Mitochondrial phospholipid hydroperoxide glutathione peroxidase suppresses apoptosis mediated by a mitochondrial death pathway.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a key enzyme in the protection of biomembranes exposed to oxidative stress. We investigated the role of mitochondrial PHGPx in apoptosis using RBL2H3 cells that overexpressed mitochondrial PHGPx (M15 cells), cells that overexpressed non-mitochondrial PHGPx (L9 cells), and control cells (S1 cells). The morphological changes and fragmentation of DNA associated with apoptosis occurred within 15 h in S1 and L9 cells upon exposure of cells to 2-deoxyglucose (2DG). The release of cytochrome c from mitochondria was observed in S1 cells after 4 h and was followed by the activation of caspase-3 within 6 h. Overexpression of mitochondrial PHGPx prevented the release of cytochrome c, the activation of caspase-3, and apoptosis, but non-mitochondrial PHGPx lacked the ability to prevent the induction of apoptosis by 2DG. An ability to protect cells from 2DG-induced apoptosis was abolished when the PHGPx activity of M15 cells was inhibited by diethylmalate, indicating that the resistance of M15 cells to apoptosis was indeed due to the overexpression of PHGPx in the mitochondria. The expression of members of the Bcl-2 family of proteins, such as Bcl-2, Bcl-xL, Bax, and Bad, was unchanged by the overexpression of PHGPx in cells. The levels of hydroperoxides, including hydrogen and lipid peroxide, in mitochondria isolated from S1 and L9 cells were significantly increased after the exposure to 2DG for 2 h, while the level of hydroperoxide in mitochondria isolated from M15 cells was lower than that in S1 and L9 cells. M15 cells were also resistant to apoptosis induced by etoposide, staurosporine, UV irradiation, cycloheximide, and actinomycin D, but not to apoptosis induced by Fas-specific antibodies, which induces apoptosis via a pathway distinct from the pathway initiated by 2DG. Our results suggest that hydroperoxide, produced in mitochondria, is a major factor in apoptosis and that mitochondrial PHGPx might play a critical role as an anti-apoptotic agent in mitochondrial death pathways.     

Changes in plasma thyroid hormones, luteinizing hormone (LH), estradiol, progesterone and corticosterone of laying hens during a forced molt

1. Circulating concentrations of iodothyronines, luteinizing hormone(LH), estradiol(E2), progesterone and corticosterone were measured in hens before, during, and after a forced molt induced by fasting. 2. Corticosterone increased at the onset of molt, peaked at the maximal molt and returned to pre- and post-molt levels. LH, E2 and progesterone declined during the molt, and the decline was coincident with the cessation of egg production. 3. Thyroxine(T4), triiodothyronine (T3) and reverse triiodothyronine(rT3) increased during the molt. The increases of T4 and T3 were not abolished even if the forced molt was conducted in mild weather.     

Evaluation of the antimicrobial activity of methylglyoxal bis(guanylhydrazone) analogues, the inhibitors for polyamine biosynthetic pathway

Metabolic and antiproliferative effects of methylglyoxal bis(butylamidinohydrazone) (MGBB) and methylglyoxal bis(cyclopentylamidinohydrazone) (MGBCP), inhibitors for polyamine biosynthetic pathway, on Escherichia coli, Shigella sonnei, Aeromonas sobria, Aeromonas hydrophila and Vibrio cholerae were investigated. MGBB at the concentration of 100 mumol/l depleted intracellular putrescine and spermidine concentrations of E. coli to 25 and 20% of the controls, respectively, while MGBCP depressed their concentrations to 38 and 24%, respectively. In these polyamine-depleted E. coli cells the syntheses of RNA, DNA and protein decreased to 13, 54 and 29% of the control, respectively, with MGBB and to 23, 71 and 55%, respectively, with MGBCP. The minimum inhibitory concentrations (MIC) of MGBB for the growth of A. sobria, E. coli, A. hydrophila, V. cholerae and Sh. sonnei were estimated to be 50, 160, 240, 285 and 320 mumol/l, respectively, whereas those of MGBCP were slightly higher for respective bacteria.     

Solid-state modifications of poly(γ-ethyl-L-glutamate)

Several modification of the arrangements of α-helical molecules were found in the solid films of poly (γ-ethyl-L -glutamate), depending on the casting solvent and the temperature. The helical conformation is somewhat looser than the normal 18-residue, 5-turn α-helix. Using x-ray diffraction, the types of molecular arrangements were classified into tetragonal, pseudohexagonal, and hexagonal ones. Tetragonal packing was observed in the filmm (form T) prepared by casting the solution in trifluorethanol or dichlorethane. The sample obtained from chloroform solution is a well-ordered, pseudohexagonal modification (form I). Forms I and T change into a poorly crystalline form III by annealing at temperatures above 130° C. It is particularly noteworthy that the less-ordered form III exhibits a thermoreversible transition around 110°C into a well-ordered form H with the hexagonal molecular packing.     

A macrophage receptor for liver lysosomal beta-glucosidase

beta-Glucosidase was purified from lysosomal membranes isolated from rat liver. Binding and uptake of the purified beta-glucosidase were mediated via an apparently single binding site on rat peritoneal macrophages. The number of sites and the Kd were 4.20 X 10(4)/cell and 1.00 X 10(-7) M, respectively. Neither of the processes was inhibited by ligands for mannose/fucose receptors, mannose 6-phosphate receptors, or scavenger receptors, or by other glycoproteins and sugar compounds. A portion of the beta-glucosidase taken up into the macrophages was degraded rapidly. These results suggested that liver lysosomal beta-glucosidase was endocytosed via a receptor not previously described.     

Alcohol dehydrogenase isozymes in the mouse: Genetic regulation, allelic variation among inbred strains and sex differences of liver and kidney A2 isozyme activity

Genetic analysis of a proposed cis-acting temporal locus ( Adh-3t ), which regulates alcohol dehydrogenase C₂ (ADH-C₂) acitivity in mouse epididymis extracts, among F₁ (ddN × BALB/c) × ddN male backcross progeny provided evidence for genetic distinctness between the structural ( Adh-3 ) and temporal ( Adh-3t ) loci on chromosome 3. Genetic analysis also confirmed the close, linkage of Adh-1 (encoding liver and kidney ADH-A₂) and Adh-3 (encoding stomach ADH-C₂) to within 0.3 centimorgans on the mouse genome. Evidence is presented for a proposed closely linked cis-acting temporal locus (designated Adh-1t ) for the A₂ isozyme (encoded by Adh-1 ) controlling the activity of this enzyme in mouse kidney extracts, but having no apparent affect on liver and intestine ADH-A₂ activities. An extensive survey of the distribution of Adh-1, Adh-3 and Adh-3t alleles among 65 strains of mice is reported — with the exception of two Japanese strains (ddN and KF), linkage disequilibrium between Adh-3 and Adh-3t was observed. Sex differences in mouse liver and kidney ADH-A₂ activities were observed, with male/female ratios of approximately 0.6 and 3 respectively for these tissue extracts.     

Polyclonal Antibody Production in Murine Spleen Cells Induced by Staphylococcus

Polyclonal plaque-forming cell (PFC) responses in murine spleen cells induced by Staphylococcus aureus and S. epidermidis were studied. Injection of Balb/c mice with S. aureus strain 248βH resulted in the generation of anti-trinitrophenyl (TNP) and anti-sheep red blood cell PFC in their spleens. Cultures of Balb/c spleen cells in the presence of S. aureus 248βH, Cowan I, or a protein A-deficient mutant yielded many anti-TNP PFC. The larger the number of organisms that were added to the cultures, the better was the PFC response. Both living and killed organisms, were capable of inducing the response, but an excess of living 248βH organisms in the cultures abrogated the response. All of the organisms (12 strains of S. aureus and 11 strains of S. epidermidis) freshly isolated from patients had the ability to induce the polyclonal PFC response in cell cultures. These organisms stimulated cultured C3H/HeJ mouse spleen cells, which were unresponsive to bacterial lipopolysaccharide (LPS). Cultured cells from the spleens of athymic nu/nu mice also responded to these organisms, and the number of PFC in nu/nu cell cultures was always greater than that in nu/+ cells prepared from a haired litter mate. Moreover, the responses of nu/nu spleen cell cultures to which nylon wool column-filtered splenic nu/+ T cells were added were lower than expected. These findings suggest that the polyclonal PFC response to staphylococci is thymus independent, but that the magnitude of the response is regulated by mature T cells. Cultures of macrophage-depleted spleen cells responded to the organisms to an extent similar to that of the control. The 248βH organisms were less capable of stimulating spleen cells of 2-week-old mice (i.e., early maturing B cells) than LPS. However, spleen cells from adult (7-week-old) and aged (9-month-old) mice responded well to both the organisms and LPS. Previous sensitization with the organisms in vivo did not affect any polyclonal responses of spleen cells in vitro to either the organisms or LPS. The role of staphylococcal protein A in the polyclonal PFC response to staphylococci is discussed.     

Settling-site selection and survival of two scale insects,Ceroplastes rubens andC. ceriferus,on citrus trees

We studied settling-site selection and the resulting survival of two sessile scale insects, Ceroplastes rubens and C. ceriferus, in the citrus tree, Citrus unshiu, in central Japan. C. rubens preferred 0-year-old twigs most as a settling-site; the density of nymphs settling on 0-year-old twigs was significantly higher than those on ≥1-year-old twigs, and few nymphs settled on ≥3-year-old twigs. The mean survival rates from settling until reproduction in the next year were significantly higher on more preferred twigs than on less preferred ones. In C. ceriferus, nymphs significantly preferred 1- and 2-year-old twigs to 0- and ≥3-year-old ones, and the mean survival rates on the more preferred 1- and 2-year-old twigs were significantly higher than those on less preferred ≥3-year-old twigs. However, the survival rate on less preferred 0-year-old twigs was slightly higher than those on 1- and 2-year-old ones. Thus, in both species of scale, it was the preferred twigs which were more profitable sites for survival after settling, except for less preferred 0-year-old twigs for C. ceriferus. In both scale species, most mortality was due to growth cessation, which is believed to be related to the twig quality as a food source. Predators and parasitoids were minor mortality factors. Both species showed constant survival rates until the density of settled nymphs exceeded double the “upper-limit” density, whereupon they decreased drastically. Nymphs of C. rubens settling on twigs of high scale density showed a spacing-out distribution, those of C. ceriferus did not. In C. rubens, an increase in preference for originally less profitable twigs at the later stage of the settling season was observed, but not in C. ceriferus. Accordingly, individuals of C. rubens showed a stronger tendency to avoid conspecifics than did C. ceriferus. Although nymphs of the two scales clearly preferred more profitable sites, their settling-site selection did not agree with the predictions from the ideal free distribution theory (Fretwell and Lucas, 1970). The discrepancies were (1) frequent settling on less profitable sites at the early stage of the settling season, (2) insufficient utilization of the most profitable twigs, and (3) virtually 100% mortality on overcrowded twigs under conditions where unoccupied profitable twigs still remained. These discrepancies are thought due to the limited dispersal time of nymphs.     

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.