Steroid hormones, thyroid hormones, retinoic acids, and vitamin D bind to their receptors, which are now called steroid/nuclear receptors, and liganded receptors translocate either intracellularly or intranuclearly and form large protein complexes with cofactors to induce or repress gene transcription. Therefore, steroid/nuclear receptors are ligand-dependent transcription factors.
  With the advent of green fluorescent protein (GFP) and its color variants, the subcellular distribution of many steroid/nuclear receptors has been found to be much more dynamic than previously thought, with some of the receptors shuttling between the cytoplasm and nucleus. Steroid/nuclear receptors can be divided into three categories based on their unliganded distribution: those that are primarily in the nucleus, those in the cytoplasm, and those with mixed cytoplasmic and nuclear distributions. However, in all cases, the addition of a ligand leads to almost complete nuclear translocation of the receptors. Hormonal stimulation induces intranuclear receptor distribution from a homogeneous pattern to a heterogeneous dot-like image. Ligand binding to steroid/nuclear receptors leads to the recruitment of many proteins including cofactors to provoke the redistribution of receptor complexes in the nucleus. This focal organization could involve more complex events than simple DNA binding sites for transcription. Protein activities and interactions of steroid/nuclear receptors can be imaged and localized in a single cell.

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The postnatal expression of heat shock protein (Hsp) 25 during the amelogenesis of rat molars was investigated by immunocytochemistry and confocal microscopy. The localization pattern of Hsp 25-immunoreactivity in the inner enamel epithelium and ameloblast cell layer of the rat molars was almost identical to that in the rat incisors which we have previously reported: an intense Hsp 25-immunoreactivity, which first appeared in the preameloblasts, was recognized in secretory ameloblasts and ruffle-ended ameloblasts with stage-specific immunointensity. Confocal microscopy with Hsp 25-antibody and rhodamine-labeled phalloidin clearly demonstrated the co-localization of Hsp 25 and actin filaments in the ameloblast layer, supporting our hypothesis that this molecule might serve to reinforce the ameloblast layer during enamel formation as well as the formation and maintenance of the ruffled border in ruffle-ended ameloblasts. Interestingly, the enamel free area cells, which essentially lack the ability for enamel formation, showed the Hsp 25-immunoreactivity during 4-11 days when they developed a ruffled border, but decreased in that immunoreactivity after postnatal 15 days following apoptosis. Since Hsp 25 has been shown to be a specific inhibitor of apoptosis, the enamel-free area cells contribute to determine the outline of dentin at the cusped area. These data support our previous hypothesis on the diverse functions of Hsp 25 in amelogenesis.

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Using a novel immunocytochemical staining method, we aimed to characterize the phase transition zone (PTZ) (∼100 μm) in adult ocular lenses and the process of terminal differentiation (denucleation) within normal fiber cells. The binding to DNA of zeta(ζ)-crystallin (Z-DNA-binding protein) and anti-double-stranded (ds-)-B-DNA antibody probes was found to decline gradually throughout denucleating fibers, with a precipitous decrease occurring at about 100 μm (PTZ). Nuclei of superficial fiber cells (in front of the PTZ) showed the highest DNA probe-binding values, followed by middle fibers (MF) and deep fibers (DF). With the use of ζ-crystallin, anti-ds-B-DNA antibody, and anti-single stranded (ss-) DNA antibody probes, it was possible to reveal a loss of reactivity of fiber cell ds-DNA. Ss-DNA antibody binding was seen initially in the MF and reached its highest intensity level in the DF. The pattern of ζ-crystallin probe-DNA reactivity correlates with the loss of anti-B-DNA antibody staining and decreased eosin-protein staining. These data suggest that a reorganization of DNA and intracellular protein supramolecular order in normal adult lenses occurs at a depth of about 100 μm (PTZ).

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To study injury and subsequent changes in skeletal muscles, the rat sciatic nerve was electrically stimulated at 50 Hz and muscle contraction was induced for 30 min. Muscle damage was classified into five types (hypercontraction, hyperstretching, Z band disorders, misalignment of myofilament and regions of scarce myofilaments) by electron microscopy and quantified by ultrastructural assessment. After electrical nerve stimulation, the percentages of the injured areas of the soleus muscle were 18.8 ± 15.8% (mean ± SD) at O h, 9.7 ± 1.0% at 6 h, 22.0 ± 23.6% at 12 h, 13.1 ± 3.2 % at 24 h, 4.9 ± 6.0% at 3 days and 0.5 ± 0.4% at 7 days. At 0 h, the vast majority of ultrastructural alterations were sarcomere hypercontraction. At 6 h, hypercontraction was not recognizable and sarcomere hyperstretching and Z band disarrangement constituted the major findings. At 12 h, when the injury reaehed its maximum, myofilament disorganization and hyperstretching were predominant. At 24 h or afterwards, the injury began to decrease and recovered to almost normal conditions by 7 days. There were very few necrotic muscle fibers in all specimens. It is considered that the muscle lesions in the present study were reversible, and recovered through changes in various types of sarcomere alterations. Z band streaming and free ribosomes were frequently found at 12 and 24 h, which may indicate repair processes rather than newly formed lesions.

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We used α-gustducin, a taste-cell-specific G protein to investigate the onset of taste transduction and its relation to the development of the palatal and lingual taste buds. Frozen cryostat and paraffin sections were prepared from the palatal and lingual gustatory epithelium of the rat from birth till postnatal day 21 (PN 2ld). At PN 1-7d, α-gustducin-immunoreactive solitary ovoid or bipolar cells were scattered among the oral epithelium either horizontally along the oral surface or vertically oriented between the basal lamina and oral surface. In the circumvallate and foliate papillae, these cells became wrapped in α-gustducin-immunonegative cells surrounded by an extracellular space forming a bud-like structure. Simultaneously, different stages of typical taste buds were recognized, but α-gustducin was only expressed in some neonatally developed pored buds. At PN 1d, α-gustducin was expressed in pored taste buds with a relatively higher frequency recorded in the soft palate as compared with the nasoincisor, circumvallate, and foliate papillae. The immunoreactive cells were spindle shaped with elongated processes extending from the base to the pore of the taste buds. During the second week, the solitary cells could no longer be recognized while the total counts of immunoreactive cells within the taste buds gradually increased. We argue that taste transduction is essentially required from the time of birth and can be fulfilled by both of the solitary chemosensory cells, which are immunoreactive for α-gustducin and scattered in the oral epithelium, and the taste cells within the mature taste buds. Moreover, the onset of taste transduction accomplished by the palatal taste buds developed earlier than that achieved by taste buds in the circumvallate and foliate papillae.

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Membrane-type 1 matrix metalloproteinase (MT1-MMP), a membrane-bound matrix metalloproteinase, plays crucial roles in cellular migration through the matrix during embryogenesis, wound healing, and the invasion of host tissues by cancer cells. Mammalian trophoblast cells exhibit different degrees of invasiveness towards the endometrium in different species during gestation. The highly invasive trophoblast cells of primates and rodents which form hemochorial placentae have often been compared to metastatic cancer cells, and are known to express MT1-MMP at their invasive edge. So far, however, little is known about MT1-MMP expression in the placenta of non-invasive type including the synepitheliochorial placepta of bovidae. As an approach to assess the role played by MT1-MMP in the non-invasive synepitheliochorial placentation, we determined the open reading frame (ORF) base sequence of caprine MT1-MMP (DDBJ/EMBL/ GenBank database: AB010921); this sequence is the first registered MT1-MMP ORF sequence of artyodactyls which develop placentae of the non-invasive type. The deduced amino acid sequence of caprine MT1-MMP exhibited 92, 87 and 89% identity with its human, mouse and rat counterparts, respectively. Availability of the cloned caprine MT1-MMP cDNA allowed us to carry out Northern blot analysis which revealed that in the placentome, the expression levels of MT1-MMP mRNA were very low on Day 35 of gestation (peri-implantation stage), while the levels gradually increased from Day 75 to Day 100. In the interplacentome regions of the placenta and the uterus, the signal levels were higher than those in the placentome, and increased from Day 35 onward, peaking on Day 75. In situ hybridization experiments revealed that the binucleate trophoblast cells reacted with the MT1-MMP cRNA probe throughout the period examined while the uninuclear principal trophoblast cells did so only on Day 100. Of particular interest is the expression of MT1-MMP transcripts in the luminal and glandular epithelial cells of the gestational endometrium, since epithelial cells in general have been noted to lack MMP expression, including MT-MMPs. The high levels of MT1-MMP expression in the endometrial epithelial cell populations might reflect extensive remodeling during gestation.

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The regeneration process of dental pulp following tooth replantation in rat molars was investigated by immunocytochemistry for heat shock protein (Hsp) 25 and protein gene product 9.5 (PGP 9.5). In control teeth at postnatal 4 weeks, the odontoblasts showed intense Hsp 25-immunoreactivity in the coronal dental pulp, but little or no immunoreactivity in the root and floor pulp. In contrast, the Hsp 25-negative odontoblasts in the latter areas displayed immunoreactivity for PGP 9.5. Tooth replantation caused loss of Hsp 25- and PGP 9.5-immunoreactions in the dental pulp during postoperative days 1-3. At postoperative day 5, plump cells with clear nucleoli and several fine processes - presumably newly differentiated odontoblasts - at the pulp-dentin border became immunopositive for Hsp 25. These data suggest that the expression of Hsp 25- and PGP 9.5-immunoreactivity reflects the status of differentiation of the odontoblasts. Furthermore, some pulpal nerve fibers as well as the Schwann cells in the dental pulp, ordinarily negative in Hsp 25-immunoreaction, acquired their immunoreactivity by postoperative day 5, but lost it thereafter, suggesting the involvement of Hsp 25 in the regeneration of pulpal nerve fibers. In the case of bone-like tissue formation in the pulp space, on the other hand, no Hsp 25-immunoreactive odontoblasts were recognized in the pulp-dentin border. Thus, the alignment of Hsp 25-immunopositive odontoblasts along the pulpdentin border indicates a decisive factor for inducing the reparative dentin formation after tooth replantation.

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The three-dimensional distribution of the biliary tract in the rat was studied by scanning electron microscopy of biliary casts. The casts were prepared by a retrograde infusion of a low viscosity or monomeric methacrylate resin mixture into the common bile duct. No resin flow from the bile canaliculi to sinusoidal capillaries was ever noted. Bile canaliculi formed intricate meshworks and drained via the Hering’s canals into the bile ductules. The bile canalicular meshworks of adjacent lobules intercommunicated with each other. The bile ductules formed a marked periportal plexus around the portal vein branch, and drained into the intrahepatic bile duct running along the portal vein branch. The junctional zone of the Hering’s canal and bile ductule usually showed an ampullary dilation. When the Hering’s canal directly drained into a thick bile ductule or into a periportal plexus of bile ductules, such an ampullary dilation at the origin of the bile ductule was never replicated. The extrahepatic bile duct protruded many crypt-like projections which presumably corresponded to parietal glands. It is suggested that the periportal plexus of bile ductules may store the bile as a substitute for the gallbladder.

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The diazepam binding inhibitor (DBI), initially isolated as an endogenous 10-kDa polypeptide from the brain, has the ability to displace ligands from benzodiazepine binding sites on γ-aminobutyric acid (GABA) receptors. However, DBI is widely distributed outside the brain, with the highest expression in the intestine. The present in situ hybridization study revealed the cellular expression of DBI mRNA throughout the gastrointestinal tract of mice, showing it to be intensely expressed in the spinous layer in the stratified squamous epithelium of the oral cavity, esophagus and forestomach, in surface mucous cells in the glandular stomach, and in columnar (absorptive) cells of the intestinal villi. A precise identification of DBI-expressing cell types was confirmed immunohistochemically, although the expressing cells detectable by the two histochemical methods differed slightly in their extension. Noteworthily, DBI always coexisted with the fatty acid binding protein (FABP), which participates in the uptake and metabolic processing of long chain fatty acids. In addition to the biochemical finding that DBI is identical with the acyl-CoA binding protein (ACBP), the distributional patterns of DBI and its colocalization with FABPs suggests its involvement in the absorption and metabolism of lipid in the epithelia of the digestive tract.

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