Non-secretory exocytoses in the brain.

Regulated exocytosis, the process by which the membrane of specific cytoplasmic organelles fuse with the plasma membrane in response to adequate stimulation, is most often considered to serve only for the discharge of secretory products, in the brain especially neurotransmitters and peptides. Growing evidence demonstrates however that non-secretory exocytoses, aimed at the insertion at the cell surface of the organelle membrane, are of great physiological importance and may also have critical roles in specific diseases. Recently, two groups of non-secretory exocytoses have been identified: those aimed at the transfer to the cell surface of specific proteins, that we have proposed to be called the protein-exposing exocytoses; and those aimed at the enlargement of the surface itself, the expansive exocytoses. Here we present the existing knowledge about three types of non-secretory exocytoses that occur in the brain: the protein-exposing exocytoses that transfer ionic receptors to the postsynaptic membrane, the best known example being that of the glutamatergic AMPA receptor, a main actor of synaptic plasticity; the expansive exocytosis necessary for the growth of nerve fibres; and the rapid exocytosis of enlargeosomes, that can induce considerable expansion of the cell surface area in a variety of cells types, including the astrocytes.     

Bone marrow stromal cell antigen 2 is a specific marker of type I IFN-producing cells in the naive mouse, but a promiscuous cell surface antigen following IFN stimulation

Type I IFN-producing cells (IPC) are sentinels of viral infections. Identification and functional characterization of these cells have been difficult because of their small numbers in blood and tissues and their complex cell surface phenotype. To overcome this problem in mice, mAbs recognizing IPC-specific cell surface molecules have been generated. In this study, we report the identification of new Abs specific for mouse IPC, which recognize the bone marrow stromal cell Ag 2 (BST2). Interestingly, previously reported IPC-specific Abs 120G8 and plasmacytoid dendritic cell Ag-1 also recognize BST2. BST2 is predominantly specific for mouse IPC in naive mice, but is up-regulated on most cell types following stimulation with type I IFNs and IFN-gamma. The activation-induced promiscuous expression of BST2 described in this study has important implications for the use of anti-BST2 Abs in identification and depletion of IPC. Finally, we show that BST2 resides within an intracellular compartment corresponding to the Golgi apparatus, and may be involved in trafficking secreted cytokines in IPC.     

Controlled loading of oligodeoxyribonucleotide monolayers onto unoxidized crystalline silicon; fluorescence-based determination of the surface coverage and of the hybridization efficiency; parallel imaging of the process by Atomic Force Microscopy

Unoxidized crystalline silicon, characterized by high purity, high homogeneity, sturdiness and an atomically flat surface, offers many advantages for the construction of electronic miniaturized biosensor arrays upon attachment of biomolecules (DNA, proteins or small organic compounds). This allows to study the incidence of molecular interactions through the simultaneous analysis, within a single experiment, of a number of samples containing small quantities of potential targets, in the presence of thousands of variables. A simple, accurate and robust methodology was established and is here presented, for the assembling of DNA sensors on the unoxidized, crystalline Si(100) surface, by loading controlled amounts of a monolayer DNA-probe through a two-step procedure. At first a monolayer of a spacer molecule, such as 10-undecynoic acid, was deposited, under optimized conditions, via controlled cathodic electrografting, then a synthetic DNA-probe was anchored to it, through amidation in aqueous solution. The surface coverage of several DNA-probes and the control of their efficiency in recognizing a complementary target-DNA upon hybridization were evaluated by fluorescence measurements. The whole process was also monitored in parallel by Atomic Force Microscopy (AFM).     

Phenological models for blooming of apple in a mountainous region

Six phenological series were available for ‘Golden Delicious’ apple blooming at six sites in Trentino, an alpine fruit-growing region. Several models were tested to predict flowering dates, all involving a “chilling and forcing” approach. In many cases, application of the models to different climatic conditions results in low accuracy of prediction of flowering date. The aim of this work is to develop a model with more general validity, starting from the six available series, and to test it against five other phenological series outside the original area of model development. A modified version of the “Utah” model was the approach that performed best. In fact, an algorithm using “chill units” for rest completion and a thermal sum for growing-degree-hours (GDH), whose efficiency changes over time depending on the fraction of forcing attained, yielded a very good prediction of flowering. Results were good even if hourly temperatures were reconstructed from daily minimum and maximum values. Errors resulting from prediction of flowering data were relatively small, and root mean square errors were in the range of 1–6 days, being <2 days for the longest phenological series. In the most general form of the model, the summation of GDH required for flowering is not a fixed value, but a function of topoclimatic variables for a particular site: slope, aspect and spring mean temperature. This approach allows extension of application of the model to sites with different climatic features outside the test area.     

Mechanical resistance of proteins explained using simple molecular models

Recent experiments have demonstrated that proteins unfold when two atoms are mechanically pulled apart, and that this process is different to when heated or when a chemical denaturant is added to the solution. Experiments have also shown that the response of proteins to external forces is very diverse, some of them being "hard," and others "soft." Mechanical resistance originates from the presence of barriers on the energy landscape; together, experiment and simulation have demonstrated that unfolding occurs through alternative pathways when different pairs of atoms undergo mechanical extension. Here we use simulation to probe the mechanical resistance of six structurally diverse proteins when pulled in different directions. For this, we use two very different models: a detailed, transferable one, and a coarse-grained, structure-based one. The coarse-grained model gives results that are surprisingly similar to the detailed one and qualitatively agree with experiment; i.e., the mechanical resistance of different proteins or of a single protein pulled in different directions can be predicted by simulation. The results demonstrate the importance of pulling direction relative to the local topology in determining mechanical stability, and rationalize the effect of the location of importation/degradation tags on the rates of mitochondrial import or protein degradation in vivo.     

Multilocus patterns of nucleotide diversity, linkage disequilibrium and demographic history of Norway spruce [Picea abies (L.) Karst

DNA polymorphism at 22 loci was studied in an average of 47 Norway spruce [Picea abies (L.) Karst.] haplotypes sampled in seven populations representative of the natural range. The overall nucleotide variation was limited, being lower than that observed in most plant species so far studied. Linkage disequilibrium was also restricted and did not extend beyond a few hundred base pairs. All populations, with the exception of the Romanian population, could be divided into two main domains, a Baltico-Nordic and an Alpine one. Mean Tajima's D and Fay and Wu's H across loci were both negative, indicating the presence of an excess of both rare and high-frequency-derived variants compared to the expected frequency spectrum in a standard neutral model. Multilocus neutrality tests based on D and H led to the rejection of the standard neutral model and exponential growth in the whole population as well as in the two main domains. On the other hand, in all three cases the data are compatible with a severe bottleneck occurring some hundreds of thousands of years ago. Hence, demographic departures from equilibrium expectations and population structure will have to be accounted for when detecting selection at candidate genes and in association mapping studies, respectively.     

Overweight and obesity prevalence in urban Sardinian children

The aim of this study is to evaluate the prevalence of overweight and obesity in children (6-10 years) of the city of Cagliari (Sardinia, Italy) with different socioeconomic status. The sample is composed by 1000 children, 500 males and 500 females, 6 to 10 years old, attending primary schools in Cagliari during 2003. For uniformity with similar Italian studies, in this study overweight and obesity were defined as calculated from the charts published by Tanner et al. (1966). In the Cagliari children, the prevalence of obesity is 22.70%. The percentages of overweight and obese children increase with age: respectively from 11.5% and 14.0% at 6 years to 15.4% and 22.7% at 10 years. There are higher numbers of overweight and obese boys than girls in all the age classes. Both males and females show an increasing percentage of overweight and obesity as the socioeconomic level decreases. Males present higher percentages of overweight and obesity than females of the same social level, i.e. in the lowest social category overweight is 18.68% in males and 13.60% in females and obesity 26.46% in males and 23.62% in females. The standard multivariate regression analysis with the indicator of overweight and obesity as dependent variable showed that the sex (male), socio-economic status, maternal schooling and sums of the limb and trunk skinfolds have the greatest influence on overweight and obesity. The results suggest that overweight and obesity are becoming serious social and health problems in Sardinia.     

In silico analysis of the two tandem somatomedin B domains of ENPP1 reveals hints on the homodimerization of the protein

The homodimerization of ENPP1 is mediated by the two somatomedin B (SMB) domains of the protein through a mechanism that is yet unknown at the atomistic level. The tandem arrangement of these domains without an intermediate spacer implies their possible packing into a functional assembly, which we explored by rigid docking. To exclude potential bias in the docking search we assessed the absence of flexible protein regions by evaluating the normalized B-factors calculated from the Cα atom displacements derived from molecular dynamics simulations. After filtering the docking results exploiting the criterion that residues located at the inter-domain interfaces are more conserved than non-interface residues, the resulting best model of the tandem SMB domains revealed the presence of two large conserved surface patches not engaged in the inter-domain contact. The largest patch is flat and contains all the invariant positively charged residues characterized by fully solvent-exposed side chains within the tandem SMB domains, suggesting as a possible role its interaction with the negative phospholipids on the cell surface. We envisage that an ENPP1 monomer bound to the cell membrane via the transmembrane segment can also interact with the cell surface through the largest conserved patch favoring a specific geometry of the tandem SMB module on the cell that optimally exposes the second conserved patch for the symmetric interaction with another membrane-bound ENPP1 monomer, finally promoting the homodimerization. Biological implications of this model and insights into the effects of the K173Q variant associated with insulin resistance and related abnormalities are presented.     

Prying open single GroES ring complexes by force reveals cooperativity across domains

Understanding how the mechanical properties of a protein complex emerge from the interplay of intra- and interchain interactions is vital at both fundamental and applied levels. To investigate whether interdomain cooperativity affects protein mechanical strength, we employed single-molecule force spectroscopy to probe the mechanical stability of GroES, a homoheptamer with a domelike quaternary stucture stabilized by intersubunit interactions between the first and last β-strands of adjacent domains. A GroES variant was constructed in which each subunit of the GroES heptamer is covalently linked to adjacent subunits by tripeptide linkers and folded domains of protein L are introduced to the heptamer's termini as handle molecules. The force-distance profiles for GroES unfolding showed, for the first time that we know of, a mechanical phenotype whereby seven distinct force peaks, with alternating behavior of unfolding force and contour length (ΔL(c)), were observed with increasing unfolding-event number. Unfolding of (GroES)(7) is initiated by breakage of the interface between domains 1 and 7 at low force, which imparts a polarity to (GroES)(7) that results in two distinct mechanical phenotypes of these otherwise identical protein domains. Unfolding then proceeds by peeling domains off the domelike native structure by sequential repetition of the denaturation of mechanically weak (unfoldon 1) and strong (unfoldon 2) units. These results indicate that domain-domain interactions help to determine the overall mechanical strength and unfolding pathway of the oligomeric structure. These data reveal an unexpected richness in the mechanical behavior of this homopolyprotein, yielding a complex with greater mechanical strength and properties distinct from those that would be apparent for GroES domains in isolation.