Scavenger receptor for malondialdehyde-modified high density lipoprotein on rat sinusoidal liver cells

We report here the presence of a membrane-associated receptor which mediates endocytic uptake of malondialdehyde-modified high density lipoprotein (MDA-HDL) on sinusoidal liver cells. Binding of [125I]MDA-HDL to the cells was followed by internalization and degradation in lysosomes. The binding and lysosomal degradation of [125I]MDA-HDL were effectively inhibited by unlabeled MDA-HDL and acetyl-HDL. However, formaldehyde-treated serum albumin or low density lipoprotein modified either by acetylation or malondialdehyde, ligands known to undergo receptor-mediated endocytosis by sinusoidal liver cells, did not affect the binding of [125I]MDA-HDL to the cells. These results indicate that a receptor for MDA-HDL is described as a distinct member among the scavenger receptors for chemically modified proteins.     

Effect of catecholamine on aldosterone release in isolated rat glomerulosa cell suspensions

Effect of adrenergic activity on the adrenal steroidogenesis and the modulation by catecholamines of aldosterone release were studied in isolated rat adrenal cell suspensions. Isoproterenol, norepinephrine and epinephrine, but not dopamine, caused statistically significant increase in aldosterone release. Both prazosin (alpha 1 antagonist) and yohimbine (alpha 2 antagonist) suppressed the norepinephrine-induced aldosterone release in a dose dependent manner, respectively. Both atenolol (beta 1 antagonist) and ICI 118-551 (beta 2 antagonist) also blocked (-)-isoproterenol-induced aldosterone release in a dose dependent manner, respectively. Neither (-)-isoproterenol nor (+/-)-norepinephrine at concentrations of 10(-6) M potentiated aldosterone release stimulated by angiotensin II or ACTH. These results suggest that catecholamines stimulate aldosteroidogenesis, but it appears unlikely that aldosterone release induced by ACTH or angiotensin-II is modulated by adrenergic stimulation.     

Interaction between the replication origin and the initiator protein of the filamentous phage f1. Binding occurs in two steps

The replication initiator protein of bacteriophage f1 (gene II protein) binds to the phage origin and forms two complexes that are separable by polyacrylamide gel electrophoresis. Complex I is formed at low gene II protein concentrations, and shows protection from DNase I of about 25 base-pairs (from position +2 to +28 relative to the nicking site) at the center of the minimal origin sequence. Complex II is produced at higher concentrations of the protein, and has about 40 base-pairs (from -7 to +33) protected. On the basis of gel mobility, complex II appears to contain twice the amount of gene II protein as does complex I. The 40 base-pair sequence protected in complex II corresponds to the minimal origin sequence as determined by in-vivo analyses. The central 15 base-pair sequence (from +6 to +20) of the minimal origin consists of two repeats in inverted orientation. This sequence, when cloned into a plasmid, can form complex I, but not complex II. We call this 15 base-pair element the core binding sequence for gene II protein. Methylation interference with the formation of complex I by the wild-type origin indicates that gene II protein contacts six guanine residues located in a symmetric configuration within the core binding sequence. Formation of complex II requires, in addition to the core binding sequence, the adjacent ten base-pair sequence on the right containing a third homologous repeat. A methylation interference experiment performed on complex II indicates that gene II protein interacts homologously with the three repeats. In complex II, gene II protein protects from DNase I digestion not only ten base-pairs on the right but also ten base-pairs on the left of the sequence that is protected in complex I. Footprint analyses of various deletion mutants indicate that the left-most ten base-pairs are protected regardless of their sequence. The site of nicking by gene II protein is located within this region. A model is presented for the binding reaction involving both protein-DNA and protein-protein interactions.     

A novel actinomycete derived from wheat heads degrades deoxynivalenol in the grain of wheat and barley affected by Fusarium head blight

Deoxynivalenol (DON) is a hazardous and globally prevalent mycotoxin in cereals. It commonly accumulates in the grain of wheat, barley and other small grain cereals affected by Fusarium head blight (caused by several Fusarium species). The concept of reducing DON in naturally contaminated grain of wheat or barley using a DON-degrading bacterium is promising but has not been accomplished. In this study, we isolated a novel DON-utilising actinomycete, Marmoricola sp. strain MIM116, from wheat heads through a novel isolation procedure including an in situ plant enrichment step. Strain MIM116 had background degradation activity, and the activity was enhanced twofold by the consumption of DON. Among Tween 20, Triton X-100 and Tween 80, we selected Tween 80 as a spreading agent of strain MIM116 because it promoted DON degradation and the growth of strain MIM116 in the presence of DON. The inoculation of MIM116 cell suspension plus 0.01% Tween 80 into 1,000 harvested kernels of wheat and barley resulted in a DON decrease from approximately 3 mg kg^?1 to less than 1 mg kg^?1 of dry kernels, even when cells had only basal levels of DON-degrading activity. To the best of our knowledge, this is the first report that describes (1) the isolation of a DON-degrading bacterium from wheat heads, (2) the effects of surfactants on the biodegradation of DON and (3) the decrease of DON levels in naturally contaminated wheat and barley grain using a DON-degrading bacterium.     

Birth weight and birth rate of heavy calves conceived by transfer of in vitro or in vivo produced bovine embryos

The aim of this study was to evaluate the difference in birth weight and gestation length between Japanese Black calves obtained from transfer of bovine embryos produced in vitro (IVP) and those developed in vivo (IVD). An additional objective was to clarify the sire effect on birth weight and gestation length and to examine the birth rate of heavier calves. Two Japanese Black bulls breed at our experimental station were used as a semen source for production of IVP and IVD embryos. Thirty-eight Japanese Black heifers and cows of various genetic backgrounds were used as embryo donors for IVD embryos. Ovaries for IVP embryos were collected at random at a local slaughterhouse from Japanese Black cattle of various genetic backgrounds. IVP embryos were produced using co-culturing with cumulus cells in 5% CS+TCM 199. Both the IVD and IVP embryos were transferred non-surgically to Holstein recipients on day 7+/-1 of estrous cycle. In this study, the birth weights and gestation lengths of half-sib single calves for bull A and B were analyzed.The numbers of single calves born by transfer of IVP and IVD embryos for bull A and B were 133 and 121, 243 and 465, respectively. The birth weight of the IVP calves was significantly higher (P<0.01) than that of the IVD (bull A: 31.0+/-0.4 kg versus 27.2+/-0.4 kg and bull B: 29.9+/-0.6 kg versus 26.6+/-0.2 kg). Gestation length of the IVP calves for bull A was significantly longer (P<0.01) than that of the IVD (291.9+/-0.9 days versus 283.6+/-0.5 days). However, for bull B, there were no differences in gestation length between the IVP and IVD calves (285.9+/-0.7 days versus 286.2+/-0.3 days). These results clearly indicated that IVP calves had heavier birth weights than IVD calves but that the average gestation length of IVP calves was not always longer than that of IVD calves. Furthermore, the birth rate of heavier calves and the incidence of stillbirth and perinatal mortality up to 48 h post partum in IVP calves (bull A: 11.3%, bull B: 7.8%) were greater (P<0.05) than those in IVD calves from both bulls (bull A: 4.1%, bull B: 3.7%).     

Influences of the priming procedure and saline circulation conditions on polyvinylpyrrolidone in vitro elution from polysulfone membrane dialyzers

In hemodialysis (HD), the patient's blood is purified via circulation in an extracorporeal circuit containing a dialyzer. In the manufacturing process of polysulfone (PSu) membrane dialyzers, the membranes are hydrophilized via the addition of the hydrophilic agent polyvinylpyrrolidone (PVP) to increase their hydraulic permeability. The elution of PVP from the membrane reduces the membrane's hydraulic permeability, and the eluted PVP could cause adverse effects in the human body. Therefore, it is important to identify the factors that induce PVP elution from PSu dialyzer membranes to improve the efficiency and safety of HD. In the present study, experimental circuits connecting each of the three types of PSu membrane dialyzers that had been sterilized, using gamma irradiation, autoclaving, or in-line steam methods, were prepared. After the dialyzers were primed, saline was circulated in the circuits at a flow rate of 100 mL/min or 200 mL/min. At 0, 2, 4, 6, and 8 h after circulation was initiated, the amount of PVP eluted from the PSu membranes in vitro was determined. In this experimental setting, longer the circulation duration, greater the amount of PVP eluted from the PSu membranes of the tested dialyzers; however, the flow rate did not influence the in vitro elution of PVP. Furthermore, the immersion of the dialyzer membranes in saline for 24 h strongly facilitated the in vitro elution of PVP. In sum, these results suggest that the duration of PSu membrane incubation in saline is a determinant of the level of PVP elution from the PSu membrane dialyzers.     

Dehydroepiandrosterone increased oxidative stress in a human cell line during differentiation

Dehydroepiandrosterone (DHEA), a reversible inhibitor of glucose-6-phosphate dehydrogenase (G6PD), is increasingly taken as an antioxidative and anti-ageing supplement. This study investigated the effects of DHEA on the expression of G6PD and on the state of oxidative stress in a human promyelocytic leukaemia cell line, HL60, during the differentiation to neutrophil-like cell. This study differentiated HL60 with dimethyl sulfoxide (DMSO) in the presence (DMSO-HL60/DHEA) or absence (DMSO-HL60) of DHEA. During the differentiation, activity, mRNA and protein levels of G6PD were increased. DHEA increased these levels further. DHEA by itself suppressed the production of superoxide from DMSO-HL60 upon stimulation with phorbol myristate acetate (PMA). However, DMSO-HL60/DHEA stimulated with PMA in the absence of DHEA produced superoxide and 8-oxo-deoxyguanosine more than PMA-stimulated DMSO-HL60. After addition of H₂O₂, the ratio of reduced glutathione to oxidized glutathione was lower in DMSO-HL60/DHEA than in DMSO-HL60. These findings indicate that DHEA acts both as an antioxidant and as a pro-oxidant.     

Cholesterol transport between red blood cells and lipoproteins contributes to cholesterol metabolism in blood

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1^+/+ and Abca1^−/− mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.     

Base composition and nucleosome density in exonic and intronic regions in genes of the filamentous ascomycetes Aspergillus nidulans and Aspergillus oryzae

We sequenced nucleosomal DNA fragments of the filamentous ascomycetes Aspergillus nidulans and Aspergillus oryzae and then mapped those sequences on their genomes. We compared the GC content and nucleosome density in the exonic and intronic regions in the genes of A. nidulans and A. oryzae. Although the GC content and nucleosome density in the exonic regions tended to be higher than those in the intronic regions, the difference in the distribution of the GC content was more notable than that of the nucleosome density. Next, we compared the GC content and nucleosome density in the exonic regions of 9616 orthologous gene pairs. In both Aspergillus species, the GC content did not correlate with the nucleosome density. In addition, the Spearman's rank correlation coefficient (ρ = 0.51) between the GC content of the exonic regions of the 9616 orthologous gene pairs was higher than that (ρ = 0.31) of the nucleosome densities of A. nidulans and A. oryzae. These results strongly suggest that the GC content in the exons of the orthologous gene pairs has been conserved during evolution but the nucleosome density has varied throughout.     

Human Parainfluenza Virus Type 2 V Protein Inhibits TRAF6-Mediated Ubiquitination of IRF7 To Prevent TLR7- and TLR9-Dependent Interferon Induction

Paramyxovirus V proteins block Toll-like receptor 7 (TLR7)- and TLR9-dependent signaling leading to alpha interferon production. Our recent study has provided evidence that interaction of the V proteins with IRF7 is important for the blockade. However, the detailed mechanisms still remain unclear. Here we reexamined the interaction of the human parainfluenza virus type 2 (HPIV2) V protein with signaling molecules involved in TLR7/9-dependent signaling. Immunoprecipitation experiments in HEK293T cells transfected with V protein and one of the signaling molecules revealed that the V protein interacted with not only IRF7 but also TRAF6, IKKα, and MyD88. Whereas overexpression of TRAF6 markedly enhanced the level of V protein associating with IRF7, IKKα, and MyD88 in HEK293T cells, the level of V protein associating with TRAF6 was little affected by overexpression of IRF7, IKKα, and MyD88. Moreover, knockdown or knockout of endogenous TRAF6 in HEK293T or mouse embryonic fibroblast cells resulted in dissociation of the V protein from IRF7, IKKα, and MyD88. These results demonstrate that binding of the V protein to IRF7, IKKα, and MyD88 is largely indirect and mediated by endogenous TRAF6. It was found that the V protein inhibited TRAF6-mediated lysine 63 (K63)-linked polyubiquitination of IRF7, which is prerequisite for IRF7 activation. Disruption of the tryptophan-rich motif of the V protein significantly affected its TRAF6-binding efficiency, which correlated well with the magnitude of inhibition of K63-linked polyubiquitination and the resultant activation of IRF7. Taken together, these results suggest that the HPIV2 V protein prevents TLR7/9-dependent interferon induction by inhibiting TRAF6-mediated K63-linked polyubiquitination of IRF7.