Structural and functional characterization of subunits of the F0 sector of the mitochondrial F0F1-ATP synthase.

Proteolytic digestion of F1-depleted submitochondrial particles (USMP), reconstitution with isolated subunits and titration with inhibitors show that the nuclear-encoded PVP protein, previously identified as an intrinsic component of bovine heart F0 (F01) (Zanotti, F. et al. (1988) FEBS Lett. 237, 9-14), is critically involved in maintaining the proper H+ translocating configuration of this sector and its correct binding to the F1 catalytic moiety. Trypsin digestion of USMP, under conditions leading to cleavage of the carboxyl region of the PVP protein and partial inhibition of transmembrane H+ translocation, results in general loss of sensitivity of this process to F0 inhibitors. This is restored by addition of the isolated PVP protein. Trypsin digestion of USMP causes also loss of oligomycin sensitivity of the catalytic activity of membrane reconstituted soluble F1, which can be restored by the combined addition of PVP and OSCP, or PVP and F6. Amino acid sequence analysis shows that, in USMP, modification by [14C] N,N'-dicyclohexylcarbodiimide of subunit c of F0 induces the formation of a dimer of this protein, which retains the 14C-labelled group. Chemical modification of cysteine-64 of subunit c results in inhibition of H+ conduction by F0. The results indicate that proton conduction in mitochondrial F0 depends on interaction of subunit c with the PVP protein.

Correlation between rat liver regeneration and mitochondrial energy metabolism.

The time course of changes in mitochondrial energy metabolism during liver regeneration, following partial hepatectomy, is analyzed. For 24 h after surgical operation, a lag phase in the time course of the growth of liver is observed. In this period mitochondria showed a decrease of: (1) the respiratory control index; (2) the rate of oxidative phosphorylation; (3) the amount of immunodetected beta-F1 and F01-PVP subunits of F0F1-ATP synthase. No decrease, but instead a small increase in the content of mRNA for beta-F1 was observed in this phase. After this lag phase the growth of liver started, the content of mRNA for beta F1, as well as the level of immunodetected mitochondrial beta-F1 and F01-PVP subunits, increased and oxidative phosphorylation recovered. Analysis of the relative beta F1 protein/mRNA ratio indicates a decrease of beta F1 translational efficiency which remained low up to 72 h after partial hepatectomy and reached the same ratio of control at 96 h. It is concluded that the regenerating capability of rat liver is correlated with the efficiency of oxidative phosphorylation.

[Biological risk in the operating room: microbiological monitoring of the environment and analysis of the associated variables]. / Rischio biologico in sala operatoria: monitoraggio micro- biologico ambientale ed analisi delle variabili associate.

The authors present an environmental microbiological monitoring programme carried out over a period of 15 months in 16 operating theatres performing specific types of surgery. The levels of microbial contamination of the air and of four of the most representative surfaces of the clean area were determined at 3 different times for each theatre, both before and during surgery. For the air assessment, the results obtained with three different samplers, Sed-3 Unit, SAS and RCS, were compared. The results were on the whole acceptable, but some poor conditions were detected during the theatres in use, especially in general surgery theatres; in some of these the floors showed levels of contamination consistently exceeding the reference limits. As the monitoring programme proceeded, the microbiological quality of the air and of the surfaces in the theatres notably improved. The three air samplers showed different conditions expressed with units of measure not always readily comparable. For active samplers, the bacterial load determined by RCS, although less variable, were always higher (even 2-3 fold) than those obtained with the SAS. Passive sampling takes longer but determines the real risk of infection for the patients; contemporary determination of the fall-out and the CFU/m3 helps to identify the occupational risks. Since the limit values established by the ISPESL guidelines for the operating theatres have been defined only for active samplers, there is urgent need for more exhaustive national guidelines to define similar values also for passive sampling. The Authors conclude stressing the importance of promoting continuing information-education programmes to heighten the awareness of all those involved in operating theatre activities.

The effect of F0 inhibitors on the Vibrio alginolyticus membrane ATPase.

The inhibition of membrane ATPase from the marine alkalotolerant bacterium Vibrio alginolyticus by DCCD, triphenyltin and venturicidin was studied. DCCD proved to be an irreversible inhibitor, while venturicidin and triphenyltin produced a reversible inhibitory effect. The DCCD-binding proteolipid was identified in the membrane preparations. The effect of the inhibitors on ATPase activity and ATP-dependent Na(+)-transport in V. alginolyticus subcellular vesicles is discussed.

Identification of nucleus-encoded F0I protein of bovine heart mitochondrial H+-ATPase as a functional part of the F0 moiety.

The F0I protein of apparent Mr 27,000, previously characterized [(1988) Eur. J. Biochem. 173, 1-8] as a genuine component of bovine heart F0, has been sequenced and shown to be identical with the nucleus encoded 24,668 Da protein characterized earlier [(1987) J. Mol. Biol. 197, 89-100]. It is directly shown by proteolytic cleavage and reconstitution experiments that this protein, denoted here as PVP from the single-letter codes of the last three residues of the N-terminus, is involved in proton conduction by F0 and in its sensitivity to oligomycin.

Activation of a complex of ATPase with the natural protein inhibitor in submitochondrial particles.

Almost all ATPase molecules in submitochondrial particles, isolated from beef heart mitochondria in the presence of MgATP, are in an active complex with the natural protein inhibitor (IF1). In de-energized particles at high ionic strength a slow and irreversible ATPase activation is found to occur due to a dissociation of the enzyme-inhibitor complex. The pH-dependence of this process points out that deprotonation of IF1 molecule is an essential step in the dissociation of the complex. Zn2+ sharply accelerates ATPase activation, probably via binding with the deprotonated form of IF1. ATPase activation is completely prevented by MgATP, indicating the formation of a transient enzyme-inhibitor complex retaining ATPase activity.

The gamma subunit of F1 and the PVP protein of F0 (F0I) are components of the gate of the mitochondrial F0F1 H(+)-ATP synthase.

The gamma subunit of the F1 moiety of the bovine mitochondrial H(+)-ATP synthase is shown to function as a component of the gate. Addition of purified gamma subunit to F0-liposomes inhibits transmembrane proton conduction. This inhibition can be removed by the bifunctional thiol reagent diamide. Immunoblot analysis shows that the diamide effect is likely due to disulphide bridging of the gamma subunit with the PVP protein of the F0 sector.

Role of the carboxyl-terminal region of the PVP protein (F0I subunit) in the H+ conduction of F0F1 H+-ATP synthase of bovine heart mitochondria.

By means of protein sequencing, labelling with thiol reagents and reconstitution studies it is shown that the carboxyl-terminal region of the PVP protein (F0I subunit, nuclear-encoded protein of Mr 25,000) of mitochondrial F0 promotes transmembrane proton conduction by F0 and the sensitivity of this process to oligomycin.

Mitochondrial F0F1 H+-ATP synthase. Characterization of F0 components involved in H+ translocation.

The membrane F0 sector of mitochondrial ATP synthase complex was rapidly isolated by direct extraction with CHAPS from F1-depleted submitochondrial particles. The preparation thus obtained is stable and can be reconstituted in artificial phospholipid membranes to result in oligomycin-sensitive proton conduction, or recombined with purified F1 to give the oligomycin-sensitive F0F1-ATPase complex. The F0 preparation and constituent polypeptides were characterized by SDS-polyacrylamide gel electrophoresis and immunoblot analysis. The functional role of F0 polypeptides was examined by means of trypsin digestion and reconstitution studies. It is shown that, in addition to the 8 kDa DCCD-binding protein, the nuclear encoded protein [(1987) J. Mol. Biol. 197, 89-100], characterized as an intrinsic component of F0 (F0I, PVP protein [(1988) FEBS Lett. 237,9-14]) [corrected] is involved in H+ translocation and the sensitivity of this process to the F0 inhibitors, DCCD and oligomycin.

Age related changes of the mitochondrial energy metabolism in rat liver and heart.

The influence of aging on mitochondrial energy metabolism of rat liver and rat heart has been studied by analysis of (i) respiratory rate of succinate-supplemented mitochondria in state III (coupled state in the presence of ADP + Pi); (ii) the rate of synthesis of ATP in succinate-supplemented mitochondria; (iii) the ATP hydrolase activity of sonicated submitochondrial particles. The results indicate a decrease of the F(0)F(1)-ATP synthase activity in mitochondria isolated from both organs of aged (24-month-old) as compared to young (3-month-old) rats which was accompanied by a decrease of immunodetected amount of the beta-F(1) (subunit of the catalytic F(1) sector of F(0)F(1)-ATP synthase). These effects were more evident in heart than in liver mitochondria. Analysis of the mitochondrial content of glutathione (GSH), an essential intracellular antioxidant agent, shows a decrease in mitochondria of both tissues of aged animals. Exposure of submitochondrial particles to free radicals, produced either by (60)Co or by respirtory chain (in presence of the inhibitor antimycin A) mimicked the alterations of F(0)F(1) ATP synthase obsreved in submitochondrial particles of aged rats. The possible relationship between aging process, free radical production and alteration of mitochondrial oxidative phosphorylation is discussed.

Age-dependent changes in the mitochondrial F0F1 ATP synthase.

The age dependence of ATP hydrolase activity and oligomycin sensitive passive proton conduction in sonicated submitochondrial particles of rat brain and rat heart has been investigated. The results show an increase of Vmax of the ATP hydrolase activity and decrease of oligomycin sensitive passive proton conduction with increase of the age of rats from 3 to 6 months. Decrease of ATPase activity and increase of oligomycin sensitive proton conduction occur with further aging to 24 months. Immunoblot analysis shows that both the F(1) and F(0) contents of mitochondria vary with the age of rats, the former exhibiting relatively larger changes.

Evaluation and comparison of hemocoagulation parameters both in serum and in ascites from patients with gynecologic malignancies.

The authors report the results obtained investigating PT - PTT - Fbg - Alcohol test - FDP - AT III in the serum and FDP in the ascitic fluid of 18 patients with ovarian cancer, 2 patients with pelvic relapse of cervical cancer and 10 patients with cirrhosis of the liver. It is pointed out the presence of FDP in the ascitic fluid in large amounts, so it not seems to be an ovarian cancer specific marker, but an aspecific marker of malignancy.

[Clinical investigation of hyaluronidase and nerve growth factor (NGF) at the ocular level: detailed review of the international literature]. / Sperimentazione clinica di ialuronidasi e del nerve growth factor (NGF) a livello oculare: revisione accurata della bibliografia internazionale.

In this paper the authors analyse the international bibliography about clinic tests on hyaluronic acid and Nerve Growth Factor at ocular level. This study was performed on request of a private oculistic clinic in the centre of Rome about the clinic test of the drugs mentioned above. Therefore, this paper is a joint work of Public Health physicians and oculists.

[Preventive and therapeutic approach to tuberculosis in the general, and high-risk populations]. / Approccio preventivo e terapeutico della malattia tubercolare nella popolazione generale e nei soggetti a rischio.

Authors in the present contribute illustrate the tuberculosis disease in all aspects, the infection, the disease in active phase and in the resistant phase, the law, the prevention and the professional risk in exposed subject, and to consider the epidemiological aspect. Authors hope a coordinate approach between the different figure involved in the therapy and prevention of tuberculosis disease.

[Delphi study in the identification of research need in occupational medicine in Italy]. / Studio Delphi per l'identificazione delle priorità di ricerca in medicina del lavoro in Italia.

The aim of the study was to identify the research priorities and strategies in Occupational Safety and Health (OSH) in Italy with a high degree of consensus. Based on the Delphi technique, a two-phase questionnaire was sent to experts at Universities, Local Health Units (ASLs) and Trade Unions. In the first phase, experts were requested to indicate three research topics in the OSH sector. A total of 27 topics for both universities and ASLs and 18 topics for Trade Unions were identified. In the second phase, experts were requested to assign a score from one (low relevance) to five (high relevance) to each topic previously identified. On the basis of a mean score reported for each topic, two ranked lists of priorities, one referred to universities and ASLs the other referred to Trade Unions, were formulated. The highest priority identified by universities and ASLs was occupational carcinogenesis, followed by quality in occupational medicine. Workers' information, education and participation was also identified with a high degree of priority. For Trade Unions, occupational cancers as well as training, information, participation and prevention awareness had the highest priority. Trade Unions also identify small-industries and occupational accidents as topics with high priority for research development. This study allowed a high degree of consensus to be reached regarding the research priorities in the OSH sector in Italy. Differences in the topics identified, or regarding the mean score of topics commonly identified, were related to the origin of the expert recruited to this study (University, ASLs or Trade Unions) and, for universities and ASLs, to the geographical area. In the authors' opinion, the full transfer of existing scientific data to occupational health practice and the harmonization of the priorities identified by this investigation are crucial if the research needs in the OSH sector in Italy are to be met.

Crossed beam studies of radical-radical reactions: O(3P) + C3H5 (allyl).

The dynamics of the radical-radical reaction O((3)P) + C(3)H(5) has been investigated by means of the crossed molecular beam technique with mass spectrometric detection at a collision energy of 73.0 kJ mol(-1); the reaction mechanism of the H-displacement channel has been elucidated, while experimental evidence of the occurrence of one or more C-C bond-breaking channels at this collision energy has been obtained.

[Isoproterenol causes changes in the mitochondrial energy metabolism in the rat heart]. / Isoproterenolo causa alterazioni del metabolismo energetico mitocondriale nel cuore del ratto.

In this paper we report observations on kinetic and structural characteristics of mitochondrial ATP synthase of rat-heart after subcutaneous injection of isoproterenol. The results obtained indicate: a decrease of respiratory rate either in absence (state 4) or in presence (state 3) of oxidative phosphorylation; decrease of respiratory control ratio; decrease of ATP hydrolase activity in sonic submitochondrial particles; decrease of relative content of the catalytic subunit F1 with respect to the membrane sector F0. The data obtained are in favour of the hypothesis that isoproterenol causes structural and functional alterations of mitochondrial ATP synthase.