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Biomedical subjects

P K Müller

Publications and source records attributed to P K Müller.

At least 19 recordsLinked to original sources
PubMed2001

Interactions of fibrillin-1 with heparin/heparan sulfate, implications for microfibrillar assembly.

Fibrillin-1 is a major constituent of the 10-12 nm extracellular microfibrils. Here we identify, characterize, and localize heparin/heparan sulfate-binding sites in fibrillin-1 and report on the role of such glycosaminoglycans in the assembly of fibrillin-1. By using different binding assays, we localize two calcium-independent heparin-binding sites to the N-terminal (Arg(45)-Thr(450)) and C-terminal (Asp(1528)-Arg(2731)) domains of fibrillin-1. A calcium-dependent-binding site was localized to the central (Asp(1028)-Thr(1486)) region of fibrillin-1. Heparin binding to these sites can be inhibited by a highly sulfated and iduronated form of heparan sulfate but not by chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate, demonstrating that the heparin binding regions represent binding domains for heparan sulfate. When heparin or heparan sulfate was added to cultures of skin fibroblasts, the assembly of fibrillin-1 into a microfibrillar network was significantly reduced. Western blot analysis demonstrated that this effect was not due to a reduced amount of fibrillin-1 secreted into the culture medium. Inhibition of the attachment of glycosaminoglycans to core proteins of proteoglycans by beta-d-xylosides resulted in a significant reduction of the fibrillin-1 network. These studies suggest that binding of fibrillin-1 to proteoglycan-associated heparan sulfate chains is an important step in the assembly of microfibrils.

Base Sequence↗
PubMed2001

Different pattern of collagen cross-links in two sclerotic skin diseases: lipodermatosclerosis and circumscribed scleroderma.

Changes in the process of cross-linking of collagen molecules are associated with defects in the biomechanical stability of the extracellular matrix. Fibrosis of skin is characterized by an increase in pyridinolines, which are hydroxylysine aldehyde derived cross-links usually absent in healthy skin. In this study, we analyzed cross-links in lipodermatosclerosis and localized scleroderma to address the question whether all the mature cross-links currently characterized are increased in fibrosis in addition to the increase in pyridinolines. As psoralen plus ultraviolet A treatment leads to clinical improvement of fibrotic plaques in localized scleroderma we analyzed the cross-link content in lesional skin after bath psoralen plus ultraviolet A therapy. In skin from patients with localized scleroderma an increase in the total number of mature cross-links was found to be due to an increase in both pyridinolines and dehydro-histidinohydroxymerodesmosine. The concentration of histidinohydroxylysinonorleucine was unchanged. By contrast, the total number of mature cross-links was decreased in lipodermatosclerosis. This decrease was caused by a decrease of lysine aldehyde derived cross-links (dehydro-histidinohydroxymerodesmosine and histidinohydroxylysinonorleucine), whereas the concentration of pyridinolines increased. A decrease in the content of pyridinolines after bath psoralen plus ultraviolet A treatment was found in six out of nine patients with localized scleroderma, which might reflect a remodeling of the extracellular matrix. Our data provide evidence that sclerosis of skin is associated with either an increase in the number of cross-links per molecule of collagen or a change in the molecular nature of the cross-links formed.

Amino Acids↗
PubMed2001

Estradiol has a direct impact on the exocrine pancreas as demonstrated by enzyme and vigilin expression.

BACKGROUND: Estrogen receptors have been found in the exocrine pancreas; however, the exact role of estrogen in pancreatic enzyme synthesis and secretion remains to be elucidated. Vigilin, a multi-KH domain protein, is part of a tRNA-containing ribonucleoprotein complex and may be a suitable marker for stimulation of the translational machinery. In the present study, we investigated the influence of estradiol and compared it to CCK on the expression of vigilin, trypsin and amylase in rat pancreatic acini. METHODS: Acini were isolated and incubated with CCK or estradiol. The change in amylase and trypsin levels in the medium and in cell extracts were determined using a photometric method. The change in vigilin mRNA and protein expression were determined by RT-PCR and Western blotting, respectively. RESULTS: Treatment of isolated exocrine pancreatic cells with estradiol caused stimulation of amylase and trypsin production and inhibition of secretion, while treatment with CCK showed only a minor effect on enzyme production and resulted mainly in a stimulation of secretion. Further we found an increase in vigilin mRNA and protein expression in acini stimulated with both CCK-8 and estradiol. CONCLUSION: Our data suggest that estradiol may play a role in inducing exocrine enzyme production but not secretion, and that vigilin, as a marker for translational activity, is stimulated in parallel to the pancreatic enzymes: amylase and trypsin.

Amylases↗
PubMed2000

Mutations in calcium-binding epidermal growth factor modules render fibrillin-1 susceptible to proteolysis. A potential disease-causing mechanism in Marfan syndrome.

Most extracellular proteins consist of various modules with distinct functions. Mutations in one common type, the calcium-binding epidermal growth factor-like module (cbEGF), can lead to a variety of genetic disorders. Here, we describe as a model system structural and functional consequences of two typical mutations in cbEGF modules of fibrillin-1 (N548I, E1073K), resulting in the Marfan syndrome. Large (80-120 kDa) wild-type and mutated polypeptides were recombinantly expressed in mammalian cells. Both mutations did not alter synthesis and secretion of the polypeptides into the culture medium. Electron microscopy after rotary shadowing and comparison of circular dichroism spectra exhibited minor structural differences between the wild-type and mutated forms. The mutated polypeptides were significantly more susceptible to proteolytic degradation by a variety of proteases as compared with their wild-type counterparts. Most of the sensitive cleavage sites were mapped close to the mutations, indicating local structural changes within the mutated cbEGF modules. Other cleavage sites, however, were observed at distances beyond the domain containing the mutation, suggesting longer range structural effects within tandemly repeated cbEGF modules. We suggest that proteolytic degradation of mutated fibrillin-1 may play an important role in the pathogenesis of Marfan syndrome and related disorders.

Amino Acid Sequence↗
PubMed2000

Export and transport of tRNA are coupled to a multi-protein complex.

Vigilin is a ubiquitous multi heterogeneous nuclear ribonucleoprotein (hnRNP) K homologous (KH)-domain protein. Here we demonstrate that purified recombinant human vigilin binds tRNA molecules with high affinity, although with limited specificity. Nuclear microinjection experiments revealed for the first time that the immuno-affinity-purified nuclear vigilin core complex (VCC(N)) as well as recombinant vigilin accelerate tRNA export from the nucleus in human cells. The nuclear tRNA receptor exportin-t is part of the VCC(N). Elongation factor (EF)-1alpha is enriched in VCC(N) and its cytoplasmic counterpart VCC(C), whereas EF-1beta, EF-1gamma and EF-1delta are basically confined to the VCC(C). Our results suggest further that vigilin and exportin-t might interact during tRNA export, provide evidence that the channeled tRNA cycle is already initiated in the nucleus, and illustrate that intracellular tRNA trafficking is associated with discrete changes in the composition of cellular cytoplasmic multi-protein complexes containing tRNA.

Animals↗
PubMed2000

Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4.

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies was established as a suitable model to study development in vitro. Here, we show that differentiation of ES cells in vitro into chondrocytes can be modulated by members of the transforming growth factor-beta family (TGF-beta(1), BMP-2 and -4). ES cell differentiation into chondrocytes was characterized by the appearance of Alcian blue-stained areas and the expression of cartilage-associated genes and proteins. Different stages of cartilage differentiation could be distinguished according to the expression pattern of the transcription factor scleraxis, and the cartilage matrix protein collagen II. The number of Alcian-blue-stained areas decreased slightly after application of TGF-beta(1), whereas BMP-2 or -4 induced chondrogenic differentiation. The inducing effect of BMP-2 was found to be dependent on the time of application, consistent with its role to recruit precursor cells to the chondrogenic fate.

Aggrecans↗
PubMed2000

Age-related differences in the expression of receptors for TGF-beta in human osteoblast-like cells in vitro.

The cell surface expression of receptors for TGF-beta was studied in human osteoblasts derived from femoral trabecular bone of a total of 19 patients aged 2-83 years. All cell populations investigated showed a similar profile of expression of TGF-beta receptors (TbetaR) I, II and III (betaglycan). There were no significant differences in cell differentiation or proliferative behaviour between the age groups. The TGF-beta receptor number per cell significantly increased with age, while the receptor affinity tended to decrease. IGF-I did not influence TbetaR expression in vitro. The results indicate an age-dependent and IGF-I independent increase of osteoblastic TGF-beta receptors in human osteoblast-like cells in vitro.

Adolescent↗
PubMed1999

Recombinant human type II collagens with low and high levels of hydroxylysine and its glycosylated forms show marked differences in fibrillogenesis in vitro.

Type II collagen is the main structural component of hyaline cartilages where it forms networks of thin fibrils that differ in morphology from the much thicker fibrils of type I collagen. We studied here in vitro the formation of fibrils of pepsin-treated recombinant human type II collagen produced in insect cells. Two kinds of type II collagen preparation were used: low hydroxylysine collagen having 2.0 hydroxylysine residues/1,000 amino acids, including 1.3 glycosylated hydroxylysines; and high hydroxylysine collagen having 19 hydroxylysines/1,000 amino acids, including 8.9 glycosylated hydroxylysines. A marked difference in fibril formation was found between these two kinds of collagen preparation, in that the maximal turbidity of the former was reached within 5 min under the standard assay conditions, whereas the absorbance of the latter increased until about 600 min. The critical concentration with the latter was about 10-fold, and the absorbance/microgram collagen incorporated into the fibrils was about one-sixth. The morphology of the fibrils was also different, in that the high hydroxylysine collagen formed thin fibrils with essentially no interfibril interaction or aggregation, whereas the low hydroxylysine collagen formed thick fibrils on a background of thin ones. The data thus indicate that regulation of the extents of lysine hydroxylation and hydroxylysine glycosylation may play a major role in the regulation of collagen fibril formation and the morphology of the fibrils.

Collagen↗
PubMed1999

[Therapeutic options in non-resectable liver metastases. Percutaneous radiological interventions].

In patients with irresectable liver metastases the following spectrum of oncological concepts is in use. Percutaneous interventional methods allows for an optimized local control rate in strictly intrahepatic disease. Regional short-term time chemotherapy and transarterial chemoembolization (TACE) belong to regional methods. As local ablative methods, intratumoral drug application, endotumoral chemotherapy and alcohol instillation can be used. The most promising thermotherapeutic strategies are radiofrequency and laser-induced thermotherapy (LITT). In a prospective study 278 patients suffering from liver metastases were treated with MR-guided LITT and exact data for the local control rate and survival rate were evaluated. The overall cumulative survival rate (Kaplan-Meier) of patients with liver metastases was 40.8 months (median: 40.97 months, 95% confidence interval 36.3-45.2).

Adult↗
PubMed1999

Liver metastases: interventional therapeutic techniques and results, state of the art.

The liver is the most common site of metastatic tumour deposits. Hepatic metastases are the major cause of morbidity and mortality in patients with gastrointestinal carcinomas and other malignant tumours. The rationale and results for interventional therapeutic techniques in the treatment of liver metastases are presented. For the treatment of patients with irresectable liver metastases, alternative local ablative therapeutic modalities have been developed. Technique and results of local interventional therapies are presented such as microwave-, radiofrequency (RF)- and ultrasound ablation, and laser-induced interstitial therapy (LITT), cryotherapy and local drug administration such as alcohol injection, endotumoral chemotherapy and regional chemoembolisation. In addition to cryotherapy, all ablative techniques can be performed percutaneously with low morbidity and mortality. Cryotherapy is an effective and precise technique for inducing tumour necrosis, but it is currently performed via laparotomy. Percutaneous local alcohol injection results in an inhomogeneous distribution in liver metastases with unreliable control rates. Local chemotherapeutic drug instillation and regional chemoembolisation produces relevant but non-reproducible lesions. Laser-induced interstitial thermotherapy (LITT) performed under MRI guidance results in precise and reproducible areas of induced necrosis with a local control of 94%, and with an improved survival rate. Interventional therapeutic techniques of liver metastases do result in a remarkable local tumour control rate with improved survival results.

Antineoplastic Agents↗
PubMed1999

Interventional MR: interstitial therapy.

The rationale and results for interstitial therapies via interventional MRI in the treatment of tumors in various regions are presented. Different interstitial treatment techniques are presented based on varying technologies both for tumor ablation and treatment monitoring. Data are presented based on 335 patients, 29-84 years of age (mean age 59 years, 196 men and 139 women) with a total of 932 liver tumors, 16 head and neck tumors and 14 abdominal recurrent pelvic and lymphatic tumors. All lesions had been treated with MR-guided laser-induced interstitial thermotherapy (LITT) via 2516 laser applications and 1856 cannulations. Data in the literature are extremely varying depending on author experience, treatment technique, and the included patient material. In our patient material we were able to achieve a local tumor control of 96.7% depending on the size of the tumorous lesion, the topographical relationship, and the applied laser parameters. The overall cumulative survival rate of patients with liver metastases was 45.74 months (median 40.97 months, 95 % confidence interval 31.42-50.52). The cumulative survival rate of the patient group with hepatic metastases of colorectal carcinoma was 42.71 months (median 39.33 months, 95% confidence interval 33.26-45.37). In patients with head and neck tumors a relevant reduction in clinically relevant symptoms such as pain, swallowing disorders, or nervous compression was achieved in 11 of 15 patients treated with LITT. In 14 soft tissue tumors, such as pelvic tumor recurrence and lymph node metastases, a local tumor control was obtained in 68% of lesions. Interstitial therapies under interventional MRI guidance, such as LITT, results in a high local tumor control with an improved survival rate.

Abdominal Neoplasms↗
PubMed1999

Overhydroxylation of lysyl residues is the initial step for altered collagen cross-links and fibril architecture in fibrotic skin.

In fibrotic skin of lipodermatosclerosis a substantial increase of the cross-link hydroxylysylpyridinoline is observed. Hydroxylysylpyridinoline is a typical cross-link of skeletal tissue and is thought to play a major part in the hardening of sclerotic tissue. We investigated whether the increase in hydroxylysylpyridinoline is due to overhydroxylation of lysyl residues in the collagen molecule, which may also be associated with an increase of glycosylated hydroxylysine residues. Furthermore, we determined whether the collagen fibrils in lipodermatosclerosis showed a decrease of the diameter in the tissue as well as in vitro after fibrillogenesis of pepsin-solubilized collagens. Isolated alpha-chains of pepsin solubilized collagen I showed an increase in lysyl hydroxylation (hyl/(hyl + lys)) as compared with normal control [alpha1(I): lipodermatosclerosis 0.18 +/- 0.01; control 0.12 +/- 0.01; alpha2(I): lipodermatosclerosis 0.36 +/- 0.02; control 0. 25 +/- 0.03, p < 0.001]. Furthermore, the content of enzymatic glycosylated hydroxlysine residues increased. This increase is associated with a decrease of fibril diameter of both tissue and fibrils formed in vitro of pepsin-solubilized collagens. In the same pool of collagens an increase in collagen III content was observed as compared with controls (lipodermatosclerosis 14.5% +/- 1.6, control 10.3% +/- 1.6, p < 0.001). Our results showed that the overhydroxylation of lysyl residues, which is required for the generation of hydroxylysylpyridinoline, is not only restricted to the telopeptides but also affects the helical part of the molecule. This process is further associated with an increase of glycosylated hydroxylysyl residues. These changes along with the increase in collagen III content seem to be responsible for the observed alteration in the architecture of collagen fibrils in sclerotic skin.

Aged↗
PubMed1999

Hypergravity stimulates collagen synthesis in human osteoblast-like cells: evidence for the involvement of p44/42 MAP-kinases (ERK 1/2).

The formation and organization of skeletal tissue is strongly influenced by mechanical stimulation. There is increasing evidence that gravitational stress has an impact on the expression of early response genes in mammalian cells and may play a role in the formation of extracellular matrix. In particular, osteoblasts may be unique in their response to gravitational stimuli since in these cells microgravity has been reported to reduce collagen synthesis, while in fibroblasts the opposite effect was observed. Here, we have investigated the influence of hypergravity induced by centrifugation on the collagen synthesis of human osteoblast-like cells (hOB) and studied the possible involvement of the mitogen-activated protein (MAP) kinase signaling cascade. Collagen synthesis was significantly increased by 42+/-16% under hypergravity at 13 x g, an effect paralleled by the enhanced expression of the collagen I alpha 2 (COL1A2) mRNA. No difference was seen in the proportion of collagen types I, III, and V synthesized by hOB. Hypergravity induced a markedly elevated phosphorylation of the p44/42 MAP kinases (ERK 1/2). The inhibition of this pathway suppressed the hypergravity-induced stimulation of both collagen synthesis as well as COL1A2 mRNA expression by about 50%. Our results show that the collagen synthesis of non-transformed hOB is stimulated under hypergravitational conditions. This response appears to be partially mediated by the MAP kinase pathway.

Base Sequence↗
PubMed1999

Ligand-induced downregulation of receptors for TGF-beta in human osteoblast-like cells from adult donors.

High concentrations of transforming growth factor b (TGF-beta) are found in the bone matrix, reflecting a pivotal role of this growth factor in the coupling of bone resorption and formation. TGF-beta strongly stimulates the synthesis of extracellular matrix proteins, but in vitro studies show an inhibitory effect on the final mineralization process, which in vivo occurs despite high concentrations of TGF-beta. Little is known about how bone-forming cells respond to different concentrations of TGF-beta and if they can transiently adapt receptor numbers in order to modulate cellular activity. Against this background, we studied the cell-surface expression of TGF-beta receptors (TbetaR) I, II and III (betaglycan) on human osteoblast-like cells from adult donors, and examined the TbetaR presentation on these cells after a preceding exposure to TGF-beta1. Affinity crosslinking studies with disuccinimidylsuberate showed the presence of all three receptor types. Preincubation with TGF-beta1 markedly reduced 125I-TGF-beta1 binding in a time-dependent and dose-dependent manner and revealed a 95% reduction after an 18-h preincubation with 200 pM TGF-beta1. In parallel, Scatchard analysis showed that the binding affinity did not change as a consequence of TGF-beta1 preincubation. Immunoblotting analyses revealed an almost complete disappearance of immunoreactive TbetaR-II and TbetaR-III proteins after a 24-h preincubation with TGF-beta1. Using semi-quantitative reverse transcription PCR, no effect of TGF-beta1 on the expression of TbetaR-II mRNA was observed. These studies demonstrate a ligand-induced downregulation of TbetaRs-II and -III on human osteoblast-like cells, without any evidence for recovery within the first 24 h, both in the presence and after the removal of the ligand. The underlying mechanism appears to be based on post-transcriptional events. The results suggest that high concentrations of active TGF-beta1 decrease the responsiveness of osteoblasts towards this growth factor.

Activin Receptors, Type I↗
PubMed1998

Production of trypsin by cells of the exocrine pancreas is paralleled by the expression of the KH protein vigilin.

Vigilin, a protein with a continuous series of 14 KH motifs, forms part of a multiprotein complex containing tRNA. Several lines of evidence have suggested that vigilin expression is enhanced in those cells which were actively engaged in protein synthesis. Accordingly, we show here by immunoelectronmicroscopy a close association of vigilin with the rough endoplasmic reticulum in rat pancreatic cells. Histological examination of these cells furthermore demonstrates the highest intensity of vigilin staining in the perinuclear, intranuclear, and basolateral regions where the endoplasmic reticulum is mainly amassed. In vivo challenge of starving rats fed prior to sacrifice raised in parallel the protein levels of both trypsin and vigilin when compared to unchallenged animals and was associated with enhanced expression of the vigilin gene. In contrast, in human and rat cell lines of pancreatic tumors with a constitutively high expression of vigilin no further stimulation by cholecystokinin treatment could be achieved. Our data provide circumstantial evidence that vigilin may play a crucial role in the ability of an organ, e.g., pancreas, to cope with the physiological demand to upregulate protein synthesis.

Animals↗
PubMed1998

tRNA is entrapped in similar, but distinct, nuclear and cytoplasmic ribonucleoprotein complexes, both of which contain vigilin and elongation factor 1 alpha.

Vigilin, which is found predominantly in cells and tissues with high levels of protein biosynthesis, was isolated in its native form from human HEp-2 cells (A.T.C.C. CCL23) by immunoaffinity chromatography. Here we demonstrate that vigilin is part of a novel large tRNA-binding ribonucleoprotein complex (tRNP), found not only in the cytoplasm, but also in the nuclei of human cells. Compositional differences in the protein pattern were detected between the nuclear and cytoplasmic tRNPs, although some properties of the purified nuclear tRNP, such as tRNA protection against nuclease attack, were identical with those of the cytoplasmic tRNP. By using either a pool of total human nuclear RNA or radioactively labelled yeast tRNAAsp in rebinding experiments, we could show that tRNA is specifically recaptured by the RNA-depleted, vigilin-containing nuclear complex. We could also show that vigilin is capable of binding tRNA in vitro. Another tRNA-binding protein is elongation factor 1 alpha, which appears to be enriched in the cytoplasmic and nuclear tRNP complexes. This suggests that the cytoplasmic tRNP may be involved in the channelled tRNA cycle in the cytoplasm of eukaryotic cells. Our results also suggest that the nuclear vigilin-containing tRNP may be related to the nuclear export of tRNA.

Biotinylation↗
PubMed1998

Identification of a new heterozygous point mutation in the COL1A2 gene leading to skipping of exon 9 in a patient with joint laxity, hyperextensibility of skin and blue sclerae. Mutations in brief no. 166. Online.

A heterozygous deletion of exon 9 in the COL1A2-mRNA of a patient with symptoms of both the Ehlers-Danlos-Syndrome and the Osteogensis Imperfecta is described. In the genomic DNA of the patient, exon 9 is homozygously present. We identified a novel heterozygous point mutation in the splice donor site of intron 9, leading to a G-->A substitution in position +5. This mutation leads to heterozygous skipping of exon 9 in the COL1A2-mRNA of this patient. The deletion results in a shortened (by 18 amino acids) but in frame 12(1) chain, which probably leads to the formation of abberantly processed triple helices.

Alternative Splicing↗