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Biomedical subjects

P Kugler

Publications and source records attributed to P Kugler.

At least 91 records · Page 5Linked to original sources
PubMed1982

Ultracytochemistry of aminopeptidase A (angiotensinase A) in the kidney glomerulus and juxtaglomerular apparatus.

Ultracytochemical studies of the Aminopeptidase A (APA; angiotensinase A; E.C. 3.4.11.7) were performed on perfusion fixed rat and mouse kidneys (low concentration aldehydes) using simultaneous azo coupling with alpha-L-Glu-MNA as substrate and HPR as well as HNF as coupling agents. The studies of glomeruli and juxtaglomerular apparatus (JGA) show that APA is localized mainly at the cell membranes of podocytes and endothelial cells (rat and mouse) and of epitheloid cells (mouse) and Goormaghtigh's cells (rat). Increased APA activities are found in the region of cell contacts of epitheloid cells (mouse) and Goormaghtigh's cells (rat). In the epitheloid cells of mice, reaction product is also observed intracellularly in lysosomal structures. Concerning the functional significance of APA in the glomerulus and JGA, it would appear that this enzyme modifies or regulates angiotensin effects in the glomerulus and JGA through the angiotensin degradation.

Adrenalectomy↗
PubMed1982

Aminopeptidase A is angiotensinase A. II. Biochemical studies on aminopeptidase A and M in rat kidney homogenate.

Biochemical fluorometric methods were used to investigate aminopeptidase A (APA; E.C.3.4.11.7) in the rat kidney homogenate and glomeruli and to compare it with aminopeptidase M (APM; E.C.3.4.11.2). It is shown that APA is a calcium-ion-dependent enzyme, while APM is not. To clarify the functional importance of APA and APM in the kidney, their activities were measured under the influence of angiotensins. Fluorimetric measurements in renal homogenate (with 2-naphthylamide derivatives as substrates), which represents mixed-enzyme tissue preparations containing a variety of peptidases besides APA and APM, showed a Km of 0.13 mM for APA and competitive inhibition of ANG II (K1 = 0.015 mM), and a Km of 0.24 for APM and competitive inhibition by ANG III (K1 = 0.003 mM). The remaining two angiotensins showed non-competitive inhibition of APA (ANG I, III) and APM (ANG I, II) in this preparation. For comparison purposes, fluorometric measurements were performed in microdissected glomeruli which contain only APA. A Km of 0.23 mM for the APA and a competitive inhibition of APA by ANG I and II were determined. Thus it was possible to show biochemically that APA is equivalent to angiotensinase A and that both APA and APM participate in angiotensin degradation in the kidney. APA initiating the breakdown of ANG I and II, and APM possibly continuing it in sequential fashion.

Aminopeptidases↗
PubMed1982

Fluorescent histochemical demonstration of cathepsin B in the rat yolk sac.

Fluorescence demonstration of cathepsin B (E.C. 3.4.22.1) activity was performed in the yolk sac of rats near term (18th day of gestation). The enzyme demonstration was performed on freeze-dried and celloidin mounted yolk-sac sections using different substituted beta-naphthylamide derivatives as substrates and nitrosalicylaldehyde as coupling agent. The discrete reaction products are localized preferentially in the apical part of the visceral yolk-sac epithelium. There is little doubt that cathepsin B is contained here in the well developed lysosomal apparatus of the yolk-sac epithelium.

Animals↗
PubMed1982

Histochemistry of angiotensinase A in the glomerulus and the juxtaglomerular apparatus.

The aminopeptidase A (APA; angiotensinase A, E.C. 3.4.11.7) can be demonstrated histochemically (by simultaneous azo coupling) in the glomerulus and JGA of the rat and mouse kidney by light and electron microscopy. In the rat JGA, the APA is localized in Goormaghtigh's cells (mainly at the cell membranes), and in the mouse it is to be found in epitheloid cells (at the cell membranes and in lysosomal structures). From quantitative histochemical investigations of subcortical glomeruli, it can be proven that the aminopeptidase A determined by histochemical means is the angiotensinase A that splits N-terminal aspartate from AI or AII. Animal experiments (sodium depletion, sodium loading, adrenalectomy) bring about marked APA alterations in the glomeruli and in the JGA. The strongest APA alterations can be seen in the adrenalectomized animals, namely an increase of the APA activity in the glomeruli and parts of the JGA. The findings suggest that this enzyme is important for the regulation of the glomerular blood flow and the renin secretion or production by means of angiotensin degradation.

Aminopeptidases↗
PubMed1981

[The histochemistry of human endosalpingeal ciliated cells (author's transl)].

The human endosalpingeal ciliated cells are not a homogeneous cell population. They can be distinguished into mitochondria prominent and normal cells. The morphological appearance of ciliated cells was studied in organ culture using different hormones and hormone combinations in the culture media. Histological (HE, semithin sections) and histochemical methods (PAS, Alcian Blue, SDH, LDH, ATPase, 5'-Nucleotidase, acid and alkaline Phosphatase) were applied. Those mitochondria prominent ciliated cells which are seen in the native endosalpinx can in vitro also be determined mainly under the influence of steroid hormones (hydrocortisone and progesterone). In hormone free incubation media the apices of ciliated cells are separated. This will happen in hormone containing media, too, but with delay, however. Some results are in agreement with the possible transformation from ciliated cells to secretory cells.

Cilia↗
PubMed1981

Kinetic and morphometric measurements of enzyme reactions in tissue sections with a new instrumental setup.

An instrumental setup is described for the measurement of enzyme kinetics and morphometry in tissue sections. It consists of a Vickers M85 microdensitometer and computer-assisted Kontron Videoplan system. The Videoplan system consists of a minicomputer with two mini-floppy disks, a keyboard, a graphic tablet, a TV monitor and a printer/plotter. The measuring component of the M85 is linked to the minicomputer via a BCD interface, and the optical system of the M85 is coupled to a TV camera for display on the monitor screen. The enzyme-kinetic data obtained with the M85 in a specified area of the tissue section (density values as a function of reaction time) are stored in the minicomputer. The measurement process is controlled by a corresponding measuring program. Through correlation analysis (a component of the commercial software) between density values and reaction time, the initial and thus maximum enzyme activity is determined. Upon completion of the kinetic measurements, the measured area of tissue is transferred by the TV camera to the monitor, and the reaction area is described and measured with the graphic tablet in video dialogue and related to the initial enzyme activity. With the setup described, it is possible to make microdensitometric measurements of enzyme activities in a specified tissue area while morphometrically analyzing the associated reaction area. To illustrate the use of the system, enzyme-kinetic (succinate dehydrogenase) and morphometric measurements are performed in tissue sections from the proximal tubule of the rat nephron. Additional applications of the system are discussed.

Animals↗
PubMed1981

Localization of aminopeptidase A (angiotensinase A) in the rat and mouse kidney.

Aminopeptidase A (E.C.3.4.11.7; APA) can be demonstrated histochemically in the rat and mouse kidney by light microscopy (simultaneous azo coupling with alpha-Glu-MNA as substrate and high-purity FBB as coupling agent) mainly in the brush borders, glomeruli and portions of the juxtaglomerular apparatus. Sex and species differences are found with regard to enzyme activity and localization. The relation of aminopeptidase A to angiotensinase A was established by inhibition experiments with angiotensin II and III. The following significant differences exist with respect to other aminopeptidases (aminopeptidase M and gamma-glutamyl transferase), which were also demonstrated: APM shows no dependence on calcium ions; APM and gamma-GT are not demonstrable in the glomerulus or juxtaglomerular apparatus.

Aminopeptidases↗
PubMed1979

A gel-sandwich technique for the qualitative and quantitative determination of dehydrogenases in the enzyme histochemistry. I. Development of the new methods on the example of LDH (E.C. 1.1.1.27).

A gel-sandwich technique for the histochemical demonstration of dehydrogenase is introduced with LDH set up as an example. Especially suitable, of the gels examined, for this technique is 1.5% W/V agar-agar low gel strength. In it several reaction ingredients for the histochemical reaction are dissolved. Considering LDH the following gel composition showed good results: 1.5% W/V agar-agar low gel strength, 5 mM TNBT in 150 microliter DMF, 120 mM L-lactate, 3--5 mM NAD+, 10 mM amytal, 22,4--32 X 10(-5) M Meldola Blue, 160 mM soldium phosphate buffer pH 7.6 (total solution of 1 ml). After the solidification of the gel, gel-bars were frozen with CO2-snow. The 40--80 micrometer thick gel slices were gained in the cryostat. Of the three different arrangement possibilities of the gel slices and the tissue-sections a sandwich arrangement (cover-gel slice--tissue section--ground-gel slice) produced the best results. The enzyme reaction is started by thawing of the gel slices (together with the tissue sections) and by putting them between the hotplate and the evaporator-head-piece, especially developed for this technique. The gel slices also remain in combination with the tissue sections after the reaction. The influence of the gel in combination with the electron carrier Meldola Blue on the spontaneous reduction rates of ditetrazolium salts in day light, were examined as well as the diffusion rates of TNBT and NADH out of gel slices and the influence of DMF and DMSO on the LDH activity. This technique prevents both, the loss of enzymes and the loss of reduction equivalents. There are given presuppositions for qualitative and quantitative histochemical investigations as well. The advantages of the new gel technique are discussed.

Adrenal Glands↗
PubMed1978

Meldola blue: a new electron carrier for the histochemical demonstration of dehydrogenases (SDH, LDH, G-6-PDH).

A new electron carrier, Meldolar Blue (8-dimethylamino-2,3-benzophenoxazine; Boehringer Mannheim GmbH, Deutsche Patentschrift P 1959410) was tested for its usefulness in the histochemical demonstration of dehydrogenase activity in adrenal cortex, liver, heart muscle of guinea pig and human oviduct and compared with PMS. For demonstrating SDH activity Meldola Blue (MB) is as efficient as PMS. A decisive advantage of MB as compared with PMS is its low sensitivity to light exposure, facilitating direct visualisation of histochemical reaction processes. Generally, a high diffusion rate of reduced electron carriers (PMS and MB) from the section into the incubation medium (PVA) leads to a loss of reduction equivalents, particularly in the demonstration of NAD- or NADP-dependent dehydrogenases (LDH, G-6-PDH) with lower TNBT concentrations. However, no inhibition of SDH-, LDH- and G-6-PDH activities was observed with incubation media containing the tested concentrations of PMS and MB.

Adrenal Glands↗
PubMed1978

Studies on the optimalisation and standardisation of the light microscopical succinate dehydrogenase histochemistry.

The present investigation was undertaken in order to establish an optimal tissue pretreatment and an optimal incubation medium for the histochemical demonstration of succinate dehydrogenase (E.C. 1.3.99.1). The investigations were performed on steroid producing (testicle, adrenal gland) and steroid dependent (Fallopian tube) tissues. We studied the influences fo formalin fixation, acetone, magnesium ions, cyanides, electron carries (phenazine methosulfate, menadione coenzyme Q10), osmolarity, substrate concentration and inhibitors (oxalacetate, oxalate, malonate, 4-chloromercuribenzoic acid). The following procedure yields blameless morphological integrity and enzyme localization as well as optimal SDH-activity: Freezing of tissue cubes (diameter less than 5 mm) in propane cooled with liquid nitrogen or in melting freon. Incubation of 5 micrometer cryostat sections in narrow jars in the following medium (38.5 ml):--10 ml of 0.2 M sodium phosphate buffer pH 7.6 (52 mM).--18 mg tetranitro-BT in 0.5 ml dimethylformamide and aqua bidest. ad 10 ml (0.5 mM).--2.6 mg KCN in 16 ml aqua bidest. (1 mM).--540 mg succinate (disodium salt, hexahydrate) in 2 ml aqua bidest. (52 mM).--3 mg PMS (phenazine methosulfate) in 0.5 ml aqua bidest. (0.25 mM). The incubation medium has an osmolarity of 440 mosm. The incubation is carried out for 10 min at 37 degree C in darkness. To avoid non specific formazan deposits in lipid containing tissues a preincubation of the cryostat sections in 100% acetone at--22 degree C or--40 degree C for 7--10 min and an incubation time of 20--30 min is recommended. Control incubations adduced proof at the specificity of the SDH demonstration. Parallel incubation without PMS in order to determine indirectly the content of endogenous CoQ10 is further recommended.

Adrenal Glands↗
PubMed1976

[Histochemical and histological investigations on the human fallopian tube under different hormonal influences. I. Demonstration of ATPase with special reference to reactive ciliated cells (author's transl)].

The localization of the Mg++-activated adenosine triphosphatase (ATPase) in the human Fallopian tube has been studied by means of histochemical methods. The samples were obtained from 18 women in the age from 23--62 years. Some of them were treated by various steroid hormones. Endosalpinx ciliary ATPase-activity represents dynein and is therefore an indicator of ciliary motility. Estrogens and gestagens have a different influence on the ATPase-activity. All cilia of one ciliated cell react in the same manner and may be regarded as a reaction unit. The relation of negative to positive ciliary borders differs characteristically in the tubal isthmus, ampulls and infundibulum and coincides with commonly known phenomena of egg transport through the oviduct. Postovulatory, reaction units increase in ampulla and infundibulum compared with the proliferative phase. The oviducts of postmenopausal women possess but a scanty outfit of reaction units. Short-time treatment with estrogen in the early secretory phase results in a great number of reaction units in all tubal segments; a similar treatment in the proliferative phase diminishes the reaction units in the ampulla. Midcycle progesterone treatment activates the ciliary ATPase in the isthmus. Low doses of lynestrenol (minipill) in the proliferative phase leads to a decrease of reaction units in all tubal segments; the pattern of ciliary reaction under low doses of lynestrenol at the time of ovulation coincides with that of the proliferative phase. Treatment with a contraceptive steroid (0,05 mg ethinylestradiol and 0,25 d-norgestrel) causes a considerable activation of the ciliary ATPase in all portions of the oviduct.

Adenosine Triphosphatases↗