Histochemical quantification of dopa-oxidase (tyrosinase) activity in epidermal melanocytes.

The graphical method of Lineweaver-Burk was applied to the histochemical system to obtain quantitative information on the dopa-oxidase activity in human epidermal melanocytes. In vitro experiment showed that the incubation time necessary for yielding a certain amount of dopa-melanin is related to the amount of enzyme present. In the case of histochemical experiment, the time required for the first appearance of dopa-melanin deposition in melanocytes at various substrate concentrations was plotted against the reciprocal of the substrate concentration. This technique made it possible to compare the dopa-oxidase activity at various sites of the tissue.     

The existence of apotyrosinase in the cytosol of Harding-Passey mouse melanoma melanocytes and characteristics of enzyme reconstitution by Cu(II)

This paper reports the effect of Cu(II) supplementation on the tyrosinase isozymes from Harding-Passey mouse melanoma. The dopa-oxidase activity of the microsomal and soluble isozymes is increased by incubation with Cu(II), whereas the activity of the unique 'in vivo' melanin-forming isozyme, bound to melanosomes, is not. Other divalent cations are ineffective in increasing the dopa-oxidase activity of tyrosinases. These results indicate the existence of a mixture of tyrosinase and apotyrosinase in the cytosol of melanocytes before reaching the melanosome. The paucity of Cu(II) in the cytosol could be one of the mechanisms of regulation contributing to avoid the formation of melanin outside the melanosome. Some kinetic characteristics of the enzymatic reconstitution of soluble and microsomal isozymes by Cu(II) are also studied, and the results suggest that the glycosylation of apotyrosinase during its maturation yields a conformational change favouring the binding of Cu(II) at the enzyme active site, by lowering the activation energy of the reconstitution reaction.     

Biochemical investigation to the reliability of the histochemical demonstration of microsomal arylsulphatase activity in cryostat sections

Summary Polyacrylamide gel-electrophoresis was performed with an extract from cultivated skin fibroblasts. Arylsulphatase activity is measured and visualised using the biochemical substrate dehydroepiandrosterone sulphate and the histochemical substrate 6-bromo-2-naphthyl sulphate respectively. The histochemical substrate was hydrolysed at Rf=0.49 and 0.58 while the biochemical substrate was hydrolysed only at 0.49. We conclude that two different microsomal arylsulphatases exist: a sulphatase able to hydrolyse steroid sulphatases (Rf=0.49) and one unable to hydrolyse steroid sulphatases (Rf=0.58). In consequence it is recommended to carry out an electrophoresis experiment after the histochemical investigation, in order to discriminate between these two types of sulphatase.     

A HISTOCHEMICAL ENZYME KINETIC SYSTEM APPLIED TO THE TRYPSIN-LIKE AMIDASE AND ESTERASE ACTIVITY IN HUMAN MAST CELLS

A method for the determination of enzyme kinetic constants V_m, K_m, and K_i in a histochemical system has been devised. As a substitute for the reciprocal of the reaction velocity, the times necessary to reach a fixed amount of end product (the initial visible color) in a tissue site at various substrate concentrations are plotted, according to the method of Lineweaver and Burk, against the reciprocal of the substrate concentrations. The technique as applied to trypsin-like esterase and amidase activities in human mast cells indicates that a single enzyme or closely related enzymes in this site are responsible for the hydrolysis of both the amide and ester substrates and that typical trypsin substrates act as competitive inhibitors of their hydrolysis. Parallel biochemical studies were performed to evaluate the effect of certain aspects of the experimental histochemical method on a purified homospecific enzyme. The relative kinetic constants derived by the histochemical method afford a further means of characterizing enzymic activity in a histochemical system.     

Origin of melanosome structures and cytochemical localizations of tyrosinase activity in differentiating epidermal melanocytes of newborn mouse skin

The mode of differentiation of epidermal melanocytes was studied by ultrastructural cytochemistry in the skin of newborn mice of strain C57BL/10J. From observations of epidermal melanoblasts and melanocytes, stage I melanosomes, including both unit membranes and inner matrices, appear to be formed from Golgi vacuoles or rough endoplasmic reticulum (RER). Stage I melanosomes were positive to ammoniacal silver-nitrate reaction in the melanoblasts of 1-day-old mice. All stages of melanosomes were similarly positive in the differentiating melanocytes of 2-day-old mice. However, Golgi apparatus, RER, and vesicles were negative. Therefore, it is conceivable that structural proteins, originated from Golgi vacuoles or RER, are developed into specialized proteins and are detected by this reaction in stage I melanosomes. Stage I melanosomes were dopa-negative in the melanoblasts. Stage I and II melanosomes were similarly negative in the differentiating melanocytes. Thus, the melanoblasts are thought to begin production of stage I melanosomes prior to the onset of tyrosinase activity. In the differentiating melanocytes, dopa-melanin depositions were observed in stage III and IV melanosomes, trans Golgi saccules, and small vesicles derived from these saccules, but not in RER. These vesicles were in contact with, or fused to, melanosomes. These findings suggest that tyrosinase may be transferred by Golgi vesicles into stage I and II melanosomes originating from Golgi vacuoles or RER.     

Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the experimentally injured rabbit cornea and tear fluid using the sensitive substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC)

Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the rabbit eye after various experimental injuries were performed using the same sensitive fluorogenic substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC). The aim of the study was to examine whether the severity of corneal damage corresponds with the level of the enzyme activity in the tear fluid. As until recently the substrate beta-galactoside-4-HFC had not been used for the histochemical detection of acid beta-galactosidase in the cornea, results obtained with this substrate in a fluorescent method were compared in parallel cryostat sections with results obtained using the substrate 5-bromo-4-chloro-3-indoxyl beta-galactoside in the indigogenic method (previously shown to be very sensitive for the detection of acid beta-galactosidase activity in the cornea). Both methods revealed similar localization and changes in enzyme activity; using beta-galactoside-4-HFC an acceptable cellular localization was achieved. For the measurement of acid beta-galactosidase activity in the tear fluid a semiquantitative biochemical method was elaborated using filter paper punches with the substrate (beta-galactoside-4-HFC) soaked with tears and incubated at 37 degrees C. The time of the first appearance of a greenish-yellow fluorescence (enzyme positivity) was recorded by UV lamp and compared with the appearance of fluorescence in calibrated punches containing known acid beta-galactosidase activities. The results show that beta-galactoside-4-HFC is useful for the biochemical assessment of acid beta-galactosidase activity in the tear fluid. Comparing histochemical and biochemical results, it can be concluded that increased enzymatic activity in tears parallels the severity of corneal damage. Further studies are necessary to evaluate whether the detection of acid beta-galactosidase activity in tears might be useful for diagnostic purposes in humans.     

Testosterone regulation of pigmentation in scrotal epidermis of the rat

Summary The effects of testosterone on melanocyte number, morphology, melanin content and tyrosinase activity were studied in epidermis from several body regions of the black-pelted Long-Evans rat. Determinations were made in epidermal sheets processed for histochemical analysis by incubation in the presence of the melanin precursor, 3,4-dihydroxyphenylalanine (DOPA). Melanin content, cell volume, dendritic branching and tyrosinase activity of scrotal epidermal melanocytes all decreased progressively with time following castration. Daily testosterone injection, begun 14 days after castration, increased tyrosinase activity in 4 days, and dendritic branching in 6 days, of treatment; melanin content, cell volume and enzyme activity were restored to normal intact levels within 14 days of treatment, at which time newly synthesized melanin was evident in keratinocytes. The total number of scrotal epidermal melanocytes was not changed by castration or testosterone administration. Neither castration nor testosterone replacement affected any parameter of epidermal melanocytes in preputial, perianal or eyelid skin which, together with the scrotum, are the animals' only pigmented areas. Androgen control of epidermal pigmentation in the male rat is therefore specific for the scrotum and is manifested through regulation of melanin synthesis in stable populations of melanocytes rather than through increases in numbers of melanocytes.This work was supported in part by research grant no. HD 00446, and training grant no. HD 00152, from the Institute of Child Health and Human Development, Public Health Service.     

Histochemical identification of non-culturable mycobacteria (M. leprae and M. lepramurium)

Summary The possibility of the histochemical demonstration of the dopa-oxidase reaction in Mycobacterium leprae from the granulomas of untreated cases of human lepromatous leprosy was studied. Fresh frozen sections were taken, fixed for one hour, and incubated with DOPA (3,4-dihydroxyphenylalanine) for two hours at pH 6.8 and at 37° C. The results obtained did not confirm the biochemical phenomena observed in human liver and spleen.Investigation supported by Grant No. DF. S1-153 from Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICIT)     

Studies on the histochemical “leucine aminopeptidase” reaction

Summary A correlative chemical and histochemical study on the leucyl--naphthyl-amide-splitting activity is presented.In the first part comparison is made between homogenates and smears of strain L cells and ELD ascites tumor cells. It was concluded that only the available enzyme activity could be visualized by histochemical means. The growing tumor cells appeared to have more activity available than the strain L cells. The intensity of the histochemical LNAse reaction bore no correlation to the total enzymatic activity extractable. Most of the available activity seemed to reside in lysosome-like structures. Cell damage by repeated freezing and thawing increased the amount of activity available to substrate interaction.In the second part a comparison is made between chemical and histochemical results in four types of progressively growing transplanted or induced mouse tumors. Chemical data showed the occurrence of a mixed pattern of LNA-splitting enzymes, among which the previously described metal-dependent group constituted the main component in the tumor cells. In all cases and types of tumors a positive histochemical LNAse reaction was noticed both in growing tumor cells as well as in different parts of the stromal compartment. The intracellular reaction in tumor cells seemed confined mainly to lysosomal-like bodies. A positive stromal reaction seemed associated with the destructive (cytolytic and collagenolytic) activity at the tumor periphery. The stromal LNAse reaction was probably due to enzymes partly different from the intracellular ones. Additional enzymatic terms, including a chymotrypsin-like and/or a carboxypeptidase activity, may originate from various host cells. These contributions may be greatly increased by accumulation of inflammatory host cells as previously noted by Hess and Mottet.     

Quantitative measurement by microdensitometry of tyrosinase (Dopa oxidase) development in whole small ascidian embryos

Summary Embryos of the ascidian, Ciona intestinalis, were fixed in either cold (5° C) 70% ethanol or cold absolute methanol during their tyrosinase development phase and incubated in buffered (pH 7.2) solutions of the enzyme substrate l-dihydroxyphenylalanine. Optical density of the reaction product (melanin) was measured in the whole small embryos at 450 nm with a Vickers M85 scanning and integrating microdensitometer. The frequency distribution of the reaction density in embryos of a population was Gaussian, and the mean optical density in embryo samples (N=25) increased linearly with incubation time when a saturation level of substrate was used. Absolute optical density units of dopa oxidase activity in embryos increased linearly in proportion to the development time preceding melanin granulogenesis thereby suggesting that the enzyme activity measured by this procedure is proportional to the amount of tyrosinase present. Since this developmental increase in activity was blocked by treatment of the embryos with puromycin, an inhibitor of protein synthesis, the change is apparently caused by new enzyme synthesis. The microdensitometry assay also confirmed results obtained previously with a radiometric assay: embryos cleavage-inhibited at 7 h development time with cytochalasin B to produce giant melanocytes developed only the same amount of enzyme activity as control embryos.     

Histological,Biochemical, and Ultrastructural Studies on Hyperpigmented Human Skin Xenografts

The mechanisms for hyperpigmentation observed in human cutaneous xenografts placed on athymic nude mice was investigated. Histologic, biochemical, histochemical, and ultrastructural examinations were performed on human skin prior to grafting and at various times ranging from 2 weeks to 30 weeks post-grafting (PG). Hyperpigmentation was macroscopically visible on the graft as early as 4–6 weeks. The number of Dopa-positive melanocytes per unit area was increased at 2 weeks PG and remained elevated until 20 weeks PG. The surface area of the melanocytes, a measure of the activity of the cells, also increased significantly and remained above the pre-grafting size throughout the study. Western blot analysis using tyrosinase specific antibody (αTy-SP) revealed the presence of tyrosinase exclusively in the grafted skin from 2 weeks to 12 weeks PG tested. Histological and ultrastructural observations revealed the presence of numerous dendritic melanocytes, indeterminant clear cells suggestive of Langerhans cells, and dermal melanophages. The results of this study suggest that the observed hyperpigmentation in grafted tissue is caused by an increase in the number of Dopa-positive melanocytes and probably from enhanced melanin production. Extracts of proteins from the xenografts exhibited prominent differences in low and high molecular proteins between pre- and post-grafted skin. Among them, the exclusive appearance of a protein doublet with apparent mw ～14 kDa was found in grafted skin, and subsequent studies indicate it has potent effects on melanocyte function.     

Comparison of kinetic and end-point microdensitometry for the direct quantitative histochemical assessment of cytochrome oxidase activity

Synopsis Cytochrome oxidase activity has been assessed by a method of kinetic microdensitometry which involves applying tissue sections to gel films containing phenylamine substrates and measuring the rate of azine dye production by continuously recording the rate of change in extinction. Optimum conditions for the technique were defined, and the results compared with those obtained by conventional end-point microdensitometry in which sections are incubated in histochemical substrate solutions and azine dye production estimated by a single measurement of extinction at the end of the incubation period. When compared with biochemically-determined enzyme activity, kinetic microdensitometry gave a better index of the proportionate activity of cytochrome oxidase in various normal tissues than did end-point microdensitometry. In addition, the degree of inhibition of cytochrome oxidase activity in tissues removed from cyanide-poisoned animals was assessed more reliably by kinetic microdensitometry than by end-point measurements. With end-point microdensitometry, the reaction is non-linear over the comparatively long incubation times required and there is also a spontaneous reactivation of cyanide-inhibited cytochrome oxidase during incubation and thus a progressively increased rate of substrate utilization. In contrast, with kinetic microdensitometry the initial linear reaction rate is measured before significant reactivation occurs. Kinetic microdensitometry can be used for direct dynamic quantitation of enzyme activity in tissues or cells; it may be a valuable technique for quantitative histochemical confirmation or extension of biochemical studies; and it appears to be a reliable direct quantitative histochemical method for investigatingin vivo inhibition of enzyme activity, where spontaneous reactivation of the enzyme-inhibitor complex may occur.     

Simultaneous azo-coupling method for steroid acetate hydrolyzing enzyme

A simultaneous azo-coupling method for histochemical localization of steroid acetate hydrolyzing enzyme is described. It is based on the observation that d-equilenin, a natural oestrogenic steroid hormone, forms deeply coloured insoluble reaction products with diazonium salts under reaction conditions suitable for histochemical purposes. An acetate at position 3 of d-equilenin is rapidly hydrolysed by tissue esterase and the liberated d-equilenin couples with a diazonium salt to form a coloured precipitate. Steroid acetate hydrolyzing enzyme activity was observed in various tissues of the rat; a comparison with nonspecific esterase activity using alpha-naphthyl acetate as substrate suggested that steroid acetate hydrolyzing enzyme activity represents the activity of one or several isozymes of classical nonspecific esterase. This conclusion has also been drawn previously from biochemical studies using esters of other steroids.     

Steady-state study of the mechanism of dopa-oxidase activity of tyrosinase

The mechanism of the dopa-oxidase activity of frog epidermis tyrosinase has been studied. Initial reaction rates have been measured as function of substrate concentrations, L-dopa and oxygen, in the presence and absence of an inhibitor, product of the reaction. Initial reaction rates versus substrate concentrations, without inhibitor, show a linear dependence in the double-reciprocal space, that discarded Ordered and Random mechanisms. Initial reaction rates versus substrate concentrations, in the presence of an inhibitor product of the reaction, show a non-linear dependence in the double-reciprocal space. This point, joined to the former one, indicates a Ping-Pong mechanism, different of the Hexa-Uni type. The reaction is discussed for first time taking into account a trisubstrate mechanism. The experimental results lead to an (Uni Uni Bi Uni) Ping-Pong mechanism. On the other hand, they can explain the differences between known data of tyrosinases from several sources. Michaelis constant have been calculated for both substrates. The values are 0.16 and 7.14 mM for oxygen and L-dopa respectively.     

Localization and activity of cholinesterases in Bidder's ganglion cells and nerve fibers of the frog heart

Summary The histochemical and cytochemical localization of cholinesterases in Bidder's ganglion of the frog heart was investigated and the activity of cholinesterases measured microgasometrically.When either acetylthiocholine or propionylthiocholine was used as substrate, the end product of histochemical and cytochemical reaction was found in all ganglion cells and nerve fibers, the only difference being in the amount of the accumulated precipitate. The end product was localized on the axolemma and in the smooth endoplasmic reticulum of nerve fibers; in the smooth and granulated endoplasmic reticulum of ganglion cells; in the synaptic region between the nerve ending and Schwann cells membrane; and, occasionally, in discontinous streches between the pre- and postsynaptic membrane. The nerve endings of the neurons of the Bidder's ganglion contain predominantly agranular vesicles with occasional clusters of granular ones.Neither the histochemical nor the cytochemical localization of enzyme activity could be detected when butyrylthiocholine was used as substrate. In microgasometric experiments splitting of butyrylcholine was hardly, if at all, measurable. Cholinesterases of the ganglion cells and of the nerve fibers of Bidder's ganglion hydrolyze propionylcholine but at a somewhat lower rate than acetylcholine.A part of this work has been presented at the VI. Congress of the Yugoslav Physiological Society, Ohrid, 1969.This work was supported by the Boris Kidri Foundation, Ljubljana, and by the Federal Research Council, Beograd.     

Microspectrophotometric data on the kinetics of acid phosphatase activity in cells removed from the peritoneal cavity of the rat

Synopsis The extinctions (optical densities) of cells incubated for acid phosphatase in a histochemical azo-coupling procedure (naphthol AS-BI prosphate-hexazotized pararosaniline) have been measured microspectrophotometrically as a function of the incubation time and substrate concentration. A microcuvette was designed for the incubation. The amount of the reaction product in the cells at 23±1°C was found to be proportional to the incubation time for at least 40 min. Lineweaver-Burk plots were linear for some cells while in others nonlinear plots suggested the presence of two or more enzymes. This suggestion was supported by results obtained from polyacrylamide gel electrophoresis experiments.     

Melanosome capping of keratinocytes in pigmented reconstructed epidermis--effect of ultraviolet radiation and 3-isobutyl-1-methyl-xanthine on melanogenesis

Reconstructed pigmented epidermis was established by co-seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air-liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3-isobutyl-1-methyl-xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.     

Melanosome Capping of Keratinocytes in Pigmented Reconstructed Epidermis – Effect of Ultraviolet Radiation and 3‐Isobutyl‐1‐Methyl‐Xanthine on Melanogenesis

Reconstructed pigmented epidermis was established by co‐seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air–liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3‐isobutyl‐1‐methyl‐xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.