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.     

Release of lysosomal enzymes in Candida albicans phagocytosis by rat peritoneal macrophages

The present paper reports the in vitro release of lysosomal enzymes in the supernatant of cultures of rat peritoneal macrophages, with the addition of Candida albicans cells. Macrophages were taken from the rat peritoneal cavity 72 hr after non-specific activation with Brain-Heart-Infusion (B.H.I.) broth containing 10% proteose-peptone No. 3. They were then cultured in Parker medium No. 199 (TC 199). After 24 hr a suspension of Candida albicans cells, in a determined concentration, was added to the peritoneal macrophage cultures. At that time, and during pre-determined periods, the following enzymes in the culture supernatants were studied using colorimetric methods: -glucuronidase, -galactosidase and acid phosphatase. It is concluded that, under identical conditions, the release of -galactosidase and acid phosphatase is higher than for -glucuronidase. The release rate of all three enzymes is the highest at a 6 hr incubation period, after which, a gradual decrease leads the rate down to 50% at 24 hr.     

A study of the strength and stability of gas vesicles isolated from a blue-green alga

Summary Acid phosphatase was studied by means of electron microscope cytochemistry in glutaraldehyde-fixed myxamoebae of Dictyostelium discoideum grown on dead bacteria. The enzyme activity was localized to the digestive vacuoles in vegetative as well as in aggregating cells. Biochemical experiments showed that the enzyme was not inactivated by fixation in 2% purified glutaraldehyde.Abbreviations used NPP p-nitrophenyl phosphate - NP p-nitrophenol - GP -glycerophosphate - glc-6-P glucose-6-phosphate - Pi orthophosphate     

Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting gene transfer efficiency during early transformation steps

The factors influencing transfer of an intron — containing -glucuronidase gene to apple leaf explants were studied during early steps of an Agrobacterium tumefaciens-mediated transformation procedure. The gene transfer process was evaluated by counting the number of -glucuronidase expressing leaf zones immediately after cocultivation, as well as by counting the number of -glucuronidase expressing calli developing on the explants after 6 weeks of postcultivation in the presence of 50 mg/l kanamycin. Of three different tested disarmed A. tumefaciens strains, EHA101(pEHA101) was the most effective for apple transformation. Cocultivation of leaf explants with A. tumefaciens on a medium with a high cytokinin level was more conducive to gene transfer than cocultivation on media with high auxin concentrations. Precultivation of leaf explants, prior to cocultivation, slightly increased the number of -glucuronidase expressing zones measured immediately after cocultivation, but it drastically decreased the number of transformed calli appearing on the explants 6 weeks after infection. Other factors examined were: Agrobacterium cell density during infection, bacterial growth phase, nature of the carbon source, explant age, and explant genotype.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - CaMV35S 35S RNA of cauliflower mosaic virus - EDTA ethylenediaminetetraacetate - FeNaEDTA ethylenediaminetetraacetate ferric-sodium salt - GusA -glucuronidase - gusA ß-glucuronidase gene of Escherichia coli - gusA-intron ß-glucuronidase gene containing an intron in the coding region - IBA indole butyric acid - 2iP N6-2-isopentenyl adenine - NAA naphthaleneacetic acid - nptII neomycinphosphotransferase II gene - X-Gluc 5-bromo-4-chloro-3-indolyl ß-D-glucuronide     

Semi-constitutive expression of an Arabidopsis thaliana α-tubulin gene

In Arabidopsis tissues, the pool of tubulin protein is provided by the expression of multiple -tubulin and -tubulin genes. Previous evidence suggested that the TUA2 -tubulin gene was expressed in all organs of mature plants. We now report a more detailed analysis of TUA2 expression during plant development. Chimeric genes containing TUA2 5-flanking DNA fused to the -glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Second-generation progeny of regenerated plants were analyzed by histochemical assay to localize GUS expression. GUS activity was seen throughout plant development and in nearly all tissues. The blue product of GUS activity accumulated to the highest levels in tissues with actively dividing and elongating cells. GUS activity was not detected in a few plant tissues, suggesting that, though widely expressed, the TUA2 promoter is not constitutively active.     

β-Glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells

In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are β-glucuronidase (β-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that β-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas β-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, β-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that β-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed. This study was funded by the University of the Basque Country through a grant to Consolidated Research Groups. U.I. is a recipient of a pre-doctoral fellowship from the Basque Government.     

Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

Acid hydrolases in the perinuclear secretion granules of the rat preputial gland. A light and electron microscope study

Synopsis Four acid hydrolases in the secretory cells and the sebum of the preputial sebaceous gland of the rat were incestigated cytochemically. A strong -glucuronidase activity was found to occur in the matrix of the perinuclear secretion granules, whereas the granule crystalloids were unreactive. The distribution of acid phosphatase at the light microscope level was similar, though the intensity of the reaction was lower and the number of positive granules smaller. By electron microscopy, the final reaction product of acid phosphatase occurred in patches at the periphery of the granule matrix, as well as in the vesicles adjoining the Golgi stacks, from which the perinuclear granules seemed to arise. In the sebum, the two hydrolases occurred in the background material between the unstained crystalloid masses. There was noN-acetyl--glucosaminidase or aryl sulphatase activity in the gland. The perinuclear granules appear to be secretory lysosomes which, after discharge from the disaggregating cell, release their acid hydrolases into the sebum.     

Distribution of acid phosphatase and β-glucuronidase in the hypoplastic cerebellum of jaundiced Gunn rats

Summary Activities of acid phosphatase and -glucuronidase in the cerebella of young jaundiced (j/j) and non-jaundiced (j/+; control) Gunn rats were studied with the enzyme histochemical method. The cerebellum of j/+ rats showed high acid phosphatase activities in Purkinje cells and neurons in the cerebellar nuclei. In j/j rats, a number of neurons were lost and numerous microglialike cells with a high acid phosphatase activity appeared in the hypoplastic cerebellum. Although -glucuronidase activity was rarely detected in the control cerebellum, a high enzyme activity was observed associated with microglialike cells in j/j rats. The present results provide a cytological basis for the reported differential increase in the activities of these lysosomal enzymes in the j/j rat cerebellum.     

Ultrastructural localization of esterase and acid phosphatase in digestive gland cells of fed and starvedCepaea nemoralis (L.) (Mollusca: Helicidae)

Summary The ultrastructural localizations of thiolacetic acid esterase, indoxyl acetate esterase and acid -glycerophosphatase have been studied in the digestive gland cells of fed and starvedCepaea nemoralis. In fed snails the major localization of all three enzymes was in the green granule vacuoles of digestive cells. In addition, the cytoplasm of calcium cells and the Golgi apparatus and GERL (?) of all cell types were acid phosphatase positive. Many digestive cells of starved snails showed a similar enzyme distribution to that found in fed snails but other digestive cells showed a very high cytoplasmic activity of all three enzymes. It is suggested that these cells are in the process of autolysis. New light is also thrown on the process by which food is transported from the digestive gland lumen to the phagosomes of digestive cells.     

Histochemical studies on the distribution of β-glucuronidase and succinic dehydrogenase in young and old spinal ganglion cells of the rat

Summary The present study compares the distribution of -glucuronidase and succinic dehydrogenase in young and old spinal ganglion cells of rat. In young cells there are indications of cyclic activity of these enzymes, i.e., in some stages there are perinuclear concentrations of the enzymes, at other times -glucuronidase and succinic dehydrogenase are uniformly distributed throughout the cytoplasm. These stages have been discussed with the identical distribution of mitochondria. However, in old spinal ganglion cells both -glucuronidase and succinic dehydrogenase become mainly concentrated in the pigment areas, suggesting thereby their possible role in the production of pigment, through the medium of the mitochondria.     

A histochemical study of lysosomal enzymes in the retina of the rat

Summary Sections of retinas from albino and pigmented rats were studied histochemically by the naphthol and lead methods for lysosomal enzymes (acid phosphatase, aryl sulfatase, glucosaminidase and -glucuronidase). Activity of the enzymes studied (except -glucuronidase) is demonstrable in granules which by their staining properties are identified as lysosomes. The matrix of the lysosome stains positively in PAS preparations and is diastaae resistant. The organelles are distributed mainly in the pigment epithelium, outer limiting membrane, inner nuclear layer, ganglion layer and inner limiting membrane of the retina.This investigation was supported by a generous grant from the Nuffield Foundation.     

The structure of the basement membrane of human lymph node high endothelial venules: An ultrastructural, histochemical and immunocytochemical study

Summary The structure of the basement membrane of the high endothelium of reactive human lymph nodes was investigated by techniques selective for carbohydrates (periodic acid-Schiff; critical electrolyte concentration staining with Alcian Blue; lectin histochemistry), specific proteins (immunohistochemistry for laminin and fibronectin) and by conventional techniques of light and transmission electron microscopy. Adjacent small lymphocytes were assigned to B and T cell subsets by use of monoclonal antibodies and they were analysed for non-specific esterase,-glucuronidase,-N-acetylglucaminidase and proteolytic activities. The basement membranes were shown to be distinctive and to contain three layers, of differing laminin, glycosaminoglycan and glycoprotein oligosaccharide content. Certain lymphocytes (probably T) contained enzymes potentially able to degrade some components of these basement membranes.     

The relation between pituitary gland and thyroid growth during the lifespan of the annual fish Cynolebias whitei and Nothobranchius korthausae: gonadotropic and thyrotropic cells

Summary In the annual cyprinodont Cynolebias whitei the cell types responsible for the increase of pituitary growth at the onset of maturation and for pituitary hyperplasia in old specimens were identified as gonadotropic cells and thyrotropic cells, respectively. The gonadotropic cells showed a high affinity to anti-carp -GTH serum, both at light- and electron-microscopical levels. The allometric relation of total gonadotropic cell volume to body length, determined for fish from six weeks up to six months of age, showed no inflections. Therefore pituitary growth in maturing fish may be partly a result of proliferation of gonadotropes, although gonadotropic cells do not contribute to pituitary hyperplasia in old fish. Thyrotropic cells showed a weak affinity to anti-carp -GTH serum at light-microscopical level. Under the electron microscope thyrotropic cells displayed signs of activation in maturing fish and signs of proliferation in old fish. The allometric relation of thyroid gland volume to body length paralleled that of pituitary volume to body length. Histologically the thyroid gland showed signs of inactivity in adult fish and of hyperplasia in old fish. The possibility, that gonadal maturation, pituitary thyrotropic activity, and growth of the thyroid in maturing fish are related through the inhibitory action of gonadal steroids on thyroid hormone release, is discussed. Pituitary hyperplasia in old fish is the result of proliferation of thyrotropic cells. Similar hyperplasia of pituiary and thyroid glands was observed in old Nothobranchius korthausae.     

Developmental regulation of expression of the malate synthase gene in transgenic plants

The cucumber malate synthase (MS) gene, including 1856 bp of 5 non-trnascribed sequence, has been transferred into Petunia (Mitchell) and Nicotiana plumbaginifolia plants using an Agrobacterium binary vector. The transferred gene is found in variable copy number in different transformants, and is stably transmitted in each case as a single Mendelian character. Transgene mRNA accumulates in the seedling during the first three days of germination, then declines in amount as the cotyledons emerge from the seed. The decline is more pronounced in light-grown seedlings than in dark-grown seedlings. Expression of the MS transgene is also detected at a low level in petals of transformed Petunia plants. In these respects the pattern of MS gene expression is similar in cucumber and in trnasformed plants, showing that the transferred DNA fragment contains a functional MS gene. A 1076 bp fragment of 5 sequence was linked to the -glucuronidase reporter gene and transferred into Nicotiana, where it was shown to direct temporal and spatial patterns of expression similar to that of the complete MS gene. However, histochemical localisation of -glucuronidase activity demonstrated that the chimaeric gene is expressed not only in cotyledons of transgenic plants, but also in endosperm and some hypocotyl cells during early germination. The relevance of these findings to the control of malate synthase gene expression is discussed.     

Lysosomal changes in the response of molluscan hepatopancreatic cells to extracellular signals

Summary Marine mussels (Mytilus edulis, a bivalve mollusc) are increasingly used as environmental sentinels in pollution biomonitoring. Pathological reactions of the lysosomal system in hepatopancreatic cells have proven to be sensitive bioindicators of pollutant effect. However, if such reactions are to be used as biomarkers, then they must be clearly distinguishable from any hormonally-induced changes linked to normal seasonal activity such as the reproductive cycle. The aim of the present study was to test the effects of several cell-to-cell signalling compounds on the lysosomes of the hepatopancreatic digestive cells.In vitro incubation of tissue slices showed that epinephrine, acetylcholine and prostaglandin F₂ reduced lysosomal membrane stability and latency of -N-acetylhexosaminidase and -glucuronidase. These results indicate the presence of cell surface receptors for all three hormones. The observed changes in lysosomal fragility were less pronounced than those induced by pollutants and when considered together with other published data, indicate that hormonal regulation of digestive cell lysosomes is unlikely to pose a problem for the use of lysosomal reactions as biomarkers for chemical insult.     

Stable transformation of the food yam Dioscorea alata L. by particle bombardment

Summary A biolistic particle gun was used to deliver genetic material into intact yam cells. Cultured suspension cells of D. alata were bombarded with microprojectiles coated with pBI221.2 DNA and histochemical assays were carried out to show transient GUS expression in bombarded cells. Stably transformed D. alata cells were recovered from cultured cells after bombardment with microprojectiles coated with pRT99gus harbouring both the nptII and uidA genes. Bombarded cells were selected on a medium containing geneticin (G418). Two months after bombardment, calli resistant to G418 were assayed for GUS expression. There was a 100% correlation between resistance to G418 and GUS expression. From these calli, four cell lines were established and GUS activity in each line was determined fluorometrically. The use of a specific GUS inhibitor showed that the GUS activity was due to the introduced uidA gene rather than to any intrinsic GUS-like activity originating from the plant. Incorporation of the introduced DNA into the plant genomic DNA was confirmed by Southern analysis.Abbreviations GUS -glucuronidase - MU 4-Methylumbelliferone - MUG 4-Methylumbelliferyl--D-glucuronide - PVP Polyvinylpyrrolidone - SDS Sodium dodecyl sulphate - TAE Tris-acetate-EDTA buffer - X-Gluc 5-Bromo-4-chloro-3-indolyl--D-glucuronide     

Cytochemical markers of bladder carcinogenesis

Summary Enzyme cytochemical and immunocytochemical techniques at the light and electron microscope levels were used to study the distribution of potential markers of chemical transformation in rodent bladders. In rat tumours induced byin vivo treatment with methylnitrosourea, alkaline phosphatase localization was normal on the external surface of the plasma membranes of some cells but abnormal in others where reaction product was seen only on intracellular membranes. 5-Nucleotidase localization was abnormal in all cells, being seen on endoplasmic reticulum and nuclear membranes only, while in normal bladders only ectoenzyme localization was seen. Heterogeneity of alkaline phosphatase and 5-nucleotidase localization was seen on the plasma membranes of these tumours after 15 days in organ culture. Some cells produced enzyme and others did not; in other cells only parts of the membrane reacted heavily, while other regions were negative.In transformed cell cultures and tumours of mouse bladder derived byin vitro treatment of explants with dimethylbenz (a) anthracene, a bimodal pattern of alkaline phosphatase localization was seen. Cells had either normal ectoenzyme reaction product or abnormal intracellular membrane reaction product. 5-Nucleotidase and ADPase were lost after transformation while cAMP-phosphodiesterase was retained as an ectoenzyme. Mg.ATPase and a cAMP-independent, calcium-insensitive protein phosphatase were induced in transformed cell cultures. An epithelial antigen was detected in the cytoplasm of both normal and transformed cells associated with reticular cytoplasmic ground substance, plasma membrane vesicles and cytoskeletal elements.     

Transient expression of the β-glucuronidase gene introduced into barley coleoptile cells by microinjection

A -glucuronidase gene was introduced directly into barley (Hordeum vulgare L. cv. Kobinkatagi) coleoptile cells by microinjection and transient expression of the gene was examined. Inner epidermis tissue of coleoptiles was excised and injected with plasmid DNA, pBI221, carrying cauliflower mosaic virus 35S promoter, -glucuronidase gene, and a nopaline synthase polyadenylation region. Histochemical assay for -glucuronidase production showed positive enzyme activity only in coleoptile cells injected with plasmid DNA. Expression of the -glucuronidase gene was examined chronologically using honogenates of injected coleoptile tissues. Glucuronidase activity first appeared after 6 hr, reached the maximum level 24 hr after injection, and decreased afterwards. These results suggest that microinjection of coleoptile tissues may be a useful approach for the genetic engineering of Gramineae plants in which protoplast regeneration is difficult.     

On the nature of the opaque and translucent enamel regions of some macropodinae (Macropus giganteus,Wallabia bicolor and Peradorcas concinna)

Summary Teeth of three macropod species, M. giganteus, W. bicolor and P. concinna, have been studied using the techniques of light microscopy, scanning- and transmission-electron microscopy and hardness measurement. Light microscope observations showed that the teeth of these species had a translucent enamel region close to the dentine and an outer opaque enamel region at the tooth's surface. These regions were not related to the presence or absence of tubules which are a characteristic feature of marsupial enamel. Hardness tests showed that the opaque enamel was softer than the translucent enamel. Scanning electron microscope observations revealed that there was no correlation between any particular prism packing or orientation and the opaque and translucent enamel regions. Transmission electron microscope observations showed that the translucent enamel region consisted of well defined prisms and well packed, lath-like crystals, whereas the opaque enamel was disrupted by voids (which ranged in size from enlarged micropores to about 2 m in diameter in extreme cases) between crystals and some randomly oriented, loosely packed crystals. This disruption within the opaque enamel region was more common at prism boundaries but pockets of disrupted enamel were also found within prisms and interprismatic regions. The opacity of the enamel was caused by scattering of light from the voids. The ultrastructure of the opaque enamel region indicated that this region was hypomineralized; hardness tests and polarized light microscope observations were consistent with these results.