The Distribution of BrdU- and TUNEL-positive Cells During Odontogenesis in Mouse Lower First Molars

This study investigated the minute distribution of both proliferating and non-proliferating cells, and cell death in the developing mouse lower first molars using 5-bromo-2-deoxyuridine (BrdU) incorporation and the terminal deoxynucleotidyl transferase-mediated deoxyuridine-5-triphosphate (dUTP)-biotin nick end labeling (TUNEL) double-staining technique. The distribution pattern of the TUNEL-positive cells was more notable than that of the BrdU-positive cells. TUNEL-positive cells were localized in the following six sites: (1) in the most superficial layer of the dental epithelium during the initiation stage, (2) in the dental lamina throughout the period during which tooth germs grow after bud formation, (3) in the dental epithelium in the most anterior part of the antero-posterior axis of the tooth germ after bud formation, (4) in the primary enamel knot from the late bud stage to the late cap stage, (5) in the secondary enamel knots from the late cap stage to the late bell stage, and (6) in the stellate reticulum around the tips of the prospective cusps after the early bell stage. These peculiar distributions of TUNEL-positive cells seemed to have some effect on either the determination of the exact position of the tooth germ in the mandible or on the complicated morphogenesis of the cusps. The distribution of BrdU-negative cells was closely associated with TUNEL-positive cells, which thus suggested cell arrest and the cell death to be essential for the tooth morphogenesis.     

Inhibitory effect of presenilin inhibitor LY411575 on maturation of hepatitis C virus core protein,production of the viral particle and expression of host proteins involved in pathogenicity

Hepatitis C virus (HCV) core protein is responsible for the formation of infectious viral particles and induction of pathogenicity. The C‐terminal transmembrane region of the immature core protein is cleaved by signal peptide peptidase (SPP) for maturation of the core protein. SPP belongs to the family of presenilin‐like aspartic proteases. Some presenilin inhibitors are expected to suppress HCV infection and production; however, this anti‐HCV effect has not been investigated in detail. In this study, presenilin inhibitors were screened to identify anti‐HCV compounds. Of the 13 presenilin inhibitors tested, LY411575 was the most potent inhibitor of SPP‐dependent cleavage of HCV core protein. Production of intracellular core protein and supernatant infectious viral particles from HCV‐infected cells was significantly impaired by LY411575 in a dose‐dependent manner (half maximum inhibitory concentration = 0.27 μM, cytotoxic concentration of the extracts to cause death to 50% of viable cells > 10 μM). No effect of LY411575 on intracellular HCV RNA in the subgenomic replicon cells was detected. LY411575 synergistically promoted daclatasvir‐dependent inhibition of viral production, but not that of viral replication. Furthermore, LY411575 inhibited HCV‐related production of reactive oxygen species and expression of NADPH oxidases and vascular endothelial growth factor. Taken together, our data suggest that LY411575 suppresses HCV propagation through SPP inhibition and impairs host gene expressions related to HCV pathogenicity.     

Discrimination of mother by infant among Japanese Macaques (Macaca fuscata)

Experiments on five mother-infant pairs of Japanese monkeys (Macaca fuscata)living together in a captive group were conducted during the first 12 weeks after birth in order to assess the time at which infants begin to discriminate their own mothers from other adult females. After removal from their social group, infants exposed to their mothers and three unfamiliar adult females at a distance of 150 cm failed to orient visually toward their mothers. However, when the infants were allowed to approach the four females, they responded preferentially to their mothers during the third month of life. We concluded that by 8–12 weeks of age, infant Japanese macaques are able to discriminate between their mothers and other adult females.     

Molecular Mechanisms for Sweet-suppressing Effect of Gymnemic Acids

Gymnemic acids are triterpene glycosides that selectively suppress taste responses to various sweet substances in humans but not in mice. This sweet-suppressing effect of gymnemic acids is diminished by rinsing the tongue with γ-cyclodextrin (γ-CD). However, little is known about the molecular mechanisms underlying the sweet-suppressing effect of gymnemic acids and the interaction between gymnemic acids versus sweet taste receptor and/or γ-CD. To investigate whether gymnemic acids directly interact with human (h) sweet receptor hT1R2 + hT1R3, we used the sweet receptor T1R2 + T1R3 assay in transiently transfected HEK293 cells. Similar to previous studies in humans and mice, gymnemic acids (100 μg/ml) inhibited the [Ca²⁺]_i responses to sweet compounds in HEK293 cells heterologously expressing hT1R2 + hT1R3 but not in those expressing the mouse (m) sweet receptor mT1R2 + mT1R3. The effect of gymnemic acids rapidly disappeared after rinsing the HEK293 cells with γ-CD. Using mixed species pairings of human and mouse sweet receptor subunits and chimeras, we determined that the transmembrane domain of hT1R3 was mainly required for the sweet-suppressing effect of gymnemic acids. Directed mutagenesis in the transmembrane domain of hT1R3 revealed that the interaction site for gymnemic acids shared the amino acid residues that determined the sensitivity to another sweet antagonist, lactisole. Glucuronic acid, which is the common structure of gymnemic acids, also reduced sensitivity to sweet compounds. In our models, gymnemic acids were predicted to dock to a binding pocket within the transmembrane domain of hT1R3.     

Expression of amiloride-sensitive epithelial sodium channels in mouse taste cells after chorda tympani nerve crush

Our previous electrophysiological study demonstrated that amiloride-sensitive (AS) and -insensitive (AI) components of NaCl responses recovered differentially after the mouse chorda tympani (CT) was crushed. AI responses reappeared earlier (at 3 weeks after the nerve crush) than did AS ones (at 4 weeks). This and other results suggested that two salt-responsive systems were differentially and independently reformed after nerve crush. To investigate the molecular mechanisms of formation of the salt responsive systems, we examined expression patterns of three subunits (alpha, beta and gamma) of the amiloride-sensitive epithelial Na(+) channel (ENaC) in mouse taste cells after CT nerve crush by using in situ hybridization (ISH) analysis. The results showed that all three ENaC subunits, as well as alpha-gustducin, a marker of differentiated taste cells, were expressed in a subset of taste bud cells from an early stage (1-2 weeks) after nerve crush, although these taste buds were smaller and fewer in number than for control mice. At 3 weeks, the mean number of each ENaC subunit and alpha-gustducin mRNA-positive cells per taste bud reached the control level. Also, the size of taste buds became similar to those of the control mice at this time. Our previous electrophysiological study demonstrated that at 2 weeks no significant response of the nerve to chemical stimuli was observed. Thus ENaC subunits appear to be expressed prior to the reappearance of AI and AS neural responses after CT nerve crush. These results support the view that differentiation of taste cells into AS or AI cells is initiated prior to synapse formation.     

Low-pressure,microwave-induced plasma spectrometry method for the determination of subnanogram quantities of mercury: Application to mercury-carboxypeptidase analysis

A simple, sensitive, and rapid method for the determination of subnanogram quantities of mercury has been devised using a tantalum-filament vaporization system and low-pressure, microwave-induced emission spectrometry. This method is applied to the analysis of mercury-substituted carboxypeptidase with a sample volume of 5 μl and an analysis time of 3 min. The coefficient of variation for 2 × 10⁻¹⁰ g of mercury in mercury-carboxypeptidase is 6.0%, and the detection limit is 3 × 10⁻¹² g.     

SRC kinase activator CDCP1 promotes hepatocyte growth factor–induced cell migration/invasion of a subset of breast cancer cells

Cancer invasion and metastasis are the major causes of cancer patient mortality. Various growth factors, including hepatocyte growth factor (HGF), are known to promote cancer invasion and metastasis, but the regulatory mechanisms involved are not fully understood. Here, we show that HGF-promoted migration and invasion of breast cancer cells are regulated by CUB domain–containing protein 1 (CDCP1), a transmembrane activator of SRC kinase. In metastatic human breast cancer cell line MDA-MB-231, which highly expresses the HGF receptor MET and CDCP1, we show that CDCP1 knockdown attenuated HGF-induced MET activation, followed by suppression of lamellipodia formation and cell migration/invasion. In contrast, in the low invasive/nonmetastatic breast cancer cell line T47D, which had no detectable MET and CDCP1 expression, ectopic MET expression stimulated the HGF-dependent activation of invasive activity, and concomitant CDCP1 expression activated SRC and further promoted invasive activity. In these cells, CDCP1 expression dramatically activated HGF-induced membrane remodeling, which was accompanied by activation of the small GTPase Rac1. Analysis of guanine nucleotide exchange factors revealed that ARHGEF7 was specifically required for CDCP1-dependent induction of HGF-induced invasive ability. Furthermore, immunofluorescence staining demonstrated that CDCP1 coaccumulated with ARHGEF7. Finally, we confirmed that the CDCP1-SRC axis was also crucial for HGF and ARHGEF7-RAC1 signaling in MDA-MB-231 cells. Altogether, these results demonstrate that the CDCP1-SRC-ARHGEF7-RAC1 pathway plays an important role in the HGF-induced invasion of a subset of breast cancer cells.