Purification of hepatitis B virus gene X product synthesized in Escherichia coli and its detection in a human hepatoblastoma cell line producing hepatitis B virus

A fused gene containing 94% of the hepatitis B virus (HBV) open reading frame X was expressed in Escherichia coli, and its 17-kDa product was purified by ion-exchange chromatography. Antibody elicited against the X-gene product reacted with materials proximal to the nuclear membrane of a human hepatoblastoma cell line producing HBV particles. No such reaction was observed with the same cell line that did not produce HBV particles.     

Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development

In ventricular cells of the mouse telencephalon, differential expression of cadherin cell adhesion molecules defines neighbouring regions; R-cadherin delineates the future cerebral cortex, while cadherin-6 delineates the lateral ganglionic eminence. By using cell labelling analyses in the whole embryo culture system, we demonstrated that the interface between R-cadherin and cadherin-6 expression is a boundary for cell lineage restriction at embryonic day 10.5. Interestingly, when a group of cells with exogenous cadherin-6 were generated to straddle the cortico-straital boundary by electroporation at embryonic day 11.0, ectopic cadherin-6-expressing cortical cells were sorted into the striatal compartment, and the reverse was the trend for ectopic R-cadherin-expressing striatal cells. Although cadherin-6 gene knockout mice engineered in this study showed no obvious phenotype in telencephalic compartmentalisation, the preferential sorting of ectopic cadherin-6-expressing cells was abolished in this mutant background. Thus, the differential expression pattern of cadherins in the embryonic telencephalon is responsible for maintaining the cortico-striatal compartment boundary.     

Physiological properties of rod photoreceptor cells in green-sensitive cone pigment knock-in mice

Rod and cone photoreceptor cells that are responsible for scotopic and photopic vision, respectively, exhibit photoresponses different from each other and contain similar phototransduction proteins with distinctive molecular properties. To investigate the contribution of the different molecular properties of visual pigments to the responses of the photoreceptor cells, we have generated knock-in mice in which rod visual pigment (rhodopsin) was replaced with mouse green-sensitive cone visual pigment (mouse green). The mouse green was successfully transported to the rod outer segments, though the expression of mouse green in homozygous retina was approximately 11% of rhodopsin in wild-type retina. Single-cell recordings of wild-type and homozygous rods suggested that the flash sensitivity and the single-photon responses from mouse green were three to fourfold lower than those from rhodopsin after correction for the differences in cell volume and levels of several signal transduction proteins. Subsequent measurements using heterozygous rods expressing both mouse green and rhodopsin E122Q mutant, where these pigments in the same rod cells can be selectively irradiated due to their distinctive absorption maxima, clearly showed that the photoresponse of mouse green was threefold lower than that of rhodopsin. Noise analysis indicated that the rate of thermal activations of mouse green was 1.7 x 10(-7) s(-1), about 860-fold higher than that of rhodopsin. The increase in thermal activation of mouse green relative to that of rhodopsin results in only 4% reduction of rod photosensitivity for bright lights, but would instead be expected to severely affect the visual threshold under dim-light conditions. Therefore, the abilities of rhodopsin to generate a large single photon response and to retain high thermal stability in darkness are factors that have been necessary for the evolution of scotopic vision.     

Amphibian teeth: current knowledge, unanswered questions, and some directions for future research

Elucidation of the mechanisms controlling early development and organogenesis is currently progressing in several model species and a new field of research, evolutionary developmental biology, which integrates developmental and comparative approaches, has emerged. Although the expression pattern of many genes during tooth development in mammals is known, data on other lineages are virtually non-existent. Comparison of tooth development, and particularly of gene expression (and function) during tooth morphogenesis and differentiation, in representative species of various vertebrate lineages is a prerequisite to understand what makes one tooth different from another. Amphibians appear to be good candidates for such research for several reasons: tooth structure is similar to that in mammals, teeth are renewed continuously during life (=polyphyodonty), some species are easy to breed in the laboratory, and a large amount of morphological data are already available on diverse aspects of tooth biology in various species. The aim of this review is to evaluate current knowledge on amphibian teeth, principally concerning tooth development and replacement (including resorption), and changes in morphology and structure during ontogeny and metamorphosis. Throughout this review we highlight important questions which remain to be answered and that could be addressed using comparative morphological studies and molecular techniques. We illustrate several aspects of amphibian tooth biology using data obtained for the caudate Pleurodeles waltl. This salamander has been used extensively in experimental embryology research during the past century and appears to be one of the most favourable amphibian species to use as a model in studies of tooth development.     

Molecular properties of rod and cone visual pigments from purified chicken cone pigments to mouse rhodopsin in situ.

We have investigated the molecular properties of rod and cone visual pigments to elucidate the differences in the molecular mechanism(s) of the photoresponses between rod and cone photoreceptor cells. We have found that the cone pigments exhibit a faster pigment regeneration and faster decay of meta-II and meta-III intermediates than the rod pigment, rhodopsin. Mutagenesis experiments have revealed that the amino acid residues at positions 122 and 189 in the opsins are the determinants for these differences. In order to study the relationship between the molecular properties of visual pigments and the physiology of rod photoreceptors, we used mouse rhodopsin as a model pigment because, by gene-targeting, the spectral properties of the pigment can be directly correlated to the physiology of the cells. In the present paper, we summarize the spectroscopic properties of cone pigments and describe our studies with mouse rhodopsin utilizing a high performance charge coupled device (CCD) spectrophotometer.     

Generation of knock-in mice carrying third cones with spectral sensitivity different from S and L cones

Red-green color vision in primates is unique in the sense that it is mediated by two photoreceptor cells that are indistinguishable in all aspects except for their visual pigments. In order to generate an animal model for investigation of the interaction between red-green inputs at the molecular level, we applied knock-in technology and X-chromosome inactivation machinery to make a mouse model with cone cells possessing visual pigments with different spectral sensitivities. We introduced a S308A point mutation into the Green opsin gene allele on the X-chromosome. This manipulation generated a 24 nm red-shift of absorption maximum in the cone pigment with negligible functional differences in other molecular properties. Amplitudes of responses in ERG and ganglion cell recordings of homozygotes were similar to those of wild-types, although the spectral sensitivities differed. Heterozygotes showed variable spectral sensitivities of ganglion cell responses due to the different integration of the native and the S308A cone inputs on the dendritic fields. In situ hybridization experiments showed that cone cells with respective pigments formed patch-like clusters of specific L cone-types, approximately 30 mum in diameter, which were randomly distributed in the dorsal region of the retinas. Since the patch-like clustering was arranged by X-inactivation, such clustering could be present in the peripheral retinas of New World monkeys with polymorphic L pigments, indicating that our mice would be a suitable model to study evolution of the mammalian color vision system.     

Effect of the interaction between lipoxygenase pathway and progesterone on the regulation of hydroxysteroid 11-Beta dehydrogenase 2 in cultured human term placental trophoblasts

Placental hydroxysteroid 11-beta dehydrogenase 2 (HSD11B2) plays an important role in pregnancy maintenance and fetal maturation. In the event of intrauterine infection, lipoxygenase (LOX) metabolites are produced in the placenta and contribute to preterm labor and adverse fetal outcomes. On the other hand, LOX metabolites are involved in production of progesterone, which is required for pregnancy maintenance. In this study, we evaluated the interaction between the LOX pathway, progesterone, and HSD11B2. Specifically, we hypothesized that LOX metabolites would alter HSD11B2 and this effect would be mediated by progesterone. We cultured human term placental trophoblasts in the presence and absence of the LOX inhibitors Nordihydroguaiaretic acid (NDGA), AA861, and Baicalein; the LOX metabolites Leukotriene B(4) and 12(S)-Hydroxyeicosatetraenoate (12-HETE); and progesterone and progesterone receptor antagonist RU486. By radiometric conversion assay, real-time quantitative PCR, Western blot analysis, and ELISA, we examined HSD11B2 enzyme activity, HSD11B2 mRNA and HSD11B2 protein expression, and progesterone output. LOX metabolites down-regulated HSD11B2 activity and HSD11B2 expression. LOX inhibitors up-regulated HSD11B2 activity and HSD11B2 and HSD11B2 expression, and these effects were attenuated by addition of LOX metabolites. Net progesterone output was increased by LOX metabolites and decreased by LOX inhibitors. Progesterone down-regulated HSD11B2 activity and HSD11B2 and HSD11B2 expression, and these effects were blocked by RU486. Furthermore, the suppressive effect of 12-HETE on HSD11B2 activity was also reversed by RU486. We conclude that HSD11B2 in human placental trophoblasts is decreased by progesterone and increased by inhibition of endogenous LOX metabolites, and that a component of the effect of LOX metabolites on HSD11B2 is mediated by their stimulation of endogenous progesterone output.