Apolipoprotein E levels in cerebrospinal fluid and the effects of ABCA1 polymorphisms

Background

Animal studies suggest that brain apolipoprotein E (apoE) levels influence amyloid-β (Aβ) deposition and thus risk for Alzheimer's disease (AD). We have previously demonstrated that deletion of the ATP-binding cassette A1 transporter (ABCA1) in mice causes dramatic reductions in brain and cerebrospinal fluid (CSF) apoE levels and lipidation. To examine whether polymorphisms in ABCA1 affect CSF apoE levels in humans, we measured apoE in CSF taken from 168 subjects who were 43 to 91 years old and were either cognitively normal or who had mild AD. We then genotyped the subjects for ten previously identified ABCA1 single nucleotide polymorphisms (SNPs).

Results

In all subjects, the mean CSF apoE level was 9.09 μg/ml with a standard deviation of 2.70 μg/ml. Levels of apoE in CSF samples taken from the same individual two weeks apart were strongly correlated (r² = 0.93, p < 0.01). In contrast, CSF apoE levels in different individuals varied widely (coefficient of variation = 46%). CSF apoE levels did not vary according to AD status, APOE genotype, gender or race. Average apoE levels increased with age by ~0.5 μg/ml per 10 years (r² = 0.05, p = 0.003). We found no significant associations between CSF apoE levels and the ten ABCA1 SNPs we genotyped. Moreover, in a separate sample of 1225 AD cases and 1431 controls, we found no association between the ABCA1 SNP rs2230806 and AD as has been previously reported.

Conclusion

We found that CSF apoE levels vary widely between individuals, but are stable within individuals over a two-week interval. AD status, APOE genotype, gender and race do not affect CSF apoE levels, but average CSF apoE levels increase with age. Given the lack of association between CSF apoE levels and genotypes for the ABCA1 SNPs we examined, either these SNPs do not affect ABCA1 function or if they do, they do not have strong effects in the CNS. Finally, we find no evidence for an association between the ABCA1 SNP rs2230806 and AD in a large sample set.     

Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.     

Extracellular polysaccharides of a copper-sensitive and a copper-resistant Pseudomonas aeruginosa strain: synthesis,chemical nature and copper binding

Extracellular polysaccharides (EPS) of a copper-sensitive (Cu^s) and a copper-resistant (Cu^r) Pseudomonas aeruginosa strain were investigated in terms of their production, chemical nature and response towards copper exposure. The extent of EPS synthesis by the resistant strain (4.78 mg mg^–1 cell dry wt.) was considerably higher over its sensitive counterpart (2.78 mg mg^–1 dry wt.). FTIR-spectroscopy and gas chromatography revealed that both the polymers were acidic in nature, containing alginate as the major component along with various neutral- and amino-sugars. Acid content in the Cu^r EPS (480.54 mg g^–1) was greater than that in the Cu^s EPS (442.0 mg g^–1). Presence of Cu²⁺ in the growth medium caused a dramatic stimulation (approximately 4-fold) in EPS synthesis by the Cu^r strain, while in a similar condition, the Cu^s failed to exhibit such response. The polymer of the resistant strain showed elevated Cu²⁺ binding (320 mg g^–1 EPS) compared to that of the sensitive type (270 mg g^–1). The overall observations show the potential of the Cu^r EPS for its deployment in metal bioremediation.     

Nickel Uptake by Pseudomonas aeruginosa: Role of Modifying Factors

Pseudomonas aeruginosa cells growing in minimal medium were 40-fold more sensitive to Ni²⁺ than cells growing in enriched medium, suggesting a possible protective role of medium ingredients. Likewise, cells pre-grown in enriched medium showed a high K _m (6.15 mM) and increased Ni²⁺ uptake (950 nmol mg⁻¹ protein, 1h) over cells pre-sown in minimal medium (K _m , 0.48 mM; 146 nmol mg⁻¹ protein, 1 h). The overall pattern indicates that cells pre-grown in enriched medium were characterized by having lowered affinity towards Ni²⁺ than those with minimal medium background. The enhanced Ni²⁺ uptake by enriched medium-grown cells can be correlated with the improved metabolic state of the cells. Ni²⁺ uptake was optimum at neutrality (pH 7.0). A major Ni²⁺ transport system was competitively inhibited by Mg²⁺, Zn²⁺, Cd²⁺, or Co²⁺ (400 μM each). Noticeably, a minor Ni²⁺ transport pathway was still operative even in the higher concentration range of Mg²⁺ (4 mM and 40 mM). The stimulation of Ni²⁺ uptake monitored in the presence of different carbon sources (0.5% wt/vol, each) showed the sequence: glucose (1.6-fold) > phenol = gallic acid (1.5-fold). Succinate, in comparison, reduced Ni²⁺ uptake (0.5-fold) possibly because of its acting as a metal chelator as well. Sensitivity of Ni²⁺ transport towards methyl viologen, azide, 2-4 DNP, and DCCD suggested that transport was energy-linked. Received: 13 January 1998 / Accepted: 21 May 1998     

A study to determine the impact of IMPT optimization techniques on prostate synthetic CT image sets dose comparison against CT image sets

BackgroundThe objective of this study is to determine the impact of intensity modulated proton therapty (IMPT) optimization techniques on the proton dose comparison of commercially available magnetic resonance for calculating attenuation (MRCA T) images, a synthetic computed tomography CT (sCT) based on magnetic resonance imaging (MRI) scan against the CT images and find out the optimization technique which creates plans with the least dose differences against the regular CT image sets.Material and methodsRegular CT data sets and sCT image sets were obtained for 10 prostate patients for the study. Six plans were created using six distinct IMPT optimization techniques including multi-field optimization (MFO), single field uniform dose (SFUD) optimization, and robust optimization (RO) in CT image sets. These plans were copied to MRCA T, sCT datasets and doses were computed. Doses from CT and MRCA T data sets were compared for each patient using 2D dose distribution display, dose volume histograms (DVH), homogeneity index (HI), conformation number (CN) and 3D gamma analysis. A two tailed t-test was conducted on HI and CN with 5% significance level with a null hypothesis for CT and sCT image sets.ResultsAnalysis of ten CT and sCT image sets with different IMPT optimization techniques shows that a few of the techniques show significant differences between plans for a few evaluation parameters. Isodose lines, DVH, HI, CN and t-test analysis shows that robust optimizations with 2% range error incorporated results in plans, when re-computed in sCT image sets results in the least dose differences against CT plans compared to other optimization techniques. The second best optimization technique with the least dose differences was robust optimization with 5% range error.ConclusionThis study affirmatively demonstrates the impact of IMPT optimization techniques on synthetic CT image sets dose comparison against CT images and determines the robust optimization with 2% range error as the optimization technique which gives the least dose difference when compared to CT plans.     

A lipopolysaccharide mutant of Bradyrhizobium japonicum that uncouples plant from bacterial differentiation.

The Tn5-containing fragment from a non-nodulating mutant of Bradyrhizobium japonicum, strain ML142, was introduced into B. japonicum strain 61A101c by marker exchange to construct strain JS314. Strain JS314 failed to nodulate several soybean varieties tested. However, on a few varieties nodulelike structures were induced to a frequency of 54% of the plants inoculated. The ultrastructure of these nodules was studied in detail by light and electron microscopy. The nodules were devoid of internal bacteria, possessed central vascular tissue (unlike the lateral vascular tissue of a normal nodule), and exhibited localized cell death of epidermal cells. Study of the cell surface polysaccharides of strain JS314 revealed that the exopolysaccharide of this strain was identical to that of the wild type. However, the lipopolysaccharide (LPS) of strain JS314 showed gross differences from that isolated from the wild-type strain. Specifically, the LPS of strain JS314 appeared to lack the high molecular weight LPS I form, strongly suggesting that the LPS lacks the O-chain. Glycosyl-composition analysis showed that the LPS of mutant JS314 lacked 2,3-di-O-methylrhamnose, 3-O-methylrhamnose, fucose, and quinovosamine. These results indicate that LPS I in B. japonicum is essential for bacterial infection of soybean, but is not required to initiate plant cortical cell division, an early plant response to infection.     

The effects of different decalcification protocols on TUNEL and general cartilage staining

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.     

Relationships between lake ecology and morphological characters in Icelandic Arctic charr,Salvelinus alpinus

The common occurrence of parallel phenotypic patterns suggests that a strong relationship exists between ecological dynamics and micro‐evolution. Comparative studies from a large number of populations under varying sets of ecological drivers could contribute to a better understanding of this relationship. We used data on morphology of arctic charr (Salvelinus alpinus) and ecological factors from 35 Icelandic lakes to test the hypothesis that morphological patterns among monomorphic charr populations from different lakes are related to interlake variation in ecological characteristics. There is extensive phenotypic diversity among populations of Icelandic charr, and populations are easily distinguished based on overall body morphology. The results obtained in the present study showed that the morphological diversity of charr was related to large‐scale diversity in lake ecology. Variation in charr morphology was related to water origin (e.g. spring fed versus run‐off), bedrock age, and fish community structure. The present study shows how various ecological factors can shape the biological diversity that we observe. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 761–771.     

Molecular evolution of a multigene family in group A streptococci

The emm genes are members of a gene family in group A streptococci (GAS) that encode for antiphagocytic cell-surface proteins and/or immunoglobulin-binding proteins. Previously sequenced genes in this family have been named "emm," "fcrA," "enn," "arp," "protH," and "mrp"; herein they will be referred to as the "emm gene family." The genes in the emm family are located in a cluster occupying 3-6 kb between the genes mry and scpA on the chromosome of Streptococcus pyogenes. Most GAS strains contain one to three tandemly arranged copies of emm-family genes in the cluster, but the alleles within the cluster vary among different strains. Phylogenetic analysis of the conserved sequences at the 3' end of these genes differentiates all known members of this family into four evolutionarily distinct emm subfamilies. As a starting point to analyze how the different subfamilies are related evolutionarily, the structure of the emm chromosomal region was mapped in a number of diverse GAS strains by using subfamily-specific primers in the polymerase chain reaction. Nine distinct chromosomal patterns of the genes in the emm gene cluster were found. These nine chromosomal patterns support a model for the evolution of the emm gene family in which gene duplication followed by sequence divergence resulted in the generation of four major-gene subfamilies in this locus.