ABSTRACT
Monitoring of microbiological contaminants in water supplies requires fast and sensitive methods for the specific detection of indicator organisms or pathogens. We developed a protocol for the simultaneous detection of E. coli and coliform bacteria based on the Fluorescence in situ Hybridization (FISH) technology. This protocol consists of two approaches. The first allows the direct detection of single E. coli and coliform bacterial cells on the filter membranes. The second approach includes incubation of the filter membranes on a nutrient agar plate and subsequent detection of the grown micro-colonies. Both approaches were validated using drinking water samples spiked with pure cultures and naturally contaminated water samples. The effects of heat, chlorine and UV disinfection were also investigated. The micro-colony approach yielded very good results for all samples and conditions tested, and thus can be thoroughly recommended for usage as an alternative method to detect E. coli and coliform bacteria in water samples. However, during this study, some limitations became visible for the single cell approach. The method cannot be applied for water samples which have been disinfected by UV irradiation. In addition, our results indicated that green fluorescent dyes are not suitable to be used with chlorine disinfected samples.
Subject(s)
Enterobacteriaceae/isolation & purification , In Situ Hybridization, Fluorescence/methods , Colony Count, Microbial , Enterobacteriaceae/growth & development , Escherichia coli/growth & development , Escherichia coli/isolation & purification , Ultraviolet Rays , Water MicrobiologyABSTRACT
Apolipoprotein E (apoE) is a regulator of peripheral cholesterol homeostasis, and the apoE-isoform E4 is a major risk factor for the development of Alzheimer's disease (AD). Accumulating evidence suggests a key role for aberrant cholesterol metabolism in AD. We hypothesized that apoE-deficiency in mice not only affects cholesterol homeostasis in the periphery, but also in the brain, and that this can be restored by astrocyte-specific expression of human apoE3, but not apoE4. Using gas-chromatography mass-spectrometry, we found that absence of apoE in mice does not affect brain cholesterol homeostasis although serum sterol levels increase dramatically, especially when the apoE-knockout mice are fed a high fat diet. We provide evidence suggesting that apoD and the ATP-binding Cassette Transporter A1 (ABCA1) play a compensatory role in the apoE-deficient brain. Surprisingly, astrocyte-specific expression of human apoE3 or apoE4 in brains of apoE-knockout mice significantly increases brain levels of cholesterol and its precursors compared to control mice, indicative of an increased cholesterol synthesis rate in the brain. This increase is independent of the apoE-isoform, suggesting that the detrimental effect of apoE4 on the pathogenesis of AD is unlikely to be due to an apoE-isoform effect on brain cholesterol homeostasis.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Apolipoproteins D/metabolism , Apolipoproteins E/deficiency , Brain/metabolism , Cholesterol/metabolism , Sterols/metabolism , ATP Binding Cassette Transporter 1 , Animals , Apolipoprotein E3/genetics , Apolipoprotein E3/metabolism , Apolipoprotein E4/genetics , Apolipoprotein E4/metabolism , Apolipoproteins D/classification , Gas Chromatography-Mass Spectrometry/methods , Gene Expression Regulation/genetics , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/metabolismABSTRACT
Cholesterol is a prominent modulator of the integrity and functional activity of physiological membranes and the most abundant sterol in the mammalian brain. DHCR24-knock-out mice lack cholesterol and accumulate desmosterol with age. Here we demonstrate that brain cholesterol deficiency in 3-week-old DHCR24(-/-) mice was associated with altered membrane composition including disrupted detergent-resistant membrane domain (DRM) structure. Furthermore, membrane-related functions differed extensively in the brains of these mice, resulting in lower plasmin activity, decreased beta-secretase activity and diminished Abeta generation. Age-dependent accumulation and integration of desmosterol in brain membranes of 16-week-old DHCR24(-/-) mice led to the formation of desmosterol-containing DRMs and rescued the observed membrane-related functional deficits. Our data provide evidence that an alternate sterol, desmosterol, can facilitate processes that are normally cholesterol-dependent including formation of DRMs from mouse brain extracts, membrane receptor ligand binding and activation, and regulation of membrane protein proteolytic activity. These data indicate that desmosterol can replace cholesterol in membrane-related functions in the DHCR24(-/-) mouse.
Subject(s)
Aging/metabolism , Desmosterol/metabolism , Membrane Microdomains/physiology , Nerve Tissue Proteins/genetics , Oxidoreductases Acting on CH-CH Group Donors/genetics , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/biosynthesis , Amyloid beta-Protein Precursor/metabolism , Animals , Brain/metabolism , Cerebral Cortex/cytology , Cholesterol/deficiency , Fibrinolysin/metabolism , G(M1) Ganglioside/metabolism , Membrane Lipids/metabolism , Membrane Proteins/metabolism , Mice , Mice, Knockout , Neurons/metabolism , Plasminogen/metabolism , Protein Binding , Proteolipids/metabolismABSTRACT
Presenilins (PSs) are components of the gamma-secretase complex that mediates intramembranous cleavage of type I membrane proteins. We show that gamma-secretase is involved in the regulation of cellular lipoprotein uptake. Loss of gamma-secretase function decreased endocytosis of low-density lipoprotein (LDL) receptor. The decreased uptake of lipoproteins led to upregulation of cellular cholesterol biosynthesis by increased expression of CYP51 and enhanced metabolism of lanosterol. Genetic deletion of PS1 or transgenic expression of PS1 mutants that cause early-onset Alzheimer's disease led to accumulation of gamma-secretase substrates and mistargeting of adaptor proteins that regulate endocytosis of the LDL receptor. Consistent with decreased endocytosis of these receptors, PS1 mutant mice have elevated levels of apolipoprotein E in the brain. Thus, these data demonstrate a functional link between two major genetic factors that cause early-onset and late-onset Alzheimer's disease.
Subject(s)
Amyloid Precursor Protein Secretases/genetics , Brain/metabolism , Cholesterol/metabolism , Lipoproteins/metabolism , Membrane Lipids/metabolism , Receptors, LDL/metabolism , Animals , Apolipoproteins E/metabolism , Endocytosis/physiology , Female , Homeostasis/physiology , Humans , Lanosterol/metabolism , Male , Membrane Lipids/genetics , Mice , Mice, Knockout , Neurons/metabolism , Up-Regulation/physiologyABSTRACT
Filamentous members of the Bacteroidetes are commonly observed in activated sludge samples originating from both municipal and industrial wastewater treatment plants (WWTP), where they occasionally can cause bulking. Several oligonucleotide 16S rRNA-targeted probes were designed to target filaments with a needle-like appearance similar to Haliscomenobacter hydrossis. The design of these probes was based on an isolate and a sequence obtained from a micromanipulated filament. The abundance of filamentous Bacteroidetes was determined in 126 industrial samples applying already published and the newly developed probes. Small populations were found in 62 % of the WWTP investigated. However, only relatively few WWTP (13 %) contained large populations of filamentous Bacteroidetes potentially responsible for bulking incidences. The identity of the most abundant filamentous Bacteroidetes with H. hydrossis morphology could be detected by probes CFB719, SAP-309 and the newly designed probe HHY-654. A comprehensive study on the ecophysiology of probe-defined Bacteroidetes populations was conducted on Danish and Czech samples. The studies revealed that they were specialized bacteria involved in degradation of sugars, e.g. glucose and N-acetylglucosamine, and may participate in the conversion of lipopolysaccharides and peptidoglycan liberated by decaying cells. Many surface-associated exo-enzymes were excreted, e.g. chitinase, glucuronidase, esterase and phosphatase, supporting conversion of polysaccharides and possibly other released cell components. The role of filamentous bacteria with a H. hydrossis-like morphology in the activated sludge ecosystem is discussed.
Subject(s)
Bacteroidetes/classification , Bacteroidetes/genetics , Biodiversity , Sewage/microbiology , Amino Acids/metabolism , Bacteroidetes/isolation & purification , Bacteroidetes/metabolism , Carbohydrate Metabolism , DNA Primers/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Ethanol/metabolism , Fatty Acids/metabolism , In Situ Hybridization, Fluorescence , Industrial Waste , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Sequence Homology, Nucleic AcidABSTRACT
Sterol regulatory element-binding proteins (SREBPs) are transcription factors governing transcription of genes related to cholesterol and fatty acid metabolism. To become active, SREBPs must undergo a proteolytic cleavage to allow an active NH(2)-terminal segment to translocate into the nucleus. SKI-1/S1P is the first protease in the proteolytic activation cascade of SREBPs. SREBP inhibition may be useful, for example, in the treatment of liver steatosis caused by homocysteine-induced lipid synthesis. Accordingly, we overexpressed inhibitory prodomains (proSKI) of SKI-1/S1P in HepG2 cells to block SREBP activation to evaluate the potential of SKI-1/S1P in controlling cellular cholesterol synthesis. SKI-1/S1P inhibition resulted in reduced cholesterol synthesis and mRNA levels of the rate-limiting enzymes, HMG-CoA reductase and squalene epoxidase, in the cholesterol synthetic pathway. The inhibitory effect was maintained in the presence of homocysteine-induced endoplasmic reticulum stress. A gene set enrichment analysis was performed to elucidate other metabolic effects caused by SKI-1/S1P inhibition. SKI-1/S1P inhibition was observed to affect a number of other metabolic pathways, including glycolysis and citric acid cycle. These results demonstrate that inhibition of SREBPs decreases cholesterol synthesis in HepG2 cells both in the absence and presence of homocysteine. SKI-1/S1P inhibition may cause widespread changes in other key metabolic pathways.
Subject(s)
Cholesterol/metabolism , Homocysteine/metabolism , Proprotein Convertases/antagonists & inhibitors , Sterol Regulatory Element Binding Proteins/antagonists & inhibitors , Cell Line , Glycolysis , Humans , Metabolic Networks and Pathways , Oligonucleotide Array Sequence Analysis , Proprotein Convertases/pharmacology , RNA, Messenger/metabolism , Serine EndopeptidasesABSTRACT
Niemann-pick type C (NPC) disease is characterized by endosomal and lysosomal accumulation of lipids, impaired tubulovesicular trafficking, and neurodegeneration leading to premature death. Current treatment options are limited to mainly symptomatic treatments. Thus, new and efficient drug targets are needed, and therefore we performed a Gene Set Enrichment Analysis (GSEA) on NPC and healthy fibroblasts to identify globally affected pathways in NPC that could serve as targets for later drug discovery programs. Cell lines were characterized by analyzing cellular concentrations of cholesterol, its precursors and metabolites, as well as cellular plant sterol levels. Gene expression analyses were performed with Sentrix Human-8 Expression BeadChips, analyzing 23,000 transcripts. Pathway analysis of the expression data was performed using the GSEA method. Twenty-seven upregulated and 33 downregulated pathways emerged as globally affected in the GSEA analysis. These pathways included, for example, mitochondrial pathway, caspase cascade, as well as prostaglandin and leukotriene metabolism. Based on the present results and earlier published data, anti-inflammatory and antiapoptotic treatment could be beneficial in NPC.
Subject(s)
Fibroblasts/metabolism , Gene Expression Profiling , Gene Expression Regulation , Niemann-Pick Disease, Type C/genetics , Oligonucleotide Array Sequence Analysis/methods , Signal Transduction , Biomarkers/metabolism , Case-Control Studies , Cells, Cultured , Fibroblasts/cytology , Humans , Lipid Metabolism , Niemann-Pick Disease, Type C/metabolismABSTRACT
OBJECTIVE: Atherosclerotic lesions are characterized by an accumulation of inflammatory cells and lipids. Osteopontin (OPN) is a cell-binding phosphoprotein, and it seems to promote the development of atherosclerosis. The purpose of our study was to find out whether plasma levels of OPN are associated with cholesterol metabolites in plasma or tissues. METHODS AND RESULTS: Forty-three normal or mildly hypercholesterolaemic subjects, aged 31 to 69, were studied. The plasma level of OPN correlated negatively with muscle lathosterol (r = -0.52, P < 0.0001) and with the muscle lathosterol to muscle cholesterol ratio (r = -0.48, P = 0.001). Lathosterol concentrations in muscle (P = 0.003) and in relation to cholesterol (P = 0.005) were also significantly different among the OPN tertiles. OPN correlated negatively and significantly with muscle lathosterol in men (r = -0.58, P = 0.001, n = 29) but not in women (r = -0.21, P = 0.48, n = 14). Correspondingly, it also correlated negatively and significantly with the muscle lathosterol to muscle cholesterol ratio (r = -0.60, P = 0.001) in men but not in women (r = -0.13, P = 0.65). Plasma levels of OPN had a non-significant inverse correlation with plasma lathosterol and the plasma lathosterol to plasma cholesterol ratio. Plasma OPN concentrations were not related to plant sterols, cholesterol and 27-hydroxycholesterol. CONCLUSIONS: Tissue markers of cholesterol synthesis were related to plasma OPN, particularly in men. This suggests that there is interplay between OPN and cholesterol metabolism in human cells.
Subject(s)
Cholesterol/blood , Hypercholesterolemia/blood , Osteopontin/blood , Adult , Aged , Analysis of Variance , Apolipoprotein A-I/blood , Apolipoproteins B/blood , Biomarkers/blood , Cholesterol/biosynthesis , Cholesterol, HDL/blood , Cholesterol, LDL/blood , Enzyme-Linked Immunosorbent Assay , Female , Finland , Humans , Hydroxycholesterols/blood , Hypercholesterolemia/metabolism , Male , Middle Aged , Muscle, Skeletal/metabolism , Phytosterols/blood , Severity of Illness Index , Sex Factors , Triglycerides/bloodABSTRACT
Cholesterol has been implicated to play an important role in the generation of Abeta peptides, which are the main component of beta-amyloid plaques in the brains of patients suffering from Alzheimer's disease (AD). Epidemiological data implicate that lowering cholesterol levels has beneficial effects on the extent of beta-amyloid pathology. Thus therapeutic intervention using cholesterol lowering drugs like statins seems to be a promising approach. A couple of studies, in vitro or in vivo by the use of AD transgenic mouse models, focused on the manipulation of cholesterol levels and the resulting effects on Abeta generation. In contrast, there is not much known about the effect of the amyloid precursor protein (APP) on cholesterol levels. In the present report, we transfected human neuroblastoma cells with human APP695 and compared cellular cholesterol levels with the respective levels in Mock-transfected control cells. Furthermore, we determined the levels of diverse cholesterol precursors and metabolites using gas chromatography-mass spectrometry (GC-MS). Significant differences in the levels of the respective cholesterol precursors were observed, whereas inhibition of gamma-secretase activity by the gamma-secretase inhibitor DAPT did not have a significant effect on cellular cholesterol metabolism.
Subject(s)
Amyloid beta-Protein Precursor/biosynthesis , Cholesterol/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/genetics , Cell Line, Tumor , Cell Proliferation , Cholesterol/biosynthesis , Dipeptides/pharmacology , Humans , TransfectionABSTRACT
Filamentous Chloroflexi species are often present in activated sludge wastewater treatment plants in relatively low numbers, although bulking incidences caused by Chloroflexi filaments have been observed. A new species-specific gene probe for FISH was designed and using phylum-, subdivision-, morphotype 1851- and species-specific gene probes, the abundance of Chloroflexi filaments were monitored in samples from 126 industrial wastewater treatment plants from five European countries. Chloroflexi filaments were present in 50% of the samples, although in low quantities. In most treatment plants the filaments could only be identified with phylum or subdivision probes, indicating the presence of great undescribed biodiversity. The ecophysiology of various Chloroflexi filaments was investigated by a suite of in situ methods. The experiments revealed that Chloroflexi constituted a specialized group of filamentous bacteria only active under aerobic conditions consuming primarily carbohydrates. Many exo-enzymes were excreted, e.g. chitinase, glucuronidase and galactosidase, suggesting growth on complex polysaccharides. The surface of Chloroflexi filaments appeared to be hydrophilic compared to other filaments present. These results are generally supported by physiological studies of two new isolates. Based on the results obtained in this study, the potential role of filamentous Chloroflexi species in activated sludge is discussed.
Subject(s)
Chloroflexi/isolation & purification , Sewage/microbiology , Waste Management , Aerobiosis , Bacterial Proteins/classification , Bacterial Proteins/metabolism , Bacterial Typing Techniques/methods , Carbohydrate Metabolism , Chloroflexi/classification , Chloroflexi/physiology , Colony Count, Microbial , DNA Probes , Enzymes/classification , Enzymes/metabolism , Genes, Bacterial/genetics , In Situ Hybridization, Fluorescence/methods , Industrial Waste/analysis , Molecular Sequence Data , Phylogeny , Sewage/analysis , Species SpecificityABSTRACT
OBJECTIVES: CD40 is a marker of immunological activation and is expressed in the atherosclerotic lesions. We studied whether CD40 and cholesterol synthesis pathways are associated with each other. DESIGN: Forty-three subjects were randomly assigned to receive either simvastatin (n = 14), atorvastatin (n = 15), or placebo (n = 14) for eight weeks. Plasma samples were obtained before and at the end of the follow-up. sCD40 levels were measured in duplicate using an enzyme-linked immunosorbent assay. Cholesterol, its precursor lathosterol, the plant sterols campesterol and sitosterol as well as 27-hydroxycholesterol were quantified by gas-liquid chromatography-mass spectrometry. RESULTS: sCD40 was inversely correlated with the lathosterol to cholesterol ratio (r = - 0.47, p = 0.002), an indicator of cholesterol synthesis rate, as well as apolipoprotein A-I (r = - 0.38, p = 0.01) in addition to being directly correlated with 27-hydroxycholesterol (r = 0.40, p = 0.008). In multivariate linear regression analysis these three predictors explained 37% of the total variability of sCD40 levels. Simvastatin or atorvastatin treatment had no significant effect on sCD40 levels. CONCLUSION: These results indirectly suggest that sCD40 concentrations are related to cellular cholesterol levels. This may be a novel indication for the relationship between immunological processes and cholesterol metabolism.
Subject(s)
CD40 Antigens/blood , Cholesterol/blood , Cholesterol/metabolism , Hypercholesterolemia/blood , Hypercholesterolemia/metabolism , Anticholesteremic Agents/pharmacology , Atorvastatin , Biomarkers/blood , Cholesterol/pharmacology , Cholesterol, Dietary/blood , Cholesterol, Dietary/metabolism , Female , Heptanoic Acids/pharmacology , Humans , Hypercholesterolemia/drug therapy , Male , Pyrroles/pharmacology , Severity of Illness Index , Simvastatin/pharmacology , SolubilityABSTRACT
The ecophysiology of five filamentous species affiliated to the Alphaproteobacteria was investigated in industrial activated sludge systems. The five species, 'Candidatus Alysiosphaera europaea', 'Candidatus Monilibacter batavus', 'Candidatus Alysiomicrobium bavaricum', 'Candidatus Sphaeronema italicum' and Meganema perideroedes, are very abundant in industrial wastewater treatment plants and are often involved in bulking incidents. The morphology of these filamentous bacterial species resembled Eikelboom's Nostocoida limicola, or Type 021N, and could only be correctly identified by using fluorescence in situ hybridization (FISH), applying species-specific gene probes. Two physiological groupings of the five species were found using microautoradiography combined with FISH. Group 1 ('Ca. Monilibacter batavus' and 'Ca. Sphaeronema italicum') utilized many short-chained fatty acids (acetate, pyruvate and propionate), whereas Group 2 ('Ca. Alysiosphaera europaea', 'Ca. Alysiomicrobium bavaricum' and Meganema perideroedes) could also exploit several sugars, amino acids and ethanol. All species had polyhydroxyalkanoate granules present and several of the species had a very large storage capacity. No activity was found under strict anaerobic conditions, while uptake of substrate was observed in the presence of nitrate or nitrite as potential electron acceptor. However, for all species a reduced number of substrates could be consumed under these conditions compared to aerobic conditions. Only a little exo-enzymic activity was found and nearly all species had a hydrophobic cell surface. Based on knowledge of the ecophysiological potential, control strategies are suggested.
Subject(s)
Alphaproteobacteria/physiology , Sewage/microbiology , Water Microbiology , Water Purification , Aerobiosis , Alphaproteobacteria/classification , Alphaproteobacteria/cytology , Alphaproteobacteria/genetics , Amino Acids/metabolism , Anaerobiosis , Bacterial Proteins/analysis , Bacterial Typing Techniques , Carbohydrate Metabolism , Cytoplasmic Granules/chemistry , Ecosystem , Enzymes/analysis , Ethanol/metabolism , Fatty Acids/metabolism , In Situ Hybridization, Fluorescence , Nitrates/metabolism , Nitrites/metabolism , Polymers/analysisABSTRACT
Twelve strains of filamentous bacteria morphologically identified as 'Microthrix parvicella' were isolated from industrial activated sludge wastewater treatment plants. 16S rRNA gene sequences analysis showed that these strains were all closely related to 'Candidatus Microthrix parvicella'. Six of them, however, had a 16S rRNA gene similarity of only 95.7% and 96.7% to 'Candidatus Microthrix parvicella' suggesting the presence of a new species. The name 'Candidatus Microthrix calida' is proposed for this new microorganism. The physiological properties of these six isolates supported the description of a new taxon. The 'Candidatus Microthrix calida' strains produced thin filaments (0.3-0.7 microm diameter), they did not grow on the media supporting the growth of 'Candidatus Microthrix parvicella' and could be cultivated at higher temperature (up to 36.5 degrees C). Preliminary data on substrate uptake were obtained by microautoradiography on pure culture. Two new fluorescence in situ hybridization (FISH) probes, Mpa-T1-1260 specific for 'Candidatus Microthrix calida' and Mpa-all-1410 targeting both Microthrix species, were designed. The presence of Microthrix spp. was investigated in 114 activated sludge plants. 'Microthrix parvicella' morphotype was detected in 23% of the analysed samples and FISH analysis revealed that 'Candidatus Microthrix calida' was present in 5% of them. The remaining 'M. parvicella' filaments were positive with probe Mpa-all-1410 but could not all be identified as 'Candidatus Microthrix parvicella' suggesting the presence of more hitherto undescribed biodiversity within this morphotype.
Subject(s)
Actinobacteria , Manure/microbiology , Sewage/microbiology , Actinobacteria/classification , Actinobacteria/physiology , Animals , In Situ Hybridization, Fluorescence , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/classificationABSTRACT
Prion diseases are fatal and at present there are neither cures nor palliative therapies known/available, which delay disease onset or progression. Cholesterol-lowering drugs have been reported to inhibit prion replication in infected cell cultures and to modulate inflammatory reactions. We aimed to determine whether simvastatin-treatment could delay disease onset in a murine prion model. Groups of mice were intracerebrally infected with two doses of scrapie strain 139A. Simvastatin-treatment commenced 100 days postinfection. The treatment did not affect deposition of misfolded prion protein PrP(res). However, expression of marker proteins for glia activation like major histocompatibility class II and galectin-3 was found to be affected. Analysis of brain cholesterol synthesis and metabolism revealed a mild reduction in cholesterol precursor levels, whereas levels of cholesterol and cholesterol metabolites were unchanged. Simvastatin-treatment significantly delayed disease progression and prolonged survival times in established prion infection of the CNS (p < or = 0.0003). The results suggest that modulation of glial responses and the therapeutic benefit observed in our murine prion model of simvastatin is not due to the cholesterol-lowering effect of this drug.
Subject(s)
Central Nervous System Infections/drug therapy , Prion Diseases/diet therapy , Simvastatin/therapeutic use , Animals , Blotting, Western , Brain/metabolism , Brain Chemistry , Central Nervous System Infections/mortality , Cholesterol/metabolism , Disease Models, Animal , Mice , Prion Diseases/mortality , Survival RateABSTRACT
Statins inhibit endogenous cholesterol synthesis, up-regulate low-density lipoprotein (LDL) receptor expression in mammalian liver cells, and thus decrease circulating LDL-cholesterol concentrations. As cholesterol seems to play a role in the development of neurodegenerative diseases, it is of interest to evaluate the effect of high dosages of statins (eg, atorvastatin or simvastatin) on brain cholesterol metabolism. Plasma samples from 44 participants (aged 30-69 years, 16 men and 18 women) of an earlier randomized, placebo-controlled, double-blind trial, who took 40 mg atorvastatin or 80 mg simvastatin daily for 2 months, were used to analyze total cholesterol, its precursor lathosterol, and its metabolites 24(S)-hydroxycholesterol and 27-hydroxycholesterol. Despite a significant decrease in absolute plasma concentrations of oxysterols, total cholesterol, and its endogenous synthesis rate, indicated by a decreased ratio of lathosterol to cholesterol, the plasma 24(S)-hydroxycholesterol to cholesterol ratio, a surrogate marker of brain cholesterol homeostasis, remained unchanged. Short-term high-dose atorvastatin and simvastatin treatment does not seem to influence brain cholesterol metabolism in patients with moderately elevated plasma cholesterol levels.
Subject(s)
Brain/drug effects , Cholesterol/metabolism , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Hypercholesterolemia/drug therapy , Adult , Aged , Atorvastatin , Biomarkers/blood , Brain/metabolism , Cholesterol/blood , Female , Heptanoic Acids/pharmacology , Heptanoic Acids/therapeutic use , Humans , Hydroxycholesterols/blood , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Hypercholesterolemia/blood , Hypercholesterolemia/metabolism , Male , Middle Aged , Pyrroles/pharmacology , Pyrroles/therapeutic use , Randomized Controlled Trials as Topic , Simvastatin/pharmacology , Simvastatin/therapeutic useABSTRACT
BACKGROUND: Enterobacter sakazakii is a foodborne pathogen that has been associated with sporadic cases and outbreaks causing meningitis, necrotizing enterocolitis and sepsis especially in neonates. The current FDA detection method includes two enrichment steps, the subculturing of the second enrichment broth on a selective agar (VRBG), a further subculturing of selected grown colonies on TSA and the subsequent biochemical identification of yellow-pigmented colonies by API20E. However, there is a strong need for simplified methods for isolation and identification of E. sakazakii. In this study, two chromogenic media, which allow to indicate presumptive E. sakazakii colonies by the alpha glucosidase activity, as well as a newly developed 1,6-alpha-glucosidase based conventional PCR assay and a rRNA oligonucleotide probe based commercial test system for identification of presumptive E. sakazakii were evaluated on 98 target and non-target strains. The methods were compared with respect to specificity aspects. RESULTS: A total of 75 presumptive E. sakazakii and 23 non-target strains were analysed by using chromogenic media, alpha-glucosidase based PCR assay, and the VIT assay. For most presumptive E. sakazakii strains on the chromogenic media, the PCR and VIT assay confirmed the identification. However, for a number of presumptive E. sakazakii isolates from fruit powder, the alpha-glucosidase PCR and VIT assay did not correspond to the typical E. sakazakii colonies on DFI and ESIA. Further characterization by API32E identification, phylogenetic analysis of partial 16S rRNA sequences and ribotyping strongly suggested, that these strains did not belong to the species E. sakazakii. The newly developed alpha-glucosidase based PCR assay as well as the commercially available VIT Enterobacter sakazakii identification test showed an excellent correlation with the 16S rRNA data, and are thus well suited for identification of E. sakazakii. CONCLUSION: The results indicate that presumptive colonies on ESIA and DFI media need further species identification. Both evaluated molecular methods, the alpha-glucosidase PCR and the 16S RNA in situ hybridisation test (VIT), although based on completely different target regions and methodologies performed equally well in terms of specificity.
Subject(s)
Cronobacter sakazakii/isolation & purification , In Situ Hybridization/methods , Polymerase Chain Reaction , Base Sequence , Chromogenic Compounds , Cronobacter sakazakii/classification , Cronobacter sakazakii/genetics , Culture Media , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/analysis , Ribotyping , alpha-Glucosidases/genetics , alpha-Glucosidases/metabolismABSTRACT
The cholesterol-synthesizing enzyme seladin-1, encoded by the Dhcr24 gene, is a flavin adenine dinucleotide-dependent oxidoreductase and regulates responses to oncogenic and oxidative stimuli. It has a role in neuroprotection and is downregulated in affected neurons in Alzheimer's disease (AD). Here we show that seladin-1-deficient mouse brains had reduced levels of cholesterol and disorganized cholesterol-rich detergent-resistant membrane domains (DRMs). This was associated with inefficient plasminogen binding and plasmin activation, the displacement of beta-secretase (BACE) from DRMs to APP-containing membrane fractions, increased beta-cleavage of APP and high levels of Abeta peptides. In contrast, overexpression of seladin-1 increased both cholesterol and the recruitment of DRM components into DRM fractions, induced plasmin activation and reduced both BACE processing of APP and Abeta formation. These results establish a role of seladin-1 in the formation of DRMs and suggest that seladin-1-dependent cholesterol synthesis is involved in lowering Abeta levels. Pharmacological enhancement of seladin-1 activity may be a novel Abeta-lowering approach for the treatment of AD.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Brain/metabolism , Cholesterol/biosynthesis , Nerve Tissue Proteins/metabolism , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Peptide Fragments/metabolism , Receptors, Cell Surface/metabolism , Amyloid Precursor Protein Secretases , Animals , Aspartic Acid Endopeptidases , Blotting, Western , Cell Line , Cell Membrane/metabolism , Cholesterol/metabolism , DNA Primers , Endopeptidases/metabolism , Fibrinolysin/metabolism , Humans , Mice , Mice, Transgenic , Plasminogen/metabolism , Protease Nexins , Reverse Transcriptase Polymerase Chain Reaction , Statistics, NonparametricABSTRACT
Sterol regulatory element binding proteins-1 and -2 (SREBPs) are transcription factors controlling lipid homeostasis in human cells. The G-allele carriers of the SREBF-1 gene C-G polymorphism in exon 18c and coding for glycine at the protein level (G952G) have shown to associate more frequently with obesity and type 2 diabetes than the C-allele carriers. However, the C-allele has suggested to be linked to dyslipidemia. Thus, our aim was to study effect of the SREBF-1 gene polymorphism (G952G) on sterol metabolism in man. Ninety-five subjects with moderate hypercholesterolemia participated in this study and 14 homozygous CC carriers of the SREBF-1 (G952G) gene were found. Plasma lathosterol concentration and lathosterol-to-cholesterol ratio, markers of endogenous cholesterol synthesis, were significantly higher in CC homozygous subject compared to others. Similarly muscle cholesterol (p=0.045) and lathosterol (p=0.054) concentrations were elevated in the CC homozygotes supporting the view that endogenous cholesterol synthesis rate is SREBF-1 genotype-dependent.
Subject(s)
Cholesterol/biosynthesis , DNA/genetics , Hypercholesterolemia/genetics , Polymorphism, Genetic , Sterol Regulatory Element Binding Protein 1/genetics , Adult , Aged , Alleles , Biopsy , Cholesterol/blood , Female , Genetic Markers , Genotype , Humans , Hypercholesterolemia/metabolism , Isomerism , Male , Middle Aged , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Polymerase Chain Reaction , Sterol Regulatory Element Binding Protein 1/metabolismABSTRACT
On a global scale, there is an increasing tendency for a more aggressive treatment of hypercholesterolemia. Minor effects of statins on brain cholesterol metabolism have been reported in some in vivo animal studies, and it seems that this is due to a local effect of the drug. We treated male mice of the inbred strain C57/BL6 with a high daily dose of lipophilic simvastatin (100 mg/kg b.wt.) or hydrophilic pravastatin (200 mg/kg b.wt.) or vehicle (controls) by oral gavage for 3 days. To compare the impact of both statins on brain cholesterol synthesis and degradation, levels of cholesterol, its precursor lathosterol, and its brain metabolite 24(S)-hydroxycholesterol as well as statin concentrations were determined in whole-brain lipid extracts using mass spectrometry. The expression of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase mRNA and of other target genes were evaluated using real-time reverse transcription-polymerase chain reaction. In addition, analysis of liver and serum samples was performed. Similar levels of simvastatin and pravastatin were detected in whole-brain homogenates. Cholesterol contents in the brain, liver, and serum were not affected by high-dose statin treatment. Whereas brain cholesterol precursor levels were reduced in simvastatin-treated animals only, no effect was observed on the formation of the brain cholesterol metabolite, 24(S)-hydroxycholesterol. Polymerase chain reaction analysis revealed that mRNA expression of HMG-CoA reductase and ATP-binding cassette transporter A1 in the brain was significantly up-regulated in simvastatin-treated animals compared with pravastatin-treated or control animals. We conclude that, under the present experimental conditions, brain cholesterol synthesis is significantly affected by short-term treatment with high doses of lipophilic simvastatin, whereas whole-brain cholesterol turnover is not disturbed.
Subject(s)
Brain/metabolism , Cholesterol/biosynthesis , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Pravastatin/pharmacology , Simvastatin/pharmacology , Animals , Hydroxymethylglutaryl CoA Reductases/metabolism , Liver/metabolism , Male , Mice , Phytosterols/analysisABSTRACT
A large number of studies deals with the association of cholesterol and Abeta levels, however, the results are so far controversial. Whereas some studies report on increased cholesterol levels, other authors refer to an association of decreased peripheral cholesterol and the incidence of Alzheimer's disease. It is also questionable whether plasma cholesterol levels could be used as a predictive biomarker for the incidence of Alzheimer's disease. In the present report, we studied the relationship between these two parameters during aging in different transgenic mouse models of Alzheimer's disease, expressing both mutant human amyloid precursor protein and mutant human presenilin-1. Measurements of plasma cholesterol levels revealed a significant reduction in aged APP/PS1 and APP/PS1ki mice, whereas plasma levels in young and aged control mice remained almost unchanged. Furthermore, statistical analysis revealed a significant negative correlation between plasma cholesterol and brain Abeta42 levels during aging in the mice expressing both APP and PS1.