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Biomedical subjects

A Roscher

Publications and source records attributed to A Roscher.

At least 19 recordsLinked to original sources
PubMed2001

Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells.

The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P2) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P2 were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P2 had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-13C]glucose, in the lines possessing the highest amounts of Fru-2,6-P2, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of 14C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P2 can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates.

Animals↗
PubMed2000

Strategies for metabolic flux analysis in plants using isotope labelling.

Flux measurements through metabolic pathways generate insights into the integration of metabolism, and there is increasing interest in using such measurements to quantify the metabolic effects of mutation and genetic manipulation. Isotope labelling provides a powerful approach for measuring metabolic fluxes, and it gives rise to several distinct methods based on either dynamic or steady-state experiments. We discuss the application of these methods to photosynthetic and non-photosynthetic plant tissues, and we illustrate the different approaches with an analysis of the pathways interconverting hexose phosphates and triose phosphates. The complicating effects of the pentose phosphate pathway and the problems arising from the extensive compartmentation of plant cell metabolism are considered. The non-trivial nature of the analysis is emphasised by reference to invalid deductions in earlier work. It is concluded that steady-state isotopic labelling experiments can provide important information on the fluxes through primary metabolism in plants, and that the combination of stable isotope labelling with detection by nuclear magnetic resonance is particularly informative.

Biotechnology↗
PubMed2000

Genome-wide, large-scale production of mutant mice by ENU mutagenesis.

In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).

Animals↗
PubMed1999

Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Ceramide (CER) is an important signaling molecule involved in a variety of cellular processes, including differentiation, cell growth, and apoptosis. Currently, different techniques are applied for CER quantitation, some of which are relatively insensitive and/or time consuming. Tandem mass spectrometry with its high selectivity and sensitivity is a very useful technique for detection of low abundant metabolites without prior purification or derivatization. In contrast to existing mass spectrometry methods, the developed electrospray tandem mass spectrometry (ESI-MS/MS) technique is capable of quantifying different CER species from crude cellular lipid extracts. The ESI-MS/MS is performed with a continuous flow injection and the use of an autosampler, resulting in a high throughput capability. The collision-induced fragmentation of CER produced, in addition to others, a characteristic fragment of m/z 264, making a precursor ion scan of 264 well suited for CER quantitation. Quantitation is achieved by use of a constant concentration of a non-naturally occurring internal standard C8-CER, together with a calibration curve established by spiking different concentrations of naturally occurring CER. The calibration curves showed linearity over a wide concentration range and sample volumes equivalent to 10 microg of cell protein corresponding to about 20, 000 fibroblasts were sufficient for CER analysis. Moreover this assay showed a detection limit at the subpicomole level. In summary, this methodology enables accurate and rapid analysis of CER from small samples without prior separation steps, thus providing a useful tool for signal transduction research.

Ceramides↗
PubMed1998

Unidirectional steady state rates of central metabolism enzymes measured simultaneously in a living plant tissue.

The unidirectional steady state reaction rates of several enzymes and metabolic fluxes of distinct processes were measured simultaneously in hypoxic maize root tips using two-dimensional phosphorus NMR exchange spectroscopy. A single spectrum monitors ATP synthesis and hydrolysis as well as the activities of four enzymes involved in key pathways of central metabolism: UDP-glucose pyrophosphorylase, phosphoglucomutase, hexose-phosphate isomerase, and enolase. The corresponding unidirectional reaction rates and net metabolic fluxes were calculated from spectral intensities. This method provides a unique picture, at enzyme resolution, of how metabolism reacts in a concerted fashion to changes in external parameters such as temperature and oxygen concentration. By increasing hypoxia via an increase in temperature, we measured the expected increase in glycolysis through enolase activity while total ATP synthesis settled. At the same time, we observed a net flux through phosphoglucomutase and UDP-glucose pyrophosphorylase toward carbohydrate synthesis. This result is discussed in relation to the current hypothesis on the turnover of cell walls and sucrose. This reaction also produces a net flux of pyrophosphate, which is needed by pyrophosphate:fructose-6-phosphate 1-phosphotransferase to work as a glycolytic enzyme.

Adenosine Triphosphate↗
PubMed1998

External quality assessment of molecular biology-based methods used in laboratories of clinical chemistry and human genetics.

The Reference Institute of Bioanalysis of the German Society of Clinical Chemistry has performed the first external assessment of molecular genetics methods used in medical diagnosis. The following procedures were tested: (I) DNA preparation from whole blood, (II) PCR amplification using "standard" primers, and (III) submarine agarose gel electrophoresis. Out of 50 participants, 45 returned samples for evaluation.

Chemistry, Clinical↗
PubMed1995

Association of X-linked adrenoleukodystrophy with HLA DRB1 alleles.

Inflammatory demyelination in the central nervous system in the childhood cerebral phenotype of X-linked adrenoleukodystrophy (X-ALD) bears resemblance to that of multiple sclerosis. With a view to an association of HLA class II genes, specifically HLA-DRB1 subtype DRB1*15 to multiple sclerosis we investigated the HLA class II DR haplotype in 29 unrelated X-ALD patients including 17 childhood cerebral phenotype patients. Our results did not show an association of DRB1*15 and X-ALD, but disclosed a significant association of HLA DRB1*16 alleles and X-ALD in general. This finding suggests that in addition to the X-chromosomal ALD gene an autosomal gene linked to the HLA class II region is involved in the pathogenesis of X-ALD. This gene should affect a pathomechanism common to all ALD variants, such as defective peroxisomal metabolism of very long chain fatty acids.

Adrenoleukodystrophy↗
PubMed1994

Bradykinin induces translocation of the protein kinase C isoforms alpha, epsilon, and zeta.

Bradykinin exerts a broad spectrum of cellular effects on different tissues. It is believed that these effects are predominantly mediated by the recently cloned B2 receptor. The mechanism of post-receptor signal transduction is not known in detail. Involvement of protein kinase C (PKC) was suggested and activation of the classical PKC isoforms alpha and beta was recently demonstrated. The aim of the present study was to investigate whether the B2 receptor also activates new (delta, epsilon) and atypical (zeta) PKC isoforms. To investigate this, chinese hamster ovary (CHO) cells, stably transfected with human B2 receptor, were used. In these cells the PKC isoforms alpha, delta, epsilon and zeta were detected by immunoblotting with specific antibodies. To monitor hormone-induced PKC translocation plasma membranes were prepared. Stimulation of the cells with bradykinin resulted in a rapid (30-60 s) translocation of the PKC isoforms alpha, epsilon, and zeta. Translocation of PKC delta was not detected. The effect of bradykinin was reduced by simultaneous addition of the receptor antagonist HOE 140, a bradykinin-related decapeptide. The data show that the B2 receptor in this cell model is able to activate, in addition to the classical PKC isoform alpha, the new PKC isoform epsilon and the atypical PKC isoform zeta. To test whether these effects are as well observed in a non-transfected cell, the experiments were repeated in human foreskin fibroblasts which naturally express high levels of B2 receptors. In this cell system similar results on PKC alpha, epsilon, and zeta were observed, suggesting that all three PKC isoforms are involved in signal transduction of the B2 receptor.

Animals↗
PubMed1993

Thromboxane A2 analogue U 46619 enhances tumour cell proliferation in HeLa cells via specific receptors which are apparently distinct from TXA2 receptors on human platelets.

In this study, we have demonstrated for the first time by using U 46619, a stable analogue of thromboxane A2 (TXA2), that TXA2 exerts a cell proliferative effect on HeLa cells which is mediated by specific TXA2 receptors, inasmuch as the cell proliferation could be dose-dependently suppressed by TXA2 receptor antagonist BM 13177. The investigation of the phospholipase C pathway by U 46619 and prostaglandin H2 (PGH2) in the presence and absence of BM 13177 in cells with or without pertussis toxin pretreatment, as well as radioligand receptor binding studies, revealed that, in contrast to TXA2 receptors on human platelets, where TXA2 and PGH2 share the same receptor binding sites, HeLa cells possess distinct receptors for TXA2 and PGH2.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1992

Sparse hair and multiple endocrine disorders in two women heterozygous for adrenoleukodystrophy.

We describe two sisters (40 and 42 years old) heterozygous for adrenoleukodystrophy who have multiple endocrine disorders. In addition to the characteristic neurological symptoms, the younger patient has Addison disease and primary hypothyroidism attributable to autoimmune thyroiditis, and the older one has Graves disease. Both patients have loss of body hair and sparse scalp hair, which have not been reported previously in women heterozygous for adrenoleukodystrophy. After the institution of glucocorticoid replacement therapy, the younger sister, who has adrenal insufficiency, has shown unexpected neurological improvement.

Addison Disease↗
PubMed1992

Clinical significance of prostacyclin and thromboxane in cancer of the female breast and genital tract.

Studies investigating the role of thromboxane A2 and prostacyclin in cancer of the female breast and genital tract are reviewed. Whereas thromboxane A2 was found to promote the tumour growth and metastasis, prostacyclin exerted a protective effect in maintaining vascular and platelet homeostasis. Thus, monitoring of prostacyclin and thromboxane levels in plasma and urine of cancer patients may be essential for the evaluation of tumour growth and metastasis. Of all modulators of thromboxane and prostacyclin biosynthesis, nafazatrom was found to exhibit promising results for the treatment of advanced breast cancer, although its use in the routine therapy is questionable at this stage.

Breast Neoplasms↗
PubMed1992

Hartnup syndrome, progressive encephalopathy and allo-albuminaemia. A clinico-pathological case study.

Clinical, biochemical, neuropathological and neurochemical findings in a case of Hartnup syndrome are reported. After initially normal development, the affected girl suffered progressive neuropsychiatric decline with statomotor and mental retardation and intractable seizures and died at the age of 2 years. Postmortem neuropathological and neurochemical investigations showed a combination of extensive neuronal degeneration and cerebral dysmyelination. Pathogenetic hypotheses and the relationship between neuropsychiatric disease and Hartnup syndrome are discussed. Additionally, a fast type bisalbuminaemia present in the girl and her mother is described.

Amino Acids↗
PubMed1992

Loss of cytosolic 15-lipoxygenase activity in A23187-stimulated human leukocytes: involvement of a translocation process?

We focused our study on the subcellular redistribution of 15-lipoxygenase in human leukocytes challenged with A23187 and arachidonic acid (AA). We found that in cytosolic fractions of stimulated cells the 15-lipoxygenase (15-LO) activity, measured as 15-HETE, was 65% less than in controls. However, no activity was found in cell membranes. This effect was complete within 2 min of incubation and was correlated in a dose dependent manner to exogenously added AA. No significant difference in cytosolic distribution of 15-LO activity was observed when cells were stimulated in presence of various concentrations of Ca++. Immunoblot analysis showed that the loss of cytosolic 15-LO activity registered after challenging with A23187 was associated with a concomitant loss of the enzyme content in the cytosol, suggesting the possibility of a translocation process. Neither the 15-LO activity nor the enzyme was, however, found in the cell membrane under our present experimental conditions. But, addition of protease inhibitors showed a slight increase of 15-LO activity in the membrane fraction. Despite the small effect, this may indicate a translocation of 15-LO following challenge of human leukocytes with A23187.

Adult↗
PubMed1989

Influence of plasmalogen deficiency on membrane fluidity of human skin fibroblasts: a fluorescence anisotropy study.

The influence of plasmalogen deficiency on membrane lipid mobility was determined by measuring fluorescence anisotropy of trimethylammoniumdiphenylhexatriene (TMA-DPH) and diphenylhexatrienylpropanoylhydrazylstachyose (glyco-DPH) inserted in the plasma membranes of human skin fibroblasts deficient in plasmalogens. The cells used were from patients affected with cerebrohepatorenal (Zellweger) syndrome (CHRS) or rhizomelic chondrodysplasia punctata. Their plasmalogen content (0-5% of total phospholipid) is significantly reduced compared with that of control cells from healthy donors (13-15% of total phospholipid) or of CHRS fibroblasts supplemented with the plasmalogen precursor, hexadecylglycerol. Plasmalogen-deficient cells consistently showed lower fluorescence anisotropies of membrane-bound DPH fluorophores corresponding to higher membrane lipid mobilities as compared to controls. However, very similar lipid mobilities were found for sonicated aqueous dispersions of phospholipids extracted either from CHRS or control cells. Therefore, the differences observed with living cells are not due to differences in the overall physical properties of the membrane lipid constituents. Other phenomena such as lipid asymmetry and/or plasmalogen-protein interactions may be responsible for the effects observed in the biomembranes.

Cell Membrane↗
PubMed1989

Plasmalogen biosynthesis in the diagnosis of peroxisomal disorders.

Fibroblasts of patients suffering from peroxisomal disorders such as chondrodysplasia punctata (rhizomelic type), neonatal adrenoleukodystrophy, Zellweger syndrome and control fibroblasts were used for the evaluation of a procedure suitable for pre- and postnatal diagnosis. This technique is based on the detection of impaired peroxisomal plasmalogen synthesis by means of a double substrate, double labelling technique using 14C-labelled hexadecanol and 3H-labelled hexadecylglycerol as precursors for peroxisomal and microsomal plasmalogen synthesis. Pathological cells are characterized by a decreased utilization of hexadecanol, thus resulting in an increased 3H/14C ratio within plasmalogens. Sensitivity and reproducibility of this method were improved by changing both the chromatographic conditions and the calculation of the diagnostic parameters.

Adrenoleukodystrophy↗
PubMed1987

The effect of D-(+)-glucosamine on levels of free N-acetylneuraminic acid and UDP-N-acetylhexosamines in infantile sialic acid storage disease (ISSD) fibroblasts.

An impairment in the regulation of N-acetylneuraminic acid (NANA) biosynthesis might potentially contribute to accumulation of free NANA in fibroblasts of patients with sialic acid storage disease (ISSD). By the use of a glucosamine loading test an increase in uridine-diphosphate-N-acetyl-hexosamines (UDP-HexNAc) but not in free NANA was found. NANA biosynthesis therefore appears to be under normal regulatory control in ISSD.

Fibroblasts↗