Quantitative Trait Locus Analysis of Plasma Cholesterol and Triglyceride Levels in KK × RR F2 Mice

A previous quantitative trait locus (QTL) study on hyperlipidemia in C57BL/6J x KK-Ay/a F2 mice identified three significant cholesterol QTLs (Cq1 and Cq2 on chromosome 1, and Cq3 on chromosome 3), and a suggestive triglyceride QTL on chromosome 9. An alternative analysis of this study identified a novel cholesterol QTL on chromosome 9 (Cq4), and a significant triglyceride QTL on chromosome 9 (Tgq1). In the present study, QTL analysis was performed on KK x RR F2 mice. A significant cholesterol QTL (Cq5, lod score 5.6) was identified on chromosome 9, and a significant triglyceride QTL (Tgq2, lod score 4.7) was identified on chromosome 8. The Cq5 locus was mapped to a region similar to the Cq4 locus. On the other hand, the Tgq2 locus overlapped with the QTL region responsible for glucose intolerance (Giq1) that was identified in a previous study. The results suggest that a different combination of QTLs is involved in the trait when a different counterpart strain is used. Identification of distinct, but related traits in an identical chromosomal region will facilitate revealing the responsible gene.     

Iron Deficiency Increases Blood Cadmium Levels in Adolescents Surveyed in KNHANES 2010–2011

Discrepancies have been reported in the relationships between iron and cadmium concentrations. The distribution of blood cadmium concentrations was assessed in a representative sample of Korean adolescents participating in the Korean National Health and Nutritional Examination Survey (KNHANES) 2010–2011, and the association between blood cadmium and iron concentrations was determined. This study was based on data from KNHANES, in which a rolling sampling design was used to perform a complex, stratified, multistage probability cluster survey of a representative sample of the noninstitutionalized civilian population in South Korea. Serum ferritin was categorized as low (<15.0 μg/L), low normal (15.0–<30.0 μg/L for girls, 15.0–<50.0 μg/L for boys), or normal (≥30.0 μg/L for girls, ≥50.0 μg/L for boys), and the association between serum ferritin and blood cadmium concentrations was assessed after adjustment for various demographic and lifestyle factors. The geometric mean (GM) of blood cadmium was significantly higher among both boys and girls in the low than in the normal ferritin group. After controlling for covariates, multiple regression analysis showed that blood cadmium concentration was inversely correlated with serum ferritin concentration in both boys and girls. In conclusion, iron deficiency is associated with increased blood cadmium concentrations in a representative sample of Korean adolescents, as evaluated in KNHANES.     

Sodium Chloride Increases Aβ Levels by Suppressing Aβ Clearance in Cultured Cells

Recent studies suggest that high-salt diet is associated with cognitive decline in human and mouse. The fact that genetic factors account for less than 50% cases of sporadic Alzheimer’s disease (AD) highlights the important contribution of environmental factors, such as high-salt diet, in AD pathogenesis. However, whether and how high-salt diet fits the “amyloid cascade” hypothesis remains unexplored. Here, we show sodium chloride (NaCl) could increase Aβ levels in the medium of HEK293 cells overexpressing amyloid precursor protein (APP) or C99 fragment. NaCl treatment dose not affect APP level, gamma secretase level or activity. Instead, NaCl treatment suppresses the capacity of cells to clear Aβ and reduces Apolipoprotein E (ApoE) level. Finally, NaCl treated THP-1 or BV2 cells are inefficient in clearing Aβ when co-cultured with rat primary neurons. Our study suggests that high-salt diet may increase AD risk by directly modulating Aβ levels.     

Quantitative Trait Locus Analysis of Plasma Cholesterol and Triglyceride Levels in C57BL/6J × RR F2 Mice

A highly significant cholesterol quantitative trait locus (QTL) (Cq6) was identified on chromosome 1 in C57BL/6J x RR F2 mice. The Cq6 was located over the gene for apolipoprotein A-Il (Apoa2), and the RR allele was associated with increased plasma cholesterol. C57BL/6J has Apoa2a alleles and RR has Apoa2b alleles. Three different Apoa2 alleles are known on the basis of amino acid substitutions at four residues. Analysis with partial Apoa2 congenic strains possessing Apoa2a, Apoa2b, and Apoa2C alleles revealed that the Apoa2b allele is unique in the ability to increase cholesterol among the three Apoa2 alleles, and that the Ala-to-Val substitution at residue 61 may be crucial as far as cholesterol metabolism is concerned. We also investigated the question of whether the Apoa1 gene is responsible for the cholesterol QTLs (Cq4 and Cq5) that had been identified previously on chromosome 9 in C57BL/6J x KK-Ay/a F2 and in KK x RR F2, but not in C57BL/6J x RR F2 mice. Similar to Apoa2 alleles, three different Apoal alleles with two successive amino acid substitutions were revealed among the strains. However, we could not correlate Apoal polymorphisms with the occurrence of QTLs in these three sets of F2 mice.     

Quantification of Imatinib Plasma Levels in Patients with Chronic Myeloid Leukemia: Comparison Between HPLC–UV and LC–MS/MS

Measurement of imatinib plasma concentration can be useful to evaluate patient adherence to daily oral therapy, potential drug–drug interaction, treatment efficacy, and severe drug-related adverse events. The aim of this study was to correlate the two different blood level test methods, HPLC–UV and LC–MS/MS. We analyzed 162 plasma samples from patients treated with imatinib. We estimated the correlation between the two analytical methods on total data set and on five sets of patients grouped into different categories based on the drug-dose therapy. Finally, imatinib quantification was correlated with genetic data on the molecular response in monitoring follow-up of CML patients.     

LPS Exposure Increases Maternal Corticosterone Levels,Causes Placental Injury and Increases IL-1Β Levels in Adult Rat Offspring: Relevance to Autism

Maternal immune activation can induce neuropsychiatric disorders, such as autism and schizophrenia. Previous investigations by our group have shown that prenatal treatment of rats on gestation day 9.5 with lipopolysaccharide (LPS; 100 μg/kg, intraperitoneally), which mimics infections by gram-negative bacteria, induced autism-like behavior in male rats, including impaired communication and socialization and induced repetitive/restricted behavior. However, the behavior of female rats was unchanged. Little is known about how LPS-induced changes in the pregnant dam subsequently affect the developing fetus and the fetal immune system. The present study evaluated the hypothalamic-pituitary-adrenal (HPA) axis activity, the placental tissue and the reproductive performance of pregnant Wistar rats exposed to LPS. In the adult offspring, we evaluated the HPA axis and pro-inflammatory cytokine levels with or without a LPS challenge. LPS exposure increased maternal serum corticosterone levels, injured placental tissue and led to higher post-implantation loss, resulting in fewer live fetuses. The HPA axis was not affected in adult offspring. However, prenatal LPS exposure increased IL-1β serum levels, revealing that prenatal LPS exposure modified the immune response to a LPS challenge in adulthood. Increased IL-1β levels have been reported in several autistic patients. Together with our previous studies, our model induced autistic-like behavioral and immune disturbances in childhood and adulthood, indicating that it is a robust rat model of autism.     

VGF Peptide Profiles in Type 2 Diabetic Patients’ Plasma and in Obese Mice

To address the possible involvement of VGF peptides in obesity and diabetes, we studied type 2 diabetes (T2D) and obese patients, and high-fat diet induced obese mice. Two VGF peptides (NAPP-19 and QQET-30) were identified in human plasma by HPLC-ESI-MS. The VGF C-terminus, the above two cleaved peptides, and the TLQP-21 related peptide/s were studied using ELISA and immunohistochemistry. In euglycemic patients, plasma NAPPE and TLQP like peptides were significantly reduced with obesity (74±10 vs. 167±28, and 92±10 vs. 191±19 pmol/ml, mean+SEM, n = 10 and 6, obese vs. normal BMI, respectively, p<0.03). Upon a standard glucose load, a distinct response was shown for VGF C-terminus, TLQP and QQET-like (ERVW immunoreactive) peptides in euglycemic normal BMI patients, but was virtually abolished in euglycemic obese, and in T2D patients independently of BMI. High-fat diet induced obese mice showed reduced plasma VGF C-terminus, NAPPE and QQET-like (ERVW) peptide/s (3±0.2 vs. 4.6±0.3, 22±3.5 vs. 34±1.3, and 48±7 vs. 100±7 pmol/ml, mean+SEM, n = 8/group, obese vs. slim, respectively, p<0.03), with a loss of the response to glucose for all VGF peptides studied. In immunohistochemistry, TLQP and/or VGF C-terminus antibodies labelled VGF containing perikarya in mouse celiac ganglia, pancreatic islet cells and thin beaded nerve fibres in brown adipose tissues, with fewer in white adipose tissue. Upon the glucose load, tyrosine hydroxylase and VGF C-terminus immunoreactive axons became apparent in pancreatic islets of slim animals, but not in obese animals. Alltogether, a significant loss of VGF peptide immunoreactivity and/or their response to glucose was demonstrated in obese patients, with or without T2D, in parallel with a similar loss in high-fat diet induced obese mice. An involvement of VGF in metabolic regulations, including those of brown and/or white adipose tissues is underlined, and may point out specific VGF peptides as potential targets for diagnosis and/or treatment.     

Beam–Plasma Discharge in Space and in a Lab

Plasma Physics Reports - The process and applications of a specific type of gaseous discharge—beam–plasma discharge (BPD)—are reviewed. A brief survey of the BPD theory is...     

Effect of Animal and Industrial Trans Fatty Acids on HDL and LDL Cholesterol Levels in Humans – A Quantitative Review

Background

Trans fatty acids are produced either by industrial hydrogenation or by biohydrogenation in the rumens of cows and sheep. Industrial trans fatty acids lower HDL cholesterol, raise LDL cholesterol, and increase the risk of coronary heart disease. The effects of conjugated linoleic acid and trans fatty acids from ruminant animals are less clear. We reviewed the literature, estimated the effects trans fatty acids from ruminant sources and of conjugated trans linoleic acid (CLA) on blood lipoproteins, and compared these with industrial trans fatty acids.

Methodology/Principal Findings

We searched Medline and scanned reference lists for intervention trials that reported effects of industrial trans fatty acids, ruminant trans fatty acids or conjugated linoleic acid on LDL and HDL cholesterol in humans. The 39 studies that met our criteria provided results of 29 treatments with industrial trans fatty acids, 6 with ruminant trans fatty acids and 17 with CLA. Control treatments differed between studies; to enable comparison between studies we recalculated for each study what the effect of trans fatty acids on lipoprotein would be if they isocalorically replaced cis mono unsaturated fatty acids. In linear regression analysis the plasma LDL to HDL cholesterol ratio increased by 0.055 (95%CI 0.044–0.066) for each % of dietary energy from industrial trans fatty acids replacing cis monounsaturated fatty acids The increase in the LDL to HDL ratio for each % of energy was 0.038 (95%CI 0.012–0.065) for ruminant trans fatty acids, and 0.043 (95% CI 0.012–0.074) for conjugated linoleic acid (p = 0.99 for difference between CLA and industrial trans fatty acids; p = 0.37 for ruminant versus industrial trans fatty acids).

Conclusions/Significance

Published data suggest that all fatty acids with a double bond in the trans configuration raise the ratio of plasma LDL to HDL cholesterol.     

Effects of Murine Norovirus on Atherosclerosis in Ldlr–/– Mice Depends on the Timing of Infection

We previously reported that murine norovirus (MNV), a virus prevalent in United States research institutions, increased atherosclerotic lesion size in Ldlr^−/− mice when the mice were infected 8 wk after feeding an atherogenic diet. To determine whether the timing of MNV infection relative to atherosclerosis development altered the disease phenotype and to examine potential mechanisms by which MNV influences the disease process, we fed Ldlr^−/− mice an atherogenic diet for 16 wk. Three days after initiating the atherogenic diet, half of the mice received MNV4 and the other half vehicle only (clarified cell-culture lysate; controls). Both groups of mice developed large aortic sinus lesions (control compared with MNV4: 133 ± 8 × 10³ µm² compared with 140 ± 7 × 10³ µm²) that were not significantly different in size. Because the timing of MNV infection relative to atherosclerosis development and hypercholesterolemia differed between our previous and the current studies, we examined whether hypercholesterolemia altered MNV4-induced changes in bone-marrow–derived macrophages. MNV4 infection increased the potential of macrophages to take up and store cholesterol by increasing CD36 expression while suppressing the ABCA1 transporter. Thus, the effects of MNV4 infection on atherosclerotic lesion size appear to be dependent on the timing of the infection: MNV4 infection promotes only established lesions. This effect may be due to MNV4’s ability to increase cholesterol uptake and decrease efflux by regulating CD36 and ABCA1 protein expression.Abbreviations: ABCA1, ATP-binding cassette A1; BMDM, bone-marrow–derived macrophage; iNOS, inducible nitric oxide synthase; MNV, murine norovirusChronic viral infection, such as occurs with HIV and hepatitis C virus, has been associated with an increased risk for atherosclerosis,^2,19,46,48 although the mechanisms by which this occurs are not clearly defined. Our laboratory has been studying the effects of murine norovirus (MNV), which chronically infects immunocompetent mice, on murine models of inflammatory diseases, including atherosclerosis. MNV is a single-stranded RNA intestinal virus that belongs to the family Caliciviridae and has shown tropism toward antigen-presenting cells such as dendritic cells and macrophages.⁵⁴ Whereas human norovirus is a major cause of nonbacterial acute gastroenteritis,⁵² MNV does not cause clinical disease in immunocompetent mice.⁵⁵ However, the high prevalence of MNV in biomedical research facilities throughout the world,^42,55 combined with its tropism for antigen-presenting cells, has prompted concern regarding potential effects on disease phenotypes in murine models of human diseases. Therefore, we previously examined 2 diseases, obesity and atherosclerosis, where macrophages have critical roles.^41,42 We found that MNV infection did not influence glucose metabolism and weight gain,⁴¹ but it significantly increased the size and macrophage content of aortic sinus lesions in Ldlr^−/− mice fed an atherogenic diet.⁴² These findings suggest that MNV might be a potential tool to determine how viral infection alters the risk of atherosclerosis.Many factors influence the progression of atherosclerosis. Accordingly, we examined whether the timing of MNV infection relative to the stage of atherosclerosis progression influenced disease phenotype and evaluated potential mechanisms by which MNV could affect the disease process. To this end, we modeled the infection of macrophages by using in vitro cultures of bone-marrow–derived macrophages (BMDM).     

A fish oil-rich diet reduces vascular oxidative stress in apoE–/– mice

Abstract

Oxidative stress contributes to lipid peroxidation and decreases nitric oxide (NO) bioavailability in atherosclerosis. While long-chain (n-3) polyunsaturated fatty acids (PUFA) are easily oxidized in vitro, they improve endothelial function. Hence, this study postulates that long-chain (n-3) PUFA decrease atherogenic oxidative stress in vivo. To test this, apoE^–/– mice were fed a corn oil- or a fish oil (FO)-rich diet for 8, 14 or 20 weeks and parameters related to NO and superoxide (O₂^.–) plus markers of lipid peroxidation and protein oxidative damage in the aortic root were evaluated. The FO-rich diet increased NO production and endothelial NO synthase (NOS) expression and lowered inducible NOS, p22^phox expression and O₂^.–production after 14 and 20 weeks of diet. Protein lipoxidative damage (including 4-hydroxynonenal) was decreased after a long-term FO-diet. This supports the hypothesis that a FO-rich diet could counteract atherogenic oxidative stress, showing beneficial effects of long-chain (n-3) PUFA.     

Expression of Bacterial l-aspartate-α-decarboxylase in Tobacco Increases β-Alanine and Pantothenate Levels and Improves Thermotolerance

l-Aspartate-α-decarboxylase catalyzes the decarboxylation of l-aspartate to generate β-alanine and carbon dioxide. This is an unusual pyruvoyl-dependent enzyme unique to prokaryotes that undergoes limited self-processing. The Escherichia coli panD gene encoding l-aspartate-α-decarboxylase was expressed under a constitutive promoter in transgenic tobacco. Transgene expression was verified by assays based on RNA blots, immunoblots and enzyme activity in vitro. The panD lines had increased levels of leaf β-alanine (1.2- to 4-fold), pantothenate (3.2- to 4.1-fold) and total free amino acids (up to 3.7-fold) compared to wild-type and vector controls. Growth of homozygous lines expressing E. coli l-aspartate-α-decarboxylase was less affected than that of the control lines when the plants were stressed for 1 week at 35 °C. When transferred from 35 to 30 °C for 3 weeks, the PanD transgenic lines recovered significantly (P ≤ 0.001) better than the controls: PanD lines had on an average 54% and 84% greater fresh and dry weights respectively, compared to the controls. Homozygous lines expressing E. coli l-aspartate-α-decarboxylase had significantly greater thermotolerance (P ≤ 0.05) during germination. At 42 °C, 95% of two T3 PanD transgenic line seeds germinated after 12 days compared to 73% for the wild-type seeds. Our results indicated that E. colil-aspartate-α-decarboxylase was correctly processed and active in the transgenic eukaryotic host and its expression resulted in increased thermotolerance in tobacco. This is Florida Agricultural Experiment Station journal series number R-10355. W.M.F. was supported by the Egypt Development Training fellowship and by the UF College of Agriculture and Life Sciences assistantship.     

Apolipoprotein A-I Deficiency Increases Cerebral Amyloid Angiopathy and Cognitive Deficits in APP/PS1ΔE9 Mice

A hallmark of Alzheimer disease (AD) is the deposition of amyloid β (Aβ) in brain parenchyma and cerebral blood vessels, accompanied by cognitive decline. Previously, we showed that human apolipoprotein A-I (apoA-I) decreases Aβ₄₀ aggregation and toxicity. Here we demonstrate that apoA-I in lipidated or non-lipidated form prevents the formation of high molecular weight aggregates of Aβ₄₂ and decreases Aβ₄₂ toxicity in primary brain cells. To determine the effects of apoA-I on AD phenotype in vivo, we crossed APP/PS1ΔE9 to apoA-I^KO mice. Using a Morris water maze, we demonstrate that the deletion of mouse Apoa-I exacerbates memory deficits in APP/PS1ΔE9 mice. Further characterization of APP/PS1ΔE9/apoA-I^KO mice showed that apoA-I deficiency did not affect amyloid precursor protein processing, soluble Aβ oligomer levels, Aβ plaque load, or levels of insoluble Aβ in brain parenchyma. To examine the effect of Apoa-I deletion on cerebral amyloid angiopathy, we measured insoluble Aβ isolated from cerebral blood vessels. Our data show that in APP/PS1ΔE9/apoA-I^KO mice, insoluble Aβ₄₀ is increased more than 10-fold, and Aβ₄₂ is increased 1.5-fold. The increased levels of deposited amyloid in the vessels of cortices and hippocampi of APP/PS1ΔE9/apoA-I^KO mice, measured by X-34 staining, confirmed the results. Finally, we demonstrate that lipidated and non-lipidated apoA-I significantly decreased Aβ toxicity against brain vascular smooth muscle cells. We conclude that lack of apoA-I aggravates the memory deficits in APP/PS1ΔE9 mice in parallel to significantly increased cerebral amyloid angiopathy.     

Pyometra in Bitches Induces Elevated Plasma Endotoxin and Prostaglandin F2α Metabolite Levels

   

Endotoxemia in bitches with pyometra can cause severe systemic effects directly or via the release of inflammatory mediators. Plasma endotoxin concentrations were measured in ten bitches suffering from pyometra with moderately to severely deteriorated general condition, and in nine bitches admitted to surgery for non-infectious reasons. Endotoxin samples were taken on five occasions before, during and after surgery. In addition, urine and uterine bacteriology was performed and hematological, blood biochemical parameters, prostaglandin F_2α metabolite 15-ketodihydro-PGF_2α (PG-metabolite), progesterone and oestradiol (E₂-17β) levels were analysed.     

Nur77 Decreases Atherosclerosis Progression in apoE?/? Mice Fed a High-Fat/High-Cholesterol Diet

Rationale

It is clear that lipid disorder and inflammation are associated with cardiovascular diseases and underlying atherosclerosis. Nur77 has been shown to be involved in inflammatory response and lipid metabolism.

Objective

Here, we explored the role of Nur77 in atherosclerotic plaque progression in apoE^−/− mice fed a high-fat/high cholesterol diet.

Methods and Results

The Nur77 gene, a nuclear hormone receptor, was highly induced by treatment with Cytosporone B (Csn-B, specific Nur77 agonist), recombinant plasmid over-expressing Nur77 (pcDNA-Nur77), while inhibited by treatment with siRNAs against Nur77 (si-Nur77) in THP-1 macrophage-derived foam cells, HepG2 cells and Caco-2 cells, respectively. In addition, the expression of Nur77 was highly induced by Nur77 agonist Csn-B, lentivirus encoding Nur77 (LV-Nur77), while silenced by lentivirus encoding siRNA against Nur77 (si-Nur77) in apoE^−/− mice fed a high-fat/high cholesterol diet, respectively. We found that increased expression of Nur77 reduced macrophage-derived foam cells formation and hepatic lipid deposition, downregulated gene levels of inflammatory molecules, adhesion molecules and intestinal lipid absorption, and decreases atherosclerotic plaque formation.

Conclusion

These observations provide direct evidence that Nur77 is an important nuclear hormone receptor in regulation of atherosclerotic plaque formation and thus represents a promising target for the treatment of atherosclerosis.     

Development of a Highly Sensitive Immuno-PCR Assay for the Measurement of α-Galactosidase A Protein Levels in Serum and Plasma

Fabry disease is an X-linked genetic disorder caused by defects in the α-galactosidase A (GLA) gene, and heterogeneous mutations lead to quantitative and/or qualitative defects in GLA protein in male patients with Fabry disease. Random X-chromosomal inactivation modifies the clinical and biochemical features of female patients with Fabry disease. Functional polymorphisms have been frequently reported in recent times, and these increase the difficulty of understanding the pathogenetic basis of the disease. To date, GLA protein level has been measured using an enzyme-linked immunosorbent assay (ELISA). However, ELISA is not highly sensitive due to the high background noise. In this paper, we introduce a novel application of the immuno-polymerase chain reaction (PCR) method (termed Multiple Simultaneous Tag [MUSTag]) for measurement of the GLA protein level in blood samples. We compared the sensitivities of the MUSTag method with plates or magnetic beads with those of ELISA for recombinant human GLA and found that the apparent maximal sensitivity was higher for the former than for the latter. We then measured the GLA concentrations in serum and plasma from male patients with classic Fabry disease (Male Fabry), females with Fabry disease (Female Fabry), male subjects harboring the functional polymorphism p.E66Q (E66Q), and control (Control) subjects. Our results revealed that compared to the MUSTag plate and ELISA, the MUSTag beads assay afforded a clearer estimation of the GLA protein levels in the serum and plasma with minimal or no background noise, although all the methods could differentiate between the Male Fabry, E66Q, and Control groups. The Female Fabry group showed characteristic heterogeneity, which was consistent with the X-linked inheritance. This novel method is expected to be useful for the sensitive determination of GLA level in blood and elucidation of the pathogenetic basis of Fabry disease.