Biomedical subjects
R Müller
Publications and source records attributed to R Müller.
CDF-1, a novel E2F-unrelated factor, interacts with cell cycle-regulated repressor elements in multiple promoters.
The cdc25C , cdc2 and cyclin A promoters are controlled by transcriptional repression through two contiguous protein binding sites, termed the CDE and CHR. In the present study we have identified a factor, CDF-1, which interacts with the cdc25C CDE-CHR module. CDF-1 binds to the CDE in the major groove and to the CHR in the minor grove in a cooperative fashion in vitro , in a manner similar to that seen by genomic footprinting. In agreement with in vivo binding data and its putative function as a periodic repressor, DNA binding by CDF-1 in nuclear extracts is down-regulated during cell cycle progression. CDF-1 also binds avidly to the CDE-CHR modules of the cdc2 and cyclin A promoters, but not to the E2F site in the B- myb promoter. Conversely, E2F complexes do not recognize the cdc25C CDE-CHR and CDF-1 is immunologically unrelated to all known E2F and DP family members. This indicates that E2F- and CDF-mediated repression is controlled by different factors acting at different stages during the cell cycle. While E2F-mediated repression seems to be associated with genes that are up-regulated early (around mid G1), such as B- myb , CDE-CHR-controlled genes, such as cdc25C , cdc2 and cyclin A , become derepressed later. Finally, the fractionation of native nuclear extracts on glycerol gradients leads to separation of CDF-1 from both E2F complexes and pocket proteins of the pRb family. This emphasizes the conclusion that CDF-1 is not an E2F family member and points to profound differences in the cell cycle regulation of CDF-1 and E2F.
The differential binding of E2F and CDF repressor complexes contributes to the timing of cell cycle-regulated transcription.
B- myb and cdc25C exemplify different groups of genes whose transcription is consecutively up-regulated during the cell cycle. Both promoters are controlled by transcriptional repression via modules consisting of an E2F binding site (E2FBS) or the related CDE plus a contiguous CHR co-repressor element. We now show that the B- myb repressor module, which is derepressed early (mid G1), is preferentially recognized by E2F-DP complexes and that a mutation selectively abolishing E2F binding impairs regulation. In contrast, the cdc25C repressor module, which is derepressed late (S/G2), interacts selectively with CDE-CHR binding factor-1 (CDF-1). E2F binding, but not CDF-1 binding, requires specific nucleotides flanking the E2FBS/CDE core, while CDF-1 binding, but not E2F binding, depends on specific nucleotides in the CHR. Swapping these nucleotides between the two promoters profoundly changes protein binding patterns and alters expression kinetics. Thus predominant CDF-1 binding leads to derepression in late S, predominant E2F binding results in up-regulation in late G1, while promoters binding both E2F and CDF-1 with high efficiency show intermediate kinetics. Our results support a model where the differential binding of E2F and CDF-1 repressor complexes contributes to the timing of promoter activity during the cell cycle.
CDF-1-mediated repression of cell cycle genes targets a specific subset of transactivators.
The cdc25C , cyclin A and cdc2 genes are regulated during the cell cycle through two contiguous repressor binding sites, the CDE and CHR, located in the region of transcription initiation and interacting with a factor termed CDF-1. The target of this repression seems to be transcriptional activation of these promoters by transcription factors bound upstream. The majority of these factors falls into the class of glutamine-rich activators, suggesting that CDF-1-mediated repression might be activation domain specific. In the present study we have used chimeric promoter constructs to demonstrate that the cdc25C UAS, but not the core promoter, is crucial for repression. In addition, we show that only specific transcription factors and activation domains are responsive to CDE-CHR-mediated cell cycle regulation. These observations clearly indicate that CDF-1 interferes with activation of transcription by a specific subset of transactivators. The repressible activation domains belong to the same class of glutamine-rich activators, pointing to specific interactions of CDF-1 with components of the transcription machinery. In agreement with this conclusion we find that a simple inversion of the CDE-CHR module completely abrogates cell cycle-regulated repression.
Characterization of the TATA-less core promoter of the cell cycle-regulated cdc25C gene.
The TATA- and Inr-less promoter of the human cdc25C gene is regulated during the cell cycle through binding of a repressor to two contiguous promoter-proximal elements, the CDE and CHR. In this study we have characterized in detail the region of the cdc25C promoter immediately downstream of these elements. Several lines of evidence suggest that this region of approximately 60 bp acts as the core promoter. This sequence: (i) harbors most of the transcription initiation sites; (ii) possesses basal promoter activity in vivo ; (iii) shows no stable protein binding in vivo as indicated by genomic dimethyl sulfate and phenanthroline copper footprinting; (iv) contains single-stranded regions in vivo as shown by potassium permanganate footprinting; (v) is hypersensitive to DNase I cleavage in permeabilized cells. Mutational analysis of the core promoter revealed the presence of three sites which play a role in transcription. Two of these sites were found to represent low affinity binding sites for transcription factors of the Sp1 family. Mutation of these sites led to decreased levels of transcription, while their alteration to canonical Sp1 sites impaired cell cycle regulation. Thus the transient interaction of Sp1 with the core promoter appears to be necessary for maximal transcription without perturbing cell cycle regulation.
Chromophore-anion interactions in halorhodopsin from Natronobacterium pharaonis probed by time-resolved resonance Raman spectroscopy.
Halorhodopsin of Natronobacterium pharaonis which acts as a light-driven chloride pump is studied by time-resolved resonance Raman spectroscopy. In single-beam experiments, resonance Raman spectra were obtained of the parent state HR578 and the first thermal intermediate HR520. The parent state is structural heterogeneous including ca. 80% all-trans and 20% 13-cis isomers. The resonance Raman spectra indicate that the all-trans conformer exhibits essentially the same chromophoric structure as in the parent states of bacteriorhodopsin or halorhodopsin from Halobacterium salinarium. Special emphasis of the resonance Raman spectroscopic analysis was laid on the C=C and C=N stretching region in order to probe the interactions between the protonated Schiff base and various bound anions (chloride, bromide, iodide). These investigations were paralleled by spectroscopic studies of retinal Schiff base model complexes in different solvents in an attempt to determine the various parameters which control the C=C and C=N stretching frequencies. From these data, it was concluded that in the parent state the anion is not involved in hydrogen bonding interactions with the Schiff base proton but is presumably bound to a nearby (positively charged) amino acid residue. On the other hand, the anion still exerts an appreciable effect on the chromophore structure which is, for instance, reflected by the variation of the isomer composition in the presence of different anions and in the anion-depleted form. In contrast to the parent state, the intermediate HR520 reveals frequency shifts of the C=N stretching in the presence of different anions. These findings indicate a closer proximity of the bound anion to the Schiff base proton which is sufficient for hydrogen bonding interactions. These changes of the anion-chromophore interaction upon transition from HR578 to HR520 may be related to the coupling of the chromophore movement with the anion translocation.
Endosomal targeting and the cytoplasmic tail of membrane immunoglobulin.
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Cyclin ET, a new splice variant of human cyclin E with a unique expression pattern during cell cycle progression and differentiation.
Cyclin E is the regulatory subunit of the cdc2-related protein kinase cdk2 and is a rate limiting factor for the entry into S phase. To date, cyclin E is the only cyclin for which alternative splicing has been described. We report here the isolation of a new splice variant of cyclin E, termed cyclin ET, which has an internal deletion of 45 amino acids compared with the full-length cyclin E protein. Even though cyclin ETcontains an intact cyclin box, it is unable to complement a triple cln mutant strain of Saccharomyces cerevisiae or to interfere with rescue by cyclin E, indicating that an intact cyclin box is functionally insufficient. The expression pattern of cyclin ET during cell cycle entry, progression and differentiation differs from that of cyclin E. Thus, ET expression precedes that of the other isoforms during the G0-->S progression; it shows a sharp peak in early G1 in cells released from a mitotic block and is strongly down-regulated in terminally differentiated myeloid cells. These observations point to different functions for cyclin ET and E and show for the first time that the alternative splicing of cyclin E is a regulated mechanism governed by the cell cycle and differentiation.
Endosomal targeting by the cytoplasmic tail of membrane immunoglobulin.
Membrane-bound immunoglobulin (mIg) of the IgG, IgA, and IgE classes have conserved cytoplasmic tails. To investigate the function of these tails, a B cell line was transfected with truncated or mutated gamma2a heavy chains. Transport to the endosomal compartment of antigen bound by the B cell antigen receptor did not occur in the absence of the cytoplasmic tail; and one or two mutations, respectively, in the Tyr-X-X-Met motif of the tail partially or completely interrupted the process. Experiments with chimeric antigen receptors confirmed these findings. Thus, a role for the cytoplasmic tail of mIg heavy chains in endosomal targeting of antigen is revealed.
Influence of advanced glycation end-products and AGE-inhibitors on nucleation-dependent polymerization of beta-amyloid peptide.
Nucleation-dependent polymerization of beta-amyloid peptide, the major component of plaques in patients with Alzheimer's disease, is significantly accelerated by crosslinking through Advanced Glycation End-products (AGEs) in vitro. During the polymerization process, both nucleus formation and aggregate growth are accelerated by AGE-mediated crosslinking. Formation of the AGE-crosslinked amyloid peptide aggregates could be attenuated by the AGE-inhibitors Tenilsetam, aminoguanidine and carnosine. These experimental data, and clinical studies, reporting a marked improvement in cognition and memory in Alzheimer's disease patients after Tenilsetam treatment, suggest that AGEs might play an important role in the etiology or progression of the disease. Thus AGE-inhibitors may generally become a promising drug class for the treatment of Alzheimer's disease.
The catalytic domain of activated collagenase I (MMP-1) is absolutely required for interaction with its specific inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1).
Here, we describe the production of recombinant human tissue inhibitor of metalloproteinases-1 (rTIMP-1) and wild-type and mutant human collagenase type I (rMMP-1) proteins in SF9 cells by the baculovirus expression system. Wild-type MMP-1, as well as the MMP-1 mutant lacking the C-terminal hemopexin-like domain [des-(248-450)-MMP-1], exhibit enzymatic activity upon cleavage of the prodomain by treatment with trypsin or 4-aminophenylmercuric acetate. Enzyme activity of both proteins can be inhibited by addition of rTIMP. Deletion of the complete active-site [des-(161-228)-MMP-1] within the catalytic domain, or mutation of a single His residue of the Zn2+ binding domain (His199), generates stable forms of MMP-1 proteins which are unable to digest collagen type I or beta-casein. In addition to co-immunoprecipitation analysis, we have established a rapid and sensitive ELISA assay using immobilized rTIMP to determine the structural requirements of MMP-1 to form complexes with its inhibitor. Only the activated and not the latent forms of wild-type and C-terminal mutant des-(248-450)-MMP-1 proteins are able to form complexes with TIMP. Neither mutation of His199, nor deletion mutants des-(161-228)-MMP-1 and des-(161-228/248-450)-MMP-1, interact with TIMP. This demonstrates that the C-terminal hemopexin domain of MMP-1, in contrast to the corresponding regions of gelatinase A and gelatinase B, does not interact with TIMP-1. In summary, we have shown that the integrity of the catalytic domain of MMP-1 and its ability to bind Zn2+ is absolutely required for complex formation with TIMP-1, which further underlines the importance of this region for proper regulation of enzymatic activity of MMP-1.
Hemodynamic action of captopril in coronary patients with heart failure tolerant to nitroglycerin.
BACKGROUND: At present there is little dispute that clinical tolerance of organic nitrates occurs during long-term treatment of patients with stable angina pectoris and congestive heart failure. HYPOTHESIS: Captopril exerts a favorable hemodynamic effect in coronary patients with heart failure who are clinically tolerant to nitroglycerin. METHODS: Development of nitrate tolerance was observed during intravenous nitroglycerin treatment (10 mg/h) in 16 of 19 patients (7 women, 12 men; mean age 56 +/- 8 years) with coronary heart disease [stenosis > or = 75%, New York Heart Association (NYHA) classes II-III). The criterion applied was a loss of efficacy of at least 50% with regard to mean pulmonary capillary wedge pressure compared with the maximum effect of nitrate. The effect of captopril (50 mg p.o.) was determined in a blank test. Captopril (50 mg p.o.) was administered again at the stage of clinically manifest nitrate tolerance. RESULTS: Compared with the effect of captopril alone, significantly more pronounced reductions in mean pulmonary capillary wedge pressure (33% compared with 27%) and in mean pulmonary arterial pressure (36% compared with 17%) and significantly greater increases in cardiac index (14% compared with 7%) and stroke work index (34% compared with 18%) (p < 0.05 in each case; Wilcoxon test for linked random samples) were measured. Maintaining nitroglycerin infusion, the effect of captopril (at least 90% of the maximum effect) lasted for 123 +/- 24 min. The baseline values (at least 75% decline in the effect of captopril) were only reached after 369 +/- 34 min. CONCLUSION: The results document a favorable hemodynamic effect of captopril in nitrate tolerance which is significantly better than that of captopril alone.
[Sports medicine and rehabilitation. Surface and footwear].
Load on the human body can be influenced by shoes and surfaces which is important both in sports and rehabilitation. The loading which can primarily be influenced are impact situations and friction as well as the stability of the foot. This stability is of main interest in the prevention of pain and injury. However, a certain amount of loading is necessary to improve strength and structure of biological materials. In the past, the development of shoe and surface materials has hardly been perceived under this aspect, but may be increasingly important in the future.
Prevalence of trabecular microcallus formation in the vertebral body and the femoral neck.
Trabecular microcallus formation (TMF) has been described previously in the human vertebra and femur, but the difference in TMF prevalence at these two sites has not been studied and the role of TMF remains controversial. In this study, the 4th lumbar vertebra (L4) and right proximal femur were removed from 27 male and 23 female cadavers. A 2 cm cube cut from the center of L4 and a 1 cm-thick slice cut from the femoral neck were cleaned, defatted, and dried. The apparent density of the L4 cubes was determined as dry weight/bulk bone volume. Using a dissecting microscope at low magnification (4-60x), TMF were identified and counted in both the vertebral and femoral samples. A 8 mm diameter core was then cut from the center of the L4 cubes in the vertical direction, and selected histomorphometric parameters of the core were evaluated with an X-ray microcomputed tomography system (micro-CT). There was a significantly greater prevalence of TMF in vertebral cubes (82%) than in the femoral slices (11%) (P < 0.001). TMF prevalence did not differ significantly between males and females, but the mean number of TMF in the vertebra was significantly (P < 0.05) greater in females (15.0/vertebra) than in males (7.7/vertebra). In the vertebra, the majority of the observed TMF were in vertical trabeculae. Subjects over 60 years old had a higher TMF prevalence than those under 60 years old (P < 0.01). TMF numbers increased with decreasing apparent density (P < 0.05), whereas no significant correlations were found between TMF and bone volume (BV/TV), trabecular number (Tb.N), or trabecular thickness (Tb.Th) as assessed by micro-CT. In two fractured vertebra, very few TMFs (2 and 4, respectively) were observed. These results demonstrated that the occurrence of TMF is strongly related to the anatomical site, probably due to differences in the applied loads and the trabecular structure between sites. The results were consistent with the hypothesis that TMF is a mechanism acting to maintain bone strength, but further studies are needed to clarify this important issue.
Expression of microtubule-associated proteins MAP2 and tau in cultured rat brain oligodendrocytes.
Oligodendrocytes in culture are characterized by large membranous sheets containing an elaborate network of microtubules. Microtubule-associated proteins (MAPs) participate in microtubule stability and the regulation of the cellular architecture. We have investigated the expression of two major groups of MAPs, MAP2 and tau, in cultured rat brain oligodendrocytes. Alternatively spliced isoforms of mRNAs encoding MAP2 and tau were assessed by means of reverse transcription and polymerase chain reaction using a newly designed set of MAP2- and tau-specific primers. The data were compared with data obtained with cultures of rat brain astrocytes and rat cerebral neurons, and adult rat brain. The results show that oligodendrocytes, similarly to neurons, express mainly MAP2c transcripts containing three microtubule-binding repeats. They also contain small amounts of MAP2b mRNA. Six low molecular weight tau isoforms, namely tau 1-6, have been described in the brain (Goedert et al. 1991). The major isoform of tau mRNA in oligodendrocytes was found to be tau 1, which represents a marker typical for immature neurons. Tau 2 and tau 4 isoforms were also detected, albeit at a very low level. Immunoblot analysis of oligodendroglia cell extracts confirmed the presence of tau protein. It migrates as a single polypeptide with an apparent molecular weight of approximately 55 kDa. In addition, oligodendrocytes express MAP2c protein, which migrates as a close double band with an apparent molecular weight around 70 kDa. Indirect immunofluorescence staining indicated that tau and MAP2 immunoreactivity was expressed in oligodendrocytes of immature and mature morphologies in the cell somata and cellular processes. Tau was particularly found in the end of the cellular extensions, and both proteins exhibited a distribution similar to myelin basic protein. Thus, oligodendroglia, like neuronal cells, contain microtubule-associated proteins, mainly MAP2c and the tau 1 isoform, although at a much lower level. The presence of these MAPs in myelin-forming cells further points to the functional significance of the cytoskeleton during oligodendrocyte differentiation, process outgrowth, and myelin formation.
THEMPO: a knowledge-based system for therapy planning in pediatric oncology.
This article describes the knowledge-based system THEMPO (Therapy Management in Pediatric Oncology), which supports protocol-directed therapy planning and configuration in pediatric oncology. THEMPO provides a semantic network controlled by graph grammars to cover the different types of knowledge relevant in the domain, and offers a suite of acquisition tools for knowledge base authoring. Medical problem solvers, operating on the oncological network, reason about adequate therapeutic and diagnostic timetables for a patient. Furthermore, a corresponding patient record, also based on semantic networks and graph grammars, has been implemented to represent the course of therapy of an oncological patient.
Use of conditional promoters for expression of heterologous proteins in Saccharomyces cerevisiae.
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Cell-cycle regulation of gene expression by transcriptional repression.
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