Early development and neurogenesis of Temnopleurus reevesii

Sea urchins are model non‐chordate deuterostomes, and studying the nervous system of their embryos can aid in the understanding of the universal mechanisms of neurogenesis. However, despite the long history of sea urchin embryology research, the molecular mechanisms of their neurogenesis have not been well investigated, in part because neurons appear relatively late during embryogenesis. In this study, we used the species Temnopleurus reevesii as a new sea urchin model and investigated the detail of its development and neurogenesis during early embryogenesis. We found that the embryos of T. reevesii were tolerant of high temperatures and could be cultured successfully at 15–30°C during early embryogenesis. At 30°C, the embryos developed rapidly enough that the neurons appeared at just after 24 h. This is faster than the development of other model urchins, such as Hemicentrotus pulcherrimus or Strongylocentrotus purpuratus. In addition, the body of the embryo was highly transparent, allowing the details of the neural network to be easily captured by ordinary epifluorescent and confocal microscopy without any additional treatments. Because of its rapid development and high transparency during embryogenesis, T. reevesii may be a suitable sea urchin model for studying neurogenesis. Moreover, the males and females are easily distinguishable, and the style of early cleavages is intriguingly unusual, suggesting that this sea urchin might be a good candidate for addressing not only neurology but also cell and developmental biology.     

Temperature-gradient DNA probe chromatography of nucleic acids on nonporous supports.

Temperature-gradient DNA probe chromatography of nucleic acids on a nonporous support with a homogeneous particle size of 2.5 microns showed a higher base sequence discriminating power and a larger linear capacity than that on a porous support with a larger and less homogeneous particle size. The resolution on the nonporous support was high enough to separate samples with a single-base mismatch of the less destabilizing groups, including G-G and G-T base mismatches, even when it locates very near the end of the probe.     

In vitro culture of human chondrocytes (1): A novel enhancement action of ferrous sulfate on the differentiation of human chondrocytes

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. The aim of our investigation was to characterize the influences of basic fibroblast growth factor (bFGF) and ferroussulfate (FeSO₄) on proliferation and differentiation of human articular chondrocytes (HAC). This is the first report of the effects of FeSO₄ on chondrogenesis of HAC. Multiplied chondrocytes of hip and shoulder joints were cultured in chondrocyte growth medium supplemented with bFGF, FeSO₄, or both bFGF + FeSO₄ for4weeks. A 20 μl aliquot of a cell suspension containing2 × 10⁷ cells ml⁻¹ was delivered onto each well of 24-well tissue culture plates. Cells cultured with the growth medium only was used as a control. Alamar blue and alcian blue staining were done to determine the chondrocyte proliferation and differentiation, respectively, after 4 weeks. The samples exposed to bFGF, FeSO₄, and combination of both indicated sufficient cell proliferation similar to the control level. Differentiations of the HAC exposed to bFGF, FeSO₄,and bFGF + FeSO₄ were 1.2-, 2.0-, and 2.2-fold of the control, respectively. Therefore, chondrocyte differentiation was significantly enhanced by the addition of FeSO₄ andbFGF + FeSO₄. The combined effects of bFGF and FeSO₄ were additive, rather than synergistic. These results suggest that treatment with ferrous sulfate alone or in combination with basic fibroblast growth factor etc, is a powerful tool to promote the differentiation of HAC for the clinical application. This revised version was published online in August 2006 with corrections to the Cover Date.     

Quantitation of beta-tubulin mRNA in mouse brain by RNA-RNA hybridization kinetics with single-stranded riboprobes

A quantitative procedure involving RNA-RNA hybridization kinetics was developed for measurement of specific mRNA accumulated in particular tissues and cells. Two types of riboprobes for quantitating mouse beta-tubulin mRNA were prepared; one was a truncated RNA covering only the coding portion of beta-tubulin cDNA and the other was a non-truncated RNA covering the vector portion as well as the coding portion. These antisense RNAs were hybridized with the mouse brain RNA, yielding heat-stable hybrids. The truncated and non-truncated antisense RNA probes showed similar hybridization kinetics. Hybridization of the sense RNA, consisting of the beta-tubulin coding portion, with the antisense RNA probe gave standards for determining the proportion of beta-tubulin mRNA in total brain RNA. By this method, the amounts of beta-tubulin mRNA included in the brains of mice of 10 and 50 days old were quantitated.     

Cell surface glycolipids of transformed NIH 3T3 cells transfected with DNAs of human bladder and lung carcinomas.

Neutral glycolipids and gangliosides of NIH 3T3 cells oncogenically transformed by transfection of DNAs from human lung carcinoma (Lx-1) and human bladder carcinoma (Ej) have been investigated. The chemical quantity and the degree of cell surface exposure of gangliotriaosylceramide (Gg3) were greatly enhanced in NIH 3T3 cells transformed by transfection of DNAs of either Lx-1 or Ej carcinoma cells. An identical but more conspicuous change in cell surface exposure of Gg3 was observed in BALB/c 3T3 cells transformed by murine sarcoma virus Kirsten strain, but the same glycolipid was absent in the original Lx-1 or Ej human carcinomas. The mechanism that defines the chemical quantity and the organization of glycolipids is controlled by multiple factors. These include not only the quantity but also the organization of glycosyl transferases and hydrolases in membranes. This also involves membrane dynamics regulated through a cytoskeletal-membrane conjunction which may determine the degree of glycolipid exposure at the cell surface. The similarity of the chemical and organizational change of a single glycolipid, Gg3, between 3T3 transformants by Kirsten murine sarcoma virus and those by transfection of human cancer DNAs may indicate a common biochemical basis triggered by activation of the oncogene.     

Specific uptake of asialofetuin-tacked liposomes encapsulating interferon-gamma by human hepatoma cells and its inhibitory effect on hepatitis B virus replication.

Asialofetuin-tacked liposomes (AF-liposomes) encapsulating interferon (IFN)-gamma were bound and internalized into a human hepatoma cell line, HepG2 cells, selectively through asialoglycoprotein receptor, but not non-tacked liposomes (N-liposomes). AF-liposomal IFN-gamma was more effective for inhibition of viral DNA replication in hepatitis B virus (HBV)-producing clone from HepG2 cells transfected with HBV-DNA than N-liposomal IFN-gamma. AF-liposomes may increase the therapeutic potential of IFN-gamma through asialoglycoprotein receptor in treating HBV-infected hepatocytes.     

ATP synthesis by an artificial proton gradient in right-side-out membrane vesicles of Escherichia coli.

Membrane vesicles formed from spheroplasts of E. coli lysed in the presence of ADP and P_i produced ATP when an artificial proton gradient (acid outside) was formed across the membrane. ATP synthesis required Mg²⁺ and ADP, was inhibited by dicyclohyxylcarbodiimide and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and stimulated by valinomycin in the presence of KCl. Synthesis was absent in a mutant lacking the Mg²⁺-ATPase. The optimum external pH was 2.5 when the internal pH was 8.2. Oxidative phosphorylation driven by D-lactate or succinate was also observed.