Calbindin D28k-like immunoreactivity during the formation of the enamel-free area in the rat molar teeth

Previous studies have demonstrated the presence of calbindin D28k in the ameloblasts derived from the inner enamel epithelium. The occlusal surfaces of the rodent molars partly lack the enamel covering, which is referred to as enamel-free area (EFA). In the present study, we compared the immunohistochemical localization of calbindin D28k-like immunoreactivity (CB-LI) in the cells at the EFA (EFA cells) and ameloblasts of the rat molar teeth at the light microscopic level. CB-LI was strong in the ameloblasts of the presecretory through the protective stages, while it was faint at the late secretory to transitional stages. However, some mature ameloblasts lacked the immunoreactivity. On the other hand, the majority of EFA cells showed distinct polarization and elongation that were absent in few cells at the early stage of EFA formation. At all stages, the EFA cells adjacent to the ameloblasts showed CB-LI, however, some cells adjacent to the mature ameloblasts lacked the reaction. Intensive CB-LI was demonstrated in EFA cells at the reduced enamel epithelium. These immunohistochemical findings suggest EFA cells have cytochemical properties similar to those of ameloblasts.     

Calbindin D28k‐like immunoreactivity during the formation of the enamel‐free area in the rat molar teeth

Previous studies have demonstrated the presence of calbindin D28k in the ameloblasts derived from the inner enamel epithelium. The occlusal surfaces of the rodent molars partly lack the enamel covering, which is referred to as enamel‐free area (EFA). In the present study, we compared the immunohistochemical localization of calbindin D28k‐like immunoreactivity (CB‐LI) in the cells at the EFA (EFA cells) and ameloblasts of the rat molar teeth at the light microscopic level. CB‐LI was strong in the ameloblasts of the presecretory through the protective stages, while it was faint at the late secretory to transitional stages. However, some mature ameloblasts lacked the immunoreactivity. On the other hand, the majority of EFA cells showed distinct polarization and elongation that were absent in few cells at the early stage of EFA formation. At all stages, the EFA cells adjacent to the ameloblasts showed CB‐LI, however, some cells adjacent to the mature ameloblasts lacked the reaction. Intensive CB‐LI was demonstrated in EFA cells at the reduced enamel epithelium. These immunohistochemical findings suggest EFA cells have cytochemical properties similar to those of ameloblasts. Anat Rec 258:384–390, 2000. © 2000 Wiley‐Liss, Inc.     

Ultrastructural Localization of Endothelin-1 in Nonneoplastic, Hyperplastic, and Neoplastic Adrenal Gland

Endothelin (ET)-1 is a 21-amino acid peptide with potent vasopressor and vasoconstrictive properties. Biochemical and recent histochemical studies have shown that this peptide is present in human adrenal cortex. This study was intended to determine ET-1 immunoreactivity in human adrenal cortex and cortical adenoma, and hyperplasia ultrastructurally. Light microscopical examination confirmed recent findings of ET-1 immunoreactivity in the three cortical zones (but not in the medulla) as well as in cortical adenoma and cortical hyperplasia. The immunoreactivity in the cortex and adenoma appeared in the cytoplasm in the form of vacuolar structures and grains. Focally, the cell membranes also showed immu-noreactive staining. Electronmicroscopical investigation revealed ET-1 immunoreactive products adjacent to the outer surface of the membrane of lipid bodies, in mitochondria and rough endoplasmic reticulum and focally on the cell membrane, but no immunolabeling was seen in the medulla. The localization of ET-1 in the endoplasmic reticulum indicates that this peptide is synthesized in the cortical cells. Its localization in the membrane of the lipid bodies and in the mitochondria suggests that it takes part in synthesis and/or secretion of steroid hormones. The focally immunolabeled cell membranes may be dependent on ET-1 binding to ET receptors.     

Morphological and functional responses of rat Leydig cells to a prolonged treatment with human chorionic gonadotropins

The morphological and functional responses of rat Leydig cells to a 3- and 6-day treatment with human chorionic gonadotropins (hCG) (10 IU/kg/day) were investigated by morphometric and radioimmunological techniques. hCG-administration induced a notable time-dependent enhancement in the steroidogenic capacity and growth of Leydig cells; this last was almost exclusively due to hypertrophy (and not to hyperplasia). The volume of mitochondrial and peroxisome compartments, as well as the surface area per cell of mitochondrial cristae and smooth endoplasmic reticulum (SER) were significantly increased after hCG treatment, and showed a highly significant positive linear correlation with both basal and stimulated testosterone production by isolated Leydig cells of the contralateral testis. Also the volume of nuclei and lipid-droplet compartment and the surface area per cell of Golgi apparatus displayed a notable hCG-induced rise, but they did not correlate with testosterone secretion. These findings suggest that, in addition to mitochondria and SER, in which the enzymes of steroid synthesis are located, peroxisomes are also specifically involved in the secretory activity of rat Leydig cells.     

IQ-ArfGEF/BRAG1 is a guanine nucleotide exchange factor for Arf6 that interacts with PSD-95 at postsynaptic density of excitatory synapses

ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates dendritic differentiation possibly through the organization of actin cytoskeleton and membrane traffic. Here, we characterized IQ-ArfGEF/BRAG1, a guanine nucleotide exchange factor (GEF) for Arf6, in the mouse brain. In vivo Arf pull down assay demonstrated that IQ-ArfGEF/BRAG1 activated Arf6 more potently than Arf1. IQ-ArfGEF/BRAG1 mRNA was abundantly expressed in the brain with higher levels in forebrain structures and cerebellar granule cells. In hippocampal neurons, IQ-ArfGEF/BRAG1 mRNA was localized not only at neuronal cell bodies but also at dendritic processes, indicating its dendritic transport and localization. Immunoprecipitation and in vitro binding experiments revealed that IQ-ArfGEF/BRAG1 formed a protein complex with N-methyl-d-aspartate (NMDA)-type glutamate receptors through the interaction with a postsynaptic density (PSD) scaffold protein, PSD-95. Immunohistochemical analysis demonstrated that IQ-ArfGEF/BRAG1 was localized preferentially at the postsynaptic density of asymmetrical synapses on dendritic spines, but was lacking at GABAa receptor-carrying inhibitory synapses. Taken together, IQ-ArfGEF/BRAG1 forms a postsynaptic protein complex containing PSD-95 and NMDA receptors at excitatory synapses, where it may function as a GEF for Arf6.     

Testicular feminization of the mouse: paucity of peroxisomes in Leydig cell of the testis.

The testes of mice with the X-linked testicular feminization (Tfm/y hermaphrodite) mutation are very small and cryptorchid. The spermatogenesis in the adult Tfm/y hermaphrodite mouse testes is arrested, and the testosterone biosynthesis is significantly reduced due to deficiency of 17-keto-steroid reductase, the enzyme essential for the conversion of androstanedione to testosterone. In this study the distribution of peroxisomes in the Leydig cells of adult Tfm/y hermaphrodite mice was investigated because of the suggestion that peroxisomes may participate in lipid metabolism and/or androgen biosynthesis is steroidogenic cells. Aldehyde-fixed testicular tissue of Tfm/y hermaphrodite mice was processed for the cytochemical localization of peroxisome catalase to facilitate identification of these organelles in the Leydig cells. Testes from Blo/y and CSa strain normal adult mice served as controls. Testicular Leydig cells of normal adult Blo/y and CSa mice contained abundant smooth endoplasmic reticulum (SER) in the form of complex interconnected tubules and double-walled membranous vesicles. Numerous peroxisomes, often in continuity with SER channels or in close association with lipid droplets, were observed in Leydig cells of normal males. In contrast, the peroxisomes in the Leydig cells of adult Tfm/y hermaphrodite mouse testes were either undiscernible or greatly reduced in number and size. SER in these cells was sparse, whereas mitochondria were numerous. In addition, abundant clusters of lipid droplets were encountered in a majority of Leydig cells of Tfm/y hermaphrodite mouse testes. Peroxisome and SER paucity in Leydig cells of Tfm/y hermaphrodite mice may be a reflection of reduced testosterone production. Whether excessive accumulation of lipid in the Leydig cells of Tfm/y hermaphrodite mouse testes is due to reduced utilization of cholesterol for the biosynthesis of testosterone or to impaired lipid metabolism due to reduction in peroxisome population in these cells remains to be ascertained.     

Chronic treatment with sildenafil stimulates Leydig cell and testosterone secretion

The phosphodiesterase type 5 (PDE5) inhibitor, Sildenafil, is a novel, oral treatment approach for pulmonary hypertension. As Leydig cells present PDE5, this study was conducted to investigate the effects of the chronic treatment with Sildenafil (25 mg/kg) on male Swiss Webster mice steroidogenesis. After a 4-week long experimental design, Leydig cells were analysed by morphological and immunocytochemical procedures. Serum testosterone was assayed by radioimmunoassay. Leydig cells presented noteworthy ultrastructural alterations, such as a vesicular smooth endoplasmic reticulum, large vacuoles scattered through the cytoplasm, enlarged mitochondria with discontinue cristaes and whorle membranes with vesicles at the periphery, which are typical characteristics of an activated steroid-secreting cell. Important immunocytochemical labelling for steroidogenic acute regulatory protein, cytochrome P450 side-chain cleavage enzyme and testosterone were detected in isolated Leydig cells. In addition, Sildenafil-treated mice showed significant increased levels of total testosterone. The results obtained in the present study are consistent with the hypothesis that the accumulation of cyclic guanosine monophosphate by PDE5 inhibition could be involved in the androgen biosynthesis stimulation. Important clinical implications of hormonal disorders should be taken into account for patients with pulmonary hypertension.     

Localization of phospholipase C β3 in the major salivary glands of adult mice

Phospholipase C (PLC)β has a role in saliva secretion by controlling intracellular Ca²⁺ via its product, IP₃. The present study was attempted to localize PLCβ isoforms in mouse salivary glands in situ. A single major band was detected for PLCβ3 in immunoblots of the parotid and sublingual glands (PG, SLG), while no such band was seen in the submandibular gland (SMG). No bands were detected for PLCβ1 or 4 in the three glands. In immuno-light microscopy of PG and SLG, substantial immunoreactivity for PLCβ3 was seen in the cytoplasm including the plasmalemma of almost all ductal cells, while no distinct immunoreactivity was discerned in most acinar cells except for sublingual demilune cells. Numerous ductal cells exhibited higher immunoreactivity for PLCβ3 in their apical/supranuclear cell domain including the plasmalemma than in the basal/infranuclear domain, indicating an apico-basal polarity. In immuno-gold electron microscopy of PG ducts and SLG ducts and demilunes, most gold particles were found in association with plasma membranes as well as various intracellular membranes, most of which formed small oblong or flattened vesicles and vacuoles. A few particles were seen without association with any membranous structures. The present finding supports the previous physio-pharmacological result that Ca²⁺-signaling proteins as well as initial intracellular Ca²⁺ changes occur in the apical cell domain including the plasma membranes of the exocrine cells.     

Substance P- and neuron-specific enolase-like immunoreactivity of rodent Leydig cells in tissue section and cell culture.

Comparative immunocytochemical studies concerning the presence of a neurotransmitter (substance P), and a marker of neuroendocrine cells (neuron-specific enolase), in the Leydig cells of 3 mammalian species (golden hamster, guinea pig, and rat) were carried out on tissue sections and cell cultures. Substance P(SP)-like immunoreactivity (-LI) was found to be present in both fetal and adult generation of Leydig cells in hamster and guinea pig, while neuron-specific enolase (NSE)-LI was detected in Leydig cells of the 3 species at all stages studied: fetal, neonatal and adult. In primary cultures of Leydig cells isolated from adult hamster testes, SP- and NSE-LI was also established. This result was considered as an indirect evidence for the synthesis of the substances under study by the steroidogenic cells of the testis. A comparison of these results with data obtained in vivo suggests that Leydig cells may be related to the APUD- or the diffuse neuroendocrine system.     

Distinct roles of Rab11 and Arf6 in the regulation of Rab11-FIP3/arfophilin-1 localization in mitotic cells

Rab11 family interacting protein 3/arfophilin-1 is a dual effector of Rab11 and Arf6 and exhibits Rab11-dependent localization to recycling endosomes in interphase. Furthermore, FIP3 undergoes dynamic redistribution to the intercellular bridge during cytokinesis. However, regulation of FIP3 redistribution and its local function by Rab11 and Arf6 has remained controversial. In this study, we developed a procedure for detecting endogenous FIP3, Arf6, and Rab11 and determined that FIP3 is localized near the intercellular bridge during cytokinesis, and to the Flemming body (the midbody) immediately before abscission; Rab11 is localized near the intercellular bridge, but not to the Flemming body; and Arf6 is localized to the Flemming body. Time-lapse analyses showed that FIP3 is transported to the intercellular bridge during cytokinesis, together with Rab11; before abscission, FIP3 becomes localized to the Flemming body, where Arf6 is already present. After abscission, FIP3 and Arf6 are incorporated into one of the daughter cells as a Flemming body remnant. Based on these observations, we propose that FIP3 localization to recycling endosomes in interphase and their transport to the intercellular bridge during cytokinesis depend on Rab11, and targeting of FIP3-positive endosomal vesicles to the Flemming body in the abscission phase depends on Arf6.     

Electron microscopic cytochemical localization of a basolateral calcium adenosine triphosphatase in vitamin D replete chick enterocytes

A cytochemical technique for the electron microscopic localization of calcium adenosine triphosphatase (Ca-ATPase) was utilized to localize this enzyme in the enterocytes of rachitic and vitamin D-replete chicks. In animals treated with cholecalciferol (CC, vitamin D3), an electron-dense reaction product was located along the basolateral membranes of the absorptive cells within 72 hr after injection. Similarly, a reaction product was identified in association with the basolateral membranes within 24 hr after injection of 1,25-dihydroxycholecalciferol, the active metabolite of vitamin D. A microvillar reaction product was not seen in either of these two groups. Electron-dense reaction products were also seen in association with mitochondria and scattered throughout the cytoplasm of these enterocytes. The Ca-ATPase reaction product was dependent upon the presence of medium calcium and substrate (ATP), was inhibited by vanadate, and was heat labile. In the rachitic animals, a reaction product indicative of Ca-ATPase activity was not seen in association with either the basolateral membranes or the mitochondria. These data appear to indicate that an energy-requiring calcium-activated membrane pump plays a role in the flux of calcium across the enterocytes of the small intestine.     

The functional development of Leydig cells in a marsupial

Leydig cells are the major source of androgen in the male mammal. We describe here for the first time the development of the Leydig cell in a macropodid marsupial, the tammar wallaby, Macropus eugenii. Leydig cells are first recognized morphologically 2 days after birth with the appearance of lipid droplets in the cytoplasm of certain interstitial cells. Lipid content closely matches the steroid content of the developing testis and marks the maturation of the steroid synthesis pathway in the tammar testis. Morphologically mature Leydig cells, marked by distinct mitochondria with tubular cristae and an extensive anastomosing network of smooth endoplasmic reticulum, are developed by day 10 after birth - the time of peak testosterone content in perinatal tammar testes. The volume percentage of each cell type in the testis does not change over time so the growth of each cellular component keeps pace with growth of the whole testis. There was no morphological or quantitative evidence of a change from one population of Leydig cells to another in the tammar testis as has been reported in several other species including the rat, mouse and human. Maturation of the testis is also marked by the development of tight junctions between the cell membranes of adjacent Sertoli cells. These appear around day 30 after birth and coincide with the onset of mitotic arrest in male germ cells. Overall, the development of the Leydig cell in the tammar wallaby follows a similar pattern to that seen in other mammals, although the start of Leydig cell differentiation is, like many other organ systems in marsupials, post natal, not fetal and there appears to be only a single population of Leydig cells.     

Aromatase in human endometrial carcinoma and hyperplasia. Immunohistochemical, in situ hybridization, and biochemical studies.

The expression of P450 aromatase and other steroidogenic enzymes were evaluated in 42 endometrioid endometrial carcinomas, 23 endometrial hyperplasias, and 7 normal endometrial specimens. These findings were correlated with clinicopathological findings to elucidate the possible biological significance of in situ estrogen production in the development of human endometrial carcinoma. Only weak aromatase immunoreactivity was observed in vascular walls and myometrial cells. In contrast, strong aromatase stromal immunoreactivity was observed in 28 of 42 (66.7%) endometrial carcinomas. However, no stromal immunoreactivity was seen in normal or hyperplastic endometrial specimens. Immunoreactivity in the carcinoma stromal cells was significantly increased at sites of invasion. These aromatase-positive cells were immunohistochemically negative for other steroidogenic enzymes involved in estrogen biosynthesis. In situ hybridization studies revealed aromatase mRNA hybridization signals in stromal cells but not in carcinoma cells. The distribution of aromatase mRNA correlated well with the immunohistochemical localization of aromatase enzyme. Quantitation of aromatase activity demonstrated 8.75 +/- 2.75 pmol/hour/mg of protein for endometrial carcinomas (22 specimens) and 0.98 +/- 1.95 pmol/hour/mg of protein for normal endometrial specimens (4 specimens). Aromatase activity was found in both estrogen receptor-positive and -negative endometrial carcinomas. Aromatase did not vary with respect to the menopausal status of patients with endometrial carcinoma. These results suggest that estrogen is produced in situ in endometrial carcinoma but not in benign endometrial lesions. Such locally synthesized estrogen may act on carcinoma cells in a paracrine fashion to promote tumor growth. Additional investigations are necessary, but increased aromatase expression in the stromal cells of endometrial carcinoma may therefore play an important role in the development of human endometrioid endometrial carcinoma.     

The changed immunolocalization of START-domain-containing 6 (StarD6) during the development of testes in rat perinatal hypothyroidism

Thyroid hormones have an essential role in maintaining the normal developmental structure of testes during the neonatal stage. START-domain-containing 6 (StarD6) is exclusively expressed in germ cells during spermatogenesis; however, its biological role in rat perinatal hypothyroidism is not clear. After hypothyroidism was induced by daily administration of 0.05% 6-propyl-2-thiouracil (PTU), the pattern of StarD6 immunolocalization was examined from gestation day 15 to postnatal day 49. In normal rats, the labelling of StarD6 was confined to the germ cells from the third-week postpartum. In contrast, its immunoreactivity in hypothyroidal rats was not detected until the fourth-week postpartum. The immunolocalization pattern of StarD6 differed from that of normal adult rats during the seventh-week postpartum. StarD6 was clearly detected in the Leydig cells of the perinatal hypothyroid rats from the fifth-week postpartum. Therefore, StarD6 may play a pivotal role, not only in the spermatogenesis of normal rats, but also in the steroidogenesis of Leydig cells under perinatal hypothyroidism.     

Ultrastructural localization of the NADPH-diaphorase activity in the Leydig cells of aging mice

Recently, it has been shown that nitric oxide may inhibit the Leydig cell steroidogenesis. The present paper describes, by means of NADPH-diaphorase histochemistry, the ultrastructural localization of the enzyme nitric oxide synthase in the Leydig cells of young adult and aging mice. In the young adult mice, the enzymatic reaction was mainly located in the mitochondria and in some clustered cistern? of the smooth endoplasmic reticulum. The nuclear envelope was faintly labeled. In the aging mice, most Leydig cells showed an enhanced enzymatic reaction. Labeled mitochondria were increased in number, and labeled areas of the smooth endoplasmic reticulum were more numerous and extended. In addition, a strong enzymatic reaction was recognized in the nuclear envelope. We conjecture that the impaired steroidogenesis observed in the testis of aging mammals might, at least in part, depend on the increased nitric oxide production in the Leydig cells. Accepted: 15 January 2001     

Ultracytochemistry of 3β-hydroxysteroid dehydrogenase in Leydig cell precursors and vascular endothelial cells of the postnatal rat testis

 In the biosynthesis of steroid hormones 3β-hydroxysteroid dehydrogenase (3β-HSD) is a key enzyme. The present report describes the subcellular localization of the enzyme in the fetal-type Leydig cells, the fibroblast-like precursors of adult-type Leydig cells and in endothelial cells of interstitial capillaries. Histochemical methods for light microscopy and ultracytochemical methods for electron microscopy were used on rat testes of postnatal day 15. 3β-HSD reactivity was located at subcellular levels by means of the ferricyanide method. A specific, distinct localization of reaction product in the form of copper ferrocyanide precipitates was observed on the membranes of the smooth endoplasmic reticulum not only in the fetal-type Leydig cells and the fibroblast-like precursors of adult-type Leydig cells, but also focally in the endothelial cells of interstitial blood capillaries. Topographically, the 3β-HSD-positive precursors were most often found in the outer layer of the boundary tissue and surrounding interstitial blood vessels. The capillaries with 3β-HSD-positive endothelial cells were usually located in the vicinity of 3β-HSD-positive Leydig cells. For the first time, 3β-HSD has been located at the subcellular level in precursors of adult-type Leydig cells and focally in capillary endothelial cells associated with them. Due to the close association between 3β-HSD-positive vascular endothelial cells and Leydig cells a paracrine relationship between the two cell types may be involved in the acute regulation of steroidogenesis by blood-borne luteinizing hormone. Accepted: 10 March 1998     

Epithelial cytodifferentiation and extracellular matrix formation in enamel-free areas of the occlusal cusp during development of mouse molars: Light and electron microscopic studies

Mandibular first molars in mice ranging in age from 18 days prenatal to 5 days postnatal were used for light and electron microscopic examinations of the enamel-free area (EFA) during development of the occlusal cusp (mesiobuccal cusp). Notable morphological changes in the inner enamel epithelium and the cells of the stratum intermedium were observed. At prenatal age of 18 days, the inner enamel epithelium of the EFA (EFA epithelium) was composed of a layer of columnar cells and covered by the cells of the stratum intermedium. Two days after birth, the EFA epithelium was made up largely of preameloblasts, with mitochondria located in the proximal side of the cells toward the stratum intermedium. The cells of the stratum intermedium were irregularly shaped, with wide intercellular spaces between them. At a postnatal age of 3 days, most of the EFA epithelial cells resembled maturation-stage ameloblasts, being short and columnar in shape and having nuclei located in their proximal side. Distal cell membranes were folded, and mitochondria were scattered throughout the cytoplasm. In 4-day-old mice, the EFA epithelium was found to be formed of short columnar or cuboidal cells with distinct intercellular spaces. The cells of the stratum intermedium could no longer be detected, and cells of the EFA epithelium could not be distinguished from those of the stellate reticulum. Odontoblasts of the EFA were arranged and polarized parallel to the basal lamina, and odontoblastic processes extended toward the cusp tip. The orientation of thin and thick collagen fibers within predentin and dentin was also parallel to the basal lamina. Even after dentin mineraliza tion, disrupted basal lamina and long, aperiodic, fine fibrils were found between the epithelium and the dentin. Following the disappearance of the basal lamina and fine fibrils, stippled material and crystals appeared on the dentin surface. The mineralized matrix, which x-ray microanalytical energy peaks identified as containing calcium and phosphorus, was continuous with enamel in the distal slope of the cusp at the cusp tip. Thus, the inner enamel epithelium of the EFA differentiated into secretory cells capable of enamel-like matrix formation. In conclusion, development of EFA in mouse molars seems to be a system suitable for use in studying both the involvement of extracellular matrix, including basal lamina constituents, in ameioblast differentiation, and the role of the cells of the stratum intermedium, together with the ameloblasts, in comprising a single functional unit responsible for enamel formation.     

Utility of immunostaining for S-100 protein subunits in gonadal sex cord-stromal tumors, with emphasis on the large-cell calcifying Sertoli cell tumor of the testis

This study concerns the immunohistochemical localization of S-100 alpha, S-100 beta, and whole brain S-100 (wbS-100) in testicular large-cell calcifying Sertoli cell tumor (LCCSCT). We examined 8 LCCSCTs (7 benign and 1 malignant), 6 Sertoli cell tumors not otherwise specified (SCTs-NOS), 6 Leydig cell tumors (LCTs), 5 ovarian Sertoli-Leydig cell tumors (SLCTs), and 7 gonadoblastomas (GBLs). The 8 LCCSCTs showed immunoreactivity for S-100 alpha, S-100 beta, and wbS-100. Five of the 6 LCTs and the Leydig cell components in the ovarian SLCTs stained positively for S-100 alpha and wbS-100 but were negative for S-100 beta. SCTs-NOS and the Sertoli cell components in the SLCTs occasionally showed focal and weak/moderate positivity for S-100 alpha, S-100 beta, and wbS-100. Sex cord cells of the GBLs were positive for S-100 beta and wbS-100 and negative for S-100 alpha. Germ cell elements of the GBLs were negative for S-100 alpha, S-100 beta, and wbS-100. In nonneoplastic testicular parenchyma adjacent to the above-mentioned tumors, there was S-100 alpha reactivity in Leydig cells, rete testis, and a few Sertoli cells. S-100 beta reactivity was seen in a few Sertoli cells, Schwann cells, and some endothelial cells. WbS-100 reactivity was present in Leydig cells, a few Sertoli cells, rete testis, Schwann cells, and some endothelial cells. The results indicate that S-100 alpha and S-100 beta can potentially be used as immunohistochemical markers for LCCSCT, especially when differentiating it from LCT, which may mimic LCCSCT on routine histopathology. Although the biological significance of both S-100 subunits expression in LCCSCT remains unknown, these notable calcium-binding proteins may be associated with the characteristic calcification in LCCSCT through regulation of calcium levels in the tumor cells.     

Localization of phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K) α, β, γ in the three major salivary glands in situ of mice and their response to β‐adrenoceptor stimulation

Phosphatidylinositol 4‐phosphate 5‐kinase (PIP5K), which is composed of three isozymes (α, β and γ), catalyzes the production of phosphatidylinositol bisphosphate (PIP2). This phospholipid functions in membrane trafficking, as an anchor for actin cytoskeletons and as a regulator of intramembranous channels/transporters. It is also a precursor of such second messengers as diacylglycerol, inositol triphosphate and phosphatidylinositol (3,4,5)‐triphosphate. In the present study, the expression and localization of endogenous PIP5Ks were examined in the three major salivary glands of young adult mice in situ. In western blotting of normal control glands, immunoreactive bands for individual PIP5Ks were detectable, with the highest density in the parotid gland and the weakest density in the submandibular gland. In immuno‐light microscopy under non‐stimulated condition, weak immunoreactivity for PIP5Kα was confined to the apical plasmalemma in parotid, but not sublingual or submandibular, acinar cells. Immunoreactivity for PIP5Kβ was weak to moderate and confined to ductal cells but not acinar cells, whereas that for PIP5Kγ was selectively and intensely detected in myoepithelial cells but not acinar cells, and it was weak in ductal cells in the three glands. In western blot of the parotid gland stimulated by isoproterenol, a β‐adrenoceptor agonist, no changes were seen in the intensity of immunoreactive bands for any of the PIP5Ks. In contrast, in immuno‐light microscopy, the apical immunoreactivity for PIP5Kα in parotid acinar cells was transiently and distinctly increased after the stimulation. The increased immunoreactivity was ultrastructurally localized on most apical microvilli and along contiguous plasma membrane, where membranous invaginations of various shapes and small vesicles were frequently found. It was thus suggested that PIP5Kα is involved in post‐exocytotic membrane dynamics via microvillous membranes. The present finding further suggests that each of the three isoforms of PIP5K functions through its product PIP2 discretely in different cells of the glands to regulate saliva secretion.