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Biomedical subjects

U Schmitz

Publications and source records attributed to U Schmitz.

At least 19 recordsLinked to original sources
PubMed2004

[Chorioallantoic membrane of fertilized avian eggs as a substrate for assessment of cancerous invasiveness].

PURPOSE: Invasiveness is a characteristic feature of malignant tumors considerably determining the prognosis of affected patients. For assessment, apart from in vitro procedures with limited validity, tests on animal models have been established which certainly should be replaced by alternative methods whenever possible. The chorioallantoic membrane (CAM) of fertilized avian eggs represents an epithelial-lined membrane composed of all three blastodermic germ layers. In an "in ovo" assay cancer cells can be applied to this membrane after sinking (CAM assay). Tumor growth and invasiveness should be monitored in succession. MATERIAL AND METHODS: Hybrid chorionic carcinoma trophoblast cells were expanded in cell culture and spread over the CAM of hen's eggs after sinking followed by further incubation at 37 degrees C. The growth and development of the tumors were assessed macroscopically and finally (immuno-)histologically. Additionally, cytokeratin 19 was determined by enzyme-linked immunosorbent assay following homogenization of the tumor cells. RESULTS. Macroscopically, development of solid tumors was evident. Histological and immunohistochemical analysis revealed initial intraepithelial followed by cone-shaped infiltration of the CAM by the tumor cells. Tumor growth could be correlated with quantitative cytokeratin 19 measurements. CONCLUSIONS: Histomorphological appearance of the tumors was comparable with those results achieved in an immunodeficient mouse model. In addition, the CAM assay can be used for qualitative assessment of invasiveness of malignant tumors and yields quantitative results regarding growth kinetics. In contrast to conventional animal models, there is no need for official approval. Finally, this method is economical and facilitates processing many cases within a short time.

Animals↗
PubMed2002

Mechanisms of syncytial fusion: a review.

Syncytial fusion of trophoblast is a key process in placental morphogenesis and physiology. Disturbed syncytial fusion may lead to a number of pregnancy-associated pathologies. The mechanisms regulating syncytial fusion are only partly understood. This review tries to summarize the available knowledge on trophoblast fusion, originating from different scientific disciplines. Among the themes addressed in this paper are: morphogenesis and functions of syncytiotrophoblast; early apoptotic events and changes in plasmalemmal phospholipid orientation; proteins involved in membrane fusion: ADAMs and retrovirally-derived proteins and short-lived proteolipid intermediates in membrane fusion. Deeper understanding of syncytiotrophoblast fusion in future studies is only to be anticipated from collaborative studies focusing in parallel on physicochemical events in the participating plasmalemmas, early apoptotic/differentiation events preceding the fusion and role of the fusogenic membrane proteins.

Adult↗
PubMed2001

A novel dicationic polyamide ligand binds in the DNA minor groove as a dimer.

We have investigated DNA binding properties of a dicationic polyamide molecule (GL020924) that has exhibited unique protein displacement and gene regulation activities. Fluorescence, thermal melting and electrospray ionization mass spectrometry experiments showed that the binding stoichiometry of GL020924 is 2:1 to various DNA oligomers with 8-11 contiguous A/T bp. In accordance with those findings, circular dichroism experiments showed GL020924 binds as a partially staggered side-by-side dimer spanning 10-12 bp. These observations and molecular modeling studies demonstrate that the 2:1 GL020924-DNA complex may exhibit a novel form of stacking orientation involving at least partially parallel peptide groups.

Cations↗
PubMed2001

The hybridization-stabilization assay: a solution-based isothermal method for rapid screening and determination of sequence preference of ligands that bind to duplexed nucleic acids.

The gene-to-drug quest will be most directly served by the discovery and development of small molecules that bind to nucleic acids and modulate gene expression at the level of transcription and/or inhibit replication of infectious agents. Full realization of this potential will require implementation of a complete suite of modern drug discovery technologies. Towards this end, here we describe our initial results with a new assay for identification and characterization of novel nucleic acid binding ligands. It is based on the well recognized property of stabilization of hybridization of complementary oligonucleotides by groove and/or intercalation binding ligands. Unlike traditional thermal melt methodologies, this assay is isothermal and, unlike gel-based footprinting techniques, the assay also is performed in solution and detection can be by any number of highly sensitive, non-radioisotopic modalities, such as fluorescence resonance energy transfer, described herein. Thus, the assay is simple to perform, versatile in design and amenable to miniaturization and high throughput automation. Assay validation was performed using various permutations of direct and competitive binding formats and previously well studied ligands, including pyrrole polyamide and intercalator natural products, designed hairpin pyrrole-imidazole polyamides and furan-based non-polyamide dications. DNA specific ligands were identified and their DNA binding site size and sequence preference profiles were determined. A systematic approach to studying the relationship of binding sequence specificity with variation in ligand structure was demonstrated, and preferred binding sites in longer DNA sequences were found by pseudo-footprinting, with results that are in accord with established findings. This assay methodology should promote a more rapid discovery of novel nucleic acid ligands and potential drug candidates.

Base Pairing↗
PubMed2001

Angiotensin II-induced stimulation of p21-activated kinase and c-Jun NH2-terminal kinase is mediated by Rac1 and Nck.

p21-activated kinase (PAK) has been shown to be an upstream mediator of JNK in angiotensin II (AngII) signaling. Little is known regarding other signaling molecules involved in activation of PAK and JNK by AngII. Rho family GTPases Rac and Cdc42 have been shown to enhance PAK activity by binding to p21-binding domain of PAK (PAK-PBD). In vascular smooth muscle cells (VSMC) AngII stimulated Rac1 binding to GST-PAK-PBD fusion protein. Pretreatment of VSMC by genistein inhibited AngII-induced Rac1 activation, whereas Src inhibitor PP1 had no effect. Inhibition of protein kinase C by phorbol 12,13-dibutyrate pretreatment also decreased AngII-mediated activation of Rac1. The adaptor molecule Nck has been shown previously to mediate PAK activation by facilitating translocation of PAK to the plasma membrane. In VSMC AngII stimulated translocation of Nck and PAK to the membrane fraction. Overexpression of dominant-negative Nck in Chinese hamster ovary (CHO) cells, stably expressing the AngII type I receptor (CHO-AT1), inhibited both PAK and JNK activation by AngII, whereas it did not affect ERK1/2. Finally, dominant-negative Nck inhibited AngII-induced DNA synthesis in CHO-AT1 cells. Our data provide evidence for Rac1 and Nck as upstream mediators of PAK and JNK in AngII signaling and implicate JNK in AngII-induced growth responses.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Mutational analysis of INI1 in sporadic human brain tumors.

The INI1/SMARCB1/hSNF5 gene on chromosome 22 is frequently mutated in rhabdoid tumors. An association of INI1 mutations with allelic losses on chromosome 22 supports a classical tumor suppressor mechanism. Several brain tumor entities including astrocytomas, glioblastomas and ependymomas are characterized by allelic losses on chromosome 22. In the present study we examined a series of 200 brain tumors by Single-strand conformation polymorphism analysis and direct sequencing for point mutations in INI1. In addition, all tumors were analyzed for homozygous deletions spanning both exons 3 and 8 of INI1. No mutations or homozygous deletions were detected in astrocytomas, glioblastomas, oligodendroglial tumors, neurinomas or medulloblastomas. However, a point mutation could be identified in the single case of plexus carcinoma. Our data suggest that INI1 mutations are involved in the pathogenesis of plexus carcinoma; however, INI1 alterations are not a frequent event in the majority of brain tumor entities.

Base Sequence↗
PubMed2001

INI1 mutations in meningiomas at a potential hotspot in exon 9.

Rhabdoid tumours have been shown to carry somatic mutations in the INI1 (SMARCB1/hSNF5) gene. A considerable fraction of these tumours exhibit allelic losses on chromosome 22. Allelic loss on 22q also is characteristic for meningiomas, however most of these alterations are considered to be associated with mutations of the NF2 gene. We examined a series of 126 meningiomas for alterations in the INI1 gene. Four identical somatic mutations in exon 9 were detected resulting in an exchange of Arg to His in position 377 of INI1. Our observations were reproduced both by using DNA from a new round of extraction and by employing overlapping primers. This mutational hotspot therefore appears to be an important target in the formation of a fraction of meningiomas. In addition, 4 novel polymorphisms of INI1 were characterized. Our data indicate that the INI1 is a second tumour suppressor gene on chromosome 22 that may be important for the genesis of meningiomas.

Alleles↗
PubMed2000

One-dimensional imaging with a palm-size probe.

A new portable magnetic resonance imaging device was built. Spatially resolved NMR was achieved by placing a gradient coil pair and a Helmholtz pair type radio-frequency probe in the gap between two antiparallel polarized permanent magnets. The flat face of the low-field (nu(proton) = 20 MHz) apparatus allowed for the study of arbitrarily large objects in situ. Relaxation time weighted 1D images were achieved over a 15-mm field of view by a single-point spin-echo sequence. A phase encoding time on the order of 200 micros permited the scanning of a wide range of heterogeneous materials.

Cost-Benefit Analysis↗
PubMed2000

Angiotensin II-induced growth of vascular smooth muscle cells requires an Src-dependent activation of the epidermal growth factor receptor.

BACKGROUND: Angiotensin II (Ang II) is a potent stimulus of vascular smooth muscle cell (VSMC) growth. Activation of extracellular signal-regulated kinase (ERK), the archetypal mitogen-activated protein (MAP) kinase, and phosphatidylinositol 3 (PI3) kinase are critical steps in Ang II-induced mitogenic signaling. However, the mechanism involved in the activation of these kinases upon binding of Ang II to its receptor is poorly understood. METHODS: In the present study, we examined the role of the epidermal growth factor receptor (EGFR) in Ang II signaling in VSMCs employing immunoprecipitation, Western blot analysis, kinase immunocomplex assay, and [3H]-thymidine incorporation. RESULTS: A time-dependent tyrosine phosphorylation of the EGFR in response to Ang II was observed that was mediated by the Ang II type 1 receptor. This transactivation of the EGFR was blocked in the presence of PP1, an inhibitor of the intracellular Src-like tyrosine kinases. The tyrphostin AG 1478, a selective EGFR antagonist, inhibited both Ang II- and EGF-induced tyrosine phosphorylation of the EGFR. Furthermore, Ang II induced the binding of the adaptor protein Shc to the EGFR, leading to phosphorylation of Shc. In addition, the same nanomolar concentrations of AG 1478 that were effective in EGF signaling blocked the Ang II-induced activation of ERK and PI3 kinase in a dose-dependent manner. Proliferation of VSMCs, detected by measurements of DNA synthesis, following stimulation with Ang II was potently inhibited in the presence of AG 1478 or PP1. CONCLUSION: Our data suggest that EGFR serves as a role in mitogenic signaling following stimulation with Ang I through a ligand-independent and Src-dependent transactivation of the EGFR. Furthermore, we demonstrate this transactivation as a pivotal step in Ang II-induced activation of MAP kinase and PI3 kinase, as well as growth of VSMCs.

Angiotensin II↗
PubMed1999

Effects of captopril on fibrinolytic function in healthy humans.

Several lines of evidence point to an interrelation of the renin angiotensin system (RAS) with the endogenous fibrinolytic system. In the present study, we have therefore investigated the effect of the ACE-inhibitor captopril on various parameters of the fibrinolytic system in healthy volunteer subjects. 10 male subjects aged 28-38 years were given captopril 25 mg b.i.d. over 2 weeks. Venous blood was drawn before and at the end of the treatment period at 09.00 AM, after the volunteers had received their last dose of captopril by 07. 30 AM. Blood samples were processed for the determination of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1). Both parameters were determined with respect to their abundance (as antigen concentrations) and function (activity). In addition, the concentration and activity of the von Willebrand factor were also determined. Two weeks of captopril treatment had no significant effect on any of the above mentioned parameters. Our results thus show that short-term treatment with the ACE-inhibitor captopril, at least in healthy subjects on an unrestricted NaCl intake, does not affect the fibrinolytic balance between t-PA and PAI-1 or the von Willebrand factor.

Adult↗
PubMed1999

[Reliability of functional x-ray analysis of cervical vertebrae flexion and extension].

Functional radiographic analysis of the cervical spine in flexion and extension position is increasingly used as screening method for the diagnosis of segmental functional disorders. The objective of this study was to prove Penning's evaluation method for the metrical recording of segmental angles in the sagittal plane for selectivity, reliability and usability. Passive functional radiographs of the cervical spine in flexion and extension were taken of 20 patients with painful limitation of mobility of the cervical spine and 20 subjects, similar in sex and age, without complaints. The radiographs were duplicated. Five physicians measured the angles of segmental mobility in a blind study. Statistical analysis was conducted using the t-test and calculating the correlation coefficient "r". The results of the study prove that the evaluation method by Penning shows a usable and, for segments C3/C4 to C6/C7, significant selectivity. The selectivity of p < or = 0.05 and p < or = 0.01 is sufficient to distinguish patients from healthy subjects. The correlation between the five reviewers showed good to very good results (0.6 < r < or = 0.8; r > 0.8). The measured values, however, have to be considered, in connection with the appropriate clinical symptoms, as still "normal" or "functionally disordered" in the context of segmental hypo-resp. hypermobility.

Adult↗
PubMed1999

Lysophosphatidic acid stimulates protein kinase C isoforms alpha, beta, epsilon, and zeta in a pertussis toxin sensitive pathway in vascular smooth muscle cells.

The natural phospholipid lysophosphatidic acid (LPA) has been characterized as an important vascular smooth muscle cell (VSMC) mitogen whose effects are mainly mediated by pertussis toxin (PTX)-sensitive guanosine triphosphate (GTP)-binding protein (Gi-protein). Protein kinase C (PKC) isoforms play an important role in intracellular signaling cascades and in growth of VSMC. In the present study we investigated the effect of LPA on activation of PKC isoforms alpha, beta, epsilon, and zeta in VSMC by Western blot of cytosolic and membrane fractions. Furthermore, we examined the role of PKC activation on LPA-induced growth of VSMC using PKC inhibitor 19-27. Stimulation of VSMC by 5 microg/mL LPA for 10 min increased the amount of PKC alpha, beta, epsilon, and zeta in the particulate fraction by 689%, 285%, 424%, and 510%, respectively, and returned to control level after 30 min. Correspondingly, the amount of PKC alpha, beta, epsilon, and zeta in the cytosolic fraction decreased by 32%, 94%, 44%, and 95%, respectively, compared to control. Furthermore, we could show that LPA-induced activation of PKC alpha, beta, epsilon, and zeta isoforms was PTX sensitive. Incubation of VSMC with nonspecific PKC inhibitor 19-27 (10 micromol/L) for 24 h resulted in a 30% inhibition of LPA-induced DNA synthesis as measured by [3H]thymidine incorporation. In conclusion, in VSMC LPA stimulated translocation of PKC isoforms alpha, beta, epsilon, and zeta in a PTX-sensitive manner. Furthermore stimulation of PKC might be critically involved in LPA-induced mitogenesis in VSMC.

Animals↗
PubMed1999

Structure of the phylogenetically most conserved domain of SRP RNA.

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein required for cotranslational targeting of proteins to the membrane of the endoplasmic reticulum of the bacterial plasma membrane. Domain IV of SRP RNA consists of a short stem-loop structure with two internal loops that contain the most conserved nucleotides of the molecule. All known essential interactions of SRP occur in that moiety containing domain IV. The solution structure of a 43-nt RNA comprising the complete Escherichia coli domain IV was determined by multidimensional NMR and restrained molecular dynamics refinement. Our data confirm the previously determined rigid structure of a smaller subfragment containing the most conserved, symmetric internal loop A (Schmitz et al., Nat Struct Biol, 1999, 6:634-638), where all conserved nucleotides are involved in nucleotide-specific structural interactions. Asymmetric internal loop B provides a hinge in the RNA molecule; it is partially flexible, yet also uniquely structured. The longer strand of internal loop B extends the major groove by creating a ledge-like arrangement; for loop B however, there is no obvious structural role for the conserved nucleotides. The structure of domain IV suggests that loop A is the initial site for the RNA/protein interaction creating specificity, whereas loop B provides a secondary interaction site.

Base Sequence↗
PubMed1999

Structure of the most conserved internal loop in SRP RNA.

The signal recognition particle (SRP) directs translating ribosomes to the protein translocation apparatus of endoplasmic reticulum (ER) membrane or the bacterial plasma membrane. The SRP is universally conserved, and in prokaryotes consists of two essential subunits, SRP RNA and SRP54, the latter of which binds to signal sequences on the nascent protein chains. Here we describe the solution NMR structure of a 28-mer RNA composing the most conserved part of SRP RNA to which SRP54 binds. Central to this function is a six-nucleotide internal loop that assumes a novel Mg2+-dependent structure with unusual cross-strand interactions; besides a cross-strand A/A stack, two guanines form hydrogen bonds with opposite-strand phosphates. The structure completely explains the phylogenetic conservation of the loop bases, underlining its importance for SRP54 binding and SRP function.

Base Sequence↗
PubMed1999

Early intracellular signalling pathway of ethanol in vascular smooth muscle cells.

1. ERKs belong to MAP kinase family and are activated by several growth and stress factors. Although ethanol has been shown to modulate ERK1 and ERK2 (p44(mapk) and p42(mapk)) activity, it can also act as an antiproliferative agent in various mammalian cells. Since the nature of the antiproliferative effect of ethanol in VSMCs has not been defined, we examined its effects on growth and on early intracellular events normally induced by growth factors in VSMCs. 2. Measurement of cytosolic Ca(2+) and pH in cell monolayers was performed using fura-2/AM and BCECF/AM, respectively. The effect of ethanol on VSMCs growth was assessed by [(3)H]-thymidine incorporation, by cell counting and by determination of the caspase 3 activity. Stimulation of ERK1 and ERK2 was examined by the chemiluminescence Western blotting method. The expression of c-fos was quantitated by Northern blotting. Determination of inositolphosphates was performed after labelling of VSMCs with myo-[2-(3)H]-inositol and separation of inositolphosphates by HPLC. 3. Ethanol (0.3 - 1.0% v v(-1), 17 - 170 mM) induced a dose-dependent maximal stimulation of p44(mapk)/p42(mapk) at 30 min and expression of c-fos mRNA with a maximum at 120 min. Intracellular events upstream to MAP kinase, like an increase in [Ca(2+)](i), activation of the Na(+)/H(+) exchanger and formation of phosphoinositol metabolites were also markedly activated by ethanol. Treatment of VSMCs with ethanol for 3 - 5 min induced an increase in DNA synthesis whereas treatment of the cells for more than 30 min was toxic. Caspase 3 activity was not modulated by ethanol treatment of VSMCs. 4. We may postulate that the activation of these mitogenic signals including the elevation of DNA synthesis reflects a cell effort to protect itself against the toxic effects of ethanol.

Animals↗