Stage- and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis

DNA methylation is essential for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, contribute to the creation of DNA methylation patterns in embryos. We demonstrated that the Dnmt3a and Dnmt3b proteins are expressed at different stages of embryogenesis. Dnmt3b is specifically expressed in totipotent embryonic cells, such as inner cell mass, epiblast and embryonic ectoderm cells, whilst Dnmt3a is significantly and ubiquitously expressed after E10.5. The difference in the expression stages of the Dnmt3a and Dnmt3b proteins may contribute to their distinct functions during the embryogenesis.     

Adrenomedullin stimulates the growth of cultured normal human osteoblasts as an autocrine/paracine regulator

Adrenomedullin (AM) is a 52 amino acid peptide that is synthesized in a variety of tissues, including the vessels and bones. This study showed that normal human osteoblast (NHOst) secreted immunoreactive AM and that AM stimulated intracellular cAMP production in these cells. An anti-AM monoclonal antibody, which inhibited endogenous AM, caused the number of NHOst to decrease. The effect of a low concentration AM was inhibited by addition of a cAMP-dependent protein kinase A inhibitor (H89). These data suggest that AM is an autocrine or paracrine regulator that promotes the proliferation of NHOst via the cAMP pathway.     

Effects of tungstosilicate on strains of methicillin-resistant Staphylococcus aureus with unique resistant mechanisms

In a previous study, it was found that polyoxotungstates such as undecatungstosilicate (SiW11) greatly sensitized strains of methicillin-resistant Staphylococcus aureus (MRSA) to beta-lactams. In this report, the effects of SiW11 on several MRSA strains with unique resistant mechanisms were studied. SiW11 was still effective to MRSA mutants with higher beta-lactam resistance due to reduced cell-lytic activity. Since the antimicrobial effect of TOC-39 (a cephem antibiotic with strong affinity to penicillin-binding protein (PBP) 2') was not strongly enhanced in any case, it was confirmed that the sensitizing effect of SiW11 is due to reduced expression of PBP2'. However, the sensitizing effect of SiW11 was relatively weak in MRSA strains with lowered susceptibility to glycopeptide antibiotics. A certain resistant mechanism other than the mecA-PBP2' system worked in such a strain. Interestingly, an MRSA mutant with the Eagle-type resistance was dramatically sensitized. This result suggests that SiW11 has another site of action besides reducing the expression of PBP2'.     

Multiple four-stranded conformations of human telomere sequence d(CCCTAA) in solution

By detailed NMR analysis of a human telomere repeating unit, d(CCCTAA), we have found that three distinct tetramers, each of which consists of four symmetric single-strands, slowly exchange in a slightly acidic solution. Our new finding is a novel i-motif topology (T-form) where T4 is intercalated between C1 and C2 of the other duplex. The other two tetramers have a topology where C1 is intercalated between C2 and C3 of the other parallel duplex, resulting in the non-stacking T4 residues (R-form), and a topology where C1 is stacked between C3 and T4 of the other duplex (S-form). From the NMR denaturation profile, the R-form is the most stable of the three structures in the temperature range of 15–50°C, the S-form the second and the T-form the least stable. The thermodynamic parameters indicate that the T-form is the most enthalpically driven and entropically opposed, and its population is increased with decreasing temperature. The T-form structure determined by restrained molecular dynamics calculation suggests that inter-strand van der Waals contacts in the narrow grooves should contribute to the enthalpic stabilization of the T-form.     

Point mutation in kit receptor tyrosine kinase reveals essential roles for kit signaling in spermatogenesis and oogenesis without affecting other kit responses

The Kit receptor tyrosine kinase functions in hemato- poiesis, melanogenesis and gametogenesis. Kit receptor-mediated cellular responses include proliferation, survival, adhesion, secretion and differentiation. In mast cells, Kit-mediated recruitment and activation of phosphatidylinositol 3'-kinase (PI 3-kinase) produces phosphatidylinositol 3'-phosphates, plays a critical role in mediating cell adhesion and secretion and has contributory roles in mediating cell survival and proliferation. To investigate the consequences in vivo of blocking Kit-mediated PI 3-kinase activation we have mutated the binding site for the p85 subunit of PI 3-kinase in the Kit gene, using a knock-in strategy. Mutant mice have no pigment deficiency or impairment of steady-state hematopoiesis. However, gametogenesis is affected in several ways and tissue mast cell numbers are affected differentially. While primordial germ cells during embryonic development are not affected, Kit(Y719F)/Kit(Y719F) males are sterile due to a block at the premeiotic stages in spermatogenesis. Furthermore, adult males develop Leydig cell hyperplasia. The Leydig cell hyperplasia implies a role for Kit in Leydig cell differentiation and/or steroidogenesis. In mutant females follicle development is impaired at the cuboidal stages resulting in reduced fertility. Also, adult mutant females develop ovarian cysts and ovarian tubular hyperplasia. Therefore, a block in Kit receptor-mediated PI 3-kinase signaling may be compensated for in hematopoiesis, melanogenesis and primordial germ cell development, but is critical in spermatogenesis and oogenesis.     

Ganglioside GT1b in rat brain binds to p58, a brain-specific sodium-dependent inorganic phosphate cotransporter: expression cloning with a specific monoclonal antibody to ganglioside GT1b-binding protein

To evidence the notion that gangliosides involve neuronal cell interactions in the brain, we surveyed the presence of ganglioside-binding proteins in membrane lysates of adult rat cerebellum. Three proteins (p58, p90, and p160) were identified as GT1b-binding proteins by incubation of the blot of the membrane lysate with GT1b micelles. We generated a monoclonal antibody (mAb) specific to the polypeptide portion of the GT1b-binding proteins (YAK-2). The YAK-2 mAb specifically reacted with all three proteins on blots of proteins pretreated under nonreducing conditions for SDS-PAGE, but reacted mainly with p58 under reducing conditions, showing that p90 and p160 are oligomeric forms of p58. The binding activity of the YAK-2 mAb was completely inhibited by the presence of GT1b micelles, indicating the specificity of YAK-2 mAb for p58 and its oligomers. Immunohistochemical investigations revealed that both p58 and GT1b colocalize within the granular layer of adult rat cerebellum. Expression cloning of p58 cDNA was performed using YAK-2 mAb, and five putative clones were obtained. Among them, the nucleotide sequence of one cDNA completely matched that of rat brain-specific sodium-dependent inorganic phosphate cotransporter (rBNPI), a 61 kDa membrane protein. COS7 cells were transfected with a Flag-chimeric construct containing the rBNPI/p58 cDNA, and the membrane lysate was subjected to immunoprecipitation with anti-Flag antibody. One protein (64 kDa) was detected only with YAK-2 mAb, and the membrane lysate specifically bound to GT1b micelles. Taking together, we propose that rBNPI/p58 functions as a GT1b-binding protein in neuronal cells.     

Phenotypic Characterization of Polysaccharidases Produced by Four Prevotella Type Strains

Four ruminal Prevotella type strains, P. ruminicola JCM8958^T, P. bryantii B₁4^T, P. albensis M384^T, and P. brevis ATCC19188^T, were characterized for polysaccharide-degrading activities with the reducing sugar release assay and zymogram analyses. Carboxymethylcellulase, xylanase, and polygalacturonate (PG)-degrading enzyme activities were determined in cultures grown on oat spelt xylan, xylose, arabinose, cellobiose, and glucose as sole growth substrates. P. ruminicola and P. albensis showed carboxymethylcellulase induction patterns. When xylan was supplied as a sole growth substrate, xylanase activities produced by P. bryantii and P. albensis were at least 18- and 11-fold higher, respectively, than during growth on other carbohydrates, suggesting that the regulation of the xylanases was highly specific to xylan. All strains constitutively produced PG-degrading enzymes. The corresponding activity of P. bryantii was more than 40-fold higher than in other strains. Zymogram analyses routinely detected the presence of high-molecular-weight (100–170 kDa) polysaccharide-degrading enzymes in ruminal Prevotella. Characteristics of the polysaccharide-degrading activities showed diversity of ruminal Prevotella species. Received: 29 November 1999 / Accepted: 1 February 2000     

Cloning of a cDNA for Xenopus prolactin receptor and its metamorphic expression profile

A pituitary hormone, prolactin (PRL) shows various effects on cellular metabolism in amphibians, such as stimulation of larval tissue growth and inhibition of metamorphic changes. All these effects are mediated by its cell surface receptor. However, lack of information on PRL receptor (PRL-R) gene expression has made the physiological importance of the PRL/PRL-R system obscure in amphibian metamorphosis. Hence, a Xenopus PRL-R cDNA was cloned, its structure was characterized, and specific binding of PRL to Xenopus PRL-R expressed in COS-7 cells was confirmed. In adult tissues, high level expression was found in the lung, heart, brain, thymus and skin, and low level in the oviduct, kidney and spinal cord. The developmental expression pattern showed that PRL-R messenger ribonucleic acid (mRNA) was expressed in the brain and tail from premetamorphosis and the level increased toward late metamorphosis, suggesting that PRL may inhibit the metamorphic changes in those organs. The level of brain PRL-R mRNA reached a peak just at the start of the metamorphic climax stages and then decreased, whereas in the tail, mRNA expression peaked at late metamorphosis. In the kidney, mRNA expression increased and reached a maximum level at the end of metamorphosis. The results obtained were discussed in relation to metamorphosis.     

Engineering of the hydrophobic core of an alpha-helical coiled coil

The amino acid sequence that forms the alpha-helical coiled coil structure has a representative heptad repeat denoted by defgabc, according to their positions. Although the a and d positions are usually occupied by hydrophobic residues, hydrophilic residues at these positions sometimes play important roles in natural proteins. We have manipulated a few amino acids at the a and d positions of a de novo designed trimeric coiled coil to confer new functions to the peptides. The IZ peptide, which has four heptad repeats and forms a parallel triple-stranded coiled coil, has Ile at all of the a and d positions. We show three examples: (1) the substitution of one Ile at either the a or d position with Glu caused the peptide to become pH sensitive; (2) the metal ion induced alpha-helical bundles were formed by substitutions with two His residues at the d and a positions for a medium metal ion, and with one Cys residue at the a position for a soft metal ion; and (3) the AAB-type heterotrimeric alpha-helical bundle formation was accomplished by a combination of Ala and Trp residues at the a positions of different peptide chains. Furthermore, we applied these procedures to prepare an ABC-type heterotrimeric alpha-helical bundle and a metal ion-induced heterotrimeric alpha-helical bundle.