Biomedical subjects

U Stahl

Publications and source records attributed to U Stahl.

At least 19 recordsLinked to original sources

PubMed2006

Expression of calcitonin receptor-like receptor in human vascular tumours.

BACKGROUND: Vascular tumours such as Kaposi's sarcoma and capillary haemangioma are characterised by abnormal vascularisation and proliferation of endothelial cells or neoplastic cells. Adrenomedullin, a potent vasodilative peptide, and its receptor, calcitonin receptor-like receptor (CRLR), play an important part in angiogenesis. AIM: To establish whether this system also plays a part in vascular diseases, showing abnormal proliferation such as vascular tumours. METHODS: CRLR expression was investigated in several specimens of Kaposi's sarcoma and other vascular tumours, using immunohistochemical analysis with a previously described CRLR-specific polyclonal antibody and reverse transcriptase-polymerase chain reaction. RESULTS: Intense and specific CRLR-immunoreactive staining of neoplastic cells was observed in all specimens, which was of greater intensity than similar staining of adjacent normal endothelium. CONCLUSIONS: CRLR is expressed in vascular tumours and, with adrenomedullin, may have a role in neoplastic vascular growth.

Adrenomedullin↗

PubMed2004

Antifungal proteins: targets, mechanisms and prospective applications.

All organisms have evolved several defence systems in order to protect themselves against bacteria, fungi and viruses. Higher organisms have developed a complex network of humoral and cellular responses, called adaptive immunity. A second defence system, innate immunity, was discovered in the early 1980s, consisting of small cationic peptides with a broad antimicrobial spectrum. These proteins act immediately at sites of infection or inflammation. The production of proteins with antimicrobial activity was not limited to higher organisms but was also found in insects, plants and microorganisms. During the last 2 decades a broad range of proteins with very different structural features have been isolated and characterised from differing organisms ranging from bacteria to human beings. Over 500 cationic membrane-acting proteins with antimicrobial and antifungal activities have been identified to date. Apart from these proteins, a very large number of antifungal proteins active on the fungal cell wall, on enzymes of the cell wall synthesis machinery, the plasma membrane and on intracellular targets have been characterised.

Animals↗

PubMed2004

[Phosphoinositide 3-kinase (PI3-K) expression. Tumorigenesis of epithelial carcinoma of the mouth].

Phosphoinositide 3-kinase (PI3-K) is a heterodimeric enzyme involved in the regulation of mitogenesis, apoptosis, cell adhesion, and motility. PI3-K was suggested as a protooncogene in human cancer. To determine the expression of PI3-K during cancerogenesis and tumor invasion of HNSCC, we investigated normal and dysplastic epithelium of the oral cavity, squamous cell carcinoma and lymph node metastasis by immunohistochemistry. The strongest immunoreactivity for p85alpha and p110alpha was found in invasive tumors and their metastases. Carcinomas in situ showed a focal positivity. Dysplasias and normal epithelium reacted predominantly negatively. The PI3-K inhibitor LY294002 inhibited proliferation and invasion of the HNSCC cell line CAL-27 and induced apoptosis in vitro. Our data suggest PI3-K as a marker of malignancy and tumor invasion. We suggest including PI3-K in the multistep carcinogenesis model of HNSCC. In addition, PI3-K is a potential target for pharmacological intervention.

Apoptosis↗

PubMed2004

Engineering of Saccharomyces cerevisiae for the production of L-glycerol 3-phosphate.

L-glycerol 3-phosphate (L-G3P) was accumulated in Saccharomyces cerevisiae by pathway engineering. Intracellular concentration of this metabolic intermediate could be increased more than 20 times compared to the wild type by overexpressing GPD1 encoding the glycerol 3-phosphate dehydrogenase in a gpp1 Delta gpp2 Delta mutant which lacks both isoenzymes of glycerol 3-phosphatase. Investigation of cellular pattern of triacylglycerols and glycerophospholipids did not reveal considerable changes due to accumulation of their precursor L-G3P. Hyperosmotic stress did not affect the L-G3P pool in the gpp1 Delta gpp2 Delta mutant overexpressing GPD1 despite an about 4-fold increase of specific GPD activity. In contrast, oxygen limitation improved intracellular L-G3P concentration by enhancing the availability of cytosolic NADH. The reduction of pyruvate decarboxylase activity by deleting PDC2 led to an additional increase. In fact, the triple mutant gpp1 Delta gpp2 Delta pdc2 Delta overexpressing GPD1 accumulated 17 mg L-G3P/g dry weight during glucose batch fermentation under oxygen limitation. This value corresponds to an about 100-fold increase compared to that found in the wild type.

Genetic Enhancement↗

PubMed2003

The antifungal protein from Aspergillus giganteus causes membrane permeabilization.

We investigated the inhibitory effects of the antifungal protein (AFP) from Aspergillus giganteus on the growth of several filamentous fungi. For this purpose, the MICs of AFP were determined and ranged from 0.1 micro g/ml for Fusarium oxysporum to 200 micro g/ml for Aspergillus nidulans. The antifungal activity of AFP was diminished in the presence of cations. We were able to show that incubation of AFP-sensitive fungi with the protein resulted in membrane permeabilization using an assay based on the uptake of the fluorescent dye SYTOX Green. No permeabilization by AFP could be detected at concentrations below the species-specific MIC. Furthermore, AFP-induced permeabilization could readily be detected after 5 min of incubation. Localization experiments with fluorescein isothiocyanate-labeled AFP and immunofluorescence staining with an AFP-specific antibody supported the observation that the protein interacts with membranes. After treatment of AFP-sensitive fungi with AFP, the protein was localized at the plasma membrane, whereas it was mainly detected inside the cells of AFP-resistant fungi. We conclude from these data that the growth-inhibitory effect of AFP is caused by permeabilization of the fungal membranes.

Aspergillus↗

PubMed2001

Transcriptional regulation of the Antifungal Protein in Aspergillus giganteus.

Regulation of the expression of the afp gene that codes for the Antifungal Protein of Aspergillus giganteus was investigated using a reporter system. For this purpose, the E. coli reporter gene uidA encoding beta-glucuronidase (GUS) was placed under the control of the afp promoter. No homologous integration of the reporter construct into the afp site was observed among 156 transformants tested. In one of the transformants carrying a single, ectopically integrated, copy of the construct, GUS and AFP both displayed exactly the same temporal expression patterns under various cultivation conditions, as assayed by Northern and protein analyses. Thus, this transformant was used to identify factors that are involved in the transcriptional regulation of afp expression. Expression is only detectable in the vegetative mycelium, whereas no expression occurs in aerial hyphae or conidia, indicating that afp expression is developmentally regulated. Transcription of afp is regulated by ambient pH, being suppressed under acidic conditions and strongly induced under alkaline conditions. This observation suggests that PacC regulates the afp gene, which is consistent with the presence of two putative PacC binding sites within the 5' upstream region. Transcription is not subject to carbon catabolite repression or nitrogen metabolite repression. The expression of afp is up-regulated by heat shock, upon growth in the presence of excess NaCl and ethanol, and under conditions of carbon starvation. In contrast, expression decreases slightly in the presence of hydrogen peroxide and under nitrogen starvation. These data are compatible with the presence of a putative heat shock element (NTTCNNGANTTCN) and five putative C(4)T stress-responsive elements within the afp promoter.

Antifungal Agents↗

PubMed2001

[Cellular senescence: a mechanism of the development of osteoporosis?].

Osteoporosis is one of the most common diseases of the elderly. This leads to the hypothesis that the ageing of the organism is reflected as a cytogerontological effect in a specific loss of bone cell function. Three underlying pathogenetic mechanisms need to be considered: (1) cellular aging in general, (2) impairment of the systemic stimulation of bone formation by e.g. decreasing hormone levels, and (3) lower cellular effectiveness of cytokines and growth factors. Cellular aging consists of replicative and postmitotic senescence. While the replicative senescence limits only the number of cell cycles, the postmitotic aging is influenced by endo- and exogenous factors. These lead to genetic alterations known as delayed persistent genomic instability and to an increasing impairment of specific cellular functions. In the postmitotic phase, osteopenia caused by the decrease of systemically available sexual hormones is a major field of research. Osteopenia caused by a decreased activity of locally effective cytokines and growth factors is becoming increasingly understood. New therapeutic strategies, which modulate the local osteoblast activity, e.g. in bone defect healing, are under development. In conclusion, cellular senescence is considered to be one element in the development of bone loss. Potential therapeutic targets may open up an additional path in the treatment of local and systemic osteopenias.

Adult↗

PubMed2001

Insights into the genetic diversity of initial dioxygenases from PAH-degrading bacteria.

Alpha subunit genes of initial polyaromatic hydrocarbon (PAH) dioxygenases were used as targets for the PCR detection of PAH-degrading strains of the genera Pseudomonas, Comamonas and Rhodococcus which were obtained from activated sludge or soil samples. Sequence analysis of PCR products from several Pseudomonas strains showed that alpha subunits (nahAc allele) of this genus are highly conserved. PCR primers for the specific detection of alpha subunit genes of initial PAH dioxygenases from Pseudomonas strains were not suitable for detecting the corresponding genes from the genera Comamonas and Rhodococcus. Southern analysis using a heterologous gene probe derived from the P. putida OUS82 PAH dioxygenase alpha subunit identified segments of the PAH-degradation gene cluster from C. testosteroni strain H. Parts of this gene cluster containing three subunits of the initial PAH dioxygenase were isolated. These three subunits [ferredoxin (pahAb), alpha (pahAc) and beta (pahAd) subunit] were amplified by PCR as one fragment and expressed in Escherichia coli DH5alpha, resulting in an active initial dioxygenase with the ability to transform indole and phenanthrene. The DNA sequence alignment of alpha subunits from C. testosteroni H and various PAH-degrading bacteria permitted the design of new primers and oligonucleotide probes which are useful for the detection of the initial PAH dioxygenases from strains of Pseudomonas, Comamonas and Rhodococcus.

Bacterial Proteins↗

PubMed2001

Phase-specific gene expression in Saccharomyces cerevisiae, using maltose as carbon source under oxygen-limiting conditions.

The transcription of ten stress-response genes was investigated under oxygen-limiting conditions with maltose and glucose, respectively. Six of these genes (HSP12, HSP26, HSP30, HSP78, HSP82 and HSP104) showed expression only during the stationary phase. HSP12 and HSP104 were transcribed 10 h earlier with maltose than with glucose. Fermentation in wort yielded similar results to the maltose-based medium. HSP12, HSP26 and HSP30 were highly expressed. Thus, the HSP26 and the HSP30 promoter can be used for late, phase-specific expression of the desired genes with glucose or maltose as carbon source, and HSP12 with glucose only. MET14, a gene important for sulfite formation, was overexpressed in wort, using the HSP26 promoter during the stationary phase.

Blotting, Northern↗

PubMed2000

Cells of the yeast Saccharomyces cerevisiae are transformable by DNA under non-artificial conditions.

Transformants of bakers' yeast (Saccharomyces cerevisiae) can be generated when non-growing cells metabolize sugars (without additional nutrients) in the presence of plasmid DNA. These results suggest that there is a mechanism by which DNA can naturally be taken up by the yeast cell. Natural transformation does not take place in common complete or minimal yeast culture media such as YPD and YNB. The starvation conditions used in our experiments thus seem to be an important prerequisite for such transformation events.

Culture Media↗

PubMed2000

Hirudin reduces tissue factor expression and attenuates graft arteriosclerosis in rat cardiac allografts.

BACKGROUND-Intravascular clotting has been implicated in the pathogenesis of cardiac allograft vasculopathy (CAV). We previously identified the expression of tissue factor (TF), the primary cellular initiator of blood coagulation, within the coronary intima, which was associated with neointimal thickening. In the present study, the effect of recombinant hirudin on CAV was assessed in Lewis to Fisher rat heterotopic cardiac allografts. METHODS AND RESULTS-Transplant recipients were randomized to a control group (n=10) and a hirudin-treated group (n=12; 2 mg. kg(-1). d(-1) SC). Histological evaluations of rejection, CAV, and TF staining were performed 120 days after transplantation. No significant differences were observed between the 2 groups with respect to the degree of rejection. Hirudin significantly (P<0.05) suppressed the development of CAV in the graft microvessels, but it was less effective in large coronary arteries. Graft intimal cells, isolated by laser-assisted cell picking, showed a marked upregulation of TF gene transcription, which was prevented by hirudin (P<0.01). As demonstrated by immunohistochemistry and quantitative analyses of TF mRNA levels by real-time polymerase chain reaction, hirudin treatment resulted in a significant reduction of TF protein and mRNA expression (P<0.001). CONCLUSIONS-Treatment with hirudin in this rat cardiac transplant model inhibited TF expression and decreased neointimal hyperplasia. These results suggest that TF inhibition by hirudin, in addition to its direct effect on thrombin, may attenuate the hypercoagulable state and prevent the development of CAV at least in restricted sites of the graft coronary vasculature.

Animals↗

PubMed2000

Phospholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants.

Triacylglycerol (TAG) is known to be synthesized in a reaction that uses acyl-CoA as acyl donor and diacylglycerol (DAG) as acceptor, and which is catalyzed by the enzyme acyl-CoA:diacylglycerol acyltransferase. We have found that some plants and yeast also have an acyl-CoA-independent mechanism for TAG synthesis, which uses phospholipids as acyl donors and DAG as acceptor. This reaction is catalyzed by an enzyme that we call phospholipid:diacylglycerol acyltransferase, or PDAT. PDAT was characterized in microsomal preparations from three different oil seeds: sunflower, castor bean, and Crepis palaestina. We found that the specificity of the enzyme for the acyl group in the phospholipid varies between these species. Thus, C. palaestina PDAT preferentially incorporates vernoloyl groups into TAG, whereas PDAT from castor bean incorporates both ricinoleoyl and vernoloyl groups. We further found that PDAT activity also is present in yeast microsomes. The substrate specificity of this PDAT depends on the head group of the acyl donor, the acyl group transferred, and the acyl chains of the acceptor DAG. The gene encoding the enzyme was identified. The encoded PDAT protein is related to lecithin:cholesterol acyltransferase, which catalyzes the acyl-CoA-independent synthesis of cholesterol esters. However, budding yeast PDAT and its relatives in fission yeast and Arabidopsis form a distinct branch within this protein superfamily, indicating that a separate PDAT enzyme arose at an early point in evolution.

Acyl Coenzyme A↗

PubMed1999

Enhanced sterol-acyl transferase activity promotes sterol accumulation in Saccharomyces cerevisiae.

The sterol-acyl transferase encoded by the gene ARE2 was transcriptionally deregulated in the yeast Saccharomyces cerevisiae to understand its role in sterol storage and sterol enrichment. Our results show that sterols can indeed be enriched in yeast by enhancing the capacity of the cells to esterify sterols. ARE2 overexpression had no impact on the accumulation of the early sterols such as lanosterol, but influenced the later intermediates and the end product ergosterol. Thus an enhanced conversion of free sterols to their esterified counterparts may provide a tool to increase the overall sterol content of the yeast cell. We have previously shown that the overexpression of a truncated version of the key enzyme of the early sterol pathway, HMG-CoA reductase (HMG1), leads to an increase in the early sterols such as lanosterol and zymosterol. The simultaneous deregulation of both genes in one strain produces a cumulative effect in that both early and late sterols are enhanced. Karmellae-like structures can be detected when Are2p is overexpressed. Are2p therefore constitutes a new member of the karmellae-inducing protein family.

Acyltransferases↗

PubMed1999

In situ detection of tissue factor within the coronary intima in rat cardiac allograft vasculopathy.

Cardiac allograft vasculopathy is a major cause of morbidity and mortality of cardiac transplant recipients. The underlying cause of this disease remains unclear. Histological studies have implicated accelerated hemostasis and intravascular fibrin deposition in its pathogenesis. In the present study a defined model of this disease in the rat was used to elucidate the implication of tissue factor in the production of the hypercoagulable state observed in cardiac allograft vessels. Tissue factor protein and mRNA expression were studied in rat heart allografts developing allograft vasculopathy resembling human disease. Immunohistochemistry demonstrated tissue-factor-positive cells present in the allograft coronary intima and adventitia. Significant staining for tissue factor was detected in the endothelium lining coronary lesions in cardiac allografts and in interstitial mononuclear cells, respectively. Both transplant coronary endothelial cells and mononuclear cells contained tissue factor mRNA as indicated by oligo-cell reverse transcription polymerase chain reaction after laser-assisted cell picking. In contrast, tissue factor mRNA and protein were not or negligibly detectable within the coronary intima of nontransplanted control hearts. Thus, the present study clearly demonstrates that aberrant tissue factor expression occurs within the coronary intima after cardiac transplantation. Tissue factor, activating downstream coagulation mechanisms, may account for the intravascular clotting abnormalities observed in cardiac allografts and may represent a key factor in transplant atherogenesis.

Animals↗

PubMed1999

Rat porphobilinogen deaminase gene: a pseudogene-free internal standard for laser-assisted cell picking.

Analysis of gene expression and its transcriptional regulation requires a reliable access to target mRNA. However, mRNA extractions from homogenized tissue are limited because only average data are obtained, and cell-specific expression may not be addressed. Here, we describe a new method that combines the microscopic selection of oligocellular clusters or a few isotypic cell profiles from complex tissues by UV-laser-assisted cell picking with a simplified and highly efficient protocol for mRNA amplification. For positive control and quantitation reference, a reliable housekeeping gene is needed. For this purpose, we designed primers of the rat porphobilinogen deaminase (PBGD) gene. In contrast to many commonly used housekeeping primer pairs that co-amplify processed pseudogenes, this sequence allowed detection of a pseudogene-free rat cDNA sequence, thus eliminating the need for a DNase-digestion step. PBGD mRNA was consistently expressed in all complex tissues investigated and in 21 specific cell types harvested by laser-assisted cell picking. PBGD is suggested as a reliable new rat housekeeping gene, particularly suitable for analysis of oligocellular samples such as those obtained by laser-assisted cell picking in complex tissues.

Animals↗

PubMed1998

Mutant alleles of the MRS2 gene of yeast nuclear DNA suppress mutations in the catalytic core of a mitochondrial group II intron.

Previous studies show that some yeast strains carrying point mutations of domain 5 that block splicing of a mitochondrial group II intron yield spontaneous revertants in which splicing is partially restored by dominant mutations of nuclear genes. Here we cloned and sequenced the suppressor allele of one such gene, and found it to be a missense mutation of the MRS2 gene (MRS2-L232F). The MRS2 gene was first implicated in group II intron splicing by the finding that overexpression of the wild-type gene weakly suppresses the splicing defect of a mutation of another intron. Tetrad analysis showed that independently isolated suppressors of two other domain 5 mutations are also allelles of the MRS2 gene and DNA sequencing identified a new missense mutation in each strain (MRS2-T230I and MRS2-L213M). All three suppressor mutations cause a temperature-sensitive respiration defect that is dominant negative in heterozygous diploids, but those strains splice the mutant intron at the elevated temperature. The three mutations are in a domain of the protein that is likely to be a helix-turn-helix region, so that effects of the mutations on protein-protein interactions may contribute to these phenotypes. These mutations suppress the splicing defect of many, but not all, of the available splicing defective mutations of aI5gamma, including mutations of several intron domains. Protein and RNA blot experiments show that the level of the protein encoded by the MRS2 gene, but not the mRNA, is elevated by these mutations. Interestingly, overexpression of the wild-type protein restores much lower levels of splicing than were obtained with similar elevated levels of the mutated Mrs2 proteins. The splicing phenotypes of these strains suggest a direct role for Mrs2 protein on group II intron splicing, but an indirect effect is not yet ruled out.

Alleles↗

PubMed1998

Human transferrin receptor is active and plasma membrane-targeted in yeast.

The human transferrin receptor, a type II plasma membrane protein which mediates iron transport in human cells, was expressed in the yeast Saccharomyces cerevisiae. The transferrin receptor synthesized by yeast cells was posttranslationally modified comparable to the native receptor with respect to glycosylation and dimer formation. The location of the expressed receptor in the yeast plasma membrane indicates that the targeting of this type II membrane protein shares similar mechanisms in yeast and mammalian cells. The yeast-expressed transferrin receptor showed binding activity towards its natural ligand, transferrin in an ELISA binding assay.

Cell Membrane↗

PubMed1998

Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast.

The enzyme 3-hydroxy-3-methylglutaryl-co-enzyme-A (HMG-CoA) reductase is known as the rate limiting enzyme in early sterol biosynthesis in eukaryotic cells. To eliminate this regulation in the yeast Saccharomyces cerevisiae, a truncated HMG1 gene, producing a form of the enzyme that lacks the membrane-binding region (i.e. amino acids 1-552), was constructed and overexpressed in this yeast. The transformed strains accumulated large amounts of the sterol precursor squalene, while the levels of ergosterol and a number of other sterol compounds were only slightly elevated. These findings suggest that HMG-CoA reductase is not the only rate-limiting step in sterol synthesis and its overexpression cannot significantly influence this pathway beyond the sterol precursor squalene.

Cytosol↗