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S Hein

Publications and source records attributed to S Hein.

52 records · Page 3Linked to original sources
PubMed1994

Altered expression of titin and contractile proteins in failing human myocardium.

Our own previous ultrastructural studies in human hearts with dilated cardiomyopathy and heart failure showed sarcomeric and cytoskeletal disarrangement. On the basis of these findings we tested the hypothesis that in cardiomyopathic failing hearts not only the sarcomere structure but also the organization and the amount of numerous contractile proteins are disturbed. Titin was included in this study because it is the elastic "third" filament of the sarcomere and also plays an important role as template for myosin and actin filaments in sarcomerogenesis. Human cardiac tissue obtained at the time of transplantation surgery was investigated using immunohistochemistry with monoclonal antibodies against titin, myosin, actin, tropomyosin, and troponin T. Additionally, isolated myocytes from rat or pig heart were used for the standardization of the localization pattern. In normal tissue, myosin and the thin filament complex showed a regular cross striation that was wider in myosin staining than for actin, troponin T, and tropomyosin corresponding with the different width of the A and I bands in the sarcomere. Titin localization in normal human and animal myocardium showed a regular cross striation pattern. In diseased cardiac tissue titin fluorescence intensity was reduced and frequently disorganization or almost complete loss of titin from many myocytes were present. Severe abnormalities of contractile proteins consisting of disarrangement or lack of filaments were also observed. Double staining procedures showed that in the same myocyte defects of the contractile apparatus were accompanied by a simultaneous reduction of titin indicating that the "third" sarcomeric filament system is involved in heart failure. Abnormalities of titin expression may be especially important because titin significantly influences sarcomeric elastic behaviour and is necessary as template for the organization of newly synthesized myosin and actin filaments. The loss of titin may contribute to the altered compliance in failing hearts. It is concluded that disorganization and loss of titin, myosin, and the thin filament complex are severe in the failing human heart because of dilated cardiomyopathy and that these changes may represent several of the most important components of the structural correlate of reduced cardiac function.

Adult↗
PubMed1994

Biochemical and molecular characterization of the Alcaligenes eutrophus pyruvate dehydrogenase complex and identification of a new type of dihydrolipoamide dehydrogenase.

Sequence analysis of a 6.3-kbp genomic EcoRI-fragment of Alcaligenes eutrophus, which was recently identified by using a dihydrolipoamide dehydrogenase-specific DNA probe (A. Pries, S. Hein, and A. Steinbüchel, FEMS Microbiol. Lett. 97:227-234, 1992), and of an adjacent 1.0-kbp EcoRI fragment revealed the structural genes of the A. eutrophus pyruvate dehydrogenase complex, pdhA (2,685 bp), pdhB (1,659 bp), and pdhL (1,782 bp), encoding the pyruvate dehydrogenase (E1), the dihydrolipoamide acetyltransferase (E2), and the dihydrolipoamide dehydrogenase (E3) components, respectively. Together with a 675-bp open reading frame (ORF3), the function of which remained unknown, these genes occur colinearly in one gene cluster in the order pdhA, pdhB, ORF3, and pdhL. The A. eutrophus pdhA, pdhB, and pdhL gene products exhibited significant homologies to the E1, E2, and E3 components, respectively, of the pyruvate dehydrogenase complexes of Escherichia coli and other organisms. Heterologous expression of pdhA, pdhB, and pdhL in E. coli K38(pGP1-2) and in the aceEF deletion mutant E. coli YYC202 was demonstrated by the occurrence of radiolabeled proteins in electropherograms, by spectrometric detection of enzyme activities, and by phenotypic complementation, respectively. A three-step procedure using chromatography on DEAE-Sephacel, chromatography on the triazine dye affinity medium Procion Blue H-ERD, and heat precipitation purified the E3 component of the A. eutrophus pyruvate dehydrogenase complex from the recombinant E. coli K38(pGP1-2, pT7-4SH7.3) 60-fold, recovering 41.5% of dihydrolipoamide dehydrogenase activity. Microsequencing of the purified E3 component revealed an amino acid sequence which corresponded to the N-terminal amino acid sequence deduced from the nucleotide sequence of pdhL. The N-terminal region of PdhL comprising amino acids 1 to 112 was distinguished from all other known dihydrolipoamide dehydrogenases. It resembled the N terminus of dihydrolipoamide acyltransferases, and it contained one single lipoyl domain which was separated by an adjacent hinge region from the C-terminal region of the protein that exhibited high homology to classical dihydrolipoamide dehydrogenases.

Alcaligenes↗
PubMed1992

Identification of a lipoamide dehydrogenase gene as second locus affected in poly(3-hydroxybutyric acid)-leaky mutants of Alcaligenes eutrophus.

From a genomic library of Alcaligenes eutrophus strain H16 in the broad-host range cosmid pVK100 a 6300-bp EcoRI-fragment was cloned, which restored the wild-type phenotype in transposon-induced poly(3-hydroxybutyric acid)-leaky mutants, derived from A. eutrophus. Nucleotide sequence analysis of the region adjacent to the transposon insertion revealed an open reading frame which complied with all criteria for a coding region. This region was referred to as phbL, and the deduced amino acid sequence from this part of phbL showed 60% amino acid identity in an overlap of 98 residues to the lipoamide dehydrogenase gene (lpd) of Escherichia coli. In addition, the 6300-bp EcoRI-fragment conferred expression of lipoamide dehydrogenase activity to E. coli.

3-Hydroxybutyric Acid↗
PubMed1991

Identification and partial characterization of the secretory glycoproteins of the bovine subcommissural organ-Reissner's fiber complex. Evidence for the existence of two precursor forms.

The subcommissural organ (SCO) is a brain gland whose secretory material is released into the cerebrospinal fluid where it condenses into a thread-like structure known as Reissner's fiber (RF). This fiber extends along the aqueduct, fourth ventricle and central canal of the spinal cord. The present investigation was designed to identify and partially characterize the secretory products of the bovine SCO in their intracellular location and after they have been released and packed into RF form. 5,000 SCOs were dissected out under a microscope, whereas RF of 30,000 cows were collected by perfusing the central canal of the spinal cord with artificial cerebrospinal fluid. Extracts of SCO and RF were used for (i) raising polyclonal antibodies; (ii) immunoblotting; (iii) lectin binding on electrotransfers: concanavalin A (affinity = mannose, glucose) and Limax flavus agglutinin (affinity = sialic acid); (iv) immunoaffinity chromatography; (v) preparative SDS-PAGE and raising of polyclonal antibodies against each of the secretory glycoproteins identified in the immunoblots. All antibodies and the two lectins were also applied to tissue sections of the SCO and RF of several species. The immunocytochemical study of the bovine SCO using an anti-RF serum showed that the secretory material present in the rough endoplasmic reticulum (RER), secretory granules and in RF is strongly immunoreactive. Con A binding sites were only found in the endoplasmic reticulum, whereas Limax flavus agglutinin revealed secretory granules and RF, only. In the blots the immunostaining was used to identify secretory polypeptides. The glycosylated nature of the latter was established by their affinity for Con A and/or Limax flavus agglutinin. Furthermore, this latter lectin allowed us to distinguish whether the intracellular source of a secretory glycoprotein is from a pre-Golgi (RER) or a post-Golgi (secretory granules) compartment. Four glycoproteins were identified in the SCO with apparent molecular weights of 540, 450, 320 and 190 kDa. The three former were also purified by immunoaffinity chromatography. The 540 and 320 kDa forms are present in the SCO but missing in RF, have affinity for Con A, but not for LFA. It is suggested that these two compounds correspond to two precursor forms. The 450 and 190 kDa glycoproteins are present in both, the SCO and RF, and have affinity for Con A and Limax flavus agglutinin. These most likely correspond to processed forms. The presence of more than one precursor was further substantiated by immunocytochemical findings using antisera against the 540, 450 and 320 kDa forms.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Identification and molecular characterization of the Alcaligenes eutrophus H16 aco operon genes involved in acetoin catabolism.

Acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) and the fast-migrating protein (FMP) were purified to homogeneity from crude extracts of acetoin-grown cells of Alcaligenes eutrophus. Ao:DCPIP OR consisted of alpha and beta subunits (Mrs, 35,500 and 36,000, respectively), and a tetrameric alpha 2 beta 2 structure was most likely for the native protein. The molecular weight of FMP subunits was 39,000. The N-terminal amino acid sequences of the three proteins were determined, and oligonucleotides were synthesized on the basis of the codon usage of A. eutrophus. With these, the structural genes for the alpha and beta subunits of Ao:DCPIP OR and FMP, which were referred to as acoA, acoB, and acoC, respectively, were localized on one single EcoRI restriction fragment which has been cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The nucleotide sequences of a 5.3-kbp region of this fragment and one adjacent fragment were determined, and the structural genes for acoA (1,002 bp), acoB (1,017 bp), and acoC (1,125 bp) were identified. Together with the gene acoX, whose function is still unknown and which is represented by a 1,080-bp open reading frame, these genes are probably organized in one single operon (acoXABC). The transcription start site was identified 27 bp upstream of acoX; this site was preceded by a region which exhibited complete homology to the enterobacterial sigma 54-dependent promoter consensus sequence. The amino acid sequences deduced from acoA and acoB for the alpha subunit (Mr, 35,243) and the beta subunit (Mr, 35,788) exhibited significant homologies to the primary structures of the dehydrogenase components of various 2-oxo acid dehydrogenase complexes, whereas those deduced from acoC for FMP (Mr, 38,941) revealed homology to the dihydrolipoamide acetyltransferase of Escherichia coli. The occurrence of a new enzyme type for the degradation of acetoin is discussed.

Acetoin↗
PubMed1991

Impairment of the myocardial ultrastructure and changes of the cytoskeleton in dilated cardiomyopathy.

This study was designed to determine the morphological correlate of chronic heart failure. Myocardial tissue from eight patients undergoing transplantation surgery because of end-stage dilated cardiomyopathy was investigated by electron microscopy and immunocytochemistry using monoclonal antibodies against elements of the cytoskeleton: desmin, tubulin, vinculin, and vimentin. The tissue showed hypertrophy, atrophy of myocytes, and an increased amount of fibrosis. Ultrastructural changes consisted of enlargement and varying shape of nuclei, numerous very small mitochondria, proliferation of T tubules, and accumulation of lipid droplets and glycogen. The most obvious ultrastructural alteration was the decrease of myofilaments, ranging from rarefication to complete absence of sarcomeres in cells filled with unspecified cytoplasm. Immunocytochemistry showed that desmin was localized at the Z lines. In diseased myocardium, the amount of desmin was increased, but it was disorderly arranged. Tubulin formed a fine network throughout the myocytes and was significantly increased in cardiomyopathic hearts. Vinculin, a protein closely associated with the cytoskeleton, occurred not only at the sarcolemma and the intercalated disc but also within the myocardial cells. Ultrastructural changes and alterations of the cytoskeleton were severe in about one third of all cells. About one third of all cells showed moderately severe changes, and the remaining cells were normal. Vimentin was present in the interstitial cells and was increased in relation to the increase of fibrosis. We conclude that the increase of fibrosis, the degeneration of hypertrophied myocardial cells, and the alterations of the cytoskeleton are the morphological correlates of reduced myocardial function in chronic heart failure.

Antibodies, Monoclonal↗
PubMed1988

Pinealocytes immunoreactive with antisera against secretory glycoproteins of the subcommissural organ: a comparative study.

By means of light-microscopic immunocytochemistry two polyclonal antibodies (AFRU, ASO; see p. 470) directed against secretory glycoproteins of the subcommissural organ were shown to cross-react with cells in the pineal organ of lamprey larvae, coho salmon, a toad, two species of lizards, domestic fowl, albino rat and bovine (taxonomic details, see below). The AFRU-immunoreactive cells were identified as pinealocytes of the receptor line (pineal photoreceptors, modified photoreceptors or classical pinealocytes, respectively) either due to their characteristic structural features or by combining AFRU-immunoreaction with S-antigen and opsin immunocytochemistry in the same or adjacent sections. Depending on the species, AFRU- or ASO-immunoreactions were found in the entire perikaryon, inner segments, perinuclear area, and in basal processes facing capillaries or the basal lamina. In most cases, only certain populations of pinealocytes were immunolabeled; these cells were arranged in a peculiar topographical pattern. In lamprey larvae, immunoreactive pinealocytes were observed only in the pineal organ, but not in the parapineal organ. In coho salmon, the immunoreaction occurred in S-antigen-positive pinealocytes of the pineal end-vesicle, but was absent from S-antigen-immunoreactive pinealocytes of the stalk region. In the rat, AFRU-immunoreaction was restricted to S-antigen-immunoreactive pinealocytes found in the deep portion of the pineal organ and the habenular region. These findings support the concept that several types of pinealocytes exist, which differ in their molecular, biochemical and functional features. They also indicate the possibility that the AFRU- and ASO-immunoreactive material found in certain pinealocytes might represent a proteinaceous or peptide compound, which is synthesized and released from a specialized type of pinealocyte in a hormone-like fashion. This cell type may share functional characteristics with peptidergic neurons or paraneurons.

Animals↗
PubMed1986

Light- and electron-microscopic immunocytochemistry and lectin histochemistry of the subcommissural organ: evidence for processing of the secretory material.

The subcommissural organ (SCO) of the rat was investigated by use of histochemical and immunocytochemical methods at the light- and electron-microscopic levels. Consecutive thin methacrylate sections were stained with the pseudoisocyanin (Psi), immunoperoxidase (IMC; employing an antiserum against Reissner's fiber, AFRU), periodic acid-Schiff (PAS) and periodic acid-silver methenamine (SM) techniques, and reacted with six types of lectins. Psi, SM, concanavalin A (Con A) and IMC were also used for double and triple sequential staining of the same section. Increasing dilutions of AFRU (from 1:1000 to 1:200 000) were used for immunostaining of serial paraffin sections. In addition, ultrastructural localization of (i) Con A-binding sites and (ii) immunoreactive secretory material was performed. Some of these procedures were also applied to the ophidian and canine SCO. Con A-positive, Psi-positive and immunoreactive materials coexisted within the same cisternae of the rough endoplasmic reticulum. The Golgi apparatus lacked Con A-positive and immunoreactive substances. Apical secretory granules and secreted material lying on the surface of the SCO showed (i) the highest affinity for AFRU, but were (ii) Con A-negative, and (iii) wheat-germ agglutinin-, PAS- and SM-positive. Reissner's fiber displayed a low affinity for AFRU. It is suggested that the SCO secretes N-linked glycoproteins, the carbohydrate and protein moieties of which undergo (i) a maturation process before being released, and (ii) some kind of modification(s) after their release into the ventricle. The perivascular secretory cells of the dog SCO might secrete a material different from that secreted by the ependymal cells.

Animals↗
PubMed1985

Reissner's fiber and the wall of the central canal in the lumbo-sacral region of the bovine spinal cord. Comparative immunocytochemical and ultrastructural study.

Reissner's fiber (RF) of the subcommissural organ (SCO), the central canal and its bordering structures, and the filum terminale were investigated in the bovine spinal cord by use of transmission electron microscopy, histochemical methods and light-microscopic immunocytochemistry. The primary antisera were raised against the bovine RF, or the SCO proper. Comparative immunocytochemical studies were also performed on the lumbo-sacral region of the rat, rabbit, dog and pig. At all levels of the bovine spinal cord, RF was strongly immunoreactive with both antisera. From cervical to upper sacral levels of the bovine spinal cord there was an increasing number of ependymal cells immunostainable with both antisera. The free surface of the central canal was covered by a layer of immunoreactive material. At sacral levels small subependymal immunoreactive cells were observed. From all these structures sharing the same immunoreactivity, only RF was stained by the paraldehyde-fuchsin and periodic-acid-Schiff methods. At the ultrastructural level, ependymal cells with numerous protrusions extending into the central canal were seen in the lower lumbar segments, whereas cells displaying signs of secretory activity were principally found in the ependyma of the upper sacral levels. A few cerebrospinal fluid-contacting neurons were observed at all levels of the spinal cord; they were immunostained with an anti-tubulin serum. The lumbo-sacral segments of the dog, rat and rabbit, either fixed by vascular perfusion or in the same manner as the bovine material, did not show any immunoreactive structure other than RF. The possibilities that the immunoreactive ependymal cells might play a secretory or an absorptive role, or be the result of post-mortem events, are discussed.

Animals↗
PubMed1984

Comparative immunocytochemical study of the subcommissural organ.

The subcommissural organs (SCO) of 76 specimens belonging to 25 vertebrate species (amphibians, reptiles, birds, mammals) were studied by use of the immunoperoxidase procedure. The primary antiserum was obtained by immunizing rabbits with bovine Reissner's fiber (RF) extracted in a medium containing EDTA, DTT and urea. Antiserum against an aqueous extract of RF was also produced. The presence of immunoreactive material in cell processes and endings was regarded as an indication of a possible route of passage. Special attention was paid to the relative development of the ventricular, leptomeningeal and vascular pathways established by immunoreactive structures. The SCO of submammalian species is characterized by (i) a conspicuous leptomeningeal connection established by ependymal cells, (ii) scarce or missing hypendymal cells, and (iii) a population of ependymal cells establishing close spatial contacts with blood vessels. The SCO of most mammalian species displays the following features: (i) ependymal cells lacking immunoreactive long basal processes, (ii) hypendymal secretory cells occurring either in a scattered arrangement or forming clusters, (iii) an occasional leptomeningeal connection provided by hypendymal cells, and (iv) in certain species numerous contacts of secretory cells with blood vessels. In the hedgehog immunoreactive material was missing in the ependymal formation of the SCO, but present in hypendymal cells and in the choroid plexuses. The SCO of several species of New and Old-World monkeys displayed immunoreactive material, whereas that of anthropoid apes (chimpanzee, orangutan) and man was completely negative with the antisera used.

Animals↗
PubMed1984

Spatial and structural interrelationships between secretory cells of the subcommissural organ and blood vessels. An immunocytochemical study.

In 76 specimens (amphibians, reptilians, mammals) belonging to 25 different vertebrate species, the region of the subcommissural organ (SCO) was investigated with the use of a primary antiserum raised against an extract of bovine Reissner's fiber + the immunoperoxidase procedure according to Sternberger et al. (1970). In the SCO of a toad (Bufo arenarum) and several species of reptiles (lacertilians, ophidians, crocodilians), the ependymal cells were the only type of secretory cell displaying vascular contacts, whereas in mammals ependymal and hypendymal cells established intimate spatial contacts with blood vessels. In Bufo arenarum, but especially in the reptilian species examined, the ependymo-vascular relationship was exerted by a population of ependymal cells having a rather constant location within the SCO and projecting to capillaries that showed a remarkably constant pattern of anatomical distribution. In the SCO of mammals the modality and degree of the structural relationships between secretory cells and blood vessels varied greatly from species to species. In the SCO of the armadillo and dog the secretory tissue was organized as a thick, highly vascularized layer with most of the cells oriented toward the capillaries. A rather opposite situation was found in the SCO of New- and Old-World monkeys, where vascular contacts were restricted to a few ependymal cells.

Animals↗
PubMed1977

Carp (Cyprinus carpio) muscle fructose 1,6-bisphosphatase: purification and some properties.

1. Fructose 1,6-bisphosphatase from the white muscle tissue of the carp, Cyprinus carpio L. was purified. 2. The mol. wt of the enzyme was 145,000. Its subunit mol. wt was ca. 35,000. 3. The enzyme exhibited neutral pH optimum, activation by monovalent cations, and temperature-dependent allosteric AMP inhibition. 4. Carp muscle fructose 1,6-bisphosphatase was 10- to 30-fold more sensitive to AMP inhibition than the carp liver enzyme. 5. The carp muscle enzyme was less sensitive to AMP inhibition than the muscle enzyme from a homeothermic mammal. These results are interpreted as an example of temperature-adaptation of an enzyme regulatory property.

Adenosine Monophosphate↗