Abeta-degrading endopeptidase,neprilysin, in mouse brain: synaptic and axonal localization inversely correlating with Abeta pathology

Metabolism of amyloid-beta peptide (Abeta) is closely associated with the pathology and etiology of Alzheimer's disease (AD). Since neprilysin is the only rate-limiting catabolic peptidase proven by reverse genetics to participate in Abeta metabolism in vivo, we performed detailed immunohistochemical analysis of neprilysin in mouse brain using neprilysin-deficient mice as a negative control. The aim was to assess, at both the cellular and subcellular levels, where Abeta undergoes neprilysin-dependent degradation in the brain and how neprilysin localization relates to Abeta pathology in amyloid precursor protein (APP)-transgenic mice. In hippocampus, neprilysin was present in the stratum pyramidale and stratum lacunosum-moleculare of the CA1-3 fields and the molecular layer of the dentate gyrus. Confocal double immunofluorescence analyses revealed the subcellular localization of neprilysin along axons and at synapses. This observation suggests that after synthesis in the soma, neprilysin, a type II membrane-associated protein, is axonally transported to the terminals, where Abeta degradation is likely to take place. Among various cell types, GABAergic and metabotropic glutamate 2/3 receptor-positive neurons but not catecholaminergic or cholinergic neurons, expressed neprilysin in hippocampus and neocortex, implying the presence of a cell type-specific mechanism that regulates neprilysin gene expression. As expected, Abeta deposition correlated inversely with neprilysin expression in TgCRND8 APP-transgenic mice. These observations not only support the notion that neprilysin functions as a major Abeta-degrading enzyme in the brain but also suggest that down-regulation of neprilysin activity, which may be caused by aging, is likely to elevate local concentrations of Abeta at and around neuronal synapses.     

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     

A Comparative Analysis of the Murine Thymic Microenvironment in Normal,Autoimmune, and Immunodeficiency States

It is widely accepted that the thymic microenvironment regulates normal thymopoiesis through a highly coordinated and complex series of cellular and cytokine interactions. A direct corollary of this is that abnormalities within the microenvironment could be of etiologic significance in T-cell-based diseases. Our laboratory has developed a large panel of monoclonal antibodies (mAbs) that react specifically with epithelial or nonepithelial markers in the thymus. We have taken advantage of these reagents to characterize the thymic microenvironment of several genetic strains of mice, including BALB/cJ, C57BL/6J, NZB/BlnJ, SM/J, NOD/Ltz, NOD/Ltz-scid/sz, C57BL/6J-Hcph m^e/Hcph m^e, and ALY/NscJcl-aly/aly mice, and littermate control animals. We report herein that control mice, including strains of several backgrounds, have a very consistent phenotypic profile with this panel of monoclonal antibodies, including reactivity with thymic epithelial cells in the cortex, the medulla and the corticomedullary junction, and the extracellular matrix. In contrast, the disease-prone strains studied have unique, abnormal staining of thymic cortex and medulla at both the structural and cellular levels. These phenotypic data suggest that abnormalities in interactions between developing thymocytes and stromal cells characterize disease-prone mice.     

Prediction of sensitivity of advanced non-small cell lung cancers to gefitinib (Iressa, ZD1839)

Gefitinib (Iressa, ZD1839), an inhibitor of epidermal growth factor receptor-tyrosine kinase, has shown potent anti-tumor effects and improved symptoms and quality-of-life of a subset of patients with advanced non-small cell lung cancer (NSCLC). However, a large portion of the patients showed no effect to this agent. To establish a method to predict the response of NSCLC patients to gefitinib, we used a genome-wide cDNA microarray to analyze 33 biopsy samples of advanced NSCLC from patients who had been treated with an identical protocol of second to seventh line gefitinib monotherapy. We identified 51 genes whose expression differed significantly between seven responders and 10 non-responders to the drug. We selected the 12 genes that showed the most significant differences to establish a numerical scoring system (GRS, gefitinib response score), for predicting response to gefitinib treatment. The GRS system clearly separated the two groups without any overlap, and accurately predicted responses to the drug in 16 additional NSCLC cases. The system was further validated by the semi-quantitative RT-PCR, immunohistochemistry and ELISA for serological test. Moreover, we proved that the anti-apoptotic activity of amphiregulin, a protein that was significantly over-expressed in non-responders but undetectable in responders, leads to resistance of NSCLC cells to gefitinib in vitro. Our results suggested that sensitivity of a given NSCLC to gefitinib can be predicted according to expression levels of a defined set of genes that may biologically affect drug sensitivity and survival of lung cancer cells. Our scoring system might eventually lead to achievement of personalized therapy for NSCLC patients.     

Immunohistochemical expressions of cytokeratins, mucin core proteins, p53, and neuroendocrine cell markers in epithelial neoplasm of appendix

Epithelial neoplasms of appendix are infrequent, and their pathological features are not fully characterized. We collected 33 cases of appendiceal tumors and examined immunohistochemically the expression of cytokeratins (CK, CK7, and CK20), mucin core protein (MUC1, MUC2, MUC5AC, and MUC6), E-cadherin, chromogranin A, and p53 protein. Gene analysis of TP53 was also conducted on exons 5 to 8. Clinically, mucinous tumors were predominant in females. Immunohistochemically, all the tumors expressed CK20, whereas CK7 was positive in one third of the cases. Similarly, MUC2 was expressed in all the tumors, whereas MUC1 and MUC5AC were detected in about a half of the cases. Although chromogranin A-positive cells are generally sparse in normal appendix, they were more common in mucinous tumors than in nonmucinous tumors. Contrary to the previous data reported (Mod Pathol 2002;15:599-605), mucinous carcinoma exhibited a higher frequency of p53-positive cells (mean 29%) compared with mucinous adenoma (2.8%) (P < .001), whereas nonmucinous tumors showed high levels of p53-positive cells to similar extent (51%-67%) in both adenoma and carcinoma. The high expression of p53 protein coincided with the presence of mutations in multiple sites of TP53 gene in mucinous tumors. This is the first report that characterized the immunophenotypic profile of appendiceal epithelial neoplasms with an emphasis of a higher frequency of p53 positivity in mucinous carcinoma cases compared with mucinous adenoma in the appendix.     

Genetic and phenotypic characteristics of enterovirus 71 isolates from patients with encephalitis and with hand,foot and mouth disease

Summary Biological and biochemical characters of seven enterovirus 71 (E71) isolates were compared. Four isolates (two from patients with hand, foot and mouth disease [HFMD] and two from patients with encephalitis) grew in cynomolgus monkey kidney cells both at 39.5 and 35° C. However, the remaining three strains (from patients with HFMD) grew at 35° C, but not at 39.5° C. Three temperature-resistant and two temperature-sensitive strains were tested for neurovirulence in monkeys. Temperature-resistant strains were shown to be neurovirulent, whereas temperature-sensitive strains were less neurovirulent. The results suggest correlation between temperature-sensitive growth and neurovirulence in monkeys of E71. Variation in the electrophoretic mobility of the viral polypeptides was detected in three out of seven strains. The fingerprinting of oligonucleotides generated from the viral genome showed similar patterns in two isolates from patients with HFMD and one from patient with encephalitis and variable patterns in each genomic map of remaining four strains. These variations of polypeptide patterns and of oligonucleotide maps could not be correlated with pathogenicity (encephalitis or HFMD), temperature-sensitive growth and neurovirulence in monkeys.With 4 Figures     

Anionic polymerization of fluorine-containing vinyl monomers, 6. Polymerizations of fluoroalkyl acrylates and methacrylates initiated by organoaluminium compounds

The anionic polymerizations of 2,2,2-trifluoroethyl acrylate ( 1a ), 2,2,2-trifluoroethyl methacrylate ( 1b ), 2,2,2-trifluoro-1-trifluoromethylethyl acrylate ( 2a ), and 2,2,2-trifluoro-1-trifluoromethylethyl methacrylate ( 2b ) were examined with triethylaluminium and diethylaluminium active methylene chelate compounds as initiators. 2b was polymerized with triethylaluminium. Diethyl(ethyl cyanoacetato)aluminium was found to produce polymers of 1a and 1b . Diethyl(acetylacetonato)aluminium and diethyl (dimethyl malonato)aluminium, however, show low reactivity towards these four monomers. The resulting polymers show unimodal molecular weight distribution with number-average molecular weights of 10⁴?10⁵, measured by GPC. The initiation reaction and the copolymerization reactivity with styrene support the anionic polymerization mechanism.     

Immunohistochemical and electron microscopic observations of stromal cells in the human oviduct mucosa

Stromal cells in the lamina propria of the human oviduct mucosa are unique cells that can differentiate into decidual cells during ectopic pregnancy in the oviduct. The nature of stromal cells is still unknown. In the present study, we investigated human oviductal stromal cells with transmission electron microscopy and immunohistochemistry and revealed that they had ultrastructural features similar to myofibroblasts and expressed alpha-smooth muscle actin, a marker used to identify myofibroblasts. Primary cilia were also one of the characteristic profiles of the stromal cells. These findings showed that the connective tissue-stromal cells in the human oviduct mucosa are myofibroblasts. They are considered to play an important role in the transport of oocytes by bringing about contraction of the mucosal folds.