The time during which beta-ecdysone is required for the differentiation in vitro and in situ of wing imaginal discs of Drosophila melanogaster.

The time during which β-ecdysone is required for the apolysis and imaginal differentiation of wing discs of Drosophila both in vitro and in situ has been examined, and it is concluded that β-ecdysone is required as a sustained stimulus rather than as a trigger for differentiation. These results are compared with the requirement for β-ecdysone for the puffing of salivary gland polytene chromosomes during the prepupal stage (Richards, G. P., 1976, Develop. Biol.48, 191–195). It is suggested that imaginal discs and larval salivary glands require different exposures to β-ecdysone to fulfill their developmental commitments and that the drop in β-ecdysone titer during the early prepupal stage, which is necessary for the subsequent puffing of the polytene chromosomes, plays little or no part in imaginal disc differentiation.     

Morphofunctional changes of salivary glands of female ixodid ticks of subfamilies Ixodinae and Amblyomminae (Acari: Ixodidae) during feeding and their significance

Using light and electron microscopy, a dynamics of functioning of salivary glands of female ticks of the genus Ixodes was studied. Comparative analysis of morphofunctional changes and role of the salivary gland secretions of females of subfamilies Ixodinae and Amblyomminae during feeding was performed on the basis of literature data and our own studies.     

Xylose-linked proteoglycan synthesis does not have a primary role in the control of secretory cell differentiation in salivary glands

Xylose-linked proteoglycans, particularly chondroitin sulfate proteoglycan, have been shown to play a significant role in the regulation of salivary gland morphogenesis. The purpose of this study was to determine if xylose-linked proteoglycans are involved in the regulation of differentiation of salivary gland secretory cells. Embryonic rat submandibular salivary gland rudiments were cultured for 120 hr in the presence or absence of 0.75 to 1.0 mM p-nitrophenyl-beta-D-xylopyranoside (beta-D-xyloside), an inhibitor of xylose-linked proteoglycan assembly. beta-D-Xyloside has been shown to block submandibular gland morphogenesis (Thompson and Spooner, 1982). In the present study glandular morphogenesis was blocked in 93.3% of the rudiments cultured in the presence of beta-D-xyloside. However, secretory cell differentiation was observed in 71.4% of those rudiments in which morphogenesis had been inhibited. Biochemical evaluation confirmed that xylose-linked proteoglycan assembly had been inhibited by xyloside. These results indicate that while xylose-linked proteoglycans play a significant role in the control of salivary gland morphogenesis these molecules are not primary regulators for secretory cell differentiation within developing salivary glands.     

Developmental puffing activity in the salivary gland and Malpighian tubule chromosomes ofAcricotopus lucidus (Diptera,Chironomidae)

A detailed map of the salivary gland chromosomes ofAcricotopus lucidus is presented. Differences in puffing and developmental Puffing sequences of the three salivary gland lobes were investigated from mid fourth larval instar to pupation and compared with the puffing pattern of the Malpighian tubules. The intraglandular differentiation is quite extensive; the differences in the pattern of gene activity between the anterior lobe and the main and side lobes are as great as between the salivary gland and the Malpighian tubules. In the main and side lobes all developmental puffing changes proceed synchronously whereas in the anterior lobe both asynchronous and synchronous changes occur. In the anterior lobe the asynchronous regression of BR 3 and BR 4 is followed by a characteristic sequence of activation and inactivation of puffs.     

Functional transplantation of salivary gland cells differentiated from mouse early ES cells in vitro

Atrophy or hypofunction of the salivary gland because of aging or disease causes hyposalivation and has an effect on the quality of life of patients, for example not only dry mouth but deterioration in mastication/deglutition disorder and the status of oral hygiene. Currently conducted therapies for atrophy or hypofunction of the salivary gland in clinical practice are only symptomatic treatments with drugs and artificial saliva, and therefore it is preferable to establish a radical therapy. At this time, as a fundamental investigation, by co-culturing mouse early ES (mEES-6) cells with human salivary gland-derived fibroblasts (hSG-fibro), differentiation of mEES-6 cells to salivary gland cells has been attempted. Also, the possibility of cell engraftment was examined. After identifying the cells which were co-cultured with GFP-transfected mEES-6 cells and hSG-fibro, the cells were transplanted into the submandibular gland of SCID mice, and the degree of differentiation into tissues was examined. The possibility of tissue functional reconstitution from co-cultured cells in a three-dimensional culture system was examined. Our results confirmed that the co-cultured cells expressed salivary gland-related markers and had an ability to generate neo-tissues by transplantation in vivo. Moreover, the cells could reconstitute gland structures in a three-dimensional culture system. By co-culture with hSG-fibro, mEES-6 cells were successfully differentiated into salivary gland cells which were transplantable and have tissue neogenetic ability.     

Immunolocalization patterns of cytokeratins during salivary acinar cell development in mice

Embryonic development of the mouse salivary glands begins with epithelial thickening and continues with sequential changes from the pre-bud to terminal bud stages. After birth, morphogenesis proceeds, and the glands develop into a highly branched epithelial structure that terminates with saliva-producing acinar cells at the adult stage. Acinar cells derived from the epithelium are differentiated into serous, mucous, and seromucous types. During differentiation, cytokeratins, intermediate filaments found in most epithelial cells, play vital roles. Although the localization patterns and developmental roles of cytokeratins in different epithelial organs, including the mammary glands, circumvallate papilla, and sweat glands, have been well studied, their stage-specific localization and morphogenetic roles during salivary gland development have yet to be elucidated. Therefore, the aim of this study was to determine the stage and acinar cell type-specific localization pattern of cytokeratins 4, 5, 7, 8, 13, 14, 18, and 19 in the major salivary glands (submandibular, sublingual, and parotid glands) of the mouse at the E15.5, PN0, PN10, and adult stages. In addition, cell physiology, including cell proliferation, was examined during development via immunostaining for Ki67 to understand the cellular mechanisms that govern acinar cell differentiation during salivary gland morphogenesis. The distinct localization patterns of cytokeratins in conjunction with cell physiology will reveal the roles of epithelial cells in salivary gland formation during the differentiation of serous, mucous or seromucous salivary glands.     

Serial cultivation of epithelial cells from human and macaque salivary glands

Summary To study the regulation of human salivary-type gene expression we developed cell culture systems to support the growth and serial cultivation of salivary gland epithelial and fibroblastic cell types. We have established 22 independent salivary gland epithelial cell strains from parotid or submandibular glands of human or macaque origin. Nineteen strains were derived from normal tissues and three from human parotid gland tumors. Both the normal and the tumor-derived salivary gland epithelial cells could be serially cultivated with the aid of a 3T3 fibroblast feeder layer in a mixture of Ham’s F12 and Dulbecco’s modified Eagle’s media supplemented with fetal bovine serum, calcium, cholera toxin, hydrocortisone, insulin, and epidermal growth factor. Salivary gland epithelial cells cultured under these conditions continued to express the genes for at least two acinar-cell-specific markers at early passages. Amylase enzyme activity was detected in conditioned media from cultured rhesus parotid epithelial cells as late as Passage 5. Proline-rich-protein-specific RNAs were detected in primary cultures of both rhesus and human parotid epithelial cells. Neither amylase enzyme activity nor PRP-specific RNAs were detected in fibroblasts isolated from the same tissues. In addition, salivary gland epithelial cells cultured under our conditions retain the capacity to undergo dramatic morphologic changes in response to different substrata. The cultured salivary gland epithelial cells we have established will be important tools for the study of salivary gland differentiation and the tissue-specific regulation of salivary-type gene expression.     

Possible role of nitric oxide in radiation-induced salivary gland dysfunction

In this study, we developed a murine model of xerostomia to elucidate the mechanism of radiation-induced salivary gland dysfunction and determined the levels of nitric oxide (NO) in the salivary glands to assess its involvement in the salivary dysfunction induced by radiation. In addition, an inhibitor of NO synthesis was administered to the model in vivo, and its effect on saliva secretion was investigated. Salivary gland irradiation at a dose of 15 Gy caused a significant decrease in secretion compared to unirradiated salivary glands. There were no marked differences between the irradiated mice and unirradiated mice in water or food consumption or in body weight changes. The NO levels in the cultured salivary gland epithelial cells were increased by treatment with a combination of interferon gamma (Ifng), interleukin 1-beta (Il1b), and tumor necrosis factor alpha (Tnfa). Irradiation increased the NO level in the salivary gland tissue. The presence of N(G)-monomethyl-l-arginine acetate (l-NMMA), an inhibitor of NO synthesis, caused a decrease in the NO level in cultured salivary gland tissues after irradiation. Administration of l-NMMA to irradiated mice improved saliva secretion. These results suggest that excessive production of NO induced by radiation is involved in the formation of radiation-induced xerostomia. The finding that administration of an inhibitor of NO synthesis ameliorated the dysfunction of irradiated salivary glands indicates that NO plays a role as a mediator of the dry mouth symptoms that occur after irradiation.     

Influence of thyroid function upon "Substance P" induced secretion of saliva by submaxillary glands

The effects of changes in thyroid function on the action of "Substance P" upon the secretion of saliva by the submaxillary glands was studied in male Wistar rats, with parasympathetic decentralization on the left side. The dose-response curves to increasing doses of "Substance P" showed in hyperthyroid animals increased salivary secretion while in hypothyroid animals the dose-response curve to the drug was decreased. Every animal showed supersentivity to "Substance P" in the decentralized gland. The influence of changes in thyroid function in the denervated glands was the same as that in the unoperated side, increased salivary secretion in hyperthyroidism and decreased in hypothyroidism.     

THE EFFECT OF CHANGES IN THE RESPIRATORY METABOLISM UPON GENOME ACTIVITY : A Correlation between Induced Gene Activity and an Increase in Activity of a Respiratory Enzyme

The in vitro regression of experimentally induced chromosome puffs was investigated in explanted salivary gland chromosomes of Drosophila hydei. It was observed that the regression of the puffs 2-32A, 2-36A, 2-48C, and 4-81B is accelerated if substrates for the respiratory metabolism are supplied to the cells. A similar effect can be produced by addition of KCN or oligomycin to medium in which intact salivary glands are incubated. The acceleration of puff regression by these substances occurs not only if the puff-inducing stimulus is removed but as well under conditions in which the stimulus is maintained. Regression of the puffs 2-32A, 2-36A, and 4-81B is inhibited if cycloheximide is present in the incubation medium. Chloramphenicol has no effect on puff regression. Measurements on nicotinamide adenine dinucleotide-dehydrogenase activity in homogenates of salivary glands revealed an increase in enzyme activity of 41 %. Maximum increase is attained at 30 min after the induced puffs have reached their maximum size. The increase in enzyme activity does not occur if the glands are kept in a medium containing either actinomycin D or cycloheximide. Chloramphenicol does not inhibit the increase in enzyme activity. The possible relationship between puff activity and its control as a result of changes in the respiratory metabolism is discussed.     

Human salivary gland branching morphogenesis: morphological localization of claudins and its parallel relation with developmental stages revealed by expression of cytoskeleton and secretion markers

Development of salivary glands is a highly complex and dynamic process termed branching morphogenesis, where branched structures differentiate into mature glands. Tight junctions (TJ) are thought to play critical roles in physiological functions of tubular organs, contributing to cell polarity and preventing lateral movement of membrane proteins. Evidence demonstrated that claudins are directly involved in TJ formation and function. Using immunohistochemistry and immunofluorescence we have mapped the distribution of claudins-1, 2, 3, 4, 5, 7 and 11 and compared it with the expression of differentiation markers in human salivary glands obtained from foetuses ranging from weeks 4 to 24 of gestation. Expression of all claudins, except claudin-2 was detected in the various phases of human salivary gland development, up to fully mature salivary gland. The expression of all claudins increased according to the progression of salivary gland maturation evidenced by the classical markers-cytokeratin 14, cytokeratin low molecular weight, smooth muscle actin and human secretory component. Tight junction proteins-claudins appear to be important in the final shape and physiological functions of human salivary glands and are parallel related with markers of salivary gland differentiation.     

An indirect mechanism by which a protein from the male gonad hastens salivary gland degeneration in the female ixodid tick, Amblyomma hebraeum.

In the adult female tick, Amblyomma hebraeum Koch (Acari: Ixodidae), salivary gland degeneration is triggered by an ecdysteroid, provided the female is above a critical weight (approximately 300-400 mg). In mated females, salivary gland degeneration is virtually complete within 4 days of detachment from the host. In virgin females, salivary gland degeneration is delayed by 4 days. This delay can be reversed by the injection of a male reproductive tract homogenate directly into the hemocoel. In this study, we consider a possible mechanism of action for this "male factor." Once mated, male factor likely gains access to its target tissue(s) as a humoral factor. Male factor, however, appears not to act by sensitizing the salivary glands to the action of ecdysteroids. Instead, it appears to act by accelerating the appearance of ecdysteroids in the hemolymph.     

捕食性蝽类唾液腺解剖方法—以叉角厉蝽为例

唾液腺的解剖是研究捕食性蝽类唾液成分及功能的关键。以叉角厉蝽Eocanthecona furcellata为例,报道了解剖获得其完整唾液腺的方法。该方法与其他解剖方法相比明显不同之处在于使用镊子固定虫体,更有利于唾液腺的解剖与收集。通过拍照观察,叉角厉蝽的唾液腺由主腺、副腺、肺门和导管组成。主腺可分为主腺前叶和主腺后叶,它们之间通过肺门相连。主腺肺门处着生有两根导管,其中一根导管与口器相连,而另一根导管与副腺连接。本文描述的解剖方法以及叉角厉蝽唾液腺形态结构可用于指导解剖获得其它捕食性蝽类的唾液腺,进而提取唾液开展成分鉴定和功能研究。     

Branching morphogenesis of mouse salivary epithelium in basement membrane-like substratum separated from mesenchyme by the membrane filter

Branching morphogenesis of mouse salivary gland has been studied with organ-culture system. We developed a novel transfilter culture system for analyzing branching morphogenesis of the salivary epithelium. The submandibular salivary epithelium from early 13-day mouse fetus, clotted with Matrigel and separated from the mesenchyme by membrane filter, showed extensive growth and branching morphogenesis, morphological differentiation of lobules and stalks, and a typical cleft shape. The epithelium showed little growth and no branching without Matrigel clot or without the mesenchyme. This branching morphogenesis was induced even when the pore size of the filter was reduced to 0.05 microns. Use of type I collagen gel instead of Matrigel mostly induced incomplete morphogenesis with various histological abnormalities. These results suggest that the salivary epithelium can undergo branching morphogenesis in the absence of the mechanical action of mesenchymal cells although it needs an appropriate extracellular matrix and some mesenchymal factors transmitted through the filter.     

Human submandibular gland (HSG) cell line as a model for studying salivary gland Ca2+ signalling mechanisms

The human submandibular gland cell line (HSG) has been used as a model for studying the molecular mechanisms of salivary cells. The aim of this study was to investigate some aspects of salivary Ca2+ signalling. We focused on the presence and function of specific molecular markers of salivary cells to see whether this cell line retained normal salivary characteristics, despite the neoplastic changes. We detected the M3 acetylcholine receptor and intracellular salivary amylase mRNA with RT-PCR. Carbachol treatment caused a rapid, transient elevation of [Ca2+]i, showing that the cholinergic receptors are functional in HSG cells. Protein kinase C activation by phorbol-esther PMA, prior to carbachol treatment, inhibited the normal Ca2+ signalling pathway in HSG cells. Using selective antagonists, we also identified the dominant muscarinic receptor subtype M3 on HSG cells. We also observed that functional extracellular purinergic receptors were present on HSG cells and coupled to intracellular Ca2+ signalling. Our results suggested that the coupling mechanisms of these receptors remained relatively intact despite the neoplastic transformation. This enables us to use this cell line to model the role of muscarinic and purinergic control of salivary gland function, cell proliferation and differentiation.     

Caffeine-induced calcium release from the endoplasmic reticulum of acinar cells of the submandibular salivary gland

Ryanodine receptors (RyRs) play a key role in the generalization and spreading of calcium waves in excitable cells; however, the question of the existence of functionally active RyRs in nonexcitable cells demonstrating the capacity for exocytosis (e.g., salivary gland acini) remains open. We studied changes in the total amount of calcium stored in the endoplasmic reticulum (ER) of acinar cells of the submandibular salivary gland of rats and changes in the concentration of ionized Ca²⁺ inside the ER ([Ca²⁺]_ER) using, respectively, a metallochrome dye, arsenazo III, and a low-affinity fluorescent dye, mag-fura 2/AM. In permeabilized cells, caffeine caused dose-dependent decreases in the total amount of calcium and concentration of ionized calcium. The effective concentration of caffeine providing a 50% drop in the [Ca²⁺]_ER (EC₅₀) was, on average, 7.3 ± 1.1 mM. The caffeine-induced drop in the [Ca²⁺]^ER was insensitive to heparin; in addition, it was blocked by high concentrations (100 μM) of ryanodine, potentiated by ryanodine applied in mild concentrations (10 μM), and also demonstrated a bell-shaped dependence on the concentration of cytoplasmic Ca²⁺. Such peculiarities are typical characteristics of the RyR-mediated reaction. Therefore, functional RyRs whose activation results in a transient release of calcium from the ER are present in acinar cells of the submandibular salivary gland. Neirofiziologiya/Neurophysiology, Vol. 39, No. 2, pp. 107–112, March–April, 2007.     

Evaluation of the Effects of Quercetin on Damaged Salivary Secretion

With the aim of discovering an effective method to treat dry mouth, we analyzed the effects of quercetin on salivary secretion and its mechanism of action. We created a mouse model with impaired salivary secretion by exposure to radiation and found that impaired secretion is suppressed by quercetin intake. Moreover, secretion levels were enhanced in quercetin-fed normal mice. To elucidate the mechanisms of these effects on salivary secretion, we conducted an analysis using mouse submandibular gland tissues, a human salivary gland epithelial cell line (HSY), and mouse aortic endothelial cells (MAECs). The results showed that quercetin augments aquaporin 5 (AQP5) expression and calcium uptake, and suppresses oxidative stress and inflammatory responses induced by radiation exposure, suggesting that quercetin intake may be an effective method to treat impaired salivary secretion.     

Integrin expression in developing human salivary glands

The development and complete differentiation of salivary glands is a complex process that involves a large number of co-ordinated events. Little is known about the molecular basis for salivary gland development. However, we have reported previously that integrins appear to play a role. Integrins are heterodimeric transmembrane receptors consisting of one α and one β subunit that play a pivotal role in the interaction of cells with the extracellular matrix. Such interactions regulate the organisation of cells of tissues and organs during development as well as cell proliferation and differentiation. Using immunohistochemistry and Western and Northern blot analysis, we mapped the localisation and expression of integrins β1, β3 and β4 in human salivary glands obtained from foetuses ranging from weeks 4–24 of gestation and compared it with adult salivary glands. Integrin β1 first appeared during the canalisation stage and during the differentiation stage. A message first appeared at week 6 of development. The expression of β4 integrin protein and message was observed only in the late stage of differentiation. Integrin β3 was not detected in the developing glands; however, integrins β1, β3 and β4 were all expressed in adult salivary gland tissues. The data suggest that integrins, particularly β1, have a role to play in salivary gland development and differentiation.