Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apolipoprotein B mRNA editing

Apolipoprotein B (apoB) mRNA editing catalyzed by apoB mRNA editing catalytic subunit 1 (APOBEC-1) has been proposed to be a nuclear process. To test this hypothesis, the subcellular distribution of hemagglutinin-(HA) tagged APOBEC-1 expressed in transiently transfected hepatoma cells was determined by indirect immunofluorescence microscopy. HA-APOBEC-1 was detected in both the nucleus and cytoplasm of rat and human hepatoma cells. Mutagenesis of APOBEC-1 demonstrated that the N-terminal 56 amino acids (1–56) were necessary for the nuclear distribution of APOBEC-1, but this region did not contain a functional nuclear localization signal (NLS). However, we identified a 24-amino acid domain in the C terminus of APOBEC-1 with characteristics of a cytoplasmic retention signal (CRS) or a nuclear export signal (NES). These data suggest, therefore, that the nuclear import of APOBEC-1 may not be mediated by a positive NLS; rather, it may be achieved by overcoming the effect of a CRS/NES. We also demonstrated that the nuclear distribution of APOBEC-1 occurred only in cell lines that were capable of editing apoB RNA. We propose that the cellular distribution of APOBEC-1 is determined by multiple domains within this protein, and a nuclear localization of the enzyme may be regulated by cell type-specific factors that render these cells uniquely editing competent.

Chromosomal arm replacement generates a high level of intraspecific polymorphism in the terminal inverted repeats of the linear chromosomal DNA of Streptomyces ambofaciens

The chromosomal DNA of the bacteria Streptomyces ambofaciens DSM40697 is an 8-Mb linear molecule that ends in terminal inverted repeats (TIRs) of 210 kb. The sequences of the TIRs are highly variable between the different linear replicons of Streptomyces (plasmids or chromosomes). Two spontaneous mutant strains harboring TIRs of 480 and 850 kb were isolated. The TIR polymorphism seen is a result of the deletion of one chromosomal end and its replacement by 480 or 850 kb of sequence identical to the end of the undeleted chromosomal arm. Analysis of the wild-type sequences involved in these rearrangements revealed that a recombination event took place between the two copies of a duplicated DNA sequence. Each copy was mapped to one chromosomal arm, outside of the TIR, and encoded a putative alternative sigma factor. The two ORFs, designated hasR and hasL, were found to be 99% similar at the nucleotide level. The sequence of the chimeric regions generated by the recombination showed that the chromosomal structure of the mutant strains resulted from homologous recombination events between the two copies. We suggest that this mechanism of chromosomal arm replacement contributes to the rapid evolutionary diversification of the sequences of the TIR in Streptomyces.

Endothelin ETA receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice

This study investigated whether endothelin-1 (ET-1), a potent vasoconstrictor, which also stimulates cell proliferation, contributes to endothelial dysfunction and atherosclerosis. Apolipoprotein E (apoE)-deficient mice and C57BL/6 control mice were treated with a Western-type diet to accelerate atherosclerosis with or without ETA receptor antagonist LU135252 (50 mg/kg/d) for 30 wk. Systolic blood pressure, plasma lipid profile, and plasma nitrate levels were determined. In the aorta, NO-mediated endothelium-dependent relaxation, atheroma formation, ET receptor-binding capacity, and vascular ET-1 protein content were assessed. In apoE-deficient but not C57BL/6 mice, severe atherosclerosis developed within 30 wk. Aortic ET-1 protein content (P < 0.0001) and binding capacity for ETA receptors was increased as compared with C57BL/6 mice. In contrast, NO-mediated, endothelium-dependent relaxation to acetylcholine (56 ± 3 vs. 99 ± 2%, P < 0.0001) and plasma nitrate were reduced (57.9 ± 4 vs. 93 ± 10 μmol/liter, P < 0.01). Treatment with the ETA receptor antagonist LU135252 for 30 wk had no effect on the lipid profile or systolic blood pressure in apoE-deficient mice, but increased NO-mediated endothelium-dependent relaxation (from 56 ± 3 to 93 ± 2%, P < 0.0001 vs. untreated) as well as circulating nitrate levels (from 57.9 ± 4 to 80 ± 8.3 μmol/liter, P < 0.05). Chronic ETA receptor blockade reduced elevated tissue ET-1 levels comparable with those found in C57BL/6 mice and inhibited atherosclerosis in the aorta by 31% without affecting plaque morphology or ET receptor-binding capacity. Thus, chronic ETA receptor blockade normalizes NO-mediated endothelial dysfunction and reduces atheroma formation independent of plasma cholesterol and blood pressure in a mouse model of human atherosclerosis. ETA receptor blockade may have therapeutic potential in patients with atherosclerosis.

Apolipoprotein E is essential for amyloid deposition in the APPV717F transgenic mouse model of Alzheimer's disease

We quantified the amount of amyloid β-peptide (Aβ) immunoreactivity as well as amyloid deposits in a large cohort of transgenic mice overexpressing the V717F human amyloid precursor protein (APPV717F+/− TG mice) with no, one, or two mouse apolipoprotein E (Apoe) alleles at various ages. Remarkably, no amyloid deposits were found in any brain region of APPV717F+/− Apoe−/− TG mice as old as 22 mo of age, whereas age-matched APPV717F +/− Apoe+/− and Apoe+/+ TG mice display abundant amyloid deposition. The amount of Aβ immunoreactivity in the hippocampus was also markedly reduced in an Apoe gene dose-dependent manner (Apoe+/+ > Apoe+/− ≫ Apoe−/−), and no Aβ immunoreactivity was detected in the cerebral cortex of APPV717F+/− Apoe−/− TG mice at any of the time points examined. The absence of apolipoprotein E protein (apoE) dramatically reduced the amount of both Aβ1–40 and Aβ1–42 immunoreactive deposits as well as the resulting astrogliosis and microgliosis normally observed in APPV717F TG mice. ApoE immunoreactivity was detected in a subset of Aβ immunoreactive deposits and in virtually all thioflavine-S-fluorescent amyloid deposits. Because the absence of apoE alters neither the transcription or translation of the APPV717F transgene nor its processing to Aβ peptide(s), we postulate that apoE promotes both the deposition and fibrillization of Aβ, ultimately affecting clearance of protease-resistant Aβ/apoE aggregates. ApoE appears to play an essential role in amyloid deposition in brain, one of the neuropathological hallmarks of Alzheimer's disease.

Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation

The structure of truncated human apolipoprotein A-I (apo A-I), the major protein component of high density lipoprotein, has been determined at 4-Å resolution. The crystals comprise residues 44–243 (exon 4) of apo A-I, a fragment that binds to lipid similarly to intact apo A-I and that retains the lipid-bound conformation even in the absence of lipid. The molecule consists almost entirely of a pseudo-continuous, amphipathic α-helix that is punctuated by kinks at regularly spaced proline residues; it adopts a shape similar to a horseshoe of dimensions 125 × 80 × 40 Å. Four molecules in the asymmetric unit associate via their hydrophobic faces to form an antiparallel four-helix bundle with an elliptical ring shape. Based on this structure, we propose a model for the structure of apo A-I bound to high density lipoprotein.

Plasmon analyses of Triticum (wheat) and Aegilops: PCR–single-strand conformational polymorphism (PCR-SSCP) analyses of organellar DNAs

To investigate phylogenetic relationships among plasmons in Triticum and Aegilops, PCR–single-strand conformational polymorphism (PCR-SSCP) analyses were made of 14.0-kb chloroplast (ct) and 13.7-kb mitochondrial (mt)DNA regions that were isolated from 46 alloplasmic wheat lines and one euplasmic line. These plasmons represent 31 species of the two genera. The ct and mtDNA regions included 10 and 9 structural genes, respectively. A total of 177 bands were detected, of which 40.6% were variable. The proportion of variable bands in ctDNA (51.1%) was higher than that of mtDNA (28.9%). The phylogenetic trees of plasmons, derived by two different models, indicate a common picture of plasmon divergence in the two genera and suggest three major groups of plasmons (Einkorn, Triticum, and Aegilops). Because of uniparental plasmon transmission, the maternal parents of all but one polyploid species were identified. Only one Aegilops species, Ae. speltoides, was included in the Triticum group, suggesting that this species is the plasmon and B and G genome donor of all polyploid wheats. ctDNA variations were more intimately correlated with vegetative characters, whereas mtDNA variations were more closely correlated with reproductive characters. Plasmon divergence among the diploids of the two genera largely paralleled genome divergence. The relative times of origin of the polyploid species were inferred from genetic distances from their putative maternal parents.

Dramatically decreased high density lipoprotein cholesterol, increased remnant clearance, and insulin hypersensitivity in apolipoprotein A-II knockout mice suggest a complex role for apolipoprotein A-II in atherosclerosis susceptibility

Apolipoprotein (apo) A-II is the second most abundant apolipoprotein in high density lipoprotein (HDL). To study its role in lipoprotein metabolism and atherosclerosis susceptibility, apo A-II knockout mice were created. Homozygous knockout mice had 67% and 52% reductions in HDL cholesterol levels in the fasted and fed states, respectively, and HDL particle size was reduced. Metabolic turnover studies revealed the HDL decrease to be due to both decreased HDL cholesterol ester and apo A-I transport rate and increased HDL cholesterol ester and apo A-I fractional catabolic rate. The apo A-II deficiency trait was bred onto the atherosclerosis-prone apo E-deficient background, which resulted in a surprising 66% decrease in cholesterol levels due primarily to decreased atherogenic lipoprotein remnant particles. Metabolic turnover studies indicated increased remnant clearance in the absence of apo A-II. Finally, apo A-II deficiency was associated with lower free fatty acid, glucose, and insulin levels, suggesting an insulin hypersensitivity state. In summary, apo A-II plays a complex role in lipoprotein metabolism, with some antiatherogenic properties such as the maintenance of a stable HDL pool, and other proatherogenic properties such as decreasing clearance of atherogenic lipoprotein remnants and promotion of insulin resistance.

A common polymorphism associated with antibiotic-induced cardiac arrhythmia

Drug-induced long QT syndrome (LQTS) is a prevalent disorder of uncertain etiology that predisposes to sudden death. KCNE2 encodes MinK-related peptide 1 (MiRP1), a subunit of the cardiac potassium channel IKr that has been associated previously with inherited LQTS. Here, we examine KCNE2 in 98 patients with drug-induced LQTS, identifying three individuals with sporadic mutations and a patient with sulfamethoxazole-associated LQTS who carried a single-nucleotide polymorphism (SNP) found in ≈1.6% of the general population. While mutant channels showed diminished potassium flux at baseline and wild-type drug sensitivity, channels with the SNP were normal at baseline but inhibited by sulfamethoxazole at therapeutic levels that did not affect wild-type channels. We conclude that allelic variants of MiRP1 contribute to a significant fraction of cases of drug-induced LQTS through multiple mechanisms and that common sequence variations that increase the risk of life-threatening drug reactions can be clinically silent before drug exposure.

Isoform-specific effects of human apolipoprotein E on brain function revealed in ApoE knockout mice: Increased susceptibility of females

Apolipoprotein E (apoE) mediates the redistribution of lipids among cells and is expressed at highest levels in brain and liver. Human apoE exists in three major isoforms encoded by distinct alleles (ɛ2, ɛ3, and ɛ4). Compared with APOE ɛ2 and ɛ3, APOE ɛ4 increases the risk of cognitive impairments, lowers the age of onset of Alzheimer’s disease (AD), and decreases the response to AD treatments. Besides age, inheritance of the APOE ɛ4 allele is the most important known risk factor for the development of sporadic AD, the most common form of this illness. Although numerous hypotheses have been advanced, it remains unclear how APOE ɛ4 might affect cognition and increase AD risk. To assess the effects of distinct human apoE isoforms on the brain, we have used the neuron-specific enolase (NSE) promoter to express human apoE3 or apoE4 at similar levels in neurons of transgenic mice lacking endogenous mouse apoE. Compared with NSE-apoE3 mice and wild-type controls, NSE-apoE4 mice showed impairments in learning a water maze task and in vertical exploratory behavior that increased with age and were seen primarily in females. These findings demonstrate that human apoE isoforms have differential effects on brain function in vivo and that the susceptibility to apoE4-induced deficits is critically influenced by age and gender. These results could be pertinent to cognitive impairments observed in human APOE ɛ4 carriers. NSE-apoE mice and similar models may facilitate the preclinical assessment of treatments for apoE-related cognitive deficits.

Opposite roles of apolipoprotein E in normal brains and in Alzheimer’s disease

We have characterized the interaction between apolipoprotein E (apoE) and amyloid β peptide (Aβ) in the soluble fraction of the cerebral cortex of Alzheimer’s disease (AD) and control subjects. Western blot analysis with specific antibodies identified in both groups a complex composed of the full-length apoE and Aβ peptides ending at residues 40 and 42. The apoE–Aβ soluble aggregate is less stable in AD brains than in controls, when treated with the anionic detergent SDS. The complex is present in significantly higher quantity in control than in AD brains, whereas in the insoluble fraction an inverse correlation has previously been reported. Moreover, in the AD subjects the Aβ bound to apoE is more sensitive to protease digestion than is the unbound Aβ. Taken together, our results indicate that in normal brains apoE efficiently binds and sequesters Aβ, preventing its aggregation. In AD, the impaired apoE–Aβ binding leads to the critical accumulation of Aβ, facilitating plaque formation.

Lamellar lipoproteins uniquely contribute to hyperlipidemia in mice doubly deficient in apolipoprotein E and hepatic lipase

Remnants of triglyceride-rich lipoproteins containing apolipoprotein (apo) B-48 accumulate in apo E-deficient mice, causing pronounced hypercholesterolemia. Mice doubly deficient in apo E and hepatic lipase have more pronounced hypercholesterolemia, even though remnants do not accumulate appreciably in mice deficient in hepatic lipase alone. Here we show that the doubly deficient mice manifest a unique lamellar hyperlipoproteinemia, characterized by vesicular particles 600 Å–1,300 Å in diameter. As seen by negative-staining electron microscopy, these lipoproteins also contain an electron-lucent region adjacent to the vesicle wall, similar to the core of typical lipoproteins. Correlative chemical analysis indicates that the vesicle wall is composed of a 1:1 molar mixture of cholesterol and phospholipids, whereas the electron-lucent region appears to be composed of cholesteryl esters (about 12% of the particle mass). Like the spherical lipoproteins of doubly deficient mice, the vesicular particles contain apo B-48, but they are particularly rich in apo A-IV. We propose that cholesteryl esters are removed from spherical lipoproteins of these mice by scavenger receptor B1, leaving behind polar lipid-rich particles that fuse to form vesicular lipoproteins. Hepatic lipase may prevent such vesicular lipoproteins from accumulating in apo E-deficient mice by hydrolyzing phosphatidyl choline as scavenger receptor B1 removes the cholesteryl esters and by gradual endocytosis of lipoproteins bound to hepatic lipase on the surface of hepatocytes.