Fibroblastic cells from human periapical granulation tissue preferentially form calcified matrices in decalcified boiled rat bone

We have been studying the potential of human fibroblastic cells (HFC) from periapical granulation tissue to form a calcified matrix. Recently, we reported that inflamed periapical granulation tissue contains osteogenic cells. In the present study, we tested the hypothesis that HFC, cultured with decalcified bone (DB) of rat, might form much greater calcified matrices than with rat decalcified boiled bone (DBB), which was originally prepared as a negative control. HFC were cultured with DB or DBB in the presence or absence of 2 mM -glycerophosphate (-GP) and 50 g/ml ascorbic acid. After six weeks of culture, a number of von Kossa-positive globular structures were unexpectedly observed inside DBB, but not DB. Without HFC, such structures were never seen in DBB incubated with 2 mM -GP and 50 g/ml ascorbic acid. DB cultured with HFC under the same conditions did not show these structures. Electron-microscopic observation revealed that matrix vesicles aggregated on collagen fibrils around globular structures in DBB. Energy dispersive X-ray microanalysis confirmed that these structures were calcified matrices composed of calcium and phosphate. These results suggest that human periapical granulation tissue contains cells responsible for the formation of calcified matrices in DBB, and that DBB could serve as an excellent scaffold for the calcification of HFC, rather than DB.This work was supported by grant-in-aid (project 15689024) for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.     

Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture

Bipotent mesendoderm that can give rise to both endoderm and mesoderm is an established entity from C. elegans to zebrafish. Although previous studies in mouse embryo indicated the presence of bi-potent mesendoderm cells in the organizer region, characterization of mesendoderm and its differentiation processes are still unclear. As bi-potent mesendoderm is implicated as the major precursor of definitive endoderm, its identification is also essential for exploring the differentiation of definitive endoderm. In this study, we have established embryonic stem (ES) cell lines that carry GFP gene in the goosecoid (Gsc) gene locus and have investigated the differentiation course of mesendodermal cells using Gsc expression as a marker. Our results show that mesendoderm is represented as a Gsc-GFP+ E-cadherin(ECD)+ PDGFRalpha(alphaR)+ population and is selectively induced from ES cells under defined conditions containing either activin or nodal. Subsequently, it diverges to Gsc+ ECD+ alphaR- and Gsc+ ECD- alphaR+ intermediates that eventually differentiate into definitive endoderm and mesodermal lineages, respectively. The presence of mesendodermal cells in nascent Gsc+ ECD+ alphaR+ population was also confirmed by single cell analysis. Finally, we show that the defined culture condition and surface markers developed in this study are applicable for obtaining pure mesendodermal cells and their immediate progenies from genetically unmanipulated ES cells.     

Disturbance of sphingolipid biosynthesis abrogates the signaling of Mss4, phosphatidylinositol-4-phosphate 5-kinase, in yeast

The functional relationships between phosphoinositides and sphingolipids have not been well characterized to date. ISP-1/myriocin is a potent inhibitor of sphingolipid biosynthesis and induces severe growth defects in eukaryotic cells because of the sphingolipid deprivation. We characterized a novel multicopy suppressor gene of ISP-1-mediated cell death in yeast, MSS4. MSS4 encodes a phosphatidylinositol-4-phosphate 5-kinase that synthesizes phosphatidylinositol (4,5)-bisphosphate (PI4,5P(2)). We demonstrate here that ISP-1 treatment of yeast causes defects both in the activity and subcellular localization of Mss4. The effect of the Mss4 defect on the downstream signaling was examined, because interaction between the Mss4 product, PI4,5P(2), and the pleckstrin-homology domain of Rom2 mediates recruitment of Rom2 to the membrane, which is the crucial step for subsequent Rho1/2 activation. Indeed, failure of Rom2 recruitment was observed in ISP-1-treated cells as well as in csg2-deleted cells, which have reduced mannosylated inositolphosphorylceramide. These data suggested that proper sphingolipids are required for the signaling pathway involving Mss4.     

Contribution of non-extensor muscles of the leg to maximal-effort countermovement jumping

Background  

The purpose of this study was to determine the effects of non-extensor muscles of the leg (i.e., muscles whose primary function is not leg extension) on the kinematics and kinetics of human maximal-effort countermovement jumping. Although it is difficult to address this type of question through experimental procedures, the methodology of computer simulation can be a powerful tool.     

Involvement of hedgehog,wingless, and dpp in the initiation of proximodistal axis formation during the regeneration of insect legs,a verification of the modified boundary model

To understand the mechanism of regeneration, many experiments have been carried out with hemimetabolous insects, since their nymphs possess the ability to regenerate amputated legs. We first succeeded in observing expression patterns of hedgehog, wingless (wg), and decapentaplegic (dpp) during leg regeneration of the cricket Gryllus bimaculatus. The observed expression patterns were essentially consistent with the predictions derived from the boundary model modified by Campbell and Tomlinson (CTBM). Thus, we concluded that the formation of the proximodistal axis of a regenerating leg is triggered at a site where ventral wg-expressing cells abut dorsal dpp-expressing cells in the anteroposterior (A/P) boundary, as postulated in the CTBM.     

Functional characterization of betaine/proline transporters in betaine-accumulating mangrove

Betaine is an important osmoprotectant in many plants, but its transport activity has only been demonstrated using a proline transporter from tomato, a betaine-nonaccumulating plant. In this study, two full-length and one partial transporter genes were isolated from betaine-accumulating mangrove Avicennia marina. Their homologies to betaine transporters from bacteria and betaine/4-aminobutyrate transporters from mammalian cells were low but were high to proline transporters from Arabidopsis and tomato. Two full-length transporters could complement the Na(+)-sensitive phenotype of the Escherichia coli mutant deficient in betT, putPA, proP, and proU. Both transporters could efficiently take up betaine and proline with similar affinities (K(m), 0.32-0.43 mm) and maximum velocities (1.9-3.6 nmol/min/mg of protein). The uptakes of betaine and proline were significantly inhibited by mono- and dimethylglycine but only partially inhibited by betaine aldehyde, choline, and 4-aminobutyrate. Sodium and potassium chloride markedly enhanced betaine uptake rates with optimum concentrations at 0.5 m, whereas sucrose showed only modest activation. The change of amino acids Thr(290)-Thr-Ser(292) in a putative periplasmic loop to Arg(290)-Gly-Arg(292) yielded the active transporter independent of salts, suggesting the positive charge induced a conformational change to the active form. These data clearly indicate that the betaine-accumulating mangrove contains betaine/proline transporters whose properties are distinct from betaine transporters of bacteria and mammalian cells.     

Comparison of the mutations induced by p-benzoquinone, a benzene metabolite, in human and mouse cells

Benzene is one of the chemicals widely contaminating the environment. Benzene is suggested to be a human leukemogen. When benzene is absorbed in the human body, it is metabolized firstly in the liver and subsequently in the bone marrow where it provokes initiation of leukemia. In the present study, we analyzed mutations induced by p-benzoquinone (p-BQ), a benzene metabolite, in human cells using a shuttle vector plasmid pMY189, and compared frequencies, types and spectra of the mutations with those of the mutations previously revealed in mouse cells using a similar plasmid pNY200. We found that p-BQ induces mutations in human and mouse cells at similar frequencies but with different types of mutagenesis. The proportion of tandem base mutations was significantly lower in human cells than in mouse cells. Most base substitutions were induced in G:C base pairs in both human and mouse cells. However, the proportion of G:C→C :G transversion is significantly higher in human cells. These findings indicate that the p-BQ-induced DNA damage in human and mouse cells is processed in a different manner, and that extrapolation of mice findings on experimental benzene carcinogenesis to human cancer risk assessment should be conducted carefully.     

Household Demography and Early Childhood Mortality in a Rice-Farming Village in Northern Laos

This paper extends Alexandr Chayanov’s model of changing household demography (specifically the ratio of food consumers to food producers) and its influence on agricultural behavior so that it includes possible adverse effects of a rising ratio on nutritional status and early childhood mortality within the household. We apply the model to 35 years’ worth of longitudinal demographic and economic data collected in the irrigated-rice growing village of Na Savang in northern Laos. When appropriate controls are included for other household variables, unobserved inter-household heterogeneity, and changes in local conditions and national policy over the study period, the analysis suggests that a unit increase in the household’s consumer/producer ratio induces something like a nine-fold increase in the risk of death among household members aged less than five years. Monte Carlo simulation studies suggest that this may be an over-estimate but also that the effect is probably real and likely to be an important factor in household demography. At the very least, the results suggest that Chayanov’s model still has theoretical relevance and deserves to be revived.     

Ultrastructural localization of immunolabeled substance P and methionine-enkephalin-octapeptide in the surface layer of the dorsal horn of rat spinal cord

Summary A preembedding dual immunolabeling technique and electron microscopy were utilized to demonstrate the localization of immunoreactive substance P and methionine-enkephalin-octapeptide (Enk-8) in ultrathin sections of the surface layer (laminae I and II) of rat spinal dorsal horn. The immunoreaction of Enk-8 was visualized as goldtoned silver particles and that of substance P as diaminobenzidine reaction products. Axonal terminals with immunoreactive substance P, and also unlabeled axonal terminals, formed synaptic junctions with the perikarya and dendritic processes of Enk-8-containing neurons. Dendritic profiles immunolabeled for substance P were synaptically linked with unlabeled axons but not with Enk-8-positive ones. Furthermore, it was found that Enk-8 axons and substance P axons terminated synaptically in juxtaposition to one another on the same immunonegative dendrites. Among the Enk-8-containing neurons axonal profiles also appeared to be synaptically associated with immunoreactive Enk-8 dendritic processes.