Cytochemical demonstration of enzymatic activities of the parotid, submandibular, and sublingual glands of Praomys (Mastomys) Natalensis

Histochemical techniques were applied to salivary glands removed from adult multimmate rodents (Praomys) of either sex to detect and localize the following enzymatic activities: acid and alkaline phosphatase, arylsulphatase, ali-esterases, beta-glucuronidase, N-acetyl-betaglucosaminidase, and L-leucyl-aminopeptidase. No reaction was observed for alkaline phosphatase and glucuronidase. The glands reacted differently to the other enzymatic activities. Alkaline phosphatase and glucosaminidase were present only in one glandular type whereas arysulphatase and esterases were present in all types although demonstrating a variable staining intensity in different glands. Sharp differences in some enzymatic activities of the submandibular and parotid glands were related to the sex of the animal.     

Markers of cell death in salivary glands of males of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari, Ixodidae)

The salivary glands of Rhipicephalus sanguineus males at stages: unfed (control), at day seven post-attachment, and at days three and seven post-detachment from the host were examined using methods of enzymatic analysis and cell viability. At these stages of feeding, different staining patterns were observed in the cells of type IV, III, II and I acini, which were affected by degeneration in this sequence. Acid phosphatase reaction was inversely proportional to that of ATPase, while ATPase reaction was proportional to membrane integrity. Salivary gland cells of unfed males exhibited intact nucleus and plasma membrane, suggesting that the acid phosphatase detected may participate in the normal physiology of acini. In males at day seven post-attachment, intact membranes were observed in almost all types of acini, as well as stronger reaction for acid phosphatase, nuclear changes, and decrease in ATPase reaction, changes associated with the degenerative process. At days three and seven post-detachment degeneration progress, being observed loss of membrane integrity, nuclear changes, prominent decrease in ATPase reaction, and an increase in acid phosphatase reaction in the first case and a decreased of it at day seven post-detachment from the host. During cell death, alterations occurred in the following sequence: a) nuclear changes, b) loss of ATPase reaction, c) loss of integrity of the plasma membrane, and d) increase of acid phosphatase. The latter might be associated with the late degradation of cytoplasmic remnants, characterizing the process of cell death in glands of R. sanguineus males as atypical or non-classic apoptosis.     

Protein phosphatase activity is required for prothoracicotropic hormone-stimulated ecdysteroidogenesis in the prothoracic glands of the tobacco hornworm,Manduca sexta

The multiple phosphorylation of ribosomal protein S6 appears to be required for prothoracicotropic hormone (PTTH)-stimulated protein synthesis and ecdysteroidogenesis by the prothoracic glands of Manduca sexta. The present study investigated the role of protein phosphatase in these phenomena by analyzing the effects of pretreatment of prothoracic glands with the phosphatase inhibitors okadaic acid and calyculin A in both basal and PTTH-stimulated glands. Okadaic acid or calyculin A treatment enhanced ribosomal S6 phosphorylation in control glands to a level similar to that observed with PTTH-stimulated glands. This treatment also prevented S6 dephosphorylation but had no apparent synergistic effect on S6 phosphorylation in PTTH-stimulated glands. Most importantly, okadaic acid or calyculin A treatment inhibited, rather than augmented, ecdysteroidogenesis in both PTTH-stimulated and non-stimulated glands. The composite data suggest that protein phosphatase activity sensitive to okadaic acid or calyculin A is required for PTTH-stimulated ecdysteroidogenesis. © 1996 Wiley-Liss, Inc.     

AN ULTRASTRUCTURAL AND BIOCHEMICAL STUDY OF THE EFFECTS OF THREE INHIBITORS OF CHOLESTEROL BIOSYNTHESIS UPON MURINE ADRENAL GLAND AND TESTIS : Histochemical Evidence for a Lysosome Response

Triparanol and 20,25-diazacholesterol inhibit cholesterol biosynthesis and result in the accumulation of desmosterol. AY-9944, another inhibitor, produces an accumulation of 7-dehydrocholesterol. Adult male C3H mice receive one of these drugs intraperitoneally. Livers, adrenal glands, and testes from each drug group are excised, and portions of each are analyzed by a modified Liebermann-Burchard reaction for quantitation of sterols. Adrenals and testes are examined also by electron microscopy. Fine-structural localization of acid phosphatase has been studied in triparanol-treated adrenal glands. Biochemical analysis reveals that 14–64% of the sterols occurs as desmosterol or 7-dehydrocholesterol. Fine-structural alterations in the adrenal glands and testes from each drug group are essentially identical. The predominant cytological feature is the occurrence of increased numbers of pleomorphic, unit-membrane-limited, electron-opaque, cytoplasmic inclusions. Hence, the cellular modifications following triparanol administration are not unique, as has been suggested. They represent a generalized phenomenon, probably related to inhibition of cholesterol biosynthesis, which is an effect common to each drug. Lead phosphate reaction product (indicating acid phosphatase activity) is demonstrable within these membrane-limited cytoplasmic bodies, identifying them as morphological lysosomes. The utilization of a lysosomal mechanism in sterol-synthesizing cells, which are accumulating cholesterol intermediates, is discussed.     

Histochemistry of hydrolytic enzymes in resting submandibular glands of rabbits

Summary The distribution of several hydrolytic enzymes was investigated in rabbit submandibular glands at both the light and electron microscopical levels. Glands were fixed by either immersion or perfusion fixation with a variety of fixatives containing 1–2% glutaraldehyde and 2–4% formaldehyde in 0.1m cacodylate buffer at pH 7.2. Light microscopically, the acinar cells showed some staining for ATPase, acid phosphatase and nonspecific esterases but showed weak staining for thiamine pyrophosphatase. Acid phosphatase staining occurred most strongly in granular tubule cells. Staining for esteroproteases was confined to the periluminal rims of intercalary and striated ducts. Alkaline phosphatase was very sensitive to glutaraldehyde and was confined to myoepithelial cells.Electron microscopical observations revealed the presence of acid phosphatase reaction product in lysosomes, immature granules and in GERL-like structures, the last being much more conspicuous in the granular tubule cells. ATPase reaction product was localized to the basal and luminal plasma membranes and lumina of both acinar and granular tubule cells. The Golgi complex of these two types of cells exhibited only moderate amounts of reaction product for thiamine pyrophosphatase. Alkaline phosphatase activity, on the other hand, was exclusively localized to myoepithelial cells in their plasma membranes and sometimes in the nuclear envelope.     

Effect of juvenile hormone on acid phosphatase activity in six tissues of the milkweed bug

After juvenile hormone treatment on day of ecdysis, the haemolymph, salivary glands, gut, cuticle, testes, and fat body of the fifth instar male milkweed bug were assayed for acid phosphatase activity at daily intervals throughout the instar. Increased acid phosphatase activity after juvenile hormone treatment was found in the haemolymph at the beginning of the instar, in the haemolymph and salivary glands in the middle of the instar, and in the testes near the end of the instar. The significance of these findings is discussed.     

A cytochemical study of the leaf-gland enzymes of insectivorous plants of the genus Pinguicula

Summary Cytochemical methods have been used to study the distribution of acid phosphatase, esterase, ribonuclease, amylase and protease activity in the stimulated and unstimulated leaf glands of Pinguicula grandiflora, P. vulgaris, P. lusitanica, and P. caudata. Two gland types are present, stalked and sessile. The stalked glands bear a muco-polysaccharide secretion droplet, and are concerned with capture of the prey; the sessile glands are specialised for digestion. In unstimulated glands of both classes, acid phosphatase, esterase and ribonuclease activity is associated with the anticlinal walls of the head cells, which have a characteristic spongy inner surface, comparable with that of transfer cells. Acid phosphatase and esterase activity was also detected in the vacuoles of the head cells of the sessile glands. Substrate film tests showed that amylase is readily released from the stalked glands but not from the sessile ones, while in contrast proteolytic activity is mainly associated with the sessile glands.On stimulation by suitable nitrogenous materials, the glands begin to sectete fluid onto the leaf surface within 1 hr. During the process the enzymes held in the spongy walls are discharged, and activity is also lost from the intracellular sites in the sessile glands.Digestion on the leaf surface and resorption of the products has been followed autoradiographically after feeding of ¹⁴C-labelled protein. Within 2 hr, digestion products enter the leaf, and move towards the margin in the vascular system. Movement out of the leaf begins within 12 hr. Microautoradiographs showed a concentration of products around the bases of the sessile glands and in the cells of the gland head, showing that these glands are involved in resorption as well as secretion.A possible mechanism of gland function is discussed.     

Acid phosphatase activity in the larval salivary glands of developing Drosophila melanogaster.

Both the biochemical profile and the optical and fine structural localization of acid phosphatase activity in the larval salivary glands of developing Drosophila melanogaster is described. Biochemically, acid phosphatase shows peak activity in the glands of feeding larvae, followed by a marked decline. Directly preceding the onset of cell histolysis however, enzyme activity increases 1.5 fold and is maintained at this level. Histochemically, acid phosphatase activity initially appears as discrete point or lysosomal sources. As development proceeds, an intense and diffuse form of enzyme is seen, accompanying an extremely vacuolated cytoplasm. Ultrastructurally, the enzyme is located in lysosomes, Golgi elements, multivesicular bodies and both within, and on the extracisternal surface of the rough endoplasmic reticulum. This extracisternal or cytosolic form appears directly preceding cell lysis and eventually shows a comprehensive cellular distribution. Large numbers of acid phosphatase positive haemocytes are attached to the basal glandular surface at all developmental stages. In morphologically intact gland cells, discrete extracisternal enzyme activity appears associated with local areas of degradation.     

Death by apoptosis in salivary glands of females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

The salivary glands of females of the tick Rhipicephalus sanguineus at three feeding stages: unfed, engorged, and at day three post-engorgement, were subjected to cytochemical methods of enzymatic analysis and cell viability. Comparing glands at these stages, was observed distinct staining patterns in cells of different types of acini, specially in degenerating types III, II, I, which were affected in this sequence by cell death. This study also revealed changes in: nuclei, staining intensity for acid phosphatase and ATPase activities, and permeability of the plasma membrane. Acid phosphatase activity was inversely proportional to that of ATPase, while ATPase activity was always proportional to membrane integrity. The glands of unfed females exhibited high metabolic activity and cells with intact nucleus and plasma membrane, suggesting that the presence of acid phosphatase detected in these individuals may participate in the normal physiology of some acini, as they were not undergoing degeneration. In acini I and II of engorged females, we observed cells with intact membranes, as well as changes characterized by nuclear changes, decrease in ATPase activity, and stronger acid phosphatase activity. At day three post-engorgement, degeneration progressed to more advanced stages, loss of membrane integrity was observed in most cells (of some type I acini, most type II acini, and all type III acini), as well as prominent nuclear changes, decrease in ATPase activity, and intense acid phosphatase activity, resulting in apoptotic bodies. During the death of cells nuclear changes preceded cytoplasmic ones in the following sequence: nuclear changes, loss of ATPase activity, loss of integrity of the plasma membrane, increase in acid phosphatase activity, and formation of apoptotic bodies. The presence of acid phosphatase with a secondary role (late) during cell death, degrading final cell remnants, characterized this process in the glands of R. sanguineus females as atypical or non-classic apoptosis.     

Hormonal control of salivary gland degeneration in the ixodid tick Amblyomma hebraeum

Under normal circumstances, salivary glands of female ixodid ticks begin degenerating within hours of completing the blood meal. We have monitored cytological, functional and biochemical changes in the tissue which are diagnostic of the degenerative process. Although ultimately degeneration also befalls salivary glands of partially fed ticks removed prematurely from the host, the process is considerably delayed. When we transplanted salivary glands from partially fed ticks into the haemocoels of replete specimens, autolysis was induced in the donor tissue, whereas such was not the case when similar glands were transplanted to the haemocoels of other partially fed ticks. We thus suggest that a humoral factor is involved in postprandial resorption of the salivary glands. Succinate dehydrogenase activity decreases, and acid phosphatase activity increases in the salivary glands as a function of time post-engorgement. However, these enzyme assays are not sensitive enough to detect the earliest stages of autolysis.     

Ultrastructural cytochemistry of phosphatases in the ductal component of pleomorphic adenoma of human parotid and submandibular salivary glands

Summary Some ductal cells of pleomorphic adenomas showed evidence of secretory activity, with apical secretory granules, thiamine pyrophosphatase activity in the Golgi apparatus, and acid phosphatase activity in GERL-like structures and in immature secretory granules. Alkaline phosphatase activity was demonstrated rarely at the luminal plasma membrane and in intracellular vesicles, suggesting resorptive activity. ATPase reaction product was associated with contiguous surfaces of tumour cells, particularly of those cells adjacent to the basement membrane, these latter cells apparently differentiating in a different manner to the luminal cells. A comparison of luminal ductal cells of the tumours with normal salivary glands revealed most similarity with intercalary ductal cells.     

Fine structural and histochemical studies on salivary glands of Peripatoides novae-zealandiae (Onychophora) with special reference to acid phosphatase distribution

The Onychophora feed on small arthropods and produce saliva when ingesting prey. Although saliva undoubtedly helps to liquefy the food its constituents have not yet been fully described. The salivary glands, two long tubes of glandular epithelium, are known to secrete a powerful protease, however, besides other enzymes and mucus. In Peripatoides novae-zealandiae there are protein-secreting cells of three types, referred to here as columnar, cuboidal and modified cells, and mucus cells. The anterior two-thirds of the gland show most cell diversity, while the posterior region consists mainly of columnar cells. These are the most numerous elements overall and they probably secrete salivary protease. In thick resin sections the granules of all protein-secreting cells stain strongly with methylene blue. Those of columnar cells are markedly uneven in size and accumulate distally, eventually filling the cytoplasm. More proximal Golgi regions may be discernible. Mucus cells are all of one type and their secretion droplets are stained lightly by methylene blue. The electron microscope shows that distal microvilli, desmosomes and septate junctions are common to all gland cells. In columnar cells, secretory material is contributed by Golgi complexes and by rough endoplasmic reticulum. Early secretory vacuoles containing dense material are seen in the concavity of Golgi regions. They are precursors to larger condensing vacuoles whose contents have a more flocculent appearance, and which may attain 3–4 μm in diameter. These evolve into secretory granules, usually of uneven texture, which are up to 2–5 μm in diameter. Histochemical tests for acid phosphatase show moderate amounts of enzyme throughout the gland. In whole mounts and sections the strongest reaction is in a band of cuboidal cells along the anterior median border. Columnar cells show a diffuse cytoplasmic reaction towards the base and sometimes distal to the nucleus, and mucus cells may also react strongly round the nucleus. Cytoplasm near the lumen shows little reaction. The secretory granules do not appear to contain active enzyme. Under the electron microscope a positive reaction for acid phosphatase is seen in lysosomal derivatives near the base and lateral periphery of gland cells. These bodies are probably autophagic vacuoles and they may contain membranous whorls and possibly old secretion granules. Acid phosphatase is involved also in the elaboration of new secretory granules in both columnar and mucus cells. Dense reaction product is found in a system of interconnected tubules and cisternae near the innermost face of the Golgi complex, which is interpreted as GERL. Acid phosphatase is present in the peripheral zone of adjacent early secretory vacuoles, and interconnections occur between GERL and secretory vacuoles. It is suggested that GERL tubules containing the enzyme may fuse with early secretory vacuoles and release acid phosphatase at their periphery. The acid phosphatase reaction is negative in large condensing vacuoles and most secretory granules. These findings are consistent with what is known from mammalian cells, including those of salivary glands.     

Immunocytochemical localization of seminal proteins in salivary and lacrimal glands of the rat

Antibodies against 10 different secretory proteins from the accessory sex glands of the male rat were used for immunohistochemical studies of salivary and lacrimal glands from intact and castrated rats, at the light- and electron-microscopic levels. In the parotid gland, secretory acinar cells showed immunoreactivity with antibodies against prostatic binding protein, cystatin-related peptide and acid phosphatase (isoenzyme pI 8.0; 5.6) typical of ventral prostate, and seminal vesicle secretion VI. Western blotting analysis indicated that immunoreactivity against prostatic binding protein was attributable to a subunit, presumably C3. Acid phosphatase pI 5.6 showed a molecular weight of 66 kDa, which is at variance with the prostatic form. Immunoreactivity for secretory transglutaminase, derived from the coagulating gland, was restricted to myoepithelial and stromal cells. In castrated animals, the immunoreactivity of acinar cells was reduced to the background level, whereas stromal transglutaminase immunoreactivity was unaltered. The distribution pattern of immunoreactivity for the proteins mentioned was almost identical in the lacrimal gland. Significant differences were however observed in the immunoreactivity of the inframandibular gland, where serous glandular cells were non-immunoreactive for seminal proteins, with the exception of acid phosphatase isoenzyme pI 8.0. Granules present in the convoluted granular ducts were immunoreactive particularly for acid phosphatase (isoenzyme pI 5.6)but much less for cystatin-related peptide; immunoreactivity was reduced after castration. The straight portion of the inframandibular duct system was immunoreactive for transglutaminase, but no influence of castration was visible. The distribution of immunoreactivity for seminal proteins present in the salivary and lacrimal glands and the pronounced androgen-dependence of their expression point to functional relationships of the respective proteins at both glandular sites.     

Immunohistochemistry of acid phosphatase in the human prostate: normal and pathologic. Cytochemistry and biochemistry of acid phosphatases II

Three different antisera against human prostatic acid phosphatase were used for direct and indirect immunohistochemical demonstration of acid phosphatase in paraffin sections of infantile and adult normal, hyperplastic and carcinomatous prostatic tissue. All antisera were prepared in rabbits. Antiserum A was prepared from highly purified acid phosphatase extracted from autopsy specimens. Antiserum B was a concentrate of a commercial antiserum used in radioimmunoassay and was prepared from purified extracts of human seminal fluid. Antiserum C was a peroxidase-conjugated antiserum prepared from purified extracts of human seminal fluid. The specificity of the three antisera was compared using different immunohistochemical methods and tissues. It was comparably high in all three antisera which gave only slightly different staining results in prostatic tissue. The staining results in prostatic carcinoma were only dependent on the titer of the respective antiserum. Carcinomas with a cribriform growth pattern showed variable staining, but always had a positive immunoreactions, provided the titer of the antiserum was sufficiently high. Striking differences were observed in metaplastic, atrophic and hyperplastic prostatic epithelium. The most intense reaction was observed in atrophic glands: it was much less intense in hyperplastic and normal epithelium and negative or slightly positive in metaplastic epithelium.     

Ultrastructural localization of phosphohydrolases in gametes, zygotes and zoospores of Ulva mutabilis Foyn.

Gametes, zoospores, and zygotes of the multicellular, green alga Ulva mutabilis showed acid phosphatase reaction product in Golgi vesicles and on the membrane lining the vacuole. In addition gametes and zoospores showed enzyme reaction product on the entire surface membrane including the flagellar membrane. The surface membrane enzyme activity disappears from the zygote shortly after copulation and at the same time lysosome-like bodies start to appear in the cytoplasm. No alkaline phosphatase activity could be detected. The distribution of acid phosphatase is discussed in relation to the events taking place during and shortly after fertilization.     

HISTOPATHOLOGICAL AND HISTOCHEMICAL CHANGES IN HONEYBEE LARVAE (APIS MELLIFERA L.) AFTER INFECTION WITH BACILLUS LARVAE,THE CAUSATIVE AGENT OF AMERICAN FOULBROOD DISEASE

Morphological, histochemical and cytochemical changes were examined in honeybee larvae after infection with the bacterium Bacillus larvae. The results indicate cell necrosis in the midgut epithelium accompanied by increasing cell vacuolization and nuclear pyknosis following per os inoculation with B. larvae. Many autolysosomes were positive for acid phosphatase. Non-vacuolar acid phosphatase activity was also found in lysed cell compartments. No such activity was found in regenerative epithelial cells. Degradation of haemocytes, salivary glands and other tissues was also observed. Histochemical analyses after per cutaneous inoculation with B. larvae of three- and five-day-old honeybee larvae show intense non-vacuolar acid phosphatase activity followed by disintegration of infected salivary glands, epithelial cell cytoplasm and haemocytes.     

Histochemistry of the chick esophagus and trachea. II. Enzymes, nucleic acids, proteins, carbohydrates, and fats

Tissues of White Leghorn embryos of stages 17–45 and chicks of one day, two days, and three weeks of age were frozen, sectioned in a cryostat and, where appropriate, were fixed in cold calcium formol. Acid phosphatase, non-specific esterase, adenosine triphosphatase, 5-nucleotidase, non-specific glycerophosphatase, nucleotidediphosphatase, and glucose-6-phosphatase were localized in these tissues. Ribonucleic acid, acid mucopolysaccharides, triglycerides, and neutral fats were localized in tissues fixed with FAA and embedded in paraffin. Positive acid phosphatase reactions were obtained in the epithelium of the trachea and esophagus at all stages of development. 5-nucleotidase was found in the muscularis mucosae of the esophagus at all stages. Non-specific esterase appeared with histodifferentiation of the esophageal epithelium. Ribonucleic acid was localized in the basal regions of the epithelium. Mucous glands of the esophagus are rich in ribonucleic acid and acid phosphatase at all stages of development. With histodifferentiation and the onset of secretion of sulfated acid mucopolysaccharides, the glands and their ducts become highly reactive for adenosine triphosphatase and nucleotide-diphosphatase, indicating a role of these enzymes in secretion.     

Observations under the electron microscope on the localization of phosphatases in epithelial cells of Cowper's glands in rats

The positive results of the reactions for alkaline and acid phosphatases were obtained in epithelial cells of Cowper's glands in rats. Observations under the electron microscope allowed us to state that alkaline phosphatase was localized in the cell's areola, in membranes of smooth intraplasmic reticulum and in the basement membrane of the epithelium. The acid phosphatase was seen in primary lysosomes as well as in the secondary ones, which are seldom seen on unincubated specimens.     

Salivary glands of the cat: A histochemical study

Synopsis The submandibular, sublingual and parotid glands of the cat have been studied. Mucosubstance histochemistry demonstrated acidic mucosubstances with varying properties in the acini. Thiamine pyrophosphatase and nucleoside diphosphatase reaction products were seen with a Golgi-like appearance in acinar cells. Granules of acid phosphatase, -glucuronidase and E600-resistant esterase reaction products, presumably representing lysosomal enzyme activities, were seen in acinar and ductal cells. Diffuse acid phosphatase and -glucuronidase reaction products were seen in central cells of the submandibular acini, and diffuse non-specific esterase reaction product was seen in acinar and ductal cells. Arylamidase reaction product was associated with some acinar cells. Reaction product from a peroxidase technique was seen in demilunar cells of the submandibular acini, in parts of the sublingual acini, in parotid acini, and in ductal cells. Cytochrome oxidase and succinate dehydrogenase reaction products were seen most strongly in striated ducts, whereas NADH- and NADPH-diaphorase reaction products were seen at a high level throughout the ducts.     

Lysosomal enzymes in relation to the isoproterenol-induced secretory cycle in rat parotid gland

Two lysosomal enzymes, cathepsin D and acid phosphatase, were detected in significant amounts in the lysosome-containing subcellular fractions of rat parotid tissue and found to have dissimilar distributions in these fractions. The total levels of these enzymes were measured at various times throughout a complete secretory cycle induced synchronously by fasting rats overnight and administering isoproterenol at time zero. The results showed a 30% increase in cathepsin D activity in the glands by 10 h post-stimulation, and a 20% decrease in acid phosphatase activity 7 h after stimulation. These results suggest that there are cyclic changes in lysosomal enzymes during the secretory cycle of this gland, but that these changes are complex ones and cannot be related to specific cellular processes at this time.