Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage, which induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in the perivascular microenvironment and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in postnatal bone marrow generation. Using histological analysis, we observed that periosteum-derived PDPN-expressing stromal cells infiltrated the cartilaginous anlage of the postnatal epiphysis and populated on the primitive vasculature of secondary ossification center. Furthermore, immunophenotyping and cellular characteristic analyses indicated that the PDPN-expressing stromal cells constituted a subpopulation of the skeletal stem cell lineage. In vitro xenovascular model cocultured with human umbilical vein endothelial cells and PDPN-expressing skeletal stem cell progenies showed that PDPN-expressing stromal cells maintained vascular integrity via the release of angiogenic factors and vascular basement membrane-related extracellular matrices. We show that in this process, Notch signal activation committed the PDPN-expressing stromal cells into a dominant state with basement membrane-related extracellular matrices, especially type IV collagens. Our findings suggest that the PDPN-expressing stromal cells regulate the integrity of the primitive vasculatures in the epiphyseal nascent marrow. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.     

Apoptotic cells of an epithelial cell line, AsPC-1, release monocyte chemotactic S19 ribosomal protein dimer

A pancreatic carcinoma cell line, AsPC-1, underwent apoptosis in vitro when heat-treated for 60 min at 43 degrees C. Apoptotic AsPC-1 cells liberated a monocyte chemotactic factor into the culture supernatant 24 to 30 h after the heat-treatment. This factor was immunologically identified as the cross-linked homodimer of S19 ribosomal protein (RP S19), since the majority of the chemotactic activity was absorbed by both anti--RP S19 rabbit antibodies and an anti--isopeptide bond monoclonal antibody immobilized on agarose beads. Intracellular transglutaminase activity increased during the apoptotic process, reaching the peak strength between 18 and 24 h after the heat-treatment. A recombinant RP S19 acquired the monocyte chemotactic capacity when incubated with the apoptotic cell extract obtained at the 18th hour. The chemotactic activity acquirement as well as the transglutaminase activity were blocked by treatment of the extract with anti--type II transglutaminase rabbit antibodies. When the recombinant RP S19 was treated with an authentic type II transglutaminase, the dimerization of RP S19 concomitant with the generation of the monocyte chemotactic activity was observed. Peptide-map analyses involving amino acid sequencing demonstrated that the inter-molecular isopeptide bond was heterogeneous: Gln12 or Gln137 and Lys29 or Lys122 were cross-linked. Site-directed mutagenic analysis indicated that the cross-linking of Gln137, but not other residues such as Gln12, Lys29, and Lys122, was essential for expression of the chemotactic activity.     

Involvement of histone phosphorylation in thymocyte apoptosis by protein phosphatase inhibitors

Incubation of rat thymocytes with the inhibitors of protein phosphatase such as calyculin A and okadaic acid resulted in an increase in DNA fragmentation. These effects were dependent on the concentration of the inhibitors and the incubation time. Analyses of the fragmented DNA revealed the production of approximately 50 kbp of DNA and a 180 bp DNA ladder. In addition, a laser scanning-microscopic analysis showed that these compounds caused nuclear condensation. Thus, these results demonstrated that protein phosphatase inhibitors induced thymocyte apoptosis. The inhibitors of protein phosphatase increased the phosphorylation of proteins of approximately 15 kDa. The phosphorylation of proteins preceded the DNA fragmentation induced by these inhibitors. Judging from acetic acid-urea-Triton X-100 gel electrophoresis, the phosphorylated proteins were histone H1 and H2A/H3. Therefore, these results suggest that phosphorylation of histones triggers the DNA fragmentation of thymocytes undergoing apoptosis.     

DNA sequence comparison considering both amino acid and nucleotide insertions/deletions because of evolution and experimental error

Amino acid similarity often needs to be considered in DNA sequence comparison to elucidate gene functions. We propose a Smith-Waterman-like algorithm which considers amino acid similarity and insertions/deletions in sequences at the DNA level and at the protein level in a hybrid manner. The algorithm is applied to cDNA sequences of Oryza sativa and those of Arabidopsis thaliana. The results are compared with the results of application of NCBI's tblastx program (which compares the sequences in the BLAST manner after translation). It is shown that the present algorithm is very helpful in discovering nucleotide insertions/deletions originating from experimental errors as well as amino acid insertions/deletions due to evolutionary reasons.     

Synthesis and physical and physiological properties of 4'-thioRNA: application to post-modification of RNA aptamer toward NF-kappaB

We report herein full details of the preparation of 4′-thiouridine, -cytidine, -adenosine and -guanosine phosphoramidites based on our synthetic protocol via the Pummerer reaction. Fully modified 4′-thioRNAs containing four kinds of 4′-thioribonucleoside units were prepared according to the standard RNA synthesis. The T_m values and thermodynamic parameters of a series of duplexes were determined by UV melting and differential scanning calorimetry (DSC) measurements. The resulting overall order of thermal stabilities for the duplexes was 4′-thioRNA:4′-thioRNA >> 4′-thioRNA:RNA > RNA:RNA > RNA:DNA > 4′-thioRNA:DNA. In addition, it was shown that the dominant factor in the stability of the duplexes consisting of 4′-thioRNA was enthalpic in character. The CD spectra of not only 4′-thioRNA:RNA and 4′-thioRNA:4′-thioRNA but also 4′-thioRNA:DNA were all similar to those of duplexes in the A-conformation. The stability of 4′-thioRNA in human serum was 600 times greater than that of natural RNA. Neither the RNA:RNA nor the 4′-thioRNA:4′-thioRNA duplexes were digested under the same conditions. The first example of a post-modification of an RNA aptamer by 4′-thioribonucleoside units was demonstrated. Full modification of the aptamer thioRNA3 resulted in complete loss of binding activity. In contrast, modifications at positions other than the binding site were tolerated without loss of binding activity. The post-modified RNA aptamer thioRNA5 was thermally stabilized and resistant toward nuclease digestion. The results presented in this paper will, it is hoped, contribute to the development of 4′-thioRNA as a new generation of artificial RNA.     

Changes in receptor activator of nuclear factor-kappaB, and its ligand, osteoprotegerin, bone-type alkaline phosphatase, and tartrate-resistant acid phosphatase in ovariectomized rats

We investigated time-course changes in the expression of receptor activator of nuclear factor-kappaB (RANK), its ligand (RANKL), osteoprotegerin (OPG), bone-type alkaline phosphatase (BAP), and tartrate-resistant acid phosphatase (TRAP) in ovariectomized (OVX) rats. Samples of sera and coccyges were used for analysis of the enzyme activities and expression levels of proteins and mRNAs, and an immunohistochemical analysis was also performed. Serum BAP activity increased to 158.6% of the pre-operation value at 1 week after OVX, and then decreased to 38.7% at 8 weeks after OVX. On the other hand, the serum TRAP activity increased to 130.9% of the pre-operation level at 1 week after OVX, and was maintained at a high level, compared with the pre-operation level. The patterns of BAP and TRAP activity in the coccyges specimens were similar to those seen in the sera. The expression profiles of TRAP, RANK, and RANKL proteins in the coccyx specimens were similar to the pattern of serum TRAP activity, while the profiles of the BAP and OPG proteins were similar to the pattern of serum BAP activity in OVX rats. The changes in the mRNA expression levels of the osteogenic proteins were similar to those for protein expression. These biochemical changes in OVX rats were confirmed by immunohistochemical studies. Our results suggest that not only osteoclastogenesis accelerated but also osteoblastogenesis transiently increased during the early phase of osteoporosis.     

Ethacrynic-acid-induced glutathione depletion and oxidative stress in normal and Mrp2-deficient rat liver

Oxidative stress in the liver is sometimes accompanied by cholestasis. We investigated the localization and role of multidrug-resistance-associated protein (Mrp) 2, a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress. Normal Sprague-Dawley rat (SDR) and Mrp2-deficient Eisai hyperbilirubinemic rat (EHBR) livers were perfused with 500 microM EA. The release of glutamic pyruvic transaminase (GPT) and thiobarbituric-acid-reactive substances (TBARS) from EHBR liver was markedly delayed compared with that from SDR liver. This is mainly due to the higher basal level of glutathione (GSH) in EHBR liver (59.1 +/- 0.3 nmol/mg protein) compared with SDR liver (39.7 +/- 1.5 nmol/mg protein). EA similarly induced a rapid reduction in GSH followed by mitochondrial permeability transition in the isolated mitochondria from both SDR and EHBR. Internalization of Mrp2 was detected before nonspecific disruption of the canalicular membrane and GPT release in SDR liver perfused with 100 microM EA. SDR liver preperfused with hyperosmolar buffer (405 mosmol/L) for 30 min induced internalization of Mrp2 without changing the basal GSH level, while elimination of hepatic GSH by 300 microM EA perfusion was significantly delayed thereafter. Concomitantly, hepatotoxicity assessed by the release of GPT and TBARS was also significantly attenuated under hyperosmolar conditions. In conclusion, preserved cytosolic and intramitochondrial GSH is the key factor involved in the acute hepatotoxicity induced by EA and its susceptibility could be altered by the presence of Mrp2.     

Novel chiral sulfur-containing ferrocenyl ligands for palladium-catalyzed asymmetric allylic substitution

New chiral ferrocenyl ligands having chiral sulfinyl and phosphinyl groups were prepared. The palladium-catalyzed allylic substitution reaction using these chiral ligands showed good enantioselectivity. The mechanism for the asymmetric reaction is proposed on the basis of the stereochemical outcome.