Hansjörg Schäfer

Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system.

A method has been developed for culturing cardiac myocytes in a collagen matrix to produce a coherently contracting 3‐dimensional model heart tissue that allows direct measurement of isometric contractile force. Embryonic chick cardiomyocytes were mixed with collagen solution and allowed to gel between two Velcro‐coated glass tubes. During culture, the cardiomyocytes formed spontaneously beating cardiac myocyte‐populated matrices (CMPMs) anchored at opposite ends to the Velcro‐covered tubes through which they could be attached to a force measuring system. Immunohistochemistry and electron microscopy revealed a highly organized tissue‐like structure of α‐actin and α‐tropomyosin‐positive cardiac myocytes exhibiting typical cross‐striation, sarcomeric myofilaments, intercalated discs, desmosomes, and tight junctions. Force measurements of paced or unpaced CMPMs were performed in organ baths after 6–11 days of cultivation and were stable for up to 24 h. Force increased with frequency between 0.8 and 2.0 Hz (positive “staircase”), increasing rest length (Starling mechanism), and increasing extracellular calcium. The utility of this system as a test bed for genetic manipulation was demonstrated by infecting the CMPMs with a recombinant β‐galactosidase‐carrying adenovirus. Transduction efficiency increased from about 5% (MOI 0.1) to about 50% (MOI 100). CMPMs display more physiological characteristics of intact heart tissue than monolayer cultures. This approach, simpler and faster than generation of transgenic animals, should allow functional consequences of genetic or pharmacological manipulation of cardiomyocytes in vitro to be studied under highly controlled conditions.—Eschenhagen, T., Fink, C., Remmers, U., Scholz, H., Wattchow, J., Weil, J., Zimmermann, W., Dohmen, H. H., Schäfer, H., Bishopric, N., Wakatsuki, T., Elson, E. L. Three‐dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system. FASEB J. 11, 683–694 (1997)

Distinction of nephroblastomas from other childhood tumors using antibodies to intermediate filaments

SummaryTen nephroblastomas were investigated by antibodies to inter-mediate filaments. In seven cases, which in light microscopy were characterized by the presence of blastema and tubules, immunofluorescence microscopy with IF-specific antibodies reveals expression of cytokeratin and vimentin in blastema cells, while tubules were only labelled by the cytokeratin antibodies. This result was independent of whether the conventional cytokeratin antibody or monoclonal antibodies specific for cytokeratin 18 were used. Stroma cells were vimentin-positive. In two cases nephroblastomas were undifferentiated and also lacked tubuli formation. In both these tumors blastema cells were vimentin-positive and cytokeratin-negative. Finally one case of clear cell sarcoma of the kidney could only be labelled by the vimentin antibody. Thus antibodies to intermediate filaments seem to be useful tools to distinguish nephroblastomas from neuroblastomas or rhabdomyosarcomas, especially in cases of metastasis.

Prenatal diagnosis of carbamoyl phosphate synthetase I deficiency by identification of a missense mutation in CPS1

Carbamoyl phosphate synthetase I (CPS1) deficiency is an autosomal recessive metabolic disorder affecting the first enzymatic step of urea cycle. We report a consanguineous family in which the index patient died at 11 days of age from a severe form of CPS1 deficiency. Initial diagnosis was based on clinical histopathological, and enzymatic investigations. Direct sequencing of the complete CPS1 coding region revealed a disease‐associated homozygous Thr544Met mutation in CPS1. On the basis of the molecular data, prenatal diagnosis was established for genomic DNA and performed at gestational week 12, after chorionic villus sampling. The fetus was homozygous for the Thr544Met mutation, and termination of pregnancy was elected. Histopathological signs of the hepatocellular metabolic disorder similar to that of the index patient were found in fetal liver thus giving morphological evidence for this hereditary error of urea cycle function as early as gestational week 12. Hum Mutat 12:206–211, 1998.

The significance of calcium in insulin secretion

Calcium plays an important role in the release of insulin. When the GBHA [glyoxal bis (2-hydroxyanil)] reaction is employed, calcium can be clearly demonstrated by light microscope in pancreatic islets of mice. The specificity of this finding has now been proved by elemental X-ray analysis. Electron microscopically, certain cations can be visualized by a precipitation technique using potassium pyroantimonate as the precipitating agent. In the B cell of mice this technique reveals a characteristic precipitation pattern. Elemental X-ray analysis suggests that the precipitates contain high amounts of calcium. The pattern of the precipitates changes dependent on the functional state of the B cell. In normoglycemia the deposits are mainly associated with the granule membranes, the cell membranes and the cytoplasmic matrix. In hypoglycemia there is a shift of precipitates into the endoplasmic reticulum and the mitochondria, which are thought to be storage organelles for intracellular calcium. The deposits within the halos of the numerous secretory granules, are diminished. In hyperglycemia there is a marked ion translocation across the cell membrane to its inner surface and particularly into the halos of the secretory granules, while the deposit content of mitochondria and endoplasmic reticulum is decreased. Within the saccules of the secretory granules, the deposits sometimes seem to impregnate a filamentous network, which encloses the secretory granule and cannot be seen by conventional electron microscopical preparations. The morphological data suggest that emiocytosis of hormone granules is associated with a release of cellular calcium. The presented observations in treated and untreated animals extend and support the conceptions on the specific role of calcium within the insulin releasing mechanism of the B cell. Calcium spielt eine wichtige Rolle bei der Insulinsekretion. Lichtmikroskopisch läßt sich unter Anwendung der GBHA [Glyoxal bis (2-hydroxyanil)]-Methode Calcium eindeutig in den Pankreasinseln der Maus nachweisen. Die Spezifität dieses Befundes wurde durch die Röntgenelementaranalyse gesichert. Elektronenmikroskopisch können bestimmte Kationen durch eine Präzipitationstechnik mit Hilfe von Kaliumpyroantimonat sichtbar gemacht werden. In der B-Zelle der Maus ergibt diese Technik ein charakteristisches Verteilungsmuster von Präzipitaten. Die Röntgenelementaranalyse zeigt, daß die Präzipitate große Calciummengen enthalten. Das Verteilungsmuster der Niederschläge verändert sich in Abhängigkeit von dem funktionellen Zustand der B-Zelle. Bei Normoglykämie treten die Ausfällungen hauptsächlich in Verbindung mit den Membranen der Hormongranula, den Zellmembranen und der Matrix des Cytoplasmas auf. Bei Hypoglykämie zeigt sich eine Verschiebung der Präzipitate in das endoplasmatische Retikulum und in die Mitochondrien, die als Speicherorganellen für das intracelluläre Calcium angesehen werden. Die Ausfällungen innerhalb der Halos der zahlreichen Sekretgranula sind vermindert. Bei Hyperglykämie ergibt sich eine erhebliche Ionenverlagerung durch die Zellmembran zu deren innerer Oberfläche und besonders in die Halos der Sekretgranula, während die Präzipitatmengen in Mitochondrien und endoplasmatischem Retikulum vermindert sind. Innerhalb der Vesikel der Sekretgranula scheinen die Präzipitate manchmal ein filamentöses Netzwerk zu imprägnieren, welches das Sekretgranulum umhüllt und bei konventioneller elektronenmikroskopischer Präparation nicht sichtbar ist. Die morphologischen Befunde deuten darauf hin, daß die Emiocytose der Hormongranula mit einer Ausschleusung von cellulärem Calcium einhergeht. Die vorliegenden Beobachtungen an intakten Tieren erweitern und bestätigen die Konzeptionen über die spezifische Rolle des Calciums im Insulinsekretionsmechanismus der B-Zelle.

The selective estrogen receptor-beta agonist biochanin A shows vasculoprotective effects without uterotrophic activity.

Objective: Current hormone therapy in postmenopausal women is associated with uterotrophic activity and cancer-promoting effects. In this experimental study, we compared the effects of the selective estrogen-receptor (ER) &bgr; agonist biochanin A, and the selective ER&agr; agonist ethinylestradiol, on the development of intimal hyperplasia after balloon injury and on uterus morphology. Design Female F344 rats with or without prior ovariectomy were used for aortic denudations. Animals remained untreated or received oral biochanin A (100 mg/kg) or ethinylestradiol (100 &mgr;g/kg). After 14 days, aortas and uteri were harvested for histologic and immunohistochemical analyses. Computerized assessments of aortic adhesion molecule expression, and isometric relaxation experiments, and uteri were analyzed. In vitro studies with smooth muscle cells and endothelial cells were performed to further investigate the effects of hormone treatment on cell proliferation, migration and adhesion molecule expression. Results: Among untreated rats, ovariectomized animals tended to show greater neointimal hyperplasia and increased expression of the adhesion molecules 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). Biochanin A treatment reduced neointima formation, inhibited VCAM-1 up-regulation, and improved the vascular relaxation response. No effect was observed on uterus growth or histology. Ethinylestradiol also reduced aortic neointima formation and inhibited VCAM-1 up-regulation, but failed to improve endothelial function and significantly induced uterus growth. Both agents showed antiproliferative and weak antimigratory effects on smooth muscle cells, and reduced VCAM-1 expression on stimulated endothelial cells in vitro. Conclusions: The ER&bgr; agonist biochanin A shows vasculoprotective effects without uterotrophic activity. Because hormone therapy may have cancer-promoting side effects, administration of ER&bgr;-selective agents might be alternatively used to reduce the risk of cardiovascular disease in postmenopausal women.

Enrichment of G protein α-subunit mRNAs in the AV-conducting system of the mammalian heart

We investigated the expression pattern of the heterotrimeric G proteins Gsα, Giα-2 and Goα in rat and guinea-pig heart by in situ hybridization. Cryosections were hybridized with single-stranded 35S-cRNA probes complementary to subtype-specific sequences of the respective mRNAs. Hybridization signals were visualized by exposition to X-ray films and dipping autoradiography. The rank order of abundance was Giα-2∼Gsα>>Goα. In general, G protein α-subunit mRNAs were evenly distributed in the heart including endo- and epicardium, large vessels and valves. Goα-mRNA levels were significantly higher in atria than in ventricles. In contrast to the rather uniform labeling of working myocardium, expression of all three G proteins was enriched in small intramural blood vessels and in subendocardial Purkinje fibers of septum and papillary muscles. A more marked enrichment of Gsα-, Giα-2- and especially Goα-mRNA was seen in neuronal ganglionic cells in the atrial septum and posterior regions of the atrium. The main finding, however, was an enrichment of all three G protein mRNAs in the atrioventricular conductive tissue. The accumulation was strictly co-localized with acetylcholinesterase-positive regions identified as the atrioventricular node, the bundle of His and the right and left bundle branches and was seen similarly in rat and guinea-pig hearts. Quantitative in situ hybridization revealed Gsα-, Giα-2- and Goα-mRNA levels in the bundle of His to be 206±0.13%, 191±0.15% and 165±0.06%, respectively, of that in the surrounding interventricular working myocardium. These findings indicate that heterotrimeric G proteins play an important role in modulation of electrical conductance in the heart.

Immunocytochemical studies on parafollicular cells of various mammals.

Using specific antisera, calcitonin, calcitonin gene-related peptide (CGRP), somatostatin as well as neuron-specific enolase, chromogranin, secretory peptide I and calbindin (vitamin D-dependent calcium-binding protein) were looked for in parafollicular cells of rats, Syrian hamsters, Mongolian gerbils, mice, guinea pigs, rabbits and pigs. Calcitonin and CGRP were most invariably present in various species. Somatostatin was absent in mice and Mongolian gerbils and present in variable amounts in the remaining species. Neuron-specific enolase could not be detected in rabbits, while in the pigs and the Mongolian gerbils it could be demonstrated only in some parafollicular cells. Calbindin was present exclusively in parafollicular cells of guinea pigs. Chromogranin and secretory protein-I were present only in some animal species.

FK778, a novel immunosuppressive agent, reduces early adhesion molecule up-regulation and prolongs cardiac allograft survival.

The adhesion molecules, P‐selectin, ICAM‐1, and VCAM‐1 are important mediators of T‐cell adhesion and T‐cell co‐stimulation. We investigated the effect of the malononitrilamide FK778 on cardiac allograft survival, acute allograft rejection, and adhesion molecule up‐regulation in a heterotopic, cardiac transplantation model. Rats received low‐ or high‐dose FK778 or no treatment. Grafts were harvested on the fifth postoperative day for histologic examinations. To assess allograft survival, recipients were treated for a maximum of 10 days and grafts were harvested after cessation of the contractile activity. FK778 low dose showed a mild but significant decrease in mononuclear infiltration but failed to markedly reduce histologic rejection, adhesion molecule up‐regulation, or to prolong allograft survival. However, high‐dose FK778 treatment significantly reduced early up‐regulation of P‐selectin, ICAM‐1, and VCAM‐1, abolished infiltration, reduced histologic rejection and resulted in prolonged cardiac allograft survival. Therefore, FK778 is a novel, highly desirable immunosuppressive drug for transplantation medicine.

Morphologic effects of diazoxide and diphenylhydantoin on insulin secretion and biosynthesis in b cells of mice

The action of diazoxide, an antidiuretic agent, and diphenylhydantoin, an antiepileptic (DPH), both with strong hyperglycemic side effects on the pancreatic B cells, was examined by electron microscopy and cytochemistry, with the following findings. 1. Effects on secretory apparatus: the severe hyperglycemic syndrome following a single injection of diazoxide (200 mg/kg) or DPH (150 mg/kg) did not change the granularity of the B cells. Ultrastructurally a marked increase of lysosomal digestion of secretory granules (crinophagy) was observed in almost all B cells. Crinophagy may be regarded as a result of an impaired discharge of secretory granules during simultaneous maintenance of biosynthesis. It is also possible that changes of the electrophysical properties of the granule surfaces may play an additional role in crinophagy. 2. Effect on synthesizing apparatus: in B cells subtotally degranulated by the injection of anti-insulin serum (AIS), regranulation occurred more rapidly after the additional administration of diazoxide or DPH than without these compounds. This fact may imply that, under the hyperglycemic conditions tested, diazoxide or DPH have no effect on the synthesizing capacity of the B cells. Effects on secretory apparatus: the severe hyperglycemic syndrome following a single injection of diazoxide (200 mg/kg) or DPH (150 mg/kg) did not change the granularity of the B cells. Ultrastructurally a marked increase of lysosomal digestion of secretory granules (crinophagy) was observed in almost all B cells. Crinophagy may be regarded as a result of an impaired discharge of secretory granules during simultaneous maintenance of biosynthesis. It is also possible that changes of the electrophysical properties of the granule surfaces may play an additional role in crinophagy. Effect on synthesizing apparatus: in B cells subtotally degranulated by the injection of anti-insulin serum (AIS), regranulation occurred more rapidly after the additional administration of diazoxide or DPH than without these compounds. This fact may imply that, under the hyperglycemic conditions tested, diazoxide or DPH have no effect on the synthesizing capacity of the B cells.

Structure of rat liver nuclei after depletion and reassociation of histone H1.

Abstract Chromatin in isolated rat liver nuclei was compared with chromatin in ( i ) nuclei depleted of H1 by acid extraction; ( ii ) nuclei treated at pH 3.2 (without removal of H1), and ( iii ) depleted nuclei following reassociation of H1. Electron microscopy and digestion by DNase I, micrococcal nuclease and endogenous Ca/Mg endonuclease were used for this comparative examination. Electron micrographs of H1-depleted nuclei showed a dispersed and finely granular appearance. The rate of DNA cleavage by micrococcal nuclease or DNase I was increased several-fold after H1 removal. Discretely sized intermediate particles produced by Ca/Mg endonuclease in native nuclei were not observed in digests of depleted nuclei. Digestion by micrococcal nuclease to chromatin particles soluble in 60 mM NaCl buffer appeared not to be affected in depleted nuclei. When nuclei were treated at pH 3.2, neither the appearance of chromatin in electron micrographs nor the mode or rate of nuclease digestion changed appreciably. Following reassociation of H1 to depleted nuclei, electron micrographs demonstrated the reformation of compacted chromatin, but the lower rate of DNA cleavage in native nuclei was not restored. Further, H1 reassociation produced a significant decrease in the solubility of nuclear chromatin cleaved by micrococcal nuclease or Ca/Mg endonuclease. In order to evaluate critically the reconstitution of native chromatin from H1-depleted chromatin we propose the use of digestion by a variety of nucleases in addition to an electron microscopic examination.