Complexes of matrilin-1 and biglycan or decorin connect collagen VI microfibrils to both collagen II and aggrecan

Native supramolecular assemblies containing collagen VI microfibrils and associated extracellular matrix proteins were isolated from Swarm rat chondrosarcoma tissue. Their composition and spatial organization were characterized by electron microscopy and immunological detection of molecular constituents. The small leucine-rich repeat (LRR) proteoglycans biglycan and decorin were bound to the N-terminal region of collagen VI. Chondroadherin, another member of the LRR family, was identified both at the N and C termini of collagen VI. Matrilin-1, -3, and -4 were found in complexes with biglycan or decorin at the N terminus. The interactions between collagen VI, biglycan, decorin, and matrilin-1 were studied in detail and revealed a biglycan/matrilin-1 or decorin/matrilin-1 complex acting as a linkage between collagen VI microfibrils and aggrecan or alternatively collagen II. The complexes between matrilin-1 and biglycan or decorin were also reconstituted in vitro. Colocalization of collagen VI and the different ligands in the pericellular matrix of cultured chondrosarcoma cells supported the physiological relevance of the observed interactions in matrix assembly.     

Generalism in the interaction of Tulasnellaceae mycobionts with orchids characterizes a biodiversity hotspot in the tropical Andes of Southern Ecuador

Biotic interactions play an important role in the assembly and stability of communities. All orchids depend on mycobionts for early establishment, but whether individual orchid species depend on a specific or broad spectrum of mycobionts is still a matter of debate. Tulasnellaceae (Basidiomycota) is the richest and most widespread mycobiont worldwide. We assessed Tulasnellaceae richness in epiphytic and terrestrial orchids in different habitats, and evaluated the degree of generalism in orchid-Tulasnellaceae interactions and the robustness of this mutualistic system to the extinction of mycobiont partners. We sampled 114 orchid individuals including all common and rare species in 56 plots of 1 m² in 3 habitats: pristine forest, regenerating forest and a landslide site in a tropical montane rainforest in Southern Ecuador. We found 52 orchid and 29 Tulasnellaceae species. The composition of Tulasnellaceae OTUs was moderately to highly similar across habitats and between orchid growth forms. A significantly nested network architecture indicated the existence of a core of generalist Tulasnellaceae OTUs interacting with both rare and common orchids. Terrestrial and epiphytic orchids showed significant differences in robustness to the extinction of their Tulasnellaceae mycobionts. Thus, generalist mycobionts may be relevant for the preservation of hyperdiverse orchid communities in the tropics.     

Constitutive upregulation of the transforming growth factor-β pathway in rheumatoid arthritis synovial fibroblasts

Genome-wide gene expression was comparatively investigated in early-passage rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts (SFBs; n = 6 each) using oligonucleotide microarrays; mRNA/protein data were validated by quantitative PCR (qPCR) and western blotting and immunohistochemistry, respectively. Gene set enrichment analysis (GSEA) of the microarray data suggested constitutive upregulation of components of the transforming growth factor (TGF)-β pathway in RA SFBs, with 2 hits in the top 30 regulated pathways. The growth factor TGF-β1, its receptor TGFBR1, the TGF-β binding proteins LTBP1/2, the TGF-β-releasing thrombospondin 1 (THBS1), the negative effector SkiL, and the smad-associated molecule SARA were upregulated in RA SFBs compared to OA SFBs, whereas TGF-β2 was downregulated. Upregulation of TGF-β1 and THBS1 mRNA (both positively correlated with clinical markers of disease activity/severity) and downregulation of TGF-β2 mRNA in RA SFBs were confirmed by qPCR. TGFBR1 mRNA (only numerically upregulated in RA SFBs) and SkiL mRNA were not differentially expressed. At the protein level, TGF-β1 showed a slightly higher expression, and the signal-transducing TGFBR1 and the TGF-β-activating THBS1 a significantly higher expression in RA SFBs than in OA SFBs. Consistent with the upregulated TGF-β pathway in RA SFBs, stimulation with TGF-β1 resulted in a significantly enhanced expression of matrix-metalloproteinase (MMP)-11 mRNA and protein in RA SFBs, but not in OA SFBs. In conclusion, RA SFBs show broad, constitutive alterations of the TGF-β pathway. The abundance of TGF-β, in conjunction with an augmented mRNA and/or protein expression of TGF-β-releasing THBS1 and TGFBR1, suggests a pathogenetic role of TGF-β-induced effects on SFBs in RA, for example, the augmentation of MMP-mediated matrix degradation/remodeling.     

The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics

A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a “buffering” effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (~pH 6.0). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0–7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination.     

Recombinant expression and characterization of a l-amino acid oxidase from the fungus Rhizoctonia solani

Applied Microbiology and Biotechnology - l-Amino acid oxidases (L-AAOs) catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids, ammonia, and hydrogen peroxide....     

Post-lesional cerebral reorganisation: Evidence from functional neuroimaging and transcranial magnetic stimulation

Reorganisation of cerebral representations has been hypothesised to underlie the recovery from ischaemic brain infarction. The mechanisms can be investigated non-invasively in the human brain using functional neuroimaging and transcranial magnetic stimulation (TMS). Functional neuroimaging showed that reorganisation is a dynamic process beginning after stroke manifestation. In the acute stage, the mismatch between a large perfusion deficit and a smaller area with impaired water diffusion signifies the brain tissue that potentially enables recovery subsequent to early reperfusion as in thrombolysis. Single-pulse TMS showed that the integrity of the cortico-spinal tract system was critical for motor recovery within the first four weeks, irrespective of a concomitant affection of the somatosensory system. Follow-up studies over several months revealed that ischaemia results in atrophy of brain tissue adjacent to and of brain areas remote from the infarct lesion. In patients with hemiparetic stroke activation of premotor cortical areas in both cerebral hemispheres was found to underlie recovery of finger movements with the affected hand. Paired-pulse TMS showed regression of perilesional inhibition as well as intracortical disinhibition of the motor cortex contralateral to the infarction as mechanisms related to recovery. Training strategies can employ post-lesional brain plasticity resulting in enhanced perilesional activations and modulation of large-scale bihemispheric circuits.     

Mosaic chromosomal aberrations in synovial fibroblasts of patients with rheumatoid arthritis, osteoarthritis, and other inflammatory joint diseases

Chromosomal aberrations were comparatively assessed in nuclei extracted from synovial tissue, primary-culture (P-0) synovial cells, and early-passage synovial fibroblasts (SFB; 98% enrichment; P-1, P-4 [passage 1, passage 4]) from patients with rheumatoid arthritis (RA; n = 21), osteoarthritis (OA; n = 24), and other rheumatic diseases. Peripheral blood lymphocytes (PBL) and skin fibroblasts (FB) (P-1, P-4) from the same patients, as well as SFB from normal joints and patients with joint trauma (JT) (n = 4), were used as controls. Analyses proceeded by standard GTG-banding and interphase centromere fluorescence in situ hybridization. Structural chromosomal aberrations were observed in SFB (P-1 or P-4) from 4 of 21 RA patients (19%), with involvement of chromosome 1 [e.g. del(1)(q12)] in 3 of 4 cases. In 10 of the 21 RA cases (48%), polysomy 7 was observed in P-1 SFB. In addition, aneusomies of chromosomes 4, 6, 8, 9, 12, 18, and Y were present. The percentage of polysomies was increased in P-4. Similar chromosomal aberrations were detected in SFB of OA and spondylarthropathy patients. No aberrations were detected in i) PBL or skin FB from the same patients (except for one OA patient with a karyotype 45,X[10]/46,XX[17] in PBL and variable polysomies in long-term culture skin FB); or ii) synovial tissue and/or P-1 SFB of normal joints or of patients with joint trauma. In conclusion, qualitatively comparable chromosomal aberrations were observed in synovial tissue and early-passage SFB of patients with RA, OA, and other inflammatory joint diseases. Thus, although of possible functional relevance for the pathologic role of SFB in RA, these alterations probably reflect a common response to chronic inflammatory stress in rheumatic diseases.     

Studies on the root associations of the truffle Terfezia terfezioides

The paper contains an overview of the results of the studies made on the truffle Terfezia terfezioides, particularly the investigations related to the associations of this fungus with plants. Twelve plant species originated from a natural habitat of the fungus were supposed to be connected with T. terfezioides based on the anatomy of the endogenous fungal structures in their roots. Aseptic experiments were carried out on modified MMN substrates with different phosphate concentrations to study the interaction of T. terfezioides with Robinia pseudoacacia and Helianthemum ovatum. The colonization of the roots of black locust was always weaker than that of Helianthemum. The main characteristics were the intracellular coiled, branched, frequently septated hyphae in dead root cells. The intercellular hyphae formed Hartig-net with finger like structures only in Helianthemum, the interactions could not be considered unambiguously as mycorrhizae. There was no difference between the RFLP profiles of the nr DNA ITS of nineteen fruit bodies collected at the same time from the habitat and the ITS of three randomly chosen specimens were identical on sequence level, too. These invariability makes to design species specific PCR primers possible to check unambiguously the host plants.     

Alteration of biliary ursodeoxycholic acid in guinea pig during early stages of cholestyramine feeding

The effects of cholestyramine feeding on biliary ursodeoxycholic acid, fecal excretion of bile acids and neutral sterols on cholesterol 7α-hydroxylase and hepatic HMG-CoA reductase were examined in the guinea pig. In the bile there was a 57% decrease in the concentration of ursodeoxycholic acid while an increase was observed in the concentration of chenodeoxycholic acid. Cholestyramine feeding for ten days resulted in a decrease in plasma cholesterol levels and an increase in both hepatic HMG-CoA reductase and cholesterol 7α-hydroxylase activities. The fecal excretion of both bile acids and neutral sterols was significantly increased.