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Biomedical subjects

R Müller

Publications and source records attributed to R Müller.

At least 307 records · Page 17Linked to original sources
PubMed1995

Significance of prophylactic urodilatin (INN: ularitide) infusion for the prevention of acute renal failure in patients after heart transplantation.

Acute renal failure is a serious problem following heart transplantation. In first uncontrolled clinical trials, Urodilatin revealed beneficial effects in the prophylaxis and therapy of acute renal failure following heart and liver transplantation. Here, we present the first randomized, placebo-controlled, double-blind study on 24 patients following heart transplantation to investigate whether prophylactic i.v. Urodilatin infusion can prevent acute renal failure requiring renal replacement therapy. Postoperative drug management was characterized by intravenous application of high furosemide, cyclosporine, and vancomycin doses. Urodilatin infusion was started postoperatively with a dose of 40 ng / kg bw / min for 6 days. 6 of the 12 patients in the Urodilatin group and 6 of the 12 patients in the placebo group had a stable diuresis (3 - 4 l / day) during the study period of 6 days. In contrast, the remaining 6 patients of each group developed oliguria / anuria and required subsequent hemofiltration / hemodialysis. Cumulative duration of hemofiltration (88 +/- 7.39 hours in the placebo treated patients versus 44 +/- 5.35 hours in the Urodilatin treated patients, p < 0. 05) as well as frequency of hemodialysis (3.0 +/- 0.49 times in the placebo group vs 1.2 +/- 0.29 times in the Urodilatin group, p < 0. 05) were significantly reduced using Urodilatin. Mean arterial blood pressure was stable during the Urodilatin infusion period and was not different to that observed in placebo patients. We conclude that Urodilatin does not reduce the incidence of acute renal failure and the subsequent requirement for hemofiltration / hemodialysis in our patient population, but seems to reduce the duration of hemofiltration and frequency of hemodialysis compared to the placebo group.

Acute Kidney Injury↗
PubMed1995

Structure of the human TIMP-3 gene and its cell cycle-regulated promoter.

The gene encoding tissue inhibitor of metalloproteinases-3 (TIMP-3) is regulated during development, mitogenic stimulation and normal cell cycle progression. The TIMP-3 gene is structurally altered or deregulated in certain diseases of the eye and in tumour cells. A detailed knowledge of the TIMP-3 gene and its regulatory elements is therefore of paramount importance to understand its role in development, cell cycle progression and disease. In this study, we present the complete structure of the human TIMP-3 gene. We show that TIMP-3 is a TATA-less gene, which initiates transcription at one major site, is composed of five exons and four introns spanning a region of approximately 30 kb, and gives rise to three distinct mRNAs, presumably due to the usage of alternative polyadenylation signals. Using somatic cell hybrids the TIMP-3 locus was mapped to chromosomal location 22q13.1 We also show that the TIMP-3 5' flanking region is sufficient to confer both high basal level expression in growing cells and cell cycle regulation in serum-stimulated cells. While the first 112 bases of the promoter, which harbour multiple Sp1 sites, were found to suffice for high basal level activity, the adjacent region spanning positions -463 and -112 was found to be a major determinant of serum inducibility. These results provide an important basis for further investigations addressing the role of TIMP-3 in physiological processes and pathological conditions.

3T3 Cells↗
PubMed1995

Cell cycle regulation of cdc25C transcription is mediated by the periodic repression of the glutamine-rich activators NF-Y and Sp1.

The late S/G2-specific transcription of the human cdc25C gene is dependent on an initiator-proximal repressor element (CDE) and an upstream activating sequence (UAS) of undefined nature. We now show that these upstream sequences harbour multiple in vivo protein binding sites that interact with transcriptional activators and form separable, context-independent functional modules. Major components of the UAS are a bona fide Sp1 site and three direct sequence repeats (Yc-boxes). The Yc-boxes interact with the CCAAT-box binding protein NF-Y and are critically dependent on synergistic interactions for efficient transcription activation. The NF-Y complexes, as well as Sp1, are constitutive activators, whose activation function is periodically repressed through the CDE. These observations indicate that the cell cycle regulation of cdc25C transcription is mainly due to the CDE-mediated repression of glutamine-rich activators.

3T3 Cells↗
PubMed1995

Cell cycle regulation of the cyclin A, cdc25C and cdc2 genes is based on a common mechanism of transcriptional repression.

The S/G2-specific transcription of the human cdc25C gene is due to the periodic occupation of a repressor element ('cell cycle-dependent element'; CDE) located in the region of the basal promoter. Protein binding to the major groove of the CDE in G0 and G1 results in a phase-specific repression of activated transcription. We now show that CDE-mediated repression is also the major principle underlying the periodic transcription of the human cyclin A and cdc2 genes. A single point mutation within the CDE results in a 10- to 20-fold deregulation in G0 and an almost complete loss of cell cycle regulation of all three genes. In addition, the cdc25C, cyclin A and cdc2 genes share an identical 5 bp region ('cell cycle genes homology region'; CHR) starting at an identical position, six nucleotides 3' to the CDE. Strikingly, mutation of the CHR region in each of the three promoters produces the same phenotype as the mutation of the CDE, i.e. a dramatic deregulation in G0. In agreement with these results, in vivo DMS footprinting showed the periodic occupation of the cyclin A CDE in the major groove, and of the CHR in the minor groove. Finally, all three genes bear conspicuous similarities in their upstream activating sequences (UAS). This applies in particular to the presence of NF-Y and Sp1 binding sites which, in the cdc25C gene, have been shown to be the targets of repression through the CDE.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Mimics of the binding sites of opioid receptors obtained by molecular imprinting of enkephalin and morphine.

Molecular imprinting of morphine and the endogenous neuropeptide [Leu5]enkephalin (Leu-enkephalin) in methacrylic acid-ethylene glycol dimethacrylate copolymers is described. Such molecular imprints possess the capacity to mimic the binding activity of opioid receptors. The recognition properties of the resultant imprints were analyzed by radioactive ligand binding analysis. We demonstrate that imprinted polymers also show high binding affinity and selectivity in aqueous buffers. This is a major breakthrough for molecular imprinting technology, since the binding reaction occurs under conditions relevant to biological systems. The antimorphine imprints showed high binding affinity for morphine, with Kd values as low as 10(-7) M, and levels of selectivity similar to those of antibodies. Preparation of imprints against Leu-enkephalin was greatly facilitated by the use of the anilide derivative rather than the free peptide as the print molecule, due to improved solubility in the polymerization mixture. Free Leu-enkephalin was efficiently recognized by this polymer (Kd values as low as 10(-7) M were observed). Four tetra- and pentapeptides, with unrelated amino acid sequences, were not bound. The imprints showed only weak affinity for two D-amino acid-containing analogues of Leu-enkephalin. Enantioselective recognition of the L-enantiomer of phenylalanylglycine anilide, a truncated analogue of the N-terminal end of enkephalin, was observed.

Amino Acid Sequence↗
PubMed1995

Proto-oncogenic properties of the DP family of proteins.

The cellular transcription factor DRTF1/E2F is implicated in the control of cellular proliferation due to its interaction with key regulators of cell cycle progression, such as the retinoblastoma tumour suppressor gene product, cyclins and cyclin-dependent kinases. DRTF1/E2F is a heterodimeric DNA binding activity which arises when a member of two distinct families of proteins, DP and E2F, interact as DP/E2F heterodimers, for example, DP-1 and E2F-1. In DRTF1/E2F the activity of DP-1 is under cell cycle control, possibly by phosphorylation, and in many types of cells it is a frequent, if not general DNA binding component of DRTF1/E2F. The expression of other DP proteins, such as DP-2, is tissue-restricted. Here, we show that DP-1 and DP-2 are integrated with another growth regulating pathway which involves signal transduction emanating from activated Ras protein. Thus, activated Ha-ras can co-operate with DP-1 or DP-2 in the transformation of rat embryo fibroblasts, establishing for the first time that DP proteins are endowed with proto-oncogenic activity. Moreover, an analysis of a dominant-negative and mutant DP-1 proteins suggests that the primary target through which DP-1 mediates its oncogenic activity is unlikely to be due to the regulation of E2F site-transcription, suggesting an E2F-independent effector function for DP-1. These results therefore establish DP genes as proto-oncogenes and thus argue that deregulating the normal control of DP protein activity will be important in promoting aberrant cellular proliferation.

Animals↗
PubMed1995

Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds.

An expression system for Saccharomyces cerevisiae (Sc) has been developed which, depending on the chosen vector, allows the constitutive expression of proteins at different levels over a range of three orders of magnitude and in different genetic backgrounds. The expression system is comprised of cassettes composed of a weak CYC1 promoter, the ADH promoter or the stronger TEF and GPD promoters, flanked by a cloning array and the CYC1 terminator. The multiple cloning array based on pBIISK (Stratagene) provides six to nine unique restriction sites, which facilitates the cloning of genes and allows for the directed cloning of cDNAs by the widely used ZAP system (Stratagene). Expression cassettes were placed into both the centromeric and 2 mu plasmids of the pRS series [Sikorski and Hieter, Genetics 122 (1989) 19-27; Christianson et al., Gene 110 (1992) 119-122] containing HIS3, TRP1, LEU2 or URA3 markers. The 32 expression vectors created by this strategy provide a powerful tool for the convenient cloning and the controlled expression of genes or cDNAs in nearly every genetic background of the currently used Sc strains.

Cloning, Molecular↗
PubMed1995

[Flow cytometry identification of CD34-positive cells].

In the present study three clinical applications of flow-cytometric quantification of CD34-positive cells in the field of blood and marrow transplantation are evaluated. By daily determination of CD34-positive cells in the peripheral blood after mobilization of stem cells with chemotherapy or growth-factors it is possible to set the optimal time for collection of the transplant material. Thereafter the yield of CD34-positive cells within the harvested material is determined. Finally, the efficacy of positive CD34 selection using a biotin-avidin column is evaluated. Initial experience is presented here.

Antigens, CD↗
PubMed1995

Expression of the messenger RNAs for luteinizing hormone-releasing hormone (LHRH) and its receptor in human ovarian epithelial carcinoma.

Recently we reported the presence of specific high affinity binding sites for luteinizing hormone-releasing hormone (LHRH) and its analogues (Kd = 1.5 or 1.7 nM) in the human epithelial ovarian cancer cell lines EFO-21 and EFO-27. The proliferation of these cell lines was inhibited by nM concentrations of a LHRH agonist. This study was performed to ascertain whether these ovarian cancer cell lines produce LHRH and whether the high affinity LHRH binding site found previously was identical to the pituitary LHRH receptor. Significant amounts of immunoreactive LHRH were found in the extracts of both the EFO-21 cell line (449 +/- 56 fmol/10(6) cells) and the EFO-27 line (409 +/- 76 fmol/10(6) cells). LHRH bioactivity of these extracts, assessed in terms of release of luteinizing hormone by rat pituitary cells, was comparable to that of authentic LHRH. EFO-21 and EFO-27 cells expressed the mRNAs for both human LHRH and human LHRH receptor as assessed by reverse transcriptase-PCR using oligonucleotide primers according to published sequences. In addition, in eight of eight biopsy samples of human epithelial ovarian cancers we detected mRNA for LHRH, six of these specimens expressed the mRNA representing the LHRH receptor. These data support the concept that human epithelial ovarian cancers might have a local system based on LHRH to regulate cell proliferation. It is still obscure at present whether LHRH produced locally has a stimulatory, inhibitory, or no impact on the proliferation of ovarian cancer cells. However, exogenous LHRH agonists seem to have clear antiproliferative activity, probably mediated through LHRH receptors. This finding might provide the base for novel approaches in the therapy of epithelial ovarian cancer.

Animals↗
PubMed1995

Deregulation of genes encoding microfilament-associated proteins during Fos-induced morphological transformation.

The mechanism of Fos-induced transformation is still poorly understood. In the present study, we have asked whether genes whose products play a role in determining cell morphology might become deregulated in the course of Fos-induced transformation. A clear up-regulation in Fos-transformed rat fibroblasts was seen with ezrin, as well as tropomyosin (TM) -3 and -5B, while TM-1 was down-regulated. Significantly, the same genes were deregulated in a very similar, but hormone-inducible way in cells expressing a Fos-estrogen receptor fusion protein. In agreement with these results, Fos-expressing cells showed decreased levels of two TM isoforms of 36 and 38 kDa, and showed an impaired TM network. The significance of these observations is strengthened by the fact that the deregulation of TM expression has been shown to contribute to morphological transformation in other experimental systems. Deregulation of the TM and ezrin genes preceeds the induction of morphological transformation suggesting that this deregulation is not merely a consequence of transformation. On the other hand, deregulation follows the induction of direct Fos target genes. We therefore propose that a cascade of regulatory events is triggered by Fos oncoproteins which eventually leads to the deregulation of genes encoding cytoskeleton-associated proteins.

Animals↗
PubMed1995

Cell proliferation and cell cycle progression are not impaired in fibroblasts and ES cells lacking c-Fos.

The transcription factor AP-1 is thought to play an important role in the control of cell proliferation, but the function of individual Fos and Jun family members is a largely unresolved issue. To directly analyse the function of c-Fos in the control of cell proliferation we have used embryonic stem (ES) cells and fibroblasts lacking c-Fos due to a disruption of the c-fos gene by homologous recombination. Our results demonstrate that proliferation of normally cycling cells and reentry of quiescent cells into the cell cycle following serum stimulation are not c-Fos-dependent and occur with similar efficiency in c-fos-/- and control cells. We also show that there is no compensatory overexpression or activation of other known Fos or Jun family members. On the contrary, the c-fos-/- cells showed a reduced induction of fra-1 after serum stimulation which is in agreement with the previous identification of fra-1 as a c-Fos target gene. Comparison of the AP-1 binding and transactivation activities in c-fos-/- and +/+ fibroblasts by electrophoretic mobility antibody supershift and CAT assays suggests that c-Fos is not a major component of AP-1 complexes in these cells. It is therefore conceivable that the lack of any detectable effect on cell proliferation in c-fos-/- cells might be due to a functional redundancy among the different AP-1 family members.

3T3 Cells↗
PubMed1995

Periodic cdc25C transcription is mediated by a novel cell cycle-regulated repressor element (CDE).

We show that the cell cycle-regulated transcription of the TATA-less cdc25C gene in late S/G2 is largely mediated by a novel promoter element (CDE) located directly 5' to one of the two major transcription initiation sites. Genomic dimethylsulfate footprinting experiments, using either synchronized or sorted normally cycling cells, show the formation in vivo of a CDE-protein complex in both G0 and G1 cells and its dissociation in G2. Mutation of the CDE severely impairs cell cycle regulation of the cdc25C promoter and results in high expression in G0/G1, indicating that the CDE functions as a cell cycle-regulated cis-acting repressor element. Cell cycle regulation is also lost upon removal of the enhancer region located immediately upstream of the CDE, but is largely restored when this enhancerless minimal cdc25C promoter fragment is linked to the constitutive SV40 early enhancer. This indicates that the CDE is dependent on the presence of a transcriptional enhancer to effect cell cycle regulation. Our observations suggest that the periodic activation of the cdc25C gene in late S/G2 is brought about, at least in part, by a unique regulatory mechanism involving the cell cycle-regulated dissociation of a repressor from the CDE.

Base Sequence↗
PubMed1995

Identification of a novel mitogen-inducible gene (mig-6): regulation during G1 progression and differentiation.

We have identified a novel human gene (mig-6) that is rapidly induced upon mitogenic stimulation of quiescent fibroblasts. Serum induction is partially inhibited by protein synthesis inhibitors, indicating that mig-6 shares characteristics of both primary and secondary response genes. In contrast to most other mitogen-responsive genes, mig-6 mRNA expression is also regulated during normal cell cycle progression, showing a clear peak around mid-G1. Consistent with the regulation of mig-6 expression during the cell cycle, terminal differentiation of HL-60 cells to either granulocytic or macrophage-like cells also leads to clear changes in the levels of mig-6 mRNA. These observations suggest that the mig-6 gene represents a useful tool for the analysis of cell cycle progression and perhaps terminal differentiation. As a first step toward a functional characterization we show that the Mig-6 polypeptide is located in the cytoplasm.

Amino Acid Sequence↗