N-glycan structures of murine hippocampus serine protease, neuropsin, produced in Trichoplusia ni cells

N-glycans of neuropsin (serine protease in the murine hippocampus) expressed in Trichoplusia ni cells were released from the glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a two dimensional high-performance liquid chromatography (HPLC) mapping technique on two kinds of HPLC columns. Fourteen different major N-glycan structures were identified, of which 6 were high-mannose type (9.1%), and the remaining 8 were paucimannosidic type. The presence of insect specific N-glycan structures containing both 1,3- and 1,6- di-fucosylated innermost N-acetylglucosamine residue (23.3%), as below, was also confirmed by 600 MHz ¹H-NMR spectroscopy.     

Oligodendrocytes and radial glia derived from adult rat spinal cord progenitors: morphological and immunocytochemical characterization.

Self-renewing, multipotent neural progenitor cells (NPCs) reside in the adult mammalian spinal cord ependymal region. The current study characterized, in vitro, the native differentiation potential of spinal cord NPCs isolated from adult enhanced green fluorescence protein rats. Neurospheres were differentiated, immunocytochemistry (ICC) was performed, and the positive cells were counted as a percentage of Hoescht+ nuclei in 10 random fields. Oligodendrocytes constituted most of the NPC progeny (58.0% of differentiated cells; 23.4% in undifferentiated spheres). ICC and electron microscopy (EM) showed intense myelin production by neurospheres and progeny. The number of differentiated astrocytes was 18.0%, but only 2.8% in undifferentiated spheres. The number of differentiated neurons was 7.4%, but only 0.85% in undifferentiated spheres. The number of differentiated radial glia (RG) was 73.0% and in undifferentiated spheres 80.9%. EM showed an in vitro phagocytic capability of NPCs. The number of undifferentiated NPCs was 32.8% under differentiation conditions and 78.9% in undifferentiated spheres. Compared with ependymal region spheres, the spheres derived from the peripheral white matter of the spinal cord produced glial-restricted precursors. These findings indicate that adult rat spinal cord ependymal NPCs differentiate preferentially into oligodendrocytes and RG, which may support axonal regeneration in future trials of transplant therapy for spinal cord injury.     

Cadherin-related neuronal receptor 1 (CNR1) has cell adhesion activity with beta1 integrin mediated through the RGD site of CNR1

Cadherin-related neuronal receptor (CNR) proteins are a diverse set of synaptic protocadherins, but little is known about its adhesive properties. We found that overexpressed CNR1 protein localized on the cell surface of HEK293T cells and increased the calcium-dependent cell aggregation potential. However, we could not detect the strong homophilic binding activity of CNR1 EC-Fc fusion protein in vitro. Parental HEK293T cells adhered to Arg-Gly-Asp (RGD) motif of EC1 domain of CNR1-Fc fusion protein. The fusion protein that the Asp73 of EC1 point-mutated to Glu (RGE-Fc) lost the adhesive activity. The adhesion activity of HEK293T cells to CNR1 EC-Fc fusion protein was completely blocked by inhibitors of integrins, including RGDS peptide and anti-beta1 integrin antibodies. The increased cell-aggregative property of CNR1 transfectants was also blocked by RGDS peptides. At cell-cell junctions of the CNR1 transfectants, co-localization between CNR1 and HEK293T endogenous beta1 integrin was observed. Furthermore, the spatiotemporal expression patterns of CNR and beta1 integrin nearly overlapped in the molecular layer of the developing mouse cerebellum in the main stage of synaptogenesis. These results indicate that CNR1 has a heterophilic, calcium-dependent cell adhesion activity with the beta1 integrin subfamily, and raise the possibility of CNR-beta1 integrin association in synaptogenesis.     

Suppression of 3-deoxyglucosone and heparin-binding epidermal growth factor-like growth factor mRNA expression by an aldose reductase inhibitor in rat vascular smooth muscle cells

Reactive carbonyl compounds and oxidative stress have been recently shown to up-regulate the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen for vascular smooth muscle cells (SMCs) produced by SMC themselves. Because the polyol pathway has been reported to influence the formation of carbonyl compounds and the oxidative stress in various cells, we conducted this study to investigate whether the polyol pathway affects HB-EGF expression along with the generation of carbonyl compounds and the oxidative stress in SMCs. We found that, compared with those cultured with 5.5mM glucose, SMCs cultured with 40 mM glucose showed the accelerated thymidine incorporation, elevated levels of intracellular sorbitol, 3-deoxyglucosone (3-DG), advanced glycation end products (AGEs), and thiobarbituric acid-reactive substances (TBARS) along with the enhanced expression of HB-EGF mRNA. An aldose reductase inhibitor (ARI), SNK-860, significantly inhibited all of these abnormalities, while aminoguanidine suppressed 3-DG levels and HB-EGF mRNA expression independent of sorbitol levels. The results suggest that the polyol pathway may play a substantial role in SMC hyperplasia under hyperglycemic condition in part by affecting HB-EGF mRNA expression via the production of carbonyl compounds and oxidative stress.     

Expression of a novel secretory form (Crb1s) of mouse Crumbs homologue Crb1 in skin development

Drosophila Crumbs and the mammalian homologues encoded by the Crb genes are transmembrane proteins required for determination of retinal cell polarity. We cloned a novel variant of mouse Crb1 and termed it Crb1s. Since the 3'-end of exon 6 remained unspliced, Crb1s coded for a short secretory protein lacking the transmembrane and cytoplasmic domains required for the function of Crb1. The Crb1 expression was confined to brain and eye, whereas Crb1s was detectable in various tissues including skin, lung, and kidney in adult mice. Active expression of Crb1s, but not Crb1, was observed during the skin development, in which localization of the Crb1s protein was altered from the basal layer to the upper layers. Cultured mouse keratinocytes synthesized the Crb1s protein and secreted a 80 kDa processed form to the supernatant. After Ca(2+)-induced differentiation, Crb1s became associated with focal adhesions and cell-cell contacts. Crb1s may play a role distinct from that of Crb1 in epidermal tissue morphogenesis.     

Copper mediates dityrosine cross-linking of Alzheimer's amyloid-beta

We have previously reported that amyloid Abeta, the major component of senile plaques in Alzheimer's disease (AD), binds Cu with high affinity via histidine and tyrosine residues [Atwood, C. S., et al. (1998) J. Biol. Chem. 273, 12817-12826; Atwood, C. S., et al. (2000) J. Neurochem. 75, 1219-1233] and produces H(2)O(2) by catalyzing the reduction of Cu(II) or Fe(III) [Huang, X., et al. (1999) Biochemistry 38, 7609-7616; Huang, X., et al. (1999) J. Biol. Chem. 274, 37111-37116]. Incubation with Cu induces the SDS-resistant oligomerization of Abeta [Atwood, C. S., et al. (2000) J. Neurochem. 75, 1219-1233], a feature characteristic of neurotoxic soluble Abeta extracted from the AD brain. Since residues coordinating Cu are most vulnerable to oxidation, we investigated whether modifications of these residues were responsible for Abeta cross-linking. SDS-resistant oligomerization of Abeta caused by incubation with Cu was found to induce a fluorescence signal characteristic of tyrosine cross-linking. Using ESI-MS and a dityrosine specific antibody, we confirmed that Cu(II) (at concentrations lower than that associated with amyloid plaques) induces the generation of dityrosine-cross-linked, SDS-resistant oligomers of human, but not rat, Abeta peptides. The addition of H2O2 strongly promoted Cu-induced dityrosine cross-linking of Abeta1-28, Abeta1-40, and Abeta1-42, suggesting that the oxidative coupling is initiated by interaction of H2O2 with a Cu(II) tyrosinate. The dityrosine modification is significant since it is highly resistant to proteolysis and is known to play a role in increasing structural strength. Given the elevated concentration of Cu in senile plaques, our results suggest that Cu interactions with Abeta could be responsible for causing the covalent cross-linking of Abeta in these structures.     

p51/p63 Controls subunit alpha3 of the major epidermis integrin anchoring the stem cells to the niche

p51/p63, a member of the tumor suppressor p53 gene family, is crucial for skin development. We describe here identification of ITGA3 encoding integrin alpha(3) as a target of its trans-activating function, proposing that p51/p63 allows epidermal stem cells to express laminin receptor alpha(3)beta(1) for anchorage to the basement membrane. When activated by genotoxic stress or overexpressed ectopically in non-adherent cells, p51/p63 transduced a phenotype to attach to extracellular matrices, which was accompanied by expression of ITGA3. Motifs matching the p53-binding consensus sequence were located in a scattered form in intron 1 of human ITGA3, and served as p51/p63-responsive elements in reporter assays. In addition to the trans-activating ability of the TA isoform, we detected a positive effect of the DeltaN isoform on ITGA3. The high level alpha(3) production in human keratinocyte stem cells diminished upon elimination of p51/p63 by small interfering RNA or by Ca(2+)-induced differentiation. Furthermore, a chromatin immunoprecipitation experiment indicated a physical interaction of p51/p63 with intron 1 of ITGA3. This study provides a molecular basis for the standing hypothesis that p51/p63 is essential for epidermal-mesenchymal interactions.     

15-deoxy-delta 12,14-prostaglandin J2 and laminar fluid shear stress stabilize c-IAP1 in vascular endothelial cells

Laminar shear stress strongly inhibits vascular endothelial cell apoptosis by unknown mechanisms. We reported that shear stress stimulates endothelial cells to produce 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) by elevating the expression level of lipocalin-type prostaglandin D synthase. To investigate the role of 15d-PGJ2 produced in the vascular wall, we examined the effect of 15d-PGJ2 on endothelial cell apoptosis. We induced apoptosis in human umbilical vein endothelial cells (HUVECs) by growth factor deprivation. 15d-PGJ2 strongly inhibited DNA ladder formation, nuclear fragmentation, and caspase-3-like activity in HUVECs. To elucidate the mechanism by which 15d-PGJ2 inhibits endothelial cell apoptosis, we examined expression of the inhibitor of apoptosis proteins (IAP) cellular-IAP1 (c-IAP1), c-IAP2, x-linked IAP, and survivin in HUVECs. In parallel with the inhibition of apoptosis, 15d-PGJ2 elevated the expression level of c-IAP1 protein in a dose- and time-dependent manner without changing the mRNA level. Laminar shear stress also induced c-IAP1 expression. Chase experiments with the use of cycloheximide revealed that 15d-PGJ2 and shear stress both inhibited the proteolytic degradation of c-IAP1 protein. These results suggested that 15d-PGJ2 inhibits endothelial cell apoptosis through, at least in part, c-IAP1 protein stabilization. This mechanism might be involved in the antiapoptotic effect of laminar shear stress.     

Membrane filter (pore size, 0.22-0.45 micro m; thickness, 150 micro m) passing-through activity of Pseudomonas aeruginosa and other bacterial species with indigenous infiltration ability

Bacteria growing on MF-Millipore filters (thickness, 150 micro m) passed through the underlying membrane by their infiltration activity. Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli passed through a 0.45- micro m pore size filter within 48-96 h. Pseudomonas aeruginosa, Serratia marcescens, and Listeria monocytogenes passed through a 0.3- micro m pore size filter. P. aeruginosa passed through a 0.22- micro m pore size filter. The membranes which allowed passing-through of bacteria showed normal bubble point values in the integrity test. Studies with isogenic S. marcescens mutants indicated that flagellum-dependent motility or surface-active exolipid were important in the passing-through. P. aeruginosa PAO1 C strain defective in twitching motility was unable to pass through the 0.22- micro m filter. Scanning electron microscopy showed bacteria passing-through the 0.22- micro m filter. Millipore membrane filters having well-defined reticulate structures will be useful in the study of infiltration activity of microbes.     

The genome stability in Corynebacterium species due to lack of the recombinational repair system

Corynebacterium species are members of gram-positive bacteria closely related to Mycobacterium species, both of which are classified into the same taxonomic order Actinomycetales. Recently, three corynebacteria, Corynebacterium efficiens, Corynebacterium glutamicum, and Corynebacterium diphtheriae have been sequenced independently. We found that the order of orthologous genes in these species has been highly conserved though it has been disrupted in Mycobacterium species. This synteny suggests that corynebacteria have rarely undergone extensive genome rearrangements and have maintained ancestral genome structures even after the divergence of corynebacteria and mycobacteria. This is the first report that the genome structures have been conserved in free-living bacteria such as C. efficiens and C. glutamicum, although it has been reported that obligate parasites such as Mycoplasma and Chlamydia have the stable genomes. The comparison of recombinational repair systems among the three corynebacteria and Mycobacterium tuberculosis suggested that the absence of recBCD genes in corynebacteria be responsible for the suppression of genome shuffling in the species. The genome stability in Corynebacterium species will give us hints of the speciation mechanism with the non-shuffled genome, particularly the importance of horizontal gene transfer and nucleotide substitution in the genome.