioma model

The prognosis of patients who are diagnosed with glioblastoma multiforme is very poor, due to the difficulty of an early and accurate diagnosis and the lack of currently efficient therapeutic compounds. The efficacy of phenyl-tert-butylnitrone (PBN) as a potential anti-glioma therapeutic drug was assessed by magnetic resonance (MR) imaging (T(1)/T(2)-weighted imaging) and MR angiography (time-of-flight imaging, in conjunction with a Mathematica-based program) methods by monitoring morphologic properties, growth patterns, and angiogenic behaviors of a moderately aggressive rat C6 glioma model. MR results from untreated rats showed the diffusive invasiveness of C6 gliomas, with some associated angiogenesis. PBN administration as a pretreatment was found to clearly induce a decrease in growth rate and tumor regression as well as preventing angiogenesis. This compound even had a 40% efficiency in reducing well-established tumors. MR findings rivaled those from histology and angiogenesis marker immunostaining evaluations. In this study we demonstrated the efficiency of PBN as a potential anti-glioma drug and found it to inhibit tumor cell proliferation and prevent vascular alterations in early stages of glioma progression. The MR methods that we used also proved to be particularly suitable in following the angiogenic behavior and treatment response of a potential anti-glioma agent in a rat C6 glioma model.     

Identification of long chain alkylidenemalonates as novel small molecule modulators of histone acetyltransferases

Pentadecylidenemalonate 1b, a simplified analogue of anacardic acid, was identified as the first mixed activator/inhibitor of histone acetyltransferases (HATs). It potentiates PCAF HAT activity while inhibiting those of p300/CBP and recombinant CBP. The remarkable apoptotic effect together with the ability to selectively acetylate histone versus non-histone substrates appoint 1b as a lead for the development of anticancer drugs.     

Mapping alpha-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy

Using site-directed spin-labeling EPR spectroscopy, we mapped the region of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)) that undergoes induced folding. In addition to four spin-labeled N(TAIL) variants (S407C, S488C, L496C, and V517C) (Morin et al. (2006), J Phys Chem 110: 20596-20608), 10 new single-site cysteine variants were designed, purified from E. coli, and spin-labeled. These 14 spin-labeled variants enabled us to map in detail the gain of rigidity of N(TAIL) in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol or the C-terminal domain X (XD) of the viral phosphoprotein. Different regions of N(TAIL) were shown to contribute to a different extent to the binding to XD, while the mobility of the spin labels grafted at positions 407 and 460 was unaffected upon addition of XD; that of the spin labels grafted within the 488-502 and the 505-522 regions was severely and moderately reduced, respectively. Furthermore, EPR experiments in the presence of 30% sucrose allowed us to precisely map to residues 488-502, the N(TAIL) region undergoing alpha-helical folding. The mobility of the 488-502 region was found to be restrained even in the absence of the partner, a behavior that could be accounted for by the existence of a transiently populated folded state. Finally, we show that the restrained motion of the 505-522 region upon binding to XD is due to the alpha-helical transition occurring within the 488-502 region and not to a direct interaction with XD.     

Detection of a gamma-carboxy-glutamate as novel post-translational modification of human transthyretin

During analysis of the proteome in the cerebrospinal fluid (CSF) of the Caucasian form of moyamoya disease (MMD), a novel post-translational modification of human transthyretin was observed. Two-dimensional electrophoresis and subsequent peptide sequencing with ESI-MS/MS were performed to discover the gamma-carboxylation of the Glu-42 (Gla-42).     

Lipid droplet changes in proliferating and quiescent 3T3 fibroblasts

Lipid droplets (LDs) are fat-storing organelles present in virtually all eukaryotic cells and involved in many aspects of cell biology related to lipid metabolism and cholesterol homeostasis. In this study, we investigated the presence of LDs in proliferating and quiescent (contact-inhibited) 3T3 fibroblasts to verify a correlation with cell growth. LDs were characterized by Nile red staining, positivity to adipophilin and negativity to perilipin. LDs were numerous in proliferating cells, but very few in quiescent cells. However, the fraction of quiescent cells, which resumed proliferation after scratch-wound assay, also resumed the formation of LDs. In proliferating cells, the number of LDs correlated with the DNA content, suggesting a continuous accumulation of LDs during cell growth. These findings were supported by biochemical data showing much higher rates of cholesterol esterification and triglyceride synthesis in proliferating cells. Both filipin staining and the fluorescent cholesterol analog dehydroergosterol revealed the presence of an intense traffic of free cholesterol, mediated by acidic vesicles, in proliferating cells. Nile red ratiometric measurements revealed a different lipid composition of LDs in proliferating and quiescent cells. Changes in the number and composition of LDs were also found in growing cells treated with inhibitors of cholesterol esterification (Sandoz 58-035), endosomal cholesterol efflux (U18666A) and V-ATPase (bafilomycin-A1).     

Toponomics analysis of functional interactions of the ubiquitin ligase PAM (Protein Associated with Myc) during spinal nociceptive processing

Protein associated with Myc (PAM) is a giant E3 ubiquitin ligase of 510 kDa. Although the role of PAM during neuronal development is well established, very little is known about its function in the regulation of synaptic strength. Here we used multiepitope ligand cartography (MELC) to study protein network profiles associated with PAM during the modulation of synaptic strength. MELC is a novel imaging technology that utilizes biomathematical tools to describe protein networks after consecutive immunohistochemical visualization of up to 100 proteins on the same sample. As an in vivo model to modulate synaptic strength we used the formalin test, a common model for acute and inflammatory pain. MELC analysis was performed with 37 different antibodies or fluorescence tags on spinal cord slices and led to the identification of 1390 PAM-related motifs that distinguish untreated and formalin-treated spinal cords. The majority of these motifs related to ubiquitin-dependent processes and/or the actin cytoskeleton. We detected an intermittent colocalization of PAM and ubiquitin with TSC2, a known substrate of PAM, and the glutamate receptors mGluR5 and GLUR1. Importantly these complexes were detected exclusively in the presence of F-actin. A direct PAM/F-actin interaction was confirmed by colocalization and cosedimentation. The binding of PAM toward F-actin varied strongly between the PAM splice forms found in rat spinal cords. PAM did not ubiquitylate actin or alter actin polymerization and depolymerization. However, F-actin decreased the ubiquitin ligase activity of purified PAM. Because PAM activation is known to involve its translocation, the binding of PAM to F-actin may serve to control its subcellular localization as well as its activity. Taken together we show that defining protein network profiles by topological proteomics analysis is a useful tool to identify previously unknown protein/protein interactions that underlie synaptic processes.