Carbohydrate histochemistry of the epidermis of Natrix piscator during its sloughing cycle

Carbohydrate histochemistry of the scale and hinge epidermis of the chequered water snake, Nalrix piscator , throughout the sloughing cycle, has been described. The small amount of mucopolysaccharide present in the Oberhautchen, mesos layer, α-layer and β-Mayer (in its initial stage of differentiation) is comparable with that in amphibian epidermis and the epidermis of certain freshwater fish undergoing keratinization. Moderate amounts of mucopolysaccharide in the lacunar tissue and clear layer may protect against environmental pathogens and retain water to protect the epidermis from desiccation. Mucous cells could not be located in the epidermis throughout the sloughing cycle, contrary to some previous observations that they occur in the hinge region. The general absence of glycogen in the epidermis in most stages of the sloughing cycle suggests that the glycogen metabolized in the epidermis is utilized immediately, in view of the high energy requirements of proliferation and differentiation. Accumulation of glycogen granules in the presumptive α-layer in stage 2 and in the clear layer, presumptive Oberhautchen and presumptive β-Mayer in stage 3 is correlated with low energy requirements, indicating a slowing down of the process of keratinization of cells in these layers.     

Succinic dehydrogenase activity in the epidermis ofNatrix piscator during its sloughing cycle

Succinic dehydrogenase activity, in the epidermis of Nairix piscutor in different stages of sloughing cycle, has been localized using a nitro-BT technique with appropriate controls. The staining properties of different layers in scale epidermis are similar to the corresponding layers in hinge epidermis.
In the stratum germinativum, the layers of undifferentiated epidermal cells in all stages of the sloughing cycle, and in the lacunar tissue of Stages 3,4 and 5, a positive though weak reaction for SDH activity reflects the active metabolic state of the cells in these layers. Loss of SDH activity in Stage 6 indicates an inactive metabolic state of the lacunar tissue cells, corresponding with their disintegration owing to the cessation of nutrients as a result of keratinization of cells in the underlying layers.
The Oberhautchen, mesos and alpha layers in all Stages, and the clear layer cells in Stages 5 and 6 (outer epidermal generation), the presumptive Oberhautchen, presumptive mesos layer and presumptive alpha layer in all stages of their differentiation, and the presumptive beta layer in Stages 3 and 4 (inner epidermal generation) all stain purple with nitro-BT technique even in sections incubated in the medium without the substrate-succinate. The reaction is inhibited by prior treatment with 0.1 M N-ethyl maleimide blocking protein-bound -SH groups. This suggests that the reaction is due to the presence of protein-bound -SH groups in these sites. The reduced intensity of reaction in the mature beta layer of the outer epidermal generation, and in the presumptive beta layer in Stages 5 and 6 of the inner epidermal generation, is due to simultaneous loss of their content of -SH groups with maturation and keratinization.     

The lacunar cells and its relationship to interdigitating reticulum cells

Lacunar cells, which are characteristic of the nodular sclerosis type of Hodgkin's disease, were investigated by light and electron microscopy and by enzyme cytochemical and immunohistochemical methods. Characteristic ultrastructural features of the lacunar cells were its size, its multilobated nucleus, and the pale cytoplasm containing only a few organelles. These features distinguish the lacunar cell from typical Sternberg-Reed and Hodgkin cells. Enzyme cytochemically, lacunar cells were weakly positive for acid phosphatase and non-specific esterase. the reaction product was distributed either diffusely or more focally in the cytoplasm. By immunostaining, kappa, lambda, and IgG could be detected in some lacunar cells. The immunostaining pattern was bitypic, which might have resulted from non-specific uptake. All the results of the present study indicate that lacunar cells are non-lymphoid cells. When lacunar cells were compared with cells of normal lymphoid tissue, their ultrastructure was found to be very similar to that of interdigitating reticulum cells. Both cell types showed a bizzarrely shaped nucleus and an electron-transparent cytoplasm with only some vesicles and tubules. Furthermore, lacunar cells and interdigitating reticulum cells exhibited a similar reaction pattern of acid phosphatase and non-specific esterase. Thus, from a cytologic and enzyme cytochemical point of view, a direct relationship between the two cell types is very likely.     

Human bone cell enzyme expression and cellular heterogeneity: correlation of alkaline phosphatase enzyme activity with cell cycle

Alkaline phosphatase, long implicated in biomineralization, is a feature of the osteoblast phenotype. Yet in cultured bone cells, only a fraction stain positive histochemically. To determine whether osteoblast enzyme expression reflects cellular heterogeneity with respect to cell cycle distribution or length of time in culture, the activities of alkaline phosphatase, tartrate-resistant and -sensitive acid phosphatases, and non-specific esterases were assayed kinetically and histochemically. In asynchronous subconfluent cultures, less than 15% of the cells stained positive and assayed activity was 0.04 IU/10(6) cells/cm2. After 1 week, the percent of alkaline phosphatase positive-staining cells increased 5-fold, while activity increased 10-fold. Non-specific esterases and tartrate-sensitive acid phosphatase were constitutive throughout time in culture, whereas tartrate-resistant acid phosphatase activity appeared after 2 weeks. Cell cycle analysis of human bone cells revealed a growth fraction of 80%, an S phase of 8.5 h, G2 + 1/2 M of 4 h, and a G1 of 25-30 h. In synchronous cultures induced by a thymidine-aphidicolin protocol, alkaline phosphatase activity dropped precipitously at M phase and returned during G1. A majority of the alkaline phosphatase activity lost from the cell surface at mitosis was recovered in the medium. Tartrate-sensitive acid phosphatase and non-specific esterase levels were relatively stable throughout the cell cycle, while tartrate-resistant acid phosphatase activity was not assayable at the density used in synchronous cultures. From these data, variations in alkaline phosphatase activity appear to reflect the distribution of cells throughout the cell cycle.     

Histological and histochemical studies on the distribution of a few enzymes in the neoplastic liver of Uroloncha malabarica (Linnaeus)

The present work describes histological and histochemical observations made on the neoplastic liver of Indian silver bills, Uroloncha malabarica. The histology of neoplastic tissue as well as liver has been discussed. Further, a few enzymes like alkaline phosphatase, acid phosphatase, 5-nucleotides and non-specific esterase have been localized in the diseased liver. The occurrence of lymphocytoma caused a marked change in the localization of the enzymes. Sometimes total inhibition of the enzyme was encountered. Damaged sinusoid cells and bile canaliculi of the neoplasm as well as liver lobules show no reaction for alkaline phosphatase. However, its counterpart, acid phosphatase, exhibits intense activity in both neoplastic tissue and liver cells. Aggregates of neoplastic tissue give moderate 5-nucleotidase reaction while it gives poor activity in hepatic tissue of the diseased liver. Parenchymatous cells are able to give some activity for the non-specific esterase while it is very dull in the neoplastic tissue.     

Ultrastructure of the epidermis of the lizard (Lacerta vivipara) at the resting stage of the sloughing cycle

The ultrastructure of the epidermis of the lizard ( Lacerta vivipara ) one day after sloughing is described. The non-keratinized layers of the epidermis are essentially similar in structure to those of amphibians and mammals. The cells of the basal layer are not however separated from each other by the large spaces described in the amphibian (Farquhar & Palade, 1965). The middle layers of the epidermis at this stage of the sloughing cycle produce neither the characteristic mucous granules found in amphibians nor the keratohyalin granules of mammals. A small number of granules corresponding in size and location to the "Odland bodies" of both mammalian and amphibian epidermis are, however, present. The intermediate layer cells also contain a number of bodies similar in appearance to those described by Farquhar & Palade as lysosomes in amphibian skin. These structures are both osmium iodide and acid phosphatase positive. Unlike the condition in amphibians and mammals, the cytoplasm of cells in the layer immediately beneath the keratinized strata is honeycombed with small vesicles, and contains large irregular vacuoles of uncertain content. Certain nonkeratinizing elements within the epidermis are tentatively interpreted as nerve terminations. Two morphologically distinct keratinized strata can be distinguished, the inner stratum consisting of flattened cells similar to those of the stratum corneum of mammalian epidermis; individual cell outlines cannot be distinguished in the outer stratum, which has a structure similar to that of avian feather keratin. A shallow surface zone of the outer keratinized stratum has been identified as the Oberhautchen. This consists of longitudinally disposed leaflets or laminae which are responsible for the sculptured pattern of the epidermal surface. The observations reported here provide a basis for analysis of changes occurring at other stages of the sloughing cycle.     

Ontogeny of enzymatic activities in fed and fasting turbot, Scophthalmus maximus L.

The presence and localization of acid and alkaline phosphatase, non-specific proteases, aminopeptidase, amylase, non-specific esterase and lipase was investigated by histoenzymologic methods in fed and fasting turbot from day 1 to day 40 post-hatching and compared with published data. Alkaline phosphatase and aminopeptidase activities were delected at day 1 in the distal region of the developing digestive tube. At day 3 (opening of the mouth) aminopeptidase and alkaline phosphatase activities were found all along the intestine. Sites of non-specific esterase and protease activities became apparent in the digestive tract at days 2 and 3 respectively. Amylase was present in the exocrine pancreas at day 3 and in the lumen of the intestine at day 4. Acid phosphatase was active in the cellular structure surrounding the yolk stores and in the lipid droplets at day 1 and in the intestinal epithelium at day 3. Lipase was found at day 15 when the larvae metamorphose into juveniles.
All the investigated enzymes were detected in fasting animals, except for lipase. However, the intensities of the enzymatic activities were weaker in the fasting specimens relative to the fed specimens between days 7 and 10.     

The localization of enzyme activities in the pancreatic appendages of Sepia officinalis L. (Cephalopoda).

In this study, enzyme activities of the pancreatic appendages of the ductus hepatoPancreas (the so-called "pancreas") in Sepia officinalis L. have been demonstrated by light and electron micicroscopical methods: Malate dehydrogenase, monoamine oxidase, acid phosphatase, beta-glucuronidase, adenosine triphosphatase and carbonic anhydrase were shown by the former, and monoamine oxidase, catalase, glutamic oxalacetic transaminase, choline esterase (non-specific), alkaline phosphatase, acid phosphatase and carbonic anhydrase by the latter technique. The correlation between enzyme activity and distribution, and the presumed function of the two pancreatic epithelia is discussed.     

Epidermal keratinization in the salamander and a comparison with other amphibia

In a variety of amphibians examined the stratum corneum was one cell in depth, although in Xenopus it was up to three cells deep. The flattened horny cells were closely fused together along their lateral membranes to form a continuous sheet. Disulphide bonds of keratin were most concentrated in the peripheral cytoplasm, but the interiors of the cornified cells were sufficiently well keratinized to prevent more than slight enzymatic cytolysis of the normal cell components. Characteristically large, weakly stainable, non-shrunken nuclear remnants were found in the salamander and frog horny layers, but the clawed toad had small pyknotic (parakeratotic) nuclei. The mature amphibian keratinocytes contained free fats, bound phospholipids, calcium and sulphydryl groups, together with acid phosphatase and non-specific esterase. Cornification appears to begin by a process of separate individual cell keratinization and lateral membranes of neighbouring cells only later become fused together. This differs from the process in higher vertebrates in which the cells undergoing keratinization form a uniform transitional layer in the epidermis. In the amphibian epidermis neighbouring cells occur in different stages of keratinization.     

The enzyme histochemistry of the pineal gland in rats

Synopsis The enzyme histochemistry of the adult rat pineal is reviewed with particular reference to the probable endocrine activity of this organ. The parenchymal cells contain large amounts of oxidative enzymes and non-specific esterase, rather less leucine amino peptidase and acid phosphatase, and only small amounts of phosphorylase and alkaline phosphatase. In addition, high concentrations of alkaline phosphatase are present in the walls of capillary vessels. Leucine aminopeptidase is also seen in the connective tissue around blood vessels.     

A comparison between osmiophilic reagents and the Gomori lead method for the electron cytochemical demonstration of two lysosomal enzymes in oral epithelium

Synopsis Osmiophilic reagents were used to study the histochemical localization of acid phosphatase and non-specific esterase in the keratinized oral mucosa of rat. The reaction product from both enzymes was found in the epithelium and in cells of the corium as discrete granules, suggestive of a lysosomal localization. Treatment with E-600 before incubation for non-specific esterase did not change this localization. The osmium black end-product, due to acid phosphatase activity, was examined with the electron microscope and compared with the localization obtained by the Gomori lead phosphate technique. Both methods produced a reaction product in membrane-bounded bodies resembling lysosomes, as described in other tissues. These organelles were present in the basal prickle and granular cell layers of the epithelium. In the keratinized layer the reaction product was localized between the cell membranes of the deeper cells and no deposits were present in the cells. It is suggested that the osmiophilic reagents provide a good alternative to the Gomori method for the localization of lysosomal acid phosphatase at both the light and electron microscope levels.     

Observations on the epidermis of desert-living iguanids

The epidermis of 146 specimens of Dipsosaurus dorsalis and 182 Uma notata collected throughout the active period of the animals' year has been examined. The morphology of the epidermis is essentially similar to previously described lacertilians but differs in the relatively great degree of development of the mesos layer and the complete keratinization of the lacunar tissue prior to sloughing. Analysis of sloughing frequency throughout the year suggests that species specific patterns may exist, but these do not correlate with any particular known ecologic datum. The patterns do not reflect the reproductive activity of the two species supporting previous experimental conclusions on the lack of effect of gonadial hormones on epidermal activity. There appears to be no evidence of association of femoral gland activity with epidermal activity in D. dorsalis, but the situation is not clearcut in U. notata. These data are discussed in the light of recent studies of the evolutionary origin of epidermal glands in lizards.     

Histochemistry of the development of the digestive system of Siberian sturgeon during early ontogeny

At hatching, the yolk-sac matrix of Siberian sturgeon Acipenser baeri contained neutral glycoconjugates, glycogen, proteins rich in arginine, lysine, tyrosine, cysteine and cystine, glycoproteins containing mannose (Man) and/or glucose (Glc), N -acetyl-D-galactosamine (GalNAc), L-fucose (Fuc), sialic acid and/or N -acetyl-D-glucosamine (GlcNAc) residues, as well as neutral and acidic lipids. Buccopharyngeal and anterior oesophageal goblet cellls produced a combination of neutral and acid sialoglycoproteins, while those from the posterior oesophagus secreted only neutral glycoproteins; both types of secretions contained tryptophan and -S-S- groups and were unreactive to lectin techniques. Most intestinal goblet cells secreted mainly carboxylated and sulphated sialoglycoproteins with some rests of neutral glycoconjugates, while few of them produced only acid or neutral glycoproteins. Intestinal glycoproteins were rich in GalNAc, GlcNAc and sialic acid residues. Close relationships between digestive enzymes and morphological development of digestive organs were observed. Histochemistry of enzymes revealed that just after hatching, alkaline and acid phosphatase, ATP -ase and non-specific esterase activities were detected in the yolk sac. From the onset of exogenous feeding to the juvenile stage (30 days post-hatch), an enhancement of enzymatic activities was observed, as alkaline and acid phosphatase, ATP -ase, aminopeptidase M and nonspecific esterase sharply increased. However, lipase activity decreased in the liver and brush border of enterocytes by 13–14 days post-hatch. Two types of lipase were detected in the alimentary canal, a non-pancreatic lipase that was secreted in the cardiac stomach by gastric glands, and a pancreatic lipase, which activity was mainly detected in the brush border of the intestinal epithelium.     

Epiermal morphology and sloughing frequency in normal and prolactin treated Anolis carolinensis (Iguanidae, Lacertilia)

The morphology of the epidermal generations throughout the sloughing cycle of Anolis carolinensis is essentially the same as that described for other squamates, whether studied in control or hormone-injected animals. Readily identifiable granules in the clear layer of this and other lizard species emphasize the eventual hardening of this layer prior to its separation from the subjacent Oberhautchen, which brings about sloughing. Eosinophils are described for the first time in the lacunar tissue of lizard, but their functional role remains problematical. Daily 25 μg injections of prolactin (alone or in combination with gonadotropins) raise the frequency of sloughing by shortening the resting phase as compared with controls and animals receiving 1.0 I.U./day injections of gonadotropins. The duration of the proliferation-renewal phase and the pattern of histological changes occurring therein is unchanged by experimental treatments. The results show that neither endogenous nor exogenous gonadotropins affect the sloughing frequency, but exogenous prolactin produces a similar effect to that described for thyroxine. Although the mechanisms of action are unknown, the results provide another example of the cutaneous action of prolactin in vertebrates.     

Histochemical studies of some enzymes in the tissues of the schistosome vector snailBulinus truncatus (Audouin) with special reference to the effects of a molluscicide. II. Hydrolases

Summary Accomparative study of six hydrolases, acid and alkaline phosphatases, aryl sulphatase, -gluchronidase cholinesterase, and non-specific esterase, was carried out on the tissues of normal healthy and Frescon-treatedBulinus. The presence and activity of these enzymes in the tissues of normal animals were taken to indicate the probale functions of the tissues concerned. Frescon administration caused inhibition of acid phosphatase and also induced the release of cholinesterase and non-specific esterase in some tissue. It is concluded that the most important effects of Frescon on snail physiology are the disorganization of neuronal function and disturbance of olfactory activity.     

Qualitative and quantitative histochemical changes in the caecum and liver of turkeys infected with Histomonas meleagridis.

Changes in the amount and distribution of acid and alkaline phosphatase, non-specific esterase, glycogen, lipid and acid mucopolysaccharide in the caecal wall and liver of turkey poults infected with Histomonas meleagridis have been studied histochemically. A microdensitometer was used to measure changes in activity and distribution of acid phosphatase and non-specific esterase in the caecal mucosa. During the course of the infection there is a marked reduction in activity and distribution of acid phosphatase and non-specific esterase but little change in the amounts and distribution of alkaline phosphatase, glycogen, lipid and acid mucopolysaccharide in the wall of the main part of the caecum. Similar, but smaller, changes occurred in the wall of the neck region of the caecum. In the liver most changes occurred in the immediate vicinity of the parasites. Initially, there was a reduction in the amount of glycogen in the parasitic lesions but later in the infection there was a marked loss of glycogen in all regions of the liver. Changes in the caecum are apparently brought about by the parasite prior to and after invasion of the caecal tissues; changes in the liver occur after it has been invaded.     

Lipid histochemistry of the epidermis of Natrix piscator during its sloughing cycle

The lipid histochemistry of the scale and hinge epidermis of the chequered water snake, Natrix piscator , throughout the sloughing cycle, has been described. The presence of comparatively high concentrations of phospholipids in the mesos layer and a-layer, in comparison to neutral lipids, has been associated with a permeability barrier to transcutaneous water flux. Free fatty acids, present in almost all epidermal layers and in eosinophilic granular cells, may protect the epidermis from bacterial and fungal attacks. Cholesterol, in addition to phospholipids, in various keratinized layers, is assumed to be derived from membranous structures of epidermal cells and is regarded as a stabilizer of the phospholipids in membranes.     

Ontogeny of pituitary cell-types and the hypothalamo-hypophysial relationship during early development of chum salmon,Oncorhynchus keta

Tissue non-specific alkaline phosphatase is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic, protein phosphatase, and phosphotransferase activities. This enzyme is distributed virtually in all mammalian tissues, particularly during embryonic development. Its expression is stagespecific and can be demonstrated in the developing embryo as early as the 2-cell stage. It has been suggested that tissue non-specific alkaline phosphatase might play a role in tissue formation. In the study reported here, a genetransfer approach was employed to investigate possible roles for this enzyme by inserting the cDNA for rat tissue non-specific alkaline phosphatase into CHO and LLC-PK1 cells. Permanently transfected cell-lines expressing varying levels of alkaline phosphatase were estblished. The data showed that functional enzyme was expressed in the transfected cells. Cell spreading and attachment were enhanced in transfected CHO cells expressing high levels of tissue non-specific alkaline phosphatase but not in the LLC-PK1 cells. Further, in CHO cells, proliferation was shown to be inversely proportional to the level of the tissue non-specific alkaline phosphatase expression. Homotypic cell association was demonstrated in both alkaline phosphatase-positive and alkaline phosphatase-negative cells in both CHO and LLC-PK1 celllines. Taken together, these findings suggest that in addition to a role in mineralization of bone, tissue nonspecific alkaline phosphatase might also play a role in other cell activities, including those related to differentiation, such as cell-cell or cell-substrate interaction and proliferation.     

Multiple Hydrolases in Bean Leaves (Phaseolus vulgaris L.) and the Effect of the Halo Blight Disease Caused by Pseudomonas phaseolicola (Burkh.) Dowson

Multiple forms of hydrolytic enzymes were demonstrated in extracts of healthy bean leaves (Phascolus vulgaris L.) and bean leaves infected with the halo blight organism [Pseudomonas phaseolicola (Burkh.) Dowson] by polyacrylamide disc electrophoresis.Bean leaves contained up to 4 acid phosphatase bands, 9 esterase bands active towards alpha-naphthyl acetate, and 7 esterase bands towards alpha-naphthyl butyrate. Only low or no activity was found for alkaline phosphatase, lipase, and aminopeptidase.Two artifacts are described which were observed with the lead phosphate method for acid phosphatase and the Tween method for demonstration of lipase.After infection with the halo blight organism the major acid phosphatase of the host increased during early and decreased at later infection stages. An acid phosphatase of bacterial origin with a more neutral pH optimum could be demonstrated in infected leaves. It is suggested that the bacterial acid phosphatase plays a role in uptake of metabolites by the pathogen.Several esterase bands decreased after infection. One host band with activity towards alpha-naphthyl butyrate increased. Also the pathogen showed an esterase band with high activity towards alpha-naphthyl butyrate.     

Keratohyalin-like granules in lizard epidermis: evidence from cytochemical, autoradiographic, and microanalytic studies

Epidermal sloughing in lizards is determined by the formation of an intraepithelial shedding complex in which keratohyalin-like granules are formed. The chemical nature of these granules is unknown, as is their role in keratinization. The goal of this study was to test whether they contain some amino acids similar to those found in mammalian keratohyalin. The embryonic and regenerating epidermis of lizards are useful systems to study the formation of these granules. Histochemically keratohyalin-like granules react to histidine and contain some sulfhydryl groups (cysteine). X-ray microanalysis shows that these granules contain sulfur and often phosphorus, two elements also present in the mature clear, oberhautchen, and beta layer. Instead the mesos, alpha, and lacunar layers contain only sulfur. Most sulfur is probably in a disulfide-bonded form, particularly in mature cells of the shedding complex, in large keratohyalin-like granules, and in the beta-keratin layer. Early differentiating beta-keratin cells have the maximal incorporation of tritiated proline, whereas tritiated arginine is slightly more concentrated in the basal layer of the epidermis. A high uptake of tritiated histidine is observed mainly in keratohyalin-like granules of the clear layer, but also in the oberhautchen layer and forming the alpha-lacunar layer. Immunogold electron microscopy shows that keratohyalin-like granules do not localize keratin but are embedded within a keratin network. These results suggest that keratohyalin-like granules of lizards, like mammalian keratohyalin, contain some sulfur-rich and histidine-rich proteins. These granules participate in the process of hardening of the clear layer that molds the spinulae of the deeper oberhautchen to form the superficial microornamentation.