Isolation and characterization of tri‐ and tetra‐repeat microsatellite loci in the white‐spotted charr Salvelinus leucomaenis (Salmonidae)

Tri‐ and tetra‐motif repeat microsatellite marker loci were developed for the white‐spotted charr Salvelinus leucomaenis. The 454 pyrosequencing was used to discover repeat arrays, and eight microsatellite‐primer sets, available for the estimation of polymorphisms, were identified. The number of alleles in a wild population ranged from two to four and the observed and expected heterozygosities were 0·180–0·600 and 0·188–0·599, respectively.     

Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of proteolytic degradation

Expression of somatostatin in the brain declines during aging in various mammals including apes and humans. A prominent decrease in this neuropeptide also represents a pathological characteristic of Alzheimer disease. Using in vitro and in vivo paradigms, we show that somatostatin regulates the metabolism of amyloid beta peptide (Abeta), the primary pathogenic agent of Alzheimer disease, in the brain through modulating proteolytic degradation catalyzed by neprilysin. Among various effector candidates, only somatostatin upregulated neprilysin activity in primary cortical neurons. A genetic deficiency of somatostatin altered hippocampal neprilysin activity and localization, and increased the quantity of a hydrophobic 42-mer form of Abeta, Abeta(42), in a manner similar to presenilin gene mutations that cause familial Alzheimer disease. These results indicate that the aging-induced downregulation of somatostatin expression may be a trigger for Abeta accumulation leading to late-onset sporadic Alzheimer disease, and suggest that somatostatin receptors may be pharmacological-target candidates for prevention and treatment of Alzheimer disease.     

Involvement of calpain in osteoclastic bone resorption

There is increasing evidence that calpain contributes to the reorganization of the cytoskeleton in the integrin-mediated signaling pathway. Osteoclastic bone resorption requires cell-matrix contact, an event mediated by integrin alphavbeta3, and subsequent cytoskeletal reorganization to form characteristic membrane domains such as the sealing zone and ruffled border. In this study, therefore, we investigated whether calpain is involved in osteoclastic bone resorption. Membrane-permeable calpain inhibitors suppress the resorption activity of human osteoclasts, but an impermeable inhibitor does not. Upon the attachment of osteoclasts to bone, micro-calpain is translocated from the cytosolic to the cytoskeletal fraction and is autolytically activated. Both the activation of micro-calpain and the formation of actin-rings, the cytoskeletal structures essential for bone resorption, are inhibited by membrane-permeable calpain inhibitors. The activated micro-calpain in osteoclasts selectively cleaves talin, which links the matrix-recognizing integrin to the actin cytoskeleton. These findings suggest that calpain is a regulator of the bone resorption activity of osteoclasts through reorganization of the cytoskeleton related to actin-ring formation.     

A mode of assembly of P0, P1, and P2 proteins at the GTPase-associated center in animal ribosome: in vitro analyses with P0 truncation mutants

Ribosomal P0, P1, and P2 proteins, together with the conserved domain of 28 S rRNA, constitute a major part of the GTPase-associated center in eukaryotic ribosomes. We investigated the mode of assembly in vitro by using various truncation mutants of silkworm P0. When compared with wild type (WT)-P0, the C-terminal truncation mutants CDelta65 and CDelta81 showed markedly reduced binding ability to P1 and P2, which was offset by the addition of an rRNA fragment covering the P0.P1-P2 binding site. The mutant CDelta107 lost the P1/P2 binding activity, whereas it retained the rRNA binding. In contrast, the N-terminal truncation mutants NDelta21-NDelta92 completely lost the rRNA binding, although they retained P1/P2 binding capability, implying an essential role of the N terminus of P0 for rRNA binding. The P0 mutants NDelta6, NDelta14, and CDelta18-CDelta81, together with P1/P2 and eL12, bound to the Escherichia coli core 50 S subunits deficient in L10.L7/L12 complex and L11. Analysis of incorporation of (32)P-labeled P1/P2 into the 50 S subunits with WT-P0 and CDelta81 by sedimentation analysis indicated that WT-P0 bound two copies of P1 and P2, but CDelta81 bound only one copy each. The hybrid ribosome with CDelta81 that appears to contain one P1-P2 heterodimer retained lower but considerable activities dependent on eukaryotic elongation factors. These results suggested that two P1-P2 dimers bind to close but separate regions on the C-terminal half of P0. The results were further confirmed by binding experiments using chimeric P0 mutants in which the C-terminal 81 or 107 amino acids were replaced with the homologous sequences of the archaebacterial P0.     

A Nitrate Reductase Inactivator Protein from Spinach. Purification, Molecular Weight and Subunit Composition

A nitrate reductase-inactivator protein has been purified 16,000-foldfrom spinach leaves by pH 5 treatment, chromatography on SE53,Con A-Sepharose, and chromatofocusing. The yield was 12%, thespecific activity was 115 units mg^–1. Polyacrylamide gelelectrophoresis of the final purified inactivator fraction yielded2 major protein bands and both bands exhibited nitrate reductase-inactivatoractivity. Analysis of this inactivator protein by gel filtrationand SDS-gel electrophoresis revealed protein stainable materialonly in a molecular weight range of 110,000–115,000. SDSgel electrophoresis under reducing conditions yielded 2 proteinbands corresponding to molecular weights of 51,000 and 53,000.The proteolytic mapping for the two separated subunits appearedsimilar and possibly identical. (Received October 28, 1991; Accepted February 24, 1992)     

Life history variation among years in a brackish water type of the Ninespine Stickleback, <Emphasis Type="Italic">Pungitius pungitius</Emphasis>

The life history variation in a brackish water type of the Ninespine Stickleback Pungitius pungitius collected over 3 years was studied by examining the strontium (Sr) and calcium (Ca) concentrations in the otoliths. The life histories showed the fish were either brackish water residents or anadromous throughout those years. 74–78% of the fish had the former life history and 22–26% the latter. The composition of each life history was constant throughout the 3 years, and the occurrence of the “anadromous” novel life history found previously is a general phenomenon in this species.     

Mouse epidermal keratinocytes in three-dimensional organotypic coculture with dermal fibroblasts form a stratified sheet resembling skin

We describe an organotypic model of mouse skin consisting of a stratified sheet of epidermal keratinocytes and dermal fibroblasts within a contracted collagen gel. The model was designed to maintain the polarity of stratified keratinocytes and permit their long-term culture at an air-liquid interface. After air exposure, the thickness of the keratinocyte sheet transiently increased and then decreased to two cell layers at 2 weeks. The two-cell-layer structure is similar to that of the adult mouse epidermis. Cytokeratin 5 was localized in the lowest cell layer in the epithelial sheet, but cytokeratin 1 and loricrin were localized in the outer cell layers, resembling mouse skin. The expressions of interleukin 1alpha and 1beta in the keratinocytes and of keratinocyte growth factor 1 and 2 in the fibroblasts correlated with keratinocyte stratification. The mouse organotypic coculture is useful in studying epithelial cell-mesenchymal cell interactions in vitro.     

Signal peptide peptidase: biochemical properties and modulation by nonsteroidal antiinflammatory drugs

Signal peptide peptidase (SPP) is an intramembrane aspartyl protease that cleaves remnant signal peptides after their release by signal peptidase. SPP contains active site motifs also found in presenilin, the catalytic component of the gamma-secretase complex of Alzheimer's disease. However, SPP has a membrane topology opposite that of presenilin, cleaves transmembrane substrates of opposite directionality, and does not require complexation with other proteins. Here we show that, upon isolation of membranes and solubilization with detergent, the biochemical characteristics of SPP are remarkably similar to gamma-secretase. The majority of the SPP-catalyzed cleavages occurred at a single site in a synthetic substrate based on the prolactin (Prl) signal sequence. However, as seen with cleavage of substrates by gamma-secretase, additional cuts at other minor sites are also observed. Like gamma-secretase, SPP is inhibited by helical peptidomimetics and apparently contains a substrate-binding site that is distinct from the active site. Surprisingly, certain nonsteroidal antiinflammatory drugs known to shift the site of proteolysis by gamma-secretase also alter the cleavage site of Prl by SPP. Together, these findings suggest that SPP and presenilin share certain biochemical properties, including a conserved drug-binding site for allosteric modulation of substrate proteolysis.     

Expression screening of 17q12-21 amplicon reveals GRB7 as an ERBB2-dependent oncogene

Gene amplification is a major genetic alteration in human cancers. Amplicons, amplified genomic regions, are believed to contain "driver" genes responsible for tumorigenesis. However, the significance of co-amplified genes has not been extensively studied. We have established an integrated analysis system of amplicons using retrovirus-mediated gene transfer coupled with a human full-length cDNA set. Applying this system to 17q12-21 amplicon observed in breast cancer, we identified GRB7 as a context-dependent oncogene, which modulates the ERBB2 signaling pathway through enhanced phosphorylation of ERBB2 and Akt. Our work provides an insight into the biological significance of gene amplification in human cancers.