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S Hein

Publications and source records attributed to S Hein.

At least 37 records · Page 2Linked to original sources
PubMed2000

The cytoskeleton and related proteins in the human failing heart.

In addition to functional alterations, heart failure has a structural basis as well. This concerns all components of the cardiac myocytes as well as the extracellular space. Proteins of the cardiomyocyte can be subdivided in 5 different categories: 1) Contractile proteins including myosin, actin, tropomyosin and the troponins. 2) Sarcomeric skeleton: titin, myosin binding protein C, alpha-actinin, myomesin, and M-protein. 3) True 'cytoskeletal' proteins: tubulin, desmin and actin. 4) Membrane-associated proteins: dystrophin, spectrin, talin, vinculin, ankyrin and others. 5) Proteins of the intercalated disc: desmosomes consisting of desmoplakin, desmocollin, desmoglein and desmin; adherens junctions with N-cadherin, the catenins and vinculin, and gap junctions with connexin. Failing myocardium obtained from patients undergoing cardiac transplantation exhibits ultrastuctural degeneration and an altered nucleus/cytoplasm relationship. The contractile proteins and those of the sarcomeric skeleton, especially titin, are downregulated, the cytoskeletal proteins desmin and tubulin and membrane-associated proteins such as vinculin and dystrophin are upregulated and those of the intercalated disc are irregularly arranged. Elevation of cytoskeletal proteins correlates well with diastolic and contractile dysfunction in these patients. The enlarged interstitial space contains fibrosis, i.e. accumulations of fibroblasts and extracellular matrix components, in addition to macrophages and microvascular elements. Loss of the contractile machinery and related proteins such as titin and alpha-actinin may be the first and decisive event initiating an adaptive increase in cytoskeleton and membrane associated components. Fibrosis may be stimulated by subcellular degeneration. The hypothesis is put forward that all proteins of the different myocardial compartments contribute to the deterioration of cardiac function in heart failure.

Cardiomyopathy, Dilated↗
PubMed2000

A dynamic bubble trap reduces microbubbles during cardiopulmonary bypass: a case study.

Microemboli passing to the cerebral circulation during cardiopulmonary bypass can contribute to postoperative neurologic dysfunction. Many studies conclude that air microbubbles predominantly are responsible for this problem. A dynamic bubble trap (DBT) was developed to diminish the number of microbubbles in the arterial line of extracorporeal circulation. The DBT is able to substantially reduce the number of air microbubbles, as shown in two patients undergoing coronary artery bypass grafting, where a high number of microbubbles was assessed. Although a 40-micron arterial filter was used, many bubbles larger than 40 microns occurred in the arterial line. The DBT reduced the number of large microbubbles from 2,267 to 67 in patient 1 and from 897 to 61 in patient 2.

Aged↗
PubMed1999

Spatiotemporal development and distribution of intercellular junctions in adult rat cardiomyocytes in culture.

The mode of development of the intercalated disk (ID) is largely unknown, and the hypothesis was tested that the assembly of cell adhesion junctions may precede the formation of gap junctions (GJ) in developing ID in adult rat cardiomyocyte (ARC) in long-term culture. Immunostaining for connexin 43 (Cx43) and for cell adhesion junction proteins (N-cadherin, catenins, and desmoplakin) in single- and double-label techniques was analyzed and quantified by confocal and electron microscopy. All proteins investigated disappeared 48 hours after ARC isolation and reappeared parallel to redifferentiation of ARC. The newly formed ID, observed after 5 days, showed the presence of N-cadherin, catenins, and desmoplakin, low levels of Cx43, and absence of ultrastructurally discernible gap junctions. A progressive incorporation of Cx43 within ID was observed after 6 days, when cell adhesion junction proteins were already organized as zipper-like structures. Quantitative confocal analysis revealed a progressive augmentation of the fluorescence intensity of Cx43, associated with an increase in both the number and size of GJ, resulting in a substantial increase in the percentage of total GJ length per reassembled ID from 1.67% (day 6) to 15.58% (day 12). In the present study, we show that (1) the formation of the ID can be followed in ARC in culture and (2) the assembly of the adhering type of junction is the prerequisite for subsequent GJ formation within the ID. These findings may have clinical relevance in elaborating strategies for using myocardial grafts and for the potential restoration of GJ communication in cardiac diseases.

Age Factors↗
PubMed1999

Oral clodronate in breast cancer patients with bone metastases: a randomized study.

OBJECTIVES: To investigate the effect of the bisphosphonate clodronate on the occurrence of skeletal events (hypercalcaemia, fractures and radiotherapy) in breast cancer patients with bone metastases. DESIGN: Prospective, randomized, controlled, clinical trial. SETTING: A department of oncology in a university hospital. SUBJECTS: One hundred patients who received firstline systemic antineoplastic treatment for metastatic breast cancer with bone involvement were randomized to receive clodronate as two 400 mg capsules twice a day for 2 years or no additional therapy. RESULTS: In the clodronate group the number of skeletal events was reduced to 14 events in 48 evaluable patients as compared with 21 events in 51 evaluable control patients. The time to the first skeletal event was significantly longer in the clodronate group than in the control group (P = 0.015) and the most distinct difference was a lower occurrence of fractures in the clodronate group (P = 0.023). After 15 months the effect of clodronate tended to decline as the need for radiotherapy increased in the clodronate group compared with the control group (P = 0.069). Significant improvements in several quality-of-life aspects were seen in both groups during the first 6 months, but there was no significant difference between the groups. No effect was observed on time to radiologically evaluated disease progression in bone or on survival. The most frequent side-effects resulting in discontinuation of clodronate were nausea and diarrhoea. CONCLUSION: Oral clodronate is associated with a temporary reduction of morbidity related to bone metastases in breast cancer patients.

Adult↗
PubMed1999

Microvascular endothelial cells remodel cultured adult cardiomyocytes and increase their survival.

We investigated the paracrine effect of cardiac microvascular endothelial cells (MVEC) on cultured adult rat cardiomyocytes (ARC). ARC were exposed for 8 days to serum-free medium (CM) conditioned by MVEC. Controls were grown in FCS or FCS-free medium. Protein synthesis of CM-stimulated ARC increased twofold versus 5% FCS-stimulated cells until day 8. Seventy-nine percent of CM-treated myocytes survived, whereas only twenty-four percent of FCS-free ARC retained viability. The phenotype of myocytes exposed to CM was different from control. Analysis by confocal laser microscopy of CM-stimulated myocytes showed actin staining throughout the whole cell body up to the peripheral extensions, with concomitant appearance of myomesin in a cross-striated pattern. The reexpression of fetal alpha-smooth muscle actin determined immunohistochemically and by Western blot increased from day 6 in CM-treated cells, whereas ARC grown in up to 20% serum were negative. These effects could not be mimicked by any of the other cardioactive substances tested here, indicating a novel trophic factor in CM.

Actins↗
PubMed1998

The subcommissural organ.

The subcommissural organ (SCO) is a phylogenetically ancient and conserved structure. During ontogeny, it is one of the first brain structures to differentiate. In many species, including the human, it reaches its full development during embryonic life. The SCO is a glandular structure formed by ependymal and hypendymal cells highly specialized in the secretion of proteins. It is located at the entrance of the aqueduct of Sylvius. The ependymal cells secrete into the ventricle core-glycosylated proteins of high molecular mass. The bulk of this secretion is formed by glycoproteins that would derive from two different precursors of 540 and 320 kDa and that, upon release into the ventricle aggregate, form a threadlike structure known as Reissner's fiber (RF). By addition of newly released glycoproteins to its proximal end, RF grows caudally and extends along the aqueduct, fourth ventricle, and the whole length of the central canal of the spinal cord. RF material continuously arrives at the dilated caudal end of the central canal, known as the terminal ventricle or ampulla. When reaching the ampulla, the RF material undergoes chemical modifications, disaggregates, and then escapes through openings in the dorsal wall of the ampulla to finally reach local blood vessels. The SCO also appears to secrete a cerebrospinal fluid (CSF)-soluble material that is different from the RF material that circulates in the ventricular and subarachnoidal CSF. Cell processes of the ependymal and hypendymal cells, containing a secretory material, terminate at the subarachnoidal space and on the very special blood capillaries supplying the SCO. The SCO is sequestered within a double-barrier system, a blood-brain barrier, and a CSF-SCO barrier. The function of the SCO is unknown. Some evidence suggests that the SCO may participate in different processes such as the clearance of certain compounds from the CSF, the circulation of CSF, and morphogenetic mechanisms.

Animals↗
PubMed1998

Synechocystis sp. PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria.

During cultivation under storage conditions with BG11 medium containing acetate as a carbon source, Synechocystis sp. PCC6803 accumulated poly(3-hydroxybutyrate) up to 10% (w/w) of the cell dry weight. Our analysis of the complete Synechocystis sp. PCC6803 genome sequence, which had recently become available, revealed that not only the open reading frame slr1830 (which was designated as phaC) but also the open reading frame slr1829, which is located colinear and upstream of phaC, most probably represent a polyhydroxyalkanoic acid (PHA) synthase gene. The open reading frame slr1829 was therefore designated as phaE. The phaE and phaC gene products exhibited striking sequence similarities to the corresponding PHA synthase subunits PhaE and PhaC of Thiocystis violacea, Chromatium vinosum, and Thiocapsa pfennigii. The Synechocystis sp. PCC6803 genes were cloned using PCR and were heterologously expressed in Escherichia coli and in Alcaligenes eutrophus. Only coexpression of phaE and phaC partially restored the ability to accumulate poly(3-hydroxybutyrate) in the PHA-negative mutant A. eutrophus PHB-4. These results confirmed our hypothesis that coexpression of the two genes is necessary for the synthesis of a functionally active Synechocystis sp. PCC6803 PHA synthase. PHA granules were detected by electron microscopy in these cells, and the PHA-granule-associated proteins were studied. Western blot analysis of Synechocystis sp. PCC6803 crude cellular extracts and of granule-associated proteins employing antibodies raised against the PHA synthases of A. eutrophus (PhaC) and of C. vinosum (PhaE and PhaC) revealed no immunoreaction.

Acyltransferases↗
PubMed1998

Partial sequencing of Reissner's fiber glycoprotein I (RF-Gly I).

The bulk of the secretion of the subcommissural organ is formed by glycoproteins that appear to be derived from two precursor forms of 540 and 320 kDa. Upon release into the ventricle, these glycoproteins aggregate to form Reissner's fiber. We report the isolation of three cDNA clones from a cDNA library prepared from bovine subcommissural organ RNA, by using an anti-Reissner's fiber serum for immunoscreening. Inserts of 0.7, 1.2, and 2.5 kb were amplified by the polymerase chain reaction, subcloned into pUC18 vector, and sequenced. Although restriction mapping of the three inserts initially suggested that all of them were derived from the same mRNA, sequence analysis showed that a short non-homologous region was present in the 0.7-kb insert when compared with the 1. 2-kb and 2.5-kb inserts, suggesting that they corresponded to two different, although highly homologous, mRNAs. Northern analyses showed a single mRNA species of approximately 9.5 kb present in the subcommissural organ and missing in the choroid plexus, brain cortex, and liver. In situ hybridization confirmed that the expression of the RNA was restricted to cells of the bovine subcommissural organ. Polyclonal antibodies raised against a synthetic peptide, whose amino-acid sequence was deduced from the 2.5-kb cDNA, reacted specifically with the bovine and rat subcommissural organ-Reissner's fiber complex. In immunoblots of bovine subcommissural organ, this antibody revealed the precursor 540-kDa form and its putative processed form of 450 kDa. It is concluded that the cloned cDNA encodes for the major constitutive glycoprotein of Reissner's fiber, here designated as RF-Gly I. The sequenced region of RF-Gly I displays a high degree of homology with some regions of the von Willebrand factor and certain mucins; it also displays two motifs homologous with repeats present in proteins of the spondin family and other proteins. A core sequence of the RF-Gly I repeats suggests that this molecule displays protein-binding properties.

Amino Acid Sequence↗
PubMed1998

The internal and external protein scaffold of the T-tubular system in cardiomyocytes.

The transverse tubule system of the cardiomyocyte remains undeformed despite the extreme forces it undergoes during the contraction-relaxation cycle, but the morphological basis for its stability remains unclear. Therefore, we have investigated the architecture and subcellular protein scaffold of the cardiac T-tubules and compared it with that of the costameres and of the free sarcolemma. Tissue samples from normal rat and monkey hearts, and left ventricular tissue from normal and cardiomyopathic human hearts obtained at transplantation surgery were investigated using immunocytochemistry and confocal microscopy and by electron microscopy. In addition, we used a re-differentiation model of isolated, cultured adult rat cardiomyocytes. The cell membrane of the cardiac T-tubules was found to contain the cell-matrix focal adhesion molecules (FAMs) vinculin, talin, the alpha5beta1 integrin and the membrane-associated proteins (MAPs) dystrophin and spectrin. FAMs and MAPs were localized in the T-tubular membrane in a similar pattern: in longitudinally oriented myocytes as transverse punctate lines at the Z-level; in transversally cut myocytes a radial tubular network was found to extend throughout the interior of the cell. Immunolabeling for basement membrane components including collagen IV, fibronectin and laminin showed a colocalization with FAMs and MAPs parallel to the transverse T-tubules. The costameres of the sarcolemma showed a protein composition resembling that of the T-tubules but the intervening segments of free sarcolemma showed absence of FAMs and presence of MAPs. For the first time, we demonstrate the existence and protein composition of the T-tubular scaffold in the human heart. Furthermore, we show that cardiomyocytes from human failing hearts have less abundant but more dilated T-tubules than do experimental animals. These results indicate that the cardiac T-tubular system contains a subcellular scaffold closely resembling that of the costameres. It consists of FAMs, MAPs and basal lamina proteins that confer structural integrity to the cardiac T-tubular membrane during contraction/relaxation cycles.

Animals↗
PubMed1997

Biosynthesis of poly(4-hydroxybutyric acid) by recombinant strains of Escherichia coli.

The aim of this study was the production of the homopolyester poly(4-hydroxybutyric acid) (poly(4HB)) with recombinant strains of Escherichia coli. Wild-type strains and other widely used non-recombinant strains of E. coli are not able to produce polyhydroxyalkanoic acids (PHA) as storage compounds and cannot utilize 4-hydroxybutyric acid as sole carbon source. Accordingly, hybrid plasmids of pBluescript vectors were constructed which harbored the Alcaligenes eutrophus PHA synthase gene (phaC) and the Clostridium kluyveri orfZ putatively encoding a 4-hydroxybutyric acid-coenzyme A transferase. A 3.5-kb genomic SmaI/ApaI fragment from A. eutrophus, which comprises phaC, and a 1.8-kb genomic ApaI/EcoRI fragment from C kluyveri, which contained orfZ, were inserted into the SmaI and EcoRI sites of the vectors pKS- and pSK-, respectively. The two resulting plasmids pSKSE5.3 and pKSSE5.3 comprising phaC and orfZ colinear or antilinear to lacZ, respectively, were transformed into E. coli XL1-Blue. Recombinant strains synthesized the homopolyester poly(4HB), when the cells were cultivated in Luria-Bertani broth and if glucose and 4-hydroxybutyric acid were provided as carbon sources. If glucose was omitted, a copolyester of 3-hydroxybutyric acid and 4-hydroxybutyric acid was accumulated. The homopolyester poly(4HB) was also accumulated during cultivation of these strains in M9 mineral salts medium containing glucose plus 4-hydroxybutyric acid as carbon sources. Poly(4HB) could amount up to approximately 80% (w/w) of the cell dry matter if E. coli XL1-Blue harboring pKSSE5.3 was cultivated in M9 mineral salts medium and if the cultures were not sufficiently supplied with oxygen. 4HB was also incorporated into PHA if gamma-butyrolactone was used as carbon source. If levulinic acid, 4-hydroxyvaleric acid or gamma-valerolactone were used as carbon sources, only very low amounts of PHA were accumulated which did not contain 4-hydroxyalkanoic acids as constituents.

4-Butyrolactone↗
PubMed1997

Inhibition of carrageenan-induced spinal c-Fos activation by systemically administered c-fos antisense oligodeoxynucleotides may be facilitated by local opening of the blood-spinal cord barrier.

Proto-oncogenes of the fos and jun family are rapidly expressed in the central nervous system following various stimuli. Proto-oncogene encoded nuclear proteins such as c-Fos or c-Jun act as transcription factors that may link neuronal excitation to changes in target gene expression. However, the precise in vivo functions of proto-oncogenes in neuroplasticity are still poorly understood. In the present study the effect of systemically administered c-fos antisense oligodeoxynucleotides (ODNs) on c-Fos and dynorphin protein levels in rat L4 spinal cord has been investigated by immunohistochemistry during carrageenan-induced hindpaw inflammation. Continuous infusion of terminal-phosphorothioated c-fos antisense ODNs by subcutaneously implanted miniosmotic pumps for 3 days sequence-specifically suppressed c-Fos protein expression in dorsal horn neurons by about 50%, while the increase in c-Jun immunopositive nuclei was not affected. Digital image analysis revealed a concomitant decrease in spinal dynorphin immunoreactivity. Moreover, 48 hr after carrageenan injection into one hindpaw plasma protein extravasation was observed in numerous blood vessels in the ipsilateral dorsal horn using intravenously administered Evans Blue. Our results provide further evidence that c-Fos may contribute to the regulation of spinal dynorphin gene expression following noxious stimulation. The local increase in blood-spinal cord barrier permeability during sustained peripheral inflammation may permit penetration of hydrophilic antisense ODNs into the central nervous system.

Animals↗
PubMed1996

Alcaligenes eutrophus possesses a second pyruvate dehydrogenase (E1).

Two gene loci, which hybridized with pdhA, the structural gene of the E1 component of the Alcaligenes eutrophus pyruvate dehydrogenase complex [Hein, S. & Steinbüchel, A. (1994) J. Bacteriol. 176, 4394-4408], were identified on two nonrelated A. eutrophus chromosomal BamHI fragments by using a pdhA-specific DNA probe. These data indicated that A. eutrophus possesses, beside PdhA, two additional distinct pyruvate dehydrogenases (E1). A 6.8-kbp genomic BamHI fragment of A. eutrophus was cloned, and sequence analysis of a 3.896-kbp region revealed the structural gene pdhE (2.694 kbp) for a second pyruvate dehydrogenase (E1), which was not clustered with structural genes for other components of 2-oxo acid dehydrogenase complexes. The A. eutrophus pdhE gene product (898 amino acid residues) exhibited significant similarities to the E1 components of the pyruvate dehydrogenase complexes of A. eutrophus, Neisseria meningitidis, Escherichia coli and Azotobacter vinelandii, which are also composed of only one type of subunit. Heterologous expression of pdhE in the aceEF deletion mutant E. coli YYC202 was demonstrated by spectrometric detection of enzyme activities and by phenotypic complementation to acetate prototrophy. These complementation studies indicated that the E1 component of the A. eutrophus pyruvate dehydrogenase complex can be replaced by a functionally active pdhE gene product.

Alcaligenes↗
PubMed1996

Cloning and characterization of the Alcaligenes eutrophus 2-oxoglutarate dehydrogenase complex.

Nucleotide sequence analysis of a 3.3-kb genomic EcoRI fragment and of relevant subfragments of a genomic 13.2-kb SmaI fragment of Alcaligenes eutrophus, which were identified by using a dihydrolipoamide dehydrogenase-specific DNA probe, revealed the structural genes of the 2-oxoglutarate dehydrogenase complex in a 7.5-kb genomic region. The genes odhA (2850 bp), odhB (1248 bp), and odhL (1422 bp), encoding 2-oxoglutarate dehydrogenase (E1), dihydrolipoamide succinyltransferase (E2), and dihydrolipoamide dehydrogenase (E3), respectively, occur co-linearly in one gene cluster downstream of a putative -35/-10 promoter in the order odhA, odhB, and odhL. In comparison to other bacteria, the occurrence of genes for two E3 components for the pyruvate as well as for the 2-oxoglutarate dehydrogenase complexes is unique. Heterologous expression of the A. eutrophus odh genes in E. coli XL1-Blue and in the kgdA mutant Pseudomonas putida JS347 was demonstrated by the occurrence of protein bands in electropherograms, by spectrometric detection of enzyme activities, and by phenotypic complementation, respectively.

Alcaligenes↗
PubMed1996

Pathogenesis of dilated cardiomyopathy and heart failure: insights from cell morphology and biology.

The cause of dilated cardiomyopathy is not yet clear but the recent discovery of a chromosomal aberration as well as the presence of autoantibodies indicate a multicausal origin. Knowledge of pathogenetic mechanisms continues to evolve and includes decreased sarcoplasmic reticulum Ca2+ uptake, reduced beta-receptor density and decreased contractility, the presence of enlarged myocytes showing numerous degenerative alterations, and fibrosis. Defects in titin, a large sarcomeric protein, may be responsible for disturbances of sarcomerogenesis in dilated cardiomyopathy. New animal models are promising but significant progress will also be made using cell culture systems like adult cardiac myocytes.

Animals↗
PubMed1995

Selenium in serum and whole blood in patients with surgical interventions.

Selenium values of serum and whole blood were determined for two groups of patients undergoing large surgical interventions (e.g., gastrectomy) and receiving a total parenteral nutrition (TPN). All patients showed low selenium values at the beginning of TPN. The first group received a supplementation of selenium, and it was established that selenium levels normalized after 6-7 d. The balances of selenium turned out to be positive in all cases. The second group, which was not administered selenium supplementation, showed significantly lower values of selenium. It can, therefore, be concluded that a substitution of selenium prior to large surgical interventions following TPN clearly helps patients combat the increase in the oxidant stress reactions.

Aged↗
PubMed1995

Ischemia induces early changes to cytoskeletal and contractile proteins in diseased human myocardium.

Ischemia is known to produce damage to subcellular organelles, such as nuclei and mitochondria, in myocardial tissue. We tested the hypothesis that during myocardial ischemia various cytoskeletal and contractile proteins also undergo changes. We induced total global ischemia by incubation in buffer of tissue samples from six human left ventricles that were obtained from heart transplant recipients. Samples were removed from the incubation medium at different time intervals and investigated by immunohistochemistry using monoclonal antibodies against myosin, actin, tropomyosin, troponin T, myomesin, desmin, tubulin, and vinculin. The degree of ischemic injury was determined by electron microscopy. Ischemic cardiomyopathic human tissue showed disturbances of the localization pattern of myosin, actin, tropomyosin, and troponin T as early as 10 minutes after the onset of ischemia; this disruption was complete at 20 minutes. Tubulin also started changing at 10 minutes, but complete disruption was only evident after 120 minutes. Desmin and myomesin showed an intermediate response; changes began at 30 to 40 minutes, and disruption was complete at 90 to 120 minutes. Vinculin was most resistant to ischemia. Ultrastructurally, the tissue showed moderate reversible ischemic injury during the entire period of 180 minutes. Measuring the exposure time in seconds allowed quantitation of the intensity of the fluorescence. We reached the following conclusions: (1) Ischemia causes damage to the contractile proteins sooner than to the cytoskeleton and subcellular organelles. (2) Diseased human hearts are extremely susceptible to the effects of ischemia. These findings are important for the situation of induced cardiac arrest in heart operations and for preservation of donor hearts for transplantation.

Actins↗
PubMed1995

[Interactions between cardiomyocytes and extracellular matrix in the failing human heart].

Numerous morphological changes can be observed in human myocardium failing because of dilated cardiomyopathy. These can be observed by electron microscopy and by immunofluorescence microscopy using monoclonal antibodies. These changes include: 1) the occurrence of hypertrophied and atrophied myocytes as well as cells of normal size, 2) degenerative changes in myocytes; these consist of nuclei of varying size and shape, lack of contractile material, disorganization of the cytoskeleton, and sequestration of cellular particles into the extracellular space and 3) an enlarged extracellular space, that is, fibrosis, which contains increased amounts of the different matrix proteins such as fibronectin and laminin, the various collagens, and chondroitin sulfate, in addition to cellular debris and numerous macrophages and fibroblasts. On the basis of these findings it is hypothetized that there exists an interaction between myocytes and the extracellular matrix. The cells of the latter may be stimulated to higher rates of proteins synthesis by the presence of cellular debris. This process, in turn, may be harmful for the structural integrity of myocytes which consequently sequester more cellular particles. In this manner, a vicious circle may be started that leads to further structural and functional deterioration of the myocardium, finally resulting in failure.

Cardiomyopathy, Dilated↗