Molecular interactions between presenilin and calpain: inhibition of m-calpain protease activity by presenilin-1, 2 and cleavage of presenilin-1 by m-, mu-calpain

Several mutations of presenilin (PS)-1, 2 result in early onset Alzheimer disease. Using the yeast two-hybrid system, the interaction between PS2 loop domain and the C-terminal region of mu-calpain was previously identified. Calpain is a calcium dependent-protease and there are two isoforms, m-calpain and mu-calpain, which differ in the calcium concentration required for activation. m-Calpain needs about 10(-3) M calcium ions, whereas mu-calpain about 10(-5) M. When PS and calpain were separately expressed in COS cells by cDNA transfection and then combined in vitro, or both were co-transfected to be co-expressed in vivo in COS cells, PS1 and PS2 reduced the casein proteolysis activity of m-calpain but not that of mu-calpain. Some of the PS mutations related to Alzheimer disease decreased this inhibitory activity. On the other hand, PS1 was cleaved by m-calpain and mu-calpain at a different site from those already reported (constitutive cleavage or alternative cleavage). These results suggest a regulatory function of presenilin on the calpain system.     

Clinical significance of expression of membrane type 1 matrix metalloproteinase and matrix metalloproteinase-2 in human head and neck squamous cell carcinoma

Three different membrane-type matrix metalloproteinases (MT-MMPs) activate in vitro the latent form of matrix metalloproteinase-2 (MMP-2), which is one of the key proteinases in invasion and metastasis of various cancers. We examined the mRNA expression of MT1, 2, and 3-MMPs and MMP-2 in cell lines of head and neck squamous cell carcinoma (HNSCC) and quantitated the relative expression levels in human HNSCC tissues by Northern blotting. The tissue localization of MT1-MMP and MMP-2 was determined by immunohistochemistry and in situ hybridization. Their implications in clinicopathologic factors were statistically evaluated. All cell lines examined consistently expressed MT1-MMP and MMP-2, but not MT2, 3-MMP. In the clinical specimens, there was a significant correlation in coexpression of messenger of RNA (P = .0005) and colocalization by immunohistochemistry (P < .0001) for MT1-MMP and MMP-2. Relative mRNA expression levels of MT1-MMP and MMP-2 in the carcinoma tissues were significantly higher than those of the control tissues (P = .0045 and P = .0122, respectively). Both mRNA expression level and immunopositivity of MT1-MMP significantly correlated with lymph node metastasis (P = .0081 and P = .0193, respectively), which was confirmed by multivariate logistic regression analysis. Immunoreaction of MT1-MMP and its mRNA expression were observed in both carcinoma cells and stromal cells. The localization of MMP-2 closely corresponded to that of MT1-MMP. These observations suggest that MT1-MMP possesses a role as a determinant of lymph node metastasis in HNSCC, and that concurrent expression of MT1-MMP and MMP-2 are involved in progression of HNSCC.     

Effects of sodium, potassium and calcium ions on the slow wave in the circular muscle of the guinea-pig stomach

1. The contribution of Na, K and Ca ions to the generation of slow waves in the circular muscle of the guinea-pig stomach was studied.     

Effects of Ca removal on the smooth muscle of the guinea-pig taenia coli

1. The effects of removing the external Ca ions on the spontaneous and evoked activity of the smooth muscle of the guinea-pig taenia coli were investigated with the double sucrose-gap method.2. In Ca-free Locke solution the membrane was depolarized, the membrane resistance became low, the spike amplitude became small and the mechanical response decreased. In most preparations the electrical and mechanical activity was abolished within 10 min, but in some preparations the electrical activity continued for more than 30 min.3. In Ca-free solution containing 0.1 mM-EGTA, the membrane was depolarized and the electrical and mechanical activity was abolished within 5 min in every preparation. When NaCl was replaced with sucrose, the effects of Ca removal on the spike activity and contraction appeared very slowly and the membrane potential and membrane resistance remained unchanged.4. When Ca was replaced with Mg (2 mM) the spike was blocked within 1 min without depolarization or reduction of the membrane resistance. In Na-deficient (sucrose) solution, the presence of Mg accelerated the disappearance of the spike caused by Ca removal.5. In Ca-free solution containing 0.5 mM-Mg, a spike-like activity was observed without accompanying mechanical response. This activity was blocked by increasing the Mg concentration above 2 mM. It was Na-dependent, since it was abolished by removing Na from the external solution, but it was not influenced by tetrodotoxin (2 x 10(-6) g/ml.).6. It was concluded that calcium has at least two functions, one as current carrier for the action potential and another as controller of the Na permeability of the membrane. It was also suggested that the Ca which is bound at the membrane may be utilized as a source of Ca ions to carry the current for the action potential.     

Cartilage regeneration using slow release of bone morphogenetic protein-2 from a gelatin sponge to treat experimental canine tracheomalacia: a preliminary report

We investigated whether saber sheath-type tracheomalacia could be treated by the slow release of bone morphogenetic protein (BMP)-2 from a gelatin sponge. A 1 cm gap was made in the middle portion of each of 10 consecutive tracheal cartilage rings in the canine cervix (control group, n = 3), then a gelatin sponge containing 12 microg of BMP-2 solution was implanted in the gap (12 microg group, n = 3). In another group (120 microg + P group, n = 3), the implanted gelatin sponge contained 120 microg of BMP-2 solution, and the gap was covered with periosteum. All of the control dogs developed saber sheath-type tracheomalacia, whereas tracheomalacia was not observed in the 12 microg and 120 microg + P groups. In the 12 microg group, fibrous cartilage was observed at the ends of the cartilage stumps. In the 120 microg + P group, newly formed bone and cartilage were observed to form a bridge between the cartilage stumps. The regeneration of cartilage or bone induced by the slow release of BMP-2 from a gelatin sponge might be useful for treatment of tracheomalacia.     

Inferring alternative splicing patterns in mouse from a full-length cDNA library and microarray data

Although many studies on alternative splicing of specific genes have been reported in the literature, the general mechanism that regulates alternative splicing has not been clearly understood. In this study, we systematically aligned each pair of the 21,076 cDNA sequences of Mus musculus, searched for putative alternative splicing patterns, and constructed a list of potential alternative splicing sites. Two cDNAs are suspected to be alternatively spliced and originating from a common gene if they share most of their region with a high degree of sequence homology, but parts of the sequences are very distinctive or deleted in either cDNA. The list contains the following information: (1) tissue, (2) developmental stage, (3) sequences around splice sites, (4) the length of each gapped region, and (5) other comments. The list is available at http://www.bioinfo.sfc.keio.ac.jp/intron. Our results have predicted a number of unreported alternatively spliced genes, some of which are expressed only in a specific tissue or at a specific developmental stage.     

Spatial and temporal expression patterns of the cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 during mouse development

The cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 are thought to regulate progression of the cell cycle. We have previously shown that the phenotypes of p27-/- mice are substantially different from those of p57-/- mice, suggesting that spatial and temporal expression patterns of p27Kip1 and p57Kip2 might be distinct. In this study, the roles of p27Kip1 and p57Kip2 in development were examined by characterizing their expression patterns during mouse embryogenesis by immunohistochemical analysis. Whereas certain organs and tissues (brain, lens, ganglion, lung, heart, liver, skin and kidney) expressed both proteins, others expressed only p27Kip1 (thymus, spleen, retina, testis and ovary) or only p57Kip2 (gut, palate, pancreas, cartilage and skeletal muscle). In addition, some organs expressed both p27Kip1 and p57Kip2 but showed mutually exclusive patterns of distribution among tissues. Thus, in the adrenal gland, p57Kip2 was expressed in the cortex but not in the medulla, whereas p27Kip1 was expressed in the medulla but not in the cortex. Whereas the expression of p57Kip2 in most tissues was restricted to embryogenesis, expression of p27Kip1 in many tissues was maintained in adult animals. Double-label immunofluorescence staining with either anti-p27Kip1 or anti-p57Kip2 and anti-BrdU revealed that the expression of p27Kip1 and p57Kip2 was inversely correlated with cell proliferation, suggesting that p27Kip1 and p57Kip2 are expressed exclusively in postmitotic cells. These complex spatial and temporal patterns of expression are consistent with the phenotypes of mice deficient in p27Kip1 or p57Kip2, and they suggest that these proteins might play important roles in tissue development.     

Nucleotide sequence of genome segment 5 from Bombyx mori cypovirus 1

Summary.  The complete nucleotide sequences of the double-stranded RNA genome segments 5 (S5) from Bombyx mori cypovirus 1 (BmCPV-1) strains I and H were determined. The segments consisted of 2,852 nucleotides encoding putative proteins of 881 amino acids with molecular masses of approximately 101 kDa (p101). A homology search showed that p101 has high similarity (93%) to foot-and-mouth disease virus (FMDV) 2A protease (2A^pro) at amino acid position 219 to 235. These findings suggest the possibility that p101 encoded by BmCPV-1 S5 might be cleaved into two non-structural proteins by post-translational autocleavage involving a 2A^pro-like protease. Received February 21, 2000 Accepted June 23, 2000