Biomedical subjects

R Hardmeier

Publications and source records attributed to R Hardmeier.

11 recordsLinked to original sources

PubMed2001

Postmortem changes in the level of brain proteins.

A systematic study on postmortem changes of brain proteins has not been performed so far and information is limited to basic principles of specific or nonspecific proteolysis or proteolysis of individual proteins. We studied protein level alterations in rat brain of animals kept at 23 degrees C for several postmortem times up to 72 h. Brain tissue protein extracts were analyzed by two-dimensional electrophoresis and the proteins with different levels were identified by matrix-assisted laser desorption ionization mass spectrometry. The changes observed mainly concerned structural proteins and enzymes. The levels of dihydropyrimidinase-related protein-2 decreased within 6 h and two new spots were detected representing shorter forms of the protein. Most of the other alterations appeared about 48 h postmortem. The most significant were reduced levels of neurofilament, alpha-internexin, synaptosomal-associated protein 25, glial fibrillary acidic protein, heat shock proteins, and dynamin-1; increased levels of 14-3-3 proteins and spectrin; and generation of shorter forms of certain proteins, such as tubulins, actin, and serum albumin. The results may be useful in neuropathology and brain protein studies.

Animals↗

PubMed2001

Expression of the multidrug resistance P glycoprotein (Pgp) and multidrug resistance associated protein (MRP1) in Down syndrome brains.

Transport by ATP-dependent efflux pumps such as P glycoprotein (Pgp) and multidrug resistance associated protein (MRP), encoded by multidrug resistant (MDR) associated genes, is an increasingly recognized mechanism by which cells maintain substrate homeostasis and evade drug therapy. Pgp and MRP are members of the so-called ATP binding cassette (ABC) transporters superfamily, which are associated with many biological processes in both prokaryotes and eukaryotes, as well as clinical problems. The observation of upregulated sequences that are homologous to the Mycobacterium smegmatis phage resistance (mpr) gene and putative ABC transporters subunits in fetal Down syndrome (DS) using the gene hunting technique, subtractive hybridization formed the Rationale for this study. The expression of Pgp and MRP1 is therefore investigated in different brain regions of controls and adult DS patients with western blot technique. No apparent changes were observed between controls and DS in levels of Pgp in all brain regions examined. By contrast, MRP1 detection using the rat monoclonal antibody (MRPr1) produced a significant elevation in DS temporal cortex (P < 0.01) and parietal cortex (P < 0.05). Although MRP1 detected with the mouse monoclonal antibody (MRPm6) tended to increase in most of the regions of DS brain, it failed to reach significance level. Age or postmortem interval did not correlate with protein levels in both controls as well as DS. Taken together, the current data provide evidence for the presence of MDR related pumps in different regions of the human brain. In addition, overexpression of MRP1 in DS brain may have some relevance to the disorder either by deranging substrate homeostasis or limiting drug access.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Superoxide dismutase SOD1, encoded on chromosome 21, but not SOD2 is overexpressed in brains of patients with Down syndrome.

BACKGROUND: The antioxidant enzyme Cu/Zn-superoxide dismutase-1 (SOD1) gene is localized to chromosome 21q22.1 and catalyzes the dismutation of superoxide anions to hydrogen peroxide, which may lead to the increased production of active oxygen species in Down Syndrome (DS), trisomy 21. Although a number of studies have addressed this question, proposing the overexpression hypothesis, no specific protein-chemical data on SOD protein levels in the brains of patients with DS are available. METHODS: We therefore determined the protein SOD-1 and SOD-2 levels in the brains of controls (n = 9) and adult patients with DS (n = 9) and Alzheimer disease (AD; n = 9). Two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization-mass spectroscopy detection and identification was used for the analyses. RESULTS: We found significantly increased SOD-1 levels in DS temporal, parietal, and occipital cortex, whereas SOD-1 was decreased in the AD temporal cortex and SOD-2 was comparable between all groups. CONCLUSIONS: Increased SOD-1 levels in patients with DS may reflect the overexpression by the trisomic state, as a response to the oxidative stress, as has been proposed in DS by several authors. However, it well may be that glial proliferation, which is markedly increased in DS brain, may underlie the increased brain levels of this ubiquitous protein. The decrease of SOD1 in the temporal cortex of patients with AD may reflect an antiapoptotic mechanism or simply cell loss in the brain.

Aged↗

PubMed1999

Rat brain proteins: two-dimensional protein database and variations in the expression level.

A two-dimensional database of rat brain proteins was constructed. Brain samples from newborn animals were analyzed by two-dimensional electrophoresis and the proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry. The database comprises 210 different proteins, the majority of which are structural components, heat shock proteins and enzymes with various catalytic activities. Several minor differences in the expression level were detected, mainly of quantitative nature, which most likely represent allelic differences. The map may be useful in studies of neurological disorders in animal models of human diseases.

Animals↗

PubMed1998

mRNA levels of the hypoxia inducible factor (HIF-1) and DNA repair genes in perinatal asphyxia of the rat.

Hypoxia inducible factor 1 (HIF-1) is a transcription factor which is expressed, when mammalian cells are subjected to hypoxia, activating the transcription of genes encoding proteins thought important for maintaining oxygen hemostasis. The aim of the study was to evaluate HIF-1 mRNA levels in a non-invasive model of perinatal asphyxia (PA). Brain was taken for studies on HIF-1 alpha and beta 10 min following the asphyctic period. To rule out influences by the redox status we also determined antioxidant enzyme mRNA levels for superoxide dismutase, catalase, glutathion peroxidase and performed electron spin resonance studies. To study the link to protein phosphorylation as previously proposed, we evaluated mRNA levels for protein kinase C. As DNA breaks were reported to occur in PA, we determined mRNA levels of two genes representing DNA nucleotide excision repair, ERCC2 and ERCC3, and a DNA repair gene involved in the repair of oxidation mediated DNA damage, XRCC1. mRNAs for HIF-1 were not detectable following 5-20 minutes of asphyxia. The antioxidant enzymes did not show any changes during the asphyctic periods either and electron spin resonance failed to detect the presence of the hydroxyl radical. PKC significantly decreased with the length of the asphyctic period. ERCC2 and XRCC1 mRNAs were inducible during the acute phase of asphyxia indicating early repair phenomena. HIF-1 may not be relevant for periods of PA up to 20 minutes, the maximal survival time in our model. Neonatal factors may be responsible for that phenomenon although we cannot rule out that HIF-1 changes may occur at the protein level.

Animals↗

PubMed1998

Transcription of the XRCC1 gene in kidneys of radiosensitive and radioresistant mice following whole-body irradiation.

The XRCC1 gene was described to play a role in the sensitivity of mammalian cell lines towards ionizing irradiation. Cells with a mutation of this gene present with decreased single-strand break repair and reduced recombination repair, show increased double-strand breaks, and the sister chromatid exchange is increased up to tenfold. The goal of our study was to investigate the transcription of this gene in the kidney following ionizing irradiation in the mouse, as this could be relevant to the pathogenetic mechanisms found in radiation nephropathy. Furthermore, we intended to examine whether radiation-sensitive mice would show a transcriptional pattern different from radiation-resistant mice. Radiation-sensitive BALB/c/J/Him mice and radiation-resistant C3H/He/Him mice were whole body irradiated with X-ray at 2, 4, and 6 Gy and sacrificed 5, 15, and 30 min after irradiation. mRNA was isolated from kidney cortex and hybridized with probes for XRCC1 and beta-actin as a housekeeping gene control. Following irradiation at 2 Gy, radiation-resistant mice increased transcriptional levels of mRNA-XRCC1/mRNA-beta-actin as early as after 5 min, and 15 and 30 min after irradiation, XRCC1 transcription was still higher than in radiation-sensitive mice. At higher radiation doses, no differences were found. This finding is the first in vivo study on XRCC1 of this kind and may in part explain the differences in the radiation sensitivity between the two strains studied.

Animals↗

PubMed1997

Transcription and activity of antioxidant enzymes after ionizing irradiation in radiation-resistant and radiation-sensitive mice.

The involvement of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase in radiobiological processes has been described at the enzyme activity level. We irradiated radiation-resistant (RR) and radiation-sensitive (RS) mice and studied antioxidant enzymes at the transcriptional and activity level. In addition, aromatic hydroxylation and lipid peroxidation parameters were determined to study radiation resistance at the oxidation level. RS BALB/c/J Him mice and RR C3H He/Him mice were whole-body-irradiated with x-rays at 2, 4, and 6 Gy and killed 5, 15, and 30 min after irradiation. mRNA was isolated from liver and hybridized with probes for antioxidant enzymes and beta-actin as a housekeeping gene control. Antioxidant enzyme activities were determined by standard assays. Parameters for aromatic hydroxylation (o-tyrosine) and lipid peroxidation (malondialdehyde) were determined by HPLC methods. Antioxidant transcription was unchanged in contrast to antioxidant activities; SOD and CAT activities were elevated within 15 min in RR animals but not in RS mice, at all doses studied. Glutathione peroxidase activity was not different between RR and RS mice and was only moderately elevated after irradiation. No significant differences were found between RR and RS animals at the oxidation level, although a radiation dose-dependent increase of oxidation products was detected in both groups. We found that ionizing irradiation led to increased antioxidant activity only minutes after irradiation in the absence of increased transcription of these antioxidant enzymes. RR animals show higher antioxidant enzyme activities than do RS mice, but oxidation products are comparable in RS and RR mice. As unchanged transcription of antioxidant enzymes could not have been responsible for the increased antioxidant enzyme activities, preformed antioxidant enzymes should have been released by the irradiation process. This would be in agreement with previous studies of preformed, stored SOD. The finding of higher SOD and CAT activities in RR than in RS animals could point to a role for these antioxidant enzymes for the process of radiation sensitivity.

Animals↗

PubMed1997

The transcription of the XRCC1 gene in spleen following ionizing irradiation in radiosensitive and radioresistant mice.

The XRCC1 gene was described to play a role for the sensitivity of mammalian cell lines towards ionizing irradiation. Cells with a mutation of this gene present with decreased single strand break repair, reduced recombination repair, show increased double strand breaks and sister chromatid exchange is increased up to tenfold. The goal of our study was to investigate the transcription of this gene in the spleen following ionizing irradiation in the mouse. Furthermore, we intended to examine whether radiation sensitive (RS) mice would show a transcriptional pattern different from radiation resistant (RR) mice. Radiation sensitive BALB/c/J Him mice and radiation resistant C3H He/Him mice were untreated or whole body irradiated with X-ray at 4 and 6 Gy and sacrificed 5, 15 and 30 min after irradiation. mRNA was isolated from the spleen and hybridized with probes for XRCC1 and beta-actin as a house keeping gene control. Transcription of XRCC1 was not different in unirradiated or 4 Gy-irradiated mouse RR or RS mouse strains. When irradiated at 6 Gy, RR mice showed an approximately threefold increase of mRNA XRCC1/mRNA beta actin as early as 15 min after irradiation. We conclude that radiation resistant mice show a higher transcription level for the XRCC1 gene in the spleen early after high dose X-ray whole body irradiation. This finding is the first in vivo study on XRCC1 of this kind and may in part explain the differences in the radiation sensitivity between the two strains studied.

Animals↗

PubMed1997

The transcription of the XRCC1 gene in the heart of radiation-resistant and radiation-sensitive mice after ionizing irradiation.

The XRCC1 (X-Ray Repair Cross Complementing) gene was described to play a role for the sensitivity of mammalian cell lines toward ionizing irradiation. Cells with a mutation of this gene present with decreased single strand break repair and reduced recombination repair, they show increased double strand breaks, and sister chromatid exchange is increased up to 10-fold. The goal of our study was to investigate the transcription of this gene in the heart after ionizing irradiation in the mouse. Furthermore, we intended to examine whether radiation-sensitive mice would show a transcriptional pattern different from radiation-resistant mice. Radiation-sensitive BALB/c/J Him mice and radiation-resistant C3H He/Him mice were whole body irradiated with x-ray at 2, 4, and 6 Gy and killed 5, 15, and 30 min after irradiation. mRNA was isolated from the heart and hybridized with probes for XRCC1 and beta-actin as a housekeeping gene control. Irradiation at 2 Gy showed increased transcription of XRCC1 at 5 min in the C3H He/Him group, approached XRCC1 transcription of BALB/c J/Him mice at 15 min, and was lower in the latter at 30 min after irradiation. Irradiation at 4 Gy showed double the transcription at 5 min and an about 3-fold rapid increase of mRNA XRCC1 in the radiation-resistant group at 15 min after irradiation, returning to the transcriptional level of sensitive animals at 30 min. Irradiation at 6 Gy seemed to overwhelm the system in both groups, but resistant mice still showed higher levels of XRCC1 transcription. We conclude that radiation-resistant mice show a higher transcription level for the XRCC1 gene in the heart early after x-ray whole body irradiation. This findings is the first in vivo study on XRCC1 of this kind and may in part explain the differences in the radiation sensitivity between the two strains studied.

Animals↗

PubMed1997

The transcription of liver thioredoxin following the ionizing irradiation of radioresistant and radiosensitive mice.

The radiation protective effect of thioredoxin (TRX) in a bacterial system has been reported and based upon this observation we were interested to examine TRX transcription in the mammalian system following ionizing irradiation. In order to answer the question whether radiation sensitive mice (BALB/c) showed TRX transcription different from radiation resistant mice (C3H), we exposed these strains to X-ray doses of 2 Gy, 4 Gy and 6 Gy. Groups consisting of 6 mice were sacrificed 5, 15 and 30 minutes after irradiation and livers were immediately taken into liquid nitrogen. Total RNA was isolated from the organs by the use of a commercially available kit and used for Northern blots and slot blots with a chemiluminescence technique. Northern blots revealed a single band at 538 bp for TRX and at 1.8 kb for beta-actin. Quantification of mRNA TRX by densitometry of slot blots revealed that C3H transcribed TRX significantly higher at an earlier time point (5 min) than BALB/c. This delayed transcription of TRX in the radiosensitive mouse strain showed a comparable pattern at three different radiation doses and may well be responsible for radioresistance although no quantitative differences of TRX transcription between BALB/c and C3H mice were detectable.

Actins↗

PubMed1994

Vitamin A is excreted in the urine during acute infection.

Episodes of acute infection are thought to deplete body stores of vitamin A. The mechanism by which this might occur is not known, but increased metabolic requirements are presumed to play a role. We have found, however, that significant amounts of retinol and retinol-binding protein (RBP) were excreted in the urine during serious infections, whereas only trace amounts were found in the urine of healthy control subjects. The geometric mean excretion rate in 29 subjects with pneumonia and sepsis was 0.78 mumol retinol/d. Subjects with fever (temperature > or = 38.3 degrees C) excreted significantly more retinol (geometric mean = 1.67 mumol/d) than did those without fever (0.18 mumol/d; t = 3.53, P < 0.0015). Aminoglycoside administration and low glomerular filtration rates (< 35 mL/min) were also associated with higher rates of urinary retinol excretion. Thirty-four percent of patients excreted > 1.75 mumol retinol/d, equivalent to 50% of the US recommended dietary allowance. These data show that vitamin A requirements are substantially increased during serious infections because of excretion of retinol in the urine, and suggest that these losses are due to pathologic changes associated with the febrile response.

Adult↗