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J Seitz

Publications and source records attributed to J Seitz.

At least 127 records · Page 7Linked to original sources
PubMed1987

Cytochemistry and biochemistry of acid phosphatases VII: Immunohistochemistry of canine prostatic acid phosphatase.

Acid phosphatase (E.C. 3.1.3.2.) has been isolated from canine prostatic gland homogenates by gel permeation chromatography (AcA34 or G150), by affinity chromatography (con A-Sepharose), or by using fluid phase liquid chromatography (FPLC) using Superose 12 and Mono P columns. Acid phosphatase-enriched fractions were submitted to analytical SDS-PAGE or to analytical isoelectric focusing. A protein with a molecular weight of 30 kD (on SDS gels) was used for immunization of rabbits. The antiserum produced was cross-reactive with prostatic acid phosphatase (canine and human) as shown by immunoblotting. When applied to paraffin or plastic sections of normal canine prostate, a positive immunoreaction was found exclusively in the secretory cells. In experimentally altered glands (castration and/or hormone treatment), a varying pattern of immunoreactive cells was found. In canine prostatic carcinomas, intensively reacting cell clusters were found along with nonreactive cells. The antiserum was also slightly cross-reactive with the respective human antigen, but the cross-reactivity of an antiserum prepared against human prostatic secretory acid phosphatase with canine prostatic acid phosphatase was far more pronounced.

Acid Phosphatase↗
PubMed1986

Immunoelectron microscopic evidence for different compartments in the secretory vacuoles of the rat seminal vesicles.

Immunoelectron microscopy of the rat seminal vesicle was performed using specific antibodies to secretory proteins. Proteins were precipitated from rat seminal vesicle secretion and were separated by SDS-polyacrylamide gel electrophoresis. Among the great number of bands the two most prominent bands were selected and designated SVS II and IV. Their apparent molecular weights were 48 kDa and 16.5 kDa respectively. The bands were excised from the gels and used for antibody production in rabbits. The respective antisera were used for immunohistochemical studies both at the light and electron microscopic levels in the rat seminal vesicle and the different prostatic lobes in infantile, adult and castrated animals. A positive immunoreaction was observed in seminal vesicle and lateral prostatic epithelium of the intact adult rat, while it was lacking in prepubertal and castrated animals. The subcellular distribution of both proteins was clearly different: SVS II was exclusively confined to the electron dense core of the secretory vacuoles, while SVS IV was detected only in the clear halo surrounding the central granule. It is suggested that the spatial arrangement of both proteins in the seminal vesicle secretion vacuole reflects a particular functional significance of each of these proteins. These proteins may serve as a tool in the study of regulation of androgen-dependent protein synthesis.

Androgens↗
PubMed1986

Vaginal fluid adenosine 3',5'-cyclic monophosphate (cAMP) in the rat: interaction with sperm cAMP-dependent protein kinase regulatory subunits.

Cyclic AMP (adenosine 3',5'-cyclic monophosphate) concentrations were determined in rat vaginal fluids throughout the estrous cycle. Radioimmunoassay results demonstrated that estrus and early metestrus vaginal fluids had significantly (p less than 0.01) elevated cAMP concentrations compared to proestrus, late metestrus, and early and late diestrus. Ovariectomy reduced RIA-detectable cAMP in vaginal fluid. When cauda sperm were preincubated for 5 min with vaginal fluids from each stage of the estrous cycle, results demonstrated that only estrus- and early metestrus-stage vaginal fluids caused a decrease in [32P]-8N3cAMP (8-azido photoaffinity analogue of cAMP) photolabeling of sperm cAMP-dependent protein kinase regulatory subunits RI and RII. To examine if this reduction in [32P]-8N3cAMP photoincorporation by sperm RI and RII could be due to endogenous cAMP, vaginal fluids were boiled, trypsinized, and/or incubated with EGTA or phosphodiesterase. Only phosphodiesterase-treated vaginal fluids restored sperm regulatory subunit photoincorporation of [32P]-8N3cAMP. It is suggested that cAMP is present in rat vaginal fluids during the estrous cycle in a concentration sufficient to bind the regulatory subunits of rat sperm cAMP-dependent protein kinase.

Animals↗
PubMed1986

Biochemical and immunological relationships of prostatic and leukocytic acid phosphatases and their subcellular localization.

Human acid phosphatases from various sources were purified by a new rapid procedure including chromatofocusing and gel permeation chromatography on an FPLC system. Isoenzymes 2a and 4 (Mr 105,000 and 76,000, respectively) are the prevalent acid phosphatases in prostate, leukocytes and bone marrow. Additional forms are proteolytic products formed during isolation procedure. The source of elevated acid phosphatase in blood serum in metastasizing prostatic carcinoma can be either prostate itself, the tumour or, more likely, bone marrow cells destroyed during the invasion by the tumour cells. Distribution of isoenzymes is not organ specific. It was found using an antiserum against isoenzyme 4 that immunoreactivity is confined to the specific secretory granules both of prostatic epithelium and leukocytes.

Acid Phosphatase↗
PubMed1985

Effects of sexual rest or sexual activity on the structure and function of the ventral prostate of the rat.

Previous studies have shown that sexual activity increases the weight of the accessory sex glands significantly, while the organ weights correlate inversely with the assayable androgen receptor concentrations in the prostate of sexually active versus sexually resting rats. In an effort to determine the structural basis of this phenomenon, the ventral prostates of adult rats kept with female rats for 4-6 months (HE-rats) were compared to those kept in groups of 5 males in one cage (HO-rats) for the same period. As an estimate of the secretory function of the gland the concentration of prostatic binding protein (PBP) was determined in prostatic cytosols using a highly specific ELISA. Catecholamines were measured by means of HPLC and subsequent electrochemical detection. Morphological studies included immunocytochemistry of PBP, visualization of adrenergic nerves, stereological analysis of stromal and glandular compartments of the prostate, and electron microscopy of the epithelium. The main findings were as follows: 1) The prostates of HE-rats were 35% heavier than those of the HO-rats. 2) The content in secretion was in the same range in both HE-rats and HO-rats (1.5 and 1.44 mg PBP per 1 mg DNA). 3) Immunocytochemistry and electron microscopy demonstrated a very homogeneous secretion within the glandular lumen of HO-rats with a diminished amount of secretory material within the glandular cells. In HE-rats the glandular lumina were clearly larger in diameter and intraluminal secretion was less homogeneously stained. The height of the epithelium was increased and the individual secretory cells contained several secretory granules.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1985

Cytochemistry and biochemistry of acid phosphatases V: Electrophoretic studies on the heterogeneity of acid phosphatases from human prostate, seminal fluid, and leukocytes.

Comparison of zymograms of acid phosphatases (orthophosphoric monoester phosphohydrolase, acid optimum, E.C.3.1.3.2) from human prostate, leukocytes, seminal vesicles, and seminal fluid, separated by analytical isoelectric focusing (IEF), resulted in the identification of three individual groups, particularly of the prostate. These groups contain three molecular forms at different molecular weights and activities in varying quantities and combinations. The molecular weights, estimated by SDS gel electrophoresis, were 1) 86,000, 2) 76,000, and 3) 46,000/50,000 (in a doublet) daltons. None of these isoenzymes were restricted to the prostate, but they were present in very high concentrations in the prostate. Compared to the prostate, the seminal vesicles and isolated leukocytes had very closely related zymograms of acid phosphatases. No specific inhibitor has been found that would selectively inhibit one particular isoenzyme without affecting the others. Protein titration and staining activity of IEF gels at different pH values showed that the isoenzymes from all three groups have high hydrolytic activity of orthophosphoric monoesters beyond the acidic pH range of pH 4-5. Incubation of acidic isoforms of acid phosphatases with neuraminidase did not result in the formation of a homogeneous stem molecule. Further analysis of the secretory moiety using the western blotting method showed a binding of peroxidase-conjugated concanavalin A (Con A), indicating that these isoenzymes are glycoproteins. Moreover, isoenzymes extracted from prostate, seminal fluid, and human leukocytes are immunologically identical.

Acid Phosphatase↗
PubMed1985

Cytochemistry and biochemistry of acid phosphatases. VI: Immunoelectron microscopic studies on human prostatic and leukocytic acid phosphatases.

Using different antisera against secretory and lysosomal prostatic acid phosphatases, the localization of the respective antigens was studied in the human prostate at the ultrastructural level. Secretory acid phosphatase was confined exclusively to the secretory vacuoles of the glandular cells. Discharge of the secretory material occurs in a merocrine type of secretion. The identical antigen could be localized in the primary and secondary granules of neutrophil and eosinophil granulocytes separated from human peripheral blood. The antiserum used was also cross-reactive with the canine prostate, where a very distinct immunoreaction was observed with the secretory granules of the glandular cells. The antibodies directed against lysosomal acid phosphatases prepared from prostatic homogenates consistently gave a positive immunoreaction with dense bodies, lipofuscin, and secretory granules. The respective antigens were present also in neutrophil and eosinophil granulocytes. These findings do not identify the existence of a prostate-specific acid phosphatase, which does not exist. The secretory form of the isoenzymes, however, is clearly distinct from the lysosomal form, both of which are present in granulocytes. Therefore the origin of acid phosphatases elevated in peripheral blood in cases of metastatic prostatic cancer could be either the carcinomatous cells or leukocytes destroyed during the process of metastasis.

Acid Phosphatase↗
PubMed1985

Ultrastructural localization of uteroglobin immunoreactivity in rabbit lung and endometrium, and rat ventral prostate.

Recent biochemical studies have demonstrated amino acid sequence homologies between uteroglobin from rabbit endometrium and prostatic binding protein from rat ventral prostate. We have studied the ultrastructural distribution of uteroglobin-immunoreactive material in rabbit lung and endometrium and rat ventral prostate using an uteroglobin antibody raised in guinea pigs. Secretory granules of bronchiolar Clara cells, endometrial non-ciliated cells and rat prostate secretory cells gave a positive immunoreaction when this antibody was used. The results indicate a close relationship of immunoreactive epitopes of proteins present in those secretory cells. The functional properties of these proteins (glycoproteins, steroid binding, androgen-dependent secretion) suggest a close functional relationship, for instance a surface action such as coating, capping, masking or lubrication.

Androgen-Binding Protein↗
PubMed1985

[Immunohistochemical identification of functional relationships in the accessory sex glands].

The distribution of tissue- and organ specific marker proteins has been studied in the accessory sex glands of different mammals. Tissue specific marker proteins are desmin, actin, vimentin, laminin, keratin and related proteins which are well suited to identify smooth muscle cells, fibrocytes, basal cells of the epithelium etc. Secretory proteins were used as markers for organ specificity, e.g. secretory acid phosphatase, prostate specific antigen. In the accessory sex glands of the rat a most complicated pattern of typical secretions was identified using specific antibodies. As deduced from this pattern functional relations are likely between the lateral prostate and the seminal vesicle and the dorsal prostate and the coagulation gland respectively. The ventral prostate of the rat has a particular range among these glands and is homologous to the human prostate.

Animals↗
PubMed1983

Physical properties and function of phallolysin.

Phallolysin, a mixture of two to three cytolytic proteins (all of Mr 34 000), has been isolated from Amanita phalloides mushrooms and purified to homogeneity (specific activity 24 000 hemolytic units/mg of protein). After separation by isoelectric focusing, the amino acid composition of two of these proteins has been determined. They are rich in water-soluble amino acids and contain one tryptophan residue each, but no cysteine or methionine. Mr was determined to be 34 000 in the native form as well as under denaturing conditions, indicating that the native proteins exist as monomers. Many of the physical properties of phallolysin are strikingly similar to those of staphylococcal alpha-toxin, e.g., molecular weight, existence of multiple forms, pI values, amino acid composition, and thermolability (60 degrees C). Pure phallolysin allowed us to prepare a radioactively labeled toxin. Labeling was achieved by reaction with formaldehyde, followed by reduction with sodium [3H]borohydride. With the labeled toxin (specific activity 7-14 Ci/mmol, ca. 60% biological activity), we investigated its binding to human A2 erythrocytes. We determined the number of receptors on these cells (2 X 10(4) per cell) as well as their affinity to the toxin (KD = 4 X 10(-9) M). In studies on the mechanism of cytolytic activity, we were able to distinguish at least three sequential events: binding of the toxin to human erythrocytes, K+ release, and membrane rupture (hemoglobulin release). These steps could be characterized by different kinetics as well as by different temperature dependencies. Again, the kinetic data for phallolysin are very closely related to those obtained for staphylococcal alpha-toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amanita↗
PubMed1983

Morphological and functional characteristics of isolated acini used for in vitro studies of prostatic secretion.

Acini of the rat ventral prostate were isolated by interstitial injection of a collagenase-containing medium, subsequent incubation in the same medium and repeated aspiration through pipette tips with decreasing gauge of the tip opening. Functional integrity of the isolated acini was assessed by morphological studies, including transmission and scanning electron microscopy, freeze-fracturing, and immunocytochemistry. Incubation studies with different incubation media were performed monitoring O2-consumption as a parameter of functional activity, in addition to the incorporation rate of radioactively labeled amino acids into newly synthesized proteins. Optimal incubation conditions (shaking water bath, 20 strokes/min, 37 degrees C, gassing with carbogen at 15 min intervals) were found with M 199 medium supplemented with dihydrotestosterone. Stimulation of prostatic secretion was maximal with 10(-7) M of pilocarpin, which was more effective than carbamylcholine. Incorporation of precursors into prostatic proteins proceeded for about 2 h at a linear rate. Thereafter a rapid loss of functional and morphological integrity of the isolated acini was observed including disintegration, vacuolation and lysis of individual cells. The system of isolated prostatic epithelium developed is a useful tool in the study of prostatic secretion in vitro in short term experiments.

Acid Phosphatase↗
PubMed1982

Intracellular localization of Prostatic Binding Protein (PBP) in rat prostate by light and electron microscopic immunocytochemistry.

Extra- and intracellular distribution of Prostatic Binding Protein (PBP) was studied in the different genital organs of the male rat by immunocytochemistry at the light and electron microscopic levels. PBP was extracted from cytosols of rat ventral prostate and used for immunization of rabbits. The specificity of the antiserum raised was tested by "western blotting" and immunoelectrophoresis. From the different fixatives tested for optimal structural and antigenic preservation of the ventral prostate a mixture containing 2.5% paraformaldehyde, 0.5% glutaraldehyde and 0.5% CaCl2 in cacodylate buffer, 0.05 M, pH 7.3 was selected. Using the immunofluorescence technique and the unlabeled antibody enzyme method PBP-immunoreactivity was detected at the light microscopic level in the luminal secretions of the ventral prostate. No reaction was observed with the seminal vesicle, the coagulating gland, the dorsal and lateral prostates, the epididymis and the testis. Intracellular secretory granules reacting with PBP antiserum were exclusively found in the secretory cells of the ventral prostate. Insufficiently fixed cells showed a diffuse generalized reaction of the cytoplasm indicating a leakage of the antigen from the secretory granules. Such artifacts were common in tissue sections processed with the preembedding-staining procedure. At the ultrastructural level therefore mostly the postembedding staining method was performed using both the unlabeled antibody enzyme method and the ferritin-labeled immunoglobulin technique in osmicated, Epon-embedded tissue. Labeling with either method was intense in the secretory granules and the condensing vacuoles, while the labeling density of the rough endoplasmic reticulum and the Golgi cisternae was in the background range. Castration experiments showed that secretory material displaying PBP immunoreactivity was retained within the acinar lumen of the gland for several days after castration, but was absent from most secretory cells already by four days after castration. Immunocytochemistry of PBP therefore is a very sensitive method for analysing the secretory activity and its androgen dependence of the prostate of the rat.

Androgen-Binding Protein↗
PubMed1981

p-Chlorophenylalanine-induced proliferation of the seminal vesicle epithelium.

Intraperitoneal injection of p-chlorophenylalanine (pCPA) methylester (100 mg/kg body weight) results in an activation of the lysosomal system of the secretory cells in the rat seminal vesicle and an elevation of the activities of lysosomal enzymes within 15 min following the injection. Large autophagic vacuoles are formed, sequestering rough endoplasmic reticulum and part of the Golgi apparatus within 2 h. Shortly after the activation of the lysosomal system an elevation of both DNA- and protein synthesis is measured biochemically. 6 h subsequent to the injection a wave o mitoses of the secretory cells begins, reaching a maximum of 6 h later and then declining within 3 h. About 12 h following the injection a second rise in lysosomal activity begins, declining within 24 h. The entire sequence of lysosomal and proliferative activities is inhibited in antiandrogen-pretreated rats. Deduced from these findings the following hypothesis of growth regulation of the accessory sex glands is advanced: enhanced loss of intracellular material during autophagocytosis diminishes the intracellular concentration of a substance curtailing cell division below its effective threshold resulting in division of the secretory cells. The prerequisites of this mechanism are (i) a sufficient distributive capacity of the stroma for hormones (androgens) and metabolic precursors, and (ii) sufficient capacity of the basal cells for transporting the precursors to the secretory cells. Sloughing of the secretory cells separates them from these auxiliary structures (stroma and basal cells) and enables the basal cells to divide.

Animals↗
PubMed1981

Immunohistochemistry of acid phosphatase in the human prostate: normal and pathologic. Cytochemistry and biochemistry of acid phosphatases II.

Three different antisera against human prostatic acid phosphatase were used for direct and indirect immunohistochemical demonstration of acid phosphatase in paraffin sections of infantile and adult normal, hyperplastic and carcinomatous prostatic tissue. All antisera were prepared in rabbits. Antiserum A was prepared from highly purified acid phosphatase extracted from autopsy specimens. Antiserum B was a concentrate of a commercial antiserum used in radioimmunoassay and was prepared from purified extracts of human seminal fluid. Antiserum C was a peroxidase-conjugated antiserum prepared from purified extracts of human seminal fluid. The specificity of the three antisera was compared using different immunohistochemical methods and tissues. It was comparably high in all three antisera which gave only slightly different staining results in prostatic tissue. The staining results in prostatic carcinoma were only dependent on the titer of the respective antiserum. Carcinomas with a cribriform growth pattern showed variable staining, but always had a positive immunoreactions, provided the titer of the antiserum was sufficiently high. Striking differences were observed in metaplastic, atrophic and hyperplastic prostatic epithelium. The most intense reaction was observed in atrophic glands: it was much less intense in hyperplastic and normal epithelium and negative or slightly positive in metaplastic epithelium.

Acid Phosphatase↗
PubMed1981

Cytochemistry and biochemistry of acid phosphatases. III. Inhibition experiments of lysosomal and secretory acid phosphatases of the rat ventral prostate.

Biochemical and cytochemical inhibition experiments of rat prostatic acid phosphatase were performed using enzymes separated on isoelectric focusing (IEF) gels, and thin sections of the rat ventral prostate. Various inhibitors, including L (+) tartrate, mercuric ions and sodium fluoride were applied to electrofocused enzymes which were subsequently stained for acid phosphatase activity. Enzymes focused on IEF gels at pH 7.9 and 8.1, respectively, were inhibited with 1.8 x 10-3 M tartrate, while the enzyme activities with isoelectric points (pl) of 5.6 and 7.15, respectively, were only slightly inhibited by this compound. Using 10-3M mercuric ions, enzymes with pl of 5.6 and 7.15 were inhibited while the enzymes with pl of 7.9 and 8.1 were still active. The biochemical procedures were adapted to chopper sections of perfused-fixed ventral prostate of the rat. Preincubation of the sections with 2.4 x 10-3M mercuric chloride blocked the secretory enzyme and most of the lysosomal enzyme and resulted in an artificial staining of the Golgi apparatus and other cytoplasmic organelles. Nuclear precipitates however were prevented. L (+) tartrate could not be used at the ultrastructural level since it developed false positive results by the formation of lead tartrate. The results indicate that no selective inhibition of either secretory or lysosomal acid phosphatase can be achieved at the ultrastructural level using metal salts or tartrate, respectively.

Acid Phosphatase↗