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.     

Meal feeding schedule and ventromedial hypothalamic lesions do not affect rhythm of pineal serotonin N-acetyltransferase activity in rats

Effects of meal feeding schedule and bilateral lesions of the ventromedial hypothalamus (VMH) on the circadian rhythm of pineal serotonin N-acetyltransferase (SNAT) activity were examined in rats, under LD (12:12) condition. Neither meal feeding nor VMH lesions affected the phase of the circadian rhythm of pineal SNAT activity, but the VMH lesions reduced the level. Meal feeding caused a shift of the phases of the daily rhythms of phosphoenolpyruvate carboxykinase and tyrosine aminotransferase activities in the liver. These findings suggest that the circadian rhythm of pineal SNAT activity is not entrained by the food intake, and that the VMH does not function as a master oscillator of the rhythm.     

Photoreduction of autooxidized albumin-heme hybrid in saline solution: revival of its O(2)-binding ability.

Recombinant human serum albumin (rHSA) incorporating 2-[8-[N-(2-methylimidazolyl)]octanoyloxymethyl]-5,10,15,20-tetrakis(alpha,alpha,alpha,alpha-o-pivalamido)phenylporphinatoiron(II)s (Fe(II)Ps) [rHSA-Fe(II)P] is a synthetic hemoprotein which can bind and release O(2) reversibly under physiological conditions (saline solution [NaCl]: 150 mM, pH 7.3) as do hemoglobin and myoglobin. However, the central ferrous ions of Fe(II)Ps are slowly oxidized to O(2)-inactive ferric forms. Based on the UV-vis. absorption spectroscopy, the majority of the autooxidized Fe(III)Ps in albumin are determined to be six-coordinate high-spin complexes with a proximal imidazole and a chloride anion, which show ligand-to-metal charge transfer (LMCT) absorption at 330 nm. Interestingly, photoirradiation of this LMCT band under an argon atmosphere led to reduction of the central ferric iron of Fe(III)P, allowing the revival of the O(2)-binding ability. The ratio of the photoreduction reached a maximum of 83%, which is probably due to the partial dissociation of the axial imidazole. The same photoirradiation under a CO atmosphere provides the corresponding carbonyl rHSA-Fe(II)P. Laser flash photolysis experiments revealed that the reduction was completed within 100 ns. The quantum yields (Phi) of these photoreductions were approximately 0.01.     

Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins.

In the eucaryotic nucleus, heterogeneous nuclear RNAs exist in a complex with a specific set of proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs). The C proteins, C1 and C2, are major constituents of hnRNPs and appear to play a role in RNA splicing as suggested by antibody inhibition and immunodepletion experiments. With the use of a previously described partial cDNA clone as a hybridization probe, full-length cDNAs for the human C proteins were isolated. All of the cDNAs isolated hybridized to two poly(A)+ RNAs of 1.9 and 1.4 kilobases (kb). DNA sequencing of a cDNA clone for the 1.9-kb mRNA (pHC12) revealed a single open reading frame of 290 amino acids coding for a protein of 31,931 daltons and two polyadenylation signals, AAUAAA, approximately 400 base pairs apart in the 3' untranslated region of the mRNA. DNA sequencing of a clone corresponding to the 1.4-kb mRNA (pHC5) indicated that the sequence of this mRNA is identical to that of the 1.9-kb mRNA up to the first polyadenylation signal which it uses. Both mRNAs therefore have the same coding capacity and are probably transcribed from a single gene. Translation in vitro of the 1.9-kb mRNA selected by hybridization with a 3'-end subfragment of pHC12 demonstrated that it by itself can direct the synthesis of both C1 and C2. The difference between the C1 and C2 proteins which results in their electrophoretic separation is not known, but most likely one of them is generated from the other posttranslationally. Since several hnRNP proteins appeared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as multiple antigenically related polypeptides, this raises the possibility that some of these other groups of hnRNP proteins are also each produced from a single mRNA. The predicted amino acid sequence of the protein indicates that it is composed of two distinct domains: an amino terminus that contains what we have recently described as a RNP consensus sequence, which is the putative RNA-binding site, and a carboxy terminus that is very negatively charged, contains no aromatic amino acids or prolines, and contains a putative nucleoside triphosphate-binding fold, as well as a phosphorylation site for casein kinase type II. The RNP consensus sequence was also found in the yeast poly(A)-binding protein (PABP), the heterogeneous nuclear RNA-binding proteins A1 and A2, and the pre-rRNA binding protein C23. All of these proteins are also composed of at least two distinct domains: an amino terminus, which possesses one or more RNP consensus sequences, and a carboxy terminus, which is unique to each protein, being very acidic in the C proteins and rich in glycine in A1, and C23 and rich in proline in the poly(A)-binding protein. These findings suggest that the amino terminus of these proteins possesses a highly conserved RNA-binding domain, whereas the carboxy terminus contains a region essential to the unique function and interactions of each of the RNA-binding proteins.     

The different effects of recombinant human tumor necrosis factor on rat fibrosarcoma sublines

Summary The antitumor effect of recombinant human tumor necrosis factor (rH-TNF) on two clones of rat fibrosarcoma with different metastatic potential to lymph nodes was examined. The colony formation of clone A, which has high metastatic potential, was completely inhibited by continuous exposure to rH-TNF at 50 U/ml. In contrast, colony formation of clone G, which has low metastatic potential, was not inhibited by high concentrations of rH-TNF (10,000 U/ml). The inhibitory effect of rH-TNF on colony formation by clone A was also observed with a 1-h exposure to rH-TNF. This effect was time and concentration dependent, as determined by the colony assay, ³H-thymidine uptake assay, and ⁵¹Cr-release assay. ³H-thymidine and ³H-uridine uptake per cell of clone A exposed to rH-TNF was not decreased. This suggests that the mechanisms of the antitumor effect of rH-TNF were not due to inhibition of DNA and RNA synthesis of tumor cells. In vivo growth and lymph node metastases of clone A inoculated i.p. to Donryu strain rats were completely suppressed by 14 consecutive i.p. injections of 10⁵ or 10⁶ U/kg per day of rH-TNF. On the other hand the growth of clone G was not influenced by rH-TNF administration.     

Genotoxic effects of o-phenylphenol metabolites in CHO-K1 cells

The effects of microsomal activation and/or deactivation on the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) in cultured Chinese hamster ovary cells (CHO-K1 cells) by o-phenylphenol (OPP) were studied, and concurrently the metabolites were determined. After a 3-h incubation in the presence of 15% S9 mix (45 microliters/ml of S9), OPP (25-150 micrograms/ml) dose-independent SCEs and chromosomal aberrations were induced, while the amount of phenylhydroquinone (PHQ) metabolite produced from OPP did not increase linearly in the higher doses. The maximum induction of chromosomal aberrations was 18% at the 150 micrograms/ml dose, and of SCEs 13.8/cell at 75 micrograms/ml. The corresponding control values were 3% and 5.8/cell. The lowest dose required to induce SCEs in the presence of S9 mix was 25 micrograms/ml. Changing the percent of S9 mix (0-50%) while holding the OPP dose constant (100 micrograms/ml) produced a correlation between SCEs and the production of PHQ. PHQ caused cytogenetic effects both with and without S9 mix, however, in the absence of S9 mix it was more lethal and was oxidized to phenylbenzoquinone (PBQ). These results suggest that the enhanced cytogenetic effects of OPP by the addition of S9 mix correlated with the amount of PHQ produced or with the further oxides of PHQ such as phenylsemiquinone and/or PBQ which are capable of being produced from PHQ spontaneously or by the mixed-function oxidase system.     

N-Terminal Amino Acid Sequence and Processing Site of Sweet Potato Cytochrome c Oxidase Subunit II

The N-terminal amino acid sequence of sweet potato cytochromec oxidase subunit II polypeptide was determined. Comparisonsbetween the sequence and amino acid sequences deduced from thenucleotide sequences of other higher plant subunit II genesindicate a post-translational clevage of N-terminal extensionpart. ¹Present address: Institute of Low Temperature Science, HokkaidoUniversity, Sapporo, 060 Japan. (Received June 13, 1989; Accepted September 8, 1989)     

Effect of disuse on the ultrastructure of the achilles tendon in rats

We examined the influence exerted, through disuse of the hindlimb, on the collagen fibres of the achilles tendon in rats. With disuse the body mass decreased by 28%, and the mass of soleus muscle decreased by 20%. A decrease in the surface area and diameter was observed in the experimental group when compared to the control group. A histogram of the collagen fibres showed a decrease of the thick fibres in the experimental group. The maximum surface area of collagen fibres in the experimental group was seen to be only 43% of that of the control group. These results showed a decrease in the thickness of the collagen fibres of the achilles tendon through disuse. This seemed to suggest that resistance to tension is decreased by disuse.