Smad4 in osteoblasts exerts a differential impact on HSC fate depending on osteoblast maturation stage.

Osteoblasts (OBs) are indispensable for the maintenance of hematopoietic stem cells (HSCs) in the bone marrow microenvironment. Here we investigated how Smad4 modulates HSC fate at distinct stages of OB development. For this, we conditionally knocked out Smad4 in cells expressing type I collagen (Col1a1) and osteocalcin (OC), respectively. Col1a1-expressing OBs were widely present in both the trabecular and cortical compartment, whereas OC-expressing OBs were predominantly located in the cortical compartment. HSCs from Col1a1 mutants displayed senescence-associated phenotypes. OC mutants did not exhibit HSC senescence-related phenotypes, but instead showed preferential HSC death. Of note, stromal cell-derived factor 1 expression was lower in Col1a1 mutants than control littermates, suggesting potential impairment of CXCR4-CXCL12-mediated HSC retention. Disruption of the CXCR4-CXCL12 axis by AMD3100 administration led to an increase in the senescence-associated ß-galactosidase activity and low competitive potential. Collectively, our findings indicate that deletion of Smad4 in OBs differentially modulates HSC fate in a stage-dependent manner.

Alteration of conserved alternative splicing in AMELX causes enamel defects.

Tooth enamel is the most highly mineralized tissue in vertebrates. Enamel crystal formation and elongation should be well controlled to achieve an exceptional hardness and a compact microstructure. Enamel matrix calcification occurs with several matrix proteins, such as amelogenin, enamelin, and ameloblastin. Among them, amelogenin is the most abundant enamel matrix protein, and multiple isoforms resulting from extensive but well-conserved alternative splicing and postsecretional processing have been identified. In this report, we recruited a family with a unique enamel defect and identified a silent mutation in exon 4 of the AMELX gene. We show that the mutation caused the inclusion of exon 4, which is almost always skipped, in the mRNA transcript. We further show, by generating and characterizing a transgenic animal model, that the alteration of the ratio and quantity of the developmentally conserved alternative splicing repertoire of AMELX caused defects in enamel matrix mineralization.

A novel synthetic method for nanosized crystalline titania for use in the decomposition of dyes.

Photocatalytically active nanocrystalline titania particles were prepared using a hydrothermal process, by controlling the particle size and crystallinity. The crystalline structures and morphologies of the particles were characterized by X-ray diffraction and transmission electron microscopy. The BET method was used to determine the surface area and verify the grain size. To estimate the photocatalytic activity of the synthesized particles, a dye photodegradation experiment was carried out and the activity of the particles was compared with that of conventional titania. The results show that synthesized nanocrystalline titania particles had a higher photocatalytic activity than that of conventional titania. These findings provide a basis for the preparation of more effective and usef ul materials for use in AOP applications.

Fat body expressed yolk protein genes in Hyphantria cunea are related to the YP4 follicular epithelium yolk protein subunit gene of pyralid moths.

cDNA clones for two of the yolk proteins, YP1 and YP2, produced by the fat body of the moth, Hyphantria cunea, were sequenced and found to be homologous to the follicular epithelium yolk proteins of pyralid moths. Both cDNA clones coded for polypeptides of 290 residues and the deduced amino acid sequence identity between YP1 and YP2 was very high (79.0%). Analysis of the secondary structure of the predicted polypeptides suggests that YP1 and YP2 do not form heteromeric proteins because of differences in secondary structure due to the lack of alpha helices in YP1. Northern blot analysis showed that the transcripts for YP1 (1.2 kb) and YP2 (1.1 kb) were present primarily in the female fat body with only trace levels detectable in the ovary of the adult female. In a developmental study, the YP1 and YP2 transcripts were first detectable in 10-day-old pupae and increased into the adult stage. These results suggest that the YP1 and YP2 genes in H. cunea have been recruited to replace the vitellogenin gene as the primary source of yolk proteins. During this process they have acquired a modified pattern of expression that is different from homologous genes reported in pyralid moths. The assessment of the evolution of proteinaceous yolk in these moths should serve as an excellent model for the evolution of gene recruitment.

Local expression and distribution of a storage protein in the ovary of Hyphantria cunea.

Storage protein-1 (HcSP-1) is a major storage protein found in the hemolymph and fat body of Hyphantria cunea. HcSP-1 has a high methionine (6.0%) and low aromatic amino acid content (8.5%) (Cheon et al., 1998). In this study, the accumulation and expression of HcSP-1 in ovary was investigated using biochemical and immunocytochemical methods. HcSP-1 was detected in the ovaries in 6-day-old pupae and accumulated toward the end of pupal life, when HcSP-1 transcripts were detectable by Northern blot analysis and RT-PCR. In situ hybridization showed that the HcSP-1 mRNA was located in the nurse cells and follicular epithelial cells, but not in the oocyte. Though most of the HcSP-1 that is incorporated in the yolk bodies of the oocyte is probably sequestered from the surrounding hemolymph, HcSP-1 is an important yolk protein contributing to early yolk body formation before the development of patency by the follicular epithelium.

Characterization and immunological analysis of ferritin from the hemolymph of Galleria mellonella.

Ferritin, an iron-binding protein, was purified from the larval hemolymph of the wax moth, Galleria mellonella by KBr density ultracentrifugation and FPLC (Superose 6). The iron content of ferritin was determined by atomic emission spectroscopy and Ferene S stain. Native molecular mass of ferritin was estimated as 630 kDa. SDS-PAGE revealed that the ferritin consists of two major polypeptides of 26 and 32 kDa and one minor polypeptide of 30 kDa. An isoelectric point of ferritin was measured to be approximately 7.3 and only the 32-kDa subunit is glycosylated. The ferritin contains large amounts of lysine, glutamine, glutamic acid and leucine but tryptophan was not detected. Electron microscopic examination of negatively stained preparations showed an 11-nm particle in external diameter and 7-nm iron core. Ferritin is present in both the ovary and testis. Localization of ferritin by immunoelectron microscopy in ovary and testis revealed that the gold particles were located in vitelline membrane and yolk granules but not in follicular epithelium of ovary. In the testis, the gold particles were located in testicular fluid and lumen of vas deferens.

Cloning and expression of a ferritin subunit for Galleria mellonella.

Ferritin was purified from iron-fed Galleria mellonella hemolymph by ultra centrifugation and FPLC (Superose 6). SDS-PAGE revealed three bands of 26, 30, and 32 kDa. The ferritin 26 kDa subunit cDNA was obtained from RT-PCR using primer designed from N-terminal sequence analysis. 5'-RACE was used to obtain the complete protein coding sequence. The sequence encodes a 211 amino acid polypeptide including a 20 amino acid leader peptide. An IRE (iron-responsive element) sequence with a predicted stem-loop structure was present in the 5'-UTR of ferritin mRNA. Sequence alignment has a sequence identity with Calpodes ethlius (S)(74%), Drosophila melanogaster (50%), and Aedes aegypti (39%). Northern blot analysis indicated that there were 1.5- and 1.75-fold increases in the expression of ferritin mRNA after iron-fed fat body and midgut, respectively. Also, we confirmed that the ferritin mRNA is not expressed in adult ovary and testis. Arch.

The structure of Sky1p reveals a novel mechanism for constitutive activity.

Sky1p is the only member of the SR protein kinase (SRPK) family in Saccharomyces cerevisiae. SRPKs are constitutively active kinases that display remarkable substrate specificity and have been implicated in RNA processing. Here we present the three-dimensional structure of a fully active truncated Sky1p. Analysis of the structure and structure-based functional studies reveal that the C-terminal tail, an unusual Glu residue located in the P+1 loop, and a unique mechanism for the positioning of helix alpha C act together to render Sky1p constitutively active. We have modeled a substrate peptide bound to Sky1p. The modeled complex combined with mutagenesis studies illustrate the molecular basis for substrate recognition by this kinase and suggest a mechanism by which SRPKs catalyze a sequential phosphorylation reaction of the consecutive RS dipeptide repeats characteristic of mammalian SRPK substrates.

Conserved SR protein kinase functions in nuclear import and its action is counteracted by arginine methylation in Saccharomyces cerevisiae.

Mammalian serine and arginine-rich (SR) proteins play important roles in both constitutive and regulated splicing, and SR protein-specific kinases (SRPKs) are conserved from humans to yeast. Here, we demonstrate a novel function of the single conserved SR protein kinase Sky1p in nuclear import in budding yeast. The yeast SR-like protein Npl3p is known to enter the nucleus through a composite nuclear localization signal (NLS) consisting of a repetitive arginine- glycine-glycine (RGG) motif and a nonrepetitive sequence. We found that the latter is the site for phosphorylation by Sky1p and that this phosphorylation regulates nuclear import of Npl3p by modulating the interaction of the RGG motif with its nuclear import receptor Mtr10p. The RGG motif is also methylated on arginine residues, but methylation does not affect the Npl3p-Mtr10p interaction in vitro. Remarkably, arginine methylation interferes with Sky1p-mediated phosphorylation, thereby indirectly influencing the Npl3p-Mtr10p interaction in vivo and negatively regulating nuclear import of Npl3p. These results suggest that nuclear import of Npl3p is coordinately influenced by methylation and phosphorylation in budding yeast, which may represent conserved components in the dynamic regulation of RNA processing in higher eukaryotic cells.

cDNA cloning and antibacterial activities of cecropin D-like peptides from Agrius convolvuli.

We have characterized full-length cDNAs encoding two isoforms of agriusin, cecropin D-like antibacterial peptide, present in the hemolymph of the immunized Agrius convolvuli larvae. The cloned cDNAs of agriusins 1 and 2 contain 331 and 329 bp, respectively. The nucleotide sequencing of cDNAs showed that they encode 62 amino acids, whose mature portion was deduced to consist of 38 amino acid residues with over 94% sequence identity. In the sequence homology search, mature agriusin 1 showed over 86 and 71% amino acid sequence homology with bactericidin 4 from Manduca sexta and cecropin D from Hyalophora cecropia, respectively. Since it was demonstrated from the deduced amino acid sequences that the C-terminal residues of agriusins are followed by a Gly residue, two types of synthetic agriusin 1 (syn-agriusin 1 amide and acid) were prepared to verify if natural agriusin 1 is C-terminally amidated. From acid-urea PAGE and reversed phase HPLC profiles to compare two synthetic peptides, we could confirm that the C-terminal amino acid residue of natural agriusin 1, like several cecropins so far identified, is amidated. Finally, our antibacterial assay performed with two syn-agriusins 1 revealed that there is little difference between antibacterial activities of both peptides against Gram-positive and Gram-negative bacteria.