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Biomedical subjects

J E Fräki

Publications and source records attributed to J E Fräki.

At least 19 recordsLinked to original sources
PubMed1989

Effect of drugs on histamine radio-enzyme assay.

The effect of over 200 drugs and other compounds on histamine radio-enzyme assay was studied. Some muscle relaxants (e.g. alcuronium), some sympathomimetics (e.g. dopamine, isoxsuprine, tyramine and possibly phenylethylamine), antimalarial drugs, procaine, procainamide, Berenil and serotonin were potent compounds to interfere with this assay. In some special cases still potentially inhibitory drugs seemed to be some muscle relaxants (e.g. vecuronium, pancuronium and tubocurarine), antidepressants, antihistamines (e.g. cimetidine, ranitidine and diphenhydramine), chinidin, disopyramide, tolazoline and salazosulfapyridine.

Histamine↗
PubMed1989

Alpha-fluoromethylhistidine in the treatment of idiopathic cold urticaria.

Alpha-fluoromethylhistidine (alpha-FMH), a new irreversible inhibitor of mammalian histidine decarboxylase, was tested in the treatment of idiopathic cold urticaria in 11 patients. In the initial trial with 50 mg b.i.d., a significant decrease (about 30%) in the total blood histamine level was found after 3 weeks of treatment but clinically there was no improvement in the symptoms of ten cold urticaria patients nor in the responses to the ice-cube test. In the second trial with three patients suffering from severe idiopathic cold urticaria, a higher dose of up to 500 mg b.i.d. of alpha-FMH for 3 weeks resulted in a marked decrease in the total blood histamine level as well as in an apparent inhibition of histamine synthesis in the skin previously exposed several times to cold water. The symptoms of cold urticaria and the responses in the ice-cube tests also decreased simultaneously. No clinical side effects nor changes in laboratory analysis were seen during the treatment with alpha-FMH. These results suggest that alpha-FMH may be useful in the treatment of severe cold urticaria especially in combination with histamine exhaustion of mast cells using cold water.

Administration, Oral↗
PubMed1989

Biochemical and histochemical evaluation of tryptase in various human tissues.

The distribution of tryptase in various human tissue high-salt extracts (skin, lung, pancreas, liver, kidney, and spleen) was studied. Tryptase activity was compared with tissue histamine concentration, chymase activity, and cathepsin D, and histamine-N-methyltransferase (HMT) activities. Tryptase activity, found biochemically in tissue extracts, was localized in tissue sections by an enzyme-histochemical method using peptide 4-methoxy-2-naphthylamide substrates and Fast Garnet GBC as the chromogen. The highest levels of tryptase activity were found in lung and skin extracts. Liver, kidney, and spleen extracts displayed only a little activity. The distribution of histamine was similar to that of tryptase, whereas distributions of cathepsin D and HMT were quite different from that of tryptase. High-salt extracts of lung contained no detectable chymase activity, but in skin extracts this activity was high. Using an enzyme-histochemical method, the tryptase activity in tissue sections seemed solely to be confined to cells, which were granular and Giemsa positive after the red azo dye had been removed with Tween 20. Skin and lung sections contained the highest number of positively stained cells. The inhibition properties of tryptase, found in both tissue extracts and sections, and the substrate profile in tissue sections were identical. Human leukocyte preparation was negative for tryptase when stained enzyme-histochemically. The present results suggest that tryptase in human tissues is found only in the mast cells. The enzyme seems to be identical in the various human tissues studied because the different high-salt extracts were immunologically cross-reactive when tested with a rabbit polyclonal antibody against skin tryptase.

Histocytochemistry↗
PubMed1989

Effect of handling of plasma samples on histamine radio-enzyme assay.

1. Freeze-thawing of plasma samples increased the histamine level at physiological histamine concentrations as analyzed by radio-enzyme assay, but not by high performance liquid chromatography. 2. Heating of plasma samples decreased the histamine levels. 3. Enzymatic or non-enzymatic formation of histamine during handling of plasma samples was not detected. 4. Some albumin preparations contain histamine.

Adult↗
PubMed1989

Comparison of histamine assay methods in measuring in vitro-induced histamine release in patients with allergic rhinitis.

histamine release tests using whole blood were applied in testing in vitro Type I birch allergic reaction in two patients with allergic rhinitis. Heparinized blood was incubated with varying dilutions of allergen for 30 min at +37 degrees C. After centrifugation of the blood, plasma was separated and the liberated histamine was analysed by histamine radio enzyme assay (REA), high performance liquid chromatography (HPLC) with post-column derivatization system or radio immuno assay (RIA), and the results obtained by these methods were compared. REA and HPLC gave similar results, while RIA gave somewhat higher values, but was less sensitive.

Adolescent↗
PubMed1989

Effect of human mast cell tryptase on human plasma proenzymes.

The effect of human skin mast cell tryptase on human plasma proenzymes (prothrombin, coagulation factor XII, complement C1s, protein C and plasminogen) was investigated. Tryptase had no effect on these proenzymes, when incubated with them at 37 degrees C for up to 90 min, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by the ability to hydrolyze specific peptide p-nitroanilide substrates. After prolonged treatment with tryptase, proenzymes could be fully activated with their specific activators. The results indicate that tryptase neither activates these plasma proenzymes nor inactivates the corresponding active enzymes. As a positive control, the tryptase preparation was also incubated with human fibrinogen and rat thymus histones. Prolonged treatment with tryptase increased the thrombin-induced clotting time of fibrinogen. Tryptase also efficiently hydrolyzed histone H1 from rat thymus. Histones H3/H2B and H2A were hydrolyzed less efficiently than H1, and no hydrolysis of histone H4 by tryptase was detected under the experimental conditions.

Animals↗
PubMed1988

Human skin tryptase: purification, partial characterization and comparison with human lung tryptase.

Human skin tryptase was isolated using stepwise low- and high-salt extraction and further purified 448-fold with 33% yield using octyl-Sepharose CL-4B hydrophobic affinity chromatography, Sephacryl S-200 gel filtration and finally octyl-Sepharose CL-4B or cellulose phosphate ion exchange chromatography. The skin tryptase, which has an apparent Mr of 120,000 by gel filtration in high-salt buffer, consisted of polypeptide chains of Mr 34,000 and 38,000 when resolved on SDS gels. Both polypeptide chains, labelled with [3H]diisopropyl fluorophosphate, indicated that they were representative of subunits and that the native proteinase was an aggregate of subunits. However, in some preparations only one band with Mr 34,000 was seen. In low-salt buffer the enzyme was labile and at least 1.4 M KCl was needed to keep the enzyme stabile when incubated at 37 degrees C for 30 min. Heparin glycosaminoglycan partially stabilized the tryptase but addition of protein (e.g. albumin, 80 micrograms/ml) to the tryptase-heparin mixture was needed to keep the enzyme stabile. Tryptases purified by exactly the same method from human lung tissue and from human skin had identical molecular size in gel filtration and in SDS-polyacrylamide gel electrophoresis. They also revealed identical enzyme kinetic parameters with several synthetic peptide substrates. The inhibition profile was identical for both enzymes, and they also crossreacted completely in immunodiffusion plates. These studies strongly indicate that mast cells found in skin as well as lung contain closely related, possible identical trypsin-like proteinases.

Chromatography, Gel↗
PubMed1988

The allosteric effect of salt on human mast cell tryptase.

The inhibitory effect of potassium chloride and ammonium sulphate on purified human skin tryptase and bovine trypsin was studied enzyme-kinetically, using Z-Gly-Pro-Arg-pNA, Z-Gly-Pro-Arg-AMC, benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME) as substrates. With increasing salt concentrations, the curve of reaction velocity vs. substrate concentration changed from hyperbolic to sigmoidal when anilide substrates (Z-Gly-Pro-Arg-pNA or -AMC) were used. Only the Km value increased, while the Vmax value remained unchanged. The trend was similar with BAEE or TAME as the substrates. However, the effect of salt on the hydrolysis of these ester substrates was not as strong as on the hydrolysis of anilide substrates, and sigmoidal kinetics were not observed even at the highest KCl concentration (0.7 M) used. Heparin, used as a stabilizer, had no influence on this phenomenon, but it did slightly decrease the apparent Km and Vmax values in low-salt conditions. By comparison, trypsin was not as strongly affected by salt as tryptase, and the inhibition type was mixed competitive and non-competitive. The present results indicate that the salt acts on tryptase as an allosteric effector, and this should be carefully considered when enzyme kinetic parameters and enzyme activity of skin tryptase are measured.

Ammonium Sulfate↗
PubMed1988

Hydrolysis of histones by proteinases.

Hydrolysis of histones by proteinases from rat liver, skin and other sources was studied by using a rat thymus histone preparation as the substrate and polyacrylamide-gel electrophoresis and densitometric analysis as the methods to detect histone subtypes and their hydrolysis. The rat mast-cell proteinase I effectively hydrolysed histones except type H4. Thrombin hydrolysed effectively histones H1 and H2A, whereas plasmin hydrolysed all types of histones. Cathepsin D hydrolysed especially histone H2A. Cathepsins B and L hydrolysed all histones more slowly, and cathepsin H hydrolysed them extremely slowly. Epidermal aminoendopeptidase did not hydrolyse histones. Trypsin and chymotrypsin were used as reference enzymes, which hydrolysed all types of histones in very low concentrations. This study suggests that a variety of proteinases could play a role in histone hydrolysis. Hydrolysis of a specific subtype of histones, such as histone H2A at pH 6 by cathepsin D, may be directly involved in regulation of epidermal-cell differentiation.

Aminopeptidases↗
PubMed1988

Optimization of histamine radio enzyme assay with purified histamine-N-methyltransferase.

The radio enzyme assay for histamine based on the transmethylation with purified histamine-N-methyltransferase and utilizing [3H-methyl]-S-adenosylmethionine as the methyl donor has been optimized to measure low histamine concentrations, for example in plasma. The pH-optimum for the assay is pH 8.3 in Tris-glycine buffer at 20 degrees C. An incubation time of 90 min is necessary using an enzyme concentration of 5.8 micrograms/ml. EDTA and dithiothreitol were included in the assay to keep the histamine-N-methyltransferase active as agents that oxidize -SH groups were found to be inhibitory to the reaction. The present assay is sensitive to about 0.5 nmol/l of histamine in a sample volume of 50 microliter (about 3 pg/sample).

Animals↗
PubMed1988

Histologic characteristics of lichen planus transplanted onto nude mice and cultured in vitro.

Typical confluent lesions of lichen planus were transplanted onto nude mice and cultured in organ culture. The characteristic histologic appearance of lichen planus disappeared after grafting and became similar to normal skin within 6 weeks on nude mice; the dense lymphocyte infiltrate in dermis disappeared, the basal cell layer normalized, and the colloid bodies disappeared from epidermis, although some of them were found in dermis. The granular layer also normalized, but the stratum corneum remained hyperkeratotic 6 weeks after transplantation. In organ culture, characteristic histologic features of lichen planus disappeared in 3-5 days via a rapid necrosis of the upper part of the epidermis and formation of a new, normal-looking basal epidermis. These results suggest that lesions of lichen planus are primarily dependent on the influence of the host to maintain their typical histologic appearance.

Adult↗
PubMed1988

Immunoperoxidase and enzyme-histochemical demonstration of human skin tryptase in cutaneous mast cells in normal and mastocytoma skin.

Trypsin-like proteinase isolated from human skin was localized in cutaneous mast cells using immunoperoxidase and enzyme-histochemical techniques. Skin biopsy specimens were taken from four mastocytoma and four healthy patients. Immunoperoxidase staining was performed with protein A-sepharose purified rabbit polyclonal antibody raised against human skin tryptase and using aminoethylcarbazole as chromogen. The positively stained cells in the dermis were granular in character. Using peptide 4-methoxy-2-naphthylamide substrates (Bz-Arg-MNA, Z-Lys-Arg-MNA, Z-Gly-Arg-MNA, Z-Pro-Arg-MNA and Z-Gly-Pro-Arg-MNA) and Fast Garnet GBC as chromogen the red azo dye was found to precipitate in the cytoplasmic granules of the cutaneous mast cells. The enzymatic reaction was totally inhibited by diisopropyl fluorophosphate, leupeptin, and benzamidine. No marked inhibition was seen with soybean trypsin inhibitor and alpha-1-anti-trypsin. The best substrate was Z-Gly-Pro-Arg-MNA giving the strongest red azo dye when incubation time was 15, 30 or 60 min. These results show the localization of human skin tryptase in dermal mast cells and the usefulness of Z-Gly-Pro-Arg-MNA as a suitable substrate tested for enzyme-histochemical localization of mast cells in healthy or mastocytoma skin.

Histocytochemistry↗
PubMed1988

Purification and identification of two serine class proteinases from dog mast biochemically and immunologically similar to human proteinases tryptase and chymase.

Serine class proteinases with trypsin-like and chymotrypsin-like specificity were purified from dog mastocytoma tissue. An antiserum was produced against the chymotrypsin-like proteinase. The antiserum reacted with mast cells in skin sections prepared from normal dogs consistent with the proteinase being a mast cell constituent. The antiserum also cross-reacted with the major chymotrypsin-like proteinase isolated from normal dog skin and partially cross-reacted with human skin chymase. No cross-reaction was detected with rat chymase. The trypsin-like proteinase from dog mastocytoma tissue was similar to tryptase isolated from human skin. It had a similar subunit structure, was not inhibited by many protein proteolytic enzyme inhibitors, bound to heparin, and reacted strongly with antiserum against human tryptase. Antiserum against human tryptase also reacted with mast cells in skin sections prepared from normal dog skin. No immunocytochemical labeling of rat skin mast cells was observed with anti-human tryptase. These studies establish the presence of a trypsin-like and chymotrypsin-like proteinase in dog skin mast cells and provide immunological evidence which suggests that both proteinases are more closely related to human than rat mast cell proteinases. These immunological and biochemical relationships are important when comparing the roles of these proteinases in different animals.

Animals↗
PubMed1988

Comparison of the new antihistamine acrivastine (BW 825C) versus cyproheptadine in the treatment of idiopathic cold urticaria.

A double-blind, crossover trial with a new triprolidine derivative, acrivastine (BW 825C; 8 mg 3 times daily), cyproheptadine (4 mg 3 times daily) and placebo was carried out in 18 patients suffering from idiopathic cold urticaria. Acrivastine and cyproheptadine significantly (p less than 0.01) reduced weal areas following ice cube challenge when compared to placebo. Acrivastine was found to be significantly more effective (p less than 0.01) than cyproheptadine in reducing weal areas. Furthermore, cyproheptadine caused significantly more drowsiness than acrivastine (p = 0.021) or placebo (p = 0.013), which did not differ from each other. This study shows that acrivastine is an effective agent in the treatment of cold urticaria and suggests that acrivastine in the dose used lacks adverse effects, such as drowsiness, traditionally associated with antihistamine therapy.

Adult↗
PubMed1987

Separation and identification of cathepsins in newborn rat epidermis.

Cathepsins B, D, H, and L were identified in the extract of 2-day-old rat epidermis and separated by gel filtration from aminoendopeptidase with a Mr of 400,000 and from the low-molecular-weight cysteine proteinase inhibitor. They were further purified by ion exchange column chromatography. The final separation for cathepsins B and H was performed by gel filtration, while cathepsin D was purified by pepstatin affinity chromatography and cathepsin L by fast protein liquid chromatography (FPLC). Substrate specificity, inhibitor susceptibility, and apparent molecular weights of the separated proteinases were determined and values compared to rat liver enzymes. Apparent molecular weights for epidermal cathepsins B, H, and L were higher than those for comparable liver enzymes of adult rats. The cysteine proteinase inhibitor in epidermis was found to inhibit cathepsins B, H, and L but not cathepsin D and aminoendopeptidase of rat epidermis. This study demonstrates the presence of cathepsin L in the epidermis and describes simultaneous separation and comparison of epidermal catheptic proteinases.

Animals↗
PubMed1986

Serine proteinases in human cutaneous mastocytosis.

The main chymotryptic and tryptic proteinases of human skin were found in high-salt extracts of human dermis. The levels of these enzymes were markedly increased in salt extracts of human cutaneous mastocytosis as compared to the levels found in extracts of involved skin from the same patients, human cutaneous hemangiomas, and normal human skin. These data suggest that the chymotryptic and tryptic proteinases of human skin are primarily of mast-cell origin.

Biopsy↗