Influence of a low background radiation environment on biochemical and biological responses in V79 cells

We present the results of an experiment aimed at comparing the effects of different background radiation environments on metabolism and responses to gamma-rays and cycloheximide of cultured mammalian cells. Chinese hamster V79 cells were maintained in exponential growth in parallel for up to 9 months at the Istituto Superiore di Sanità (ISS) and at the INFN-Gran Sasso underground Laboratory (LNGS) where exposure due to gamma-rays and to radon was reduced by factors of about 70 and 25, respectively. After 9 months the cells grown at the LNGS (cumulative gamma dose about 30 microGy, average radon concentration around 5 Bq/m(3)), compared to the cells grown at the ISS (cumulative gamma-ray dose about 2 mGy, average radon concentration around 120 Bq/m(3)), exhibited i). a significant increase of the cell density at confluence, ii). a significantly higher capacity to scavenge organic and inorganic hydroperoxides but a reduced scavenging capacity towards superoxide anions and iii). an increase in both the basal hprt mutation frequency and sensitivity to the mutagenic effect of gamma-rays. The cells grown at the LNGS also showed a greater apoptotic sensitivity starting at the third month of culture, that was no longer detected after 9 months. Overall, these data suggest a role of background ionizing radiation in determining an adaptive response, although they cannot be considered conclusive.     

Some human B and T cell epitopes of bovine serum albumin,the major beef allergen

Bovine serum albumin (BSA) is the major beef allergen. Since IgE and T cell recognitions are central to the specific immune response to allergens, the identification and immunologic characterization of B and T cell epitopes of BSA represent important steps in the development of treatments for beef allergy. Prior to our experiments, we hypothesized that BSA-specific antibodies and T cells react primarily with sequential epitopes in which the amino acid sequences differ greatly between bovine and human albumin. To clarify this hypothesis, 16 peptides corresponding to such regions were synthesized as candidate epitopes. Among them, at least two regions, aa336-345 and aa451-459, were found to be B cell (IgE-binding) epitopes. In inhibition ELISA experiments, EYAV (aa338-341) and LILNR (aa453-457) bound to patient IgE antibodies and were found to be the cores of the IgE-binding epitopes. Three regions, DDSPDLPKLKPDPNTLC (aa107-123), PHACYTSVFDKLKHLVDEP (aa364-382), and LSLILNRLC (aa451-459), were found to induce T cell proliferation in more than half of the patients tested. Of interest was that these three regions were also recognized by B cells. Information concerning human B and T cells epitopes can contribute greatly to the elucidation of the etiology of beef allergy.     

Isolation of single-stranded DNA from loop-mediated isothermal amplification products.

Loop-mediated isothermal amplification (LAMP), in which a specific DNA sequence can be directly amplified under isothermal conditions, yields DNA in large quantities of more than 500 microg/ml. We have developed a method to isolate single-stranded DNA fragments from LAMP products that are stem-loop DNAs with several inverted repeats of the target DNA. This method requires the TspRI restriction enzyme, a primer hybridized to the 3' overhanging sequence at its cleavage site, and a DNA polymerase with strand displacement activity. The LAMP products are digested with TspRI and are then extended using the primer, producing the strand-specific DNA fragments. All processes, from LAMP reaction to primer extension, can be carried out at the same temperature. The use of strand-specific DNA would be conducive for detection by hybridization technique such as DNA microarrays.     

BACE1 interacts with nicastrin

Beta-amyloid peptide (Abeta) is generated through the proteolytic cleavage of beta-amyloid precursor protein (APP) by beta- and gamma-secretases. The beta-secretase, BACE1, initiates Abeta formation followed by gamma-cleavage within the APP transmembrane domain. Although BACE1 localizes in the transGolgi network (TGN), its physiological substrates and modulators are not known. In addition, the relationship to other secretase(s) also remains unidentified. Here, we demonstrate that BACE1 binds to nicastrin, a component of gamma-secretase complexes, in vitro, and that nicastrin activates beta-secretase activity in COS-7 cells.     

The effects of aggregation-inducing motifs on amyloid formation of model proteins related to neurodegenerative diseases

To examine the effects of aggregation-inducing motifs related to neurodegenerative diseases on amyloid formation of host protein, we prepared several chimera myoglobins, in which various aggregation-inducing motifs were inserted. The focused aggregation-inducing motifs included five (R5) or two (R2) oligopeptide repeats in yeast Sup35p, five octapeptide repeats (OPR) in the human prion protein, a nonamyloid beta component (NAC) in alpha-synuclein, and tandem repeats of 50 glutamines (Q50). Circular dichroism and infrared spectroscopies suggested that the OPR, R5, and Q50 motifs formed an antiparallel beta sheet as well as a random coil, whereas the R2 and NAC motifs mainly formed random coils. The OPR, R5, and Q50 mutants, but not the R2 and NAC mutants, readily formed the SDS-resistant aggregates under physiological condition, and electron microscopy revealed that the aggregates contained amyloid fibrils. The destabilization and increase in gyration radius of the OPR, R5, and Q50 mutants correlated with the tendency to form amyloid fibrils. A control mutant bearing a nonamyloidgenic sequence was also moderately destabilized but did not form amyloid fibrils. Therefore, we concluded that the OPR, R5, and Q50 motifs, even in a quite stable protein such as myoglobin, led the host protein to formation of amyloid fibrils under physiological condition.     

A new technique to co-localise membrane proteins with Homer/vesl

The minimal requirements were defined as necessary for cluster formation of the group 1 metabotropic glutamate receptor (mGluR), which is regulated by the Homer/vesl family of scaffolding proteins [Curr. Opin. Neurobiol. 10 (2000) 370]. Cluster formation of G-protein-coupled receptors (GPCRs) plays a fundamental role in signal transduction, particularly at the neuronal synapse. To understand the interaction of mGluR with PSD-Zip45, a Homer/vesl family member, we designed a series of chimeric receptor proteins, consisting of C-terminal mGluR1alpha sequences that were fused to endothelin B receptors (ET(B)Rs). In vitro and in vivo studies revealed that an extended 20 amino acid long C-terminal mGluR1alpha peptide, including the proline-rich core motif PPXXF, is sufficient to induce clustering of chimeric ET(B)R/mGluR1alpha receptors by PSD-Zip45. This result is especially important because it constitutes the basis for a new approach to form two-dimensional crystals of membrane proteins in situ, which may render unstable membrane proteins amenable to electron crystallographic structure determination.     

Migration of nerve growth cones requires detergent-resistant membranes in a spatially defined and substrate-dependent manner

Motility of nerve growth cones (GCs) is regulated by region-specific activities of cell adhesion molecules (CAMs). CAM activities could be modified by their localization to detergent-resistant membranes (DRMs), specialized microdomains enriched in signaling molecules. This paper deals with a question of whether DRMs are involved in GC migration stimulated by three CAMs; L1, N-cadherin (Ncad), and beta1 integrin. We demonstrate that L1 and Ncad are present in DRMs, whereas beta1 integrin is exclusively detected in non-DRMs of neurons and that localization of L1 and Ncad to DRMs is developmentally regulated. GC migration mediated by L1 and Ncad but not by beta1 integrin is inhibited after DRM disruption by micro-scale chromophore-assisted laser inactivation (micro-CALI) of GM1 gangliosides or by pharmacological treatments that deplete cellular cholesterol or sphingolipids, essential components for DRMs. Characteristic morphology of GCs induced by L1 and Ncad is also affected by micro-CALI-mediated DRM disruption. Micro-CALI within the peripheral domain of GCs, or even within smaller areas such as the filopodia and the lamellipodia, is sufficient to impair their migration. However, micro-CALI within the central domain does not affect GC migration. These results demonstrate the region-specific involvement of DRMs in CAM-dependent GC behavior.