Induction of migration of periodontal ligament cells by selective regulation of integrin subunits

The recruitment of tissue‐resident stem cells is important for wound regeneration. Periodontal ligament cells (PDL cells) are heterogeneous cell populations with stemness features that migrate into wound sites to regenerate periodontal fibres and neighbouring hard tissues. Cell migration is regulated by the local microenvironment, coordinated by growth factors and the extracellular matrix (ECM). Integrin‐mediated cell adhesion to the ECM provides essential signals for migration. We hypothesized that PDL cell migration could be enhanced by selective expression of integrins. The migration of primary cultured PDL cells was induced by platelet‐derived growth factor‐BB (PDGF‐BB). The effects of blocking specific integrins on migration and ECM adhesion were investigated based on the integrin expression profiles observed during migration. Up‐regulation of integrins α3, α5, and fibronectin was identified at distinct localizations in migrating PDL cells. Treatment with anti‐integrin α5 antibodies inhibited PDL cell migration. Treatment with anti‐integrin α3, α3‐blocking peptide, and α3 siRNA significantly enhanced cell migration, comparable to treatment with PDGF‐BB. Furthermore, integrin α3 inhibition preferentially enhanced adhesion to fibronectin via integrin α5. These findings indicate that PDL cell migration is reciprocally regulated by integrin α3‐mediated inhibition and α5‐mediated promotion. Thus, targeting integrin expression is a possible therapeutic strategy for periodontal regeneration.     

Intramolecular and intermolecular perturbation on electronic state of FAD free in solution and bound to flavoproteins: FTIR spectroscopic study by using the C = O stretching vibrations as probes

The intramolecular and intermolecular perturbation on the electronic state of FAD was investigated by FTIR spectroscopy by using the C=O stretching vibrations as probes in D(2)O solution. Natural and artificial FADs, i.e. 8-CN-, 8-Cl-, 8-H-, 8-OCH(3)-, and 8-NH(2)-FAD labelled by 2-(13)C, (18)O=C(2), or 4,10a-(13)C(2) were used for band assignments. The C(2)=O and C(4)=O stretching vibrations of oxidized FAD were shifted systematically by the substitution at the 8-position, i.e. the stronger the electron-donating ability (NH(2) > OCH(3) > CH(3) > H > Cl > CN) of the substituent, the lower the wavenumber region where both the C(2)=O and C(4)=O bands appear. In contrast, the C(4)=O band of anionic reduced FAD scarcely shifted. The 1,645-cm(-1) band containing C(2)=O stretching vibration shifted to 1,630 cm(-1) in the medium-chain acyl-CoA dehydrogenase (MCAD)-bound state, which can be explained by hydrogen bonds at C(2)=O of the flavin ring. The band was observed at 1,607 cm(-1) in the complex of MCAD with 3-thiaoctanoyl-CoA. The 23 cm(-1) shift was explained by the charge-transfer interaction between oxidized flavin and the anionic acyl-CoA. In the case of electron-transferring flavoprotein, two bands associated with the C(4)=O stretching vibration were obtained at 1,712 and 1,686 cm(-1), providing evidence for the multiple conformations of the protein.     

Chemical and genetic differentiation of Ligularia tsangchanensis in Yunnan and Sichuan provinces of China

The intraspecific diversity in L. tsangchanensis collected in the Chinese Provinces Yunnan and southwestern Sichuan was studied by chemical and genetic approaches. The samples collected in Yunnan were found to contain cacalol (1) as the sole major component, while samples from Sichuan contained 7alpha- and 7beta-eremophila-9,11-dien-8-one (5 and 6) as well as the 3alpha-angeloyloxy derivative 7 as major components. In addition, the sequences of the internal transcribed spacers (ITSs) of the ribosomal RNA gene indicated that the Yunnan and the Sichuan samples constitute separate clades. These results demonstrate that L. tsangchanensis in Yunnan and Sichuan are distinct.     

Chemical and genetic diversity of Ligularia latihastata and Ligularia villosa in Yunnan Province of China

The chemical constituents of the root extracts and the nucleotide sequences of the atpB-rbcL intergenic region of Ligularia latihastata and L. villosa, collected in northwestern Yunnan Province, were studied. In the twelve collected samples of L. latihastata, two major benzofurans, 5,6-dimethoxy-2-(1-methylethenyl)-1-benzofuran (1) and euparin (2) were detected as major components. The minor compound (2R*,3S*)-5-acetyl-2,3-dihydro-6-hydroxy-2-(1-methylethenyl)-1-benzofuran-3-yl (2Z)-2-[(acetoxy)methyl]but-2-enoate (4) was found to be susceptible to artifact formation upon extraction with EtOH. The intra-specific diversity in chemical composition of the samples was small, but the diversity in the atpB-rbcL sequence was fairly large. Compounds 1 and 2 were also found in the three collected samples of L. villosa, indicating that the two species are chemically close to each other, in agreement with morphological taxonomy.     

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.     

Circumnutational movement in rice coleoptiles involves the gravitropic response: analysis of an agravitropic mutant and space‐grown seedlings

Plants exhibit helical growth movements known as circumnutation in growing organs. Some studies indicate that circumnutation involves the gravitropic response, but this notion is a matter of debate. Here, using the agravitropic rice mutant lazy1 and space‐grown rice seedlings, we found that circumnutation was reduced or lost during agravitropic growth in coleoptiles. Coleoptiles of wild‐type rice exhibited circumnutation in the dark, with vigorous oscillatory movements during their growth. The gravitropic responses in lazy1 coleoptiles differed depending on the growth stage, with gravitropic responses detected during early growth and agravitropism during later growth. The nutation‐like movements observed in lazy1 coleoptiles at the early stage of growth were no longer detected with the disappearance of the gravitropic response. To verify the relationship between circumnutation and gravitropic responses in rice coleoptiles, we conducted spaceflight experiments in plants under microgravity conditions on the International Space Station. Wild‐type rice seeds were germinated, and the resulting seedlings were grown under microgravity or a centrifuge‐generated 1 g environment in space. We began filming the seedlings 2 days after seed imbibition and obtained images of seedling growth every 15 min. The seed germination rate in space was 92–100% under both microgravity and 1 g conditions. LED‐synchronized flashlight photography induced an attenuation of coleoptile growth and circumnutational movement due to cumulative light exposure. Nevertheless, wild‐type rice coleoptiles still showed circumnutational oscillations under 1 g but not microgravity conditions. These results support the idea that the gravitropic response is involved in plant circumnutation.     

Inhibition of the human chemokine receptor CXCR4 by antisense phosphorothioate oligodeoxyribonucleotides

The CXC chemokine receptor CXCR4/fusion, a major coreceptor for the T-cell line T-tropic (X4) HIV-1 virus, plays a critical role in T-tropic virus fusion and entry into permissive cells. In the present study, we describe the effects of an antisense phosphorothioate oligodeoxyribonucleotide (anti-S-ODN) on the inhibition of CXCR4 gene expression in X4 HIV-1 infected HeLa-CD4 cells, to find more efficacious therapeutic possibilities for human immunodeficiency virus type 1 (HIV-1) infection. The naked antisense phosphorothioate oligodeoxyribonucleotide (anti-S-ODN-1), containing the AUG initiation codon at the center of the oligodeoxyribonucleotide, showed a slightly higher inhibitory effect on HIV-1 gag p24 production among all sequences tested. We also examined the concomitant use of a basic peptide transfection reagent, nucleosomal histone proteins (RNP), for the delivery of the anti-S-ODN-1. The anti-S-ODN-1 encapsulated with RNP had higher inhibitory effects on p24 products than the naked anti-S-ODN-1. When the anti-S-ODN-1 encapsulated with RNP was incubated with HeLa-CD4 cells, the surface levels of this chemokine receptor showed high suppression, indicating sequence-specific inhibition. The activities of unmodified oligodeoxyribonucleotide are effectively enhanced by using a basic peptide, RNP.     

Thermococcus siculi sp. nov., a novel hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent at the Mid-Okinawa Trough

A novel coccoid-shaped, hyperthermophilic, anaerobic archaeon, strain RG-20, was isolated from a deep-sea hydrothermal vent fluid sample taken at 1394-m depth at the Mid-Okinawa Trough (27°32.7′N, 126°58.5′E). Cells of this isolate occur singly or in pairs and are about 0.8 to 2 μm in diameter. Growth was observed at temperatures between 50° and 93°C, with an optimum at 85°C. The pH range for growth is 5.0–9.0, with an optimum around 7.0. Strain RG-20 requires 1%–4% of NaCl for growth, and cell lysis occurs at concentrations below 1%. The newly isolated strain grows preferentially in the presence of elemental sulfur on proteinaceous substrates such as yeast extract, peptone, or tryptone, and no growth was observed on carbohydrates, carboxylic acids, alcohols, or lipids. This microorganism is resistant to streptomycin, chloramphenicol, ampicillin, and kanamycin at concentrations up to 150 μg/ml, but is susceptible to rifampicin. Analysis of the hydrolyzed core lipids by thin-layer chromatography (TLC) revealed the presence of archaeol and caldarchaeol. The mol% G+C content of the DNA is 55.8. Partial sequencing of the 16S rDNA indicates that strain RG-20 belongs to the genus Thermococcus. Considering these data and on the basis of the results from DNA-DNA hybridization studies, we propose that this strain should be classified as a new species named Thermococcus siculi (si′cu.li. L. gen. n. siculi, of the deep-sea [siculum, deep-sea in literature of Ovid], referring to the location of the sample site, a deep-sea hydrothermal vent). The type strain is isolate RG-20 (DSM No. 12349). Received: May 11, 1998 / Accepted: July 24, 1998