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H Schumann

Publications and source records attributed to H Schumann.

At least 19 recordsLinked to original sources
PubMed2002

Load-dependent induction of apoptosis in multicellular myocardial preparations.

Increased mechanical load has been proposed as an inductor of apoptosis, but it is unknown whether this can occur in the range of pre- and afterloads that prevail in the beating heart. We investigated apoptosis in cultured rabbit multicellular myocardial preparations over several days. Muscles contracted in absence of pre- and afterload (unloaded isotonic), in absence of preload but in presence of afterload (unloaded isometric), or in presence of both (loaded isometric). After up to 48 h of continuous contractions, apoptosis was assessed by TdT-mediated nick-end labeling (TUNEL) assay and DNA ladder analysis. In muscles that contracted loaded isometric, apoptosis was detected after 6-24 h. After 48 h, apoptosis was most prominent in this group, reflected by a high level of DNA ladder intensity (DLI; 27.8 +/- 11.5), whereas Bcl-xL (on RNA level) was significantly downregulated, and Fas remained unchanged. In unloaded isometric preparations, apoptosis was significantly less (6.9 +/- 5.9 DLI) and very similar to those contracting unloaded isotonic (6.1 +/- 5.1 DLI). We conclude that load-dependent apoptosis can occur at sarcomere lengths achievable in vivo and may mainly result from increased preload.

Animals↗
PubMed2001

A chemical definition of the effective reducing power of thulium(II) diiodide by its reactions with cyclic unsaturated hydrocarbons.

Thulium diiodide reduces cyclic aromatic hydrocarbons that have reduction potentials more positive than - 2.0 V versus SCE. Thus, TmI2 reacts with cyclooctatetraene or acenaphthylene in THF, or with lithium anthracenide in 1,2-dimethoxyethane (DME) to give thulium triiodide and the thulium(III) complexes [(eta8-C8H8)TmI(thf)2] (1), rac-ansa-[(eta5-C12H8)2TmI(thf)] (2), or [(eta2-C14H10)TmI-(dme)2] (3), respectively. The molecular structures of 1-3 were determined by single-crystal X-ray diffraction.

Journal Article↗
PubMed2001

[Computer assisted plastic closure of calvarial defects].

Large bony skull defects sometimes face problems of some pathophysiological effects, protection of the underlying brain, the impaired appearance, and from a psychological point of view. A computer-assisted method is presented, which has been successfully used in 44 patients. From 3-D-CT data a phantom was built in which Refobacin-Palacos was modeled for individual requirements. The plastic can be implanted after sterilization and leads to excellent cosmetical results. The use of a negative model reveals the possibility of an extended field of application without the necessity of highly specialized computersystems.

Adolescent↗
PubMed2000

Collagen XVII is destabilized by a glycine substitution mutation in the cell adhesion domain Col15.

Collagen XVII is a hemidesmosomal transmembrane molecule important for epithelial adhesion in the skin. It exists in two forms, as a full-length protein and as a soluble ectodomain that is shed from the keratinocyte surface by furin-mediated proteolysis. To obtain information on the conformation and the functions of this unusual collagen, its largest collagenous domain, Col15, was expressed in a eukaryotic episomal expression system and purified by DEAE and fast protein liquid- Mono S chromatography. The protein was triple-helical (T(m) of 26.5 degrees C) when produced in cultures containing ascorbic acid. When the vitamin supply was limited, the 4-hydroxyproline content was reduced from 74 to 9%, which, in turn, resulted in a drastic reduction of the stability of the triple helix. The glycine substitution mutation G627V associated with junctional epidermolysis bullosa, a human blistering skin disease, also had a striking effect on thermal stability of rCol15 causing partial unfolding already at 4 degrees C. Col15 promoted cell adhesion of epithelial and fibroblastic cell lines with a beta1 integrin-mediated mechanism. In concert with this, in acquired autoimmune blistering skin diseases, circulating IgG and IgA autoantibodies were found to target rCol15r.

Amino Acid Substitution↗
PubMed2000

The shed ectodomain of collagen XVII/BP180 is targeted by autoantibodies in different blistering skin diseases.

Collagen XVII/BP180, an epidermal adhesion molecule, exists as a full-length transmembrane protein and as a soluble 120-kd ectodomain that is shed from the keratinocyte surface by furin-mediated proteolysis. Despite a number of studies on autoantibody targets in blistering skin diseases, it has remained unclear whether the physiologically shed ectodomain of collagen XVII plays a role as an autoantigen. Here we isolated the authentic, soluble form of human collagen XVII and showed that it is an autoantigen recognized by IgG and IgA autoantibodies in different blistering skin diseases and is, in some cases, the preferential target. The ectodomain was isolated from the epidermis, keratinocyte media, amniotic fluid, and pemphigoid blister fluid, and autoantibodies affinity-purified with this ectodomain bound to the proximal surface of the epidermis in normal skin but not in collagen XVII-deficient skin. The antibody reactivity was not dependent on the native conformation or the N-glycosylation of the soluble ectodomain, but was abolished by collagenase treatment. Sera of 81 patients with a clinically active blistering skin disease were reacted with full-length collagen XVII, the authentic soluble ectodomain, and recombinant fragments. In bullous and cicatricial pemphigoid, IgG reactive with full-length collagen XVII also recognized the soluble ectodomain. In linear IgA dermatosis and chronic bullous dermatosis of childhood, IgA targeted the soluble ectodomain more efficiently than the full-length protein. The use of recombinant fragments demonstrated that epitopes were present in several noncollagenous and collagenous subdomains of the molecule, and that a significant portion of the sera targeted Col15 domain, a hitherto unrecognized epitope region.

Adolescent↗
PubMed2000

Expression of secreted frizzled related proteins 3 and 4 in human ventricular myocardium correlates with apoptosis related gene expression.

OBJECTIVE: Overload-induced heart failure is associated with myocyte apoptosis induced by unknown mechanisms. Wnt genes encode secreted signaling molecules that bind to frizzled receptors and stabilize cytosolic beta-catenin which is translocated into the nucleus, acts as transcriptional activator and imparts an apoptosis resistant phenotype. This signaling pathway is antagonized by secreted frizzled related proteins (sFRPs) which modulate apoptosis susceptibility in cell culture models. On the basis of these considerations, the present investigation compares myocardial mRNA expression of sFRPs and the level of soluble beta-catenin in tissue samples from nonfailing and failing hearts. METHODS: Nonischemic transmural samples from human failing left ventricles and from nonfailing donor ventricles were used in the present study. The mRNA concentration of the Wnt-antagonists sFRP 1-4 were determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). The myocardial localization of sFRP 3 and 4 expression was investigated using in situ RT-PCR. The pool of soluble beta-catenin was quantified by Western blot analysis of protein extracts. RESULTS: The mRNA levels of proapoptotic sFRPs 3 and 4 but not of sFRP 1 and 2 were elevated in failing ventricles compared to donor hearts. There was no significant difference between patients suffering from a dilated cardiomyopathy or a coronary heart disease. sFRPs 3 and 4 were expressed in cardiomyocytes and their expression correlated with the mRNA expression of the proapoptotic Fas/Fas-antagonist ratio, but inversely with the mRNA levels of the antiapoptotic bcl-xL. The size of the pool of 0.1% Triton soluble beta-catenin tended to decrease in myocardial samples with high sFRP 3 and 4 expression levels. CONCLUSIONS: The results support the hypothesis that in failing human myocardium the Wnt/beta-catenin pathway is attenuated by enhanced expression of two endogenous Wnt-antagonists. This might contribute to an apoptosis susceptible phenotype of overloaded human myocardium.

Apoptosis↗
PubMed2000

p0071, a member of the armadillo multigene family, is a constituent of sarcomeric I-bands in human skeletal muscle.

p0071 is a member of the armadillo gene family that is expressed in a wide variety of mammalian tissues and cell types with a prominent cell-cell contact association in epithelial cells. Here, we report the expression and localization patterns of p0071 in differentiating human skeletal muscle cells and in normal and diseased human skeletal muscle tissues. Northern blots revealed expression of p0071 mRNA in adult skeletal muscle tissue. RT-PCR analysis and Western blotting experiments identified two differentially spliced isoforms of p0071. The balance between these isoforms shifted during in vitro differentiation of isolated muscle cells from predominant expression of the short variant to a preponderance of the larger variant from day 6 onwards. Immunolocalization studies in mature skeletal muscle tissue revealed that p0071 is a constituent of myofibrils with a distinct localization at the level of sarcomeric N2-lines. During myofibrillogenesis, p0071 was not detected in non-striated nascent myofibrils, but became apparent shortly after the development of compact Z-discs in early myotubes. Furthermore, we studied the expression of p0071 in a wide variety of neuromuscular disorders by indirect immunofluorescence. Here, the myofibrillar staining of p0071 was preserved in all the disease entities included in our study. Our results provide the first evidence that a member of the armadillo multigene family is a constituent of the contractile apparatus in human skeletal muscle. The localization of p0071 at the level of I-bands and the timepoint of its integration into developing myofibrils suggest a possible role in the organization of thin filaments.

Age Factors↗
PubMed2000

Hemizygosity for a glycine substitution in collagen XVII: unfolding and degradation of the ectodomain.

Defects of collagen XVII, a keratinocyte adhesion protein, are associated with epidermal detachment in junctional epidermolysis bullosa. Although some missense mutations in the collagen XVII gene COL17A1 have been described, the molecular mechanisms leading to disease have remained elusive in these cases. Here we assessed the biologic consequences of a missense mutation by studying the folding and stability of wild-type and mutated recombinant collagen XVII domains. The mutation occurred in a junctional epidermolysis bullosa patient who was compound heterozygous for the novel glycine substitution mutation G633D and the novel nonsense mutation R145X. Collagen XVII mRNA was significantly reduced, indicating nonsense-mediated mRNA degradation and hemizygosity of the patient for the G633D substitution. As glycine residues within the collagen triple helices are important for stable conformation, the thermal stability of the wild-type and mutated eukaryotic recombinant Col15 domain of collagen XVII was assessed. The stability of the mutated fragment was clearly reduced. The midpoint of the helix-to-coil transition, Tm, was 5 degrees C lower than that of wild-type rCol15, indicating abnormal triple-helix folding and susceptibility to proteolysis. Consistently, immunoassays demonstrated reduced amounts of the full-length collagen XVII and absence of the soluble ectodomain in keratinocyte cultures, and lack of the ectodomain from the junctional epidermolysis bullosa skin. These observations show that the glycine substitution G633D in collagen XVII causes abnormal folding and susceptibility to degradation, and thus perturbs the physiologic adhesive functions of collagen XVII in the skin.

Adolescent↗
PubMed2000

IgG autoantibodies from bullous pemphigoid patients recognize multiple antigenic reactive sites located predominantly within the B and C subdomains of the COOH-terminus of BP230.

Bullous pemphigoid is a subepidermal bullous disorder characterized by an autoantibody response against the bullous pemphigoid antigen 230 (BP230) and the bullous pemphigoid antigen 180 (BP180), a cytoplasmic component and a transmembrane component, respectively, of hemidesmosomes. Although immunodominant sequences within the extracellular domain of BP180 have been identified, characterization of the antigenic sites on BP230 is still incomplete. To identify autoantibody-reactive sites on BP230 and to examine whether the targeted regions are contained within functionally important domains, recombinant fragments encompassing almost the entire BP230 were used to assess the reactivity of 25 bullous pemphigoid sera by immunoblotting. Our results demonstrate that (i) the region bearing the B and C subdomains of the COOH-terminus of BP230 contains immunodominant sequences recognized by the majority of bullous pemphigoid sera; (ii) additional autoantibody- reactive sites are present over extended regions of the NH2-terminal half of BP230 without evidence for antigenic cross-reactivity between the NH2- and COOH-termini of BP230; and, finally, (iii) autoantibodies reacting with the BP230 tail predominantly belong to the IgG4 and IgG1 subclasses, suggesting that both autoreactive TH2 and autoreactive TH1 cells regulate the autoantibody response to immunodominant sequences of BP230. As the COOH- terminus of BP230 mediates the attachment of keratin intermediate filaments to the hemidesmosomal plaque, whereas its NH2-terminus contains sequences important for its interaction with other constituents of hemidesmosomes, autoantibodies to BP230 might precipitate subepidermal blister formation and perpetuate the disease not only by eliciting an inflammatory reaction but also by interfering with the function of BP230 and thus the stability of hemidesmosomes.

Aged↗
PubMed1999

Myocardial gene expression of regulators of myocyte apoptosis and myocyte calcium homeostasis during hemodynamic unloading by ventricular assist devices in patients with end-stage heart failure.

BACKGROUND: In patients with end-stage heart failure, characterized by an increased susceptibility to cardiomyocyte apoptosis and a labile cardiomyocyte calcium homeostasis, a ventricular assist device (VAD) is implanted for bridging to cardiac transplantation and results in myocardial unloading. Although phenotype changes in the failing heart are assumed to result from hemodynamic overload, the reversibility of these changes under unloading is unknown. METHODS AND RESULTS: By use of quantitative reverse-transcription polymerase chain reaction, mRNA expression analyses were performed on left ventricular specimens obtained from 10 nonfailing donor hearts (from 8 patients with dilated cardiomyopathy and 2 patients with coronary heart disease) at the time of VAD implantation and 36 to 169 days later during VAD removal with subsequent cardiac transplantation. In terminally failing hearts before VAD support, left ventricular mRNA analyses revealed increased Pro-ANP, reduced antiapoptotic Bcl-x(L) and antiapoptotic Fas isoform FasExo6Del, and a decreased ratio of sarcoplasmic reticulum Ca(2+)-ATPase per sarcolemmal Na(+)-Ca(2+) exchanger in comparison with nonfailing ventricles. After VAD unloading, ventricular transcription of Pro-ANP was immediately normalized, and apoptotic DNA fragmentation was attenuated. In patients with dilated cardiomyopathy, mRNAs of Bcl-x(L) and FasExo6Del/Fas were enhanced depending on time on VAD. The Bcl-x(L) mRNA level correlated positively with that of the Bcl-x(L) protein. Transcription of sarcoplasmic reticulum Ca(2+)-ATPase/Na(+)-Ca(2+) exchanger demonstrated recovery in only 4 of 10 patients. CONCLUSIONS: Mechanical support of the failing heart induces a time-dependent change in myocardial gene expression compatible with a decreased susceptibility to apoptosis.

Adult↗
PubMed1999

A child with localized vulval pemphigoid and IgG autoantibodies targeting the C-terminus of collagen XVII/BP180.

Localized vulval pemphigoid of childhood (LVPC) has previously been reported in six girls. Clinical features and immunopathological data have suggested it to be a morphological variant of bullous pemphigoid. Epitope targets of the autoantibodies of these patients have not been defined in detail. We describe a 9-year-old girl with possible cicatricial LVPC and circulating IgG antibodies directed against native collagen XVII/BP180, its 120-kDa soluble ectodomain and against the C-terminus of collagen XVII/BP180. No reactivity was detected towards the NC16A domain of collagen XVII/BP180. Linear IgG and C3 deposits were found along the cutaneous basement membrane zone. On 1 mol/L salt-split skin, IgG autoantibodies were shown to bind to the epidermis, and the HLA type II allele DQB1*0301, a marker with significantly increased occurrence in patients with ocular and oral cicatricial pemphigoid, was identified in this patient. Our data suggest that LVPC is a variant of bullous pemphigoid in which direct immunofluorescence microscopy combined with immunoblot analysis can deliver valuable diagnostic information for differential diagnosis. However, differentiation between the scarring and non-scarring course of the disease cannot be made with the present diagnostic markers and therefore careful follow-up of patients with LVPC is required.

Autoantibodies↗
PubMed1999

Autoantibodies in a subgroup of patients with linear IgA disease react with the NC16A domain of BP1801.

Linear IgA disease is an autoimmune subepidermal blistering disease characterized by IgA deposits at the cutaneous basement membrane zone. IgA antibodies from linear IgA disease sera react with antigens of 97 kDa (LABD97) and 120 kDa (LAD-1), both of which appear to be fragments of the extracellular domain of bullous pemphigoid 180 (type XVII collagen). The aim of this study was to determine whether linear IgA disease sera react with the immunodominant region of BP180 (NC16A domain), which is a major target of IgG autoantibodies produced by patients with bullous pemphigoid. Indeed, 11 of 50 linear IgA disease sera were found to contain IgA autoantibodies that recognized a recombinant form of NC16A by immunoblotting. The same sera also reacted with NC16A by enzyme-linked immunosorbent assay. An epitope mapping analysis uncovered four linear IgA disease-associated epitopes located within the 45 amino acid N-terminal stretch of NC16A, all of which were previously identified as antigenic sites targeted by bullous pemphigoid autoantibodies. Eight of the linear IgA disease sera that were reactive with NC16A also recognized LAD-1 secreted by the SCC-25 cell line, and five sera recognized BP180 extracted from keratinocytes. Linear IgA disease sera depleted of reactivity to NC16A by immunoadsorption continued to react with both the LAD-1 antigen and BP180 by immunoblotting and with the basement membrane zone by indirect immunofluorescence microscopy. Our results demonstrate that IgA autoantibodies from a subset of linear IgA disease patients react with the same sites on BP180 that are targeted by IgG autoantibodies in bullous pemphigoid.

Aged↗
PubMed1999

Altered levels of mRNA of apoptosis-mediating genes after mid-term mechanical ventricular support in dilative cardiomyopathy--first results of the Halle Assist Induced Recovery Study (HAIR).

BACKGROUND: Ventricular assist devices (VADs) lead to an immediate unloading of the failing heart. Although VADs are used as a bridge to transplant, in some cases patients suffering from dilated cardiomyopathy have been weaned from the VAD without transplantation after a recovery process initiated by the cardiac support. Myocardial apoptosis is associated with inadequate myocardium and might be reverted during VAD support of the failing heart. Therefore we measured transcription of apoptosis-associated genes FasExo6 Del, Fas-receptor, and Bcl-xL as markers of a putative recovery. METHODS: Fas-receptor, its soluble isoform FasExo6Del, and Bcl-xL mRNA were quantified by standard calibrated competitive reverse-transcription polymerase chain reaction (PCR) in 6 patients suffering from dilated cardiomyopathy. RNA standards were prepared by introducing 100 bp deletions into the native cDNA, resulting in truncated PCR products with identical primer-binding sites. Standards were transcribed in vitro and the resulting RNA was quantified. RESULTS: Transcription of apoptosis-inhibiting genes FasExo6 Del and Bcl-xL were upregulated in patients supported for more than 6 weeks. Fas receptor mRNA remained unaffected by VAD support. CONCLUSIONS: Transcriptional upregulation of apoptosis-inhibiting genes might be caused by a desensitization to apoptotic stimuli and might indicate a relaxation of the diseased status of the myocardium. These data outline the first biochemical evidence of a remodelling process occurring in supported ventricular myocardium.

Apoptosis↗
PubMed1998

Two forms of collagen XVII in keratinocytes. A full-length transmembrane protein and a soluble ectodomain.

The cDNA sequence of human collagen XVII predicts an unusual type II transmembrane protein, but a biochemical characterization of this structure has not been accomplished yet. Using domain-specific antibodies against recombinant collagen XVII fragments, we identified two molecular forms of the collagen in human skin and epithelial cells. Full-length collagen XVII appeared as a homotrimeric transmembrane molecule of three 180-kDa alpha1(XVII) chains. The globular intracellular domain was disulfide-linked, and the N-glycosylated extracellular domain of three 120-kDa polypeptides was triple-helical at physiological temperatures. A second, soluble form of collagen XVII in keratinocyte culture media was recognized with antibodies to the ectodomain, but not the endodomain. The soluble form exhibited molecular properties of the collagen XVII ectodomain: a triple-helical, N-glycosylated molecule of three 120-kDa polypeptides. Northern blot analysis with probes spanning either the distal 5'or the distal 3' end of the collagen XVII cDNA revealed an identical 6-kb mRNA, suggesting that both the 180- and 120-kDa polypeptides were translated from the same mRNA, and that the 120-kDa polypeptide was generated post-translationally. In concert, keratinocytes harboring a homozygous nonsense mutation in the COL17A1 gene synthesized neither the 180-kDa alpha1(XVII) chain nor the 120-kDa polypeptide. Finally, treatment of normal keratinocytes with a synthetic inhibitor of furin proprotein convertases, decanoyl-RVKR-chloromethyl ketone, prevented the generation of the 120-kDa polypeptide. These data strongly suggest that the soluble 120-kDa polypeptide represents a specifically cleaved ectodomain of collagen XVII, generated through furin-mediated proteolytic processing. Thus, collagen XVII is not only an unusual type II transmembrane collagen, but the first collagen with a specifically processed, soluble triple-helical ectodomain.

Amino Acid Chloromethyl Ketones↗