Identification of an ADAMTS-4 cleavage motif using phage display leads to the development of fluorogenic peptide substrates and reveals matrilin-3 as a novel substrate

ADAMTS-4 and ADAMTS-5 are aggrecanases responsible for the breakdown of cartilage aggrecan in osteoarthritis. Multiple ADAMTS-4 cleavage sites have been described in several matrix proteins including aggrecan, versican, and brevican, but no concise predictive cleavage motif has been identified for this protease. By screening a 13-mer peptide library with a diversity of 10(8), we have identified the ADAMTS-4 cleavage motif E-(AFVLMY)-X(0,1)-(RK)-X(2,3)-(ST)-(VYIFWMLA), with Glu representing P1. Several 13-mer peptides containing this motif, including DVQEFRGVTAVIR and HNEFRQRETYMVF, were shown to be substrates for ADAMTS-4. These peptides were found to be specific substrates for ADAMTS-4 as they were not cleaved by ADAMTS-5. Modification of these peptides with donor (6-FAM) and acceptor (QSY-9) molecules resulted in the development of fluorescence-based substrates with a Km of approximately 35 microM. Furthermore, the role of Glu at P1 and Phe at P1' in binding and catalysis was studied by exploring substitution of these amino acids with the D-isomeric forms. Substitution of P1 with dGlu was tolerable for binding, but not catalysis, whereas substitution of P1' with dPhe precluded both binding and catalysis. Similarly, replacement of Glu with Asp at P1 abolished recognition and cleavage of the peptide. Finally, BLAST results of the ADAMTS-4 cleavage motif identified matrilin-3 as a new substrate for ADAMTS-4. When tested, recombinant ADAMTS-4 effectively cleaved intact matrilin-3 at the predicted motif at Glu435/Ala436 generating two species of 45 and 5 kDa.     

Active gamma-secretase complexes contain only one of each component

Gamma-secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin, and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional gamma-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid beta-peptides and amyloid beta-protein precursor intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by Western blot using protein standards and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of gamma-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1.     

Measurement of local strain on cell membrane at initiation point of calcium signaling response to applied mechanical stimulus in osteoblastic cells

In adaptive bone remodeling, it is believed that bone cells such as osteoblasts, osteocytes and osteoclasts can sense mechanical stimuli and modulate their remodeling activities. However, the mechanosensing mechanism by which these cells sense mechanical stimuli and transduce mechanical signals into intracellular biochemical signals is still not clearly understood. From the viewpoint of cell biomechanics, it is important to clarify the mechanical conditions under which the cellular mechanosensing mechanism is activated. The aims of this study were to evaluate a mechanical condition, that is, the local strain on the cell membrane, at the initiation point of the intracellular calcium signaling response to the applied mechanical stimulus in osteoblast-like MC3T3-E1 cells, and to investigate the effect of deformation velocity on the characteristics of the cellular response. To apply a local deformation to a single cell, a glass microneedle was directly indented to the cell and moved horizontally on the cell membrane. To observe the cellular response and the deformation of the cell membrane, intracellular calcium ions and the cell membrane were labeled using fluorescent dyes and simultaneously observed by confocal laser scanning microscopy. The strain distribution on the cell membrane attributable to the applied local deformation and the strain magnitude at the initiation point of the calcium signaling responses were analyzed using obtained fluorescence images. From two-dimensionally projected images, it was found that there is a local compressive strain at the initiation point of calcium signaling. Moreover, the cellular response revealed velocity dependence, that is, the cells seemed to respond with a higher sensitivity to a higher deformation velocity. From the viewpoint of cell biomechanics, these results provide us a fundamental understanding of the mechanosensing mechanism of osteoblast-like cells.     

Role of Src-family kinases in formation and trafficking of macropinosomes

Src-family kinases that localize to the cytoplasmic side of cellular membranes through lipid modification play a role in signaling events including membrane trafficking. Macropinocytosis is an endocytic process for solute uptake by large vesicles called macropinosomes. Although macropinosomes can be visualized following uptake of fluorescent macromolecules, little is known about the dynamics of macropinosomes in living cells. Here, we show that constitutive c-Src expression generates macropinosomes in a kinase-dependent manner. Live-cell imaging of GFP-tagged c-Src (Src-GFP) reveals that c-Src associates with macropinosomes via its N-terminus continuously from their generation at membrane ruffles, through their centripetal trafficking, to fusion with late endosomes and lysosomes. Fluorescence recovery after photobleaching (FRAP) of Src-GFP shows that Src-GFP is rapidly recruited to macropinosomal membranes from the plasma membrane and intracellular organelles through vesicle transport even in the presence of a protein synthesis inhibitor. Furthermore, using a HeLa cell line overexpressing inducible c-Src, we show that following stimulation with epidermal growth factor (EGF), high levels of c-Src kinase activity promote formation of macropinosomes associated with the lysosomal compartment. Unlike c-Src, Lyn and Fyn, which are palmitoylated Src kinases, only minimally induce macropinosomes, although a Lyn mutant in which the palmitoylation site is mutated efficiently induces macropinocytosis. We conclude that kinase activity of nonpalmitoylated Src kinases including c-Src may play an important role in the biogenesis and trafficking of macropinosomes.     

Exogenous expression of claudin-5 induces barrier properties in cultured rat brain capillary endothelial cells

Claudins are thought to be major components of tight junctions (TJs), and claudin-5 and -12 are localized at TJs of the blood-brain barrier (BBB). Claudin-5-deficient mice exhibit size-selective (<800 Da) opening of the BBB. The purpose of this study was to clarify the expression levels of claudin-5 and -12 in rat brain capillary endothelial cells, and to examine the ability of claudin-5 to form TJs in cultured rat brain capillary endothelial cells (TR-BBB). Expression of claudin-5 mRNA in rat brain capillary fraction was 751-fold greater than that of claudin-12. The level of claudin-5 mRNA in the rat brain capillary fraction (per total mRNA) was 35.6-fold greater than that in whole brain, while the level of claudin-12 mRNA was only 13.9% of that in whole brain, suggesting that expression of claudin-12 mRNA is not restricted to brain capillaries. Transfection of TR-BBB cells with the claudin-5 gene afforded TR-BBB/CLD5 cells, which showed no change in expression of claudin-12 or ZO-1, while the expressed claudin-5 was detected at the cell-cell boundaries. The permeability surface product of [(14)C]inulin at a TR-BBB/CLD5 cell monolayer was significantly smaller (P < 0.01) than that for the parental TR-BBB cells, and the values of the permeability coefficient (Pe) were 1.14 x 10(-3) and 11.6 x 10(-3) cm/min, respectively. These results indicate that claudin-5, but not claudin-12, is predominantly expressed in brain capillaries, and plays a key role in the appearance of barrier properties of brain capillary endothelial cells.     

Detection of insulin-releasing cells using in situ immunoblotting

Embryonic stem (ES) cells hold promise as a source for cell transplantation treatment of diseases such as type I diabetes. Further, cells releasing bioactive substances from ES cell progeny may be concentrated and purified for clinical applications. Although ES cell lines that express reporter genes have been established to isolate cells releasing bioactive substances, other difficulties must be overcome before these genetically modified cells can be used for gene therapy in human patients. Fluorescence- or magnetic-activated cell sorters are commonly used to isolate specific cells using antibodies against cell surface antigens. However, for some cells, such as insulin-producing beta cells, specific surface antigens have not yet been identified. In this study, we developed a simple and efficient method to identify and purify insulin- and alpha-fetoprotein-producing cells. A nitrocellulose membrane treated with anti-insulin or anti-alpha-fetoprotein antibodies was placed on a cell layer to trap insulin or alpha-fetoprotein released from the cells. The location of specific substance-producing cells was identified by immunostaining the membrane. The insulin-releasing cells were selectively collected from the culture dish using a cloning ring and transferred to another culture plate.     

Inhibition of p38 mitogen-activated protein kinase-induced apoptosis in cultured mature oligodendrocytes using SB202190 and SB203580

p38 Mitogen-activated protein kinase (p38 MAPK) is expressed in the oligodendrocyte lineage, and its activity has been implicated in the proliferation and transition of early progenitors into late progenitors. Although p38 MAPK expression has been found in the myelin sheath, however, its role in mature oligodendrocytes remains unknown. In the present study, in order to address the role of p38 MAPK in mature oligodendrocytes, selective inhibitors of p38 MAPK, SB202190, and SB203580 were added to primary cultures of mature oligodendrocytes. After 24h of exposure to the inhibitors, the appearance, and number of A2B5-positive progenitors were unchanged. However, the 2',3'-cyclic nucleotide-3'-phosphohydrolase-positive mature oligodendrocytes disappeared, and the numbers of living cells decreased in comparison to the control cells treated with SB202474, a negative analog of SB203580. Increases in the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive nuclei and in the activity of caspase-3/7 were detected 16 h after exposure to the inhibitors, thus causing the mature oligodendrocytes to die due to apoptosis. Similar results were obtained using a differentiated rat oligodendrocyte precursor cell (OPC) line, central glia-4 (CG-4). These findings indicate that p38 MAPK is vital for mature oligodendrocyte survival.     

Stem kernels for RNA sequence analyses

Several computational methods based on stochastic context-free grammars have been developed for modeling and analyzing functional RNA sequences. These grammatical methods have succeeded in modeling typical secondary structures of RNA, and are used for structural alignment of RNA sequences. However, such stochastic models cannot sufficiently discriminate member sequences of an RNA family from nonmembers and hence detect noncoding RNA regions from genome sequences. A novel kernel function, stem kernel, for the discrimination and detection of functional RNA sequences using support vector machines (SVMs) is proposed. The stem kernel is a natural extension of the string kernel, specifically the all-subsequences kernel, and is tailored to measure the similarity of two RNA sequences from the viewpoint of secondary structures. The stem kernel examines all possible common base pairs and stem structures of arbitrary lengths, including pseudoknots between two RNA sequences, and calculates the inner product of common stem structure counts. An efficient algorithm is developed to calculate the stem kernels based on dynamic programming. The stem kernels are then applied to discriminate members of an RNA family from nonmembers using SVMs. The study indicates that the discrimination ability of the stem kernel is strong compared with conventional methods. Furthermore, the potential application of the stem kernel is demonstrated by the detection of remotely homologous RNA families in terms of secondary structures. This is because the string kernel is proven to work for the remote homology detection of protein sequences. These experimental results have convinced us to apply the stem kernel in order to find novel RNA families from genome sequences.     

Nereidid polychaetes as the major diet of migratory shorebirds on the estuarine tidal flats at Fujimae-higata in Japan

The dietary items of five migratory shorebirds, Dunlin (Calidris alpina), Red-necked Stint (C. ruficollis), Grey Plover (Pluvialis squatarola), Whimbrel (Numenius phaeopus) and Black-headed Gull (Larus ridibundus), were examined by analyses of fecal droppings during the birds' migration or wintering and by surveys of macrobenthic fauna around their foraging sites on the tidal flats of Fujimae-higata, Nagoya, central Japan. Body parts of nereidid, capitellid, and spionid polychaetes and crustaceans were found in fecal droppings from all of these shorebirds. Two nereidid species (Hediste diadroma and Neanthes succinea) with relatively large body sizes seemed to be the majority dietary items. At one site, H. diadroma was dominant in terms of biomass (40-370 g/m(2)) throughout year except, for less than 1 g/m(2) in March and May (within or just after reproduction of this species). Monthly changes in the occurrence of food items in fecal droppings of C. alpina were examined in 1999 and 2000. Most (85-100%) of the fecal droppings contained nereidid body parts, including Hediste-specific simple chaetae from November to April, whereas only 23% of the droppings contained them in May. Chaetae of capitellid or spionid polychaetes were frequently found from January to April (38-86% of droppings). Crustacean body parts, including amphipod appendages, were frequently found from March to May (86-100% of droppings). The relationship between foraging habits of the shorebirds and the life history of their major prey nereidid species is discussed.