Proteomics of β2-microglobulin amyloid fibrils

Knowledge on the chemical structure of β2-microglobulin in natural amyloid fibrils is quite limited because of the difficulty in obtaining tissue samples suitable for biochemical studies. We have reviewed the available information on the chemical modifications and we present new data of β2-microglobulin extracted from non-osteotendinous tissues. β2-microglobulin can accumulate in these compartments after long-term haemodialysis but rarely forms amyloid deposits. We confirm that truncation at the N-terminus is an event specific to β2-microglobulin derived from fibrils but is not observed in the β2-microglobulin from plasma or from the insoluble non-fibrillar material deposited in the heart and spleen. We also confirm the partial deamidation of Asn 17 and Asn 42, as well as the oxidation of Met 99 in fibrillar β2-microglobulin. Other previously reported chemical modifications cannot be excluded, but should involve less than 1–2% of the intact molecule.     

Proliferation versus migration in platelet-derived growth factor signaling: the key role of endocytosis

It is common knowledge that platelet-derived growth factor (PDGF) is a critical regulator of mesenchymal cell migration and proliferation. Nevertheless, these two cellular responses are mutually exclusive. To solve this apparent contradiction, we studied the behavior of NIH3T3 fibroblasts in response to increasing concentrations of PDGF. We found that there is strong cell proliferation induction only with PDGF concentrations >5 ng/ml, whereas the cell migration response arises starting from 1 ng/ml and is negligible at higher PDGF concentrations. According to these phenotypic evidences, our data indicate that cells display a differential activation of the main signaling pathways in response to PDGF as a function of the stimulation dose. At low PDGF concentrations, there is maximal activation of signaling pathways linked to cytoskeleton rearrangement needed for cell motility, whereas high PDGF concentrations activate pathways linked to mitogenesis induction. Our results suggest a mechanism by which cells switch from a migrating to a proliferating phenotype sensing the increasing gradient of PDGF. In addition, we propose that the cell decision to proliferate or migrate relies on different endocytotic routes of the PDGF receptor in response to different PDGF concentrations.     

Are aquatic assemblages from small water bodies more stochastic in dryer climates? An analysis of ostracod spring metacommunities

Metacommunity ecology describes community organisation considering both environmental and spatial processes. We tested the relative importance of environmental and spatial factors on spring ostracod assemblages from four European regions characterised by different climatic conditions (e.g. aridity). Pure and shared effects of environment and space were calculated using redundancy analysis and variation partitioning. Both environmental and spatial variables significantly explain assemblage variation, although with different relevance among areas. The amount of variation explained by environmental factors decreased with increasing climate aridity. The reduced size of spring habitats makes them prone to drying events, which are more frequent in dryer climates. Frequent disturbances may lead to local extinctions followed by colonisations from nearby sites, in a source–sink dynamics. Early recolonisation leads to random assemblages and reduces the match between organisms and environmental conditions. As a consequence, a low amount of community variation can be explained by environmental variables. Conversely, the settled communities from wetter climates best fit the ecological characteristics of sites, and deterministic processes, such as species sorting, dominate the assemblages. In conclusion, in the studied regions, ostracod communities from small water bodies of dryer climates seem to be mainly driven by stochastic dynamics when compared to more continental areas.     

Mitochondrial permeability transition involves dissociation of F1FO ATP synthase dimers and C‐ring conformation

The impact of the mitochondrial permeability transition (MPT) on cellular physiology is well characterized. In contrast, the composition and mode of action of the permeability transition pore complex (PTPC), the supramolecular entity that initiates MPT, remain to be elucidated. Specifically, the precise contribution of the mitochondrial F₁F_O ATP synthase (or subunits thereof) to MPT is a matter of debate. We demonstrate that F₁F_O ATP synthase dimers dissociate as the PTPC opens upon MPT induction. Stabilizing F₁F_O ATP synthase dimers by genetic approaches inhibits PTPC opening and MPT. Specific mutations in the F₁F_O ATP synthase c subunit that alter C‐ring conformation sensitize cells to MPT induction, which can be reverted by stabilizing F₁F_O ATP synthase dimers. Destabilizing F₁F_O ATP synthase dimers fails to trigger PTPC opening in the presence of mutants of the c subunit that inhibit MPT. The current study does not provide direct evidence that the C‐ring is the long‐sought pore‐forming subunit of the PTPC, but reveals that PTPC opening requires the dissociation of F₁F_O ATP synthase dimers and involves the C‐ring.     

Zooplankton variability related to environmental changes in a eutrophic coastal lagoon in the Po Delta

Zooplankton variability in a lagoon of the Po Delta, the Sacca di Goro, was studied in relation to changes in its hydrodynamic and hydrological features. From 1987 to 1992 the lagoon was affected each summer by severe anoxia due to the decomposition of dense and widespread beds of Ulva rigida. In August 1992 a canal was opened through the sand bank closing off the lagoon from the sea in order to improve the water circulation. This hydraulic intervention led to significant environmental changes in the lagoon: during the two subsequent years the summer dystrophic crises were brief and less severe, due to a sharp decrease in the Ulva cover. At the same time a clear increase in phytoplankton biomass values was observed. Following the opening of the channel, the most remarkable change in zooplankton was a significant density increase of calanoids, particularly of Acartia tonsa.     

A finite element model of the L4-L5 spinal motion segment: biomechanical compatibility of an interspinous device

The biomechanical compatibility of an interspinous device, used for the "dynamic stabilization" of a diseased spinal motion segment, was investigated. The behaviour of an implant made of titanium based alloy (Ti6Al4V) and that of an implant made of a super-elastic alloy (Ni-Ti) have been compared. The assessment of the biomechanical compatibility was achieved by means of the finite element method, in which suitable constitutive laws have been adopted for the annulus fibrosus and for the metal alloys. The model was aimed at simulating the healthy, the nucleotomized and the treated L4-L5 lumbar segment, subjected to compressive force and flexion-extension as well as lateral flexion moments. The computational model has shown that both the implants were able to achieve their main design purpose, which is to diminish the forces acting on the apophyseal joints. Nevertheless, the Ni-Ti implant has shown a more physiological flexural stiffness with respect to the Ti6Al4V implant, which exhibited an excessive stiffness and permanent strains (plastic strains), even under physiological loads. The computational models presented in this paper seems to be a promising tool able to predict the effectiveness of a biomedical device and to select the materials to be used for the implant manufacturing, within an engineering approach to the clinical problem of the spinal diseases.     

Entropy calculator: getting the best from your multiple protein alignments

Amino acid sequence alignment is an extremely useful tool in protein family analysis. Most family characteristics, such as the localization of functional residues, structural constraints and evolutionary relationships may be retrieved through the observation of the conservation pattern highlighted by the alignments. A quantitative score for the conservation in the alignment allows different stages of an alignment to be compared and consequently the alignment information to be efficiently exploited. Many scoring methods have been proposed during the last three decades. Claude Shannon's theory of communication (1948) paved the way for a consistent scoring of protein alignments by considering the residue (or symbol) frequency. A number of modifications have been proposed since that time, but the core statistical approach is still considered one of the best. By combining many database managing tools for treatment of protein sequences, a ClustalW software integration, a flexible symbols treatment and gap normalization functions, Entropy Calculator software has been developed. This new tool provides a global and optimal approach to multiple sequence alignment scoring by offering an easy graphic interface and a series of modification options that help in interpreting alignments and allow conservation pattern inferences to be performed.     

Porcine liver low Mr phosphotyrosine protein phosphatase: The amino acid sequence

Porcine low M_r phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH₂-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH₂-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low Mr PTPases. This PTPase is strongly inhibited by pyridoxal 5-phosphate (K=21M) like the low Mr PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40–73 sequence with the corresponding sequence of other low Mr PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low M_r PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5-phosphate inhibition has been proposed.Abbreviations used PTPase phosphotyrosine protein phosphatase - TFA trifluoroacetic acid - SDS sodium dodecylsulfate - T tryptic peptides - SP endoproteinase Glu-C peptides - FAB fast atom bombardment - Ac acetyl - HPLC high-performance liquid chromatography - OPA o-phtaldialdehyde - PMSF phenylmethylsulfonyl fluoride - CD45 leukocyte common-antigen PTPase - LAR leukocyte-antigen-related PTPase - PTP IB human placental PTPase     

Limnological research on northern Apennine lakes (Italy) in relation to eutrophication and acidification risk

Substantial variability was found in the water chemistry of 22 northern Apennine lakes. In a group of lakes there is evidence of disturbance linked to eutrophication processes. Other lakes showed weak ion concentrations and alkalinity below the acidification risk threshold. However no acidified lakes were found. The lack of waterbodies with severely altered hydrochemistry may explain why no clear relationship between plankton community structure and water chemistry was observed.