Molecular cloning of rabbit CARP cDNA and its regulated expression in adriamycin-cardiomyopathy.

A full-length rabbit cDNA of cardiac adriamycin responsive protein (CARP) has been cloned. It shows high levels of identity at the amino acid sequence level (>86%) with the rat, mouse and human homologues. CARP mRNA levels are highly regulated in adriamycin-cardiomyopathy in rabbits.     

Identification of significant regions of transcription factor DP-1 (TFDP-1) involved in stability/instability of the protein

We previously reported the identification of DP-1 isoforms (α and β), which are structurally C-terminus-deleted ones, and revealed the low-level expression of these isoforms. It is known that wild-type DP-1 is degraded by the ubiquitin-proteasome system, but few details are known about the domains concerned with the protein stability/instability for the proteolysis of these DP-1 isoforms. Here we identified the domains responsible for the stability/instability of DP-1. Especially, the DP-1 “Stabilon” domain was a C-terminal acidic motif and was quite important for DP-1 stability. Moreover, we propose that this DP-1 Stabilon may be useful for the stability of other nuclear proteins when fused to them.     

ATM regulates Cdt1 stability during the unperturbed S phase to prevent re-replication

Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27^Kip1 was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCF^Skp2 ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCF^Skp2 pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability.     

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.     

Subunit composition and ATP site labeling of the coated vesicle proton-translocating adenosinetriphosphatase

The partially purified proton-translocating adenosinetriphosphatase [(H+)-ATPase] from clathrin-coated vesicles has been reported to contain eight polypeptides of molecular weights 15,000-116,000 [Xie, X.S., & Stone, D.K. (1986) J. Biol. Chem. 261, 2492-2495]. To determine whether these polypeptides form a single macromolecular complex, we have isolated three monoclonal antibodies which recognize the reconstitutively active (H+)-ATPase in the native, detergent-solubilized state. All three monoclonal antibodies precipitate the same set of polypeptides from either the partially purified enzyme or the detergent-solubilized coated vesicle membrane proteins. The immunoprecipitated polypeptides have molecular weights of 100,000, 73,000, 58,000, 40,000, 38,000, 34,000, 33,000, 19,000, and 17,000. These results thus indicate that this set of polypeptides forms a single macromolecular complex and suggest that they correspond to subunits of the coated vesicle (H+)-ATPase. To identify the ATP-hydrolytic subunit of the coated vesicle (H+)-ATPase, the purified enzyme was reacted with N-ethylmaleimide (NEM) and 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), both of which inhibit activity in an ATP-protectable manner. Labeling was carried out by using [3H]NEM or [14C]NBD-Cl, and the specificity of the reaction was increased by prelabeling of the protein with the nonradioactive reagents in the presence of ATP and by taking advantage of the nucleotide specificity of protection. The principal polypeptide labeled by both [3H]NEM and [14C]NBD-Cl had a molecular weight of 73,000. In addition, this protein was the only polypeptide whose labeling was significantly reduced in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estimation of Type-Specific Neutralizing Antibody to Herpes Simplex Virus Type 2 in Uterine Cervical Cancer Patients by a New Absorption Method

A simple method of estimating type-specific neutralizing antibody to type 2 herpes simplex virus (HSV-2) was devised with the use of the microneutralization system. Serially diluted serum was mixed in the well with a constant amount of type 1 virus (HSV-1), and after 3 days' incubation at 37 C, the plate was irradiated with ultraviolet light. The absorbing HSV-1 consisted of culture fluid plus an extract of infected Vero cells not especially concentrated. The well then received indicator HSV-1 or HSV-2, and after being left at 37 C for 1 hr a suspension of dispersed Vero cells was dropped into the wells, following our standard neutralization procedure. Preliminary tests with rabbit antisera showed that even a low level of HSV-2 antibody was detected by this method, unless an exceptionally high titer of HSV-1 antibody originally coexisted with the HSV-2 antibody. Sera from acutely infected persons testified to the specificity of the antibody so detected. It was revealed by means of the new technique that the rate of HSV-2 antibody was significantly higher in uterine cervical cancer patients than in control women. There was no correlation between the clinical stage of cervical cancer and the presence of HSV-2 antibody.