Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1

Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the half-type ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral cells.     

Effect of Weak Acid Hypochlorous Solution on Selected Viruses and Bacteria of Laboratory Rodents

Weak acid hypochlorous solution (WAHS) is known to have efficacy for inactivating pathogens and to be relatively safe with respect to the live body. Based on these advantages, many animal facilities have recently been introducing WAHS for daily cleaning of animal houses. In this study, we determined the effect of WAHS in inactivating specific pathogens of laboratory rodents and pathogens of opportunistic infection. WAHS with an actual chloride concentration of 60 ppm and a pH value of 6.0 was generated using purpose-built equipment. One volume of mouse hepatitis virus (MHV), Sendai virus, lymphocytic choriomeningitis virus, Bordetella bronchiseptica, Pasteurella pneumotropica, Corynebacterium kutscheri, Staphylococcus aureus, and Pseudomonas aeruginosa was mixed with 9 or 99 volumes of WAHS (×10 and ×100 reaction) for various periods (0.5, 1, and 5 min) at 25°C. After incubation, the remaining infectious viruses and live bacteria were determined by plaque assay or culture. In the ×100 reaction mixture, infectious viruses and live bacteria could not be detected for any of the pathogens examined even with the 0.5-min incubation. However, the effects for MHV, B. bronchiseptica, and P. aeruginosa were variable in the ×10 reaction mixture with the 0.5- and 1-min incubations. Sufficient effects were obtained by elongation of the reaction time to 5 min. In the case of MHV, reducing organic substances in the virus stock resulted in the WAHS being completely effective. WAHS is recommended for daily cleaning in animal facilities but should be used properly in order to obtain a sufficient effect, which includes such things as using a large enough volume to reduce effects of organic substances.     

Transferrin Receptor 1 Facilitates Poliovirus Permeation of Mouse Brain Capillary Endothelial Cells

As a possible route for invasion of the CNS, circulating poliovirus (PV) in the blood is believed to traverse the blood-brain barrier (BBB), resulting in paralytic poliomyelitis. However, the underlying mechanism is poorly understood. In this study, we demonstrated that mouse transferrin receptor 1 (mTfR1) is responsible for PV attachment to the cell surface, allowing invasion into the CNS via the BBB. PV interacts with the apical domain of mTfR1 on mouse brain capillary endothelial cells (MBEC4) in a dose-dependent manner via its capsid protein (VP1). We found that F-G, G-H, and H-I loops in VP1 are important for this binding. However, C-D, D-E, and E-F loops in VP1-fused Venus proteins efficiently penetrate MBEC4 cells. These results imply that the VP1 functional domain responsible for cell attachment is different from that involved in viral permeation of the brain capillary endothelium. We observed that co-treatment of MBEC4 cells with excess PV particles but not dextran resulted in blockage of transferrin transport into cells. Using the Transwell in vitro BBB model, transferrin co-treatment inhibited permeation of PV into MBEC4 cells and delayed further viral permeation via mTfR1 knockdown. With mTfR1 as a positive mediator of PV-host cell attachment and PV permeation of MBEC4 cells, our results indicate a novel role of TfR1 as a cellular receptor for human PV receptor/CD155-independent PV invasion of the CNS.     

A unique enzyme catalyzing the formation of 4-hydroxyaniline from 4-amino-benzoic acid in Agaricus bisporus

A unique enzyme that catalyzes the formation of 4-hydroxyaniline from 4-aminobenzoic acid was found in the homogenate of Agaricus bisporus. The enzyme was prepared from the homogenate by (NH4)2SO4 fractionation, gel filtration and ion-exchange chromatography. The products formed from 4-aminobenzoic acid by the enzyme were shown to be 4-hydroxyaniline and CO2. The reaction required FAD, NAD(P)H and O2. These results indicate that the enzyme is a new FAD-dependent monooxygenase.     

Adenovirus infection induces HuR relocalization to facilitate virus replication

HuR is an RNA-binding protein of the embryonic lethal abnormal vision (ELAV) family, which binds to the AU-rich element (ARE) in the 3′-untranslated region (UTR) of certain mRNAs and is involved in the nucleo-cytoplasmic export and stabilization of ARE-mRNAs. The cytoplasmic relocalization of ARE-mRNAs with several proteins such as HuR and pp32 increases in cells transformed by the adenovirus oncogene product E4orf6. Additionally, these ARE-mRNAs were stabilized and acquired the potential to transform cells. Although, the relocalization of HuR and the stabilization of ARE-mRNAs are crucial for cell transformation, evidence regarding the relationship of HuR and ARE-mRNAs with adenovirus replication is lacking. In this report, we demonstrate that adenovirus infection induces the relocation of HuR to the cytoplasm of host cells. Analysis using the luciferase-ARE fusion gene and the tetracycline (tet)-off system revealed that the process of stabilizing ARE-mRNAs is activated in adenovirus-infected cells. Heat shock treatment or knockdown-mediated depletion of HuR reduced adenovirus production. Furthermore, expression of ARE-including viral IVa2 mRNA, decreased in HuR-depleted infected cells. These results indicate that HuR plays an important role in adenovirus replication, at least in part, by up-regulating IVa2 mRNA expression and that ARE-mRNA stabilization is required for both transformation and virus replication.     

Neural correlates of body comparison and weight estimation in weight-recovered anorexia nervosa: a functional magnetic resonance imaging study

Background

The neural mechanisms underlying body dissatisfaction and emotional problems evoked by social comparisons in patients with anorexia nervosa (AN) are currently unclear. Here, we elucidate patterns of brain activation among recovered patients with AN (recAN) during body comparison and weight estimation with functional magnetic resonance imaging (fMRI).

Methods

We used fMRI to examine 12 patients with recAN and 13 healthy controls while they performed body comparison and weight estimation tasks with images of underweight, healthy weight, and overweight female bodies. In the body comparison task, participants rated their anxiety levels while comparing their own body with the presented image. In the weight estimation task, participants estimated the weight of the body in the presented image. We used between-group region of interest (ROI) analyses of the blood oxygen level dependent (BOLD) signal to analyze differences in brain activation patterns between the groups. In addition, to investigate activation outside predetermined ROIs, we performed an exploratory whole-brain analysis to identify group differences.

Results

We found that, compared to healthy controls, patients with recAN exhibited significantly greater activation in the pregenual anterior cingulate cortex (pgACC) when comparing their own bodies with images of underweight female bodies. In addition, we found that, compared with healthy controls, patients with recAN exhibited significantly smaller activation in the middle temporal gyrus corresponding to the extrastriate body area (EBA) when comparing their own bodies, irrespective of weight, during self-other comparisons of body shape.

Conclusions

Our findings from a group of patients with recAN suggest that the pathology of AN may lie in an inability to regulate negative affect in response to body images via pgACC activation during body comparisons. The findings also suggest that altered body image processing in the brain persists even after recovery from AN.

     

Genetic Engineering of the Processing Site of D1 Precursor Protein of Photosystem II Reaction Center in Chlamydomonas reinhardtii

The D1 protein (D1) of photosystem II (PSII) reaction centeris synthesized as a precursor (pD1) and then processed at itscarboxyl terminus to establish the function of water cleavage.The amino acid sequence of the carboxyl terminal extension excisedby this process is poorly conserved except for a residue afterthe cleavage site at position of 345. We have constructed avector for site-directed mutagenesis of the chloroplast psbAgene encoding D1 of the green alga, Chlamydomonas reinhardtii.The vector enables one to transform the chloroplasts of a psbAdeletion mutant (Fud7) and directly select transformants forresistance to spectinomycin. Using this transforming vector,we have substituted Ser345 to Gly, Cys, Val and Phe in orderto investigate effects of the amino acid side chain at thisposition on the processing rate. All of the resulting transformantsexhibited the PSII activity as wild type and grew normally underphotoautotrophic conditions even under strong light where rapidturnover of Dl protein is expected to occur. Western blottinganalysis demonstrated that mature D1 accumulates in these transformantsat wild type level. Pulse and chase labeling of chloroplast-encodedproteins using [³⁵S]sulfate revealed that the processing ofD1 precursor protein occurs in all four transformants as efficientlyas in wild type, at least under the experimental conditionsexamined. The results suggest that either the amino acid sidechain at position of 345 (+1 position) is not crucial to theenzymatic cleavage of pD1 in vivo or the apparent rate of processingin vivo is not limited by the enzymatic cleavage. (Received September 22, 1995; Accepted December 25, 1995)     

Inactivation of the MouseHPRTLocus by a 203-bp Retroposon Insertion and a 55-kb Gene-Targeted Deletion: Establishment of New HPRT-Deficient Mouse Embryonic Stem Cell Lines

To obtain useful hypoxanthine phosphoribosyltransferase (HPRT)-deficient mouse ES cell lines, two different methods were employed: (i) selection of spontaneous 6-TG-resistant mutants and (ii) gene targeting of theHPRTlocus. The first approach resulted in the establishment of E14.1TG3B1, a spontaneous HPRT-deficient cell line with an insertional mutation of 203 bp in the third exon of theHPRTgene. The insert is highly homologous to the B2 mouse repetitive element and has all the expected retroposon characteristics, thus providing an example of gene inactivation by retroposon insertion. This clone exhibited stable 6-TG resistance and high germ-line transmission frequency. Thus E14.1TG3B1 is a useful ES cell line for modifying the mouse genome using theHPRTgene as a selection marker and for transmission at a high frequency into the mouse germ line. The second approach resulted in a 55-kb deletion of the mouseHPRTlocus, demonstrating the feasibility of replacement-targeting vectors to generate large genomic DNA deletions.