A HISTOCHEMICAL STUDY OF PHAGOCYTIC AND ENZYMATIC FUNCTIONS OF RABBIT MONONUCLEAR AND POLYMORPHONUCLEAR EXUDATE CELLS AND ALVEOLAR MACROPHAGES : I. Survey and Quantitation of Enzymes, and States of Cellular Activation

The cytochrome oxidase (CO), aminopeptidase (AMP), succinic dehydrogenase (SD), acid phosphatase, esterase, and alkaline phosphatase of rabbit mononuclear (MN) and polymorphonuclear (PMN) peritoneal exudate cells and pulmonary alveolar macrophages (AM) - air dried on Mylar strips - were characterized by histochemical techniques with respect to stability, activators, inhibitors, and pH optima. A granule count method was established for the quantitation of these enzymes. For the acid phosphatase of MN, in which the most precise results were obtained, time, pH, substrate, and inhibitor curves resembled those commonly obtained biochemically. Five of these enzymes were usually more active in AM than MN, whereas the sixth, alkaline phosphatase, was not present in either cell type. AM also tended to consume more oxygen than MN and to divide more frequently. Since the most active cells in the population would be first involved in the host's defense against microbial agents, a comparison was made of the 10 per cent of the AM and MN with the highest enzymatic activities. No differences were found in the granule counts that were not reflected by the means. However, within a given AM population, cells containing ingested dust particles seemed to have higher enzymatic activities than those without particles. MN had greater acid phosphatase and SD activities than PMN and consumed more oxygen, but the CO, AMP, and esterase activites of both types of cells were of similar magnitude. PMN showed high alkaline phosphatase activity; MN showed none. A survey of the histochemical literature indicates that a positive correlation between the enzymatic and phagocytic activities of both MN and PMN exists in vivo.     

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.     

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.     

Histochemical findings in the rat gastric mucosa during starvation

Summary The influence of starving on the activity of enzymes of the rat gastric mucosa was investigated by selected histochemical methods. Beside the conventional methods of enzymatic histochemistry the technique of semipermeable membranes was used in the proof of lysosomal enzymes. Dehydrogenases were proved in aqueous and also in gel media with PMS.During the starvation in the parietal cells a marked increase took place in the activity of acid phosphatase, E-600 resistant esterase, less in -glucuronidase. High activity of the lysosomal enzymes in macrophages did not change during starvation. Nor did any changes took place in the activity of alkaline phosphatase in the endothelium of the capillaries. The chief cells in the control and starving animals, in contrast to the human gastric mucosa, did not contain any non-specific esterase. Concerning dehydrogenase, parietal cells with a different activity of these enzymes were observed both in starved and control animals.In the rat gastric mucosa starving induced changes in the activity of the enzymes which mark important organelles of the cells. Thus it is possible to consider the observed histochemical changes as a functional manifestation of morphological damage of cellular structures which are affected during starvation.     

Enzymes in the epidermis of Natrix piscator during its sloughing cycle

Acid phosphatase, non-specific esterase, alkaline phosphatase, monoamine oxidase and true lipase activities, in the epidermis of Natrix piscator in different stages of the sloughing cycle, have been localized using various histochemical techniques.
Different layers in scale epidermis have staining properties similar to corresponding layers in hinge epidermis.
Acid phosphatase and non-specific esterase activity in cell layers undergoing keratinization, and the lacunar tissue undergoing disintegration are associated with hydrolytic and catabolic wasting processes involving cell death. The activity of these enzymes in the clear layer is associated with the breaking down of the cementing substance resulting in the separation of clear layer from underlying tissue and facilitating the shedding of old slough.
Alkaline phosphatase activity in the stratum germinativum and undifferentiated epidermal cells has been associated with cell proliferation and differentiation. The presence of alkaline phosphatase in the lacunar tissue and clear layer has been correlated with the synthesis of mucopolysaccharides in these layers.
Monoamine oxidase and true lipase activity could not be located in the epidermis at any stage of the sloughing cycle.     

牛蛙消化道黏膜5种酶的分布

目的研究牛蛙(Rana catesbeinana)消化道黏膜碱性磷酸酶(ALP)、酸性磷酸酶(ACP)、ATP酶、酯酶和脂酶的分布。方法在消化道的8个部位取材,采用冰冻切片技术和酶的组织化学方法。结果 ALP主要分布于十二指肠、空肠和回肠,胃中酶反应呈弱阳性。ACP主要分布于胃中,食道和肠道酶反应呈弱阳性。ATP酶在消化道各部位均有较多分布,十二指肠、空肠和回肠显著较多,胃各部位其次。酯酶和脂酶均主要分布于肠道,胃各部位其次。结论牛蛙消化道黏膜酶的分布同其它动物有相似之处,也有其自身特点。十二指肠、空肠和回肠是牛蛙的主要消化吸收部位。     

Histochemical study of enzyme patterns in the human submaxillary gland

Summary The histochemical distribution of various enzymes, such as alkaline phosphatase, acid phosphatase, esterase, -glycosidase, aminopeptidase, succinic dehydrogenese and TPN diaphorase, in human submaxillary glands has been determined.Acini and ducts of human submaxillary gland were devoid of alkaline phosphatase activity, but this enzyme was observed in capillaries and somewhat in myoepithelium.Activities of acid phosphatase, esterase, -glucuronidase and -galactosidase were generally observed in the entire cytoplasm of serous acini; but the cytoplasm of mucous acini was either negative or showed only trace amounts.Aminopeptidase reaction of both acini and ducts was generally negative.Succinic dehydrogenase and TPN diaphorase activities were strongly active in intralobuler ducts. Serous acini exhibited less activity with these enzymes; and mucous cells showed still less and were almost negative. In serous acini, there was much greater activity of TPN diaphorase than of succinic dehydrogenase.With 7 Figures in the Text     

Histochemical localization of acid and alkaline phosphatases and glucose-6-phosphatase of the hepatopancreas of the crab, Scylla serrata (Forskål)

Simultaneous histochemical localization of non-specific monophosphate esterases, acid and alkaline phosphatases, as well as a specific monophosphate esterase, glucose-6-phosphatase has been made on the hepatopancreas of the marine crab, Scylla serrata (Forskål). Maximum activity of the 3 enzymes was observed in the juvenile and mature absorptive cells. Lining cells of the main hepatopancreatic duct exhibited a moderate activity of the 3 enzymes whereas the embryonic and fibrillar cells and connective tissue of the gland showed negative reactions for the 3 enzymes. The secretory cells showed a positive reaction for these enzymes only at the brush border.Bilateral eyestalk removed evoked a rise in the activity of the 3 enzymes within 2–4 h. The same effect was observed after injection of eyestalk extract to both normal and eyestalkless animals followed by restoration to the normal level after 24 h.The present observations indicate that glucose-6-phosphatase and acid phosphatase may be under the direct influence of eyestalk hormone(s) while alkaline phosphatase activity appears to be related to changes in the substrate. The physiological significance of the various cell types and enzymes is discussed.     

The Histochemical Localization of Several Enzymes of Soybeans Infected with the Root-knot Nematode Meloidogyne incognita acrita

The sites of activity of alkaline phosphatase, acid phosphatase, esterase, peroxidase, adenosine triphosphatase, and cytochrome oxidase were demonstrated histochemically in fresh sections of ''Lee'' soybeans infected by the root-knot nematode Meloidogyne incognita acrita. Each of the six enzymes was more active at the sites of infection than in adjacent non-infected tissue. During the early stages of infection, an increase in enzyme activity was observed in several cells in the proximity of the lip region of the nematode. However, when definite syncytia were observed, increased enzyme activity was confined primarily within the limits of the syncytium. Increased activity paralleled syncytial development and nematode maturation.     

Histological and enzyme histochemical changes in the kidney of male bullhead (Cottus gobio) during the spawning period

Histological and enzyme histochemical studies were carried out on the excretory kidney of the male bullhead ( Cottus gobio ). During the spawning season striking morphofunctional changes were observed in the second proximal segment of the kidney tubule. The tubular epithelium was greatly hypertrophied, strongly basophilic and produced a PAS-positive secretion. The enzyme histochemical pattern also changed conspicuously during this time: the alkaline phosphatase activity in the brush border was greatly reduced; the acid phosphatase and non-specific esterase activity in the cytoplasm was distinctly elevated.     

Digestive enzymes in larval Coregonus lavaretus L.

Using histochemical methods, morphofunctional aspects of the alimentary tract of larval coregonids were investigated. Larvae of Coregonus lavaretus were reared for 34 days with either zooplankton or one of two dry diets. Ontogeny, localization and diet-related modifications of the following enzymes were examined: trypsin (luminal digestion), aminopeptidase, maltase, alkaline phosphatase (brush border-bound digestion) and unspecific esterase (intracellular nutrient processing). All of the enzymes studied were present in 13-day-old larvae. Except for the intracellularly located unspecific esterase, there was an ontogenetic enhancement of enzyme staining intensities accompanied by a significant increase in the volume of the intestinal mucosa. Enzyme activities differed within and between intestinal regions. This finding suggests that a spatial gradient of nutrient breakdown and absorption already exists in the morphologically and physiologically incompletely developed digestive system of larval coregonids. Digestive enzyme activities were modified in response to the dietary regimen. There was no obvious correlation between enzymic response and growth performance of the larvae.     

Histochemical studies on the olfactory glomeruli of squirrel monkey

Summary Detailed histochemical studies on the distribution of various oxidative and dephosphorylating groups of enzymes have been made in the olfactory glomeruli of the squirrel monkey. The olfactory glomeruli showed strongly positive activity for succinic dehydrogenase, lactic dehydrogenase, monoamine oxidase, alkaline phosphatase, adenosinetriphosphatase and simple esterase. They showed moderately positive activity for cytochrome oxidase, specific cholinesterase, 5'nucleotidase; mildly positive activity for acid phosphatase; and negligible activity for nonspecific cholinesterase and glucose-6-phosphatase. The glomeruli did not show the presence of any thiamine pyrophosphatase-positive Golgi apparatus. The blood vessels surrounding the glomeruli were strongly positive for the nonspecific cholinesterase test. The significance of these results are discussed briefly.     

Histochemical demonstration of changes in liver cell enzymes in pregnant mice infected with coxsackie B3 virus

Summary Sequential changes in liver cell morphology in mice infected with Coxsackievirus B₃ have been studied using enzymic markers for early cell damage. Elevated alkaline phosphatase two days after infection was the only change identified consistent with the presence of virus in the tissue. Reduced glucose-6-phosphatase, and succinic dehydrogenase were associated with cytoplasmic vacuolation and fatty deposition which were clearly apparent 6 to 8 days after infection. Lysosomal acid phosphatase was increased at this stage in periportal areas and reduced in centrilobular regions. comparison of the Coxsackievirus induced changes with those induced by a direct liver pathogen—hepatitis virus, suggests that the histological and histochemical changes are probably of an indirect causation.     

Histochemical analysis of molluscan stomach and intestinal alkaline phosphatase: A sialoglycoprotein

Summary Though sialoprotein nature of alkaline phosphatase of certain mammalian organs has been suggested by biochemical investigations, no histochemical techniques have yet been applied to elucidate this concept. With this view, the alkaline phosphatase of stomach and intestine of a mollusc—Semperula maculata—was analysed histochemically to elucidate its sialoglycoprotein nature. The localisation of alkaline phosphatase and sialic acid was investigated by employing well known and standard histochemical techniques.Alkaline phosphatase was localised selectively in the brush border of the mucosa of stomach and intestine, it was Mg⁺⁺ nonsensitive but showed a structure-linked sensitivity to phenylalanine. The sialomucins were selectively localised in the brush border, whereas the goblet cells contained both the sialomucins and sulfomucins, and the connective tissue of lamina propria contained sulfomucins. The localisation of alkaline phosphatase and sialomucins in the brush border uniquely coincided with each other. The alkaline phosphatase activity in the brush border was completely lost after neuraminidase treatment at 37.5° C for 16 h. Such effect of neuraminidase on alkaline phosphatase activity was pH dependent and controlled by velocity of reaction. Heat-inactivated neuraminidase showed no effect on alkaline phosphatase activity.These histochemical results have been interpreted as suggesting a sialoglycoprotein nature of alkaline phosphatase in the brush border, and sialic acid somehow seems to be essential for enzyme activity. These results, thus, indicate necessity of visualising some of the sialo-glycoproteins as macromolecules with catalytic activity.     

DIFFERENCES IN ENZYME CONTENT OF AZUROPHIL AND SPECIFIC GRANULES OF POLYMORPHONUCLEAR LEUKOCYTES : I. Histochemical Staining of Bone Marrow Smears

Histochemical procedures for PMN granule enzymes were carried out on smears prepared from normal rabbit bone marrow, and the smears were examined by light microscopy. For each of the enzymes tested, azo dye and heavy metal techniques were utilized when possible. The distribution and intensity of each reaction were compared to the distribution of azurophil and specific granules in developing PMN. The distribution of peroxidase and six lysosomal enzymes (acid phosphatase, arylsulfatase, β-galactosidase, β-glucuronidase, esterase, and 5'-nucleotidase) corresponded to that of azurophil granules. Progranulocytes contained numerous reactive granules, and later stages contained only a few. The distribution of one enzyme, alkaline phosphatase, corresponded to that of specific granules. Reaction product first appeared in myelocytes, and later stages contained numerous reactive granules. The results of tests for lipase and thiolacetic acid esterase were negative at all developmental stages. Both types of granules stained for basic protein and arginine. It is concluded that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appears to be restricted to one of the granules. The findings further indicate that azurophil granules are primary lysosomes, since they contain numerous lysosomal, hydrolytic enzymes, but the nature of specific granules is uncertain since, except for alkaline phosphatase, their contents remain unknown.     

On the enzyme histochemistry of the renal interstitial cells

Summary A histochemical study of the renal interstitial cells demonstrates the absence of two of the principal enzymes involved in the citric acid cycle, the cells thus seem to lack the ability to break down fatty acids. On the other hand the renal interstitial cells demonstrate enzyme activity which could be indirect evidence of fatty acid synthesis. A pronounced nodular acid phosphatase activity and unspecific esterase activity were noticed as well as an uniform cytoplasmic esterase activity.This work was supported by a grant from the Danish State Research Foundation.     

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. V. Isocitrate: NADP+ oxidoreductase (decarboxylating) and malate: NADP+ oxidoreductase (decarboxylating).

Improved histochemical techniques for the demonstration of NADP+-specific isocitrate dehydrogenase and malate dehydrogenase in tissue sections are described. With these techniques a semipermeable membrane is interposed between the incubating solutions and the tissue sections preventing diffusion of enzymes into the medium during incubation. In the histochemical system the NADP+-dependent enzymes catalyze the electron transfer from threo-Ds-isocitrate or L-malate into NADP+. Phenazine methosulphate and menadione serve as intermediate electron acceptors between reduced coenzyme and nitro-BT. Sodium-azide and amytal are incorporated into the incubating-medium to block electron transfer to the cytochromes. For demonstrating enzyme activities in sections containing non-specific alkaline phosphatase, a phosphatase inhibitor is added into the incubation media. Problems involved in the histochemical demonstration of both enzymes are discussed.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques: a combined histochemical and biochemical investigation 2. The biochemical investigation and comparison with the histochemical observations.

The reliability of enzyme histochemical semipermeable membrane techniques for the demonstration of acid hydrolases was investigated with a combined histochemical and biochemical study. In part 1 the histochemical findings were presented. In this communication the biochemical findings are reported and compared with the histochemical findings. In m. soleus, m. plantaris, m. gastrocnemius and diaphragm of vitamin E deficient rabbits the activity of the lysosomal acid hydrolases, cathepsin D, acid maltase, acid phosphatase and beta-glucuronidase is significantly increased. This increase in activity of the investigated acid hydrolases was equal for muscles with an aerobic or an anaerobic metabolism. By means of statistical calculations the activity of the enzymes demonstrated with histochemical techniques was compared with the enzyme activity determined with biochemical techniques. From the results of this investigation it can be concluded that the histochemical semipermeable membrane techniques for the demonstration of activity of acid hydrolases are very reliable. Considering the fact that these techniques are also tissue-saving, they are therefore extremely suitable for the study of catabolic wasting processes in skeletal muscle tissues of patients with inherited or acquired muscular diseases.     

Enzyme histochemical studies on rat lungs I. An improved technique for the demonstration of enzyme activity in alveolar tissue

Synopsis Rat lungs were supported by ten different materials in an attempt to compare the quality of the sections obtained and to assess the effects of these materials on the activities of three enzyme systems in alveolar tissue as determined by histochemical techniques.Most of the supportive materials were not removed during enzyme incubation and caused some apparent inhibition of enzyme activity. Of the compounds investigated, 0.5% polyethylene glycol was found to produce the most satisfactory results with the least distortion or compression of the alveolar tissue and no apparent inhibition of the three enzymes studied, lactate dehydrogenase, non-specific esterase and alkaline phosphatase. Some of the materials investigated, notably acacia and carboxymethylcellulose, appeared to decrease the thickness of the alveolar wall. This effect was only seen in lungs supported by the most viscous materials.     

The increase in activity of acid hydrolases in muscles of rats after subcutaneous administration of dimethyl-para-phenylene diamine. A combined histochemical and biochemical investigation. I. The histochemical investigation.

The activity of acid hydrolases in skeletal muscles of normal rats and of rats after subcutaneous administration of dimethyl-para-phenylene diamine (DPPD) was studied with a combined histochemical and biochemical investigation. In this communication the histochemical findings are presented. After 4 days of DPPD treatment, coagulation necrosis, fragmentation and disintegration of fibres were seen in the muscles. An inflammatory infiltrate was seen between the muscle fibres. These pathological changes reached maximum intensity after 7 to 9 days. After 11 days the changes became less, despite continued treatment with DPPD. From the histochemical findings it appeared that the activity of acid phosphatase, beta-glucuronidase and E600 resistant non-specific esterase was increased in both a granular and a diffuse pattern in the skeletal muscles of the DPPD rats. The increase in activity of leucine aminopeptidase was much less pronounced and was mainly granular. The increase in the activity of acid hydrolases ran parallel to the severity of the pathological changes and reached a maximum after 7 to 9 days of DPPD treatment. The statistical calculations of the histochemical findings revealed that the increased activity of one acid hydrolase was significantly paralleled by an increased activity of a second hydrolase. There was a moderate probability that the activity of all other histochemically studied acid hydrolases, with the exception of leucine aminopeptidase, was increased. There was no difference in activity and localization of the acid hydrolases studied in aerobic type I and anaerobic type II fibres. The localization of acid phosphatase and beta-glucuronidase activity in muscle fibres and in inflammatory infiltrate mostly coincided. In cases where these enzymes were localized both centrally and in the subsarcolemnal areas of the muscle fibres, the activity of E600 resistant non-specific esterase was usually, and the activity of leucine aminopeptidase was exclusively located in the subsarcolemnal areas. All of the acid hydrolases examined were found to be present in the inflammatory exudate and in the connective tissue.