Cysteine proteases comprising the caspase family have been considered one of the major executioners of programmed cell death. However, detailed analyses of the programmed cell death of developing motoneurons in mice following the genetic deletion of two key caspases, casp-3 and casp-9, and in the chick embryo following treatment with caspase inhibitors, indicate that normal amounts of cell loss occur although the death process is delayed. Motoneurons undergoing programmed cell death without caspase activities exhibit a nonapoptotic morphology in which nuclear changes such as chromatin condensation are absent or reduced and which exhibit extensive cytoplasmic vacuolization such as is rarely observed in degenerating control neurons. These results suggest that caspases are involved in, but are not indispensable for, the developmental death of motoneurons, and that one function of caspases may be to facilitate the removal of cells that are destined to die. Possible alternative caspase-independent pathways for the programmed death of motoneurons are discussed.

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The structure of G-bands in human metaphase chromosomes was analyzed by comparison between light microscopic and atomic force microscopic (AFM) images of the same chromosomes. G-bands of the chromosomes were made by trypsin treatment followed by staining with a Giemsa solution. The banded chromosomes examined by light microscopy were dried either in air or in a critical point-drier, and observed by non-contact mode AFM. Air-dried chromosomes after G-band staining showed alternating ridges and grooves on their surface, which corresponded to light-microscopically determined G-positive and G-negative bands, respectively. At high magnification, the G-positive ridges were composed of densely packed chromatin fibers, while the fibers were loose in the G-negative grooves. Fibers bridging the gap between sister chromatids of a mitotic pair were often found, especially in the G-positive portions. These findings suggest that the G-banding pattern reflects the high-order structure of human metaphase chromosomes.

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Phosphatase activities associated with the intestinal brush border membrane (BBM) of the rat were examined histochemically in relation to the characteristic environment of the intestine, where luminal pH fiuctuates drastically between alkaline and acid pH ranges. Special attention was given to intestinal alkaline phosphatase (IALP) and phytase on the BBM. Whole body fresh-frozen sections of young rats and their rapidly frozen and freeze-substituted small intestines, embedded in Technovit 7100, were processed for the histochemical demonstration of phosphatase activity at three different pH values (9.2, 7.3, and 5.2), representing the deviation of luminal pH in vivo. Either an azo-dye method or lead-salt method was employed using naphthol AS-MX phosphate and ATP as substrate, respectively. With the azo-dye method, intense phosphatase reactions were demonstrated along the BBM at all three pH ranges. Phosphatase reactions of the BBM at pH 9.2 and 7.3 were abolished by L(+)-phenylalanine, heat pre-treatment, and EDTA chela-tion although some reaction remained at pH 7.3 after the treatment with EDTA or L(+)-phenylalanine. Phosphatase reactions of the BBM at pH 5.2 were resistant to L(+)-phenylalaniue, L(+)-tartrate, PCMB and EDTA chelation, implying that the characteristics of the enzyme responsible for phosphohydrolysis at acid pH values differed from those at higher pH values. The lead-salt method in which ATP was used as substrate revealed intense reactions - which were dependent on Mg⁺⁺ and stimulated by Ca⁺⁺ and resistant to L(+)-phenylalanine - to be localized along the BBM at alkaline and neutral pH values, but not at acid pH values. In vitro experiments showed progressive hydrolysis of naphthol AS-MX phosphate by purified phytase at pH 5.2, in a dose-dependent manner, and suggested the possible involvement of phytase in the phosphatase reactions of the BBM at acid pH.
These data indicate that the phosphatase reactions at alkaline and neutral pH values, associated with the BBM of the rat intestine, represent IALP and Mg⁺⁺/Ca⁺⁺-ATPase, while those at acid pH appear to correspond to phytase activity, something which has not been demonstrated by histochemical methods despite the availability of extensive data based on biochemical analyses.

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Responses of odontoblasts to cavity preparation in rat molars were investigated by immunocytochemistry for heat shock protein (Hsp) 25. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin. Confocal microscopy of Hsp 25-immunostained and rhodamine-labeled sections revealed that the immunoreactive odontoblasts were intensely labeled for phalloidin at the periphery of their cytoplasm and throughout their processes, but the reaction for phalloidin was limited within the inner half of the dentin. Cavity preparation caused an edematous reaction between the injured odontoblasts and predentin as well as a beaded swelling and successive destruction of the odontoblast processes. Immediately after cavity preparation, the odontoblasts beneath the edematous lesion showed an immunoreactivity for Hsp 25, which subsequently disappeared completely from the pulp-dentin border by 12 h after the operation. However, round cells without apparent cytoplasmic processes continued to be immunoreactive, suggesting the survival of a part of the odontoblasts against preparation stimuli. Numerous phalloidin-reactive but Hsp 25-immunonegative cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, probably categorized in a lineage of immunocompetent cells. By postoperative 72 h, newly differentiated odontoblasts with Hsp 25-immunoreactivity were arranged at the pulp-dentin border. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts, and suggest that this protein is a useful marker substance for differentiated odontoblasts.

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The expression in various cell types of peripheral tissues of glial fibrillary acidic protein (GFAP), first discovered as an intermediate filament specific for astrocytes, remains controversial owing to numerous reports of a wide distribution for GFAP-immunoreactivity in various cells. The present study employed immunohistochemistry to investigate the precise expression of GFAP in the dental pulp and trigeminal ganglion of adult rats and wild-type mice as well as GFAP-knockout mice. The exhibition of GFAP-immunoreactivity in the trigeminal ganglion was further examined by a reverse transcription polymerase chain reaction (RT-PCR) technique, and in situ hybridization histochemistry using a specific cRNA probe prepared by us. The immunoreaction for GFAP was recognizable in the axons, Schwann cells, and the fibroblasts in the dental pulp of rats and wild-type littermate mice. However, mice with null mutations in the GFAP gene remained immunoreactive for GFAP in all these locations. Intense GFAP-immunoreactivity was found in a small number of satellite cells in the trigeminal ganglion in all animals examined in this study. RT-PCR analysis demonstrated bands for the GFAP gene corresponding to the length expected from the primer design in the samples of trigeminal ganglion and dental pulp. In situ hybridization histochemistry also showed intense signals for GFAP mRNA in some satellite cells of the trigeminal ganglion, but never in the neurons. These data suggest that the GFAP-immunoreactive molecules in the pulpal axons and fibroblasts react non-specifically with the polyclonal antibody and are probably a closely related type of intermediate filament.

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This paper reports on how lymphatic vessels and their smooth muscle cells develop in the diaphragm of postnatal rats. Lymphatic endothelial cells in the diaphragm were labeled by an intraperitoneal injection of DiI-labeled acetylated low-density lipoprotein (DiI-ac-LDL). During postnatal week 1, DiI-ac-LDL was detected in many free cells in addition to distinct endothelial cells that formed lymphatic vessels. Occasionally, saccular lymphatics isolated from previously formed lymphatics were recognized; these were referred to as lymphatic islands. The DiI-ac-LDL-labeled free and lymphatic endothelial cells showed immunoreactivity for CD 34 and Flt-4, but most of them did not express either OX 62 or ED 1 immunoreactivity, with only some showing ED 1 immunoreactivity. This suggests that most of the DiI-ac-LDL-labeled elements were lymphatic endothelial cells, and that some were macrophages. After postnatal week 1, the DiI-ac-LDL positive cells were restricted to lymphatic vessels. Until postnatal week 6, Iymphatic vessels increased as the diaphragm enlarged. Towards the end of postnatal week 2, free cells expressing α-smooth muscle actin (α-SMA) immunoreactivity increased in the diaphragm, and some of these were in contact with lymphatics. A coarse plexus of smooth muscle cells surrounding the lymphatic vessels first appeared at postnatal week 2, and this plexus became denser with age. Our findings indicate that lymphatic vessels are formed not only by sprouting from previously formed lymphatic vessels but also by migrating endothelial cells, and that smooth muscle cells may be differentiated from mesenchymal cells to form a plexus surrounding the lymphatic vessels.

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The process of lymphangiogenesis was studied in the muscle coat of the rat small intestine by light and scanning and transmission electron microscopy; identification of lymphatic vessels was made by 5’-nucleotidase staining.
  Light and scanning electron microscopy demonstrated that the intramuscular lymphatic network formation, which started only postnatally, was attributable to the vascular sprouting of slender lymphatic endothelial projections and to a splitting of the vessels, causing intervascular meshes of various sizes. The growing lymphatics were consistently closed by the endothelial cells, which were characterized by an abundance of cell organelles and prominent cytoplasmic processes. The cells often revealed close contacts with the processes of developing smooth muscle cells in the jejunal muscle coat, suggesting a possible role for the latter cells in the guidance of the lymphatic extension.
  The present study is the first to suggest the closed nature of lymphatics persisting throughout their development, even at the initial stage of lymphangiogenesis.

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The basal lamina of high endothelial venules (HEVs) in human palatine tonsils was intensely stained with cationic colloidal iron at pH 1.5 and with aldehyde fuchsin. This basal lamina exhibited a thick and double- or triple-layered structure forming small compartments, in which many lymphocytes were aligned. Digestion with hyaluronidase or collagenase eliminated both the colloidal iron and aldehyde fuchsin stainings of the basal lamina of HEVs. Treatment with chondroitinase ABC reduced colloidal iron staining, but did not interfere with the aldehyde fuchsin staining. Digestion with neuraminidase, keratanase, or heparitinase did not eliminate either the cationic colloidal or the aldehyde fuchsin staining. Digestion with neuraminidase reduced the colloidal iron staining on the luminal surface coat of the HEV. Electron microscopy of ultrathin sections revealed that cationic colloidal iron particles were deposited on the basal lamina of the HEV. The basal laminae of ordinary blood vessels were thin and single-layered, and stained only weakly with cationic colloidal iron. The present study suggests that negatively charged sites in the basal lamina of HEV derive mainly from a proteoglycan complex containing hyaluronic acid and chondroitin sulfate, which firmly binds collagen. This topochemical feature is suggested to be involved in the fascilitating migration of lymphocytes after passage through the endothelial layer.

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Mucous cells have been known to occur in the terminal portions of the parotid gland in a few species of mammals during a limited period of their development. The aim of this study was to examine the occurrence and features of mucous cells in the parotid gland of the infant Japanese macaque.
  Light microscopy revealed that mucous cells in the macaque parotid gland were present in the terminal clusters and acini at postnatal day 15, were less prevalent at day 30, and continued to decrease in number over 3 months. Mucous cells were no longer recognized in the parotid gland in 6-month-old macaques.
  Electron microscopy showed that the mucous cells contained electron-lucent secretory granules and bipartite or tripartite secretory granules. By 3 months of age, there was a scarcity of mucous cells and a concomitant increase in transitional cells. These transitional cells were intermediate in structure between mucous and serous cells, and contained three types of granules: electron-lucent, bipartite or tripartite, and electron-dense. None of the cells showed apoptotic figures.
  Lectin histochemistry indicated that the mucous cells in the early postnatal period had sugar residues identical in nature to those seen in the granules from mature serous cells in the glands of 3-month-old macaques.
  Immunohistochemistry using an antibody against human α-amylase showed a weakly positive reactivity in the secretory granules of the mucous cells, starting from day 15. In the transitional cells, the electron-dense granules showed a stronger immunoreactivity than either the electron-lucent granules or the heterogeneously structured granules. These results suggest that the secretory granules of mucous cells have characteristics in common with those of serous cells, and that during the transitional period the mucous granules change from the initial electron-lucent to hetorogenous forms, finally becoming the electron-dense granules. The mucous cells in the parotid gland of the juvenile Japanese macaque are therefore suggested to be converted into serous cells.

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