Enhancement of alkaline phosphatase synthesis in pulp cells co-cultured with epithelial cells derived from lower rabbit incisors

Dental papilla mesenchymal cells differentiate into odontoblasts through epithelial-mesenchymal interactions. However, the mechanism by which enamel epithelial cells affect the differentiation of dental mesenchymal cells remains unknown. Alkaline phosphatase (ALPase) is a marker for odontoblast-like differentiation, because odontoblasts show much higher ALPase activity than dental undifferentiated mesenchymal cells. The continuously growing rabbit incisor is a good model for the epithelial-mesenchymal interaction during odontogenesis. In the present study, we isolated and maintained rabbit incisor-derived epithelial cells and rabbit incisor pulp-derived fibroblastic cells, and examined the effect of epithelial cells on ALPase activity in fibroblastic cells. Epithelial cells were stained with anti-cytokeratin 5 and 8 antibodies and showed the expression of tuftelin mRNA. In separate cultures of epithelial cells or fibroblastic cells, ALPase activity and mRNA levels were very low, but were upregulated in co-cultures of epithelial and fibroblastic cells. Histochemical analysis found high ALPase activity in fibroblastic cells close to epithelial cells. These findings suggest that epithelial cells play an important role in promoting ALPase expression in pulp fibroblastic cells. The co-culture system developed here will be useful for examining the role of the epithelial-mesenchymal interaction during odontoblast differentiation.     

Concise synthesis and biological activities of 2alpha-alkyl- and 2alpha-(omega-hydroxyalkyl)-20-epi-1alpha,25-dihydroxyvitamin D3

We found a concise route to the Trost A-ring precursor enyne for synthesizing 2alpha-alkylated 1alpha,25-dihydroxyvitamin D(3) (1) from D-glucose. The enynes were coupled with the 20-epi-CD ring part to study the effect of the double modification of 2alpha-substitution and 20-epimerization upon biological activities of 1. The novel three analogues of 2alpha-alkyl- and four analogues of 2alpha-(omega-hydroxyalkyl)-20-epi-1alpha,25-dihydroxyvitamin D(3) (5b-d and 6a-d) showed higher binding affinity for vitamin D receptor (VDR) and more potent activity in induction of HL-60 cell differentiation than those of the natural hormone 1.     

A non-sulfated form of the HNK-1 carbohydrate is expressed in mouse kidney

The HNK-1 carbohydrate, which is recognized by anti-HNK-1 antibody, is well known to be expressed predominantly in the nervous system. The characteristic structural feature of the HNK-1 carbohydrate is 3-sulfo-glucuronyl residues attached to lactosamine structures (Gal beta1-4GlcNAc) on glycoproteins and glycolipids. The biosynthesis of the HNK-1 carbohydrate is regulated mainly by two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase. In this study, we found that GlcAT-S mRNA was expressed at higher levels in the kidney than in the brain, but that both GlcAT-P and HNK-1 sulfotransferase mRNAs, which were expressed at high levels in the brain, were not detected in the kidney. These results suggested that the HNK-1 carbohydrate without sulfate (non-sulfated HNK-1 carbohydrate) is expressed in the kidney. We substantiated this hypothesis using two different monoclonal antibodies: one (anti-HNK-1 antibody) requires sulfate on glucuronyl residues for its binding, and the other (antibody M6749) does not. Western blot analyses of mouse kidney revealed that two major bands (80 and 140 kDa) were detected with antibody M6749, but not with anti-HNK-1 antibody. The 80- and 140-kDa band materials were identified as meprin alpha and CD13/aminopeptidase N, respectively. We also confirmed the presence of the non-sulfated HNK-1 carbohydrate on N-linked oligosaccharides by multistage tandem mass spectrometry. Immunofluorescence staining with antibody M6749 revealed that the non-sulfated HNK-1 carbohydrate was expressed predominantly on the apical membranes of the proximal tubules in the cortex and was also detected in the thin ascending limb in the inner medulla. This is the first study indicating the presence of the non-sulfated HNK-1 carbohydrate being synthesized by GlcAT-S in the kidney. The results presented here constitute novel knowledge concerning the function of the HNK-1 carbohydrate.     

DNA sequence recognition by NF kappa B p50 homodimer: strict and obscure recognition sites in the binding sequence

5-Formyl- and 5-(formylmethyl)-2'-deoxyuridines are introduced into a kappa B site instead of thymidine(s) in order to understand target sequence specificity of NF kappa B. It was found that one thymidine in the kappa B site is particularly important for the sequence specific recognition by NF kappa B.     

Phenotypic modulation of parietal epithelial cells of Bowman's capsule in culture

The origin of cobblestone-like polygonal cells (the most numerous in renal glomerular cell culture) remains controversial; they could be either dedifferentiated podocytes or parietal epithelial cells (PECs) of Bowman's capsule. Poor cellular outgrowth from glomeruli devoid of Bowman's capsule (decapsulated glomeruli) hinders podocytes being obtained without contamination of PECs in culture. Since podocytes are easily damaged during the isolation of glomeruli by the conventional sieving method, we devised a gentle isolation method without forced sieving, resulting in substantial numbers of arborized cells growing out from decapsulated glomeruli. The cells were distinctly different from cobblestone-like polygonal cells in their irregular and often arborized shape and extended long cytoplasmic processes that often crossed over adjacent cells. The arborized cells from decapsulated glomeruli showed intense staining for a podocyte-specific marker, podocalyxin, but no staining for markers specific to PECs (pan cadherin), mesangial cells (Thy-1) or endothelial cells (von Willebrand factor, RECA-1), indicating their podocyte origin. Polygonal cells growing out from encapsulated glomeruli were negative for podocalyxin and positive for pan cadherin at the peripheral cell-cell contact. Thus, the cell population from decapsulated glomeruli is distinctly different from that from encapsulated glomeruli, supporting the idea that polygonal cells originate from PECs, although immunocytochemical markers specific to podocytes in vivo such as WT1, synaptopodin, HSP27 and P-31 antigen were expressed significantly in the polygonal cells. Occasionally, large irregular-shaped cells appeared at the periphery of the outgrowths from encapsulated glomeruli. They were similar in shape to the arborized cells from decapsulated glomeruli but were identical in antigenic properties to cobblestone-like polygonal cells and thus may be named "pseudo-arborized cells". We conclude that PECs in culture modulate their phenotype to resemble podocytes.     

Structure of a NifS homologue: X-ray structure analysis of CsdB, an Escherichia coli counterpart of mammalian selenocysteine lyase

Escherichia coli CsdB, a NifS homologue with a high specificity for L-selenocysteine, is a pyridoxal 5'-phosphate (PLP)-dependent dimeric enzyme that belongs to aminotransferases class V in fold-type I of PLP enzymes and catalyzes the decomposition of L-selenocysteine into selenium and L-alanine. The crystal structure of the enzyme has been determined by the X-ray crystallographic method of multiple isomorphous replacement and refined to an R-factor of 18.7% at 2.8 A resolution. The subunit structure consists of three parts: a large domain of an alpha/beta-fold containing a seven-stranded beta-sheet flanked by seven helices, a small domain containing a four-stranded antiparallel beta-sheet flanked by three alpha-helices, and an N-terminal segment containing two alpha-helices. The overall fold of the subunit is similar to those of the enzymes belonging to the fold-type I family represented by aspartate aminotransferase. However, CsdB has several structural features that are not observed in other families of the enzymes. A remarkable feature is that an alpha-helix in the lobe extending from the small domain to the large domain in one subunit of the dimer interacts with a beta-hairpin loop protruding from the large domain of the other subunit. The extended lobe and the protruded beta-hairpin loop form one side of a limb of each active site in the enzyme. The most striking structural feature of CsdB lies in the location of a putative catalytic residue; the side chain of Cys364 on the extended lobe of one subunit is close enough to interact with the gamma-atom of a modeled substrate in the active site of the subunit. Moreover, His55 from the other subunit is positioned so that it interacts with the gamma- or beta-atom of the substrate and may be involved in the catalytic reaction. This is the first report on three-dimensional structures of NifS homologues.     

2',3'-Cyclic nucleotide 3'-phosphohydrolase in the developing chick brain and spinal cord

Developmental changes of the 2',3'-cyclic nucleotide 3'-phosphohydrolase activity in the chick brain and spinal cord are reported. The greater part of increase in enzyme activity occurred between 18 days of incubation and 3 days after hatching in the whole brain, and between 18 and 21 days of incubation in the spinal cord. These periods are those of active myelination in the chick brain and spinal cord, respectively. The possibility was emphasized that 2',3'-cyclic nucleotide 3'-phosphohydrolase can be used as a marker for the myelin sheaths in the developing central nervous system. Comparisons were also made among the developmental changes in the forebrain, midbrain, brain stem, cerebellum, and spinal cord.     

Isoxazolopyridone derivatives as allosteric metabotropic glutamate receptor 7 antagonists

This Letter describes the synthesis and evaluation of mGluR7 antagonists in the isoxazolopyridone series. In the course of modification in this class, novel solid support synthesis of the isoxazolopyridone scaffold was developed. Subsequent chemical modification led to the identification of several potent derivatives with improved physicochemical properties compared to a hit compound 1. Among these, 2 showed good oral bioavailability and brain penetrability, suggesting that 2 may be useful for in vivo study to elucidate the role of mGluR7.     

Application study of 1,2-α-l-fucosynthase: introduction of Fucα1-2Gal disaccharide structures on N-glycan,ganglioside, and xyloglucan oligosaccharide

We have recently generated a highly efficient 1,2-α-l-fucosynthase (BbAfcA N423H mutant) by protein engineering of 1,2-α-l-fucosidase from Bifidobacterium bifidum JCM 1254. This synthase could specifically introduce H-antigens (Fucα1-2Gal) into the non-reducing ends of oligosaccharides and in O-linked glycans in mucin glycoprotein. In the present study, we show an extended application of the engineered 1,2-α-l-fucosynthase by demonstrating its ability to insert Fuc residues into N- and O-glycans in fetuin glycoproteins, GM1 ganglioside, and a plant-derived xyloglucan nonasaccharide. This application study broadens the feasibility of this novel H-antigen synthesis technique in functional glycomics.