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M Raabe

Publications and source records attributed to M Raabe.

At least 19 recordsLinked to original sources
PubMed2006

Collection, validation and generation of bitumen fumes for inhalation studies in rats Part 3: Regeneration of bitumen fumes, inhalation setup, validation.

Undertaking a chronic inhalation study on bitumen fume presents a challenge in terms of generating sufficient amounts of representative fume. The objective of the study described in this and in previous publications was to collect sufficient fume and use this to develop a laboratory-generated exposure atmosphere, for use in chronic inhalation toxicity studies in rats that resembles, as closely as possible, personal exposures seen in workers during road paving operation. To achieve this goal, atmospheric workplace samples were collected at road paving work sites and compared with bitumen fume condensate samples collected from the headspace of hot bitumen storage tanks. In Parts 1 and 2, we described the collection and analysis of workplace samples, the strategy for in-line extraction of a suitable fraction of bitumen fume collected from the headspace of a bitumen storage tank and the comparison of the collected condensate to the workplace samples. This paper (Part 3) describes the regeneration of bitumen fume for inhalation and the exposure setup used for inhalation studies.

Aerosols↗
PubMed2006

Collection, validation and generation of bitumen fumes for inhalation studies in rats Part 2: Collection of bitumen fumes from storage tanks.

OBJECTIVES: The objective of the study described in this and an accompanying series of papers was to develop a laboratory generated exposure atmosphere to be used for chronic inhalation toxicity studies in rats that resembles, as closely as possible, personal exposures seen by workers during road paving operations. METHODS: To achieve this objective, atmospheric workplace samples were collected at road paving worksites and compared analytically with bitumen fume samples collected from the headspace of hot bitumen storage tanks. In Preiss et al. (2006) the collection and analysis of workplaces samples is described. This contribution describes the strategy for the in-line extraction of a suitable fraction of bitumen fume collected from the headspace of a bitumen storage tank and the comparison of the collected condensate to workplace samples. RESULTS: Results show that is possible to develop a collecting procedure that allows sampling from hot bitumen storage tanks in an operational asphalt mixing plant. The sampling procedure has been optimized to collect material that matches the workplace samples as closely as possible. The comparison to workplace samples has been performed using parameters that can be analyzed in both the workplace samples and the bitumen fume condensate collected from the tanks. Boiling point distribution (BPD), UV fluorescence (UV-Fl) and content of individual polycyclic aromatic hydrocarbons (PAH) were selected as parameters. The BPD of the final collected bitumen fume condensate did not differ by more than 17 degrees C from any point on the average BPD curve of the workplace samples, in the range from 5 to 95%. UV-Fl of the bitumen fume condensate nearly exactly matched the average UV-Fl of the workplace samples. However, the sum of the 17 PAHs analyzed in the test samples, compared to the mass of the condensate, is lower by a factor of approximately 3 than the sum of the 17 PAHs in some personal samples compared to the mass of Total Organic Matter (TOM). It has to be recognised that during the collection of the workplace samples, despite all efforts a number of the workers who carried a personal sampler could not be prevented from smoking.

Air Pollutants, Occupational↗
PubMed2006

Collection, validation and generation of bitumen fumes for inhalation studies in rats Part 1: Workplace samples and validation criteria.

OBJECTIVES: Undertaking a chronic inhalation study on bitumen fume presents a challenge in terms of generating large amounts of representative fume. The objective of the study described in this and the following contributions was to collect sufficient fume and develop a laboratory-generated exposure atmosphere that resembles, as closely as possible, personal exposures seen in workers during road paving operations, for use in chronic inhalation toxicity studies in rats. METHODS: To achieve this goal, atmospheric workplace samples were collected at road paving work sites both by Shell Global Solutions, Int. (Shell) and by the 'Berufsgenossenschaftliches Institut für Arbeitssicherheit' (BIA, Germany) and compared with bitumen fume condensate samples collected from the head space of hot bitumen storage tanks. Part 1 describes the collection and analysis of personal and static workplace samples. Different sampling methods were also used to allow a comparison of the standard German sampling method with the most common industry method used. Samples were analyzed by Shell, BIA and by the Fraunhofer Institute of Toxicology and Experimental Medicine (Fh-ITEM, Germany) using different methods. Parameters determined were: total particulate matter (TPM), benzene soluble matter (BSM), semi-volatiles (SV), total organic matter (TOM), boiling point distribution (BPD), polycyclic aromatic hydrocarbons (PAHs) and UV fluorescence (UVF). RESULTS: The BPD of personal and static samples had almost identical start and end points, but static samples show a tendency towards an increase in amounts of higher boiling point compounds. Personal samples generally show higher PAH concentrations than comparable static samples. The results of the analysis of personal workplace samples were used to establish validation/acceptance criteria for the bitumen fume condensate sampled from storage tanks for the inhalation study, which is described in a further publication. CONCLUSIONS: The criteria involve a range of parameters that can be analyzed in both workplace samples and samples of tank fume condensate: BPD, UVF and content of individual PAHs were selected as parameters.

Air Pollutants, Occupational↗
PubMed2002

A practical design of a cylindrical tissue-equivalent proportional counter for microdosimetry.

A 12 mm tissue-equivalent proportional counter (TEPC) was designed, constructed and successfully tested. This detector achieves features common to others TEPCs, but it does not use field shaping electrodes and it is able to work at higher bias voltages which makes it capable of measuring the whole range of energy deposition events for gamma rays. The following approach was used to design the detector: first of all the use of a cylindrical shape detector featured as simple as possible but keeping its performance as well, the next point is to avoid the use of field shaping tubes and the last one is to make the preamplifier small and as close as possible to the detector. Its construction is based on a cylindrical proportional counter with A-150 tissue-equivalent material as cathode, TE-methane gas as a proportional gas and a 20 microns diameter wire as anode.

Cesium Radioisotopes↗
PubMed2000

A deficiency of microsomal triglyceride transfer protein reduces apolipoprotein B secretion.

Microsomal triglyceride transfer protein (MTP) transfers lipids to apolipoprotein B (apoB) within the endoplasmic reticulum, a process that involves direct interactions between apoB and the large subunit of MTP. Recent studies with heterozygous MTP knockout mice have suggested that half-normal levels of MTP in the liver reduce apoB secretion. We hypothesized that reduced apoB secretion in the setting of half-normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were true, half-normal levels of MTP might have little impact on lipoprotein secretion in the setting of half-normal levels of apoB synthesis (since the ratio of MTP to apoB would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the MTP:apoB ratio would be even lower). To test this hypothesis, we examined the effects of heterozygous MTP deficiency on apoB metabolism in the setting of normal levels of apoB synthesis, half-normal levels of apoB synthesis (heterozygous Apob deficiency), and increased levels of apoB synthesis (transgenic overexpression of human apoB). Contrary to our expectations, half-normal levels of MTP reduced the plasma apoB100 levels to the same extent ( approximately 25-35%) at each level of apoB synthesis. In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum rather than the MTP:apoB ratio is the critical determinant of lipoprotein secretion. Finally, we found that heterozygosity for an apoB knockout mutation lowered plasma apoB100 levels more than heterozygosity for an MTP knockout allele. Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice.

Animals↗
PubMed1999

Chinese hamster ovary cells require the coexpression of microsomal triglyceride transfer protein and cholesterol 7alpha-hydroxylase for the assembly and secretion of apolipoprotein B-containing lipoproteins.

Due to the absence of microsomal triglyceride transfer protein (MTP), Chinese hamster ovary (CHO) cells lack the ability to translocate apoB into the lumen of the endoplasmic reticulum, causing apoB to be rapidly degraded by an N-acetyl-leucyl-leucyl-norleucinal-inhibitable process. The goal of this study was to examine if expression of MTP, whose genetic deletion is responsible for the human recessive disorder abetalipoproteinemia, would recapitulate the lipoprotein assembly pathway in CHO cells. Unexpectedly, expression of MTP mRNA and protein in CHO cells did not allow apoB-containing lipoproteins to be assembled and secreted by CHO cells expressing apoB53. Although expression of MTP in cells allowed apoB to completely enter the endoplasmic reticulum, it was degraded by a proteolytic process that was inhibited by dithiothreitol (1 mM) and chloroquine (100 microM), but resistant to N-acetyl-leucyl-leucyl-norleucinal. In marked contrast, coexpression of the liver-specific gene product cholesterol 7alpha-hydroxylase with MTP resulted in levels of MTP lipid transfer activity that were similar to those in mouse liver and allowed intact apoB53 to be secreted as a lipoprotein particle. These data suggest that, although MTP-facilitated lipid transport is not required for apoB translocation, it is required for the secretion of apoB-containing lipoproteins. We propose that, in CHO cells, MTP plays two roles in the assembly and secretion of apoB-containing lipoproteins: 1) it acts as a chaperone that facilitates apoB53 translocation, and 2) its lipid transfer activity allows apoB-containing lipoproteins to be assembled and secreted. Our results suggest that the phenotype of the cell (e.g. expression of cholesterol 7alpha-hydroxylase by the liver) may profoundly influence the metabolic relationships determining how apoB is processed into lipoproteins and/or degraded.

Animals↗
PubMed1999

Insights into apolipoprotein B biology from transgenic and gene-targeted mice.

Over the past five years, several laboratories have used transgenic and gene-targeted mice to study apolipoprotein (apo) B biology. Genetically modified mice have proven useful for investigating the genetic and environmental factors affecting atherogenesis, for defining apoB structure/function relationships, for understanding the regulation of the apoB gene expression in the intestine, for defining the "physiologic rationale" for the existence of the two different forms of apoB (apoB48 and apoB100) in mammalian metabolism and for providing mechanistic insights into the human apoB deficiency syndrome, familial hypobetalipoproteinemia. This review will provide several examples of how genetically modified mice have contributed to our understanding of apoB biology, including our new discovery that human heart myocytes secrete nascent apoB-containing lipoproteins.

Animals↗
PubMed1999

Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice.

A deficiency in microsomal triglyceride transfer protein (MTP) causes the human lipoprotein deficiency syndrome abetalipoproteinemia. However, the role of MTP in the assembly and secretion of VLDL in the liver is not precisely understood. It is not clear, for instance, whether MTP is required to move the bulk of triglycerides into the lumen of the endoplasmic reticulum (ER) during the assembly of VLDL particles. To define MTP's role in hepatic lipoprotein assembly, we recently knocked out the mouse MTP gene (Mttp). Unfortunately, achieving our objective was thwarted by a lethal embryonic phenotype. In this study, we produced mice harboring a "floxed" Mttp allele and then used Cre-mediated recombination to generate liver-specific Mttp knockout mice. Inactivating the Mttp gene in the liver caused a striking reduction in VLDL triglycerides and large reductions in both VLDL/LDL and HDL cholesterol levels. The Mttp inactivation lowered apo B-100 levels in the plasma by >95% but reduced plasma apo B-48 levels by only approximately 20%. Histologic studies in liver-specific knockout mice revealed moderate hepatic steatosis. Ultrastructural studies of wild-type mouse livers revealed numerous VLDL-sized lipid-staining particles within membrane-bound compartments of the secretory pathway (ER and Golgi apparatus) and few cytosolic lipid droplets. In contrast, VLDL-sized lipid-staining particles were not observed in MTP-deficient hepatocytes, either in the ER or in the Golgi apparatus, and there were numerous cytosolic fat droplets. We conclude that MTP is essential for transferring the bulk of triglycerides into the lumen of the ER for VLDL assembly and is required for the secretion of apo B-100 from the liver.

Alleles↗
PubMed1999

Generation of monoclonal antibodies specific for mouse apolipoprotein B-100 in apolipoprotein B-48-only mice.

Over the past 10 years, many laboratories have investigated lipid metabolism and atherogenesis with a variety of transgenic and gene knockout mouse models. Although many of these studies have yielded valuable insights, some have been hampered by a paucity of useful antibodies against mouse proteins. For example, many laboratories have analyzed genetic and dietary interventions affecting lipoprotein metabolism without useful antibodies against mouse apolipoprotein (apo) B. In this study, we sought to develop highly specific monoclonal antibodies against mouse apoB-100. To achieve this goal, gene-targeted mice that synthesize exclusively apoB-48 (apoB-48-only mice) were immunized with mouse apoB-100. The immune response against apoB-100 was robust, as judged by high titers of antibodies against mouse apoB-100. After fusing the splenic lymphocytes of the apoB-48-only mice with a myeloma cell line, we identified and cloned hybridomas that produced mouse apoB-100-specific monoclonal antibodies. Those antibodies were useful for developing sensitive and specific immunoassays for mouse apoB-100. This study illustrates the feasibility and utility of using gene-targeted mice to develop monoclonal antibodies against mouse proteins.

Animals↗
PubMed1998

Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes.

Abetalipoproteinemia, an inherited human disease characterized by a near-complete absence of the apolipoprotein (apo) B-containing lipoproteins in the plasma, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP). We used gene targeting to knock out the mouse MTP gene (Mttp). In heterozygous knockout mice (Mttp+/- ), the MTP mRNA, protein, and activity levels were reduced by 50%, in both liver and intestine. Compared with control mice (Mttp+/+), chow-fed Mttp+/- mice had reduced plasma levels of low-density lipoprotein cholesterol and had a 28% reduction in plasma apoB100 levels. On a high-fat diet, the Mttp+/- mice exhibited a marked reduction in total plasma cholesterol levels, compared with those in Mttp+/+ mice. Both the livers of adult Mttp+/- mice and the visceral endoderm of the yolk sacs from Mttp+/- embryos manifested an accumulation of cytosolic fat. All homozygous embryos (Mttp-/-) died during embryonic development. In the visceral endoderm of Mttp-/- yolk sacs, lipoprotein synthesis was virtually absent, and there was a marked accumulation of cytosolic fat droplets. In summary, half-normal MTP levels do not support normal levels of lipoprotein synthesis and secretion, and a complete deficiency of MTP causes lethal developmental abnormalities, perhaps because of an impaired capacity of the yolk sac to export lipids to the developing embryo.

Abetalipoproteinemia↗
PubMed1998

Genes for apolipoprotein B and microsomal triglyceride transfer protein are expressed in the heart: evidence that the heart has the capacity to synthesize and secrete lipoproteins.

BACKGROUND: Expression of both the apolipoprotein B (apoB) gene and the microsomal triglyceride transfer protein (MTP) gene is required for the assembly and secretion of triglyceride-rich lipoproteins in the liver and intestine. Both genes have been assumed to be silent in the heart. METHODS AND RESULTS: Northern blot and RNase protection analyses showed that the apoB and MTP genes were expressed in the hearts of mice and humans. In situ hybridization studies revealed that the apoB mRNA was produced in cardiac myocytes. Electron microscopy of human cardiac myocytes revealed lipid-staining particles of relatively small diameter (approximately 250 A) within the Golgi apparatus. CONCLUSIONS: These studies strongly suggest that the heart synthesizes and secretes apoB-containing lipoproteins.

Animals↗
PubMed1998

Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-x gene function.

Gene targeting in mice was used to investigate the unknown function of Scp2, encoding sterol carrier protein-2 (SCP2; a peroxisomal lipid carrier) and sterol carrier protein-x (SCPx; a fusion protein between SCP2 and a peroxisomal thiolase). Complete deficiency of SCP2 and SCPx was associated with marked alterations in gene expression, peroxisome proliferation, hypolipidemia, impaired body weight control, and neuropathy. Along with these abnormalities, catabolism of methyl-branched fatty acyl CoAs was impaired. The defect became evident from up to 10-fold accumulation of the tetramethyl-branched fatty acid phytanic acid in Scp2(-/-) mice. Further characterization supported that the gene disruption led to inefficient import of phytanoyl-CoA into peroxisomes and to defective thiolytic cleavage of 3-ketopristanoyl-CoA. These results corresponded to high-affinity binding of phytanoyl-CoA to the recombinant rat SCP2 protein, as well as high 3-ketopristanoyl-CoA thiolase activity of the recombinant rat SCPx protein.

Acetyl-CoA C-Acetyltransferase↗
PubMed1997

Reading-frame restoration by transcriptional slippage at long stretches of adenine residues in mammalian cells.

We previously characterized a mutant apoB allele (the apoB86 allele) that produces both a truncated apoB (apoB86) and a full-length apoB100. The mutant allele contained a deletion of a single cytosine in exon 26, creating a stretch of eight consecutive adenines in the -1 reading frame. The altered reading-frame allele was restored, with approximately 10% efficiency, by the transcriptional insertion of an extra adenine into the stretch of eight consecutive adenines, thereby accounting for the synthesis of the full-length apoB100. Here, we demonstrate that this reading-frame restoration does not occur when the long stretch of adenines is interrupted by a cytosine. To assess whether reading-frame restoration is unique to a single site in the apoB gene, the same mutation (eight consecutive adenines in the -1 reading frame) was inserted into another site within the apoB gene. Reading-frame restoration occurred at the second site and was abrogated when the stretch of adenines was interrupted by another base. Of note, a computerized analysis of human cDNA sequences revealed that long stretches of adenines in protein-coding sequences occur at a lower than predicted frequency, suggesting that evolution has selected against these sequences.

Adenine↗
PubMed1997

Effects of genotype and diet on cholesterol efflux into plasma and lipoproteins of normal, apolipoprotein A-I-, and apolipoprotein E-deficient mice.

We investigated the contribution of apoE to cholesterol efflux into plasmas of normal, apoA-I-, and apoE-deficient mice, which were fed with chow- and cholesterol-rich diets. Plasmas of normal and apoA-I-deficient mice contain apoE in pre-beta-migrating VLDL as well as in HDL-like lipoproteins, which have either electrophoretic alpha- or gamma-mobilities. The latter particle resembled gamma-LpE in human plasma also by its mobility on nondenaturing two-dimensional electrophoresis. No apoE-containing lipoproteins were found in plasmas of apoE-deficient mice. When apoA-I- and apoE-deficient mice received both chow- and fat-rich diets, their plasmas released significantly less 3H-cholesterol from radiolabeled fibroblasts than did plasma of normal mice. Removal of apoE from plasmas of normal and apoA-I-deficient mice by anti-apoE immunoaffinity chromatography decreased their cholesterol efflux capacities (per 1 minute/per 1 hour) by 26%/40% (P = 0.0092/0.0007) and 30%/26% (P = 0.0092/0.0003), respectively. Net cholesterol efflux from fibroblasts into apoA-I-deficient plasma was 45% lower compared with plasma of normal mice. Incubation of fibroblasts with apoE-deficient plasma caused net influx of cholesterol. Prior addition of human apoE to or removal of apoB-containing lipoproteins from apoE-deficient plasma restored its ability to cause net cholesterol efflux to 50% of normal plasma. Some of the differences between cholesterol efflux into normal and apoE-deficient plasmas were attributable to the failure of apoE-deficient plasmas to take up cell-derived 3H-cholesterol into gamma-LpE. Compared with normal plasma, both apoA-I-deficient and apoE-deficient plasmas were significantly decreased in their activity to esterify cell-derived 3H-cholesterol. Anti-apoE chromatography decreased significantly cholesterol esterification in normal plasma and apoA-I-deficient plasma but not in apoE-deficient plasma. Taken together, the data provide evidence that apoE is an important contributor to reverse cholesterol transport, partially because of initial uptake of cell-derived cholesterol by gamma-LpE and partially because of the contribution of apoE-containing lipoproteins to esterification of cholesterol in plasma.

Animals↗
PubMed1996

Structure and chromosomal assignment of the murine sterol carrier protein 2 gene (Scp2) and two related pseudogenes by in situ hybridization.

The murine sterol carrier protein 2 gene (Scp2) has been mapped to chromosome 4C5-->D1 by in situ hybridization. This location is syntenic with the locus of SCP2 on human chromosome 1p32. We found the overall structure of murine Scp2 to be identical to that of the human SCP2 gene. In addition, two Scp2 pseudogenes have been isolated and mapped to chromosomes 3H2-->4 and 9F.

Animals↗
PubMed1993

Cloning, expression and sequences of mouse sterol-carrier protein-x-encoding cDNAs and a related pseudogene.

The expression of the sterol-carrier protein 2 (SCP-2)-encoding gene (SCP-2) is unusually complex. At least four SCP-2-related transcripts are detected in mouse liver: two, of 1.6 and 3.0 kb, are expressed to high levels while the other two, of 0.9 and 2.2 kb, reveal relatively low expression. Hybridization with a probe which specifically hybridizes with the rat SCP-2-related cDNA encoding rat SCP-x reveals that the 2.2- and 3.0-kb transcripts encode mouse SCP-x. SCP-x transcripts are expressed predominantly in the liver, but low-level expression can be demonstrated in all tissues analyzed. Isolation and characterization of two overlapping SCP-x cDNAs indicate that the cDNAs are derived from alternatively polyadenylated transcripts spanning approx. 2.2 and approx. 2.9 kb. Nucleotide sequencing reveals that the predicted ORF, which consists of 547 codons, is composed of 143 C-terminal amino acids which are essentially identical with mouse pre-SCP-2 and 404 N-terminal residues which are specific for SCP-x. To date, it is not clear if all SCP-2-related transcripts are transcribed from a single gene. We have isolated a genomic clone containing an SCP-2-related pseudogene which has some of the characteristics expected for a truncated processed pseudogene. Therefore, our results indicate that at least some of the multiple restriction fragments which are detected by Southern hybridization analyses with SCP-x cDNA-derived probes can be explained by cross-hybridization with a pseudogene.

Acetyl-CoA C-Acetyltransferase↗