Väinö K. Hopsu-Havu

Improvements in the method for the electron microscopic localization of arylsulphatase activity

SummaryThe optimal conditions for the demonstration of arylsulphatase activity in the proximal convoluted tubule cells of the rat kidney were studied at light and electron microscopic level. 8-hydroxyquinoline sulphate, p-nitrophenyl sulphate and 2-hydroxy-5-nitrophenylsulphate were used as substrates and barium and lead as capturing ions. The effect of fixation, capturing ions, substrate concentration and pH was studied biochemically. The results of these biochemical studies were then verified histochemically. Finally a recommended method for the light and electron microscopic demonstration of arylsulphatase activity was presented.

Human skin proteases

SummaryPsoriatic scale proteases were found to be extracted effectively in salt solution (1 mol/l) containing Triton X-100 (5 g/l). The extraction in dilute buffer or sucrose yielded low activities. The acid (0.25 N H2SO4) and KSCN (2 mol/l) solutions effectively extracted plasminogen activator. Fibrinolysin was most active in salt (1 mol/l KCl) and in KSCN (2 mol/l) extracts.Psoriatic scale proteases were fractionated by Sephadex G-100 gel filtration and further by DEAE cellulose chromatography. Five different enzyme preparations were obtained. The first preparation, resembling cathepsin D, effectively hydrolysed hemoglobin at pH 3.5 and casein at pH 5.8 and was insensitive to protease modifiers. The second preparation effectively hydrolysed trypsin substrates (AGLME, TAME, BAEE and BANA) and also histone and casein at pH 7.2 and was inhibited by protease inhibitors, TLCK and E-600. The third preparation hydrolysed histone and casein at pH 10.2 and was effectively inhibited by E-600 and partially by protease inhibitors and TPCK. The fourth preparation, resembling cathepsin B1, hydrolysed BANA and BAEE at pH 5.8 and was activated by SH-reagents and EDTA. The fifth enzyme preparation hydrolysed ATEE and was inhibited by E-600 and TPCK. Plasminogen activator was found mainly in the second enzyme preparation and fibrinolysin activity in the third and fifth enzyme preparations.The second, third and fifth enzyme preparations were different from the enzymes found in healthy human skin. The proteases of psoriatic scale resemble those of tissue and cell cultures undergoing rapid cell division. The possible role of proteases in the increased cell division in psoriasis plaque is discussed.ZusammenfassungProteasen aus psoriatischen Schuppen konnten in Salzlösung (1 mol/l), welche Triton X-100 enthielt (5 g/l), extrahiert werden. Die Extraktion in verdünntem Puffer oder Saccharose zeigte niedrige Aktivitäten. Die saure Lösung (0,25 n H2SO4) und KSCN (2 mol/l) waren in der Lage, den Plasminogenaktivator zu extrahieren. Fibrinolysin war am stärksten aktiv in Salz (1 mol/l KCl) und in KSCN (2 mol/l).Die Proteasen aus psoriatischen Schuppen wurden mit Sephadex G-100 Gel Filtration und weiter durch DEAE Cellulose-Chromatographie fraktioniert. Es wurden 5 verschiedene Enzympräparationen dargestellt. Die erste Präparation, die an Kathepsin D erinnerte hydrolysierte Hemoglobin bei einem pH von 3,5 und Kasein bei einem pH von 5,8 und war unempfindlich gegenüber Proteasehemmern.Die zweite Präparation hydrolisierte Trypsinsubstrate (AGLME, TAME, BAEE und BANA) und auch Histon und Kasein bei einem pH von 7,2 und wurde durch den Proteaseinhibitor gehemmt sowie TLCK und E-600.Die dritte Fraktion hydrolisierte Histone und Kasein bei einem pH von 10,2 und wurde durch E-600 gehemmt und partiell durch Proteaseinhibitoren und TPCK. Die vierte Präparation erinnerte an Kathepsin B1 und hydrolisierte BANA und BAEE bei einem pH von 5,8 und wurde durch SH-Reagentien und EDTA aktiviert. Die fünfte Enzympräparation hydrolisierte ATEE und wurde durch E-600 und TPCK gehemmt. Der Plasminogenaktivator wurde vornehmlich in der zweiten Enzympräparation dargestellt, Fibrinolysinaktivität in der dritten und fünften Enzympräparation.Die zweite, dritte und fünfte Enzympräparation war unterschiedlich von den Enzymen, die in der gesunden menschlichen Haut gefunden wurden. Die Proteasen der psoriatischen Schuppe erinnerten an solche Enzyme, die in Geweben und Zellkulturen mit hoher Mitoserate gefunden wurden. Die mögliche Rolle der Proteasen in der erhöhten Zellteilung im psoriatischen Herd wurde diskutiert.Psoriatic scale proteases were found to be extracted effectively in salt solution (1 mol/l) containing Triton X-100 (5 g/l). The extraction in dilute buffer or sucrose yielded low activities. The acid (0.25 N H2SO4) and KSCN (2 mol/l) solutions effectively extracted plasminogen activator. Fibrinolysin was most active in salt (1 mol/l KCl) and in KSCN (2 mol/l) extracts. Psoriatic scale proteases were fractionated by Sephadex G-100 gel filtration and further by DEAE cellulose chromatography. Five different enzyme preparations were obtained. The first preparation, resembling cathepsin D, effectively hydrolysed hemoglobin at pH 3.5 and casein at pH 5.8 and was insensitive to protease modifiers. The second preparation effectively hydrolysed trypsin substrates (AGLME, TAME, BAEE and BANA) and also histone and casein at pH 7.2 and was inhibited by protease inhibitors, TLCK and E-600. The third preparation hydrolysed histone and casein at pH 10.2 and was effectively inhibited by E-600 and partially by protease inhibitors and TPCK. The fourth preparation, resembling cathepsin B1, hydrolysed BANA and BAEE at pH 5.8 and was activated by SH-reagents and EDTA. The fifth enzyme preparation hydrolysed ATEE and was inhibited by E-600 and TPCK. Plasminogen activator was found mainly in the second enzyme preparation and fibrinolysin activity in the third and fifth enzyme preparations. The second, third and fifth enzyme preparations were different from the enzymes found in healthy human skin. The proteases of psoriatic scale resemble those of tissue and cell cultures undergoing rapid cell division. The possible role of proteases in the increased cell division in psoriasis plaque is discussed.

Distribution of a dipeptide naphthylamidase in rat tissues and its localisation by using diazo coupling and labeled antibody techniques

SummarySeveral rat tissues (liver, spleen, kidney, pancreas, skin, heart, lung and brain) were shown to contain a peptidase capable of liberating naphthylamine from glycyl-dl-proline naphthylamide (Gly-Pro-NA). A single DEAE-cellulose chromatography of autodigested homogenates of the above tissues produced a partial separation of the peptidase from the enzymes hydrolysing l-leucine β-naphthylamide. The Gly-Pro-NA hydrolysing enzyme was localised in tissue sections by using diazo coupling reaction and indirect immunologic techniques. Antibodies were prepared against the enzyme purified from rat liver and kidney in the rabbit. Rabbit γ-globulin was localized by using goat anti-rabbit γ-globulin labeled with fluorescein or with peroxidase.

The decrease of hyaluronate synthesis by anti-inflammatory steroids in vitro.

The effect of anti‐inflammatory steroids (prednisolone and derivatives of hydrocortisone, dexamethasone and betamethasone) on the synthesis of hyaluronic acid and sulphated glycosaminoglycans in human skin fibroblast culture was studied. The concentrations of steroids varied between 1 × 10−10 M and 1 × 10−6 M. All tested steroids decreased the synthesis of hyaluronic acid to the same final level which was about 40–50% of the controls, but the concentrations required varied between different steroids. The relative inhibitory potencies of the steroids were calculated based on concentrations needed to decrease the synthesis of hyaluronate. When the inhibitory potency of hydrocortisone was calculated as one, the values of the other steroids were: prednisolone 5, hydrocortisone 17‐ butyrate 20, betamethasone alcohol 30, dexamethasone alcohol 38, betamethasone 17‐valerate 350–400, dexamethasone monosodium phosphate and betamethasone disodium phosphate over 400. Hydrocortisone sodium succinate was as potent an inhibitor of hyaluronate synthesis as hydrocortisone alcohol. None of the tested steroids affected the synthesis of sulphated glycosaminoglycans at these concentrations. The changes observed in the glycosaminoglycans in the medium were in accordance with the changes in the cell layer. The possible significance of hyaluronate synthesis inhibition by anti‐inflammatory steroids is discussed.

Human cystatins in normal and diseased tissues--a review.

Immunohistochemica and quantitative immunochemical methods were used to demonstrate the presence of two cysteine proteinase inhibitors, cystatins A and B, in normal and diseased tissues. Cystatin A is expressed in squamous epithelia, neutrophil granulocytes, and dendritic reticulum cells of the lymphatic tissues. Its concentration is increased in inflammatory skin diseases and decreases after the malignization of squamous epithelia. Cystatin B is seen in wet squamous epithelia, and in the cells of monocyte-macrophage series, where its concentration varies depending on the activation state of the cells. In the malignant keratinocytes cystatin B follows the behaviour of cystatin A.

Mycotic growth and soft denture lining materials.

Mycotic flora was studied from the dentures and denture bearing mucosae of 39 persons who wore soft-lined (Molloplast B) mandibular dentures and heat-cured acrylic resin maxillary dentures. Fungal growth was detected in 85% of the mandibular dentures and in 44% of the maxillary dentures (p less than 0.001). On the mandibular mucosa fungal growth was revealed in 74% and on the mucosa of the maxilla in 69%. In connection with inflamed mucosae fungal growth was always detected on the mandibular denture and on the mandibular mucosa in 93% as well as on the maxillary denture in 50% and on the maxillary mucosa in 75%. Considering the healthy mandibular mucosa fungus was found in 75% on the mandibular dentures and in 62% on the mucous membranes. In connection with healthy maxillary mucosae the corresponding figures were 42% and 68%. The specimens revealed 7 different yeasts and 2 moulds. The most common fungi were Candida albicans (86%), Torulopsis glabrata (31%), and C. tropicalis (14%). The uncured Molloplast material caused a definite inhibition of candida growth in vitro, while the cured material indicated no growth inhibition.

Demonstration of immunoreactive acid cysteine-proteinase inhibitor in reticulum cells of lymph node germinal centres.

SummarySeven human lymph nodes showing different types of reactive change, were examined for the presence of acid cysteine-proteinase inhibitor (ACPI) by the peroxidase-antiperoxidase method. A clear positive reaction was found in the germinal centres. The staining pattern indicated immunoreactivity of the dendritic reticulum cells, but the possibility that other cells, particularly histiocytic reticulum cells, may also react with antiserum raised against ACPI cannot be excluded.

Immunofluorescent localization of trypsin-like esteropeptidases in the mouse submandibular gland

SynopsisThe localization of trypsin-like esteropeptidases purified from the submandibular gland of the male mouse was studied with the indirect immunofluorescent method. The six esteropeptidases isolated previously were found to be antigenically closely related to each other and were located in the granular ducts of the gland. None of the enzymes was present in the sublingual gland.

Effect of five anti-inflammatory steroids on collagen and glycoaminoglycan synthessis in vitro

The effect of five anti‐inflammatory corticosteroids, i. e. hydrocortisone, hydrocortisone 17‐butyrate, betamethasone 17‐valerate, nicocortonide acetate and nicocortonide, on the synthesis of hyaluronic acid, sulphated glycosaminoglycans and collagen by cultured skin fibroblasts was studied. As inhibitors of all these parameters the steroids could be arranged in order hydrocortisone < hydrocortisone 17‐butyrate < betamethasone 17‐valerate, nicocortonide acetate and nicocortonide. The corticosteroid concentrations required for inhibition of hyaluronic acid were very low as compared to those required for inhibition of sulphated glycosaminoglycan and collagen synthesis.

Human skin proteases. fractionation and characterization

SummaryProteolytic enzymes of human skin extracts were fractionated by using Sephadex G-100 and Sepharose 6 B gel filtrations. The preparations obtained were characterized enzymologically. Several fractions containing proteases with clearcut enzymatic characteristics were obtained.Hydrolysis of ATEE (acetyl tyrosine ethyl ester), casein, BAPA (benzoyl arginine p-nitroanilide), BAEE (benzoyl arginine ethyl ester) and TAME (tosyl arginine methyl ester) was found to take place by the first enzyme preparation. The pH-optima were at 7.8–8.2. BAPA, BAEE and TAME were hydrolyzed by a trypsin-like protease that is different from the protease(s) hydrolysing ATEE and casein. These proteases showed a very high molecular size, estimated on the basis of elution in Sepharose 6 B gel, but were not definitely separated from each other. Separate enzymes in the first preparation hydrolyzed BANA (benzoyl arginine naphthylamide) optimally at pH 5.8 and TEE (tyrosine ethyl ester) optimally at pH 7.7.The second protease preparation hydrolyzed BAEE optimally at pH 8.2 and TEE optimally at pH 7.7. Two separate proteases were present in this preparation.The third protease preparation hydrolyzed casein optimally at pH 5.8 and hemoglobin optimally at a lower pH. The enzymes were considered to belong to cathepsin D and E group of proteases.The fourth preparation hydrolyzed BANA (and also BAEE) optimally at pH 5.8; the reaction was affected by SH-activators and chelating agents; the enzyme thus resembles cathepsin B1 (B′).The fifth preparation hydrolyzed TEE with pH-optima at pH 6.8 and 8.0. The reactions at these pH-values showed different characteristics and may thus be due to two separate esteropeptidases of a low molecular size.ZusammenfassungProteolytische Enzyme menschlicher Hautextrakte wurden unter Verwendung von Sephadex G 100 und Sepharose 6 B Gel-Filtration fraktioniert. Nach der enzymologischen Charakterisierung der erhaltenen Fraktionen handelte es sich um Proteasen mit eindeutigem enzymatischen Verhalten. Die Hydrolyse von ATEE (Acetyltyrosinäthylester), Casein, BAPA (Benzoylargininnitroanilid), BAEE (Benzoylargininäthylester) und TAME (Tosylargininmethylester) wurde mit der ersten Enzymfraktion bei einem pH-Optimum von 7,8–8,2 erzielt. BAPA, BAEE und TAME wurden durch eine trypsinähnliche Protease, die sich von den ATEE- und Casein-hydrolysierenden Proteasen unterschied, hydrolysiert. Diese Proteasen besaßen ein hohes Molekulargewicht auf der Basis der Sepharose 6 B Gel Elution, wurden jedoch nicht vollständig voneinander getrennt. Einzelne Enzyme der ersten Präparation hydrolysierten BANA (Benzoylnaphthylamid) bei einem pH-Optimum von pH 8, sowie TEE (Tyrosinäthylester) bei pH 7.7.Die zweite Proteasenfraktion hydrolysierte BAEE bei einem pH-Optimum von 8,2 und TEE bei pH 7.7. Zwei verschiedene Proteasen lagen in dieser Fraktion vor.Die dritte Proteasenpräparation hydrolysierte Casein bei einem pH-Optimum von pH 5,8 und Hämoglobin bei einem niedrigeren pH-Wert. Die Enzyme schienen der Kathepsin D- und E-Gruppe anzugehören.Die vierte Präparation hydrolysierte BANA (ebenso wie BAEE) bei einem pH-Optimum von 5.8. Die Reaktion wurde durch SH-Aktivatoren und Chelatbildner beeinflußt; das Enzym ähnelt insofern dem Kathepsin B 1.Die fünfte Fraktion hydrolysierte TEE mit einem pH-Optimum von 6,8 und 8,0. Bei diesen pH-Werten zeigten die Reaktionen unterschiedliche Charakteristika, die möglicherweise auf das Vorliegen von 2 verschiedenen Esteropeptidasen mit einem niedrigen Molekulargewicht zurückzuführen sind.