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W Schulze

Publications and source records attributed to W Schulze.

At least 55 records · Page 3Linked to original sources
PubMed1998

Adenylyl cyclase in the heart: an enzymocytochemical and immunocytochemical approach.

This review provides a discussion of the localization of adenylyl cyclase (AC) in normal mammalian heart tissue employing enzymocytochemistry (detection of the catalytic activity of AC by a metal precipitation technique) and immunocytochemistry (immunolabeling of the enzyme protein with antibodies against AC subtypes). By the metal precipitation technique, AC activity was localized in adult guinea pig cardiomyocytes along the sarcolemma and the T-tubule membranes. This reaction can be enhanced by hormones and guanylyl imidodiphosphate, fluoride, and forskolin. With this technique, no precipitates were detected at the sarcoplasmic reticulum. However, under ischemic conditions, AC activity was also found in the junctional sarcoplasmic reticulum of rat cardiomyocytes. Immunocytochemistry revealed AC in the plasma membrane of rat cardiomyocytes. Detection of AC in the perinuclear space of cardiomyocytes might reflect initiation of synthesis and processing of the enzyme protein. Colocalization of AC with cytoskeleton fibers of non-cardiomyocytes emerging in the cell culture of neonatal rat cardiocytes imply a direct cytoskeletal-AC interaction. Finally, it can be stated that the immunolabeling pattern of AC in cryosections of adult and new-born rat hearts reveals a good correspondence with the localization of AC activity in cardiomyocytes demonstrated by enzymocytochemistry.

Adenylyl Cyclases↗
PubMed1998

Immunochemical and functional characterization of an agonist-like monoclonal antibody against the M2 acetylcholine receptor.

Monoclonal antibodies were raised against a peptide corresponding to the second extracellular loop of the M2 acetylcholine receptor. One of the monoclonal antibodies, B8E5, was selected for further characterization on the basis of its high yield, its isotype (IgG2a), its dissociation kinetics and its agonist-like activity. The epitope recognized by B8E5 corresponded to the N-terminal part of the second extracellular loop of the receptor (V-R-T-V-E-) as determined by competition immunoassays and epitope scanning. The KA of B8E5 for the target peptide was assessed by surface plasmon resonance (SPR) to be 6.5x10(7) M(-1) by equilibrium and 3.7x10(7) M(-1) by kinetic analysis. B8E5 recognized the M2 acetylcholine receptor on rat cardiac tissue. It only recognized the non-reduced receptor in immunoblots. The antibody had no effect on antagonist binding but decreased the affinity for the agonist carbachol. B8E5 decreased the beating frequency of neonatal rat cardiomyocytes. The effect was specific since it was blocked by the target peptide and the antagonist atropine. The EC50 of the antibody corresponded to the KA measured by surface plasmon resonance. The physiological effect of the antibody did not lead to desensitization. The Fab fragments had no physiological effect; subsequent addition of anti-mouse IgG however restored the physiological effect. These results confirm that the N-terminus of the second extracellular loop is a functional target for antibodies against the M2 acetylcholine receptor. They suggest that the functional epitope is only accessible in the non-reduced receptor. The antibodies act through a functional dimerization of the receptor.

Amino Acid Sequence↗
PubMed1998

Differentiation-dependent expression of cardiac delta-CaMKII isoforms.

Despite their important role in controlling the cardiac Ca2+ homeostasis, presence and functions of individual isoforms of the multifunctional Ca2+/calmodulin-dependent protein kinase in the heart are not well studied. Here we report on expression of isoforms of the delta class in two differentiation states of the embryonic rat heart-derived cell line H9c2 compared to adult rat heart. Reverse transcription coupled polymerase chain reaction analysis revealed specific expression patterns of four variants of the delta class (delta B, delta C, delta 4, delta 9) in adult rat heart, H9c2 myoblasts, and skeletal muscle-like H9c2 myotubes. delta C was identified as a common isoform with higher amounts in H9c2 cells and the prominent one in myoblasts. In contrast, expression of delta 9 accompanied cardiac as well as skeletal muscle differentiation. Expression of delta B, however, was representative for differentiated cardiac muscle, whereas delta 4 expression coincided with differentiation into the skeletal muscle-like state. Our results demonstrate differentiation-dependent isoform expression of the delta class of the multifunctional Ca2+/calmodulin-dependent protein kinase of muscle. The identification of cardiac target proteins for this kinase, e.g. the alpha 1-subunit of the L-type Ca2+ channel, the sarcoplasmic reticulum Ca(2+)-ATPase, phospholamban and the ryanodine receptor define H9c2 myoblasts as a suitable model system for further functional characterization of the identified cardiac delta isoforms.

Animals↗
PubMed1998

Immunocytochemical localization of the G-protein sub-unit, G(o) alpha, in rat heart. Implications for a role of G(o) alpha in secretion of cardiac hormones.

The cellular and sub-cellular localization of the G-protein subunit, G(o) alpha, in rat heart was determined by immunofluorescence and immunoelectron microscopy. Using antibodies directed against purified G(o) alpha and an antiserum raised against the C-terminal decapeptide of G(o) alpha, strong immunoreactivity was found in the conducting system of the heart, neurons, and atrial cardiomyocytes. Labeling of ventricles was weak compared to that of atria. In neurons, immunoelectron microscopy revealed G(o) alpha was localized along the inner surface of axolemma and on axoplasmal vesicles. G(o) alpha immunoreactivity in atrial and ventricular myocytes was not restricted to sarcolemma, but was also found on sub-sarcolemmal vesicles with characteristic caveolar morphology. At the level of intercalated discs, labeling was spread over the periphery of intercalated discs avoiding its membrane structures. Additionally, in atrial endocrine cardiomyocytes, approximately 60% of secretory granules revealed G(o) alpha-labeling on the cytoplasmic surface. A small number of these granules stood out due to particularly intense labeling. The observation that these granules were found most-frequently in sub-sarcolemmal areas suggests that they may be mature granules undergoing exocytosis. Therefore, G(o) alpha found on secretory granules of endocrine cardiomyocytes may have a function in regulated exocytosis of cardiac hormones. Sarcolemmal localization of G(o) alpha in atrial and ventricular cardiomyocytes supports the role of G(o) alpha in transmembrane signal transduction. Furthermore, caveolar localization of G(o) alpha may provide a compartmental basis for integrating G(o)-mediated signaling events.

Animals↗
PubMed1998

[Functional outcome of surgical therapy of talus fractures].

Forty-five patients with fractures of the talus were treated operatively at the Department of Surgery in Bergmannsheil Hospital between 1989 and 1995. Of these, 21 patients were operated on the day of the accident. The Marti/Weber classification system was used: 9 fractures were type I, 12 type II, 18 type III and 6 type IV. Twenty-five neck-fractures were classified according to Hawkins (type I-6, type II-10, type III-6, type IV-3). The mean time of follow-up was 37.3 months (minimum of 12 months). One patient needed a primary and 2 other patients a secondary arthrodesis of the ankle and subtalar joint; 2 others required arthrodesis of the talonavicular joint. Eleven patients showed no complaints at follow-up. Twenty-five reported complaints only during strenuous exercise, 5 others during walking and 3 patients reported initial pain when walking. The mean time of therapy was 22.5 weeks. As a consequence of trauma, 7 patients changed their jobs. The most important prerequisites for successful surgery are early reduction and stable osteosynthesis. Residual complaints are frequently seen. The rate of avascular necrosis (8% in fractures of the neck of the talus) and the necessity for arthrodesis was significantly diminished.

Adolescent↗
PubMed1998

[Thymectomy in myasthenia gravis--an analysis of current status].

Between May 1992 and June 1997, 11 patients with myasthenia gravis and 1 asymptomatic patient with thymoma underwent extensive thymectomy through a median sternotomy. Seven patients were male and 5 female. The mean age at onset of myasthenia gravis was 46.5 (13-73) years. The interval between the first symptom and diagnosis was 3.6 months (1 week-7 months), between the first symptom and thymectomy 8.3 months (2 weeks-36 months) and the mean follow-up period was 28.4 months (3-57 months). Clinical improvement after extensive thymectomy was noted in 80% of patients. Four patients became asymptomatic under decreased medication. Thymectomy was found to be beneficial even in older patients or patients with the purely ocular type of myasthenia gravis. There was no perioperative mortality or long-term morbidity.

Adolescent↗
PubMed1998

Successful testicular sperm extraction (TESE) in spite of high serum follicle stimulating hormone and azoospermia: correlation between testicular morphology, TESE results, semen analysis and serum hormone values in 103 infertile men.

Spermatozoa recovered from testicular biopsies can be used through intracytoplasmic sperm injection (ICSI) to achieve a pregnancy. To assess the likelihood of successful testicular sperm extraction (TESE) in men suffering from severe oligo- or azoospermia, bilateral biopsy specimens were obtained. Following semi-thin sectioning, the morphology of testicular samples was graded according to a modified Johnsen score. TESE was performed in parallel to this histological examination. The number of isolated spermatozoa was assessed in a semiquantitative way. From 103 patients investigated, 64 (62.1%) showed azoospermia in a preceding semen analysis and 29 (28.2%) patients had sperm concentrations between 0.1 and 1 x 10(6)/ml. In 10 patients who had higher sperm counts, most spermatozoa were non-motile. Spermatozoa could be detected after TESE in the testicular tissue of 49 (77%) azoospermic men. When follicle stimulating hormone (FSH) concentration was normal, most patients had detectable spermatozoa after TESE. Nearly one-third of patients with mildly elevated FSH had no spermatozoa. Thirty-nine percent of patients in whom FSH was elevated to more than twice normal and 50% of patients with grossly elevated FSH had no detectable spermatozoa. In all, 82.8% of men with sperm concentrations between 0.1 and 1x10(6)/ml in their ejaculate showed spermatozoa in the tissue sample after TESE. Our data demonstrate that, contrary to previous recommendations, infertile men with azoospermia and high FSH values should be reconsidered for testicular biopsy, provided that tissue samples can be cryopreserved for later TESE/ICSI treatment.

Biopsy↗
PubMed1998

Ultrastructure of gametes and intracytoplasmic sperm injection: the significance of sperm morphology.

The aim of this study was to determine characteristic malformations of sperm ultrastructure in patients with severe subfertility undergoing intracytoplasmic sperm injection (ICSI). Although light microscopy (LM) can reveal major abnormalities of the three parts of the spermatozoon (head, mid-piece and flagellum), the various cell organelles of the spermatozoon and their fine structure remain unevaluated by LM. Insight into the submicroscopic organization of the spermatozoon and its complex organellar system may contribute to a better understanding of the preconditions for success or failure of fertilization. An in-depth evaluation of semen quality by transmission electron microscopy (TEM) can improve the diagnosis of male subfertility and can give substantial information about the fertilizing competence of spermatozoa. Thus, in this study 56 ejaculated sperm samples from patients with severe male subfertility or previous failed attempts at in-vitro fertilization were assessed by LM and TEM prior to ICSI to evaluate the most important sperm defects causing extreme subfertility. LM analysis was performed according to World Health Organization criteria. It could be confirmed that severe head defects are mostly involved in long-term infertility and fertilizing failure in classical IVF treatments. The most frequent head defects are disorders of the nuclear membranes and the acrosomal cap and disorganization of the chromatin structure. These defects of sperm fine structure seem to be associated with dysfunctional sperm-oocyte recognition, binding and fusion with the oolemma. Chromatin alterations and signs of decondensation or karyolysis are frequently associated with a deterioration of the nuclear membranes and may be due to impaired spermiogenesis. However, our results and the success of ICSI proved that severe sperm defects have no predictive value and do not impair the fertilization process, and also that the maturity of spermatozoa does not play an important role. Fine structure analysis revealed the pleiomorphology and heterogeneity of human spermatozoa.

Female↗
PubMed1998

Influence of different culture conditions on sarcoplasmic reticular calcium transport in isolated neonatal rat cardiomyocytes.

This study investigates sarcoplasmic reticulum (SR) calcium-(Ca2+) transport ATPase (SERCA2a) and phospholamban (PLB) in cultured spontaneously contracting neonatal rat cardiomyocytes (CM) to ascertain the function of both SR proteins under various culture conditions. The two major SR proteins were readily detectable in cultured CM by immunofluorescent microscopy using specific anti-SERCA2 and anti-PLB antibodies. Double labeling technique revealed that PLB-positive CM also labeled with anti-SERCA2. Coexpression of SERCA2 and PLB in CM was supported by measurement of cell homogenate oxalate-supported Ca2+ uptake which was completely inhibited by thapsigargin and stimulated by protein kinase A-catalyzed phosphorylation. Under serum-free conditions, incubation of CM with the SERCA2a expression modulator 3,3', 5-triiodo-L-thyronine (100 nM, 72 h) resulted in elevated Ca2+ uptake of +33%. Specific Ca2+ uptake activity was not altered if insulin was omitted from the serum-free culture medium but total SR Ca2+ transport activity was reduced under this culture condition. The results indicate that primary culture of spontaneously contracting neonatal rat CM can be employed as a useful model system for investigating both short- and long-term mechanisms determining the Ca2+ re-uptake function of the SR under defined culture conditions.

Animals↗
PubMed1998

Immunohistochemical localization of angiotensin II receptors (AT1) in the heart with anti-peptide antibodies showing a positive chronotropic effect.

Antibodies were produced against a synthetic peptide corresponding to amino acids (165-191) of the second extracellular loop of the human angiotensin II receptor subtype 1 (AT1) in rabbits. The purified antibodies had an apparent affinity of about 1 nM and were monospecific for the AT1-receptor peptide. Chemical modification of the carboxyl groups (glu at positions 173 and 185) and the sulfhydryl group (cys at position 180) of the AT1-receptor peptide did not alter the relative affinity of the coated AT1-receptor peptide to antibodies. The antibodies specifically stained CHO cells expressing the rat AT1a receptor. Immunoblots on rat kidney revealed that the antibody recognized a protein band of 59 +/- 3 kDa in a dose-dependent manner and this band was no longer detected after preincubating the antibodies with AT1-receptor peptide. Using electron microscopic and immunofluorescence immunocytochemistry techniques, angiotensin II receptors were detected in (1) the sarcolemma, T-tubules and nuclei of rat cardiomyocytes, (2) the transluminal side of endothelial cells and (3) fibroblast cells. These localizations are specific, as the immunostaining did not appear when preimmune rabbit serum was used and was blocked after preincubating antibodies with antigenic peptide. Functionally, these antibodies did not affect the ligand binding properties of the receptors but displayed agonist-like activity as shown by dose-dependent increases in beating frequency in cultured neonatal cardiomyocytes. These results suggest that the antibodies against the second extracellular loop of human AT1 receptors were able to specifically recognize AT1 receptors. In addition, they extend the observation that the second extracellular loop of the G-protein coupled membrane receptors is a specific target for antibodies with agonist-like activity.

Amino Acid Sequence↗
PubMed1997

Intracellular localization of inducible nitric oxide synthase in neonatal rat cardiomyocytes in culture.

Recognition of the role of nitric oxide (NO) in cardiovascular regulations raised an acute interest in NO-generating enzymes-nitric oxide synthases (NOS). Nevertheless, the subcellular localization of inducible isoform of NOS (NOS II) and regulation of its expression in the cardiomyocyte still remains to be elucidated. Therefore, we focused this study on the subcellular localization of NOS II in cultured neonatal rat cardiomyocytes using immunocytochemical techniques at the light and electron microscopic level as well as the demonstration of NADPH-diaphorase activity and the Griess assay for NO measurement. Cultivation of neonatal cardiomyocytes during 2 and more days induced a moderate increase in the NOS II immunolabeling in defined cytoplasmic structures and a nuclear NOS II staining in some cells. Exposure of the cell cultures to exogenous cAMP markedly stimulated NO production with a concomitant enhancement of NOS II immunolabeling of cardiomyocytes. cAMP-induced changes were significantly attenuated by dexamethasone. This report provides evidence for the localization of NOS II in the perinuclear space, Golgi complex, mitochondria, plasma membrane and along contractile fibers of cardiomyocytes, as well as for the appearance of NOS II staining of the cell nuclei in the course of cultivation. In non-cardiomyocytes contaminating the cell culture, positive immunoreaction was detected in the Golgi complex and endoplasmic reticulum. Our data point to a notable constitutive expression of NOS II in rat cardiomyocytes apparently dependent on the developmental stage.

Animals↗
PubMed1997

Relaxin-like factor: a highly specific and constitutive new marker for Leydig cells in the human testis.

The complete protein-coding region of the human relaxin-like factor (RLF; formerly Ley-I-L) was cloned by reverse transcription-polymerase chain reaction from human testis and subcloned into a bacterial expression plasmid for the production of recombinant human RLF in Escherichia coli. Polyclonal antibodies were raised against the recombinant RLF, as well as against a peptide epitope from the B-domain of the RLF polypeptide. Antibodies were used for immunohistochemistry of Bouin-fixed, paraffin-embedded samples of human testis tissues. Specific immunoreactivity was located exclusively in the Leydig cells with a consistent high intensity of staining, showing similar spatial distribution to other Leydig cell markers, such as the luteinizing hormone (LH) receptor and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and to the pattern of RLF mRNA shown by in-situ transcript hybridization. In biopsy samples from patients with severe disturbances of spermatogenesis, RLF staining intensity was consistently high in all cases, unlike staining for 3 beta-HSD which varied considerably between patients. Immunostaining for RLF would thus appear to offer an interesting new marker for Leydig cells in human testis samples.

3-Hydroxysteroid Dehydrogenases↗
PubMed1997

Cytochemical and immunocytochemical localization of Na,K-ATPase alpha subunit isoenzymes in the rat heart.

In order to understand the functional significance of Na,K-ATPase subunits as well as their isoenzymes, a precise subcellular localization of these in the myocyte is a crucial prerequisite. Cytochemical, immunofluorescence, preembedding immunogold and horse radish peroxidase-diaminobenzidine methods, demonstrated alpha 1 isoenzyme immunoreactivity on the sarcolemma, T-tubules and the subsarcolemmal cisterns of the adult cardiac myocytes. Cytochemically, ouabain resistant Na,K-ATPase precipitate was localized only in the subsarcolemmal cisterns and junctional sarcoplasmic reticulum. For alpha 2 isoenzyme, immuno-reactivity was demonstrated on the sarcolemma as well as in all areas of the myocytes in particularly a close proximation to the sarcoplasmic reticulum and microsomes. For alpha 3 isoenzyme, only a weak insignificant signal was noted on the sarcolemma, intercalated disc and sarcoplasm. It is suggested that cytochemical ouabain resistant precipitate present in subsarcolemmal cisterns and junctional sarcoplasmic reticulum represent alpha 1 isoenzyme of Na,K-ATPase. A differential as well as unique localization of alpha subunit isoenzymes of Na,K-ATPase in specific structures of cardiac myocytes may suggest importance in physiological function at these sites.

Animals↗
PubMed1996

Chemiluminescence in the Agglomeration of Metal Clusters

The agglomeration of copper or silver atoms in a matrix of noble gas atoms to form small clusters may be accompanied by the emission of visible light. Spectral analysis reveals the intermediate formation of electronically excited atoms and dimers as the source of the chemiluminescence. A mechanism is proposed, according to which the gain in binding energy upon cluster formation may even lead to the ejection of excited fragments as a result of unstable intermediate configurations. A similar concept was introduced in the field of nuclear reactions by Niels Bohr 60 years ago.

Journal Article↗
PubMed1996

A synthetic peptide corresponding to the second extracellular loop of the human M2 acetylcholine receptor induces pharmacological and morphological changes in cardiomyocytes by active immunization after 6 months in rabbits.

We have previously shown the presence of functional autoantibodies against the second extracellular loop of the human M2 acetylcholine receptor as an autoimmune epitope in 39% of patients with idiopathic dilated cardiomyopathy (DCM). To see if this autoimmune DCM can be experimentally reproduced, a synthetic peptide corresponding to the above autoimmune epitope was used as an autoantigen to immunize rabbits monthly for 6 months. Affinity-purified antibodies were able to display the negative chronotropic effects on neonatal rat cardiomyocytes in culture. The density of the M2 acetylcholine receptors was increased in the immunized rabbits compared to controls but the receptors showed only low-affinity binding sites for the agonist carbachol. Ultrastructural changes such as moderate sarcolemma alterations, mitochondrial swelling, disruption of cristae, and formation of myelin-like structures in the myocardial cells of rabbits immunized for 6 months suggest an immune-induced cardiotoxicity. With only a limited infiltration of inflammatory cells, these results point toward a pathophysiological role of anti-M2 acetylcholine receptor autoantibodies.

Amino Acid Sequence↗