Specific bindings of [3H](+)PN200-110 and [125I]ω-conotoxin to crude membranes from differentiated NG108-15 cells

The characteristics of the specific bindings of [³H](+)PN200-110 (PN: L-type Ca channel antagonist) and [¹²⁵I]-conotoxin G VI A (-CgTX: neuronal L-or N-type Ca channel antagonist) to crude membranes from undifferentiated neuroblastoma x glioma hybrid NG108-15 (NG108-15) cells and differentiated cells induced with dibutyryl cAMP (Bt₂cAMP) were examined, because we have already observed that the magnitude and rate of KCL-stimulated⁴⁵Ca uptake by NG108-15 cells increased progressively during differentiation of the cells induced with Bt₂cAMP (unpublished results). The specific binding of [³H](+)PN to these crude membranes was saturable at various concentrations of 2.5–5.0 nM [³H](+)PN. Scatchard analysis showed that the specific binding of [³H](+)PN at equilibrium was significantly increased after differentiation of the NG108-15 cells with Bt₂cAMP, but that the apparent Kd value for the specific binding of [³H](+)PN was not influenced by treatment with Bt₂cAMP. The specific binding of [³H](+)PN to crude membranes from Bt₂cAMP-treated NG108-15 cells was inhibited by a calcium agonist and antagonists, the order of their inhibitory potencies being (+)PN>nitrendipine>(–)PNBay K 8644diltiazem = verapamil. Thus, PNs showed significant stereoselective inhibition of the specific binding of [³H(+)PN. On the other hand, [¹²⁵I]-CgTX at concentrations of 0.075–0.6 nM showed scarcely any specific binding to these crude membranes, although at 0.6 nM it showed specific binding to crude membranes from rat brain in the same experimental conditions. These results suggest that the increase in magnitude or rate of KCl-stimulated⁴⁵Ca uptake during differentiation of NG108-15 cells is partially due to quantitative alteration of voltage-sensitive Ca channels in the cells, and that there are scarcely any specific binding sites for [¹²⁵I]-CgTX on Bt₂cAMP-treated or untreated NG108-15 cells.     

Evolution of the therian face through complete loss of the premaxilla

The anatomical framework of the jawbones is highly conserved among most of the Osteichthyes, including the tetrapods. However, our recent study suggested that the premaxilla, the rostralmost upper jaw bone, was rearranged during the evolution of therian mammals, being replaced by the septomaxilla at least in the lateral part. In the present study, to understand more about the process of evolution from the ancestral upper jaw to the therian face, we re-examined the development of the therian premaxilla (incisive bone). By comparing mouse, bat, goat, and cattle fetuses, we confirmed that the therian premaxilla has dual developmental origins, the lateral body and the palatine process. This dual development is widely conserved among the therian mammals. Cell-lineage-tracing experiments using Dlx1-CreER^T2 mice revealed that the palatine process arises in the ventral part of the premandibular domain, where the nasopalatine nerve distributes, whereas the lateral body develops from the maxillary prominence in the domain of the maxillary nerve. Through comparative analysis using various tetrapods, we concluded that the palatine process should not be considered part of the ancestral premaxilla. It rather corresponds to the anterior region of the vomerine bone of nonmammalian tetrapods. Thus, the present findings indicate that the true premaxilla was completely lost during the evolution of the therian mammals, resulting in the establishment of the unique therian face as an evolutionary novelty. Reconsideration of the homological framework of the cranial skeleton based on the topographical relationships of the ossification center during embryonic development is warranted.     

The role of p63 in embryonic external genitalia outgrowth in mice

Embryonic external genitalia (genital tubercle [GT]) protrude from the cloaca and outgrow as cloacal development progresses. Individual gene functions and knockout phenotypes in GT development have been extensively analyzed; however, the interactions between these genes are not fully understood. In this study, we investigated the role of p63, focusing on its interaction with the Shh–Wnt/Ctnnb1–Fgf8 pathway, a signaling network that is known to play a role in GT outgrowth. p63 was expressed in the epithelial tissues of the GT at E11.5, and the distal tip of the GT predominantly expressed the ΔNp63α isoform. The GTs in p63 knockout embryos had normal Shh expression, but CTNNB1 protein and Fgf8 gene expression in the distal urethral epithelium was decreased or lost. Constitutive expression of CTNNB1 in p63-null embryos restored Fgf8 expression, accompanied by small bud structure development; however, such bud structures could not be maintained by E13.5, at which point mutant GTs exhibited severe abnormalities showing a split shape with a hemorrhagic cloaca. Therefore, p63 is a key component of the signaling pathway that triggers Fgf8 expression in the distal urethral epithelium and contributes to GT outgrowth by ensuring the structural integrity of the cloacal epithelia. Altogether, we propose that p63 plays an essential role in the signaling network for the development of external genitalia.     

In situ analysis of centromere segregation in C57BL/6 x Mus spretus interspecific backcrosses

The analysis of major satellite sequence differences between Mus spretus and laboratory mice provides a robust method for analyzing the centromere location for the genetic maps of each mouse chromosome. Fluorescence in situ hybridization (FISH) of a genomic probe, pMR196, for the laboratory mouse major satellite sequences was used to identify C57BL/6Ros (B6) pericentromeric heterochromatin in progeny of reciprocal backcross matings. These included 80 (B6xM. spretus)F₁xM. spretus progeny (BSS) and 70 (B6xM. spretus)F₁xB6 (BSB) progeny. FISH analysis of pericentromeric heterochromatin was conducted on the same metaphase spreads that were karyotypically analyzed for chromosomespecific banding patterns. Analysis of chromosomal segregation suggested that there was not primary deviation from random assortment during meiosis in the interspecific hybrid female, because nearly all of the 190 pair-wise comparisons did not deviate from expected and because there was no consistent pattern of deviation of the same chromosomes in the reciprocal backcross progeny from similar (C57BL/6xM. spretus)F₁ hybrid females. These results affirm the value of using the major satellite to genetically mark pericentromeric heterochromatin in the analysis of the segregation and assortment of centromeres in Mus interspecific crosses.     

Increased anti-oxidative action compensates for collagen tissue degeneration in vitiligo dermis

Vitiligo is a common depigmentation disorder characterized by the selective loss of melanocytes. In our daily clinic experience, we noticed that the skin tightness of hypopigmented lesions would be more evident in comparison to that of uninvolved perilesional skin in vitiligo patients. Therefore, we hypothesized that collagen homeostasis might be maintained in vitiligo lesions, irrespective of the substantial excessive oxidative stress that occurs in association with the disease. We found that the expression levels of collagen-related genes and anti-oxidative enzymes were upregulated in vitiligo-derived fibroblasts. Abundant collagenous fibers were observed in the papillary dermis of vitiligo lesions in comparison to uninvolved perilesional skin by electron microscopy. The production of matrix metalloproteinases that degraded collagen fibers was suppressed. The deposition of acrolein adduct protein, which is a product of oxidative stress, was significantly reduced in vitiligo dermis and fibroblasts. As part of the mechanism, we found upregulation of the NRF2 signaling pathway activity, which is an important defense system against oxidative stress. Taken together, we demonstrated that the anti-oxidative action and collagen production were upregulated and that the collagen degeneration was attenuated in vitiligo dermis. These new findings may provide important clues for the maintenance of antioxidant ability in vitiligo lesions.     

Characterization of antibody against human liver guanase by immunoblotting and immunohistochemical staining of human liver guanase by a direct labeling antibody-enzyme method

Human liver guanase was purified and a specific antibody against it was raised in rabbits. The antiserum formed a single precipitin line with human liver extract, and also completely inhibited the activity of the liver enzyme. An immunoblotting study showed that the antibody bound specifically to one band of protein with guanase activity and not to other proteins. Therefore, we concluded that this antiserum against the liver enzyme was suitable for use in immunohistochemical demonstration of guanase. In tissue sections, the immunohistochemical reaction with this antibody was positive in the same locations as the histochemical guanase reaction with DAB (3,3'-diaminobenzidine tetrahydrochloride).     

Localization of the ATP-binding site in the 23-kDa and 20-kDa regions of the heavy chain of the skeletal muscle myosin head

Three kinds of ATP analogues were synthesized. These ATP analogues can be classified into two conformations, i.e. syn and anti forms with respect to the N-glycosidic bond between adenine and ribose groups of ATP. 3'-O-(N-Methylanthraniloyl)-2-azidoadenosine 5'-triphosphate (MantN2(3)ATP) is recognized as the anti form, as ATP, and the other two, 3'-O-(N-methylanthraniloyl)-8-azidoadenosine 5'-triphosphate (MantN8(3)ATP) and 1,N6-etheno-8-azidoadenosine 5'-triphosphate (epsilon N8(3)ATP) are both syn forms. Mant and etheno groups are both fluorescent which allows detection of their binding to proteins. The photochemical binding of azido groups in ATP analogues to the myosin active site, examined in the presence and absence of ATP, showed that all the analogues bound to the site of myosin ATPase. These analogues also acted as substrates of the ATPase and were hydrolyzed in the active site, as judged by competitive inhibition of the ATPase and by their ATPase activities. Of these analogues, MantN2(3)ATP is very similar to ATP in divalent-cation dependence of its hydrolysis rate and in its ability to trap ADP in the active site with vanadate, while the other two are different from ATP in these respects. The photochemical binding sites of ATP analogues were localized by gel electrophoresis of trypsinized myosin ATPase with photocross-linked ATP analogues and/or by isolating the modified peptides. MantN2(3)ATP was found in the 23-kDa fragment which has a structure common to ATP-binding proteins, i.e. Gly-Xaa-Xaa-Gly-Xaa-Gly-Lys-Thr. Mant N8(3)ATP was found in a region of the 20-kDa fragment where actin is reported to attach.     

Effect of gravity on apical dominance in Pharbitis nil.

When the upper part of main shoot of morning glory (Pharbitis nil) is gently bent down, lateral bud on the bending region is released from apical dominance and starts to elongate. But, clinorotating the bending shoots prevents the release of the lateral bud from apical dominance. These results suggest that gravity affects apical dominance in morning glory. Here we verified the gravity-regulated apical dominance by using a weeping morning glory defective in gravitropic response due to abnormal differentiation of endodermis. That is, bending main shoot of the weeping morning glory hardly caused the lateral bud to elongate. In addition, decapitation of apical bud released the lateral bud from apical dominance, and exogenous auxin applied to the cut surface of the decapitated stem was inhibitory to the outgrowth of the lateral bud in the wild type. However, the effect of auxin was much less in the weeping morning glory. Thus, apical dominance of the weeping morning glory was weaker and less influenced by gravity than that of the wild type, which could occur due to abnormal differentiation of endodermis required for graviperception.     

Effect of gravity stress on fidelity of DNA double-strand break repair.

DNA double strand break (DSB) causes many cytotoxic effects such as cellular lethality, somatic mutation, and carcinogenesis. Fidelity of DSB repair is a important factor that determines the quality of genomic stability. It is known that the most of DSBs are properly repaired on the earth, however, little is known whether those are rejoined at the same fidelity even under the space environment. One of the DSB repair pathway, homologous recombination (HR), allows the cells to repair their DSBs with error free. Therefore, the efficiency of HR is a good index to assess the fidelity of DSB repair. In order to clarify the effect of gravity stress on HR pathway, we established a cell line that can detect a site-specific DNA repair via HR. The cells carrying a reporter construct for HR were incubated under hypergravity condition after induction of site specific DSB. Our preliminary results suggest that the gravity stress may affect the HR efficiency.     

First finding of urease-positive thermophilic strains of Campylobacter in river water in the Far East, namely, in Japan and their phenotypic and genotypic characterization

Two strains of urease-positive thermophilic Campylobacter (UPTC), CF89–12 and CF89–14, which were identified as UPTC by biochemical characterization, were found for the first time in river water in the Far East, namely, in Japan. The biochemical characteristics were identical to those of strains described previously by Bolton and colleagues. Furthermore, these two strains were positive for arylsulphatase. Consequently, it was demonstrated that UPTC may possibly be differentiated phenotypically from Campylobacter lari by the arylsulphatase test, as well as urease and nalidixic acid tests. Analysis by pulsed-field gel electrophoresis (PFGE) after digestion with Apa I, Sal I and Sma I, which were found to produce distributions of DNA fragments to be suitable for analysis of the genomic DNA from the thermophilic Campylobacter , respectively, demonstrated that these three restriction enzymes produced distributions of a relatively limited number of genomic DNA fragments and also demonstrated that the PFGE profiles obtained with the three restriction enzymes were indistinguishable between the two strains, respectively. The PFGE analysis and conventional fixed-field agarose gel electrophoresis suggested that the both genomes were approximately 1862 kb in length. Even though the two isolates of UPTC were isolated from water in different rivers in Japan, the results suggested that a single strain. as opposed to two distinct strains, was isolated. PFGE profiles after digestion with Sal I and Sma I, respectively, were also demonstrated to be distinctly different among strains isolated in Japan and previously in Europe. This is the first example of the isolation of UPTC from natural sources in countries other than those in Europe.