Analysis of molecular mechanisms of ATP synthesis from the standpoint of the principle of electrical neutrality.

Theories of biological energy coupling in oxidative phosphorylation (OX PHOS) and photophosphorylation (PHOTO PHOS) are reviewed and applied to ATP synthesis by an experimental system containing purified ATP synthase reconstituted into liposomes. The theories are critically evaluated from the standpoint of the principle of electrical neutrality. It is shown that the obligatory requirement to maintain overall electroneutrality of bulk aqueous phases imposes strong constraints on possible theories of energy coupling and molecular mechanisms of ATP synthesis. Mitchell's chemiosmotic theory is found to violate the electroneutrality of bulk aqueous phases and is shown to be untenable on these grounds. Purely electroneutral mechanisms or mechanisms where the anion/countercation gradient is dissipated or simply flows through the lipid bilayer are also shown to be inadequate. A dynamically electrogenic but overall electroneutral mode of ion transport postulated by Nath's torsional mechanism of energy transduction and ATP synthesis is shown to be consistent both with the experimental findings and the principle of electrical neutrality. It is concluded that the ATP synthase functions as a proton-dicarboxylic acid anion cotransporter in OX PHOS or PHOTO PHOS. A logical chemical explanation for the selection of dicarboxylic acids as intermediates in OX PHOS and PHOTO PHOS is suggested based on the pioneering classical thermodynamic work of Christensen, Izatt, and Hansen. The nonequilibrium thermodynamic consequences for theories in which the protons originate from water vis-a-vis weak organic acids are compared and contrasted, and several new mechanistic and thermodynamic insights into biological energy transduction by ATP synthase are offered. These considerations make the new theory of energy coupling more complete, and lead to a deeper understanding of the molecular mechanism of ATP synthesis.

Mitochondria from the salt-tolerant yeast Debaryomyces hansenii (halophilic organelles?).

The yeast Debaryomyces hansenii is considered a marine organism. Sea water contains 0.6 M Na(+) and 10 mM K(+); these cations permeate into the cytoplasm of D. hansenii where proteins and organelles have to adapt to high salt concentrations. The effect of high concentrations of monovalent and divalent cations on isolated mitochondria from D. hansenii was explored. As in S. cerevisiae, these mitochondria underwent a phosphate-sensitive permeability transition (PT) which was inhibited by Ca(2+) or Mg(2+). However, D. hansenii mitochondria require higher phosphate concentrations to inhibit PT. In regard to K(+) and Na(+), and at variance with mitochondria from all other sources known, these monovalent cations promoted closure of the putative mitochondrial unspecific channel. This was evidenced by the K(+)/Na(+)-promoted increase in: respiratory control, transmembrane potential and synthesis of ATP. PT was equally sensitive to either Na(+) or K(+). In the presence of propyl-gallate PT was still observed while in the presence of cyanide the alternative pathway was not active enough to generate a Delta Psi due to a low AOX activity. In D. hansenii mitochondria K(+) and Na(+) optimize oxidative phosphorylation, providing an explanation for the higher growth efficiency in saline environments exhibited by this yeast.

10-12 years follow-up of highly bacillated BL/LL leprosy patients on combined chemotherapy and immunotherapy.

This study reports the follow-up results of 36 highly bacillated untreated BL/LL cases who were serially allocated to three treatment groups. Group I patients received a modified WHO regimen (Rifampicin 600 mg once a month supervised, 50 mg of Clofazimine and 100 mg of Dapsone daily unsupervised) and BCG 0.1 mg per dose 6 monthly; group II patients received the same multi-drug treatment (MDT) and Mw (2 x 10(8) killed bacilli per dose) 6 monthly: group III patients received the same MDT with 0.1 ml of distilled water 6 monthly and acted as a control. Treatment was continued till smear negativity. All these three groups were comparable by their initial clinical score, bacteriological index (BI), viable bacilli as assessed by the mouse foot pad (MFP), bacillary adenosine triphosphate (ATP) content and also histologically at the time of starting treatment. All these parameters were evaluated every 6 months. The vaccines were well tolerated. All the patients in group I became smear negative by 3.5 years, in group II in 3 years whereas those in group III took 5 years. The incidence of reactions was the same in all the groups during the first 2 years, however, patients of group III (MDT + placebo) continued to have reactions up to 3 years. No viable bacilli could be detected in the local and distal sites as estimated by MFP and bacillary ATP after 12 months in both the immunotherapy groups. These could be detected in patients on MDT alone up to 24 months of therapy. Histologically patients in both the immunotherapy groups (groups I and II) showed accelerated granuloma clearance, histological upgrading and non-specific healing without granuloma formation both at the local and distal sites and this was achieved much earlier compared to the MDT + placebo group. Thus, by the addition of immunotherapy the effective treatment period of achieving bacteriological negativity could be reduced by about 40%, time period of reactions reduced by 33% and there were no reactions and/or relapses in the 10-12 years post-treatment follow-up.

Limited ATP generation in cells of Mycobacterium leprae Thai-53 strain in enriched Kirchner liquid medium containing adenosine.

The ATP generation in cells of Mycobacterium leprae Thai-53 strain takes place in vitro when the cells are cultivated in Kirchner liquid medium, pH 7.0, enriched with egg-yolk solution, pyruvate, transferrin, and adenosine at 30 degrees C. Among the supplements, adenosine was key and critical for the ATP generation. The optimal concentration of adenosine was 50 micrograms/ml of the medium. ATP generation, however, was limited; the rates of increase in ATP content extracted from the cells were approximately two- to threefold compared to that of the starting samples, and the increase reached a maximum at 4 or 6 weeks after incubation. No significant ATP generation in M. leprae cells was demonstrated in medium at pH 6.2 or pH 6.6, in the original Kirchner medium with or without adenosine, or when cultured at 37 degrees C, or when containing an antileprosy drug. No detectable increase in the number of M. leprae cells was observed with the increase in intracellular ATP content and DNA replication. No effect was seen with renewal of the cultured medium by freshly prepared medium at 6 weeks' cultivation on the progressive ATP generation in M. leprae.

Effect of trifluoperazine on in vitro ATP synthesis by Mycobacterium leprae.

The effect of trifluoperazine (TFP), a calmodulin antagonist, was investigated on in vitro ATP levels of human derived Mycobacterium leprae. M. leprae were obtained from biopsies from multi-bacillary forms of leprosy and were incubated in a modified Dubos medium system which supports limited in vitro synthesis of M. leprae. This incubation was carried out in the absence and presence of different concentrations of trifluoperazine. Samples for estimation of bacillary ATP levels were taken at day 0 and at 14 days of incubation. TFP inhibited ATP levels in M. leprae and this inhibitory effect was marginal at 2.5 microg ml(-1) (35% inhibition), highly significant at 5 microg ml(-1) (87% inhibition) and almost total at 10 microg ml(-1) (98.5% inhibition). This compound appears to have potential as an anti-leprotic drug and also as a broad spectrum anti-mycobacterial agent in view of its anti-tubercular activity reported earlier.

Energy generation mechanisms in the in vitro-grown Mycobacterium lepraemurium.

Mycobacterium lepraemurium was cultivated on Ogawa egg-yolk medium and its energy coupling mechanisms were investigated. Cell-free extracts prepared from in vitro-grown cells catalyzed phosphorylation coupled to the oxidation of generated NADH, added NADH, and succinate-yielding ratios of phosphorus moles incorporated into high-energy bonds to oxygen atoms utilized (P/O ratios) of 0.75, 0.52, and 0.36, respectively. Ascorbate oxidation alone or in the presence of tetramethyl-p-phenyline-diamine (TMPD) did not yield any adenosine triphosphate (ATP). However, ascorbate in the presence of added cytochrome c was coupled to ATP synthesis and yielded a P/O ratio of 0.12. The oxidative phosphorylation was uncoupled by all of the uncouplers used without any inhibition of oxygen consumption. ATP generation coupled to NADH oxidation was completely inhibited by the flavoprotein inhibitors, such as rotenone and amytal; these inhibitors had no effect, however, on ATP synthesis associated with succinate oxidation. Antimycin A or 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO) and cyanide inhibited markedly the oxidations of NADH and succinate as well as the coupled ATP generation. The phosphorylation coupled to ascorbate plus cytochrome c was not affected by either of the flavoprotein inhibitors or by antimycin A or HQNO, but was completely inhibited by cyanide. The thiol-bearing agents p-chloromercuribenzoate (PCMB) and N-ethylmaleimide were the potent inhibitors of the phosphorylation associated with the oxidation of NADH and succinate. The results indicate that the three energy-coupling sites are functional in the respiratory chain of in vitro-grown M. lepraemurium.

Studies on energy synthesis in M. leprae.

ATP measurements have been earlier used to study the effect of various nutrients on the growth and multiplication of M. leprae. In a preliminary study, we had observed that glycerol and asparagine stimulated the ATP synthesis by M. leprae but this was marginal and not sustained. We have extended the study to investigate the role of various environmental factors which could affect this ATP synthesis. It has been observed that ATP synthesis was better and sustained for a longer period i.e. upto 2 weeks if the M. leprae were incubated at pH 6-6.5 and at 30-33 degrees C in the modified Dubos and Sauton's media. The pH and temperature above these values were suboptimal. It is concluded that temperature and pH are important factors for maintenance and synthesis of ATP by M. leprae.

Metabolic studies on mycobacteria. V. A preliminary report on the ATP synthesis by mycobacteria including M. leprae by using different substrates.

By deletion and addition of various substrates in Sauton's and Dubos media, an experimental system has been standardised in which the role of various nutrients in the energy synthesis of mycobacteria can be determined. By using this system with cultivable mycobacteria it was observed that glycerol and asparagine are the important ingredients for ATP synthesis by mycobacteria. Glucose further enhanced the ATP synthesis and growth of these mycobacteria. In the media containing asparagine or glycerol, there was marginal increase in the ATP in the M. leprae suspensions initially but this was not sustained and there was no progressive increase in biomass or multiplication. When M. leprae was incubated in the media from which both these substrates were deleted, there was progressive decline in ATP levels right from the beginning. From these preliminary results, it appears that asparagine and glycerol may be useful as substrates for ATP synthesis by M. leprae and need to be investigated further. In depth studies are necessary to find out the factors which results in the inability of M. leprae to utilise these and other substrates in a substrained manner for its multiplication and growth in artificial media.