Assessment of lysosomal function by quantitative histochemical and cytochemical methods

Summary Quantitative histochemistry and cytochemistry enables a direct link to be made between metabolic functions such as the activity of lysosomal enzymes and the morphology of a tissue or a type of cell. Several approaches exist such as microchemistry based on (bio)chemical analysis of a single cell or a small piece of tissue dissected from a freeze-dried section. This technique has been routinely used for prenatal diagnosis of inherited enzyme defects and especially of lysosomal storage diseases. Other approaches are cytofluorometry or cytophotometry, which are based on the principle that a fluorescent or coloured final reaction product is precipitated at the site of the enzyme. The amount of final reaction product is analysed per cell or per unit volume of tissue using either a microscope cytofluorometer or flow cytometer for fluorescence measurements or an image analysing system or scanning and integrating cytophotometer for absorbance measurements.In principle, fluorescence methods are to be preferred over chromogenic methods because they are more sensitive and enable multiparameter analysis. However, only a limited number of fluorogenic methods are at hand that give a final reaction product which is sufficiently water-insoluble to guarantee good localisation. The best results have been obtained with methods based on naphthol AS-TR derivatives and with methods for the demonstration of protease activity using methoxynaphthylamine derivatives as substrates and 5-nitrosalicylaldehyde as coupling reagent. Chromogenic methods are far better with respect to localisation properties and, therefore, most commonly used for quantitative histochemical analysis of lysosomal enzyme activities. Besides the measurement of enzyme reactions in tissues and cells, chromogenic methods have been applied for the analysis of kinetic parameters of lysosomal enzymesin situ which could be a better reflection of enzyme kineticsin vivo than those obtainedin vitro with biochemical means in diluted solutions. Chromogenic methods have also been used in the lysosomal fragility test which is based on the lag phase occurring when a lysosomal enzyme reaction is analysed against time. The duration of the lag phase is a parameter for the stability of the lysosomal membrane and is affected by toxic compounds or under pathological conditions. This paper reviews briefly fundamental aspects and applications of quantitative histochemical and cytochemical methods in the study of lysosomes.     

Immunocytochemical detection of sulphonylated DNA in tissue sections

Summary We report here on a new sensitive and highly specific DNA staining technique which we have called sulpho-DNA staining. DNA staining is based on a sulphonylation reaction of 2-deoxycytidine or cytidine that takes place in the 6th position of cytosine with ensuing immunodetection of the sulphonylated DNA. The specificity of DNA staining is introduced by the use of an antibody recognizing only modified DNA but not modified RNA, by recourse to an additional acid hydrolysis step which destroys RNA but not DNA. We describe here the optimal conditions for the sulphonylation of DNA using O-methylhydroxylamine and metabisulphite as reactants. The new DNA stain labels all nuclei in either normal human tissue or in tumor cells. For nuclear DNA the staining signal is higher for the sulpho-DNA staining than for the Feulgen staining for nuclear DNA. This new DNA staining technique is suitable for use on tissue sections as well as on cytosmears.     

The effects of sucrose loading on lysosomal hydrolases

Summary The addition of 88 mM sucrose to the culture medium of human skin fibroblasts from normal subjects caused remarkable increase in the intracellular lysosomal hydrolase activities. The mechanism of this induction by sucrose loading was carefully studied with several fibroblast strains of different inherited lysosomal storage disorders. In single lysosomal hydrolase defect such as GM₁-gangliosidosis, mannosidosis and Sandhoff disease, no induction of the deficient hydrolase was found with 88 mM sucrose loading. In contrast, sucrose loading caused normalization of intracellular lysosomal hydrolase activities in I-cell disease fibroblasts and cytoplasmic inclusion materials disappeared. Subsequent investigations reveal that I-cell disease cells are classified into three subgroups by the degree of hydrolase induction by sucrose loading; a high responding, an intermediate responding and a no-response group. The heterogeneity may be based upon different induction by sucrose loading of the enzyme, probably the residual phosphotransferase which is involved in the processing steps of lysosomal enzyme molecules. With the addition of mannose-6-phosphate and 10 mM NH₄Cl to cultured skin fibroblasts, it was shown that sucrose loading caused increased synthesis of lysosomal enzyme proteins. The result of the test with 2,4-dinitrophenol suggests that sucrose is indeed pinocytosed by cultured human skin fibroblasts and localized in lysosomes and that this event is the essential factor to trigger the induction of lysosomal hydrolases. Simultaneous loading of both invertase and sucrose in cultured cells caused no induction of -mannosidase activity. This result indicates that invertase is also pinocytosed, reaches the lysosomes and hydrolyzes sucrose in the lysosomes. Lysosomal overloading with sucrose resulted in induction of lysosomal hydrolases and invertase blocked the induction of -mannosidase activity. However, some induction still exists in -galactosidase and -fucosidase activity. Thus it is very likely that the induction of lysosomal hydrolases demands a complicated process.In this article, we investigated the effects of sucrose on the lysosomal hydrolases in cultured human skin fibroblasts of several inherited lysosomal storage disorders and normal subjects and discuss the possible mechanism. of the induction of lysosomal hydrolase activities by sucrose loading.     

Location of satellite DNAs in the chromosomes of the kangaroo rat (Dipodomys ordii)

The location of satellite DNA sequences in metaphase chromosomes has been studied in the kangaroo rat by the in situ hybridization technique, staining techniques and phase contrast microscopy. The HS- satellite DNA is located at the kinetochores of all but three chromosome pairs. The HD satellite is located predominantly in the short arms of the chromosomes containing HS- and in the kinetochores of chromosome pairs that lack HS-. The regions that contain the satellite DNA sequences can also be identified by the Giemsa staining technique, and can be visualized with phase contrast microscopy or following Feulgen staining of fixed chromosome preparations.     

A comparison of two cytochemical methods used to determine lysosomal function in cultured endothelial cells

Summary Two cytochemical methods were used to study the activation of acid phosphatase caused by acid incubation in intact cultured human endothelial cells. Using the Gomori lead capture method, there is a lag phase before enzyme activation, whereas using Barka & Anderson's diazonium salt simultaneous coupling method, there is not. As the lag period is due to the gradual loss of membrane restraint and indicates the original fragility of the membrane, this indicates that although the Barka & Anderson method is useful for studying the localization of lysosomal enzymes, the Gomori method should be used to study lysosomal membrane function. Pre-treatment with 100 M histamine was found to increase lysosomal activation. The mechanism is not clear, but involves a labilizing effect on the membrane, indicated by a decreased lag time in the Gomori method.     

Dual localization of acid hydrolases in endoplasmic reticulum and in lysosomes

Summary Dissimilar enzyme locations obtained on occasion by the post- and simultaneous-coupling techniques employing the substrate naphthol AS-BI -glucosiduronic acid were attributed to the inadequate incorporation of substrate into lysosomal membranes in the post-coupling technique on the one hand, as well as to the inhibition of cytoplasmic enzyme by diazotate in the simultaneous coupling technique on the other hand. The use of a fixative solvent mixture prior to the enzyme staining reaction appeared to labilize lysosomal membranes, to improve fixation and to eliminate fiber artefacts. In male mice which have been androgenized by the injection of gonadotrophin, kidney homogenates, subsequently prepared, exhibited an immediate increase in the specific activity of microsomal -glucuronidase while lysosomal -glucuronidase was unchanged for the first 36 hours.This event at 36 hours corresponded with enhanced cytoplasmic but not lysosomal staining. Diffuse reactions in enzyme morphology are discussed as well as the origin of lysosomal -glucuronidase in mouse kidney and the dual localization of hydrolases in endoplasmic reticulum and lysosomes.     

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.     

The histochemical demonstration of five lysosomal enzymes in the pars distalis of the amphibian pituitary

Summary The distribution of five lysosomal enzymes (acid phosphatase, -glucosaminidase, -glucuronidase, sulphatase and E 600-resistant esterases) was studied in the pars distalis of the urodelian pituitary. Naphthol-AS-compounds coupled with hexazonium-pararosanilin yielded particularly good localization. The typical lysosomal picture and a basically similar distribution were obtained throughout. The reaction product was mainly found in the globules of the globular basophils (-cells) reported as gonatropin producing cells by several authors. Some speculations on the possible function of the high amount of the lysosomal enzymes in the globular basophils are made.Dedicated to Professor W. Bargmann on his 60th birthday.This work was partly supported by a grant from Deutscher Akademischer Austauschdienst, Bad Godesberg.     

A quantitative immunoelectronmicroscopic study on soluble, membrane-associated and membrane-bound lysosomal enzymes in human intestinal epithelial cells

Summary We have used quantitative immunoelectronmicroscopy to compare thein situ localization of acid -glucosidase, lysosomal acid phosphatase, -hexosaminidase and glucocerebrosidase in intestinal epithelial cells of the human duodenum. Differences between these four lysosomal enzymes were observed with respect to their presence at the apical cell surface. Transport to the apical membrane seems to be a more important intracellular route for lysosomal acid phosphatase and acid -glucosidase than it is for -hexosaminidase. The membrane associated lysosomal enzyme glucocerebrosidase is not transported to the microvilli. The studies emphasize that lysosomal enzyme transport pathways are enzyme and cell type specific.     

Relationship between dough-raising activity of baker's yeast and the fraction of ‘vital’ cells as determined by methylene blue staining or by dielectrophoresis

Three methods of comparing the vitility of baker's yeast (Saccharomyces cerevisiae) were examined, and found to correlated well. The rate of dough-raising, the degree of visible staining by a reducible dye (methylene blue), and the rate of dielectrophoretic collection as the cells were subject to a nonuniform ac field all appear to agree well as indices of cell viability. The simplicity of the dielectrophoretic technique suggests its further use in cellular assays.     

The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization

Summary Conventional solution-phase polymerase chain reaction (PCR) and in situ PCR/PCR in situ hybridization are powerful tools for retrospective analysis of fixed paraffin wax-embedded material. Amplification failure using these techniques is now encountered in some centres using archival fixed tissues. Such ailures may not only be due to absent target DNA sequences in the tissues, but may be a direct effect of the type of fixative, fixation time and/or fixation temperature used. The type of nucleic acid extraction procedure applied will also influence amplification results. This is particularly true with in situ PCR/PCR in situ hybridization.To examine these effects in solution-phase PCR, -globin gene was amplified in 100 mg pieces of tonsillar tissue fixed in Formal saline, 10% formalin, neutral buffered formaldehyde, Carnoy's, Bouin's, buffered formaldehyde sublimate, Zenker's, Helly's and glutaraldehyde at 0 to 4°C, room temperature and 37°C fixation temperatures and for fixation periods of 6, 24, 48 and 72 hours and 1 week. DNA extraction procedures used were simple boiling and 5 days' proteinase K digestion at 37°C. Amplified product was visible primarily yet variably from tissue fixed in neutral buffered formaldehyde and Carnoy's, whereas fixation in mercuric chloride-based fixatives produced consistently negative results. Room temperature and 37°C fixation temperature appeared most conducive to yielding amplifiable DNA template. Fixation times of 24 and 48 hours in neutral buffered formaldehyde and Carnoy's again favoured amplification.Fixed SiHa cells (containing 1–2 copies of HPV 16) were examined using PCR in situ hybridization for the amplification of HPV 16. Discrete and diffuse amplification signals were obtained. Neutral buffered formaldehyde fixation for 12–24 hours yielded amplifiable material suitable for use with PCR in situ hybridization. Overall amplification success within cellular preparations was 40%, with non-specific background staining also seen. Possible technical problems encountered with PCR in situ hybridization are discussed.     

Microwave irradiation and in vitro release of enzymes from hepatic lysosomes

Summary An exposure system for examining in vitro effects of microwave irradiation on cellular and subcellar components has been developed. The system was used to test the effect of 2.45-GHz CW microwaves on the release of two lysosomal enzymes. At a specific absorption rate (SAR) of 10, 50 or 100 mW/g (90 min) no effects were noted at 37° C (pH of 7.3) on lysosomal fragility as determined by the release of the lysosomal enzymes cathepsin D and-glucuronidase. When the medium was adjusted to pH 5.0, microwave irradiation of the lysosomal suspension had no effect on the acid-induced enhancement of release of lysosomal enzymes. The data indicate that microwave irradiation had no labilizing effect on the lysosomal membrane, although other microwave-membrane interactions not associated with enzyme release may occur.     

Indigogenic methods for glycosidases

Summary 4-Cl-5-Br-3-indolyl--D-fucoside (IbF) was tested in histochemical and bio-chemical experiments. Sections from differently treated parts of the small intestine of suckling and adult rats, of jejunum of adult hamsters, guinea pigs, cooks, monkeys, of human jejunal biopsies, of kidney of suckling and adult rats, adult monkeys, guinea pigs and hamsters, and of some other rat organs were used in histochemical experiments. Neutral and acid -D-galactosidases prepared from homogenates of the small intestine of suckling rats by chromatography on Sephadex G 200 were used in biochemical experiments.The recommended medium for histochemical studies consists of 0.1 M citrate phosphate buffer pH 6 (brush border enzyme) and pH 4 (lysosomal enzyme), 3.6·10^–4M IbF and 3.1·10^–3 M potassium ferri- and ferrocyanide.IbF is split quite efficiently by the brush border -D-galactosidase (=lactase) and the recommended histochemical method with IbF at pH 6 is the method of choice in studies concerned with the localization of intestinal lactase. In unfixed cold microtome sections the brush border activity can be easily detected within 15–240 minutes, depending on the lactase activity of the studied sample. In this study this activity was shown to be present in the brush border of enterocytes in the upper part of crypts reaching its maximum in differentiated enterocytes covering the sides and tops of villi in the small intestine of suckling (the highest activity) and adult rats (3–4x lower activity according to the time of appearance of the staining), and of human, monkey and hamster jejunum. In the cock jejunum traces of brush border staining were seen only after 24 hours of incubation. In enterocytes of patients with celiac sprue the brush border activity was very much reduced in dependence on the stage of the disease. Brush border staining along with a diffuse cytoplasmic reaction was found in the proximal convoluted tubules of the monkey kidney. The question of localization of enzyme activity splitting IbF in the monkey kidney deserves further investigation.IbF is also split by isolated intestinal acid -D-galactosidase and histochemically a positive reaction was found in lysosomes of many cells displaying a high activity of -D-galactosidase when IbF was used in the recommended medium of pH 4. The use of aldehyde fixation (glutaraldehyde is to be preferred) is a prerequisite for the assessment of this lysosomal localization. The lysosomal activity splitting IbF is not firmly bound structurally and escapes from cold microtome sections prepared from unfixed tissue samples into the incubation solution.The recommended method is also very suitable for processing zymograms and immunoprecipitation lines of lactase with antisera obtained by Ouchterlony's technic and by immunoelectrophoresis.     

A simple and inexpensive multiple tube system for thein situ measurement of phytoplankton production by the14C method

A cheap and simple multiple tube incubation system is described for the in situ measurement of phytoplankton production by the ¹⁴C method. Incubation takes place in Pyrex glass tubes sealed with rubber suba-seal caps which are suitable for field, laboratory and incubator work. Comparison of production in the tubes with that in standard pyrex bottles showed no significant difference during both field and laboratory incubations. The in situ suspension system is transported in units and can be assembled into ladders in the field.     

Heat resistance of Klebsiella pneumoniae in media with various sucrose concentrations

Summary The heat resistance of Klebsiella pneumoniae, an organism of widespread occurrence in nature has been determined in media containing various amounts of sucrose at temperatures between 47° and 59°C.In the presence of sucrose and at all temperatures the inactivation curves show a fast initial drop (logarithmic phase) in the number of survivors followed by a less rapid one (tail phase). The influence of the sucrose concentration can be described withln k _s = ln k _O – _T [sucrose] for media with more than 0.52 mol/l sucrose for the logarithmic as well as for the tail phase of inactivation.The heat-injured cells were recovered on various media to investigate the influence of the presence of small metabolites and nutrients on the shape of the inactivation curves and on the death rate. For cells heated in media without sucrose, the recovery on a rich medium was much better than on a poor one; for cells heated in media with more than 0.26 mol/l sucrose, no difference was observed between the various recovery media.The activation energies as determined on the various media are always nearly the same, which strongly suggests that the critical sites in the heat inactivation were not enzymes playing a key role in the synthesis of small molecules such as amino acids or nucleotides.     

Enzymatic regioselective synthesis of sucrose acrylate esters

Using a protease (at 100 g l^–1) from Bacillus licheniformis, enzymatic acryloylation of sucrose (1 M) with vinyl acrylate (4 M) was carried out in anhydrous pyridine and yielded sucrose acrylate esters with more than 90% of sucrose converted in 24 h. After 5 days of reaction, the ester products consisted of 70% sucrose monoacrylate and 30% sucrose diacrylate. The monoester product was a sucrose 1-acrylate and the diester products consisted of sucrose 6,1-diacrylate and sucrose 6,1-diacrylate in the ratio of 3:2.     

Heat resistance of Salmonella senftenberg 775W at various sucrose concentrations in distilled water

Summary The heat resistance of Salmonella senftenberg 775 W, NCTC 9959, has been determined in distilled water pH 6.5 at sucrose concentrations up to 2.20 mol l^–1 at temperatures between 63 and 70°C. Surviving cells were counted on minimal and enriched agar media to investigate the influence of the various nutrients on the recovery of heat injured cells. At various sucrose concentrations and temperatures multiphasic exponential parts of inactivation curves were found. Systematic differences between the recovery media depended on sucrose concentration, temperature and phase of exponential inactivation. At 60°C and sucrose concentrations between 0.52 and 1.82 mol l^–1 the relationship between inactivation rate and sucrose concentration could be described by the equation ln k₅=ln k₀-_T [sucrose]. The activation energy of thermal inactivation reactions, substantially decreased when sucrose (1.82 mol l^–1) was added to the heating menstruum. The activation energies in different recovery agars were of the same order, which suggests that the critical sites in heat inactivation are not key enzymes of the synthetic pathways of amino-acids and nucleotides. The differences between activation energies, calculated for cells of the various exponential phases of inactivation in both non-sucrose and 1.82 mol sucrose per 1 heating media, were also small, further suggesting that these critical sites are the same in cells from the various phases. Compared to published data on the heat resistance of S. senftenberg 775 W, we found a decreased resistance in a non-sucrose medium but an equal or increased resistance, depending on the phase of exponential inactivation, at a sucrose concentration of 1.82 mol l^–1.     

Subcellular distribution of terminal α-D- and β-D-galactosyl residues in Ehrlich tumour cells studied by lectin-gold techniques

We have studied by high resolutionin situ light and electron microscopic lectin-gold techniques the subcellular distribution of -d-Gal residues using theGriffonia simplicifolia I-B₄ isolectin and compared it with that of -d-Gal residues as detected with theDatura stramonium lectin in Ehrlich tumour cells grown as ascites or monolayer. The microvillar but not the smooth plasma membrane regions were labelled with theGriffonia simplicifolia I-B₄ isolectin whereas both plasma membrane regions were equally well labelled with theDatura stramonium lectin. Elements of the endocytotic/lysosomal system such as coated membrane invaginations and vesicles, early and late endosomes and secondary lysosomes were positive for both -d-Gal and -d-Gal residues. A particular feature of Ehrlich tumour cells is an elaborate tubular membrane system located in the pericentriolar region which is labelled throughout by both lectins and represents part of the endosomal system. In the Golgi apparatus labelling with both lectins was observed to commence in trans cisternae which is indirect evidence for a joint distribution of the sequentially acting 1,4 and 1,3-galactosyl-transferases.     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Summary Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed and increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and -N-acetyl-d-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and -glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the German Research Foundation (Sfb 174)     

Studies in lipid histochemistry

Summary It was shown by experiments with staining reactions performed in situ in blood smears of adults and children, in sections prepared by various procedures, in spot tests, and by chromatographic examination of extracted lipids that haemoglobin is responsible entirely for the staining of red blood cells in the acid haematein test and for the greater part for the staining in the OTAN reaction. The acid haematein test is from the point of view of lipid histochemistry neither specific nor sensitive. On the other hand the OTAN reaction is sensitive although it is not specific. It is concluded that the extraction test with chloroform-methanol must always be performed when the lipidic nature of the demonstrated substance(s) is to be proved unequivocally and that the non-lipidic nature of the residual staining must be always considered.